Effect Of Cake-separated Moxibustion On TGF-?1/Smads Signal Pathways In Renal Interstitial Fibrosis Of CRF Rabbits

Chronic renal failure?CRF?is a series of clinical syndromes arising from progressive renal impairment caused by primary and secondary renal diseases^[1],which is clinically refractory and has a poor prognosis.Previous clinical studies have found that the cake-separated moxibustion can improve the symptoms and renal function of CRF patients in early and middle stage,improve the life quality of the patients and delay the CRF process^[2],but the mechanism is unclear.Both domestic and international studies have found that CRF is closely related to renal interstitial fibrosis,and TGF-?1/Smads signal pathway is the most important pathway of renal interstitial fibrosis^[3].Therefore,we propose this hypothesis:The cake-separated moxibustion may regulate the TGF-1/Smads signaling pathway related factors to treat CRF.Therefore,this experiment hopes to find out the therapeutic effect of the cake-separated moxibustion on the CRF model rabbit by observing the expression of positive factor TGF-?1,Smad3,CTGF,ILK and negative factor BMP-7 and Smad7 in TGF-?1/Smads signaling pathway.So the clinical value and application value of the cake-separated moxibustion for the treatment of CRF can be improved,and also provide new ideas and basis for the traditional Chinese medicine.At the same time,this study is a part of the National Natural Science Found Project"Mechanism study of cake-separated moxibustion on TGF-?1/Smads signal pathway in Chronic Renal Failure"?8166150111?.ObjectiveThrough the observation of Herbal Cake-separated Moxibustion treatment of CRF rabbits routine examination?blood pressure,body weight,serum creatinine?Scr?,blood urea nitrogen?BUN?,24h urine protein?,and the influence of TGF-?1,Smad3,CTGF,ILK,BMP-7,Smad7 mRNA and protein expression level in the TGF-?1/Smads signal pathway,to investigate the effect of herbal cake-separated moxibustion on TGF-?1/Smads signaling pathway,providing theoretical and experimental basis for the clinical use of cake-separated moxibustion in the treatment of CRF.Methods30 male healthy New Zealand rabbits?1.8kg².2kg?,adaptive feeding for 1 weeks.6 rabbits were randomly selected as group A?blank group?,and the others were given adenine model for 3 weeks.After the model was successfully built,other rabbits were randomly divided into 4 groups:model group,losartan group,cake-separated moxibustion group,inhibitor group,6 rabbits in each group.Then the blood pressure,body weight,Scr,BUN and 24h urine protein of rabbits were measured,and gave them 3courses of treatments?36 days?.The blood pressure,body weight,Scr,Urea and 24h urine protein were measured again.Then,executed all the rabbits,observed kidney tissue by HE staining and Masson staining in pathological section;measured the mRNA and protein expression level of positive factors as TGF-?1,Smad3,CTGF,ILK and negative factors as Smad7,BMP-7by Q-PCR and Western Blot;measured the serum levels of TGF-?1,CTGF and BMP-7 by ELISA;and measured the expression of TGF-?1 and Smad3 by immunohistochemical methods.Results1.Effects on the weight and blood pressureWeight:After building the model,the weight of the rabbits in each treatment group was reduced?P<0.01?when compared with blank group.Compared with the model group,there was no significant difference in the weight of rabbits in each treatment group?P>0.05?.At the end of the course of treatment,compared with the model group,all of groups were significantly increased?P<0.05?.Compared with the losartan group and inhibitor group,were no significant change in the cake-separated moxibustion group?P>0.05?.Blood pressure:After building the model,the Systolic Blood Pressure?SBP?,Diastolic Blood Pressure?DBP?,and Mean Arterial Pressure?MAP?were increased significantly in each treatment group?P<0.01?when compared with blank group.Compared with the model group,there was no significant difference in each treatment group?P>0.05?.At the end of the course of treatment,the SBP,DBP and MAP were significantly decreased in losartan group?P<0.01?,the SBP and MAP were significantly decreased in cake-separated moxibustion group?P<0.01?,except for DBP?P>0.05?.2.Effects on the Scr,BUN,24h urine proteinAfter building the model,Scr,BUN,24h urine protein are increased significantly in each treatment group?P<0.01?when compared with blank group.Compared with the model group,there was no significant difference in each treatment group?P>0.05?.At the end of the course of treatment,the Scr were significantly decreased in losartan group and cake-separated moxibustion group?P<0.01?.The BUN were significantly decreased in losartan group,and cake-separated moxibustion group?P<0.01?.Losartan had a good effect on reducing 24h urine protein?P<0.05?,but the cake-separated moxibustion was not significantly?P>0.05?.3.Effects on histopathologyHE staining:Glomerulus and renal tubular structure were intact,the capillary and renal interstitial structures were normal,and fibrous tissue hyperplasia was not obvious in blank group.Glomerular and renal interstitialhyperemia,capillariesweredestroyed,renaltubule enlargement,lumen gap narrowing,epithelial cells degeneration and enlargement,fibrous tissue was obviously proliferated in model group.The other groups showed different degrees of glomerular and renal interstitial hyperemia,renal tubule enlargement,epithelial cell degeneration and fibrous tissue hyperplasia,these were reduced when compared with the model group.Masson staining:Basal membrane of the renal tubule,the interstitium of the kidney,and the perivascular were all had small amount of collagen in blank group.The lumen of the kidney were enlarged significantly,the fibers of the renal interstitium and capillaries appeared obvious blue because of proliferation in model group.The different degrees of increase of the renal interstitial lumen and the proliferation of collagen fibers could be observed in all other groups,but they were less