七芪地黄丸对链脲佐菌素诱导糖尿病肾病的保护作用研究

Protective Effects of Qiqi Dihuang Pill on Streptozotocin-Induced Diabetic Nephropathy Rats

目的评价七芪地黄丸对糖尿病肾病(DN)大鼠肾损伤的保护作用,并探讨其可能作用机制。方法建立高脂饲料联合链脲佐菌素(STZ)诱导的DN大鼠模型,将DN模型制作成功大鼠随机分为模型组、厄贝沙坦组(39 mg/kg)和七芪地黄丸(生药9.36 g/kg)组,另取健康大鼠作为正常组,每组10只;治疗组各组大鼠分别给予相应的药物,每天1次。灌胃4周后,测量尿液中尿糖、尿微量白蛋白(mAlb)、Cr、硒结合蛋白(SBP-1)、M2型丙酮酸激酶(PKM2)、肾损伤分子(KIM-1)、中性粒细胞明胶相关脂蛋白(NGAL)含量,血液中Glu、TC、TG、LDL-C、HDL-C、BUN、SCr、TNF-α、IL-1β、IL-6、TGF-β1、IL-4、IL-10含量和肾组织中晚期糖基化终末产物(AGEs)、活性氧(ROS)、8-羟基酸脱氧写苷(8-OHdG)、羟脯氨酸(HYP)、丙二醛(MDA)含量及还原型谷光苷肽(GSH)、氧化型型谷光苷肽(GSSG)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,检测尿液、血液和肾组织中的3-硫酸吲哚酚(3-IS)含量,取肾脏进行肾脏组织形态学分析,Real-time PCR检测肾组织中TNF-α、IL-1β、IL-6、TGF-β1、IL-4、IL-10、E-cadherin、Samd6、骨形成蛋白7(BMP7)、α-微管蛋白(α-tubulin)、波形蛋白(Vimentin)、纤维链接蛋白(Fibronectin)、胶原-1(Collagen-1)、α-平滑肌肌动蛋白(α-SMA)、SIRT1、SIRT3、SIRT4、紧密连接蛋白1(Claudin-1)mRNA表达,Western Blot检测肾组织中E-cadherin、Samd6、BMP7、TGF-β1、α-tubulin、Vimentin、Fibronectin、Collagen-1、α-SMA、SIRT1、SIRT3、SIRT4、Claudin-1蛋白表达。结果与治疗前比较,七芪地黄丸及厄贝沙坦治疗1、2、3、4周空腹血糖降低,治疗2、3、4周时体重升高(P<0.05,P<0.01)。与模型组比较,七芪地黄丸治疗1周,七芪地黄丸及厄贝沙坦治疗2、3、4周时空腹血糖降低而体重增加,且治疗2、3、4周时与厄贝沙坦组比较,七芪地黄丸组空腹血糖降低,体重升高(P<0.05,P<0.01)。七芪地黄丸可显著改善肾小管上皮细胞空泡样病变以及间质纤维化和炎性浸润。与正常组比较,模型组大鼠血液中TG、TC、LDL-C、BUN、SCr、IL-1β、IL-6、TNF-α、TGF-β1以及尿液中mAlb、Cr、尿糖、SBP-1、PKM2、KIM-1、NGAL含量升高,血液、肾组织中3-IS含量明显增加,肾组织中IL-1β、IL-6、TNF-α、TGF-β1 mRNA表达明显升高,IL-4、IL-10含量和m RNA表达、血HDL-C、尿液中3-IS含量降低(P<0.01);肾组织中AGEs、ROS、8-OHdG、MDA含量和GSSG活性明显增加,Claudin-1 mRNA和蛋白表达升高(P<0.01),血GSH、CAT、SOD活性、SIRT1、SIRT3、SIRT4 mRNA和蛋白表达降低(P<0.01);与模型组比较,七芪地黄丸及厄贝沙坦均可改善上述指标,且七芪地黄丸组改善效果优于厄贝沙坦组(P<0.05,P<0.01)。结论七芪地黄丸可显著降低STZ诱导的DN大鼠Glu、AGEs、炎性细胞因子、氧化应激和肾功能生物标志物,增强内源性抗氧化系统功能,改善肾组织病理学损伤,进而发挥对STZ诱导的DN保护作用。

