HMGB1/TLR4通路在重症急性胰腺炎肠道炎症损伤及线粒体功能障碍中的作用

于克静; 张满鹤; 贾世浩; 张福梅; 郝景察

HMGB1/TLR4通路在重症急性胰腺炎肠道炎症损伤及线粒体功能障碍中的作用

Role of HMGB1/TLR4 pathway in intestinal inflammatory injury and mitochondrial dysfunction in severe acute pancreatitis

摘要

摘要:
目的： 探究高迁移率族蛋白B1（HMGB1）/Toll样受体4（TLR4）通路在重症急性胰腺炎（SAP）肠道炎症损伤及线粒体功能障碍中的作用。
方法： 将30只SD大鼠随机分为对照组、SAP组、抑制HMGB1组，每组10只。H&E染色检查大鼠胰腺及肠道组织的病理结构，RT–qPCR检测大鼠肠道组织中HMGB1、TLR4 mRNA水平，Western blotting检侧大鼠肠道组织中HMGB1、TLR4蛋白水平，试剂盒检测大鼠血清脂肪酶、淀粉酶ATP合成酶、mtROS、线粒体膜电位(Δψm)、线粒体复合体Ⅰ和Ⅲ、肿瘤坏死因子–α（TNF–α）、白细胞介素（IL）–6、IL–1β含量，透射电镜观察大鼠肠道线粒体超微结构。
结果： 与对照组相比，SAP组大鼠胰腺组织大片水肿坏死，周围可见炎性细胞浸润，肠道组织结构紊乱，肠绒毛水肿增粗并可见部分发生脱落，周围可见炎性聚集，肠道组织中HMGB1、TLR4蛋白及mRNA水平升高（P ＜ 0.01），大鼠血清脂肪酶、淀粉酶水平升高（P ＜ 0.01），血清TNF–α、IL–6、IL–1β水平升高（P ＜ 0.01），线粒体肿胀，线粒体脊及膜破裂，mtROS水平、Δψm相对荧光密度及线粒体复合体I和III水平降低（P ＜ 0.01）；与SAP组相比，抑制HMGB1组大鼠胰腺组织及肠道组织病理损伤改善，肠道组织中HMGB1、TLR4蛋白及mRNA水平降低（P ＜ 0.01），血清脂肪酶、淀粉酶水平降低（P ＜ 0.01），血清TNF–α、IL–6、IL–1β水平降低（P ＜ 0.01），线粒体结构改善，mtROS水平、Δψm相对荧光密度及线粒体复合体I和III水平升高（P ＜ 0.01）。
结论： 抑制HMGB1/TLR4通路可改善SAP肠道炎症损伤及线粒体功能障碍。

Abstract:
Objective To investigate the role of high mobility group box 1 protein (HMGB1)/Toll-like receptor 4 (TLR4) pathway in intestinal inflammatory injury and mitochondrial dysfunction in severe acute pancreatitis (SAP).
Methods Thirty Sprague-Dawley (SD) rats were randomly divided into a control group, a SAP group, and a HMGB1-inhibited group, with 10 rats in each group. H&E staining was used to examine the pathological structure of pancreatic and intestinal tissues in rats. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA levels of HMGB1 and TLR4 in intestinal tissues of rats. Western blotting was used to detect the protein levels of HMGB1 and TLR4 in intestinal tissues of rats. Reagent kits were used to detect the levels of serum lipase, amylase, ATP synthase, mitochondrial reactive oxygen species (mtROS), mitochondrial membrane potential (Δψm), mitochondrial complexes I and III, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β. Transmission electron microscopy was used to observe the ultrastructure of intestinal mitochondria in rats.
Results Compared with the control group, the pancreatic tissues of the SAP group showed extensive edema and necrosis, with inflammatory cell infiltration around the tissues. The intestinal tissue structure was disordered, and the intestinal villi were swollen and thickened, with some showing detachment. Inflammatory aggregates were visible around the tissues. The protein and mRNA levels of HMGB1 and TLR4 in intestinal tissues were increased (P ＜ 0.01). The levels of serum lipase and amylase were elevated (P ＜ 0.01), as were the levels of serum TNF-α, IL-6, and IL-1β (P ＜ 0.01). Mitochondria were swollen, with cristae and membrane ruptures. The levels of mtROS, Δψm relative fluorescence density, and mitochondrial complexes I and III were decreased (P ＜ 0.01). Compared with the SAP group, the pathological damage in pancreatic and intestinal tissues of the HMGB1-inhibited group was improved. The protein and mRNA levels of HMGB1 and TLR4 in intestinal tissues were reduced (P ＜ 0.01), as were the levels of serum lipase and amylase (P ＜ 0.01). The levels of serum TNF-α, IL-6, and IL-1β were decreased (P ＜ 0.01). Mitochondrial structure was improved, and the levels of mtROS, Δψm relative fluorescence density, and mitochondrial complexes I and III were increased (P ＜ 0.01).
Conclusions Inhibiting the HMGB1/TLR4 pathway can alleviate intestinal inflammatory injury and mitochondrial dysfunction in SAP.

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