| Objective:UUO rat model was established by unilateral ureteral obstruction.To study the effect of Chongcaoyishen decoction on vital signs,renal function and renal pathology of rats with renal interstitial fibrosis,as well as on Keap1/Nrf2 signaling pathway related protein nuclear factor E2 related factor 2(Nrf2)and Kelch-like epichlorohy drin related protein 1(Keap1).,heme oxygenase 1(HO-1),recombinant human NAD(P)H dehydrogenase quinone 1(NQO1).The purpose of this study was to investigate the effect of Chongcaoyishen decoction on renal interstitial fibrosis and its possible mechanism.Methods:A total of 120 SPF male SD rats were randomly divided into normal group(n=20),sham group(n=20)and operation group(n=80).In the sham operation group,only the left ureter was dissected and the left ureter was dissociated.The left ureteral obstruction method was used to establish renal interstitial fibrosis model in the operation group,and then they were randomly divided into model group(n=20),Chongcaoyishen decoction group(n=20),Chongcaoyishen decoction+ML385 group(n=20),and Fosinopril group(n=20).After model preparation,the Chongcaoyishen group was given 6.57g/kg/d Chongcaoyishen decoction,the Chongcaoyishen decoction+ML385 group was given 6.57g/kg/d Chongcaoyishen decoction and 30mg/kg/d ML3 85 solution,the Fosinopril group was given 0.9mg/kg/d Fosinopril solution,and the other three groups were given the same volume of distilled water by gavage once a day.On the 7th and 14th day,the general state,body weight,urine volume of the rats were observed,the urine was collected for determination of 24-hour urinary protein,and serum was collected for determination of blood urea nitrogen(BUN)and serum creatinine(Scr);renal tissue was collected for examined by HE and Masson staining.Kidney tissue at day 14 was selected for the following tests.The protein distribution of Keap1,Nrf2,HO-1 and NQO1 was determined by Immunohistochemical(IHC)method,the transcription levels of Keap1 mRNA,Nrf2 mRNA,HO-1 mRNA and NQO1 mRNA were determined by Real-time PCR,and the protein expressions of Keap1,Nrf2,HO-1 and NQO1 were determined by Western Blot.Results:1.Rats in the model group had poor vital signs,slow movement,easy to panic,curled up,fluffy fur,dark gray color,and low diet.On the contrary,mental state,reaction ability,appearance,body weight change,dietetic and other aspects of rats in Chongcaoyishen decoction group were improved to varying degrees.2.At the same time point,the levels of 24hUPQ,BUN and Scr in normal group and sham group were not statistically significant(P>0.05).The levels of 24hUPQ,BUN and Scr in model group,Chongcaoyishen decoction group,Chongcaoyishen decoction+ML385 group and Fosinopril group were different,but the model group was the most significant.The 24hUPQ,BUN and SCR levels in Fosinopril group and Chongcaoyishen decoction+ML385 group were higher than those in Chongcaoyishen decoction group(P<0.05).3.HE and Masson staining results showed that:The inflammatory infiltration of renal tubule interstitium and necrosis of renal tubule epithelial cells in the model group by microscopic observation and the degree of fibrosis was more serious than that in Chongcaoyishen decoction group and Fosinopril group(P<0.05).Chongcaoyishen decoction alleviated the pathological condition of renal tissue on the affected side of rats with unilateral ureteral obstruction,and the area of renal tissue fibrosis was decreased compared with that in Fosinopril group(P<0.05).Compared with the Chongcaoyishen decoction group,the pathological changes of the Chongcaoyishen decoction+ML385 group was more serious(P<0.05).4.IHC results show that:Take the 14th day for the following tests.Compared with normal group and sham group,the protein expressions of Nrf2,HO-1 and NQO1 in model group were higher(P<0.05),while the protein expression of Keapl in model group was lower(P<0.05).The protein expressions of Nrf2,HO-1 and NQO1 in kidney tissue of rats in Chongcaoyishen decoction group were significantly higher than those in Fosinopril group(P<0.05)and model group(P<0.05),while the protein expression level of Keapl was significantly lower than that in Fosinopril group(P<0.05)and model group(P<0.05).The protein expression levels of Nrf2,HO-1 and NQO1 in the Chongcaoyishen decoction+ML385 group were lower than those in the Chongcaoyishen decoction group(P<0.05),but there was no difference in the protein expression levels of Keapl between them(P>0.05).5.Real-time PCR results showed that:Take the 14th day for the following tests.The transcription levels of Nrf2 mRNA,HO-1 mRNA and NQO1 mRNA in model group were higher than those in normal group and sham operation group(P<0.05),while the transcription levels of Keapl mRNA were decreased(P<0.05).Compared with model group,the transcription level of Keap1 mRNA in Chongcaoyishen decoction group and Fosinopril group was decreased(P<0.05),while the transcription levels of Nrf2 mRNA,HO-1 mRNA and NQO1 mRNA were increased(P<0.05),and the gene transcription level in Chongcaoyishen decoction group was stronger than that in Fosinopril group(P<0.05).The transcription levels of Nrf2 mRNA,HO-1 mRNA and NQO1 mRNA in the Chongcaoyishen decoction+ML385 group were lower than those in the Chongcaoyishen decoction group(P<0.05),while there was no statistical significance in the transcription levels of Keapl mRNA between them(P>0.05).6.Western Blot results showed that:Take the 14th day for the following tests.The protein levels of Nrf2,HO-1 and NQO1 in model group were up-regulated,which were higher than those in normal group(P<0.05)and sham operation group(P<0.05),while the protein expression of Keapl in model group was down-regulated,which was lower than that in normal group(P<0.05)and sham operation group(P<0.05).Compared with model group,the protein expressions of Nrf2,HO-1 and NQO1 in Chongcaoy i shen decoction group and Fosinopril group were up-regulated(P<0.05),while the protein expression of Keapl was down-regulated(P<0.05).The up-regulated protein levels of Nrf2,HO-1,NQO1 and down-regulated protein levels of Keapl in Cordyceps Yishenfang group were higher than those in Fosinopril group(P<0.05).The protein levels of Nrf2,HO-1 and NQO1 in the Chongcaoyishen decoction+ML385 group were lower than those in the Chongcaoyishen decoction group(P<0.05),and the protein levels of Keap1 were almost the same as those in the Chongcaoyishen decoction group(P>0.05).The expression level of Nrf2 nucleoprotein in Chongcaoyishen decoction group was higher than that in Chongcaoyishen decoction+ML385 group(P<0.05).Conclusion:1.Chongcaoyishen decoction alleviates the physical state of UUO rats and increases the body weight and urine volume of UUO rats.2.Chongcaoyishen decoction decreased 24h urinary protein and improved BUN and Scr levels in UUO rats.3.Chongcaoyishen decoction alleviates renal pathology after obstruction in UUO rats.4.By regulating the gene level of Keap1/Nrf2 signaling pathway,Chongcaoyishen decoction can delay the process of renal interstitial fibrosis.The effect may be related to the down-regulation of Keap1 mRNA and up-regulation of Nrf2 mRNA,HO-1 mRNA and NQO1 mRNA.5.By regulating the protein expression level of Keap 1/Nrf2 signaling pathway,the Chongcaoy i shen decoction downregulates KEAP 1,promotes the nuclear transfer of Nrf2 protein,and up-regulates the protein expressions of HO-1 and NQO1.Therefore,Chongcaoy i shen decoction plays the role of anti-inflammation,anti-oxidation and anti-renal interstitial fibrosis,so as to protect renal function. |
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