Study On Anti-hepatic Fibrosis Effect And Mechanism Of Ganlong Capsule

Objective:To investigate the anti-hepatic fibrosis effect of Ganlong capsule and its mechanism,providing experimental basis for the clinical study of treating hepatic fibrosis with Ganlong Capsule.Methods:Taking 6-week-old male SD rats as experimental subjects,the rats were divided into normal group（n=8）and model group（n=40）randomly.CCl₄was used to induce chronic liver injury in rats of model group by intraperitoneal injection,establishing the hepatic fibrosis model.After successful modeling,the rats were randomly divided into normal group,model group,colchicine group（0.2 mg/kg）,low-dose Ganlong capsule group（62.5 mg/kg）,medium-dose Ganlong capsule group（125 mg/kg）and high-dose Ganlong capsule group（250mg/kg）,and 8 rats in each group.The normal group and model group were given normal saline by intragastric,and the drug group was given corresponding drugs by intragastric,both once a day.After 4 weeks of continuous drug administration,blood was collected from the rat abdominal aorta and the liver was collected.Detecting the serum biochemical indexes,serum fibrosis indexes and the Hyp content in liver tissue.HE staining and Masson staining were used to observe the tumor histopathological examination changes.ELISA was used to detect TNF-α,IL-1βand IL-6,to determine the effect of Ganlong capsule on the level of inflammation of hepatic fibrosis rats.Detecting the MDA and SOD of rat liver tissue and explore the effect of Ganlong Capsule on the level of oxidative stress in rats with hepatic fibrosis.Western blot（WB）and real time quantitative polymerase chain reaction（RT-q PCR）were used to detect the apoptosis related proteins Bax,Bcl-2,caspase 3 and the TGF-β₁/Smad pathway-related proteins TGF-β₁,Smad2,Smad3,Smad7 and their m RNA,to explore the effect of Ganlong Capsule on hepatocyte apoptosis and TGF-β₁/Smad pathway in rats with hepatic fibrosis.Immunocytochemical was used to detect the expressions ofα-SMA,COL-I,COL-Ⅲ,MMP-1 and MMP-2 in rat liver tissue,and RT-q PCR was used to detect the expressions ofα-SMA,COL-I,COL-Ⅲ,TIMP-1,MMP-2 and MMP-9 m RNA,to determine the effect of Ganlong Capsule on collagen expression and extracellular matrix degradation of hepatic fibrosis rats.Results:（1）Rats with hepatic fibrosis had messy fur,slow weight gain,severe liver adhesion,dark color and hard texture,and increased liver index（P<0.05）;After Ganlong Capsule treatment,the state of the rats was improved,the degree of liver adhesion was reduced,and the liver index gradually became normal;The serum levels of AST,ALT,ALP,HA,LN and PC-Ⅲ in the model group were significantly higher than those in the normal group（P<0.05）,while the ALB levels were lower than those in the normal group（P<0.05）.The content of Hyp in liver tissue was higher than that in the normal group（P<0.05）,and Ganlong Capsule could improve the pathological changes of the above indexes,suggesting that Ganlong Capsule has certain safety and can improve liver damage and reduce the degree of hepatic fibrosis;（2）In the pathological HE staining observation,Ganlong Capsule could reduce inflammatory cell infiltration and fiber bundles formation in the liver of hepatic fibrosis rats;In the Masson staining observation,Ganlong Capsule could improve the proliferation of hepatic collagen fibrous connective tissue in hepatic fibrosis rats;These results further suggest that Ganlong Capsule can reduce the degree of liver fibrosis by reducing collagen production;（3）ELISA results showed that the contents of TNF-α,IL-1βand IL-6 in the liver tissue of the rats in the model group were significantly higher than those in the normal group（P<0.05）.Ganlong Capsules can reduce TNF-α,IL-1βand IL-6（P<0.05）,indicating that Ganlong Capsule can reduce the production of pro-inflammatory cytokines;（4）Ganlong Capsule can reduce the content of MDA and enhance the activity of SOD in liver tissue of model group rats,indicating Ganlong Capsule can improve the peroxidative damage;（5）The results of western blotting showed that the model group had high expression of Bax,Caspase3,TGF-β₁,Smad2 and Smad3（P<0.05）,and low expression of Bcl-2 and Smad7（P<0.05）.Ganlong Capsule can down-regulate Bax,Caspase3,TGF-β₁,Smad2,Smad3 expression,up-regulated the expression of Bcl-2,Smad7.RT-q PCR experiment results showed the same trend.These show that Ganlong Capsule can inhibit hepatocyte apoptosis and TGF-β₁/Smad pathway;（6）The results of immunocytochemical staining showed thatα-SMA,COL-I,COL-Ⅲ were highly expressed in the model group（P<0.05）,MMP-1 and MMP-2 were lowly expressed（P<0.05）,and Ganlong Capsules can down-regulate the expressions ofα-SMA,COL-I and COL-Ⅲ,and up-regulate the expressions of MMP-1 and MMP-2;The results of RT-q PCR experiments showed thatα-SMA,COL-I,COL-Ⅲ,TIMP-1 m RNA relative expression increased in the model group（P<0.05）,MMP-2,MMP-9 m RNA relative expression decreased（P<0.05）,Ganlong Capsule can inhibit the expressionofα-SMA,COL-I,COL-Ⅲ,TIMP-1 m RNA and promote the expression of MMP-2 and MMP-9 m RNA.These show that Ganlong Capsule can inhibit the production of collagen and promote the degradation of extracellular matrix.Conclusions:（1）Ganlong Capsule has the effect of improving liver injury and reducing the degree of hepatic fibrosis,and has a certain dose-dependent effect;（2）The anti-hepatic fibrosis effect of Ganlong Capsule may be related to reducing the level of inflammation,reducing oxidative stress,inhibiting apoptosis of hepatocytes,inhibiting TGF-β₁/Smad pathway,inhibiting the synthesis and secretion of collagen,and promoting the degradation of extracellular matrix.