Comparative Study On The Anti-renal Fibrosis Effect Of Different Processed Products Of Rhubarb

ObjectiveTraditional Chinese physicians have been keen on the treatment of CRF with Chinese medicine rhubarb for a long time,but there are many kinds of processed products of rhubarb,and all kinds of processed products have been used in clinical treatment of CRF.In addition,the composition of raw rhubarb decoction changed with the decoction time,while the decoction time of rhubarb was not clearly defined.Therefore,this study aims to screen out the decoction time of each processed products of rhubarb by cell models,the selected drugs were then used in animal models,and the anti-renal fibrosis effect of different processed products of rhubarb were compared from the aspects of anti-oxidative stress,inhibition of TGF-β₁/p38MAPK signaling pathway,inhibition of renal tubular EMT and inhibition of inflammatory response.Thus,we can pick out the rhubarb processed products with the best curative effect on CRF and its corresponding decoction time,in order to provide a theoretical basis for the treatment of CRF with rhubarb,and further guide the clinical medication.MethodPart I:In vitro experiments.1.One hundred and forty-five male Sprague-Dawley（SD）rats were divided into 29 groups by random number table method,5 in each.The drug groups were continuously gavaged with the corresponding rhubarb decoction for 6 days,each processed rhubarb was decocted respectively for 5,7,9,10,15,20,30 minutes.And control group was gavaged with 0.9%sodium chloride solution.After the rats were anesthetized by intraperitoneal injection of chloral hydrate,blood was collected by aseptic operation of abdominal aorta to prepare drug-containing serum.Intervention of TGF-β₁-induced HRMC with drug-containing serum for 24h and 48h,detection of the cell proliferation rate,then selected the decoction time corresponding to the lowest cell proliferation rate in each processed rhubarb product.2.Hk-2 was induced by TGF-β₁ to cause renal tubular EMT,the expression ofα-SMA and E-Cadherin in HK-2 before and after induction of TGF-β₁ were detected by immunofluorescence,so as to verify the success of the cell fibrosis model.Intervention of TGF-β₁-induced HK-2 with drug-containing serum for 24h,detection the concentration ofα-SMA and E-Cadherin in each group by ELISA,then select the decoction time corresponding to the best suppressing EMT in each processed rhubarb product.PartⅡ:In vivo experiments.1.Ten of 60 male SD rats were selected as the control group by random number table method,and the remaining 50 were gavaged with adenine for 4weeks to cause a renal failure model.Then they were divided into model group,raw rhubarb group,rhubarb steamed with wine group,rhubarb stir-fried with wine group and rhubarb charcoal group by random number table method again,10 in each.The drug groups were gavaged with the decoction of drug which selected in vitro experiments,one for each processed rhubarb product,control group was gavaged with 0.9%sodium chloride solution.Comparison body weight changes in rats after 8 weeks of treatment,and collected serum and nephridial tissue.The SCr,BUN and UA of rats were detected by kit to compare the effects of four kinds of processed rhubarb products on improving renal function in rats with renal fibrosis.2.The concentration of T-SOD and MDA in rat nephridial tissue were detected by kit,the expression of NOX4 was detected by Wertern Blot,the concentration of 8-OHdG was detected by ELISA,and the effects of four kinds of processed rhubarb products on anti-oxidative stress in rats with renal fibrosis were compared.3.The protein expression of TGF-β₁,p38 and p-p38 in rat nephridial tissue were detected by Western Blot,the expression of TGF-β₁ mRNA and p38MAPK mRNA were detected by real-time fluorescent quantitative PCR,and the effects of four kinds of processed rhubarb products on inhibition of TGF-β₁/p38MAPK signaling pathway in rats with renal fibrosis were compared.4.The concentration of TNF-αand IL-1βin rat nephridial tissue were detected by ELISA,and the effects of four kinds of processed rhubarb products on suppressing expression of TNF-αand IL-1βin rats with renal fibrosis were compared.5.The expression ofα-SMA and E-cadherin in rat nephridial tissue were detected by Western Blot,and the effects of four kinds of processed rhubarb products on the inhibition of EMT in rats with renal fibrosis were compared.6.The expressions ofα-SMA and FN in rats with