基于IL-6/MMP-9/COL-Ⅳ信号通路探讨加味真武汤对慢性肾功能衰竭大鼠肾间质纤维化的影响

Effect of Modified Zhenwutang on Interstitial Fibrosis in Rats with Chronic Renal Failure Based on IL-6/MMP-9/COL-Ⅳ Signaling Pathway

目的:观察加味真武汤对慢性肾衰竭(CRF)大鼠白细胞介素-6(IL-6)、基质金属蛋白酶-9(MMP-9)、Ⅳ型胶原蛋白(COL-Ⅳ)的调控作用,探究其治疗CRF的潜在机制。方法:将50只雄性SD大鼠随机分为造模组40只和正常组10只,造模组大鼠连续腺嘌呤灌胃12周制备CRF模型。造模成功后将造模组按随机数字表法分为模型组、加味真武汤低剂量(7.2g·kg^(-1)·d^(-1))组、加味真武汤中剂量(14.4g·kg^(-1)·d^(-1))组、加味真武汤高剂量(28.8g·kg^(-1)·d^(-1))组、盐酸贝那普利(10 mg·kg^(-1)·d^(-1))组进行灌胃,正常组、模型组予等体积蒸馏水灌胃,持续干预4周。给药期间,定期检测各组大鼠体质量;给药结束后,检测大鼠血肌酐(SCr)、血尿素氮(BUN)、24 h尿蛋白定量(24 h-UTP)水平;酶联免疫吸附测定法(ELISA)检测大鼠血清IL-6水平;分别采用苏木素-伊红(HE)、马松(Masson)染色观察肾组织病理形态学变化;免疫组化法(IHC)检测大鼠肾脏细胞间黏附分子-1(ICAM-1)、IL-6、MMP-9、COL-Ⅳ蛋白表达结果;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠肾组织ICAM-1 mRNA、IL-6 mRNA、MMP-9 mRNA、COL-ⅣmRNA表达水平;蛋白免疫印迹法(Western blot)检测大鼠肾组织ICAM-1、IL-6、MMP-9、COL-Ⅳ蛋白表达水平。结果:与正常组比较,模型组大鼠SCr、BUN、24 h-UTP水平显著升高(P<0.01);血清IL-6含量显著上升(P<0.01);大鼠肾小管管腔扩张伴萎缩,小管上皮细胞坏死、肿胀、空泡变性,间质内大量炎症细胞浸润,胶原纤维沉积;肾小管间质内IL-6、ICAM-1、COL-Ⅳ呈强阳性表达,MMP-9表达减弱;模型组大鼠ICAM-1 mRNA、IL-6mRNA、COL-ⅣmRNA水平显著升高(P<0.01),MMP-9 mRNA水平显著降低(P<0.05);肾组织ICAM-1、IL-6、COL-Ⅳ蛋白表达显著升高(P<0.01),MMP-9蛋白表达显著降低(P<0.01)。与模型组比较,经加味真武汤治疗后,大鼠24 h-UTP、SCr、BUN水平均有大幅降低(P<0.01);血清IL-6含量显著下降(P<0.01);大鼠肾脏病损明显改善,胶原纤维沉积减少;IL-6、ICAM-1、COL-Ⅳ在肾小管、间质内表达减弱,MMP-9在肾小管间质内表达增强;大鼠ICAM-1 mRNA、IL-6 mRNA、COL-ⅣmRNA水平显著降低(P<0.01),MMP-9 mRNA水平明显升高(P<0.05);肾脏内ICAM-1、IL-6、COL-Ⅳ蛋白表达显著下降(P<0.01),MMP-9蛋白表达显著上升(P<0.01)。结论:加味真武汤可能通过调控IL-6/MMP-9/COL-Ⅳ信号通路,进而减轻蛋白尿,改善肾功能,减轻肾脏病理损害,延缓CRF肾间质纤维化进展。

Objective:To observe the modulatory effect of modified Zhenwutang on the interleukin-6(IL-6),matrix metallopeptidase-9(MMP-9),typeⅣcollagen(COL-Ⅳ)in rats with chronic renal failure(CRF)and to investigate the potential mechanism of its treatment of CRF.Method:Fifty male SD rats were randomly divided into a modeling group of 40 rats and a normal group of 10 rats,and the modeling group was prepared by continuous adenine gavage for 12 weeks.After successful modelling,the modelling group was divided into the model group,the low dose(7.2 g·kg^(-1)·d^(-1))group,the medium dose(14.4 g·kg^(-1)·d^(-1))group,the high dose(28.8 g·kg^(-1)·d^(-1))group and the Benadryl hydrochloride(10 mg·kg^(-1)·d^(-1))group for gavage according to the random number table method,In the normal group and the model group,equal volume of distilled water was administered by gavage for 4 weeks.After the administration,the levels of blood creatinine(SCr),blood urea nitrogen(BUN)and 24 h urine protein(24 h-UTP)were measured,the levels of serum IL-6 were measured by enzyme linked immunosorbent assay(ELISA).Immunohistochemistry(IHC)was used to detect intercellular cell adhesion molecule-1(ICAM-1),IL-6,MMP-9,and other molecules in the rat kidney.The expression of ICAM-1 mRNA,IL-6 mRNA,MMP-9 mRNA and COL-ⅣmRNA in rat kidney tissues was measured by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).The expression levels of ICAM-1,IL-6,MMP-9 and COL-Ⅳin rat kidney tissues were measured by Western blot.Result:Compared with the normal group,the levels of SCr,BUN and 24 h-UTP were significantly increased in the model group(P<0.01);the serum IL-6 level was significantly increased(P<0.01),the tubular lumen was dilated with atrophy,the tubular epithelial cells were necrotic,swollen and vacuolated,the interstitium was infiltrated by a large number of inflammatory cells and collagen fibers were deposited,the levels of IL-6,ICAM-1 and COL-Ⅳwere strongly positive in the tubular interstitium of the model group(P<0.01),The levels of ICAM-1 mRNA,IL-6 mRNA and COL-ⅣmRNA were significantly increased(P<0.01)and MMP-9 mRNA was significantly decreased(P<0.05)in the model rats.ICAM-1,IL-6 and COL-Ⅳprotein expression was significantly increased(P<0.01)and MMP-9 protein expression was significantly increased(P<0.01)in the renal tissue,and MMP-9 protein expression was significantly decreased(P<0.01).Compared with the model group,the 24 h-UTP,SCr and BUN levels of rats were significantly reduced after treatment with modified Zhenwutang(P<0.01),the serum IL-6 level was significantly decreased(P<0.01),the renal lesions of rats were significantly improved and collagen fiber deposition was reduced;the expression of IL-6,ICAM-1 and COL-Ⅳin renal tubules and interstitium was weakened,and MMP-9 in ICAM-1 mRNA,IL-6 mRNA and COL-ⅣmRNA levels were significantly reduced(P<0.01)and MMP-9 mRNA levels were significantly increased(P<0.05),ICAM-1,IL-6 and COL-Ⅳprotein expression was significantly reduced(P<0.01)and MMP-9 protein expression was significantly The expression of ICAM-1,IL-6 and COL-Ⅳproteins was significantly decreased(P<0.01)and MMP-9 protein expression was significantly increased(P<0.01).Conclusion:Modified Zhenwutang may regulate the IL-6/MMP-9/COL-Ⅳsignaling pathway,thereby reducing proteinuria,improving renal function,reducing renal pathological damage and delaying the progression of CRF interstitial fibrosis.