Escin Sodium Improves the Prognosis of Acute Pancreatitis Via Promoting Cell Apoptosis by Suppression of the ERK/STAT3 Signaling Pathway

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Hindawi Oxidative Medicine and Cellular Longevity Volume 2021, Article ID 9921839, 26 pages https://doi.org/10.1155/2021/9921839

Research Article Escin Sodium Improves the Prognosis of Acute Pancreatitis via Promoting Cell Apoptosis by Suppression of the ERK/STAT3 Signaling Pathway

Qian Zhang ,1 Chen Zhao,2 Lei Zhang,3 Kai Sun,4 Linlin Yu,5 Xianming Wang,4 Lei Ren,4 Nan Zhang,3 Chengyu Chen,6 Ju Liu,7 Haimei Wang ,3 and Hu Tian 4

1Department of General Surgery, Shandong Provincial Qianfoshan Hospital, Shandong University of Traditional Chinese Medicine, China 2Weihai Hospital Affiliated to Shandong University of Traditional Chinese Medicine, No. 29, Qingdao North Road, Huancui District, Weihai City Shandong Province, China 3College of First Clinical Medicine, Shandong University of Traditional Chinese Medicine, 16369 Jingshi Road, Lixia District, Jinan City, Shandong Province, China 4Department of General Surgery, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, No. 16766, Jingshi Road, Jinan City, Shandong Province, China 5Shandong Hospital of Traditional Chinese Medicine Affiliated to Shandong University of Traditional Chinese Medicine, 16369 Jingshi Road, Lixia District, Jinan City, Shandong Province, China 6Department of Thoracic Surgery, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, No. 16766, Jingshi Road, Jinan City, Shandong Province, China 7Laboratory of Microvascular Medicine and Medical Research Center, Shandong Provincial Qianfoshan Hospital, The First Affiliated Hospital of Shandong First Medical University, No. 16766, Jingshi Road, Jinan City, Shandong Province, China

Correspondence should be addressed to Hu Tian; [email protected]

Received 5 March 2021; Revised 31 May 2021; Accepted 8 July 2021; Published 13 August 2021

Academic Editor: Ana Lloret

Copyright © 2021 Qian Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Acute pancreatitis (AP), an inflammatory disorder of the pancreas, can cause systemic inflammatory responses. Escin Sodium (ES), a natural mixture of triterpene saponins extracted from the dry ripe fruit of Fructus Aesculi or horse chestnut crude, has been demonstrated to have antiedematous, anti-inflammatory, and antiexudative effects. We here aim to investigate the effects of ES pretreatment on AP in vivo and in vitro and explore its potential molecular mechanism. In the present study, we demonstrated that ES pretreatment could apparently decrease amylase and lipase, downregulate inflammatory cytokines, and attenuate pancreatic damage. Additionally, the increased expression of apoptotic-related proteins and the results of flow cytometry demonstrated the effects of ES on promoting apoptosis in acinar cells. Moreover, ES could enhance mitochondrial membrane potential (MMP, ΔΨm) and reactive oxygen species (ROS) level and reduce intracellular calcium concentration, which are closely related to mitochondrial-mediated death. The effect of ES pretreatment on acinar cell apoptosis was furtherly confirmed by the regulatory pathway of the ERK/STAT3 axis. These results suggest that ES attenuates the severity of AP by enhancing cell apoptosis via suppressing the ERK/STAT3 signaling pathway. These findings provide evidence for ES which is treated as a novel and potent therapeutic for the treatment of AP.

1. Introduction gastrointestinal disorders in many countries [1] and has become an economic burden to patients and health care sys- Acute pancreatitis (AP)—an inflammatory disorder of the tem [2]. According to the revision of the Atlanta (2012) [3], pancreas—is the leading cause of admission to hospital for the classification of AP defines three degrees of severity: mild 2 Oxidative Medicine and Cellular Longevity acute pancreatitis, moderately severe acute pancreatitis, and H H severe acute pancreatitis (SAP). SAP is characterized by per- O O O sistent organ failure that results in multiple organ dysfunction syndrome (MODS) and systemic inflammatory O O O response syndrome (SIRS) by activation of cytokine cascades during the early phase [4–7]. Patients who develop MODS or H SIRS within the first few days are suffering from the increased risk of death, with mortality reported to be as great as 36– 50% [4, 5, 7]. At present, many theories such as pancreatic self-digestion, inflammatory responses, oxidative stress, and calcium overloaded and cell apoptosis are supposed to explain the underlying mechanism of AP. However, the path- ogenesis of AP is complicated and has not been elucidated + completely yet. Na H O The outcome of AP is determined by the type of acinar O H cell death. The type of cell death is as follows: apoptosis, O O necrosis, autophagy, necroptosis, pyroptosis, etc. [8]. As O studies showing, the severity of acute pancreatitis is correlated with necrosis and inversely with apoptosis [9]. A shift H O O O of necrosis to apoptosis can ameliorate the prognosis of acute pancreatitis. The management of AP covers supportive care O O O H (e.g., isotonic intravenous fluids and pain control) and nutri- OOH tion [10]. Although researchers have made great efforts in H acute pancreatitis, there is still no specific drug for it. O H O H Active compounds of many plants are used for medical O demands and provide additional therapeutic effects for mod- H O H ern medicine [11]. Many agents currently used in the clinic Figure 1: Chemical structure of Escin Sodium. are from natural plants widely, such as artemisinin, aspirin, taxanes, and Vinca alkaloids. Escin Sodium (ES), C55H83NaO23 (Figure 1), a natural mixture of triterpene number: H200003239. Sodium taurocholate was purchased saponins extracted from the dry ripe fruit of Fructus Aesculi from Solarbio Science & Technology Co., Ltd. (Beijing, or horse chestnut crude [12–15], was used as medicine for China). PD98059 (ERK1/2 inhibitor) were purchased from centuries. ES has been demonstrated to have antiedematous, MCE (USA). Pentobarbital sodium was purchased from anti-inflammatory, and antiexudative effects [12, 15], having Nanjing Sunshine Biotechnology Co., Ltd. (Nanjing, China). shown excellent tolerability [15]. Recent studies have identified that ES can induce apoptosis in various diseases, such as 2.2. Animal Experiments. Adult male Sprague Dawley (SD) human osteosarcoma [16], renal cancer cells [17], human rats (250-300 g) were purchased from Jinan Pengyue Labora- acute leukemia Jurkat T cells [15], and hepatocellular carci- tory Animal Co., Ltd. (Jinan, China). Animal experiments noma [13]. However, the role of ES on AP and relevant were approved by the Animal Care and Use Committee of mechanism still remains unclear. the First Affiliated Hospital of Shandong First Medical Uni- ERK—an important molecule in the classical MAPK cas- versity (Jinan, China) according to the National Institutes cade pathway, regulating many cell functions, including apo- of Health Guidelines for the Care and Use of Laboratory Ani- ° ptosis, proliferation, and migration [18, 19]—can widely mals. Rats were housed at room temperature (25 C approxi- activate downstream molecules. STAT3 (signal transducer mately) with a 12 h light/12 h dark cycle and humidity of and activator of transcription), one of the downstream of 50%. Food and water were provided ad libitum. ERK, is also linked with survival [20]. Studies have shown A total of 50 male SD rats were randomly divided into 5 that phosphorylation of ERK can drive phosphorylation of groups: control (Con) group treated with 0.9% saline solu- STAT3 [21]. But it is still unknown whether ERK/STAT3 tion, AP group (model group), and 3 groups pretreated with gets involved in the process of AP. different concentrations of Escin Sodium (1 mg/kg, 3 mg/kg, In this study, we demonstrated that ES can optimize the and 6 mg/kg). Escin Sodium was dissolved in 0.9% saline prognosis of acute pancreatitis. We furtherly explored the solution and was intraperitoneally injected 1 h before model- underlying mechanisms and acquired the results indicating ing. Referring to the modeling method of Li et al. [22], rats that ES promotes apoptosis in STC-induced acute pancreati- were anesthetized with 3% pentobarbital sodium and the sur- tis via downregulating the ERK/STAT3 signaling pathway. gical field was completely exposed. The proximal bile duct was occluded at the hepatic portal by a vascular clamp tem- 2. Materials and Methods porarily, followed by retrograde injection of 5% sodium taurocholate (1 mg/kg body weight) in the distal end of the 2.1. Reagents. Escin Sodium was purchased from Shandong biliary-pancreatic duct. Changes in pancreatic tissues includ- Luye Pharmaceutical Co., Ltd. (Yantai, China), registration ing edema, hemorrhage, and congestion were observed for 5 Oxidative Medicine and Cellular Longevity 3 minutes. Subsequently, the vascular clamp was removed and sections were blocked by 5% normal goat serum for 45 min at sutured the incision. Rats in the control group were anesthe- room temperature and incubated with rabbit antibodies tized in the same manner, and 0.9% saline solution was against MPO (1: 400, Proteintech Group Inc., Wuhan, injected into the distal end of the biliary-pancreatic duct. China), NLRP3 (1 : 50, Proteintech Group Inc., Wuhan, Animals were sacrificed at 24 h after injection of 5% China), GSDMD (1 : 100, Proteintech Group Inc., Wuhan, sodium taurocholate. Blood samples were collected to deter- China), and caspase-1 (1 : 100, Proteintech Group Inc., ° mine serum digestive enzymes. The pancreatic tissues were Wuhan, China) in 4 C overnight. The sections were washed removed immediately and washed with cold 0.9% saline solu- in PBS 5 min/3 times and incubated with a secondary anti- tion injection 3 times. Then, the tissues were divided into 3 body for 1 hour. Then, DAB working solution (Boster Bio- sections randomly for morphological examination, Western logical Technology Co., Ltd, Wuhan, China) was dropped blotting, and real-time reverse transcription (RT) PCR. on the slides and incubated at room temperature for 1 min. Redyeing with hematoxylin was performed for 10 seconds 2.3. Cell Culture and Interventions. AR42J, rat exocrine pan- and washing with tap water for 1 min. Finally, all sections creas cells, were purchased from the Institute of Shanghai were dehydrated and sealed with neutral resin. The slides Fuheng Cell Biology (Shanghai, China) and were cultured were observed under a light microscope (×200). Data were in High-Glucose DMEM (Meilunbio, Dalian, China) con- analyzed using Image-Pro Plus software 6.0 (Media Cyber- taining 20% fetal bovine serum (FBS) (Gibco, United States) netics, Silver Spring, MD, USA). and 1% 100 U/mL penicillin and 100 μg/mL streptomycin P/S (Invitrogen, Carlsbad, CA, USA), in a humidified incuba- 2.5. Biochemical Analysis. Blood samples and cell superna- ° tor at 37 C with an atmosphere of 5% CO2. The AR42J cells tant were obtained to determine amylase and lipase levels (1×106 cells/well approximately) were seeded in 6-well after induction of acute pancreatitis with STC. The serum ° plates and washed with D-PBS (PBS without calcium and was centrifuged at 3000g for 10 min at 4 C. The levels of amy- magnesium) every 48 h before the medium changed. Two lase and lipase were analyzed by a colorimetric method using hours before intervention, the cells were incubated in a a commercial kit for amylase or lipase (Jiancheng Co., Nan- High-Glucose DMEM without FBS and P/S. jing, China). AR42J cells were randomly divided into different groups, respectively, as follows: 2.6. Reverse Transcription- (RT-) PCR and Real-Time- (q-) Control group: cultured with a 2 mL High-Glucose PCR. Real-time RT-PCR (qRT-PCR) was used to analyze DMEM. mRNA transcripts in rat pancreas and AR42J cells. The Tri- AP group: cultured with a 2 mL High-Glucose DMEM zol reagent (Invitrogen, California, USA) was used to isolated with the concentration of 390 μM STC for 4 h and 8 h, total RNA from the pancreas and AR42J cells following the respectively. manufacturer’s protocol, then converted to cDNA according AP+Escin Sodium group: cultured with a 2 mL High- to the manufacturer’s instructions (Vazyme Biotech Co., Ltd, Glucose DMEM with the concentration of 10 μg/mL, Nanjing, China). Quantitative real-time polymerase chain 20 μg/mL, and 30 μg/mL Escin Sodium for 1 h and then reaction (PCR) was performed by using SYBR Green Super- followed by 390 μM STC for 4 h and 8 h, respectively. Mix (Vazyme Biotech Co., Ltd, Nanjing, China) in the AP+PD98059 group: cultured with a 2 mL High-Glucose CFX96™ Real-time System (Bio-Rad, United States). The DMEM with the concentration of 10 μM PD98059 (an inhib- primer sequences were as follows: itor of ERK1/2 signal) for 1 h and then followed by 390 μM TNF-α: Forward: 5′-GGCGTGTTCATCCGTTCTC-3′ STC for 4 h and 8 h, respectively. Reverse: 5′-CTTCAGCGTCTCGTGTGTTTCT-3′ AP+Escin Sodium+PD98059 group: cultured with a 2 mL IL-6: Forward: 5′-ATTGTATGAACAGCGATGATG High-Glucose DMEM with the concentration of 10 μM ′ μ CAC-3 PD98059 and 20 g/mL Escin Sodium for 1 h, then followed ′ ′ by 390 μM STC for 4 h and 8 h, respectively. Reverse: 5 -CCAGGTAGAAACGGAACTCCAGA-3 IL-10: Forward: 5′-CAGACCCACATGCTCCGAGA-3′ 2.4. Histological Analysis and Immunohistochemistry. One Reverse: 5′-CAAGGCTTGGCAACCCAAGTA-3′ portion of pancreatic tissue was selected randomly and fixed IL-1β: Forward: 5′-CCCTGAACTCAACTGTGAAAT in 10% neutral-buffered formalin for 48 h, then embedded in AGCA-3′ ffi μ para n. The tissues were cut into 4 m thick sections and Reverse: 5′-CCCAAGTCAAGGGCTTGGAA-3′ stained with hematoxylin and eosin. The assessment of IL-18: Forward: 5′-GACTGGCTGTGACCCTATCTG edema, inflammation, vacuolization, and necrosis was per- ′ – TGA-3 formed according to Schmidt et al. [23 25] (Table 1). For ′ ′ immunohistochemistry, paraffin-embedded pancreas sec- Reverse: 5 -TTGTGTCCTGGCACACGTTTC-3 ° ′ tions (4 μm) were heated at 70 C for 2 h and rehydrated by GAPDH: Forward: 5 -GGCACAGTCAAGGCTGAGA ′ xylene and different concentrations of alcohol. Antigen was ATG-3 retrieved by pressure cooker in 0.01 mol/L citrate buffer at Reverse: 5′-ATGGTGGTGAAGACGCCAGTA-3′ ° constant 100 C for 2 minutes, then cooled in cold water for Relative mRNA levels were normalized to GAPDH 2 hours. Endogenous peroxidase activity was blocked by 3% mRNA, and all reactions were performed in triplicate. Melt- fi H2O2 for 20 min. Having been washed by PBS 5 min/3 times, ing curve analysis was performed to ensure the speci city of 4 Oxidative Medicine and Cellular Longevity

