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Original Article Resveratrol Alleviates Sepsis-Induced Acute Lung Injury by Suppressing Inflammation and Apoptosis of Alveolar Macrophage Cells

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Original Article Resveratrol Alleviates Sepsis-Induced Acute Lung Injury by Suppressing Inflammation and Apoptosis of Alveolar Macrophage Cells Am J Transl Res 2018;10(7):1961-1975 www.ajtr.org /ISSN:1943-8141/AJTR0057076 Original Article Resveratrol alleviates sepsis-induced acute lung injury by suppressing inflammation and apoptosis of alveolar macrophage cells Lei Yang1, Zhen Zhang2, Yuzhen Zhuo1, Lihua Cui1, Caixia Li1, Dihua Li1, Shukun Zhang1, Naiqiang Cui3, Ximo Wang2,3, Hongwei Gao4 1Tianjin Institute of Acute Abdominal Disease of Integrated Traditional Chinese and Western Medicine, Tianjin 300100, China; 2Graduate School of Tianjin Medical University, Tianjin 300070, China; 3Department of Surgery, Tianjin Hospital of Integrated Traditional Chinese and Western Medicine, Tianjin 300100, China; 4Department of Anesthesiology, Perioperative & Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston 02115, MA Received May 9, 2017; Accepted October 10, 2017; Epub July 15, 2018; Published July 30, 2018 Abstract: Sepsis is a major cause of death in intensive care units. The purpose of this study was to investigate the effect of resveratrol (RSV) on sepsis-induced acute lung injury (ALI). The underlying molecular mechanisms were deciphered by both in vitro and in vivo experiments. Polymicrobial sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). RSV pretreatment significantly attenuated CLP-induced acute lung injury, which was as- sociated with enhanced expression of VEGF-B. The protective properties of RSV were assayed in lipopolysaccharide (LPS)-stimulated MH-S cells. We determine that RSV administration inhibited the increased production of TNF-α, IL-6, and IL-1β in LPS-stimulated MH-S cells, which was associated with inhibition of the nuclear factor-κB, P38, and ERK signaling pathways. We also provide evidence that RSV administration reduced LPS-induced apoptosis of MH-S cells by altering the unbalance of Bax/Bcl-2 and inhibiting LPS-induced autophagy. The inhibitory effects of RSV on cytokine levels and apoptosis of alveolar macrophages were both blocked by VEGF-B siRNA. Furthermore, RSV administration regulated LPS-induced C5aR and C5L2 expression, revealing an additional mechanism underlying RSV’s anti-inflammatory and anti-apoptosis effects. Collectively, these results demonstrated that RSV was able to protect against sepsis-induced acute lung injury by activating the VEGF-B signaling pathway. Keywords: RSV, acute lung injury, alveolar macrophages, anti-apoptosis, anti-inflammation, VEGF-B Introduction mal models [7-9], because AMs apoptosis and dysfunction occur late in sepsis, which is impli- Despite improvements in supportive treatment cated in the increased mortality [10]. However, for acute lung injury (ALI), ALI is common and little is known about the intracellular signaling devastating clinical disorders with high morbid- pathways that regulate the fate of AMs in ALI, ity and mortality [1]. The pathophysiology of ALI and efforts directed towards the accurate me- is characterized by complex mechanisms that chanisms to improve ALI detection and treat- involve cell inflammation, cytokines, as well as ments are indispensable. Discovering new dr- abnormal apoptosis of pulmonary cells, includ- ugs and new strategies therefore remains an ing pulmonary alveolar type II epithelial cells, urgent therapeutic challenge in the treatment pulmonary vascular endothelial cells, alveolar of ALI. macrophages (AMs) [2-5]. AMs account for about 90% of the cells in the bronchoalveolar Resveratrol (3, 5, 4’-trans-trihydroxystilbene; lavage fluid (BALF) and play an important role RSV), a potent activator of SIRT-1 [11] present in the pathogenesis of ALI [6]. Many current in mulberries, peanuts, and grape skins [12, studies have indicated a consistent associa- 13], is known for its anti-inflammatory, anti-oxi- tion between AMs and ALI in humans and ani- dative and anti-apoptotic properties in multiple Resveratrol alleviates sepsis-induced acute lung injury organs, including the lungs [14, 15]. Emerging week before use. Mice were fasted but had studies have reported that RSV has protective free access to water starting 12 h before the effect in sepsis. It can significantly extend the surgery. A median incision was made in the life span of septic animals and reduce acute lower abdomen after anesthesia, skin was pre- lung injury in a LPS-induced sepsis mouse pared and disinfected, and the cecum was model [16, 17]. RSV could attenuate the apop- slightly taken out of the incision. A 4-0 braid- tosis of pulmonary microvascular endothelial ed silk suture was passed through the midpoint cells (PMVECs) and suppresse ox-LDL-induced between the colon root and cecum terminal, macrophage apoptosis [18, 19]. However, the which was used to tighten the cecum while molecular mechanisms underlying the protec- avoiding the cecum artery. After that, a 21 tive effects of RSV in septic ALI remain poorly gauge 1 inch needle was used to penetrate understood. the cecum ligation, from which a small drop of intestinal contents was squeezed after the The VEGF family includes five major factors: needle was withdrawn, followed by cecum re- VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placen- position and abdomen closure. For the sham tal growth factor [20]. VEGF-B bind to VEGF- group, the abdomen was opened, cecum ex- receptor 1 (VEGFR-1) and the neuropilin-1 posed and repositioned, and incision closed. (NRP-1) coreceptor, which are expressed in After successful modeling, mice in the CLP+ lung and lung macrophages [21, 22]. Recen- RSV group immediately received RSV (40 mg/ tly, VEGF-B was found as a key regulator of kg body weight). endothelial fatty acid uptake and insulin sensi- tivity [23, 24]. Its anti-cancer and neuroprotec- Cell culture tive effects were reported [25, 26]. In addition, VEGF-B expression is reduced in human heart MH-S cells were obtained from the American disease, and VEGFB mRNA expression is sig- Type Culture Collection (Manassas, VA, USA). nificantly increased in the hypoxic lung [27, 28]. Cells were cultured in RPMI 1640 medium sup- However, unlike VEGF-A, VEGF-B played a po- plemented with 10 mM HEPES, 2 mM l-gluta- mine, 100 U/ml streptomycin, 100 U/ml peni- tent antiapoptotic effect while lacking a gene- cillin, and 10% (v/v) FBS. Cells (approximately ral antigenic activity [29, 30]. VEGF-B treat- 1×10 6 cells/ml) were seeded in 6-well plates ment by intravitreal injection markedly reduced before being subjected to treatments. RSV at retinal apoptosis in streptozotocin (STZ)-in- 50 µM was added 2 h before LPS (from Es- duced diabetes rats [31]. However, the func- cherichia coli, Sigma) stimulation. tions of VEGFB in macrophages under septic conditions remain poorly understood. Histologic examination and grading In the previous study, we have first identified After the animals were killed, lung tissues em- the relationship between RSV treatment and bedded in paraffin and cut in 3 μm thick sec- the expression of VEGF-B [32]; we also found tions. Sections were stained with hematoxylin that RSV attenuates myocardial ischemia/re- and eosin stain. The total score was calculated perfusion injury through upregulation of VEGF- by adding up the individual scores of each cat- B [33]. The purpose of this study was to investi- egory [34]. gate the protective mechanisms of RSV in sep- sis-induced acute lung injury, particularly with Pulmonary MPO activity regard to the VEGF-B signaling. MPO activity, an indicator of polymorphonucle- Materials and methods ar leukocytes accumulation, was determined using an MPO kit produced by Jiancheng Animals and experimental designs Bioengineering Institute (Nanjing, China) ac- cording to the manufacturer’s instruction. SPF C57BL/6 male mice were purchased from China Laboratory Animal Resource Center of Cell-viability assay National Institute for Food and Drug Control. The experimental protocol was approved by the The cell viability in the various treatment groups Medicine Ethical Committee of Tianjin Nankai was assessed with an MTT assay. In this assay, Hospital. Mice were acclimatized for at least 1 MH-S cells were rested in 96-well plates (5,000 1962 Am J Transl Res 2018;10(7):1961-1975 Resveratrol alleviates sepsis-induced acute lung injury cells/well). Following incubation with RSV (10 ing to the manufacturer’s instructions. At 24 µM) for 2 h, cells were subsequently treated hr after transfection, the cells were incubated with different concentrations of LPS in com- with RSV followed by LPS treatment. plete medium for 24 h. Subsequently, 10 µl MTT was added to each well, and the plates RNA extraction and quantitative real-time poly- were incubated for an additional four hours at merase chain reaction 37°C. Subsequently, media was carefully dis- carded and 100 μl of dimethyl sulfoxide were Total RNA was isolated using trizol reagent added to dissolve the formazan crystals, then (Takara Bio, Japan) according to the manu- the 96-well plates were put on a horizontal facturer’s protocol. Reverse transcription reac- oscillator for ten minutes. The absorbance val- tions were performed using a 1 μg total RNA ues were measured by the enzyme mark instru- with TransGen Reverse Transcription Kit from ment at a wavelength of 490 and a reference Applied Biosystems. Quantitative real-time PCR wavelength of 570 nm. detection of gene expression was performed using SYBR Green Master Mix (Promega, Chi- Measurement of apoptosis by flow cytometry na) in a Bio-Rad IQ5 detection system and the analysis using the PI/Annexin V double-stain- cycle threshold (CT) values were automatical- ing method ly determined in triplicates and averaged. The
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