1.Department of Nephrology, Affiliated Hospital of Southwest Medical University;2.Department of Respiratory, The Affiliated Hospital of Southwest Medical University
目的 探讨大蒜素改善高糖诱导的人腹膜间皮细胞-间充质转化的相关机制。 方法 培养人腹膜间皮细胞（human peritoneal mesothelial cells，HPMCs）后将其进行2次分组，分组1：①对照组；②8.5 mM D-葡萄糖诱导组（8.5 mM DG组）；③17 mM D-葡萄糖诱导组（17 mM DG组）；④34 mM D-葡萄糖诱导组（34 mM DG组）；⑤68 mM D-葡萄糖诱导组（68 mM DG组）。其中除对照组外，其余组合分别用8.5 mM、17 mM、34 mM、68 mM的D-葡萄糖诱导48 h。分组2：①对照组；②34 mM D-葡萄糖诱导组（HG组）；③34 mM D-葡萄糖+低剂量大蒜素诱导组（AL-L组）；④34 mM D-葡萄糖+中剂量大蒜素诱导组（AL-M组）；⑤34 mM -葡萄糖+高剂量大蒜素诱导组（AL-H组）；⑥34 mM D-葡萄糖+JAK2抑制剂诱导组（JAK2组）。其中HG组用34 mM的D-葡萄糖诱导48 h，AL-L组、AL-M组、AL-H组用34 mM的D-葡萄糖预处理6h后分别用10 ng/mL、20 ng/mL和40 ng/mL大蒜素诱导48 h，JAK2组加入1 μmol/L AG490预处理6 h后用34 mM的D-葡萄糖诱导48 h。Elisa检测HPMCs上清的IL-6、TNF-α和IL-1β的含量；CCK-8检测细胞增殖并观察形态；Western blot检测JAK2、p-JAK2、STAT3、p-STAT3、N-cadherin、E-cadherin、Vimentin、α-SMA、MCP-1、p65、p-p65蛋白的表达情况。结果 与对照组相比，高糖诱导组HPMCs的相对存活率明显降低（P＜0.01），细胞形态表现异常，促进上皮细胞-间充质转分化（epithelial-mesenchymal transition，EMT）发生的α-SMA、N-cadherin和 Vimentin表达明显上调，抑制EMT发生的E-cadherin蛋白表达明显下调，JAK2/STAT3信号通路被激活从而导致EMT的发生（P＜0.01）；而大蒜素能明显促进高糖诱导后的HPMCs增殖，恢复异常的细胞形态，调节与EMT发生的相关蛋白水平从而改善HPMCs的上皮间质转分化；与高糖诱导组相比，大蒜素处理组HPMCs的促炎症因子IL-1β、IL-6和TNF-α明显降低，促炎症蛋白p-p50和MCP1表达明显下调，表明大蒜素能改善EMT引起的炎症。结论 大蒜素可通过抑制JAK2/STAT3信号通路调节EMT发生的标志蛋白、炎症信号蛋白及炎症因子水平从而改善高糖诱导的EMT及炎症。
Objective To investigate the mechanism of allicin in improving human peritoneal mesenchymal cell-mesenchymal transformation induced by high glucose. Methods human peritoneal mesothelial cells (HPMCs) were cultured and divided into two groups. Group 1: ①Control group; ②8.5 mM D-glucose induced group (8.5 mM DG group); ③17 mM D-glucose induced group (17 mM DG group); ④34 mM D-glucose induced group (34 mM DG group); ⑤68 mM D-glucose induced group (68 mM DG group). Except the control group, the other groups were induced with D-glucose of 8.5 mM, 17 mM, 34 mM and 68 mM, respectively, for 48 h. Group 2: ① control group; ②34 mM D-glucose induced group (HG group); ③34 mM D-glucose + low dose allicin induction group (AL-L group); ④34 mM D-glucose + medium dose allicin induction group (AL-M group); ⑤34 mm-glucose + high-dose allicin induction group (AL-H group); ⑥34 mM D-glucose + JAK2 inhibitor induction group (JAK2 group). HG group was induced with 34 mM D-glucose for 48 h, AL-L group, AL-M group and AL-H group were pretreated with 34 mM D-glucose for 6h, and then induced with 10 ng/mL, 20 ng/mL and 40 ng/mL allicin for 48 h, respectively. JAK2 group was pretreated with 1 μmol/L AG490 for 6 h and induced with 34 mM D-glucose for 48 h. The contents of IL-6, TNF-α and IL-1β in HPMCs supernatant were determined by Elisa. CCK-8 was used to detect cell proliferation and morphology. The expressions of JAK2, p-JAK2, STAT3, p-STAT3, N-cadherin, E-cadherin, Vimentin, α-SMA, MCP-1, p65 and p-p65 proteins were detected by Western blot. Results Compared with the control group, the relative survival rate of HPMCs in the high glucose induced group was significantly reduced (P<0.01), and cell morphology was abnormal, the expressions of α-SMA, N-cadherin and Vimentin that promote epithele-mesenchymal transdifferentiation were significantly up-regulated, and the expression of E-cadherin, which inhibits EMT, was significantly down-regulated. JAK2/STAT3 signaling pathway was activated, leading to the occurrence of EMT (P<0.01). Allicin can significantly promote the proliferation of HPMCs induced by high glucose, restore abnormal cell morphology, regulate the level of EMT-related proteins, and improve the epithelial-mesenchymal transdifferentiation of HPMCs. Compared with the high glucose induction group, the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α of HPMCs in allicin treatment group were significantly decreased, and the expressions of pro-inflammatory proteins p-p50 and MCP1 were significantly down-regulated, indicating that allicin could improve the inflammation caused by EMT. Conclution Allicin can improve EMT and inflammation induced by high glucose by inhibiting JAK2/STAT3 signaling pathway to regulate the levels of markers of EMT, inflammatory signaling proteins and inflammatory factors.