Table 1: Histological scoring for acute pancreatitis.

Condition Score Description 0 Absent 1Diffuse expansion of interlobular septa Edema 21+diffuse expansion of interlobular septa 32+diffuse expansion of interlobular septa 43+diffuse expansion of intercellular septa 0 Absent 1 Around ductal margin Inflammation (%) 2 In parenchyma (<50 of lobules) 3 In parenchyma (51-75 of lobules) 4 In parenchyma (>75 of lobules) 0 Absent 1 Periductal (<5) Vacuolization (%) 2 Focal (5-20) 3Diffuse (21-50) 4 Severe (>50) 0 Absent 1 <10% necrosis Cell necrosis 2 <40% necrosis 3 >40%necrosis 4 >60%necrosis

ΔΔ quantitative PCR. Data was performed using the 2- CT The strength was quantiﬁed by Image J software, and data method for analysis. analysis was used by GraphPad Prism software 8.0.

fi 2.7. Western Blotting. Total protein was extracted from the 2.8. Apoptosis Quanti ed by Flow Cytometry. Cell apoptosis fi pancreas and AR42J cells using RIPA (Beyotime, Shanghai, was quanti ed by the Annexin-V-FITC/PI Apoptosis Detec- China): cocktail (MCE, USA): phosphatase inhibitorsð tion Kit (Meilunbio, Dalian, China). After the intervention, ApexBIO, Houston, USAÞ = 100 : 1 : 1ðv/v/vÞ. Extraction of AR42J cells were digested in Accutase (Mutisciences, Hang- cytoplasmic and nuclear protein using the Cytoplasmic & zhou, China) and collected subsequently. Having been Nuclear protein extraction Kit (Beyotime, Shanghai, China). washed by D-PBS and resuspend in the Annexin V Binding Buffer, 100 μL of cell suspension was transferred into the test BCA assay kits (Beyotime, Shanghai, China) were used to μ μ detect the concentration of extracted proteins. An equal tube for staining. 5 L of Annexin-V-FITC and 5 LofPI amount of protein (30 μg) was loaded in each lane and sepa- solution were added to each sample, then incubated for 15 min at room temperature in the dark. Having been added rated by 12.5% (or 10%) sodium dodecyl sulfate- μ ff polyacrylamide gel (Epizyme, Shanghai, China). Aim pro- 400 L Annexin V binding bu er to the test tube, cell suspension was texted by the flow cytometry (BD FACSAria™ II teins were transferred to PVDF membranes (Millipore, fi USA) and blocked in 5% nonfat milk for 1 h at room temper- Sorter, USA). The results were quanti ed by FlowJo-V10.0 ature. Having been washed by TBS-T for 5 min/3 times, software. membranes were probed with the appropriate primary anti- ° bodies overnight at 4 C. The primary antibodies were pur- 2.9. Hoechst33342 and MitoTracker Red CMXRos Staining. chased from CST (Cell Signaling Technology, Danvers, Hoechst33342 and MitoTracker Red CMXRos staining were MA, USA), including antibodies against phosphor-STAT3 purchased from Beyotime (Shanghai, China). After interven- (Tyr705,1 : 2000), STAT3 (1 : 2000), and histone H3 tion, AR42J cells were incubated in a 2 mL High-Glucose (1 : 1000). Antibodies against Bax (1 : 5000), cleaved DMEM with 20 μL Hoechst33342(10 μmol/L) and 2 μL caspase-3 (1 : 1000), Bcl-2 (1 : 1000), caspase-1 (1 : 1000), MitoTracker Red CMXRos staining (200 nmol/L) in each ° P53 (1 : 1000), and GAPDH (1 : 8000) were from Proteintech plate for 15 min at 37 C as ordered by the manufacturer’s (Proteintech Group Inc., Wuhan, China). Antibodies against instructions. 2 mL of complete medium was supplemented NLRP3 (1 : 1000), GSDMD (1 : 1000), Anti-ERK1 (phosphor after being washed twice with D-PBS. The cells were visual- T202)+ERK2 (phosphorT185, 1 : 1000), and ERK (1 : 10000) ized under a Leica confocal laser scanning microscope were from Abcam (UK). Enhanced chemiluminescence (EL6000, Wetzlar, Germany). Hoechst33342 was monitored reagents (Millipore, USA) were used to detect the bands. at an excitation wavelength of 346 nm, and MitoTracker Oxidative Medicine and Cellular Longevity 5

