Clinical Observation Of Qizhu Granules In The Treatment Of Early Cirrhosis And The Mechanism Of Anti-hepatic Fibrosis Angiogenesis

In previous theoretical,clinical and experimental studies,we realized that"poison and blood stasis of liver collaterals”is the basic pathogenesis of liver fibrosis,under the guidance of this theory,Qizhu granule,a kind of medicine against liver fibrosis,was developed for the prevention and treatment of liver fibrosis and early cirrhosis.It was found that Qizhu granules could attenuate the expression of vascular endothelial growth factor(VEGF),angiotensin(Ang)/tyrosine kinase receptors 2(Tie-2),p38 mitogen activated protein kinase(P38 MAPK),phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)and so on in liver tissue,and thus play a role in alleviating liver fibrosis,which may be related to the effect of capillary in the process of liver fibrosis.Further studies are needed to determine how Qizhu granules affect capillary of liver sinusoidal endothelial cells(LSEC).This study combined with clinical,whole animal and cell experiments to further improve the clinical efficacy of Qizhu granule in the early stage of liver cirrhosis and its anti-capillary mechanism.Objective:1.Through clinical observation of the influence of Qizhu granules on TCM syndrome score,liver function,liver fibrosis and liver elastic fiber value in patients with early-stage cirrhosis of qi deficiency and blood stasis,the clinical efficacy of Qizhu granules in the treatment of early-stage cirrhosis of qi deficiency and blood stasis syndrome was studied and its safety was evaluated.2.The effects of Qizhu granules on hepatic sinusoidal endothelial cell capillary induced by Carbon tetrachloride(CCl4)in rats were investigated by in vivo experiment(Wistar Rat)and in vitro experiment(LSEC rat).Methods:1.Clinical trial:selection in March 2019-January 2021 in the Chinese Academy of Sciences hospital gate hepatitis outpatient service of traditional Chinese medicine and digestive department of 18-65 years old early cirrhosis of the liver qi deficiency and blood stasis syndrome in patients as the research object,were stochastically divided into Qizhu granule group and Compound Biejiarangan tablet group,two groups on the basis of conventional western medicine treatment,were given stilbene particles and compound turtle shell soft liver treatment,after 12 weeks of treatment two groups of patients before and after treatment respectively detect liver function,serum AST,ALT and liver fibrosis four(serum PC-?,LN and HA,IV-C),liver stiffness measurement(LSM),the TCM syndrome scores were recorded.2.Animal experiment:40 healthy male specified pathogen free(SPF)Wistar rats,weight about 180?200 g,were randomly divided into model group,compound turtle shell soft liver slices and stilbene particle group and normal group,in addition to the normal group,rats in each group were intraperitoneally injected with 40%CCl4 olive oil solution at a dose of 3 ml/kg twice a week,4 weeks continuous injection manufacturing rat liver fibrosis model,and carried out in a building with drug lavage.Building at 4 weeks,in each group rats liver tissue HE and Masson staining degree of liver injury and liver fibrosis,building successful post-processing groups of rats,take liver tissue staining immunohistochemical method to detect alpha-smooth muscle actin(a-SMA),the expression of cluster-of-differentiation-34(CD34)and Platelet endothelial cell adhesion molecule-1(CD31),image analysis,microvascular density(MVD)analysis,and Western Blot were used to detect the protein expression of Caveolin-1 and von Willebrand factor in each rat.3.Cell experiment:by intraperitoneal injection of Wistar rats by 40%CCl4 olive oil solution induced hepatic fibrosis,4 weeks LSEC separation through the generation of normal rats and liver fibrosis in rats LSEC.The drug-containing serum of normal rats,drug-containing serum of Qizhu granules and drug-containing serum of Compound Biejiaruangan tablets were prepared.Normal group(normal rat LSEC+normal rat serum),model group(liver fibrosis rat LSEC+normal rat serum),Qizhu granule group(liver fibrosis rat LSEC+Qizhu granules drug-containing serum),and Compound Biejiaruangan tablet group(liver fibrosis rat LSEC+Compound Biejiaruangan tablests drug-containing serum)were