Anti-liver Fibrosis Function And Mechanism Research Of Rhizoma Anemones Raddeanae

primary part:Extraction process research of Rhizoma Anemones Raddeanae(RAR)Objective To determined that total soap glucoside extraction best craft of RAR, provides the drug for the following pharmacology experiment.Methods Uses the Rhizoma Anemones Raddeanae-A as the control article, determines the total soap glucoside content in raw material with the method of rhodinal-glacial acetic acid.Uses the total soap glucoside content in RAR as index to determined which methods is the best. For instance, recirculation,hypersound and microwave. Adopts the orthogonal cut-and-try method to make the improvement to the extraction process. Determined that 3 factors, namely ethyl alcohol density (A), extraction time (B) and the solvent compare (C), each factor designs 3 levels, carries on the experiment with L9(34) orthogonal array.Result Three kind of extraction method's total soap glucoside extraction rate respectively is recirculation (2.30%),(hypersound 2.39%),microwave (3.69%).The variance analysis result indicated that a factor chooses 1 level, the B factor, the C factor to change the non-significance influence to the result.Conclusion The best extraction process research of RAR is, the first ether degreasing, then the ethyl alcohol microwave extraction, determined that the optimum condition is 60% ethyl alcohol,10 time of quantities, the microwave withdraws 8min,30s is a gap; Finally further purifies through the water saturated normal butyl alcohol extraction method. Extraction rate of RAR in this experiment is 2.66%.The second part: anti- hepatic fibrosis rat model induced by Pig blood serum function and mechanism research of RARObjective The rat model of porcine serum-induced hepatic fibrosis was used to evaluate the curative effect of RAR and it's extraction,and then approach its function mechanism according to the changes of histopathological examination, activation of HSC, cell factor, the extracellular matrix and so on.Methods 68 SD rats were divided into 5 groups randomly, Including the normal group,the model group,the FuZhengHuaYu group(FZHY group). the RAR group,the extraction of RAR group (EXRAR group).Rats were intraperitoneally injected with 0.5ml of porcine serum twice a week to establish immunological hepatic fibrosis model for 15 successive weeks.To begin intragastric administration after the model succeed. The model group were treated with distilled water, the FZHY group were treated with FuZhengHuaYu gelatin capsule (15.8g·kg-1) and the RAR group were treated with RAR decoctum (0.7g·kg-1), the EXRAR group were treated with the extraction of RAR (0.071g·kg-1).The course of treatment is 8 weeks. During the course of treatment, the differences in the behavior science were observed. to draw the materials from the rats at the end of 23th weeks. ALT. AST,ALB,SOD,MDA in serum and Hyp content in liver homogenate were assayed by spectrophotometry. HA. LN. PCⅢ. CⅣin serum were determined by radioimmunoassay. The level of MMP-9 measured by ELISA. HE stain and Masson stain were used to examine the histopathological change. The expression of col-Ⅰ,col-Ⅲ,α-SMA,TGF-β1 were detected by SABC and the TIMP-1mRNA by RT-PCR.Result Compared with the model group, the results indicate that the physiclal status of rats were improved remarkably in the FZHY group,RAR group and EXRAR group. At the same time, the deviation of body weight is significant (p<0.05).The HE dyeing and the masson dyeing result confirmed that the normal structure of liver is damaged completely in the model group, the hepatic cord arrangement becomes disorderly.There are many fibre hyperplasia and deposit inside abbacy and liver flocculus.collagen fibers form the interval, even has false flocculus to form. Part of hepatic cells present to analosis,engorgemen apomorphosis even punctiform necrosis. The partial liver cells present to wither, the swelling denaturation, even the punctual necrosis, enlarge of the abbacy become obvious, the inflammatory cell and necrosis cell increase.Compared with the model group, extenuation of inflammation activityand and fibrosis level is significant in FZHY group,RAR group and EXRAR group, furthermore, amelioration degree of RAR group is more significant than FZHY group.Immunohistochemistry result indicate that the expression of col-Ⅰ,col-Ⅲ,α-SMA,TGF-β1 in the model group increased, dyeing is sable and areae is asystematic. Compared with the model group, the expression of col-Ⅰ,col-Ⅲ,α-SMA,TGF-β1 in FZHY group,RAR group and EXRAR group depress obviously, dyeing is tasteless in interval, and areae is asystematic there is no false flocculus to form. Coloration exponent result indicate that, Compared with the model group, the expression of col-Ⅰ,col-Ⅲ,α-SMA,TGF-β1 in FZHY group,RAR group and EXRAR group decrease and the expression ofa-SMA in RAR group decrease than FZHY group (p<0.05).Content of ALT,AST,HA,LN,PCⅢ,CⅣ,MDA,Hyp increased (p<0.05) and the Content of Alb,SOD,MMP-9 decreased in the model group. Compared with the model group, Content of ALT,AST,HA,LN,PCⅢ,CⅣ,MDA,Hyp decreased and the Content of Alb,SOD,MMP-9 increased in FZHY group,RAR group and EXRAR group (p<0.05), except for the Content of LN in EXRAR group, as well as the the Content of SOD in FZHY group and EXRAR group. the expression of TIMP-1mRNA in model group increased (p<0.05).Compared with the model group,the expression of TIMP-1 mRNA in EXRAR group decreased (p<0.05)Conclusion It is succeed that The rat model of porcine serum-induced hepatic fibrosis. RAR and it's extraction has a significant effect on anti-fibrosis, furthermore, RAR's extraction display almost the same effect to compare with the RAR.The possible mechanism is①inhibit HSC's activation;②inhibit the composition of ECM and encourage the degradation of ECM;③inhibit the expression of TGF-β1;④defend hepatic cell and mitigate the inflammation of inflammation;⑤resist liPid PerioxidationThe third part:influence of RAR medicine serum on the generation of LX-2 and the he expression of col-Ⅰ,col-Ⅲ,α-SMA,TGF-β1Objective The multiplication of LX-2 was used to evaluate the influence of RAR and it's extraction, and then approach its function mechanism.Methods LX-2 was used to evaluate the influence of RAR and it's extraction by MTT; The expression of col-Ⅰ,col-Ⅲ,α-SMA,TGF-β1 were detected by SABC and the TIMP-1mRNA by cell immunity influence.Result Regardless of RAR group or EXRAR group has the inhibitory action to the multiplication of LX-2,and the tendency of inhibitory action become more and more obvious along with the time increase. The cell immunity influence results indicate that RAR medicine serum can make the expression of col-Ⅰ,col-Ⅲ,α-SMA in LX-2 decrease.Conclusion It is certainty that RAR medicine serum have the inhibitory effect on the multiplication of LX-2.The possible anti- hepatic fibrosis mechanism is correlated to inhibit the activation of HSC; inhibit the composition of ECM and encourage the degradation of ECM.