Department of Clinical Laboratory, Huangshi Central Hospital, Affiliated Hospital of Hubei Polytechnic University, Edong Healthcare Group, Hubei, People's Republic of China; Hubei Key Laboratory of Kidney Disease Pathogenesis and InterventionHubei, Huangshi, Hubei, People's Republic of China.
Department of Clinical Laboratory, Huangshi Central Hospital, Affiliated Hospital of Hubei Polytechnic University, Edong Healthcare Group, Hubei, People's Republic of China; Hubei Key Laboratory of Kidney Disease Pathogenesis and InterventionHubei, Huangshi, Hubei, People's Republic of China.
Toxicol In Vitro. 2022 Jun;81:105350. doi: 10.1016/j.tiv.2022.105350. Epub 2022 Mar 21.
The NLRP3 inflammasome plays a crucial role in microbially induced gastric epithelial injury, but the underlying mechanisms remain unclear. Here, we aimed to assess the impacts of puerarin on LPS-induced inflammatory damage and the involvement of the AMPK/SIRT1/NLRP3 signaling pathways in this process in GES-1 cells. Cell viability and cytotoxicity were determined using CCK-8 and lactate dehydrogenase assay kits. Apoptosis was measured using annexin staining followed by flow cytometry. Cytokine levels were detected by ELISA, and protein expression was analyzed using western blotting. Protein overexpression was achieved by transfection with relevant pcDNA3.1 vectors, and protein knockdown was achieved by transfection with relevant siRNAs. Puerarin ameliorated LPS-induced cytotoxicity and apoptosis, while repressing LPS-stimulated NLRP3 inflammasome-mediated pyroptosis in GES-1 cells, as evidenced by significantly decreased expression of NLRP3, ASC, cleaved caspase-1, IL-1β and IL-18. NLRP3 knockdown efficiently repressed LPS-induced inflammatory injury in GES-1 cells. Puerarin activated the AMPK/SIRT1 pathway in LPS-treated GES-1 cells, and knockdown of both AMPK and SIRT1 reversed the protective effects of puerarin against LPS-induced inflammatory damage. AMPK overexpression strengthened, while AMPK knockdown weakened, the ability of puerarin to inhibit NLRP3-mediated inflammatory injury in LPS-treated GES-1 cells. Our findings suggest that puerarin may ameliorate LPS-induced inflammatory injury in GES-1 cells by activating the AMPK/SIRT1 signaling pathway and thereby repressing NLRP3 inflammasome-mediated apoptosis.
NLRP3 炎性小体在微生物诱导的胃上皮损伤中起着至关重要的作用，但潜在机制尚不清楚。在这里，我们旨在评估葛根素对 LPS 诱导的炎症损伤的影响，以及 AMPK/SIRT1/NLRP3 信号通路在这个过程中的参与情况，研究对象为 GES-1 细胞。通过 CCK-8 和乳酸脱氢酶试剂盒测定细胞活力和细胞毒性。通过 Annexin 染色结合流式细胞术测定细胞凋亡。通过 ELISA 检测细胞因子水平，通过 Western blot 分析蛋白表达。通过转染相关 pcDNA3.1 载体实现蛋白过表达，通过转染相关 siRNA 实现蛋白敲低。葛根素改善 LPS 诱导的 GES-1 细胞毒性和细胞凋亡，同时抑制 LPS 刺激的 NLRP3 炎性小体介导的细胞焦亡，表现在 NLRP3、ASC、裂解的 caspase-1、IL-1β 和 IL-18 的表达显著下调。NLRP3 敲低有效地抑制 LPS 诱导的 GES-1 细胞炎症损伤。葛根素激活 LPS 处理的 GES-1 细胞中的 AMPK/SIRT1 通路，并且 AMPK 和 SIRT1 的敲低逆转了葛根素对 LPS 诱导的炎症损伤的保护作用。AMPK 过表达增强，而 AMPK 敲低削弱，葛根素抑制 LPS 处理的 GES-1 细胞中 NLRP3 介导的炎症损伤的能力。我们的研究结果表明，葛根素可能通过激活 AMPK/SIRT1 信号通路，从而抑制 NLRP3 炎性小体介导的凋亡，来改善 LPS 诱导的 GES-1 细胞炎症损伤。
