肾衰饮通过TGFβ1/MKK3/p38MAPK/CTGF途径对肾间质纤维化大鼠的影响及分子机制

Effect and Molecular Mechanism of Shenshuaiyin on Renal Interstitial Fibrosis in Rats through TGFβ1/MKK3/p38MAPK/CTGF Pathway

目的从TGFβ1/MKK3/p38MAPK/CTGF途径角度,探讨肾衰饮延缓大鼠肾间质纤维化的可能作用机制。方法将60只雄性SD大鼠适应性喂养3天后,按体重随机分为两组,即假手术组10只,造模组50只。造模采用左侧输尿管结扎术建立肾间质纤维化大鼠模型。造模成功后,将大鼠再随机分为五组:模型组,肾衰饮低剂量组,肾衰饮中剂量组,肾衰饮高剂量组及尿毒清组。分别按人与大鼠1:1/2,1:1,1:2和1:1灌胃各给药组大鼠,假手术组与模型组予生理盐水0.00015ml/10g灌胃,M组:9.56 g/(kg·d)。实验期间进行一般情况观察,灌胃治疗14天后结束实验,各组大鼠禁食、不禁水12小时,第二日早8:00称重,进行取材。收集24小时尿,全自动生化分析仪一次性检测尿肌酐(UCr)、微球蛋白(β2-MG)及尿微量白蛋白(mAlb)含量,髂总动脉分支处穿刺采血测定血肌酐(Scr)、尿素氮(BUN)含量;HE染色和Masson染色观察肾脏组织病理学改变及胶原沉积情况;免疫组织化学法检测肾脏组织相关蛋白表达;实时荧光定量聚合酶链式反应(qRT-PCR)检测肾脏组织miRNA21及相关mRNA的表达水平;免疫印迹法(Western Blot)检测肾脏组织相关蛋白表达水平。结果与假手术组大鼠比较,模型组大鼠精神倦怠,明显消瘦,排便、排尿异常。Scr、BUN、Ucr、β2-MG、mAlb显著升高(P<0.01)。HE染色模型组大鼠可见系膜增生,肾小管扩张或萎缩,肾小球硬化且形态不规则,球囊粘连,纤维化组织增多,间质增宽,可见炎性细胞浸润。Masson染色可见大鼠肾脏组织可见系膜增厚,可见大量肌成纤维广泛表达和胶原纤维沉积,模型组大鼠肾脏组织模型组大鼠肾脏组织TGF-β1、ASK-1、MKK3、CTGF、α-SMAmRNA的表达显著增高(P<0.01或P<0.05),E-cadherinmRNA的表达明显降低(P<0.05),TGF-β1、ASK-1、MKK3、p-p38MAPK、p-ATF2、CTGF、MEF2C蛋白水平显著增高(P<0.01或P<0.05);经给药治疗后,各给药组大鼠上述指标均明显好转(P<0.01或P<0.05),其中以肾衰饮高剂量组最为显著,明显优于尿毒清组(P<0.01或P<0.05)。结论肾衰饮可能通过抑制TGFβ1/MKK3/p38MAPK/CTGF途径,改善ECM降解酶系统功能异常,延缓肾间质纤维化。

Objective From the perspective of TGFβ1/MKK3/p38MAPK/CTGF pathway,to explore the possible mechanism of Shenshuaiyin delaying renal interstitial fibrosis in rats.Methods After adaptive feeding for 3 days,60 male SD rats were randomly divided into two groups according to body weight,namely 10 rats in the sham operation group and 50 rats in the model group.The left ureteral ligation was used to establish a rat model of renal interstitial fibrosis.After successful modeling,the rats were randomly divided into five groups:model group,Shenshuaiyin low-dose group,Shenshuaiyin medium-dose group,Shenshuaiyin high-dose group and Niaoduqing group.The rats in each administration group were given 1:1/2,1:1,1:2 and 1:1 of human and rat respectively,and the rats in the sham operation group and the model group were given normal saline 0.15ml/10g by gavage,M Group:9.56 g/kg/d.During the experiment,the general situation was observed,and the experiment was terminated after 14 days of gavage treatment.The rats in each group were fasted and watered for 12 hours.Urine was collected for 24 hours,and the contents of urinary creatinine(UCr),microglobulin(β2-MG)and urinary microalbumin(mAlb)were detected by automatic biochemical analyzer at one time.,blood urea nitrogen(BUN)content;HE staining and Masson staining were used to observe the pathological changes and collagen deposition in the kidney;immunohistochemistry was used to detect the expression of related proteins in the kidney tissue;real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the kidney The expression levels of miRNA21 and related mRNAs in tissues;Western blotting was used to detect the expression levels of related proteins in kidney tissues.Results Compared with the rats in the sham operation group,the rats in the model group were mentally lethargic,obviously emaciated,and had abnormal defecation and urination.Scr,BUN,Ucr,β2-MG,mAlb were significantly increased(P<0.01).In the HE staining model group,mesangial hyperplasia,renal tubule dilation or atrophy,glomerulosclerosis and irregular shape,balloon adhesion,increased fibrotic tissue,widened interstitium,and inflammatory cell infiltration were seen.Masson staining showed that the mesangium was thickened in the kidney tissue of the rat,and a large number of myoblasts were widely expressed and collagen fibers were deposited.-The expression of SMA mRNA was significantly increased(P<0.01 or P<0.05),the expression of E-cadherin mRNA was significantly decreased(P<0.05),TGF-β1,ASK-1,MKK3,p-p38MAPK,p-ATF2,CTGF,MEF2C The protein level was significantly increased(P<0.01 or P<0.05);after the drug treatment,the above indexes of the rats in each administration group were significantly improved(P<0.01 or P<0.05),among which the high-dose Shenshuaiyin group was the most,significantly better than the Niaoduqing group(P<0.01 or P<0.05).Conclusion Shenshuaiyin may improve the abnormal function of ECM degrading enzyme system and delay renal interstitial fibrosis by inhibiting TGFβ1/MKK3/p38MAPK/CTGF pathway.