汉黄芩苷抗糖尿病大鼠肾损伤及纤维化的机制研究

Study on the Mechanism of Wogonoside Protecting against Renal Injury and Fibrosis in Diabetic Rats

【目的】观察汉黄芩苷对糖尿病大鼠肾损伤及纤维化的治疗作用及机制。【方法】从50只大鼠中随机选取10只大鼠作为正常组常规饲养,剩余大鼠经6周高脂喂养后连续3 d单次腹腔注射链脲佐菌素30 mg/kg构建糖尿病模型。将建模成功的40只大鼠随机分为模型组,汉黄芩苷低、高剂量组和细胞周期素依赖蛋白激酶5(CDK5)抑制剂Roscovitine对照组,每组10只。汉黄芩苷低、高剂量组分别给予10、40 mg·kg^(-1)·d^(-1)汉黄芩苷腹腔注射,Roscovitine对照组给予Roscovitine 40 mg·kg^(-1)·d^(-1)腹腔注射,模型组和正常组给予腹腔注射等体积二甲基亚砜(DMSO),连续用药10周。给药期间,记录各组大鼠状态。给药10周后,应用全自动生化分析仪检测血清空腹血糖(FBG)、血肌酐(SCr)、血尿素氮(BUN),采用酶联免疫吸附分析(ELISA)检测尿白蛋白(Alb),苏木素-伊红(HE)染色法观察肾脏组织形态,Masson染色法评估肾间质病理情况,Western Blot法检测肾组织Ⅳ型胶原蛋白(ColⅣ)、α平滑肌肌动蛋白(α-SMA)、转化生长因子β1(TGF-β1)、丝裂原活化蛋白激酶p38(p38MAPK)、磷酸化p38MAPK(p-p38MAPK)的蛋白表达水平。【结果】与正常组比较,模型组大鼠血清FBG、SCr、BUN和尿Alb水平显著升高(P<0.05),HE染色结果显示肾小球显著肥大,间系膜基质扩张,炎性细胞浸润,Masson染色结果显示肾组织中胶原蛋白沉积显著增加,肾脏组织纤维化严重,肾组织ColⅣ、α-SMA、TGF-β1、p38MAPK和p-p38MAPK蛋白表达水平显著升高(P<0.05);与模型组比较,汉黄芩苷低、高剂量组和Roscovitine对照组大鼠血清FBG、SCr、BUN和尿Alb水平显著降低(P<0.05),肾组织损伤及纤维化程度明显减轻,肾组织ColⅣ、α-SMA、TGF-β1、p38MAPK和p-p38MAPK蛋白表达水平显著下降(P<0.05);汉黄芩苷高剂量组和Roscovitine对照组的治疗效果差异无显著性(P>0.05)。【结论】汉黄芩苷可减轻糖尿病大鼠肾损伤及肾纤维化,其机制可能与抑制TGF-β1/p38MAPK信号通路的激活有关。

Objective To observe the therapeutic effect and mechanism of wogonoside on renal injury and fibrosis in diabetic rats.Methods Ten rats were randomly selected from 50 rats as the normal group for regular feeding,and the remaining rats were fed with high fat for 6 weeks and then single intraperitoneal injection of 30 mg/kg streptozotocin for 3 days to construct a diabetes model.The 40 rats with successful modeling were randomly divided into the model group,the wogonoside low-dose and high-dose groups and the Roscovitine control group,with10 rats in each group.The wogonoside low-and high-dose groups were given 10 and 40 mg·kg^(-1)·d^(-1)of wogonoside by intraperitoneal injection,respectively,and the Roscovitine control group was given 40 mg·kg^(-1)·d^(-1)of Roscovitine by intraperitoneal injection,and the model group and normal group were given an equal volume of dimethyl sulfoxide(DMSO)intraperitoneally,the treatment lasted for 10 weeks.During the medication,the situation of all rats was recorded.After 10 weeks of treatment,serum fasting blood glucose(FBG),serum creatinine(SCr)and blood urea nitrogen(BUN)were detected by automatic biochemical analyzer,urine albumin(Alb)was detected by enzyme-linked immunosorbent assay(ELISA),renal histomorphology was observed by hematoxylin-eosin(HE)staining,and renal interstitial pathology was assessed by Masson staining,the protein expression levels of collagen typeⅣ(ColⅣ),αsmooth muscle actin(α-SMA),transforming growth factorβ1(TGF-β1),mitogenactivated protein kinase p38(p38MAPK),and phosphorylated p38MAPK(p-p38MAPK)in renal tissues were detected by Western Blot.Results Compared with the normal group,serum FBG,SCr,BUN and urinary Alb levels were significantly increased in the model groups(P<0.05),HE staining results showed significant hypertrophy of glomeruli,expansion of the mesangial matrix and inflammatory cell infiltration,Masson staining results showed a significant increase in collagen deposition and severe fibrosis in kidney tissues,and the protein expression levels of renal tissue ColⅣ,α-SMA,TGF-β1,p38MAPK and p-p38MAPK were significantly increased(P<0.05);compared with the model group,the levels of serum FBG,SCr,BUN and urinary Alb of rats in the wogonoside low-and high-dose groups and Roscovitine control group were significantly reduced(P<0.05),and renal tissue injury and fibrosis were significantly alleviated,and the protein expression levels of renal tissue ColⅣ,α-SMA,TGF-β1,p38MAPK and p-p38MAPK were significantly decresed(P<0.05);the therapeutic effect of wogonoside high-dose group and Roscovitine control group were not significantly different(P>0.05).Conclusion Wogonoside attenuated renal injury and renal fibrosis in diabetic rats,and its mechanism may be related to the inhibition of the activation of TGF-β1/p38MAPK signaling pathway.