Study On The Mechanism Of Action And Differential Metabolite Analysis Of Piper Longum Extract On CCl₄ Induced Hepatic Fibrosis In Rats

Objective:Exploring the mechanism of action of extract of Piper longum on carbon tetrachloride（CCl₄）induced hepatic fibrosis in rats.Using metabolomics to detect changes in liver tissue metabolites and exploring the efficacy of Piper longum extract on inhibiting liver fibrosis at the metabolomics level.Methods:（1）Sixty SD male rats were randomly divided into 5 groups:blank group,model group,low,medium,and high dose groups of Piper longum extract.The model group and administration group were modeled with 50%CCl₄peanut oil solution.At the same time,the blank group and model group were gavaged with 0.5%carboxymethylcellulose sodium（CMC-Na）,the treatment groups were gavaged with 0.5%CMC Na dissolved drug.After 8 weeks of administration,anesthesia was performed and blood was collected from the abdominal aorta to detect the content of aspartate transaminase（AST）and alanine aminotransferase（ALT）.The liver coefficient was calculated,HE and Masson staining was performed after liver tissue taked and the degree of liver fibrosis was observed under microscope.Immunohistochemical method was used to detect the expression of Collagen I,NLRP3,and Caspase-1 proteins in liver tissue.The tissue total RNA was extracted and q-PCR method was used to detectα-smooth muscle actin（α-SMA）、CollagenⅠ、NLRP3、Caspase-1、GSDMD、IL-1βand IL-18 m RNA content.Tissue protein was extracted and detected by Western blot method to estimate protein expression level ofα-SMA、CollagenⅠ、NLRP3、Caspase-1、GSDMD、IL-1βand IL-18.（2）Through metabonomics research,LC-MS technology combined with multivariate statistical analysis was used to screen differential metabolites from liver tissue samples of rats in each group.Pathway enrichment analysis was conducted using differential metabolites on the Metabonalyst 5.0website to further explain that the extract of Piper longum can exert a molecular mechanism of anti liver fibrosis by affecting metabolite changes.Results:（1）The liver tissue in Blank group was bright red in color,soft and smooth in texture;In Model group,the liver tissue was yellow,rough and grainy;The liver tissue in each treatment group was dark red with varying degrees of roughness.Compared with the Blank group,the liver coefficient in the Model group was significantly increased（P<0.05）,while the serum AST and ALT levels were significantly increased（P<0.001）.Compared with the Model group,the liver coefficient in the High dose group was decreased（P<0.05）,while the serum AST and ALT levels in the low,medium,and high dose groups were significantly decreased（P<0.001）.The HE staining results showed that in the Blank group,hepatocytes were neatly arranged,while in the Model group,hepatocytes were necrotic and disorderly arranged,with a large number of inflammatory cells infiltrating and forming pseudolobules.After treatment with the medication group,the pathological condition of liver fibrosis was improved and recovered.Masson staining showed that a large number of blue stained collagen fibers appeared in the Model group,with a significant increase in fiber content compared to the Blank group（P<0.001）,while collagen deposition significantly decreased in the low,medium,and high dose groups（P<0.001）,and the pathological status of liver tissue tended to be normal.The immunohistochemical results showed that compared with the Blank group,the positive expressions of collagen I,NLRP3,and Caspase-1 in the model group significantly increased（P<0.01）,compared with the model group,reduced expression of collagen I,NLRP3,and Caspase-1 in the low,medium,and high dose groups（P<0.05）.Compared with the blank group,the Q-PCR results showed that in the model group the m RNA levels ofα-SMA,Collagen I,NLRP3,Caspase-1,GSDMD,IL-1βand IL-18 were significantly upregulated（P<0.01）;Compared with the model group,in the treatment group the expression level ofα-SMA,collagen I,NLRP3,Caspase-1,GSDMD,IL-1βand IL-18 m RNA was significantly downregulated.（P<0.05）Western blot results showed that compared with the blank group,α-SMA,CollagenΙ,NLRP3,Caspase-1,GSDMD,IL-1βAnd IL-18 in the model group protein expression significantly increased（P<0.01）.Compared to the model group,each treatment groupα-SMA、CollagenΙ、NLRP3、Caspase-1、GSDMD、IL-1βAnd IL-18 protein expression were decreased to varying degrees（P<0.05）.（2）Through PCA analysis,QC samples were closely clustered,the performance of the detection system was stable,and the feasibility of the results is high.It is observed that the samples in the blank group and the model group were significantly separated,indicating significant differences between the groups,while the samples in the model group and each treatment group were not significantly separated.Therefore,PLS-DA was used for analysis,and the results showed that the differences between the model group and the blank group,as well as between the low dose group and the model group,were more significant,and the low dose group was more inclined to the blank group,the anti HF effect of Piper longum extract was verified from the perspective of metabolomics.According to ratio>=2,ratio<=1/2,q value<0.05 and VIP≥1,32 potential differential metabolites were screened from liver tissue,including cystine,cholic acid,and xanthosine.Pathway enrichment analysis found that anti HF reason of the extract of Piper longum L.maybe operated though improving metabolic disorders such as purine metabolism,glutathione metabolism,and primary bile acid biosynthesis.Conclusions:Piper longum extract can reduce the pathological characteristics of CCl₄induced liver fibrosis in rats and reduce excessive collagen deposition;By reducing the levels of AST and ALT in serum,α-SMA and CollagenΙm RNA and protein expression in the liver tissue to alleviate HF,while downregulating the pyroptosis related factors NLRP3,Caspase-1,GSDMD,and IL-1βand IL-18 m RNA and protein expression levels to play an anti HF role.The metabonomics level indicates that the anti HF effect of Piper longum extract is reflected in its ability to regulate purine metabolism,glutathione metabolism,and primary bile acid biosynthesis in rats with liver fibrosis induced by CCl₄.