Acta Medica Mediterranea, 2018, 34: 489 STUDY ON PROTECTING EFFECTS OF PARTHENOLIDE ON HEPATIC INJURY IN RATS WITH SEVE- RE ACUTE PANCREATITIS GAOZHONG LI1,2, GUANGSHENG ZHAI3, HONG TAO4, JINGMEI CAO2, XIULIAN WANG2, ZHAOSHENG CHEN1, MIN LI2, KUNMING HUANG2, JIANQIANG GUO1* 1Department of Gastroenterology, the second hospital of Shandong University, No. 247 of Beiyuan Street, Jinan 250033, Shandong Province, China - 2Department of Gastroenterology, Zibo Central Hospital, No. 54 West of Gongqingtuan Road, Zhangdian District, Zibo 255036, Shandong Province, China - 3Department of Radiotheraphy, Zibo Central Hospital, No. 54 West of Gongqingtuan Road, Zhangdian District, Zibo 255036, Shandong Province, China - 4Division of Clinical Skill Training Center, Zibo Central Hospital, No. 54 West of Gongqingtuan Road, Zhangdian District, Zibo 255036, Shandong, China ABSTRACT Objective: Our study was designed to investigate the protective effects of parthenolide on hepatic injury in rats with severe acute pancreatitis (SAP). Methods: The SAP rat models were prepared and randomly devided into five groups，the model control group, parthenolide treated group with different doses of parthenolide, and the sham operated group. The levels of AMY, ALT, AST, IL-6 and TNF-α in serum, NF-κB expression and hepatic pathological changes in all groups were detected. Results: The levels of AMY, ALT, AST, IL-6 and TNF-α in serum and expression levels of NF-κB were lower in parthenolide treated groups than that in model control group. The model control group showed obviously pathological changes compared with sham group, while the administration of parthenolide dose-dependently inhibited the changes. Conclusion: Parthenolide demonstrated a well curative capability on rats with SAP. Keywords: Severe acute pancreatitis; parthenolide; TNF-α; IL-6; NF-κB. DOI: 10.19193/0393-6384_2018_2_79 Received November 30, 2017; Accepted January 20, 2018 Introduction If the SIRS is severe, then proinflammatory mediators can cause early multiple (respiratory, car- Severe acute pancreatitis (SAP) is one of the diovascular, renal, and hepatic) organ failure. SAP most common acute abdomens in clinical practice(1), is often associated with high morbidity and mortali- which can cause systemic inflammatory response ty due to the development of pancreatic and extra- syndromes (SIRS) such as effusion of blood vessel, pancreatic necrosis, their subsequent infection and shock and multiple organ functional disturbances or multisystem organ failure (MOF)(5-7). Inflammatory even multiple organ dysfunction syndrome(2-4). SAP mediators is critical for progression of SAP and has generally undergoes two phases. During the first 1 been receiving increasing attention in recent years. or 2 week, a pro-inflammatory response occurs, SAP is a potentially lethal inflammatory condition which results in systemic inflammatory response of the pancreas that involves both peripancreatic syndrome (SIRS), a sterile response in which sepsis tissue and remote organs. Local inflammatory reac- or infection rarely occurs. tion in pancreas leads to systemic SIRS and MOF, 490 Gaozhong Li, Guangsheng Zhai et Al which is believed to be the capital cause of mortali- 60%, and with free access to food and water. All ty.8,9 Inflammatory components include interleukin procedures were in accordance with the Guidelines (IL)-6, tumor necrosis factor (TNF)-a, IL-8, IL-10, of the Animal Experiments of Yuhuangding and IL-1b are considered to be required for SAP Hospital of Yantai, Affiliated Hospital of Qingdao development(10). A recent study has shown that pro- Medical University. inflammatory stimuli such as IL-6 and TNF-a play a central role in the initiation and progression of Model of SAP SAP.11 Despite overall reduced mortality in the last The rats were fasted overnight with free access decade, SAP is a devastating disease that is associ- to water 12 h before experiments. Surgical anesthe- ated with mortality ranging from less than 10% to sia was performed by intraperitoneal injection of as high as 85%(12-16). 10% chloral hydrate. SAP was induced by retro- Parthenolide is a sesquiterpene lactone that grade infusion of 1% sodium taurocholate (0.1 was purified from the shoots of feverfew mL/100 g body weight) into the common pancreatic (Tanacetum parthenium), a traditional herbal medi- duct through epidural catheter and duodenal papil- cine that has been used for the treatment of arthritis, la.21 Five minutes later artery clamp was removed, fever and migraine in China(17). The nucleophilic tube was drawn, the abdominal wall was sutured to nature of its methylene-γ-lactone ring and epoxide establish the animal model of SAP. Duodenum and group enables rapid interactions with biological the pancreas of rats in control group was only sites. These interactions have been found to be injected saline instead of sodium taurocholate then related to its ability to induce oxidative stress, and gently put back to the abdomen. it shows multiple anti-cancer and pro-apoptotic characteristics. Previous studies have demonstrated Animal groups that parthenolide