renal fibrosis were detected by immunohistochemical staining,and the effects of four kinds of processed rhubarb products on suppressing expression of renal fibrosis markers were compared.7.Morphological changes of rat nephridial tissue including glomeruli,kidney tubules and renal interstitium which stained withPAS,Masson and HE were observed by light microscopy,ultrastructural changes of rat kidney were observed by electron microscopy,the therapeutic effects of four kinds of processed rhubarb products on CRF were compared.ResultPart I:In vitro experiments.1.The cell proliferation rate of TGF-β₁-induced HRMC which was intervened with drug-containing serum of four kinds of processed rhubarb products with different decoction time for 24h and 48h,all showed a trend of decreasing first and then increasing,with the prolongation of the decoction time.The lowest cell proliferation rate in each group intervened with drug-containing serum for 24h was 71.4%（raw rhubarb group decocting for10 mins）,63.3%（rhubarb steamed with wine group decocting for 10 mins）,70.2%（rhubarb stir-fried with wine group decocting for 9 mins）and 90.2%（rhubarb charcoal group decocting for 9 mins）.The lowest cell proliferation rates in each group intervened with drug-containing serum for 48h was 78.1%（raw rhubarb group decocting for 10 mins）,67.1%（rhubarb steamed with wine group decocting for 10 mins）,76.7%（rhubarb stir-fried with wine group decocting for 9 mins）and 91.5%（rhubarb charcoal group decocting for 9mins）.2.The concentration ofα-SMA in TGF-β₁-induced HK-2 which was intervened with drug-containing serum of four kinds of processed rhubarb products with different decoction time for 24h,all showed a trend of decreasing first and then increasing,with the prolongation of the decoction time.The lowest concentration ofα-SMA in each processed rhubarb product was 52.086 ng/ml（raw rhubarb group decocting for 10 mins）,44.596 ng/ml（rhubarb steamed with wine group decocting for 10 mins）,49.768 ng/ml（rhubarb stir-fried with wine group decocting for 9 mins）and 58.470 ng/ml（rhubarb charcoal group decocting for 9 mins）respectively.While the concentration of E-cadherin showed a trend of increasing first and then decreasing,the highest concentration of E-cadherin in each processed rhubarb product was 1.017 ng/ml（raw rhubarb group decocting for 10 mins）,1.325ng/ml（rhubarb steamed with wine group decocting for 10 mins）,1.163 ng/ml（rhubarb stir-fried with wine group decocting for 9 mins）and 0.787 ng/ml（rhubarb charcoal group decocting for 9 mins）respectively.The differences between the four kinds of processed rhubarb products were statistically significant（P<0.05）.PartⅡ:In vitro experiments.1.The values of SCr,BUN and UA of the model group were significantly increased compared with the control group（P<0.01）,the values of SCr,BUN and UA of the four drug groups were all decreased to different degrees compared with the model group（P<0.01）.Among the four drug groups,the rhubarb steamed with wine group had the largest decline,the rhubarb carbon group had the smallest decline,and the rhubarb stir-fried with wine group decreased more than the raw rhubarb group.The differences between the four drug groups were statistically significant（P<0.05）.2.The expression of NOX4 and the concentration of MDA and 8-OHdG in rat nephridial tissue of the model group were significantly increased compared with the control group（P<0.01）.Among the four drug groups,the rhubarb steamed with wine group had the largest increase,the rhubarb carbon group had the smallest increase,and the rhubarb stir-fried with wine group increased more than the raw rhubarb group.The differences between the four drug groups were statistically significant（P<0.05）.The concentration of T-SOD in rat nephridial tissue of the model group was significantly lower than that of the control group（P<0.01）.Among the four drug groups,the rhubarb steamed with wine group had the largest decline,the rhubarb carbon group had the smallest decline,and the rhubarb stir-fried with wine group decreased more than the raw rhubarb group.The differences between the four drug groups were statistically significant（P<0.05）.3.The protein expression of TGF-β₁、p38 and p-p38 in rat nephridial tissue of the model group were significantly increased compared with the control group（P<0.05）.The expressions of TGF-β₁ in the four drug groups all decreased to different degrees compared with the model group（P<0.05）,the decline was most obvious in the rhubarb