益肾泻浊方抑制裸角质层同源物2活性干预慢性肾衰竭的机制研究

Mechanism of Yishen Xiezhuo Decoction in Treatment of Chronic Renal Failure by Inhibiting NKD2 Activity

目的通过建立慢性肾衰竭(CRF)小鼠模型,以裸角质层同源物2(NKD2)为切入点,观察益肾泻浊方对CRF小鼠各项肾功能指标的改善作用,并初步探讨其作用机制。方法采用单侧输尿管梗阻法(UUO)构建小鼠CRF模型,末次给药结束后处死小鼠,观察测量小鼠肾脏形态和质量,HE和Masson染色观察肾脏病理情况,使用肾小管损伤评分和肾纤维化区域量化指标评估肾衰竭程度。运用ELISA法测定血清中IL-1β、IL-6和TNF-α的水平,试剂盒检测BUN和SCr水平。Western blot检测小鼠肾脏Collagenα1、FN1、α-SMA及NKD2蛋白含量。结果与正常组相比,模型组小鼠肾脏组织出现水肿、颜色变浅、体积增大;肾脏质量指数显著增加(P<0.01),肾小管损伤评分升高(P<0.01),肾纤维化水平和Collagenα1免疫组化表达量显著增加(P<0.01);血清BUN、SCr、IL-6、IL-1β以及TNF-α水平均明显升高(P<0.01);肾脏Collagenα1、FN1、α-SMA及NKD2蛋白表达量均增加(P<0.01)。与模型组相比,低剂量复方组小鼠肾小管损伤评分差异无统计学意义,而肾脏质量指数(P<0.01)、肾纤维化水平(P<0.05)和Collagenα1免疫组化表达量(P<0.05)均显著下降;血清BUN、SCr、IL-6、IL-1β以及TNF-α水平均明显下降(P<0.01),肾脏Collagenα1、FN1、α-SMA及NKD2蛋白表达量显著减少(P<0.01)。与模型组相比,高剂量复方组小鼠肾脏质量指数、肾小管损伤评分和肾纤维化水平均明显减少(P<0.01);血清BUN、SCr、IL-6、IL-1β以及TNF-α水平和肾脏Collagenα1、FN1、α-SMA及NKD2蛋白表达量均显著降低(P<0.01)。结论益肾泻浊方能够下调NKD2表达,减少肾脏ECM沉积,抑制炎症反应,从而延缓CRF进展。

OBJECTIVE To observe the effect of Yishen Xiezhuo Decoction on various renal function indexes by establishing a mouse model of chronic renal failure(CRF)and explore the mechanism by taking naked stratum corneum homolog 2(NKD2)as the breakthrough point.METHODS The CRF model of mice was established by unilateral ureteral obstruction(UUO).After the last administration,the mice were killed.The morphology and weight of the mouse kidneys were observed and measured.The renal pathology was observed by Hematoxylin-Eosin and Masson staining.The degree of renal failure was evaluated by renal tubular injury score and renal fibrosis area measurement.Serum IL-1β,IL-6 and TNF-αwere determined by enzyme-linked immunosorbent assay(ELISA).The levels of BUN and SCr were detected by kits.Western blot was used to detect the protein expression of Collagenα1,FN1,α-SMA and NKD2.RESULTS Compared with the control group,the kidneys of the model group showed edema,reduced color and enlarged volume;renal mass index,renal tubular injury score,renal fibrosis area and Collagenα1 immunohistochemical expression increased significantly(P<0.01).Serum levels of BUN,SCr,IL-6,IL-1βand TNF-αwere increased significantly(P<0.01),and the expression of kidney Collagenα1,FN1,α-SMA and NKD2 protein increased,too(P<0.01).Compared with the model group,no significant difference was found on the scores of renal tubular injury in the low-dose group,but the renal weight index(P<0.01),the level of renal fibrosis(P<0.05)and the immunohistochemical expression of Collagenα1(P<0.05)decreased significantly.The levels of BUN,SCr,IL-6,IL-1βand TNF-αdecreased significantly(P<0.01),the protein expression of Collagenα1,FN1,α-SMA and NKD2 also decreased significantly(P<0.01).Compared with the model group,the renal weight index,renal tubular injury scores and renal fibrosis area in the high-dose group were significantly reduced(P<0.01);The levels of BUN,SCr,IL-6,IL-1βand TNF-αdecreased significantly;the protein expression of kidney Collagenα1,FN1,α-SMA and NKD2 decreased significantly,too(P<0.01).CONCLUSION Yishen Xiezhuo Decoction can down regulate the expression of NKD2,reduce the deposition of renal ECM,inhibit inflammatory responses,and delay the progress of CRF.