Objective To evaluate the protective effect of Qiqi Dihuang Pill(QQDHP)on renal injury in diabetic nephropathy(DN)rats and explore its possible mechanism.Methods The DN rats model induced by high-fat diet combined with streptozotocin(STZ)was established,and then were randomly divided into model group,irbesartan group(39 mg/kg)and QQDHP group(crude drug 9.36 g/kg),and the healthy rats were taken as the normal group,with 10 rats in each group.Rats in the treatment group were given corresponding drugs once a day.After 4 weeks of intervention,the contents of urine Glu,mAlb,Cr,SBP-1,PKM2,KIM-1,NGAL,levels of blood glucose,TC,TG,LDL-C,HDL-C,BUN,Scr,TNF-α,IL-1β,IL-6,TGF-β1,IL-4,IL-10 and contents of AGEs,ROS,8-OHdG,HYP,MDA and activities of GSH,GSSG,SOD and CAT in renal tissue were measured.The 3-IS levels in urine,blood and renal tissue were detected by HPLC.The mRNA expressions of TNF-α,IL-1β,IL-6,TGF-β1,IL-4,IL-10,E-cadherin,Samd6,BMP7,α-tubulin,Vimentin,Fibronectin,Collagen-1,α-SMA,SIRT1,SIRT3,SIRT 4,Claudin-1 were detected by real-time PCR.The protein expressions of E-cadherin,Samd6,BMP7,TGF-β1,α-tubulin,Vimentin,Fibronectin,Collagen-1,α-SMA,SIRT1,SIRT3,SIRT4 and Claudin-1 were detected by Western Blot.Results Compared with before treatment,QQDHP and irbesartan reduced fasting blood glucose at 1,2,3and 4 weeks of treatment,and increased body weight at 2,3 and 4 weeks of treatment(P<0.05,P<0.01).Compared with the model group,QQDHP treatment for 1 week,QQDHP and irbesartan treatment for 2,3,4weeks,abdominal blood glucose decreased and weight increased,and compared with irbesartan treatment for 2,3,4 weeks,QQDHP group fasting blood glucose decreased and weight increased(P<0.05,P<0.01,respectively).QQDHP could significantly improve the vacuolar lesions of renal tubular epithelial cells,interstitial fibrosis and inflammatory infiltration.Compared with the normal group,the contents of serum TG,TC,LDL-C,BUN,SCr,IL-1β,IL-6,TNF-α,TGF-β1 and urine Glu,mAlb,Cr,SBP-1,PKM2,KIM-1,NGAL were significantly elevated,the 3-IS levels of serum and renal tissue were enhanced obviously,the mRNA expressions of IL-1β,IL-6,TNF-α,TGF-β1 were markedly increased,the serum levels and mRNA expressions in renal tissue of IL-4,IL-10,serum HDL-C and urine 3-IS contents were apparently reduced(P<0.01).The AGEs,ROS,8-OHdG,MDA contents and GSSG activity in renal tissue were markedly elevated,the mRNA and protein expressions of Claudin-1 in renal tissue were noticeably heightened(P<0.01).The serum GSH,CAT,SOD activities and the mRNA and protein expressions of SIRT1,SIRT3,SIRT4 in renal tissue were dramatically reduced(P<0.01).Compared with the model group,QQDHP and irbesartan could apparently improve all above mentioned indications,and the improvement effect of QQDHP group was better than irbesartan group(P<0.05,P<0.01).Conclusion QQDHP could significantly reduce blood glucose,AGEs,inflammatory cytokines,oxidative stress and renal function biomarkers in DN rats induced by STZ,enhance the function of endogenous antioxidant system,and improve the pathological damage of renal tissue,and then protect DN induced by STZ.