steamed with wine group,followed by the rhubarb stir-fried with wine group and the raw rhubarb group,and the rhubarb charcoal group had the smallest decline.There was significant difference in expression of TGF-β₁ between the four drug groups compared with each other（P<0.01）.The protein expressions of p38 and p-p38 in the rhubarb steamed with wine group were significantly different from those in the other three drug groups（P<0.01）,and the difference between the rhubarb stir-fried with wine group and the rhubarb carbon group was also significant（P<0.01）,while there was no statistical difference between the rhubarb stir-fried with wine group and the raw rhubarb group.The expression of TGF-β₁mRNA and p38MAPKmRNA in rat nephridial tissue of the model group were significantly increased compared with the control group（P<0.05）.The expressions of TGF-β₁mRNA and p38MAPKmRNA in the four drug groups all decreased to different degrees compared with the model group（P<0.05）,the decline was most obvious in the rhubarb steamed with wine group,followed by the rhubarb stir-fried with wine group and the raw rhubarb group,and the rhubarb charcoal group had the smallest decline.The expressions of TGF-β₁mRNA and p38MAPKmRNA in the rhubarb steamed with wine group were significantly different from those in the other three drug groups（P<0.01）.The expressions of TGF-β₁mRNA in the rhubarb stir-fried with wine group were significantly different from those in the other three drug groups（P<0.01）,the expression of p38MAPKmRNA in the rhubarb stir-fried with wine group was significantly different from that in the rhubarb stir-fried with wine group and rhubarb charcoal group（P<0.01）,while no statistical significance was found in the rhubarb group.4.The concentrations of TNF-αand IL-1βin rat nephridial tissue of the model group was significantly higher than those of the control group（P<0.01）,while the concentration of TNF-αand IL-1βin the four drug groups decreased to different degrees compared with those in the model group（P<0.01）.In the four drug groups,the descending order from more to less were rhubarb steamed with wine group,raw rhubarb group,rhubarb stir-fried with wine group and rhubarb charcoal group.There was significant difference between the rhubarb steamed with wine group and the rhubarb charcoal group（P<0.01）,and there was no statistical difference between the rhubarb steamed with wine group,the rhubarb stir-fried with wine group and the raw rhubarb group.5.The expression ofα-SMA in rat nephridial tissue of the model group was significantly increased and the expression of E-cadherin was significantly decreased compared with the control group（P<0.01）.The expression ofα-SMA of each drug group was down-regulated in different degrees and the expression of E-cadherin of each drug group was up-regulated in different degrees compared with the model group（P<0.01）.The effects of the four drug groups on inhibiting EMT were from strong to weak in order of rhubarb steamed with wine group,rhubarb stir-fried with wine group,raw rhubarb group and rhubarb charcoal group,and the difference between the groups were statistically significant（P<0.01 or P<0.05）.6.The expression ofα-SMA was observed in the whole field and the expression of FN was also significantly increased in rat kidney tissue of the model group,compared with the control group,while the expression ofα-SMA and FN were decreased to different degrees in the four drug groups compared with the model group.The expression ofα-SMA and FN in the four drug groups were from strong to weak in order of rhubarb charcoal group,raw rhubarb group,rhubarb stir-fried with wine group and rhubarb steamed with wine group.7.The pathological sections stained with PAS were observed by light microscopy.In the model group,it was found that the mesangial area of the glomerular mesangial area increased and localized glassy changes were observed,individual glomeruli had been hardened and discarded,and the renal capsules were obviously enlarged,most of the renal tubules were atrophied or disappeared,while there were also a small number of renal tubules had compensatory dilation,the renal tubular epithelial cells showed vacuolar degeneration,the wall of the small arteries were thickened and had a tendency to become glassy,the infiltration of renal interstitial inflammatory cells were increased,and the hyperplasia of fibrous tissue was obvious.The pathological changes of glomeruli,renal tubules and renal interstitium in