than those in the model group.4.Effects on the expression level of TGF-?1,Smad3,ILK,CTGF,Smad7and BMP-7mRNAThe expression level of TGF-?1,Smad3,ILK,CTGF mRNA in the model group were increased?P<0.01 or P<0.05?when compared with blank group,and BMP7,Smad7 mRNA were decreased?P<0.01 or P<0.05?.Compared with the model group,the TGF-?1 mRNA in losartan group,cake-separated moxibustion group were decreased?P<0.05?.Compared with the inhibitor group,the TGF-?1mRNA in cake-separated moxibustion group was decreased?P<0.01?.The Smad3 mRNA in losartan group and cake-separated moxibustion group were decreased?P<0.01?when compared with model group,except for inhibitor group?P>0.05?.Compared with the inhibitor group,the Smad3mRNA in cake-separated moxibustion group was decreased significantly?P<0.05?.The ILK mRNA in losartan group and cake-separated moxibustion group were decreased?P<0.05?when compared with model group,except for and inhibitor group?P>0.05?.Compared with the inhibitor group,the relative transcript level of ILK mRNA in cake-separated moxibustion group was decreased?P<0.05?.Compared with the model group,the CTGF mRNA in other groups were decreased?P<0.01 or P<0.05?.Compared with inhibitor group,the CTGF mRNA in cake-separated moxibustion group was decreased significantly?P<0.05?.The Smad7 mRNA in losartan group and cake-separated moxibustion group were increased significantly?P<0.01 or P<0.05?,when compared with model group.Compared with inhibitor group,the level of Smad7 mRNA in cake-separated moxibustion group was increased?P<0.05?.The BMP-7 mRNA in losartan group and cake-separated moxibustion group were increased?P<0.05?when compared with model group.Compared with losartan group and inhibitor group,the level of BMP-7 mRNA in cake-separated moxibustion group was not significantly?P>0.05?.5.Effects on the expression level of serum TGF-?1,CTGF and BMP-7Compared with the model group,the level of TGF-?1 in losartan group and cake-separated moxibustion group were decreased?P<0.01?.Compared with the inhibitor group,the level of TGF-?1 in cake-separated moxibustion group decreased?P<0.01?.Compared with the model group,the level of CTGF in losartan group and cake-separated moxibustion group were decreased?P<0.01?.Compared with the inhibitor group,the level of CTGF in cake-separated moxibustion group decreased?P<0.05?.Compared with the model group,the level of BMP-7 in losartan group and cake-separated moxibustion group were increased?P<0.01?.Compared with the inhibitor group,the level of BMP-7 in the cake-separated moxibustion group was increased?P<0.01?.6.Effects on the protein expression level of TGF-?1,Smad3,ILK,CTGF Smad7 and BMP-7.?1?Detect the expression of TGF-?1 and Smad3 in renal tissue by using immunohistochemistry.In the blank group,a small amount of TGF-?1 and Smad3 was expressed in the renal tissue,and it was in light yellow.In the model group,the expression of TGF-?1 and Smad3 increased significantly?P<0.05?,and was in yellowish brown.Compared with the model group,the expression of Smad3 in each treatment group decreased?P<0.05?.Compared with the western medicine group,the expression of Smad3 in cake-separated moxibustion group was not significant difference?P>0.05?.Compared with the model group,the expression of TGF-?1 in western medicine group and cake-separated moxibustion group were decreased?P<0.05?,but the difference of inhibitor group was not significantly?P>0.05?.Compared with the losartan group and inhibitor group,there was no significant difference in the expression of TGF-?1 in the cake-separated moxibustion group?P>0.05?.?2?Detect the expression of TGF-?1,Smad3,ILK,CTGF,Smad7 and BMP-7 by using Western Blot.In the model group,the protein expression of TGF-?1,Smad3,ILK,CTGF were increased?P<0.01 or P<0.05?,while Smad7 and BMP-7 were decreased?P<0.01 or P<0.05?when compared with blank group.Compared with the model group,the expression level of TGF-?1 in each treatment group was decreased?P<0.01?.Compared with the inhibitor group,the expression level of TGF-?1 in the cake-separated moxibustion group was decreased?P<0.01?.Compared with the model group,the expression level of Smad3 in each treatment group was decreased?P<0.01?,Compared with the inhibitor group,the expression level of Smad3 in the cake-separated moxibustion group was decreased?P<0.05?.Compared with the model group,the expression level of ILK in losartan group and cake-separated moxibustion group were decreased?P<0.01?.Compared with the inhibitor group,the expression level of ILK in the cake-separated moxibustion group had significantly difference?P<0.01?.Compared with the model group,the expression level of CTGF in losartan group and cake-separated moxibustion group were decreased?P<0.01 or P<0.05?.Compared with the model group,the expression of Smad7 in losartan group,cake-separated moxibustion group were increased?P<0.01 or P<0.05?.Compared with the inhibitor group,the expression of Smad7 in the cake-separated moxibustion group was not significant difference?P>0.05?.Compared with the model group,the expression of BMP-7 in each treatment group was increased?P<0.05?.Compared with the inhibitor group,the expression of BMP-7 in cake-separated moxibustion group was increased?P<0.05?.ConclusionCake-separated moxibustion has a good therapeutic effect on CRF rabbits,for it can improve the CRF rabbits'signs and symptoms,weight gain,reduce blood pressure?SBP and MAP?,decrease Scr and Urea level,and has a tendency to reduce the 24h urine protein excretion.Cake-separated moxibustion can regulate the mRNA and protein expression level of positive factors TGF-?1,Smad3,CTGF,ILK and negative factors BMP-7,and has been confirmed that the cake-separated moxibustion can treat CRF by inhibiting the correlation of TGF-?1/Smads signaling pathway.This experiment can improve the clinical and application value of the cake-separated moxibustion for the treatment of CRF,and also provide new ideas and basis for the external therapy of traditional Chinese medicine.