Research On The Effect And Mechanism With Method Of Invigorating Qi,promoting Blood Circulation,clearing Away Blood Stasis And Toxin In Rats With Hepatic Fibrosis Induced By CCl4

Objective:According to the characteristic of pathogenesis in Traditional Chinese Medicine of Hepatic Fibrosis(HF),effects were observed and evaluated with methods of invigorating QI and promoting blood circulation,clearing away blood stasis and toxin,as well as integration of these two methods on rat model of HF induced by CCl4.The mechanism of the effect was further studied and explored to provide theoretical basis for clinical application with these therapeutic methods.Methods:Study One:64 SD rats were randomly divided into normal control group(blank group,group A),model control group(group B),colchicine group(group C),Danshen group(group D),Huangqi group(group E),Danshen and Huangqi Compound Preparation Group at ratio 1:1(Group F),Danshen and Huangqi Compound Preparation Group at ratio 1:2(Group G),Danshen and Huangqi Compound Preparation at trtion2:1(Group H).In the B-H group,a HF rat model was made by intraperitoneal injection of carbon tetrachloride olive oil.After the modeling was completed,C-H group were administered with colchicine,Danshen granules,and Huangqi granules.The compound preparation group was administered with Danshen:Huangqi at a ratio of 1:1,Danshen:Huangqi at a ratio of 1:2 and Danshen:Huangqi was administered at a ratio of 2:1.After 4 weeks of intervention,serum and liver tissue specimens were collected,and the liver function and liver fibrosis indexes of the rats were measured by a biochemical analyzer.Immunohistochemical detection of ?-smooth muscle actin and type ?collagen,real-time PCR detection of TGF-?1,Smad2,?-catenin,Cyclin D1 mRNA levels,Western-blot detection of TGF-?1,Smad2,?-catenin,Cyclin D1 protein content were carried out.Study Two:60 SD rats were randomly divided into normal control group(blank group),model control group,colchicine group,Ziqi Ruangan granule low-dose group(0.3375g/kg),Ziqi Ruangan granule medium-dose group(0.675g/kg)and Ziqi Ruangan granule high dose group(1.35g/kg).Except the normal group,the other groups were made into liver fibrosis rat models by intraperitoneal injection of carbon tetrachloride olive oil.After the modeling was completed,drug intervention was given.After 4 weeks of intervention,serum and liver tissue samples were collected and biochemical analysis was applied to detect rat liver function,liver fibrosis index,SOD,HYP and MDA levels,Western-blot was uesd to detect TGF-?1,Smad2,p-Smad2 protein content.Study Three:64 SD rats were randomly divided into normal control group(blank group),model control group,colchicine group,Jiawei Ziqi Ruangan Granule in low-dose group(0.506g/kg),and Jiawei Ziqi Ruangan Granule in medium-dose Group(1.0125g/kg)and high-dose Ziqi Ruangan granule group(2.025g/kg).Except the normal group,the other groups were made into liver fibrosis rat models by intraperitoneal injection of carbon tetrachloride olive oil.After the model was completed,drug intervention was given.After 4 weeks,serum and liver tissue samples were collected and biochemical analysis was used.Instrument to detect rat liver function,liver fibrosis index,SOD,HYP and MDA levels.real-time PCR was used to detect rat liver tissue lnc-LFAR1,TGF-?1,Smad2,p-Smad2 mRNA content,Western-blot was used to detect rat liver TGF-?1,Smad2,p-Smad2 protein content.Results:Study One:Compared with group B,the ALT,ALP,and liver fibrosis indexes of each Chinese medicine group decreased significantly(P<0.05),but there was no difference between them.Compared with group B,a-SMA and coll expression were the lowest in group E and F.qPCR showed that compared with group B,TGF-?1 in group F and G decreased significantly compared with group B(P<0.05),and Cyclin D1 in each group decreased significantly(P<0.05).Western-blot results showed that Cyclin D1 decreased significantly in each treatment group(P<0.05);group C had a better effect on reducing TGF-?1 and ?-catenin than most Chinese medicine groups(P<0.05),but had comparable effects to group F;There was no significant difference in efficacy among the Chinese medicine treatment groups.Study Two:Compared with the model group,liver function indexes of all Chinese medicine treatment groups were significantly decreased(P<0.05).Compared with the model group,all indexes of liver fibrosis in each treatment group decreased(P<0.05).Compared with the model group,the lipid peroxidation indexes of each Chinese Medicine group were significantly improved(P<0.05).Compared with the model group,TGF-?1 and p-Smad2 were significantly reduced in each Chinese Medicine treatment group(P<0.05),but there was no difference among these groups.Study Three:Compared with model group,indexes of liver function,liver fibrosis and lipid peroxidation were significantly improved(P<0.05).Some indexes in jiawei Ziqi Ruangan granule group even were improved than those in colchicine group(P<0.05).qPCR results show that lnc-LFAR1,TGF-?1,Smad2,p-Smad2 mRNA content were greatly reduced in medium and high dose group(P<0.05),and there was no significant difference among colchicine group and Chinese Medicine groups.Western-blot results show that TGF-?1 and p-Smad2 decreased significantly in each treatment group(P<0.05);Smad2 in high-dose group and medium-dose group decreased significantly(P<0.05).Conclusion:1.Method of invigorating QI and promoting blood circulation can significantly reduce ALT,ALP and liver fibrosis indexes as well as the expression levels of TGF-?1,Smad2,?-catenin and Cyclin D1,especially the 1:1 compound preparation has the best effect,suggesting the equal significance of invigorating QI and promoting blood circulation,while it showed this method had a significant effect on improving liver fibrosis indexes.2.Method of clearing away blood stasis and toxin can improve liver function and liver fibrosis indexes,enhance their antioxidant capacity,and significantly reduce the expression of TGF-?1,p-Smad2 protein in rat liver,thereby blocking TGF-?/Smad signal pathway,and the efficacy is comparable to the western medicine control group,especially for liver function indicators.3.The combined method had good effect on liver function indexes,liver fiber indexes and lipid peroxidation indexes.Among them,the high-dose group had better effects on some indexes than the western medicine control group colchicine.At the same time,the experiment also found that the method of nourishing qi and activating blood circulation and clearing blood stasis and toxin can down-regulate the expression of lnc-LFAR1,which than reduced the expression of related proteins on the TGF-?/Smad pathway,indicating that Chinese Mdicine could intervene TGF-?/Smad pathway by affecting Inc-LFAR1,which provides some other ideas for further research on the target of Chinese Medicine.