the four drug groups were improved in different degrees compared with the model group.Among the four drug groups,the pathological changes in the rhubarb steamed with wine group were the most mild,and the pathological changes in the rhubarb charcoal group were the most serious.The pathological changes of the rhubarb stir-fried with wine group and the raw rhubarb group were between the rhubarb steamed with wine group and the rhubarb charcoal group.The microstructure of rat kidney was observed by electron microscopy.In the model group,it was found that the thickness of GBM was uneven,local thickening was obvious,partial GBM contour was not clear,the foot processes were diffusely fused,the mesangial matrix was severely hyperplasia,and the electron dense deposit could be seen in the mesangial area.The renal pathological changes were from heavy to light in order of rhubarb charcoal group,raw rhubarb group,rhubarb stir-fried with wine group and rhubarb steamed with wine group.ConclusionPart I:In vitro experiments.1.The four processed rhubarb products could inhibit the proliferation of TGF-β₁-induced HRMC,the overall effect of the rhubarb steamed with wine group was the most ideal,the overall effect of the rhubarb charcoal group was the worst,and the overall effect of the rhubarb stir-fried with wine group was better than that of the raw rhubarb group.The selected processed rhubarb products inhibited the proliferation of HRMC induced by TGF-β₁ from strong to weak in the following order:rhubarb steamed with wine decoction for 10minutes group,rhubarb stir-fried with wine decoction for 9 minutes group,raw rhubarb decoction for 10 minutes group,and rhubarb charcoal decoction for 9 minutes group.2.Four kinds of processed rhubarb products could inhibit the EMT of TGF-β₁-induced HK-2,among which the overall effect of rhubarb steamed with wine group was the most ideal,the overall effect of rhubarb charcoal was the worst,and the overall effect of rhubarb stir-fried with wine group was better than that of raw rhubarb group.The selected processed rhubarb products inhibited the EMT of HK-2 induced by TGF-β₁ from strong to weak in order of rhubarb steamed with wine group decoction for 10 minutes,rhubarb stir-fried with wine group decoction for 9 minutes,raw rhubarb group decoction for 10 minutes,and rhubarb charcoal group decoction for 9 minutes.PartⅡ:In vitro experiments.1.Four kinds of processed rhubarb products could effectively improve the renal function of rats with renal fibrosis,and their effects were from strong to weak in order of rhubarb steamed with wine group,rhubarb stir-fried with wine group,raw rhubarb group and rhubarb charcoal group.2.Four kinds of processed rhubarb products had the effect of anti oxidant stress,and the effects were from strong to weak in order of rhubarb steamed with wine group,rhubarb stir-fried with wine group,raw rhubarb group and rhubarb charcoal group.3.Four kinds of processed rhubarbs had inhibitory effects on the TGF-β₁/p38MAPK signaling pathway,among which rhubarb steamed with wine had the best inhibitory effect,while rhubarb carbon had the worst inhibitory effect.The inhibitory effects of rhubarb stir-fried with wine and raw rhubarb were between rhubarb steamed with wine and rhubarb carbon,and there was no difference in their inhibitory effects.4.Four kinds of processed rhubarbs could effectively inhibit the secretion of TNF-αand IL-1βin rat with renal fibrosis,while there was no significant difference in the effect of raw rhubarb group,rhubarb steamed with wine group and rhubarb stir-fried with wine group,but all of them were stronger than rhubarb charcoal group.5.Four kinds of processed rhubarbs could effectively inhibit renal tubular EMT in rats with renal fibrosis,and their effects were from strong to weak in order of rhubarb steamed with wine group,rhubarb stir-fried with wine group,raw rhubarb group and rhubarb charcoal group.6.Four kinds of processed rhubarbs all could effectively inhibit the expression ofα-SMA and FN,and their effects were from strong to weak in order of rhubarb steamed with wine group,rhubarb stir-fried with wine group,raw rhubarb group and rhubarb charcoal group.7.Four kinds of processed rhubarbs all could effectively improve the renal tissue morphology and ultrastructure of rats with renal fibrosis,and their effects were are from strong to weak in order of rhubarb steamed with wine group,rhubarb stir-fried with wine group,raw rhubarb group and rhubarb charcoal group.