人参皂苷Rg1抑制NLRP3/Caspase-1通路抵抗肾纤维化的作用研究

Effect of ginsenoside Rg1 on inhibition of NLRP3/Caspase-1 pathway against renal fibrosis

目的:通过体内外实验探讨人参皂苷Rg1对转化生长因子-β1(TGF-β1)诱导的人肾小管上皮细胞HK2纤维化细胞株中炎症及其抗纤维化的机制。方法:将HK2细胞诱导分组为对照组、纤维化模型组(2 ng/mL的TGF-β1诱导纤维化)、低剂量人参皂苷Rg1治疗组(2 ng/mL的TGF-β1+15μg/mL人参皂苷Rg1)、高剂量人参皂苷Rg1治疗组(2 ng/mL的TGF-β1+45μg/mL人参皂苷Rg1);Western印迹及免疫荧光检测结缔组织生长因子(CTGF)、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(Col-1)表达水平;Western印迹检测NOD样受体蛋白3(NLRP3)及半胱氨酸天冬氨酸蛋白水解酶-1(Caspase-1)表达水平;透射电镜检测细胞焦亡水平。动物研究将大鼠诱导分为对照组、模型组(单侧输尿管梗阻)、低剂量人参皂苷Rg1治疗组(单侧输尿管梗阻大鼠+50 mg/kg人参皂苷Rg1)、高剂量人参皂苷Rg1治疗组(单侧输尿管梗阻大鼠+100 mg/kg人参皂苷Rg1);Western印迹检测肾组织中蛋白表达水平;苏木精-伊红(HE)及Masson检测肾脏病理改变及纤维化程度;酶联免疫吸附测定法(ELISA)检测血清中炎症因子表达水平;透射电镜检测组织焦亡水平。结果:细胞实验中,人参皂苷Rg1处理后,细胞中CTGF、α-SMA、ColⅠ及NLRP3、Caspase-1的表达水平均较模型组以剂量依赖的减低(均P<0.05);细胞焦亡水平显著降低。动物实验中,人参皂苷Rg1处理后,大鼠肾组织中CTGF、α-SMA、ColⅠ及NLRP3、Caspase-1的表达水平均较模型组以剂量依赖的减低(均P<0.05);血清TGF-β1、白细胞介素-18(IL-18)、白细胞介素-1β(IL-1β)呈剂量依赖的显著下降(均P<0.05);大鼠肾脏病理改变减轻,胶原沉积减少;组织焦亡水平显著降低。结论:人参皂苷Rg1可以通过抑制NLRP3/Caspase-1通路抑制肾小管上皮细胞焦亡,进而降低CTGF、α-SMA、CollagenⅠ的表达,从而实现对肾脏的保护作用。

Objective:To investigate the mechanism of ginsenoside Rg1 on inflammatory response and anti-fibrosis in human renal tubular epithelial cell line HK2 fibrotic induced by transforming growth factor-β1(TGF-β1)in vivo and in vitro.Methods:HK2 cells were grouped into control group,fibrosis model group(2 ng/mL of TGF-β1 induced fibrosis),low-dose ginsenoside Rg1 treatment group(2 ng/mL of TGF-β1+15μg/mL ginsenoside Rg1),and high-dose ginsenoside Rg1 treatment group(2 ng/mL of TGF-β1+45μg/mL ginsenoside Rg1).Western blotting and immunofluorescence were used to detect the expression of connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA)and collagen I(Col-1).Western blotting was used to detect the expression of Nod-like receptor protein3(NLRP3)and caspase-1.The level of cellular pyroptosis was detected by transmission electron microscopy.Rats were devided into control group,model group(unilateral ureteral obstruction),low-dose ginsenoside Rg1 treatment group(unilateral ureteral obstruction rats+50 mg/kg ginsenoside Rg1),and high-dose ginsenoside Rg1 treatment group(unilateral ureteral obstruction rats+100 mg/kg ginsenoside Rg1)in animal studies.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression level of inflammatory factors in serum.Hematoxylin-eosin staining(HE)and Masson were used to detect renal pathological changes and the degree of fibrosis.Western blotting was used to detect the protein expression level in renal tissues.Transmission electron microscopy was used to detect the level of tissue pyroptosis.Results:In cellular experiments,the expression levels of CTGF,α-SMA,ColⅠ,NLRP3 and caspase-1 in cells were reduced in a dose-dependent manner after ginsenoside Rg1 treatment compared with the model group(all P<0.05);the level of cell pyroptosis was significantly reduced.In the animal test,the expression levels of CTGF,α-SMA,ColⅠ,NLRP3 and caspase-1 in the kidney tissues of rats after ginsenoside Rg1 treatment were reduced in a dose-dependent manner compared with the model group(all P<0.05);the expression levels of TGF-β1,interleukin-18(IL-18)and interleukin-1β(IL-1β)in the serum were reduced in a dose-dependent manner(all P<0.05);the pathological changes in rat kidney were alleviated and collagen deposition was reduced;the level of tissue pyroptosis decreased significantly.Conclusion:Ginsenoside Rg1 can inhibit renal tubular ep-ithelial cell pyroptosis by inhibiting NLRP3/Caspase-1 pathway,and reduce the expression of CTGF,α-SMA and ColⅠ,thus achieving a protective effect on the kidney.