| Objective:Based on the regulation of RhoA/ROCK1 pathway on its downstream factor ILK,it participates in the regulation of renal interstitial fibrosis forming substances Col-Ⅰ,Col-Ⅲ,etc,verifies the influence of Chongcaoyishen decoction on RhoA/ROCK1 signaling pathway,explores the mechanism of action of Chongcaoyishen decoction on the prevention and treatment of renal fibrosis.Methods:According to different body weight,100 male SD rats were numbered one by one and divided into three groups according to random number table:sham operation group,model group,midapril hydrochloride group,Chongcaoyishen decoction group,and high-dose Chongcaoyishen decoction group,with 20 rats in each group.Renal interstitial fibrosis model was established by left ureteral obstruction.Unilateral ureteral obstruction model was given intragastric administration the next day after surgery.The Chongcaoyishen decoction group was given 6.57g/(kg·d)water decoction daily,the high-dose Chongcaoyishen decoction group was given 13.14g/(kg·d)water decoction daily,and midapril hydrochloride group was given 0.9mg/(kg·d)water solution daily.The sham operation group and the model group were given the same volume of normal saline water by gavage every day until the completion of the experiment cycle.During the experiment,the general state and body weight of the rats were recorded regularly.The serum of the rats was collected on the 7th and 14th day of modeling respectively,and the serum urea nitrogen and creatinine were detected.Renal tissues were collected for HE staining and Masson staining to study the pathological and morphological changes of renal tissues.Immunohistochemistry was used to detect the protein expression of PDGF-BB,ROCK1,ILK,Col-Ⅰ and Col-Ⅲ.Western blot was used to detect RhoA,ROCK1,ILK,E-cad,α-SMA protein expression,and realtime PCR was used to detect RhoA,ROCK1,ILK,E-cad,α-SMA gene expression.On the 6th and 13th day of modeling,10 rats were randomly selected from each group and put into metabolic cages,and 24h urine was collected for 24h urine protein quantitative(24 HUPQ)detection.Water was allowed but cannot to eat.Results:(1)General situation of rats:The rats in model group were generally in poor condition,with dark fur,weight loss and reduced activity.The reaction ability,fur color and body weight of rats in Chongcaoyishen decoction group,midapril hydrochloride group and high dose Chongcaoyishen decoction group were improved to different degrees.(2)Quantitative levels of serum creatinine,serum urea nitrogen and 24-hour urine protein:On the 7th and 14th day after model preparation,compared with the sham operation group,the levels of serum creatinine,serum urea nitrogen and 24-hour urine protein were significantly increased in the model group,P<0.05,the difference was statistically significant.Compared with model group,midapril hydrochloride group、Chongcaoyishen decoction group and high dose Chongcaoyishen decoction group,serum creatinine level was significantly decreased,P<0.05,and the difference was statistically significant.(3)He and Masson staining showed that Chongcaoyishen decoction could improve the degree of renal pathological changes in UUO rats and reduce the percentage of renal tissue fibrosis area in UUO rats.Compared with the sham operation group,the incidence of renal interstitial fibrosis in the model group was significantly higher,P<0.05,the difference was statistically significant.Compared with the model group on day 7 and 14,the percentage of renal interstitial fibrosis area in midadepril hydrochloride group、Chongcaoyishen decoction group and high dose Chongcaoyishen decoction group decreased,P<0.05,the difference was statistically significant.Compared with the high dose group of Chongcaoyishen decoction group,the percentage of renal interstitial fibrosis area in the Chongcaoyishen decoction group decreased,P<0.05,the difference was statistically significant.Compared with the high dose group of Chongcaoyishen decoction group,the percentage of renal interstitial fibrosis area in midadepril hydrochloride group was decreased-P<0.05,and the difference was statistically significant.(4)Immunohistochemical tests showed that the protein expression levels of PDGF-BB,ILK,ROCK1,Col-Ⅰ and Col-Ⅲ in kidney tissue of rats were significantly increased in model group compared with sham operation group at the 14th day of the experiment,with statistically significant differences-P<0.05.Compared with model group,midadepril hydrochloride group、Chongcaoyishen decoction group and high dose Chongcaoyishen decoction group,the expression levels of the above proteins were significantly decreased,P<0.05,the difference was statistically significant.Compared with the high dose group of Chongcaoyishen decoction,the protein expression levels of ILK,ROCK1,Col-Ⅰ and Col-Ⅲ in the Chongcaoyishen decoction group and midapril hydrochloride group were decreased,P<0.05,the difference was statistically significant,while the expression level of PDGF-BB was not significantly different,P>0.05,the difference was not statistically significant.(5)Real-time PCR showed that the expression levels of RhoAmRNA,ROCK1mRNA,ILKmRNA,E-cadherinmRNA and a-SMAmRNA in renal tissues were detected on the 14th day of the experiment,compared with those in the sham operation group.The expression levels of RhoAmRNA,ROCK1mRNA,ILKmRNA and a-SMAmRNA in model group were significantly increased,while E-cadherinmRNA was significantly decreased,P<0.05,the difference was statistically significant.Compared with model group,the expression levels of RhoAmRNA,ROCK1mRNA,ILKmRNA and a-SMAmRNA in midadepril hydrochloride group,Chongcaoyishen decoction group and high dose Chongcaoyishen decoction group were significantly decreased,while E-cadherinmRNA was significantly increased,P<0.05,the difference was statistically significant.Compared with the high dose group of Chongcaoyishen decoction,the expression levels of RhoAmRNA,ROCK1mRNA,ILKmRNA and a-SMAmRNA in the Chongcaoyishen decoction group and midapril hydrochloride group were decreased,P<0.05,the difference was statistically significant.(6)Western Blot showed that the protein expression levels of RhoA,ROCK1,ILK,E-cadherin and a-SMA in renal tissue of rats were detected on the 14th day of the experiment.Compared with the sham operation group,the protein expression levels of ROCK1,RhoA,ILK and a-SMA in model group were increased.The expression level of E-cadherin protein was decreased,P<0.05,and the difference was statistically significant.Compared with the model group,the protein expression levels of ROCK1,RhoA,ILK and a-SMA were decreased in midadepri hydrochloride group,Chongcaoyishen decoction group and high dose Chongcaoyishen decoction group,while the protein expression level of E-cadherin was increased,P<0.05,the difference was statistically significant.Compared with the high dose group of Chongcaoyishen decoction group,the protein expression levels of ROCK1,RhoA,ILK,E-cadherin and a-SMA in midadepri hydrochloride group and Chongcaoyishen decoction group were decreased,P<0.05,and the difference was statistically significant.Conclusion:1.This study confirmed that Chongcaoyishen decoction can improve the general state of UUO model rats,reduce 24-hour urine protein quantification,serum creatinine,and serum urea nitrogen.2.Chongcaoyishen decoction can delay renal pathological changes and protect renal function biochemical indexes.3.There is activation of RhoA/ROCK1 signaling pathway during the formation of renal fibrosis.Chongcaoyishen decoction can inhibit the expression of PDGF-BB,ROCK1,RhoA,etc.in the kidney tissue of UUO model rats,and down-regulate the expression of ILK.4.Chongcaoyishen decoction inhibits the expression of ILK in renal tissues,thereby intervening ILK-mediated EMT,inhibiting the expression of E-cadherin,a-SMA,type Ⅰ and type Ⅲ collagen,and delay the progression of renal interstitial fibrosis. |
PDF Full Text Request