cultured for 48h,respectively.Trypan blue staining and flow cytometry were used to identify the LSEC in each group.The window of LSEC in each group was observed under electron microscope.The expressions of CD31,SE-1 and integrin av(33-FAK-ras/MAPK signaling pathway and related proteins in LSEC phenotypes of rats were detected by Western Blot.Results:1.Clinical trials(1)Comparison of general data between two groups:This study included 60 patients with qi deficiency and blood stasis syndrome of early liver cirrhosis,30 patients(17 males and 13 females)in Qizhu granule group,the mean age was 53.53 ±6.83 years,the course of disease was 6.23 ± 3.24 years,the Child-Pugh score was grade A,the average age was 54.13± 6.21,the duration of disease was 6.00 ± 2.83 years,and the Child-Pugh score was grade A.Both groups of patients with early cirrhosis of the liver were mainly hepatitis B virus.There was no significant difference in general data between the two groups.(2)The influence of liver function:There was no significant difference in serum AST and ALT between the two groups before treatment(P>0.05),after 12 weeks of treatment,the mean value of serum AST and ALT in Qizhu granule group was significantly lower than that before treatment(P<0.05).The mean value of serum AST and ALT compound ALT in Compound Biejiaruangan tablet group was lower than that before treatment,the difference was statistically significant(P<0.05).After treatment,the serum AST and ALT of the two groups were restored to normal level.There was no significant difference between the two groups(P>0.05).(3)The effect of Qizhu granule on four items of liver fibrosis in patients with early liver cirrhosis:There was no significant difference between two groups in serum PC-?,LN,HA and ?-C before treatment(P>0.05).After 12 weeks of treatment,the mean values of PC-?,?-C,HA and LN in Qizhu granule group were significantly lower than those before treatment(P<0.01).The mean values of Serum PC-?,?-C,HA and LN in the group of Compound Biejiaruangan tablet were significantly lower than those before treatment(P<0.01).(4)The effect of Qizhu granule on the liver elasticity of patients with qi-deficiency and blood-stasis syndrome of early cirrhosis:The liver elasticity of 60 patients with qi-deficiency and blood-stasis syndrome of early cirrhosis included in this study was higher than the normal level,liver elasticity of patients with early cirrhosis after Qizhu granules treatment was significantly lower than that before treatment(P<0.01).(5)Analysis of curative effect and safety of Qizhu granule on TCM syndromes of early cirrhosis patients with qi deficiency and blood stasis syndrome:After 12 weeks,the symptoms of Anorexia,fatigue,pain in liver region(flank area),shortness of breath,lazy speech,chest tightness and excessive rest were all improved(P<0.01),after 3 months treatment,the total effective rate of TCM syndrome of Qizhu granules was 93.33%,which was higher than that of Compound Biejiarangan tablets.Qizhu granules were superior to Compound Biejiarangan tablets(P<0.05).Qizhu granules have obvious advantages in relieving the TCM syndromes of early cirrhosis patients with qi deficiency and blood stasis and have no adverse reactions.2.Animal experiments(1)The effect on liver pathology in rats with liver fibrosis:After 4 weeks,the rats were sacrificed.The liver tissues of the normal group,the model group,and the Qizhu granule group Compound Biejia Ruangan tablet group were subjected to Masson and HE staining.The liver tissue of the model group showed proliferative fibrous tissue and fibrous septum formation,indicating In this study,the model of rat liver fibrosis was successfully established.At the same time,the degree of liver fibrosis in the Qizhu granule group and the Compound Biejiaruangan tablet group was less than that of the model group.(2)Effect of ?-SMA on the expression of liver tissue in rats with liver fibrosis:the expression of ?-SMA in liver tissue of rats with liver fibrosis was significantly greater than that in normal group.Compared with the model group,the expression of?-SMA in liver tissue of Qizhu granule group and Compound Biejiaruangan tablet group was significantly lower(P<0.05).