has strong anti-inflammatory Fifty rats were randomly divided into the effects and is a potent nuclear factor kappa-B (NF- sham-operated group, the model control group and κB) inhibitor by specifically inhibiting the IκB parthenolide treated group. In sham-operated group, kinase complex(8-20). Therefore, the present study only exploratory laparotomy was performed, name- was focused on the preventive effect of partheno- ly after entering abdominal cavity, checking pan- lide in rats model with SAP. creas and duodenum and then closing abdomen(22-23). Both sham-operated group and model control group Materials and methods were injected equivalent volume saline at the corre- sponding time points during SAP operation. After Chemicals SAP operation, rats in all groups were allowed free Parthenolide was purchased from Beijing access to water. The rats in parthenolide treated Institute of Biological Products (Beijing, China) group were injected parthenolide intraperitoneally and it’s purity was determined to be ≥ 98% by by low (100 μg/kg), middle (200 μg/kg) and high HPLC measurement. Sodium taurocholate was pur- (300 μg/kg) dose. Rats in sham-operated group and chased from USA Sigma Company. TNF-a and IL-6 model control group were intraperitoneally infused enzyme-linked immunosorbentassay (ELISA) kits with tween 80 (0.05%). were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Monoclonal antibodies ELISA assay against NF-κB p65 subunit and β-actin were pur- After the completion of treatments, the ani- chased from Santa Cruz Biotechnology, Inc. (Santa mals were sacrificed under deep anesthesia with Cruz, CA, USA). chloral hydrate (300 mg/kg, i.p.) and bloodletting via inferior vena cava. Blood samples were collect- Animals ed and centrifuged (4°C) at 3000 r/min for 15 min. Fifty healthy male Sprayer (SD) rats at 250- The upper serum was separated immediately and 280 g of body weight (7-8 weeks old) were pur- was stored at -20°C for future analysis. ELISA kits chased from the Experimental Animal Center of (BioSource International, Camarillo, CA, USA) Suzhou StairMaster technology Co. Ltd. (SPF was performed to determine serum levels of TNF-α grade, Certificate No. SCXK20140007). All rats and IL-6 according to the manufacturer’s instruc- were kept in a regulated environment with a con- tions. Changes of α-amylase (AMY), alanine trolled temperature of 22-24℃ and humidity of 50- aminotransferase (ALT) and aspartate aminotrans- Study on protecting effects of Parthenolide on hepatic injury in rats with severe acute pancreatitis 491 ferase (AST) in serum were determined by ELISA Twelve hours after modeling, the content of kits, respectively. AMY, AST and ALT in serum of model control group and parthenolide treated groups were signifi- Histopathological evaluation cantly higher than that in sham-operated group (P < The hepatic specimens was collected and fixed 0.05). The parthenolide treated groups were obvious- in 10% formalin for 24 h, embedded in paraffin, ly lower than the model control group (P < 0.05), and stained with hematoxylin-eosin (HE) and which appeared to be dose-dependent (Figure 1). examined by light microscopy. The content of IL-6 and TNF-α in serum Western Blot analysis As shown in Figure 2, 12 h after induction of Hepatic tissues were lysed by the nuclear SAP, the serum IL-6 and TNF-α level in model extract kit following the manufacturer’s instruc- control group was obviously higher than that in tions. SDS-PAGE was performed using 50 or 100 sham-operated group, whereas parthenolide treat- g/l acrylamide gels. Proteins were electrotransfered ment attenuated the increase of serum IL-6 (Figure to polyvinylidene fluoride membranes and probed 2A) and TNF-α (Figure 2B), which appeared to be with primary antibody (anti- NF-κB-p65, 1:500; β- dose-dependent. actin, 1:500). The membranes were incubated with corresponding horseradish peroxidase-linked secondary antibody. They were developed by enhanced chemiluminescence (Amersham International, Buckingham, UK), and exposure to X-ray film. Statistical analysis Data were presented as means ± standard deviation (SD). Statistical comparisons between Figure 1. Comparison of AMY, ALT, AST in serum. two groups were analyzed by t test. Values p < A: The content of AMY in serum; B: The content of ALT in 0.05 was considered statistically significant. serum; C: The content of AST in serum. *p < 0.05 compared with Sham operated group; #p < 0.05 compared with Model Results control group. Serum AMY, ALT and AST content Pathological findings of liver tissue Sham group: Twelve hours after modeling, complete structure of hepatic lobules, occasional inflammatory cell infiltration in portal area were observed. Most liver cells presented normal mor- phology (Figure 3A). In the model control group, there was obviously damaged hepatic lobule struc- ture, further increased range and area of cell necro- sis, residual metamorphic liver cells only at periph- ery of partial hepatic lobules; relatively large area of inflammatory cell infiltration within lobules or portal area, obvious congestion in sinus hepaticus (Figure 3B).