Red was monitored at an excitation wavelength of 579 nm to 4(d), and 5(a)–5(d)). IL-10, an inhibitor of inflammatory locate nuclear and mitochondria, respectively. response, decreased in the AP group in vivo but increased significantly in the ES (3 mg/kg) pretreatment group 2.10. Measure of Intracellular ROS Levels and Calcium (Figure 3(e)). However, there were no obvious changes in cell Concentration. Levels of intracellular ROS in AR42J cells experiments stimulated for 4 hours, but it decreased signifi- were detected using ROS assay kits, a chemical fluorescence cantly in the AP group stimulated for 8 hours (Figures 4(e) method (2,7-dichlorodi-hydrofluorescein diacetate, DCFH- and 5(e)). DA, Beyotime, Shanghai, China). After the intervention, These results demonstrate that ES pretreatment can AR42J cells were incubated in a 2 mL High-Glucose DMEM effectively reduce the production of lipase, amylase, and ° with 2 μL DCFH-DA (10 μmol/L) for 30 min at 37 C and inflammatory cytokines in STC-induced acute pancreatitis. washed twice in a High-Glucose DMEM, and then 2 mL of complete medium was supplemented. Cells were visualized 4.2. Escin Sodium Pretreatment Ameliorates Acinar Cell under a Leica confocal laser scanning microscope (EL6000, Injury in STC-Induced Acute Pancreatitis. To control the Wetzlar, Germany). ROS was monitored at an excitation quality of modeling, pancreatic tissues with 0.9% saline solu- wavelength of 488 nm to locate reactive oxygen. tion were used as contrast during the operation (Figure 6(a)). Fluo-4-AM was used to detect intracellular calcium con- When edema and ischemia appeared in the tissues as shown centration (Beyotime, Shanghai, China). After the interven- in Figure 6(b), we thought the modeling was successful. 24 tion, AR42J cells were washed 3 times with D-PBS. Then, hours after the operation, we observed the pancreas. In the cells were incubated in 2 mL D-PBS with 1 μL Fluo-4-AM AP group, pancreatic tissues showed ischemic necrosis and ° (0.5 μmol/L) for 20 min at 37 C. After being washed by D- bloody ascites in the abdominal cavity. However, the condi- PBS, cells were visualized under a Leica confocal laser scan- tion of the pancreas improved in ES pretreated groups com- ning microscope (EL6000, Wetzlar, Germany). Calcium con- pared with the AP group. As shown in Figure 7, the tissues centration was monitored at an excitation wavelength of suffered from less hemorrhage, necrosis, and bloody ascites 488 nm to locate. in ES pretreatment groups. In the ES (3 mg/kg) pretreatment group, pancreatic tissues were mainly characterized by inter- 3. Statistical Analysis stitial edema in contrast with ES (1 mg/kg, 6 mg/kg) pretreatment groups. The experiments were performed in triplicates and repeated We assessed the HE staining score of tissues to determine at least three times. Data are analyzed using GraphPad Prism whether ES pretreatment could ameliorate STC-induced AP statistical software (version 8, GraphPad Software, Inc., San in histomorphology. As shown in Figure 8(a), the histological Diego, CA). Data are represented as means ± standard features in the control group were normal, while in the AP deviation (SD). Unpaired Student’s t-test was used to evalu- group, histological features were characterized by edema, ate the significance between 2 groups. For comparison of inflammatory cell infiltration, and necrosis, and the patho- more than three groups, one-way analysis of variance logical score was significantly higher. However, compared (ANOVA) was applied. P <0:05 was considered statistically with the AP group, characteristics of ES pretreated groups significant. (1, 3, or 6 mg/kg) were mainly interstitial edema, with less necrosis and less infiltration of inflammatory cells, and the 4. Results obviously lower pathological score. These results indicated that ES pretreatment could reduce the severity of acute pan- 4.1. Escin Sodium Inhibits Amylase, Lipase, and creatitis induced by STC. Moreover, the ES (3 mg/kg) pre- Inflammatory Cytokines in STC-Induced Acute Pancreatitis. treatment group was more effective than the other two ES Elevation of serum amylase and/or lipase levels to at least 3 pretreated groups. times the upper limit of normal is a key component of diag- As a biomarker of activated neutrophils, MPO (myelo- nosing acute pancreatitis [26]. To access the effects of ES pre- peroxidase) could be universally applied to evaluate the infil- treatment on AP, we measured amylase, lipase, and tration of neutrophils [27]. We furtherly evaluated MPO inflammatory cytokines both in vivo and in vitro. As shown expression by immunohistochemical staining. In the control in Figures 2(a) and 2(b), ES remarkably downregulated group, MPO was not detected while intense MPO immuno- serum amylase, lipase in vivo. Amylase of cell supernatant staining was shown in pancreatic acinar cells in the AP decreased significantly in ES pretreatment groups group, whereas pretreatment with different concentrations (Figures 2(c) and 2(e)); in contrast, there was no significant of ES could obviously downregulate the MPO levels change in lipase (Figures 2(d) and 2(f)). (Figure 8(b)). In addition, the ES (3 mg/kg) pretreatment The process of acute pancreatitis is closely related to the group had a better effect of reducing MPO level in contrast release of inflammatory cytokines. We investigated the with the other two ES pretreated groups. These results were inflammatory cytokines by RT-qPCR to determine whether consistent with the HE staining. All these findings indicated pretreated with ES could downregulate the expression of that ES could ameliorate acinar cell injury in STC-induced inflammatory cytokines in AP. TNF-α, IL-6, IL-1β, and IL- acute pancreatitis. 18 increased significantly in AP groups while ES pretreated groups attenuated production of TNF-α, IL-6, IL-1β, and 4.3. Escin Sodium Promotes Acinar Cell Apoptosis but Not IL-18 both in vivo and in vitro (Figures 3(a)–3(d), 4(a)– Pyroptosis in STC-Induced AP. The type of acinar cell death, 6 Oxidative Medicine and Cellular Longevity

⁎⁎⁎ U/L ⁎ ⁎⁎⁎ U/dL ⁎⁎⁎ 40 ⁎ ⁎⁎⁎ ⁎⁎ 80 U/L ### ⁎⁎ ### # 4000 30 60

3000 20 40

2000 10 20 Lipase of blood serum blood of Lipase 1000 0 Amylase of cell supernatant of Amylase 0 Amylase of bloodAmylase of serum AP AP

0 CON CON AP CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg)

Groups (rats) AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL)

AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) Groups (AR42J 4h) Groups (rats) (a) (b) (c) ⁎⁎ U/L U/dL U/L ⁎⁎ 0.4 80 0.25 ⁎⁎ ### 0.20 0.3 60 0.15 0.2 40 0.10 0.1 20 0.05 Lipase of cell supernatant cell of Lipase supernatant cell of Lipase

0.0 cell supernatant of Amylase 0 0.00 AP AP AP CON CON CON AP+ES (10 � g/mg) AP+ES (20 � g/mg) AP+ES (30 � g/mg) AP+ES (10 � g/mg) AP+ES (20 � g/mg) AP+ES (30 � g/mg) AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) Groups (AR42J 4h) Groups (AR42J 8h) Groups (AR42J 8h) (d) (e) (f)

Figure 2: Effects of ES pretreatment on amylase and lipase in vivo and in vitro. Data are expressed as means ± SD. (a) The levels of serum amylase and lipase in the AP group increased compared with the CON (control) group, and ES pretreated groups (1, 3, and 6 mg/kg) decreased amylase significantly. (b) The ES pretreated group (1 and 3 mg/kg) decreased serum lipase. (c, e) Amylase in supernatant increased in STC-induced AP in AR42J cell, and ES pretreated groups (10, 20, and 30 μg/mL) decreased it obviously. (d, f) Lipase was not stimulated by STC, and ES pretreatment has no effect on it (#P <0:05, ##P <0:01, and ###P <0:001 versus the CON group; ∗P <0:05, ∗∗P <0:01, and ∗∗∗P <0:001 versus the AP group). like apoptosis, necrosis, and pyroptosis, will determine the pathway of apoptosis. The ratio of Bcl-2/Bax determines prognosis of acute pancreatitis. Generally, it is believed that whether the cells are apoptotic or not. When the ratio is apoptosis is beneficial when acute pancreatitis onsets, as it downregulated, apoptosis plays a crucial role and vice versa. cannot trigger the inflammatory cascade reaction. In con- As shown in Figure 9(a), the ratio of Bcl-2/Bax significantly trast, necrosis or pyroptosis was understood as upregulators decreased and cleaved caspase-3 evidently increased in the to damage response [8]. All the above results indicated that ES (3 mg/kg) pretreated group. P53 is a transcription factor the ES pretreatment had protection effects in acute pancrea- that regulates the expression of essential apoptogenic factors, titis induced by STC, and we explored whether it was related which cover both extrinsic and intrinsic apoptosis pathways to promoting apoptosis subsequently. We investigated the [28, 29]. It is also directly interacted with Bcl-2 family expression of cleaved caspase-3, Bcl-2, and Bax—biomarkers members in a transcription-independent manner [30]. As of apoptosis by Western blotting in vivo. Bcl-2 is an antia- shown in Figure 9(b), P53 was significantly upregulated poptotic marker. Bax, one of the members of the Bcl-2 family, in the ES (3 mg/kg) pretreatment group compared with is a proapoptotic effector protein that regulates the intrinsic the AP group. Oxidative Medicine and Cellular Longevity 7

