基于Nrf2-NLRP3-GSDMD信号通路探讨肾消解毒通络方含药血清抑制高糖诱导的肾小球系膜细胞焦亡的分子机制

Exploration on Molecular Mechanism of Shenxiao Jiedu Tongluo Recipe(肾消解毒通络方)Inhibiting High Glucose-Induced Pyrolysis of Glomerular Mesangial Cells

目的基于核因子E2相关因子2(nuclear factor erythroid2 related factor2,Nrf2)-Nod样受体蛋白3(nod-like receptor protein 3,NLRP3)-消皮素D(gasdermin D,GSDMD)信号通路,探究肾消解毒通络方(Shenxiao Jiedu Tongluo Recipe,SJTR)含药血清对高糖诱导的大鼠肾小球系膜细胞HBZY-1焦亡的影响及分子机制。方法金黄地鼠灌胃给药制备SJTR含药血清。体外常规培养HBZY-1细胞,随机分为对照组、模型组、抑制剂组及中药低中高剂量6组。对照组细胞加入正常血清在低糖培养液中培养,模型组加入正常血清在高糖培养液中培养,抑制剂组加入正常血清及MCC950在高糖培养液中培养,中药低中高剂量组分别加入SJTR含药血清在高糖培养液中培养,干预24 h后收集细胞及上清液,ELISA检测各组细胞上清液中白介素-1β(Interleukin,IL-1β)、IL-18(Interleukin,IL-18)、IL-6(Interleukin,IL-6)、肿瘤坏死因子(tumor necrosis factor,TNF-α)、丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD);RT-PCR检测HBZY-1细胞Nrf2、NLRP3、Caspase-1、GSDMD、IL-1β分子mRNA表达;Western blot法检测各分子蛋白的表达;免疫荧光法检测NLRP3分子蛋白的表达及分布;LDH释放实验检测细胞膜损伤情况;TUNEL染色检测DNA损伤情况。结果与对照组比较,模型组细胞上清液各炎症因子水平升高,MDA表达升高,SOD表达降低(P<0.05);细胞Nrf2分子呈低表达,IL-1β分子mRNA表达升高,NLRP3、Casepase-1、GSDMD分子mRNA及蛋白表达增强(P<0.05);上清液LDH含量增高(P<0.05);TUNEL阳性细胞增多。与模型组比较,中药中高剂量组及抑制组细胞上清液炎症因子含量下降,MDA水平降低而SOD升高(P<0.05);细胞Nrf2分子表达上调,而NLRP3、Caspase-1、GSDMD等分子表达明显降低(P<0.05);上清液LDH含量下降(P<0.05);TUNEL阳性细胞减少。结论SJTR含药血清可通过调控Nrf2-NLRP3-GSDMD信号通路各分子表达,抑制高糖诱导的HBZY-1细胞氧化应激及炎症反应,拮抗细胞焦亡,发挥细胞保护作用。

Objective Based on nuclear factor erythroid2 related factor2(Nrf2)-Nod-like receptor protein 3(NLRP3)-gasdermin D(GSDMD)signal pathway,to observe the effect of Shenxiao Jiedu Tongluo Recipe(肾消解毒通络方,SJTR)drug-containing serum on HBZY-1 pyroptosis induced by high glucose in rat mesangial cells and its molecular mechanisms.Methods SJTR drug-containing serum was prepared by intragastric administration of golden hamsters.HBZY-1 cells were routinely cultured in vitro and randomly divided into normal control group,model group,inhibitor group and three traditional Chinese medicine groups with different dosage.The cells in the control group were cultured in low glucose medium,the cells in the model group were cultured in high glucose medium,the cells in the inhibitor group were cultured in high glucose medium with normal serum and MCC950,and the cells in different dosages of traditional Chinese medicine were cultured in high glucose medium with SJTR drug-containing serum,respectively.After 24 h of drug intervention,the cells and supernatant were collected.The levels of interleukin(IL)-1β,IL-6,IL-18,tumor necrosis factor(TNF-α),malondialdehyde(MDA)and superoxide dismutase(SOD)in supernatant were detected by ELISA.The mRNA expressions of Nrf2,NLRP3,Caspase-1,GSDMD,IL-1β in HBZY-1 cells were detected by RT-PCR.The expression of each molecular protein was detected by Western Blot.The expression and distribution of NLRP3 molecular protein were detected by immunofluorescence.The damages of cell membranes were detected by LDH release tests.DNA damage was detected by TUNEL staining.Results Compared with those of the control group,the levels of inflammatory factors in the supernatant of cells in the model group were increased,the expression of MDA was increased,and the expression of SOD was decreased(P<0.05).The expression of Nrf2 molecule was low and the expression of IL-1βmRNA was increased.NLRP3,Casepase-1,GSDMD molecule mRNA and protein expression increased(P<0.05).LDH content in the supernatant increased(P<0.05).TUNEL positive cells increased.Compared with those of the model group,the contents of inflammatory factors in the supernatant of the cells in the medium and high dose Chinese medicine groups and inhibition group decreased,the level of MDA decreased and the SOD increased(P<0.05).The expressions of Nrf2 were increased,while the expressions of NLRP3,Caspase-1 and GSDMD were significantly down-regulated(P<0.05).LDH content in the supernatant decreased(P<0.05).TUNEL showed a decrease in positive cells.Conclusion SJTR-containing serum can inhibit the oxidative stress and inflammatory response of HBZY-1 cells induced by high glucose by regulating the expression of each molecule in the Nrf2-NLRP3-GSDMD signaling pathway,antagonize cell pyroptosis and exert a cytoprotective effect.