肾衰Ⅱ号方对慢性肾衰竭大鼠有氧糖酵解及肾间质纤维化的影响

Effect of Shenshuai Ⅱ Recipe on Aerobic Glycolysis and Renal Interstitial Fibrosis in Rats with Chronic Renal Failure

目的观察肾衰Ⅱ号方对慢性肾衰竭大鼠有氧糖酵解及肾间质纤维化的影响,探讨其抗肾间质纤维化的作用机制。方法体内实验以5/6(A/I)手术构建慢性肾衰竭大鼠模型,随机分为假手术组、模型组、中药组、西药组,进行相应干预8周后,取肾组织行PAS染色观察肾脏病理变化,Western blot检测α-平滑肌肌动蛋白(α-SMA)蛋白表达,免疫组化检测α-SMA、己糖激酶(HK)2蛋白定位,紫外法检测肾组织HK活性,比色法检测肾组织乳酸、丙酮酸浓度;体外实验以白细胞介素(IL)-1β刺激大鼠肾成纤维细胞NRK-49F构建NRK-49F增殖活化模型,细胞分为空白组、IL-1β组、IL-1β+肾衰Ⅱ号方低剂量组(200μg/mL)、IL-1β+肾衰Ⅱ号方高剂量组(400μg/mL)、IL-1β+2-脱氧-D-葡萄糖组(10 mmol/L),Western blot检测NRK-49F细胞α-SMA、增殖细胞核抗原(PCNA)、纤维连接蛋白(FN)、HK2蛋白表达,比色法检测细胞培养基乳酸浓度,葡萄糖氧化酶法检测细胞培养基葡萄糖浓度,免疫荧光检测HK2蛋白表达。结果体内实验:与假手术组比较,模型组PAS染色提示肾间质糖原含量增加,肾小球、肾小管形态异常;α-SMA蛋白表达明显升高(P<0.01),肾组织乳酸浓度、乳酸/丙酮酸、HK活性、HK2蛋白表达均明显升高(P<0.01)。与模型组比较,中药组和西药组肾组织病理变化改善,α-SMA蛋白表达、乳酸浓度、乳酸/丙酮酸、HK活性、HK2蛋白表达均明显降低(P<0.05,P<0.01)。体外实验:与空白组比较,IL-1β组NRK-49F细胞FN、α-SMA、PCNA、HK2蛋白表达明显升高(P<0.05,P<0.01);细胞培养基pH值、葡萄糖浓度明显降低(P<0.05),乳酸浓度明显升高(P<0.01);与IL-1β组比较,各干预组NRK-49F细胞FN、α-SMA、PCNA、HK2蛋白表达明显降低(P<0.05,P<0.01);细胞培养基pH值、葡萄糖浓度升高(P<0.05,P<0.01),乳酸浓度明显降低(P<0.01)。结论肾衰Ⅱ号方可通过抑制NRK-49F细胞有氧糖酵解反应,从而阻止成纤维细胞增殖活化,实现其抗肾间质纤维化的作用。

Objective To observe the effects of ShenshuaiⅡPrescription on aerobic glycolysis and renal interstitial fibrosisin rats with chronic renal failure;To explore its anti-renal fibrosis mechanism.Methods In vivo experiment,the rat model of chronic renal failure was established by 5/6(A/I)operation.The rats were divided into sham-operation group,model group,TCM group and Western medicine group.After 8 weeks of intervention,renal tissue was taken for PAS staining to observe the pathological changes of kidney;Western blot was used to detect the expression ofα-smooth muscle actin(α-SMA)protein;immunohistochemistry was used to detect the protein localization ofα-SMA and hexokinase(HK)2;the activity of HK in renal tissue was detected by UV;the concentration of lactic acid and pyruvate in renal tissue was measured by colorimetry.In vitro experiment,the proliferation and activation model of NRK-49F was established by stimulating rat renal fibroblasts NRK-49F with interleukin(IL)-1β.The cells were divided into blank group,IL-1β group,IL-1β+low dosage of Shenshuai Ⅱ Recipe group(200μg/mL)and IL-1β+high dosage of Shenshuai Ⅱ Recipe group(400μg/mL),IL-1β+2-deoxy-D-glucose group(10 mmol/L).The protein expression ofα-SMA,proliferating cell nuclear antigen(PCNA),fibronectin(FN)and HK2 in NRK-49F cells were detected by Western blot;the concentration of lactic acid in cell medium was detected by colorimetry;the glucose concentration in cell medium was detected by glucose oxidase method;the expression of HK2 protein was detected by immunocytofluorescence.Results Vivo experiment showed that,compared with the sham-operation group,the PAS staining in the model group indicated that the interstitial glycogen content increased,the morphology of glomerulus and renal tubule was abnormal,the expression ofα-SMA protein increased(P<0.01),and the concentration of lactic acid,lactic acid/pyruvate,HK activity and HK2 protein expression in renal tissue increased(P<0.01).Compared with the model group,the pathological changes of renal tissue in the TCM group and the Western medicine group were improved,and the expressions ofα-SMA protein,lactic acid concentration,lactic acid/pyruvate,HK activity and HK2 protein expressions decreased in the TCM group and Western medicine group(P<0.05,P<0.01).Vitro experiment showed that,compared with the blank group,the protein expressions of FN,α-SMA,PCNA and HK2 in NRK-49F cells of the IL-1β group increased(P<0.05,P<0.01),the pH value of culture medium and the concentration of glucose decreased(P<0.05),and the concentration of lactic acid increased(P<0.01).Compared with the model group,the protein expressions of FN,α-SMA,PCNA and HK2 in each intervention group decreased(P<0.05,P<0.01),the pH value of culture medium and the concentration of glucose increased(P<0.05,P<0.01),and the concentration of lactic acid decreased(P<0.01).Conclusion Shenshuai Ⅱ Prescription may inhibit the aerobic glycolysis of NRK-49F cells to prevent the proliferation and activation of fibroblasts and achieve its anti-renal fibrosis effect.