40 15 ## ## 30 10 20 � mRNA

IL-6 mRNA IL-6 5 TNF- 10 relative expression relative

⁎⁎ expression relative ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ 0 0 AP AP CON CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) Groups (rats) Groups (rats) (a) (b)

40 # 200 ### 30 150

20 100 � mRNA IL-18 mRNA IL-18 IL-1 10 50 relative expression relative relative expression relative ⁎ ⁎ ⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ 0 0 AP AP CON CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP+ES (9 mg/kg) Groups (rats) Groups (rats) (c) (d) 6 ⁎⁎⁎

2 ### IL-10 mRNA IL-10 relative expression relative

0 AP CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) Groups (rats) (e)

Figure 3: Effects of ES on inflammatory cytokines in vivo. The data were presented as the mean ± SD of three replicate experiments. (a–d) Serum levels of TNF-α, IL-6, IL-1β, and IL-18 in the AP group were higher than those in the control group, and ES pretreated with different concentrations decreased them significantly. (e) IL-10 decreased obviously in the AP group but increased in the ES (3 mg/kg) pretreatment group (#P <0:05, ##P <0:01, and ###P <0:001 versus the CON group, ∗P <0:05, ∗∗P <0:01, and ∗∗∗P <0:001 versus the AP group). 8 Oxidative Medicine and Cellular Longevity

15 10 ### ### 8 10 6

� mRNA ⁎⁎⁎ 4

5 mRNA IL-6 ⁎⁎⁎ TNF- ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ relative expression relative ⁎⁎⁎ expression relative 2

0 0 AP AP CON CON AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) Groups (AR42J 4h) Groups (AR42J 4h) (a) (b) 150 80 ### ## 60 100 ⁎⁎ 40 � mRNA 50 IL-18 mRNA IL-18 IL-1 20 relative expression relative relative expression relative ⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎ ⁎ 0 0 AP AP CON CON AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) Groups (AR42J 4h) Groups (AR42J 4h) (c) (d) 2.0

1.5

1.0

IL-10 mRNA IL-10 0.5 relative expression relative

0.0 AP CON AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) Groups (AR42J 4h) (e)

Figure 4: ES pretreatment inhibits inflammatory cytokines in vitro. AR42J cells were pretreated with different concentrations of ES for 1 h, then stimulated with STC for 4 h or 8 h. The data were presented as the mean ± SD of three replicate experiments. (a–d) Levels of TNF-α, IL-6, IL-1β, and IL-18 increased significantly in the AP group, and ES with different concentrations could downregulate them. (e) There was no significant change in IL-10 in each group stimulated for 4 h (#P <0:05, ##P <0:01, and ###P <0:001 versus the CON group, ∗P <0:05, ∗∗P <0:01, and ∗∗∗P <0:001 versus the AP group). Oxidative Medicine and Cellular Longevity 9

60 # 8 ###

6 40 4 � mRNA ⁎⁎⁎ 20 mRNA IL-6 ⁎⁎⁎ TNF- 2 relative expression relative relative expression relative ⁎⁎⁎ ⁎ ⁎ 0 0 AP AP CON CON AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) Groups (AR42J 8h) Groups (AR42J 8h) (a) (b)

100 ### 200 ### 80 150

60 100

� mRNA 40 IL-18 mRNA IL-18

IL-1 50 relative expression relative

relative expression relative 20 ⁎⁎⁎ ⁎ ⁎ ⁎ ⁎⁎⁎ ⁎⁎⁎ 0 0 AP AP CON CON AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) Groups (AR42J 8h) Groups (AR42J 8h) (c) (d) 1.5

### 1.0

0.5 IL-10 mRNA IL-10 relative expression relative

0.0 AP CON AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) Groups (AR42J 8h) (e)

Figure 5: ES pretreatment inhibits inflammatory cytokines in vitro. AR42J cells were pretreated with different concentrations of ES for 1 h, then stimulated with STC for 4 h or 8 h. The data were presented as the mean ± SD of three replicate experiments. (a–d) Levels of TNF-α, IL-6, IL-1β, and IL-18 increased significantly in the AP group, and ES with different concentrations could downregulate them. (e) IL-10 decreased obviously in the AP group stimulated for 8 h, and ES pretreatment had no effect on it (#P <0:05, ##P <0:01, and ###P <0:001 versus the CON group, ∗P <0:05, ∗∗P <0:01, and ∗∗∗P <0:001 versus the AP group). 10 Oxidative Medicine and Cellular Longevity

(a) (b)

Figure 6: Morphological changes of the pancreas before and after modeling. (a) Shows a normal pancreas. (b) Shows the state after STC stimulation within 5 minutes; the pancreas indicated by the cotton swab presents ischemic necrosis.

Con AP AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg)

Figure 7: ES pretreatment alleviated pancreatic tissue injury in visual. The pancreas of the control group was almost normal. Ischemic necrosis and bloody ascites were shown in the AP group. The blue arrows indicate typical pancreatic tissue. The ES (3 mg/kg) pretreatment group was characterized by edema. The other ES pretreated groups (1 mg/kg and 6 mg/kg) had diﬀerent degrees of tissue necrosis.

To examine the promoting apoptotic effects of ES pre- Western blotting analysis showed that there was no signifi- treatment on STC-induced AP, AR42J cells were pretreated cant difference in pyroptosis proteins in each group, both with ES (10 μg/mL, 20 μg/mL, and 30 μg/mL) for 1 h, and in vivo and in vitro. What is more, the results of immunohis- then STC was added for another 4 or 8 hours, respectively. tochemistry also verified these results (Figures 10(c)–10(e)) We explored the apoptotic effect by flow cytometric analysis However, we observed the upregulation of pyroptosis pro- in vitro. As shown in Figures 9(c) and 9(d), the early apopto- teins in individual cells by immunohistochemistry, even sis got increased in the ES (20 μg/mL, 30 μg/mL) pretreated though it is not statistically significant. group compared with the AP group. These results verified our hypothesis that ES pretreatment could ameliorate the 4.4. Escin Sodium Plays a Protective Role by Upregulating prognosis of acinar cells in STC-induced AP by promoting Mitochondrial Membrane Potential and ROS Activity but apoptosis. Downregulating Intracellular Calcium Concentration. Mito- Pyroptosis is one of programmed cell death that has chondria provide cellular energy and play a central role in received increased attention recently [31]. Studies have iden- regulating cell survival. Mitochondria are involved in cellular tified that caspase-1 is a mediator of pyroptosis and gasder- Ca2+ homeostasis and are a major source of ROS (reactive min D (GSDMD) is the executioner [32]. To determine oxygen species) [33]. Mitochondrial membrane perme- whether pyroptosis is responsible for STC-induced acute abilization (MMP, ΔΨm) is a universal trigger of both necro- pancreatitis, we detected NLRP3 (inflammasome sensor), sis and apoptosis. Loss of ΔΨm leads to ATP (adenosine caspase-1, and GSDMD expression by Western blotting triphosphate—cell energy) depletion, ultimately leading to in vivo and in vitro. We subsequently explored NLRP3, cas- necrosis [34, 35]. The accumulation of MitoTracker Red pase-1, and GSDMD expressions by immunohistochemistry CMXRos in the mitochondria depends on the ΔΨm, so it is to further identify. As indicated in Figures 10(a) and 10(b), an indicator probe for the ΔΨm. ROS usually play an Oxidative Medicine and Cellular Longevity 11

Con AP AP+ES (1 mg/kg)

### 10 ⁎⁎ ⁎⁎ ⁎⁎⁎

Score of HE 5

0 AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg)

Groups (a) Con AP AP+ES (1 mg/kg)

### 30

20 ⁎⁎ ⁎⁎

MPO activity 10 ⁎⁎⁎

0 AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg)

Groups (b)

Figure 8: ES pretreatment effectively inhibits the acinar cell necrosis and reduces inflammatory cell infiltration and MPO level in vivo. (a) HE staining was used to evaluate the pancreatic pathological scores. Representative pictures from each group and quantitative analyses of histology score were shown. (b) The levels of MPO were further examined by immunohistochemistry. Representative images from each group and quantitative analyses of MPO were shown (original magnification, ×200) (#P <0:05, ##P <0:01, and ###P <0:001 versus the CON group, ∗P <0:05, ∗∗P <0:01, and ∗∗∗P <0:001 versus the AP group). 12 Oxidative Medicine and Cellular Longevity

2.5 1.5 ⁎ Bax –21 kD ⁎ 2.0 ### Bcl-2 –26 kD 1.0 Full length –36 kD 1.5 caspase-3 1.0 0.5

