The Effect And The Mechanism Of Total C-21 Steroidal Glycosides Of Baishouwu On Hepatic And Renal Fibrosis In Rats

BackgroundBaishouwu is dried root tuber of Cynanchum Auriculatum Royle Ex Wight,C.wilfordii hemsl and C.Bungei Decne.Its taste is bitter and sweet,slightly lukewarm,non-toxic.It enters the liver and kidney,and has long been used as a nourishing,strong and tonic medicine.At present,Baishouwu has been cultivated in Shandong,Jiangsu,and Dingyuan,Anhui.The commonly used medicinal materials come from the ear leaf leather of Binhai County,Jiangsu Province.There are many chemical components in Baishouwu,the most important of which are C-21 steroidal glycosides,polysaccharides,acetophenones and other active ingredients.At present,the research on the effects of Baishouwu mainly focuses on anti-tumor,liver protection,anti-oxidation and immune regulation.Based on the previous research,this study intends to systematically study the effect of C-21 steroids,the main active component of Baishouwu,on the prevention and treatment of liver and kidney fibrosis,and to explore its function of protecting liver and kidney,the efficacy characteristics and internal laws of prevention and treatment of liver and kidney fibrosis,so as to provide scientific basis for the development and utilization of Baishouwu.Chapter one,Literature researchThis article reviews the recent research progress of Cynanchi Bungei Radix based on the difference in the pharmacological effects and contents of different chemical constituents in different species of Cynanchi Bungei Radix.The paper provides a scientific basis for further rational application of the medicinal value of different varieties of Cynanchi Bungei Radix.Chapter two,The establishment and investigation of rat liver and kidney fibrosis modelThe rats were randomly divided into a control group and a model group.The model group was injected intraperitoneally with DEN(b.w.)and subcutaneously injected with HgC12(b.w.).The orbital blood was collected every 2 weeks.After 8 weeks,the serum levels of ALT,AST,ALP,BUN,LDH,UA were measured by automatic biochemical analyzer.The indicators of fibrosis(LN?Collagen type IV?HA?PIIINP)were measured by radioimmunoassay.Six rats in the model group were randomly treated every two weeks for histopathological examination of liver and kidney,and hydroxyproline in liver and kidney was detected by alkaline hydrolysis test box.Western blot was used to detect the expression of ?-smooth muscle actin(?-SMA),transforming growth factor ?1(TGF-?1)and E-cadherin in liver and kidney.The results showed that after 2 weeks of modeling,the rats showed obvious liver and kidney functional impairment.After 4-6 weeks of modeling,there was a small amount of collagen deposition in the liver and slight interstitial fibrosis in the kidney.After 6-8 weeks of modeling,there was a large amount of collagen deposition in the liver,pseudolobules,obvious interstitial fiber hyperplasia in the kidney and local glomerular atrophy.Chapter three,Preliminary study on the pathogenesis of DEN-HgCl2 combined induction rat liver and kidney fibrosis model.The modeling method was the same as(2).6 rats in the model group were treated every 2 weeks,and the liver and kidney tissues were taken.Colorimetric method was used to detect the content of MDA and the activities of GSH-PX,T-SOD,TAOC and CAT.Real-time PCR was used to detect mRNA expressions of IL-6 and TNF-?.The protein expressions of P38 and ERK1/2,Smad2,Smad3,P-P38,P-ERK1/2,P-Smad2 and P-Smad3 were detected by Western blotting.The results showed that during the modeling period,there was obvious oxidative stress injury in the liver and kidney tissues of rats,and the mRNA expression levels of inflammatory factors in liver and kidney tissues were significantly increased,and the protein related to MAPK/TGF-?1/Smads signaling pathway was phosphorylated Level with time-dependent increasing.Chapter four,The effect of C-21 steroidal glycosides of Baishouwu on liver and kidney fibrosis in rats were studied.The rats were randomly divided into control group,model group,high-dose group(4g/kg)and low-dose group(2g/kg).Automatic biochemical analyzer was used to measure the levels of AST,ALT,ALP,TBIL,LDH,BUN and UA in serum of rats in each group.Four indexes of fibrosis in serum were measured by radioimmunoassay.The liver and kidney tissues were collected.The organ indexes of liver and kidney were calculated and the histopathology was examined.Hyp contents in liver and kidney tissue were tested by alkaline hydrolytic method.The protein expression levels of ?-SMA,E-cadherin and TGF-?1 in liver and kidney tissues were detected by Western blot and immunohistochemistry.The experimental results showed that the levels of AST,ALT,ALP,LDH,T-Bil,BUN and UA in the high-dose and low-dose group of C-21 steroside were all decreased.The levels of HA,LN and IV-C in serum and the contents of hydroxyproline in liver and kidney in C-21 steroside groups were significantly lower than those of model group.Pathological examination showed that the liver and kidney lesions of the rats in the C-21 steroside group were significantly reduced,especially in the high dose group.Chapter fiver,The mechanism of prevention and treatment of liver and kidney fibrosis in rats by C-21 steroid glycoside of Baishouwu based on MAPK/TGF-?1/Smads signaling pathwayThis chapter mainly studies the relate of prevention of C-21 steroid glycosides of Baishouwu with the regulation of MAPK/TGF-pi/Smads signaling pathway.The results showed that the activities of GSH-Px,T-SOD,T-AOC and CAT in the liver and kidney of rats in the high and low dose group were significantly increased.While the MDA content in the liver and kidney of the rats in the high and low dose group was significantly decreased,The expression levels of IL-6,TNF-?,collagen I and collagen III mRNA in the liver and kidney of rats in the high and low dose group of Baishouwu C-21 steroside were significantly lower.Western blotting results showed that the phosphorylation level of p38,ERK1/2,Smad2 and Smad3 protein in liver and kidney of rats was significantly lower.Conclusion1.Combined use of DEN-HgCl2 for 2 to 4 weeks can induce obvious liver and kidney damage in rats,collagen deposition and fibroplasia in 6 to 8 weeks,and rat liver and kidney fibrosis coexist successfully after 8 weeks modeling.2.Combined use of DEN-HgC12 can induce oxidative damage of liver and kidney tissue,activate MAPK/TGF-pi/Smads signal pathway,promote the expression of related inflammatory factors and collagen secretion,thus accelerate the process of liver and kidney fibrosis.3.C-21 steroidal glycosides of Baishouwu can significantly reduce the degree of liver and kidney fibrosis induced by DEN-HgC12,inhibit the activation of stellate cells and the expression of TGF-?1,reduce the deposition of collagen,and show a significant anti hepatorenal fibrosis effect.4.The mechanism of C-21 steroids of Baishouwu on DEN-HgC12-induced hepatic and renal fibrosis may be closely related to the regulation of MAPK/TGF-?1/Smads signal pathway,the reduction of oxidative stress injury,the inhibition of inflammatory factor expression and the inhibition of collagen deposition.