葛根素调节Nrf2/HO-1信号通路对脂多糖诱导的人牙龈成纤维细胞炎症反应的影响

Effect of Puerarin on Lipopolysaccharide-Induced Inflammatory Response in Human Gingival Fibroblasts by Regulating Nrf2/HO-1 Signaling Pathway

目的:探讨葛根素(GGS)调节核因子E2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)信号通路对脂多糖(LPS)诱导的人牙龈成纤维细胞(HGFs)炎症反应的影响。方法:体外培养HGFs细胞,将HGFs细胞分为空白对照(CK)组(0.9%氯化钠)、LPS组(100 nM LPS)、LPS+GGS低剂量(LPS+GGS-L)组(100 nM LPS+50μM GGS)、LPS+GGS高剂量(LPS+GGS-H)组(100 nM LPS+100μM GGS)、LPS+GGS-H+ML385(Nrf2/HO-1信号通路抑制剂)组(100 nM LPS+100μM GGS+10μM ML385)。采用细胞计数试剂盒-8(CCK-8)检测细胞增殖能力;酶联免疫吸附法(ELISA)检测白细胞介素(IL)-6、乳酸脱氢酶(LDH)、IL-10、IL-1β水平;试剂盒检测丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)水平;流式细胞术检测HGFs细胞凋亡率;碘化丙啶(PI)染色法检测细胞膜孔的形成;Western blot法检测B淋巴细胞瘤-2(Bcl-2)、B细胞淋巴瘤-2相关X蛋白(Bax)、NOD样受体蛋白3(NLRP3)、Nrf2、HO-1蛋白表达。结果:与CK组相比,LPS组HGFs细胞OD_(450)(24、48 h)值、IL-10水平、GSH-Px和SOD活性、Bcl-2、Nrf2和HO-1蛋白表达显著降低,IL-6、LDH、IL-1β水平、MDA含量、细胞凋亡率、细胞PI染色阳性率、Bax和NLRP3蛋白表达显著升高(P<0.05);与LPS组相比,LPS+GGS-L组和LPS+GGS-H组HGFs细胞OD_(450)(24、48 h)值、IL-10水平、GSH-Px和SOD活性、Bcl-2、Nrf2和HO-1蛋白表达显著升高,IL-6、LDH、IL-1β水平、MDA含量、细胞凋亡率、细胞PI染色阳性率、Bax和NLRP3蛋白表达显著降低(P<0.05);ML385可减轻GGS对LPS诱导的HGFs细胞炎症损伤的改善作用(P<0.05)。结论:GGS可能通过激活Nrf2/HO-1信号通路减轻LPS诱导的HGFs细胞炎症和氧化应激反应,降低细胞凋亡。

Objective:To investigate the effect of puerarin(GGS)on lipopolysaccharide(LPS)-induced inflammatory response in human gingival fibroblasts(HGFs)by regulating nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway.Methods:HGFs cells were cultured in vitro,HGFs cells were divided into blank control(CK)group(0.9%sodium chloride),LPS group(100 nM LPS),LPS+GGS low dose(LPS+GGS-L)group(100 nM LPS+50μM GGS),LPS+GGS high dose(LPS+GGS-H)group(100 nM LPS+100μM GGS),LPS+GGS-H+ML385(Nrf2/HO-1 signaling pathway inhibitor)group(100 nM LPS+100μM GGS+10μM ML385).The cell proliferation ability was detected by cell counting kit-8(CCK-8).The levels of interleukin(IL)-6,lactate dehydrogenase(LDH),IL-10 and IL-1βwere detected by enzyme-linked immunosorbent assay(ELISA).The levels of malondialdehyde(MDA),glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)were detected by kits.The apoptosis rate of HGFs was detected by flow cytometry.The formation of cell membrane pores was detected by propidium iodide(PI)staining.The expressions of B-cell lymphoma-2(Bcl-2),B-cell lymphoma-2 associated X protein(Bax),NOD-like receptor protein 3(NLRP3),Nrf2 and HO-1 were detected by Western blot.Results:Compared with CK group,the OD_(450)(24 and 48 h)value,IL-10 level,GSH-Px and SOD activity,Bcl-2,Nrf2 and HO-1 protein expression of HGFs cells in LPS group were significantly decreased,and the levels of IL-6,LDH,IL-1β,MDA content,apoptosis rate,positive rate of PI staining,Bax and NLRP3 protein expression were significantly increased(P<0.05).Compared with LPS group,the OD_(450)(24 and 48 h)value,IL-10 level,GSH-Px and SOD activity,Bcl-2,Nrf2 and HO-1 protein expression of HGFs cells in LPS+GGS-L group and LPS+GGS-H group were significantly increased,and the levels of IL-6,LDH,IL-1β,MDA content,apoptosis rate,positive rate of PI staining,Bax and NLRP3 protein expression were significantly decreased(P<0.05).ML385 could reduce the improvement of GGS on LPS-induced inflammatory injury in HGFs cells(P<0.05).Conclusion:GGS may alleviate LPS-induced inflammation and oxidative stress in HGFs cells by activating Nrf2/HO-1 signaling pathway,and reduce cell apoptosis.