Ratio of Bcl-2/Bax of Ratio 0.5

Cleaved caspase-3/GAPDH of Ratio 0.0 0.0 caspase-3 –17 kD AP AP

GAPDH –36 kD CON CON AP Con AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) Groups Groups AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) (a) ⁎⁎ 4

3 P53 –53 kD

2 Gapdh –36 kD

1 AP Ratio of P53/GAPDH of Ratio Con 0 AP CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) Groups (b) 4h CON AP AP+ES (10 �g/mL) AP+ES (20 �g/mL) AP+ES (30 �g/mL)

Q5 Q6 Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 105 105 0.095 3.13 5.01 44.4 2.18 23.2 0.25 18.4 3.00 26.9 105 105 105

104 104 104 104 104

3 3 3 10 103 10 10 103 PI

102 102 102 102 102 Q7 Q3 Q3 Q3 Q3 1 Q8 Q4 1 Q4 1 Q4 Q4 10 93.7 3.02 14.0 36.6 10 34.7 40.0 10 14.1 67.2 25.7 44.4 101 101 101 102 103 104 105 101 102 103 104 105 101 102 103 104 105 101 102 103 104 105 101 102 103 104 105

Annexin-V FITC (c)

Figure 9: Continued. Oxidative Medicine and Cellular Longevity 13

8h CON AP AP+ES (10 �g/mL) AP+ES (20 �g/mL) AP+ES (30 �g/mL)

Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 5 105 10 0.14 1.36 2.79 41.5 2.70 46.1 0.29 19.7 1.19 32.6 105 105 105

4 10 104 104 104 104

3 10 3 3 3 10 10 10 103 PI 2 10 102 102 102 102 Q3 Q3 Q3 Q3 Q3 1 Q4 1 Q4 1 Q4 1 Q4 Q4 10 97.4 1.09 10 20.4 35.3 10 11.2 40.0 10 14.0 66.0 14.5 51.7 101 101 102 103 104 105 101 102 103 104 105 101 102 103 104 105 101 102 103 104 105 101 102 103 104 105

Annexin-V FITC

⁎ ⁎⁎ ⁎⁎⁎ ⁎⁎⁎ 80 80

60 60

## ### 40 40

20 20 Ratio of early apoptosis early Ratio of apoptosis early Ratio of

0 0 AP AP CON CON AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) Groups (AR42J 4h) Groups (AR42J 8h) (d)

Figure 9: Effects of ES pretreatment on STC-induced apoptosis both in vivo and in vitro. (a, b) The expression of apoptotic-related proteins in different groups was determined by Western blotting: cleaved caspase-3, Bcl-2, Bax, and P53 in vivo. (c, d) Apoptosis of AR42J cells pretreated with different concentrations of ES for 1 h and then stimulated with STC for 4 h or 8 h, then examined using the Annexin-V-FITC/PI assay kit by flow cytometry. The ratio of early apoptosis (lower right quadrant) in different groups was quantified by FlowJo-V10 software (#P <0:05, ##P <0:01, and ###P <0:001 versus the CON group, ∗P <0:05, ∗∗P <0:01, and ∗∗∗P <0:001 versus the AP group). important role in regulating apoptosis and critically regulate To clarify whether the protective effect of ES pretreat- mitochondrial function and dysfunction [33, 36]. Ca2+ is a ment is related to ROS and DCFH-DA, they were loaded to major regulator of the acinar cell function, but an abnormal AR42J cells. As shown in Figures 12(a) and 12(b), in the con- (global and sustained) increase in cytosolic Ca2+ is a key trol group, AR42J cells grew well without obvious green fluo- pathologic signal associated with pancreatitis [37]. Abnormal rescence. However, although there was no obvious Ca2+ signal promotes acinar cell necrosis through mitochon- fluorescence in the AP group, the cells showed lysis in BF drial depolarization and subsequent ATP drop. To access (bright field), whereas ES pretreatment groups showed apoptosis, mitochondrial function, ROS activity, and intra- strong fluorescence signal no matter they were stimulated cellular calcium concentration, Hoechst33342 staining, for 4 hours or 8 hours, and the difference was statistically sig- MitoTracker Red CMXRos staining, DCFH-DA, and Fluo- nificant. Interestingly, the level of ROS is positively correlated 4-AM were used, respectively. with change of ΔΨm. The fluorescence intensity of Hoechst33342 in ES pre- And then, we furtherly explored intracellular calcium treated groups was significantly higher than the AP group concentration by Fluo-4-AM working solution. As shown at 4 h and 8 h after stimulation (Figures 11(a) and 11(b)). It in Figures 12(c), strong fluorescence signals in the AP group indicated that more apoptotic events occurred after ES pre- were detected compared with the control group. However, ES treatment. ΔΨm also increased significantly after ES pre- pretreatment could downregulate the fluorescence signal treatment which was consistent with the results of intensity. The results suggested that intracellular calcium Hoechst33342. However, compared with the control group, overloaded in the AP group, and ES pretreatment can down- the fluorescence signals of Hoechst33342 and ΔΨm in the regulate intracellular calcium concentration, which is oppo- AP group were downregulated. site to ROS and ΔΨm. 14 Oxidative Medicine and Cellular Longevity

1.5 1.5 3 NLRP3 –110 kD –53 kD GSDMD –50 kD 1.0 1.0 2 –47 kD Caspase-1 0.5 0.5 1 –35 kD Ratio of NLRP3/GAPDH of Ratio

Gapdh GSDMD/GAPDH of Ratio

–36 kD caspase-1/GAPDH of Ratio 0.0 0.0 0 AP AP AP AP Con CON CON CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP+ES (9 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP+ES (9 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) AP+ES (9 mg/kg)

Groups Groups Groups (a)

AR42J 4h AR42J 8h

NLRP3 –110 kD NLRP3 –110 kD GSDMD –53 kD GSDMD –53 kD Caspase-1 –47 kD Caspase-1 –47 kD

GAPDH –36 kD GAPDH –36 kD AP AP CON CON g/mL) g/mL) g/mL) g/mL) g/mL) g/mL) � � � � � � AP+ES (20 AP+ES (10 AP+ES (30 AP+ES (20 AP+ES (10 AP+ES (30

1.5 1.5 2.0

1.5 1.0 1.0 1.0 0.5 0.5 0.5 Ratio of of NLRP3/GAPDH Ratio Ratio of of GSDMD/GAPDH Ratio 0.0 0.0 caspase-1/GAPDH of Ratio 0.0 AP AP AP CON CON CON g/mL) g/mL) g/mL) g/mL) g/mL) g/mL) g/mL) g/mL) g/mL) � � � � � � � � � AP+ES (10 AP+ES (10 AP+ES (10 AP+ES (20 AP+ES (30 AP+ES (20 AP+ES (30 AP+ES (20 AP+ES (30 Groups (AR42J 4h) Groups (AR42J 4h) Groups (AR42J 4h)

2.0 1.5 2.0

1.5 1.5 1.0 1.0 1.0 0.5 0.5 0.5 Ratio of of NLRP3/GAPDH Ratio Ratio of of GSDMD/GAPDH Ratio 0.0 0.0 caspase-1/GAPDH of Ratio 0.0 AP AP AP CON CON CON g/mL) g/mL) g/mL) g/mL) g/mL) g/mL) g/mL) g/mL) g/mL) � � � � � � � � � AP+ES (10 AP+ES (10 AP+ES (10 AP+ES (20 AP+ES (30 AP+ES (20 AP+ES (30 AP+ES (20 AP+ES (30 Groups (AR42J 8h) Groups (AR42J 8h) Groups (AR42J 8h) (b) Figure 10: Continued. Oxidative Medicine and Cellular Longevity 15

Con AP AP+ES (1 mg/kg)

2 Expression of NLRP3 of Expression 0 AP

AP+ES (3 mg/kg) AP+ES (6 mg/kg) CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) Groups (c) Con AP AP+ES (1 mg/kg)

5 Caspase-1 activity

0 AP

AP+ES (3 mg/kg) AP+ES (6 mg/kg) CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) Groups (d) Figure 10: Continued. 16 Oxidative Medicine and Cellular Longevity

Con AP AP+ES (1 mg/kg)

GSDMD activity GSDMD 2

0 AP

AP+ES (3 mg/kg) AP+ES (6 mg/kg) CON AP+ES (1 mg/kg) AP+ES (3 mg/kg) AP+ES (6 mg/kg) Groups (e)

