| 宋剑,段吉明,岳浩浩,等.重症急性胰腺炎大鼠肠源性内毒素、 Toll样受体 4蛋白与自噬微管相关蛋白 1轻链 3、p62蛋白、溶酶体相关膜蛋白 2的变化及相关性[J].安徽医药,2024,28(10):1956-1960. |
| 重症急性胰腺炎大鼠肠源性内毒素、 Toll样受体 4蛋白与自噬微管相关蛋白 1轻链 3、p62蛋白、溶酶体相关膜蛋白 2的变化及相关性 |
| Changes and correlation of intestinal endotoxin, toll-like receptor 4 protein and autophagy microtubule-associated protein 1 light chain 3, p62 protein, lysosomal associated membrane protein 2 in rats with severe acute pancreatitis |
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| DOI：10.3969/j.issn.1009-6469.2024.10.011 |
| 中文关键词: 重症急性胰腺炎 内毒素 细胞自噬 炎症因子 肠道屏障 大鼠， Sprague-Dawley |
| 英文关键词: Severe acute pancreatitis Endotoxin Autophagy Inflammatory factors Intestinal barrier Rats, Sprague-Dawley |
| 基金项目:山西省自然科学研究面上项目( 20210302123261) |
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| 中文摘要: |
| 目的探讨重症急性胰腺炎( SAP)大鼠肠源性内毒素、 Toll样受体 4(TLR4)蛋白与自噬微管相关蛋白 1轻链 3(LC3)、 p62蛋白( p62)、溶酶体相关膜蛋白 2(LAMP-2)的变化及相关性。方法 2022年 7月至 2023年 3月进行该研究。选取 SD大鼠 36只，随机数字表法分为假手术( SO)组和 SAP组，两组以术后时间点 3h、6h、12 h各自再平均分成三组。采用胰胆管逆行注射 5％牛磺胆酸钠( 0.1 mL/100 g)制备 SAP组模型， SO组找到胰腺后关腹。酶联免疫吸附测定( ELISA)检测大鼠血清中 α-淀粉酶( AMS)、肿瘤坏死因子 -α(TNF-α)、白细胞介素 -1β(IL-1β)和脂多糖( LPS)的含量。苏木精 -伊红染色法对比各组胰腺、小肠病理学评分变化。免疫组织化学法比较各组 LC3、p62和 LAMP-2的表达。蛋白质印迹法检测 TLR4蛋白表达。结果 SAP组大鼠血清 AMS、TNF-α、IL-1β、LPS，胰腺、小肠病理学评分，胰腺 LC3、p62阳性细胞总积分，胰腺组织 TLR4蛋白表达量与 SO组相比均明显升高(均 P<0.05)SAP组各项指标随术后时间延长不断升高。 SAP组胰腺 LAMP-2阳性细胞总积分［3h:(4.23±0.29)分， 6h:(0.33±0.16)分， h:(0.20±0.13)分］与 SO组［3h:(6.27±0.10)分， 6h:(6.23±0.34)分， 12 h:(6.30±0.28)分］相比明显降低( P<0.05)。 SAP组中，大鼠血清 LPS与血清 TNF-α、IL-1β，胰腺 LC3、p62、TLR4表达变化呈正相关( LPS比 TNF-α，r=12，0.92；LPS比 IL-1β，r=0.99；LPS比 LC3，r=0.98；LPS比 p62，r=0.99；LPS比 TLR4，r=0.96；均 P<0.001)；大鼠血清 LPS与胰腺 LAMP2表达变化呈负相关(r=?0.98，P<0.001)。结论 SAP时 LPS突破肠道屏障移位至胰腺，激活 TLR4通路表达大量炎症因子促进胰腺炎症反应，使细胞自噬蛋白 LC3与 p62表达上调，但 LAMP-2表达下降致自噬通量受损，肠源性内毒素移位与细胞自噬受损可共促进 SAP病程发展。 |
| 英文摘要: |
| Objective To investigate the changes and correlation of intestinal endotoxin, Toll-like receptor 4 (TLR4) protein and autophagy microtubule-associated protein 1 light chain 3 (LC3), p62 protein (p62) and lysosome associated membrane protein 2 (LAMP-2) in rats with severe acute pancreatitis (SAP).Methods The study will be conducted from July 2022 to March 2023. Thirty-six SD rats were randomly assigned into sham operation (SO) group and SAP group. The two groups were further assigned into three groups at 3 h, 6h and 12 h after surgery. The SAP group model was prepared by retrograde injection of 5% sodium taurocholate (0.1 mL/100 g) into thepancreaticobiliary duct. The abdomen of the SO group was closed after the pancreas was found. The levels of α-amylase (AMS), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and lipopolysaccharide (LPS) in the serum of rats were detected by enzyme-linked immunosorbent assay (ELISA). The changes of pancreatic and small intestinal pathological scores among the groups were compared byhematoxylin-eosin staining. The expressions of LC3, p62 and LAMP-2 among the groups were compared by immunohistochemistry. The expression of TLR4 protein was detected by western blot detection.Results Serum AMS, TNF-α, IL-1β, LPS, pathological score ofpancreas and small intestine, total score of LC3 and p62 positive cells in pancreas, and the expression of TLR4 protein of pancreatic tissue in SAP group were significantly higher than those in SO group (P<0.05), all the indexes in SAP group increased with time. The total score of LAMP-2 positive cells in pancreas in SAP group [3 h: (4.23 ±0.29) points, 6 h: (0.33 ±0.16) points, 12 h: (0.20 ±0.13) points]was significantly lower than that in SO group [3 h: (6.27±0.10) points, 6 h: (6.23±0.34) points, 12 h: (6.30±0.28) points] (P<0.05). In the SAP group, serum LPS of rats was positively correlated with the changes in serum TNF-α, IL-1β, and the expressions of pancreatic LC3, p62, and TLR4 (LPS vs. TNF-α, r=0.92, P<0.001; LPS vs. IL-1β, r=0.99, P<0.001; LPS vs. LC3,r=0.98, P<0.001; LPS vs. p62, r= 0.99, P<0.001; LPS vs. TLR4, r=0.96, P<0.001). There was a negative correlation between serum LPS and pancreatic LAMP-2 expression in rats (r=?0.98, P<0.001).Conclusions In SAP, LPS breaks through the intestinal barrier and translocates to the pancreas, activating the TLR4 pathway to express a large number of inflammatory factors to promote pancreatic inflammatory response, upregulatingthe expression of cellular autophagy proteins LC3 and p62, but decreasing the expression of LAMP-2, resulting in impaired autophagyflux. The translocation of enteric endotoxin and impaired cellular autophagy can jointly promote the development of SAP. |
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