(3)Effect of CD31 and CD34 expression in liver tissue of rats with liver fibrosis:the expression of CD31 and CD34 in liver tissue of rats in each group was detected by immunofluorescence staining,and the staining intensity of liver tissue in model group was the strongest,compared with the model group,the average optical density of CD31 and CD34 in liver tissue of Qizhu granule group and Compound Biejiaruangan tablet group decreased significantly(P<0.05).(4)Effect of VWF and Caveolin-1 protein expression in liver tissue of rats with liver fibrosis:the expression of vWF and Caveolin-1 protein in liver tissue of normal rats was less,and the expression level of vWF and Caveolin-1 protein in liver tissue of liver fibrosis model group was increased obviously.The expression of vWF and Caveolin-1 protein in liver tissue of rats with liver fibrosis was lower than that of rats with liver fibrosis model group(P<0.05).(5)Effects on hepatogenesis MVD in rats with liver fibrosis:CD31 and CD34 labeling methods were used to analyze the MVD of liver tissues in each group.After CCl4 treatment,the MVD of the model group was significantly higher than that of the normal group(P<0.05).Qizhu granule and Compound Biejiaruangan tablet can significantly reduce the MVD of rat liver tissues in the treatment of CCl4-induced hepatic fibrosis,which is significantly different from the model group.3.Cell experiments(1)Observation of LSEC:Trypan Blue staining showed that there was no significant difference in cell survival rate between model group and normal group.The ratio of CD 146+cells and SE-1+cells in normal group and model group was above 90%,according to the results of Flow cytometry.(2)Effect of LSEC fenestrations on hepatic fibrosis in rats:under scanning electron microscope,there were many fenestrations on the surface of normal LSEC,the fenestrations of LSEC were decreased in model group,and the fenestrations of LSEC in liver fibrosis rats were increased after treated with Qizhu granules.The fenestration structure of LSEC was similar to that of Compound Biejiaruangan tablet.(3)The expression of CD31 and SE-1 in the model group was significantly higher than that in the normal group(P<0.05).The expression of CD31 and SE-1 in LSEC in the Qizhu granule group was significantly lower than that in the liver fibrosis model group(P<0.05).(4)The effect of Qizhu granule on LSEC Integrin ?V?3-FAK-R as/MAPK signal pathway:compared with normal group,the ratio of LSEC integrinaV?3/GAPDH in model group was significantly increased(P<0.01),The ratio of integrin aV?3/GAPDH in LSEC decreased after LSEC was treated with Qizhu granules medicated serum(P<0.01).Compared with the normal group,the ratios of p-FAK/FAK and p-MAPK/MAPK in LSEC of rats in the model group were significantly increased(P<0.01),and the ratios of p-FAK/FAK,RAS and p-MAPK/MAPK in LSEC of rats with liver fibrosis were all decreased after treatment with Qishu granules drug serum,and the ratios of p-FAK/FAK and p-MAPK/MAPK were decreased with statistical significance(P<0.01).Compared with Compound Biejiaruangan tablet group,the protein expression level of integrin ?V?3 in Qizhu granule group was significantly decreased(P<0.05).Conclusion:1.Qizhu granule can significantly improve the TCM syndrome scores of qi-deficiency and blood stasis syndrome in early liver cirrhosis after 3 months of treatment,can restore transaminase to normal in patients with qi-deficiency and blood stasis syndrome in early liver cirrhosis,and can improve liver fiber in patients with qi-deficiency and blood stasis syndrome in early liver cirrhosis chemical indicators can reduce the liver elasticity of patients with qi-deficiency and blood stasis syndrome in early liver cirrhosis.2.Qizhu granule can significantly inhibit CCl4-induced hepatic fibrosis in rats,reduce the expression of CD31,CD34,vWF,Caveolin-1 in liver tissue of hepatic fibrosis rats,and decrease the density of hepatic sinusoid microvessels in liver tissue of hepatic fibrosis rats,reduce the proliferation of hepatic sinusoids micro vasculature,prevent capillary.3.Qizhu granule can alleviate the capillary in the process of liver fibrosis by inhibiting the reduction of LSEC fenestrations and improving the phenotype of LSEC in the process of liver fibrosis,at the same time,the Integrin ?V?3-FAK-R as/MAPK signaling pathway in LSEC may play an anti-sinusoidal capillary role.