Figure 10: Expression of pyroptosis-related proteins in acinar cells in acute pancreatitis induced by STC in vivo and in vitro. (a, b) The expression of pyroptosis-related proteins: NLRP3, GSDMD, and caspase-1 were determined by Western blotting, and there was no significant difference in each group both in vivo and in vitro. (c–e) Levels of NLRP3, caspase-1, and GSDMD were examined by immunohistochemistry and showed no significant statistical difference, which further identified the Western blotting results. Representative pictures from each group and quantitative analyses of protein expression were shown (original magnification, ×200) (#P <0:05, ##P <0:01, and ###P <0:001 versus the CON group, ∗P <0:05, ∗∗P <0:01, and ∗∗∗P <0:001 versus the AP group). 4.5. Escin Sodium Promotes Acinar Cell Apoptosis via group (combined group). We detected relevant cellular sig- Downregulating ERK/STAT3 Phosphorylation. The above naling pathway proteins by Western blotting. In the AP results suggested that ES pretreatment could maintain a group, p-ERK and p-STAT3 were activated in contrast with higher level of ΔΨm and ROS, downregulate intracellular the control group (Figures 13(c) and 13(d)). However, ERK calcium concentration, and promote early apoptosis in phosphorylation was significantly downregulated after ES STC-induced acute pancreatitis. To elucidate the mechanism pretreatment and the same trend also appeared in STAT3 of ES promoting apoptosis in AP induced by STC, we phosphorylation. We then extracted the cytoplasmic and hypothesized that it is related to ERK/STAT3/Bcl-2 axis. To nuclear proteins from the cells and found that p-STAT3 in verify our hypothesis, we first analyzed p-ERK/ERK and p- the nucleus was increased considerably in the AP group STAT3/STAT3 in vivo by Western blotting. As shown in and decreased obviously in the ES pretreated group. But there Figure 13(a), ES pretreated groups (1 mg/kg, 3 mg/kg) dra- was still no apparent change in p-STAT3 in the cytoplasm. matically downregulated the expressions of p-ERK and p- This was synchronized with the results of ERK inhibitors. STAT3. We then extracted the cytoplasmic and nuclear pro- Moreover, the results were consistent no matter whether teins from the tissue and found that the nuclear expression of the stimulation lasted for 4 hours or 8 hours (Figures 13(e) p-STAT3 was increased in the AP group, while decreased and 13(f)). obviously in ES pretreatment groups. Interestingly, however, To further clarify the mechanisms of the ERK/STAT3 there was no significant change in the expression of p-STAT3 pathway and apoptosis, we then measured the expression of in the cytoplasm (Figure 13(b)). apoptotic-related proteins—Bcl-2, Bax, cleaved caspase-3, We further tested with pathway inhibitor—PD98059 and P53. The early apoptosis of AR42J cells in each group in vitro. According to the above results, compared with the was detected again by flow cytometry. As shown in ES (10 μg/mL, 30 μg/mL) pretreated groups, ES (20 μg/mL) Figures 14(a)–14(d), cleaved caspase-3 and P53 were upregu- pretreatment has more advantages in promoting apoptosis. lated apparently in ES (20 μg/mL), PD98059, and combined We then divided AR42J cells into 5 groups: control group, groups and were statistical significant in contrast with the AP group, AP+ES (20 μg/mL) group, AP+PD98059 AP group. Moreover, the ratio of Bcl-2/Bax showed the (10 μM) group, and AP+PD98059(10 μM)+ES (20 μg/mL) opposite trend. Flow cytometry analysis showed that the ES Oxidative Medicine and Cellular Longevity 17

AR42J 4h

BF Hoechst Mito Tracker Merge IF

CON

AP+ES (10 �g/mL)

AP+ES (20 �g/mL)

AP+ES (30 �g/mL)

(a) AR42J 8h

BF Hoechst Mito Tracker Merge IF

CON

AP+ES (10 �g/mL)

AP+ES (20 �g/mL)

AP+ES (30�g/mL)

(b)

Figure 11: ES pretreatment induces apoptosis and increases mitochondrial membrane potential (MMP, ΔΨm) in STC-induced acute pancreatitis. (a, b) AR42J cells were pretreated with different concentrations of ES for 1 h and then stimulated by STC for 4 h or 8 h, respectively. The fluorescence intensity was observed by fluorescence microscopy, and representative images are presented. Scale bar, 250 μm. IF (intensity of fluorescence) was measured by ImageJ 6 times at different points randomly, then Origin2018 was used to show the IF, blue represents Hoechst32242, and red represents MitoTracker. BF: bright field; Hoechst: apoptosis staining; MitoTracker: ΔΨm staining; merge: state after images overlay. 18 Oxidative Medicine and Cellular Longevity

4h 8h BF ROS Merge BF ROS Merge

CON CON

AP AP

� AP+ES (10 �g/mL) AP+ES (10 g/mL)

� AP+ES (20 �g/mL) AP+ES (20 g/mL)

� AP+ES (30 �g/mL) AP+ES (30 g/mL)

0.8 ⁎⁎ 1.5

0.6 ⁎⁎⁎ ⁎⁎ ⁎⁎ 1.0

0.4 ⁎⁎⁎ 0.5 ROS level (%) level ROS 0.2 (%) level ROS ⁎⁎⁎

0.0 0.0 AP AP CON CON AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) AP+ES (10 � g/mL) AP+ES (20 � g/mL) AP+ES (30 � g/mL) Groups (AR42J 4h) Groups (AR42J 8h) (a) (b) CON AP AP+ES (10 �g/mL) AP+ES (20 �g/mL) AP+ES (30 �g/mL)

CON AP AP+ES (10 �g/mL) AP+ES (20 �g/mL) AP+ES (30 �g/mL) (c)

Figure 12: ES pretreatment triggered ROS generation in STC-induced acute pancreatitis. (a, b) AR42J cells were pretreated with different concentrations of ES for 1 h and then stimulated by STC for 4 h and 8 h, respectively. ROS generation was observed by fluorescence microscopy; representative images are presented. Scale bar, 250 μm. BF: bright field; merge: state after images overlay. (c) Intracellular calcium concentration was observed by fluorescence microscopy, and representative images are presented. Scale bar, 100 μm(#P <0:05, ##P <0:01, and ###P <0:001 versus the CON group, ∗P <0:05, ∗∗P <0:01, and ∗∗∗P <0:001 versus the AP group). xdtv eiieadClua Longevity Cellular and Medicine Oxidative Histone H3 (p-T202+ p-STAT3 (Tyr705) GAPDH (p-T202+ p-T185) p-STAT3 p-STAT3 p-STAT3 (Tyr705) (Tyr705) (Tyr705) p-ERK STAT3 p-T185) Gapdh p-ERK Gapdh STAT3 ERK ERK

CON Con Con

AP AP AP AR42J 4h

AP+ES (20 �g/mL) AP+ES (1 mg/kg) AP+ES (1 mg/kg)

AP+PD98059 AP+ES (3 mg/kg) AP+ES (3 mg/kg)

AP+ES+PD98059 AP+ES (6 mg/kg) AP+ES (6 mg/kg) –36 kD –79 kD –86 kD –17 kD –79 kD –86 kD –79 kD –42 kD –42 kD –44 kD –36 kD –86 kD –79 kD –86 kD –44 kD –79 kD –42 kD –42 kD –44 kD –36 kD –86 kD –79 kD –86 kD –44 kD Figure

Ratio of p-ERK/ERK Ratio of p-STAT3/Histone H3 Ratio of p-ERK/ERK 0.0 0.5 1.0 1.5 2.0 10 6 8 0 2 4 0 1 2 3 4 3 Continued. 13: (b) (c) (a) ### ### CON ## CON CON Groups (AR42J4h) Groups (nuclear) AP AP AP ⁎⁎ ⁎ ⁎ Groups ⁎⁎⁎ ⁎⁎ AP+ES (20 �g/mL) AP+ES (1 mg/kg) AP+ES (1 mg/kg) ⁎ ⁎ ⁎ AP+PD98059 AP+ES (3 mg/kg) AP+ES (3 mg/kg)

AP+ES+PD98059 AP+ES (6 mg/kg) AP+ES (6 mg/kg)

Ratio of p-STAT3/STAT3 Ratio of p-STAT3/GAPDH Ratio of p-STAT3/STAT3 1.5 0.0 0.5 1.0 0 1 2 3 4 0 1 2 3 4 5 ###

CON CON CON ## Groups (AR42J4h) Groups (cytosolic) AP AP AP ⁎ Groups ⁎ ⁎ AP+ES (20 �g/mL) AP+ES (1 mg/kg) AP+ES (1 mg/kg) ⁎ ⁎ AP+PD98059 AP+ES (3 mg/kg) AP+ES (3 mg/kg)

AP+ES+PD98059 AP+ES (6 mg/kg) AP+ES (6 mg/kg) 19 0: 20 T o r8h epciey h xrsinlvl fpEK R,pSA3 n TT eeaaye ywsenbotn nvitro. signi in no blotting was western there by However, ( ES+PD98059. analyzed group and were each PD98059, STAT3 in ES, and cytoplasm with p-STAT3, the pretreatment ERK, after in p-ERK, decreased STAT3 of was levels it expression while increased, increased, The was signiﬁ respectively. group signiﬁ h, no AP 8 could the was ES or of there nuclears h 4 in However, for p-STAT3 ES. (20 of STC with ES expression The pretreatment with (b) pretreated after vivo. were decreased in blotting was Western it by while analyzed were STAT3 and STAT3, Figure 001 essteA group). AP the versus 3 oe fteEKSA3sgaigptwyi T-nue ct acetts a h xrsinlvl fpEK R,p- ERK, p-ERK, of levels expression The (a) pancreatitis. acute STC-induced in pathway signaling ERK/STAT3 the of Roles 13: atydwrglt h R/TT inln aha.(,f h xrsino -TT nncer fteA ru was group AP the of nuclears in p-STAT3 of expression The f) (e, pathway. signaling ERK/STAT3 the downregulate cantly Histone H3 Cytosolic p-STAT3 p-STAT3 (Tyr705) (Tyr705) GAPDH Histone H3 Nuclear Cytosolic p-STAT3 p-STAT3 (Tyr705) (Tyr705) GPDHA Nuclear (p-T202+ p-STAT3 (Tyr705) GAPDH p-T185) p-ERK STAT3 μ ERK /L,EKihbtrP909(10 PD98059 inhibitor ERK g/mL), CON CON AP CON AP AP+ES (20 �g/mL) AP # AR42J 8h P AP+ES (20 �g/mL) <0 AP+PD98059 AP+ES (20 �g/mL) :

