Study On The Mechanism Of Action Of Crocin Ⅰ Against Intrahepatic Cholestatic Liver Injury

Objective:（1）By establishing a rat model of intrahepatic cholestasis（IC）liver injury induced byα-naphthalene isothiocyanate（ANIT）,the efficacy of crocin I in anti-cholestatic liver injury was evaluated.（2）Using the non-targeted metabolomics technology of liquid chromatography-mass spectrometry（LC-MS）to study the intervention effect of crocin I on the serum metabolism of IC-type liver injury rats,and to explore potential metabolites;using transcriptomics technology（RNA-seq）to study the information of crocin I on gene expression in rats with type IC liver injury.Using the combined analysis method of metabolomics and transcriptomics,the mechanism of action of crocinⅠagainst liver injury was further explored.Method:（1）Pharmacodynamic study:48 SD rats were randomly divided into Control group,ANIT group,UDCA group,CR-L group,CR-M group and CR-H group.The representative indicators of liver injury,the levels of inflammatory factors,the oxidative stress indicators of liver tissue in the serum of each group were detected,and the pathological sections were observed to evaluate the efficacy of crocin I,and to screen out the optimal dose of crocin I.group for follow-up research.（2）Metabolomics research:Metabolomics analysis based on UPLC-Q-Exactive-MS method is to analyze the mechanism of crocin I against type IC liver injury from the level of biological small molecules.Through multivariate statistical analysis and KEGG metabolic pathway enrichment analysis of serum differential metabolites in the ANIT model group and the crocin I group with the best efficacy,the potential biomarkers that play a role in disease resistance and the significant metabolic pathways involved were identified..（3）Transcriptomic research:The blank group,the model group,and the optimal crocin I group were subjected to transcriptional sequencing analysis from the gene level to explore the mechanism of crocin I against type IC liver injury.First,the quality control analysis of the sequencing data was carried out,and the sequence comparison analysis of the quality control data was carried out.Finally,the GO and KEGG databases were used to perform functional annotation and pathway enrichment of the screened differential genes.（4）Combined metabolomics and transcriptomics analysis:First,the data obtained by metabolomics and the data obtained by transcriptomics are obtained by hypergeometric distribution algorithm to obtain significantly enriched pathways,and the genes and potential biomarkers on the common pathways are correlated Sexual heat map analysis and correlation network analysis were used to obtain key genes.Finally,molecular docking and Western blot were used to analyze and verify the proteins corresponding to the key genes,and further elaborate the action pathway of crocin I in disease resistance and the regulation ability of related proteins,and obtain the final protective mechanism.Result:（1）Pharmacodynamic analysis showed that:crocin I dose groups can reduce serum AST,ALT,TBA,TBIL,DBIL levels and MDA levels in liver tissue homogenate,increase SOD and GSH levels in liver tissue homogenate,The levels of serum TNF-α,IL-6 and IL-1βwere reduced,and histological lesions were improved.It was concluded that the crocin I high-dose（CR-H）group had the best therapeutic effect,so the high-dose crocin I was selected for further omics research and analysis.（2）After non-targeted metabolomics analysis,225 significantly different metabolites were identified between the ANIT model group and the Control group,and 194 significantly different metabolites were identified between the ANIT model group and the CR-H group.To explore possible metabolic pathways,we used the Metabo Analyst 5.0 online tool to analyze the differential metabolites between the ANIT model group and the CR-H treatment group.The differential metabolites between the CR-H group and the ANIT model group involved a total of 16 metabolic pathways,including glycerophospholipid metabolism,histidine metabolism,steroid hormone biosynthesis,tryptophan metabolism,linoleic acid metabolism,porphyrin and chlorophyll metabolism,α-Linolenic acid metabolism,β-alanine metabolism,glutathione metabolism,cytochrome P450 metabolism of xenobiotics,arachidonic acid metabolism,primary bile acid biosynthesis,purine metabolism,etc.Through venn analysis,it was found that 76 metabolites changed significantly before and after modeling,and 40 metabolites showed a reversal trend after treatment with crocin I,indicating that 40 metabolites may be potential biomarkers thing.（3）Transcriptomic studies showed that after crocin I intervention treatment,compared with the ANIT model group,473 differentially expressed genes（DEGs）were significantly changed,153 were significantly up-regulated and 320 were significantly down-regulated.The differential genes in the Crocin I＿vs＿ANIT group were enriched by GO,and were significantly enriched in the functions of inflammation,response to fatty acids,and positive regulation of leukocyte migration（Padjust<0.05）;in the KEGG pathway enrichment analysis,it was found that most genes were enriched To retinol metabolism,calcium signaling pathway,PPAR signaling pathway,circadian rhythm,chemokine signaling pathway,arachidonic acid metabolism,bile secretion,primary bile acid biosynthesis,etc.（4）Combined analysis of 76 differential metabolites obtained by metabolomics and differential genes obtained by transcriptomics to obtain common pathways.Differential genes and metabolites were concentrated in 2 pathways related to cholestatic diseases,namely bile secretion and steroid hormone biosynthesis pathway.Correlation heat map and correlation network analysis showed that four genes,Hmgcr,Abcg5,Sult2a1,and Ugt2b7,had high correlation with metabolites and were identified as key genes.Molecular docking showed that the binding energies between the small molecules and the target protein were all less than-5 kcal·mol^-1,indicating that the ligand-receptor binding activity was good.Western blot results showed that compared with the control group,the Hmgcr protein level in the liver tissue of the ANIT group was significantly increased（P<0.05）,while the Abcg5 and Sult2a1protein levels were significantly decreased（P<0.05）.,Hmgcr protein level in high-dose group was significantly decreased（P<0.05）,Abcg5 protein level was significantly increased in middle and high-dose groups of crocin I（P<0.05）,and Sult2a1 protein was significantly increased in high-dose group（P<0.05）;Ugt2b7protein was not significant.Three proteins,Hmgcr,Abcg5,and Sult2a1,all exist in the bile secretion pathway,indicating that crocin I can play an anti-IC-type liver injury by regulating the expression of Hmgcr,Abcg5,and Sult2a1.Conclusion:（1）Crocin I（90 mg/kg）has a significant therapeutic effect on intrahepatic cholestatic liver injury,and has the effect of enhancing the body’s ability to resist oxidative stress and inhibiting the secretion of inflammatory factors,fully demonstrating that crocinⅠhas potential research value in the treatment of type IC liver injury.（2）The combined analysis of metabolomics and transcriptomics showed that the mechanism of action of crocin I against cholestatic liver injury is related to the regulation of the biosynthesis of bile acids and bilirubin in the bile secretion pathway,and the regulation of Hmgcr,Abcg5 and Sult2a1.The protein expression level is related to play a protective therapeutic effect.