Study On The Anti Hepatic Fibrosis Effect And Mechanism Of Ganlong Capsule Based On Metabonomics

Objectives:Based on carbon tetrachloride（CCl₄）liver fibrosis animal model,the protective effect and mechanism of Ganlong capsule on liver fibrosis rats were investigated at the basic pharmacodynamic level and metabolomics level.Methods:1.Male SD rats were used as experimental subjects,and CCl₄was injected intraperitoneally to establish a liver fibrosis model.Different doses of Ganlong capsules and colchicine were used to intervene for 4 weeks.The general state and body weight changes of the experimental rats were recorded during the experiment.2.The main organs of rats were extracted and the organ index was calculated.3.Take pictures to record the appearance of the liver,and investigate the morphological changes of the liver.4.The left hepatic lobe of the same part was taken and trimmed for HE and Masson staining to observe the effect of experimental drugs on the histopathological changes of liver tissue in rats with hepatic fibrosis,and to score the liver fibrosis.5.Serological index detection:serum aspartate aminotransferase（AST）and alanine aminotransferase（ALT）levels by the microplate reader to reflect the liver function of the experimental rats.In addition,enzyme-linked immunosorbent assay（ELISA）was used to detect the four levels of liver fibrosis in serum to reflect the degree of liver fibrosis in experimental animals.6.Using LC-MS technology,combined with multivariate statistical analysis.Screening the potential biomarkers of anti-hepatic fibrosis by Ganlong Capsule in the liver tissue,urine and serum samples of experimental rats,and the metabolic pathway enrichment analysis on the Metaboanalyst 5.0 website platform to elucidate its metabolomic mechanism of anti-hepatic fibrosis.Results:1.The general state of the experimental animals showed:compared with the control group,the general state of the rats after CCl₄modeling became worse,mainly as follows:the hair was messy,easy to fall off,and the animal response sensitivity was reduced;After the corresponding test drug intervention,the general state of the rats in each drug group improved to different degrees compared with the model group,and tended to be normal.The results of body weight change trend showed that there was no significant difference in the body weight changes of the rats in each group during the experiment.2.The results of organ index showed that:persistent liver injury factors caused the liver index of the rats in the model group to increase significantly compared with the control group（P<0.01）.After administration of the corresponding test drugs,the liver index of the rats in each drug group was significantly lower than that of the liver fibrosis model group（P<0.01）,and tended to the control group,indicating that the experimental drug Ganlong Capsule and the positive drug colchicine had a certain protective effect on the liver of rats.Intraperitoneal injection of CCl₄led to a significant increase in the spleen index of rats（P<0.01）,and each dose of Ganlong Capsule could significantly reduce the increase in the spleen index of rats caused by CCl₄（P<0.01）,which may be related to the protective effect of Ganlong Capsule on the spleen of experimental rats.In addition,high-dose Ganlong Capsule can significantly increase（P<0.01）the thymus index in rats with liver fibrosis,which may be related to the enhanced immune function.3.The morphological results of liver appearance showed that:compared with the control group,the liver of the liver fibrosis model group rats became darker in color,hard and rough in texture,with many yellow fibers and serious adhesion.Compared with the model group,the liver color and luster of the rats in each experimental drug groups tended to be ruddy,the texture tended to be soft,and the yellow fibers and adhesions were reduced to varying degrees,with Ganlong Capsule high dose group and colchicine group improving the most.4.The histopathological results of the liver showed that:compared with the control group,the liver tissue staining of the rats showed obvious degeneration and inflammatory infiltration of the liver cells in the model group,and a large number of fibrous bundles and fibrous septa centered on the portal area appeared.The lobule structure is disordered,with occasional false lobules.Compared with the model group,after the effect of corresponding drugs,the proliferation of collagen fibers in the liver tissue was improved to varying degrees,the lobule structure was normal,and the structural degeneration and inflammatory infiltration of the liver cells were significantly reduced.The results of liver fibrosis score showed that the fibrosis score of the control group was 0,and the fibrosis score was significantly increased after CCl₄modeling（P<0.01）,after the intervention of the corresponding test drugs,the liver fibrosis scores in each drug group were reduced to varying degrees,among which Ganlong Capsule with high dose was the best（P<0.01）.5.Serological index results showed that:the levels of serum liver function index AST and ALT in liver fibrosis model rats were significantly increased after CCl₄modeling（P<0.01）,and after the intervention of the corresponding experimental drugs in each group,the levels of AST and ALT in the serum of the experimental rats all significantly decreased（P<0.01）,and tended to control,indicating that the experimental drugs all had a certian anti-CCl₄liver injury effect.In addition,compared with control group,Long-term stimulation of CCl₄can significantly increase the levels of HA,LN,PC-III and COL-IV in the serum of four items of liver fibrosis in rats（P<0.01）.After the experimental drug intervention,the serum levels of HA,LN,PC-Ⅲ,COL-IV in each administration groups exhibited varying degrees of reduction（P<0.05 or P<0.01）,among which the colchicine group and the Ganlong capsule high-dose group were the closest to the control group（P<0.01）,that may be related to the anti-hepatic fibrosis effect of the experimental drug.6.The results of metabolomics showed that:After PCA and OPLS-DA analysis:the model group was clearly separated from the control group,and the Ganlong capsule administrated group was also clearly separated from the model group and trended toward the control group,which proved the therapeutic effect of Ganlong Capsule on rats with liver fibrosis from the perspective of metabolomics.A total of 43potential biomarkers,including phosphatidylethanolamine,phosphatidylcholine,（R）-3-hydroxybutyric acid,gamma-Aminobutyricacid,deoxycytidine and uracil were screened out in liver tissue samples,21 potential biomarkers,including phosphatidylethanolamine,phosphatidylcholine,thymidine,and thymine were screened from serum samples,20 potential biomarkers including choline,betaine aldehyde and L-leucine were screened from urine samples,and found that the anti fibrotic effect of Ganlong capsule is mainly reflected in the improvement of disturbed glycerophospholipid metabolism,butyrate metabolism,pyrimidine metabolism,linoleic acid metabolism,Glycine,serine and threonine metabolism and Valine,leucine and isoleucine biosynthesis.Conclusions:1.Ganlong Capsule can improve the liver histopathological changes,the liver function and the degree of liver fibrosis of CCl₄-induced hepatic fibrosis in rats.2.Ganlong Capsules may exert anti-hepatic fibrosis effects by improving the imbalance of glycerophospholipid metabolism,linoleic acid metabolism,butyric acid metabolism,pyrimidine metabolism,Glycine,glycine,serine and threonine metabolism,and valine,leucine and isoleucine biosynthesis of CCl₄-induced hepatic fibrosis in rats.