05, AP+PD98059 AP+ES+PD98059 AP+PD98059 ## –36 kD –79 kD –86 kD –17 kD –79 kD –86 kD AP+ES+PD98059 P –36 kD –79 kD –86 kD –17 kD –79 kD –86 kD

<0 AP+ES+PD98059 –36 kD –86 kD –79 kD –86 kD –44 kD –79 kD –42 kD –42 kD –44 kD : and 01, Nuclear ratio of p-STAT3/ Histone H3 Nuclear ratio of p-STAT3/ 0.0 0.5 1.0 1.5 2.0 2.5 Histone H3 μ 1 2 3 0

) n S(20 ES and M), Ratio of p-STAT3/STAT3 ### (f) 0.0 0.5 1.0 1.5 2.0 2.5 (e) P ###

Groups (AR42J8h) CON (d) ###

Groups (AR42J4h) CON <0 AP ### Groups (AR42J8h) ⁎⁎ CON : AP 001 ⁎⁎ � ⁎⁎ AP+ES (20 g/mL) ⁎ AP ⁎⁎ �

AP+ES (20 g/mL) ⁎ ⁎⁎ essteCNgroup, CON the versus AP+PD98059 AP+ES (20 �g/mL) ⁎ ⁎⁎

atcag npSA3i h yols.(,d Cells d) (c, cytoplasm. the in p-STAT3 in change cant AP+PD98059 μ

/L+P909(10 PD98059 g/mL)+ AP+ES+PD98059 AP+ES+PD98059 AP+PD98059 AP+ES+PD98059 xdtv eiieadClua Longevity Cellular and Medicine Oxidative Cytosolic ratio of p-STAT3/ Cytosolic ratio of p-STAT3/ GAPDH GAPDH Ratio of p-ERK/ERK 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 0.0 Groups (AR42J8h) Groups (AR42J8h) Groups (AR42J4h) CON CON CON ## ∗

P AP AP AP μ ⁎ <0 )fr1h hnsiuae by stimulated then h, 1 for M) AP+ES (20 �g/mL) AP+ES (20 �g/mL) AP+ES (20 �g/mL) ⁎ ⁎ : 05, AP+PD98059 AP+PD98059 AP+PD98059 ∗∗ AP+ES+PD98059 AP+ES+PD98059 AP+ES+PD98059 P <0 ﬁ atcag np- in change cant : and 01, ∗∗∗ P < xdtv eiieadClua Longevity Cellular and Medicine Oxidative Full length caspase-3 caspase-3 Cleaved GAPDH Full length caspase-3 caspase-3 Cleaved GAPDH Bcl-2 Bax Bcl-2 Bax

CON CON

AP GAPDH AP AR42J 4h AR42J 8h P53 AP+ES (20 �g/mL) AP+ES(20 �g/mL)

CON AP+PD98059 AP+PD98059

AP+ES+PD98059 AP AP+ES+PD98059 –37 kD –17 kD –36 kD –26 kD –21 kD –36 kD –17 kD kD –36 kD –26 –21 kD

AP+ES (20 �g/mL) 4h

Figure AP+PD98059 Ratio of Bcl-2/Bax Ratio of Bcl-2/Bax 0.5 1.0 1.5 2.0 0.0 0.0 0.5 1.0 1.5

AP+ES+PD98059 4 Continued. 14: (a) (b) (c) –36 kD –53 kD CON CON Groups (AR42J4h) Groups Groups (AR42J8h) AP AP ⁎ ⁎ ⁎ AP+ES (20 �g/mL) ⁎ AP+ES (20 �g/mL) ⁎ Ratio of P53/GAPDH ⁎

0 1 2 3 4 5 AP+PD98059 AP+PD98059

AP+ES+PD98059 AP+ES+PD98059 CON Groups (AR42J4h)

⁎ Ratio of

⁎⁎ cleaved caspase-3/GAPDH AP+ES (20 �g/mL) Ratio of caspase-3/GAPDH 0.0 0.5 1.0 1.5 2.0 2.5 ⁎ 1 2 3 4 0 AP+PD98059

CON CON Groups (AR42J4h)

AP+ES+PD98059 ps(AR42J8h) Grou # AP AP ⁎⁎ ⁎⁎ ⁎⁎⁎ � � ⁎ AP+ES (20 g/mL) ⁎⁎⁎ AP+ES (20 g/mL) ⁎⁎

AP+PD98059 AP+PD98059

AP+ES+PD98059 AP+ES+PD98059 21 22 o r8h epciey h xrsinlvl fcevdcsae3 c-,Bx n 5 eeaaye yWsenbotn.(,f) (e, blotting. Western by analyzed by were kit P53 assay and Annexin-V-FITC/PI Bax, the Bcl-2, using caspase-3, examined cleaved was di of group in levels each quadrant) right expression in (lower The cells apoptosis respectively. AR42J h, of 8 Apoptosis or h 4 for (20 ES with pretreated Figure h O group, CON the 4 Spooe ppoi nSCidcdauepnraii i oneuaigteEKSA3ptwy ad R2 el were cells AR42J (a-d) pathway. ERK/STAT3 the downregulating via pancreatitis acute STC-induced in apoptosis promotes ES 14:

PI PI 4h 8h 10 10 10 10 10 10 10 10 10 10 ∗ 1 2 3 4 5 1 2 3 4 5 P 95.5 Q4 0.15 Q1 93.3 Q4 0.078 Q1 <0 10 10 1 1 : μ 10 10 05, 2 /L,EKihbtrP909(10 PD98059 inhibitor ERK g/mL), 2 CON CON 10 10 ∗∗ 3 3 P 10 10 4 4 <0 10 10 5 5 GAPDH 2.87 1.47 2.98 3.61 : Q3 Q2 Q3 Q2 and 01, P53 10 10 10 10 10 10 10 10 10 10 ﬀ 1 2 3 4 5 1 2 3 4 5 rn ruswsquantiﬁ was groups erent 14.7 Q4 3.02 Q1 2.02 Q1 20.6 Q4 10 10 1 1

∗∗∗ Ratio of early apoptosis 100 10 10 20 40 60 80 2 2 0 CON P AP 10 10 AP <0 3 3

10 AP 10 :

CON ## Groups (AR42J 4h) 001 4 4 8h 10 10 5 AP 5 38.5 43.8 ⁎⁎ 35.9 41.5 � Q3 Q2 Q3 Q2 essteA group). AP the versus AP+ES (20 g/mL) ⁎⁎⁎ � AP+ES (20 g/mL) ⁎⁎⁎ 10 10 10 10 10 10 10 10 10 10 μ 1 2 3 4 5 1 2 3 4 5 AP+PD98059 ) n S(20 ES and M), 12.6 Q4 0.20 Q1 Annexin-V FITC 13.3 Q4 0.22 Q1 Annexin-V FITC 10 AP+PD98059 10 /L PP909AP+ES+PD98059 AP+PD98059 AP+ES (20�g/mL) 1 1 AP+ES (20�g/mL) db lwoV0sfwr ( software FlowJo-V10 by ed (e) (f) 10 AP+ES+PD98059 10 AP+ES+PD98059 2 2 (d) –36 kD –53 kD 10 10 3 3 10 10 4 Ratio of early apoptosis 4 100 20 40 60 80 10 10 0 5 5 70.5 16.6 68.9 17.6 μ Q3 Q2 Q3 Q2 Ratio of P53/GAPDH 0.0 0.5 1.0 1.5 2.0 /L+P909(10 PD98059 g/mL)+ 10 10 10 10 10 10 10 10 10 10 1 2 3 4 5 1 2 3 4 5

CON ### Groups (AR42J8h) 0.097 Q1 5.33 Q4 0.064 Q1 6.69 Q4 10 10 1 1

CON ### ⁎⁎⁎ AP Groups (AR42J8h) AP+PD98059 10 10 ⁎⁎⁎ 2 2 � AP ⁎⁎⁎ AP+ES (20 g/mL) Longevity Cellular and Medicine Oxidative 10 10 ⁎ 3 3 ⁎

# � 10 10 AP+ES (20 g/mL) ⁎ P AP+PD98059 4 4 <0 10 10 AP+PD98059 5 5 10.6 84.0 11.1 AP+ES+PD98059 82.2 Q2 Q3 Q2 Q3 : 05, μ 10 10 10 10 10 10 10 10 10 10 AP+ES+PD98059 )fr1h hnsiuae ySTC by stimulated then h, 1 for M) 1 2 3 4 5 ﬂ 1 2 3 4 5 ## wctmty h ai fearly of ratio The cytometry. ow 0.022 Q1 5.62 Q4 4.94 Q4 0.011 Q1 10 10 P 1 1 AP+ES+PD98059 <0 10 10 2 2 : and 01, 10 10 3 3 10 10 4 4 10 10 ### 5 5 8.90 85.4 86.5 8.54 Q2 Q3 Q3 Q2 P <0 : 001 versus Oxidative Medicine and Cellular Longevity 23

Supramaximal STC

Escin Acinar cell sodium Ca2+ P ERK

ROS p-STAT3

Necrosis Bcl-2 Apoptosis Bax P53

Caspase-3 activated Mitochondrial depolarization

Figure 15: A brief diagram of the effects of ES on pancreatic acinar cells. pretreatment group could promote early apoptosis as well as MODS or SIRS. Various pathological factors that lead to aci- the PD98059 group. Moreover, the combined group showed a nar cell injury stimulate inflammatory responses (infiltration better trend of promoting apoptosis (Figures 14(e) and 14(f)). of neutrophils, release of cytokines TNF-α and IL-6, etc.) Taken all the results into consideration, we deduced that ES [40]. Suppression of proinflammatory cytokines could ame- promotes acinar cell apoptosis via downregulating ERK and liorate the severity of pancreatitis. Our study has shown that STAT3 phosphorylation. TNF-α, IL-6, IL-1β, and IL-18 decreased significantly after ES pretreatment both in vivo and in vitro. IL-10 has been 5. Discussion considered as an anti-inflammatory cytokine which contributes to the maintenance of immune homeostasis [41]. We Escin Sodium (ES) has been commonly used in clinical, as its explored whether the function of ES is related to the increase anti-inflammatory and antiedematous properties make it a of IL-10. The results indicated that IL-10 in the AP group was choice of therapy for chronic venous insufficiency. Nowa- significantly lower than in the control group, whereas ES days, ES has also been investigated against diverse cancers (3 mg/kg) pretreatment could increase it obviously. In vitro [38]. However, only little is known about the effect of ES on study, there was no significant change in IL-10 in each group acute pancreatitis, and the corresponding mechanism stimulated for 4 hours, but with the extension of stimulation remains obscure. (8 hours), the level of IL-10 in the AP group decreased signif- In this study, we confirmed that ES exerts a protective icantly. It was not completely consistent in vivo and in vitro effect in STC-induced AP by promoting apoptosis through experiments. Based on this phenomenon, we hold the opin- the inactivation of the ERK/STAT3 signaling pathway. AP ion that the environment of the body is complex, and cells induced by STC is a reliable and reproducible model with a in vitro cannot be fully simulated. short duration. When acute pancreatitis onsets, the increased To further assess the effects of ES pretreatment on path- permeability of acinar cells produces serum amylase and ological features in STC-induced acute pancreatitis, HE lipase that release to circulation at high levels [2]. The prac- staining and MPO activity were used for evaluation. The tice guidelines released by the ACG (American College of results suggested that ES could alleviate acinar cell damage, Gastroenterology) suggested that both serum amylase and reduce inflammatory cell infiltration, and downregulate the lipase could be applied for the diagnosis of AP [39]. Our expression of MPO. study demonstrated that STC significantly increased serum Apoptosis, a pattern of programmed cell death, is consid- amylase and lipase, whereas ES pretreatment showed con- ered to be a benefit to the prognosis of acute pancreatitis. In versed outcomes dramatically in vivo. In vitro study, STC our study, we found that ES pretreatment activated apoptotic only increased amylase in cell supernatant rather than lipase. relevant proteins in rats, and then we detected apoptosis by We do not know why there is no obvious change in lipase, flow cytometry in AR42J cells. As indicated by our results, which needs further study. However, amylase decreased sig- ES pretreatment promoted early apoptosis. Pyroptosis, nificantly after ES pretreatment in vitro. another pattern of programmed cell death, is considered to As mentioned above, acute pancreatitis not only causes be involved in the process of acute pancreatitis. In the present local inflammations but also causes systemic inflammatory study, we first identified pyroptosis-related proteins in the responses, which can induce serious complications such as pancreas and in AR42J cells. NLRP3/caspase-1/GSDMD 24 Oxidative Medicine and Cellular Longevity were detected by Western blotting and immunohistochemis- which directly promotes MOMP (mitochondrial outer mem- try. According to the above results, both the Western blotting brane permeabilization), inducing apoptosis. Hu et al. and the immunohistochemical results suggested that there showed that transcriptional activity of p53 was impaired, was no significant difference in pyroptosis-related proteins. which explained the apoptosis silencing during AP [29]. However, it was worth noting that high expression of pyrop- Our results show that ES pretreatment can induce apoptosis tosis proteins was found in individual cell by immunohisto- in AP by activating caspase-3 and P53 and downregulating chemistry. Hence, we deduce that pyroptosis does exist the Bcl-2/Bax ratio. Taken all the results into consideration, when acute pancreatitis onsets, but it is not the main way we suppose that ES may regulate ΔΨm by changing Bcl- of death in acinar cells induced by STC. 2/Bax and P53, thus promoting apoptosis. But this mecha- Mitochondria, positioned at the heart of cellular metabo- nism is complicated, appealing for deeper research. lism [34], and MMP (ΔΨm) play a decisive role in determin- To elucidate the upstream of ES on apoptotic pathways, ing the way of cell death. Loss of ΔΨm leads to ATP we furtherly explored a potential one—ERK/STAT3—which depletion, unable to maintain ionic gradients across the might be involved in this process. In the classic MAPK cas- plasma membrane, causing necrosis finally. Mitochondria cade activation pathway, ERK plays an important role and also commit the apoptotic pathway by releasing the resident its phosphorylation can affect many cellular functions, protein cytochrome c and lead to the downstream apoptotic including apoptosis [21, 47]. STAT3 (signal transducer and events [33, 35, 42]. All the above results have identified the activator of transcription 3) has also been linked with sur- effect of ES pretreatment on promoting apoptosis; we then vival, proliferation, etc. [20]. P-ERK (phosphorylation of further explored the mitochondrial function and ROS activ- ERK) is closely related to p-STAT3 (phosphorylation of ity to fully understand its apoptotic-related events. In the STAT3) [21], while Bcl-2, one of the STAT3 target genes, is present study, we found that STC caused the loss of ΔΨm, involved in mitochondrial-related cell death. We first found whereas ES pretreatment enhanced it. The fluorescence that ES could reduce the level of p-ERK and p-STAT3 consis- intensity of Hoechst32242 was attenuated in the AP group tently in vivo in STC-induced acute pancreatitis. Then, we but increased in ES pretreatment groups, which were consis- blocked the activation of ERK with PD98059 in AR42J cells tent with the changes of ΔΨm. Therefore, we speculate that to further confirm the relationship between ERK/STAT3 ES pretreatment promoted apoptosis by changing the mito- and apoptosis. As shown in the above results, the ES chondrial membrane potential. (20 μg/mL) pretreatment group could significantly downreg- ROS mainly originate from mitochondria, but little was ulate p-ERK and p-STAT3 simultaneously with PD98059, known about its properties and their role in cell death in but when used in combination, it did not further reduce. acute pancreatitis. We next detected ROS activity in Activation of caspase-3 and Bax are positively correlated with AR42J cells. As shown in the above results, ES pretreat- apoptosis [48], while the ratio of Bcl-2/Bax is negatively cor- ment can increase ROS activity, which is consistent with related with it. We next found that both ES (20 μg/mL) and the changes of ΔΨm and Hoechst32242. A study [43] by PD98059 could upregulate cleaved caspase-3 and P53 and Booth et al. has proved that ROS exert its protective effect downregulate the Bcl-2/Bax ratio apparently. What is more, in acinar cells, which is different from many other cell when applied in combination, they furtherly promoted apo- types. As for why ROS are harmful to other cells (e.g., ptosis. The results of flow cytometry are also consistent with liver cells) and beneficial to pancreatic acinar cells, we those of Western blotting. are still uninformed. In pancreatic mitochondria, supramaximal CCK, ceru- 6. Conclusion lein, or TLC-S cause Ca2+-dependent loss of ΔΨm. ΔΨm loss caused by abnormal Ca2+ signal not only promotes necrosis In summary, Escin Sodium pretreatment improves the prog- but also inhibits apoptosis by limiting cytochrome c release nosis of acute pancreatitis induced by STC via inducing apo- [44]. Our results showed that intracellular calcium over- ptosis mediated by the ERK/STAT3 signaling pathway loaded in the AP group, which was consistent with the liter- (Figure 15). The results of this study provide new insight into atures [45]. However, the group pretreated with different the potential efficacy of Escin Sodium in the treatment of concentrations of ES could reduce the intracellular calcium acute pancreatitis. concentration in different degrees. Generally, the decrease of ΔΨm and ROS and the increase of calcium concentration Abbreviations exist at the same time when acute pancreatitis onsets, which can be reversed by ES pretreatment. However, the relation- AP: Acute pancreatitis ships between calcium concentration, ROS, and ΔΨm in aci- ES: Escin Sodium nar cells are interesting and blurred and need further MMP, ΔΨm: Mitochondrial membrane potential exploration. ROS: Reactive oxygen species Apoptosis preserves the plasma membrane integrity and SAP: Severe acute pancreatitis is thus considered to be immunologically silent. This is essen- MODS: Multiple organ dysfunction syndrome tially different from cell necrosis. Necrotic cell suffers from SIRS: Systemic inflammatory response syndrome serious cell membrane rupture, causing inflammatory cas- STAT3: Signal transducer and activator of cade reactions [46]. The expression of Bcl-2 and Bax is essen- transcription tial for ΔΨm changes, and P53 can move to mitochondria ERK: Extracellular regulated protein kinases Oxidative Medicine and Cellular Longevity 25

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