肝纤维化肝阴虚证大鼠生物内涵的多组学研究OA

Objective To explore the biological significance of liver fibrosis(LF)with syndrome of liver yin deficiency in rats using metabolomics and transcriptomics,providing a scientific basis for a combined disease-syndrome model of LF.Methods According to body weight,30 male SD rats were divided into three groups(normal,model,and Zhibai Dihuang Pill groups),with 10 rats per group.The model and Zhibai Dihuang Pill groups received daily intragastric thyroxine suspension(80 mg/kg)and twice-weekly intraperitoneal injection of 50%CCl4 in olive oil solution(1.5 mL/kg)to induce LF with syndrome of liver yin deficiency.In the normal group,an equal volume of distilled water was administered by gavage,and an equal volume of olive oil solution was given by intraperitoneal injection.The Zhibai Dihuang Pill group also received daily Zhibai Dihuang Pill suspension(408 mg/kg).Experimental procedures,including modeling and drug administration,were performed over two weeks.Liver weight and organ index were measured,and serum levels of alanine amino-transferase(ALT),aspartate transferase(AST),total bilirubin(TBIL),total bile acid(TBA),γ-glutamyltransferase(γ-GT),alkaline phosphatase(ALP),laminin(LN),hyaluronic acid(HA),transforming growth factor-β1(TGF-β1),and matrix metalloproteinase-2(MMP-2)were assessed.In liver tissue,tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6,IL-10,malondialdehyde(MDA),reduced glutathione(GSH),and the activities of catalase(CAT)and total superoxide dismutase(T-SOD)were evaluated.Hematoxylin and eosin(HE)staining was performed to examine liver damage,whereas Masson and Sirius red stainings were used to observe collagen fiber formation and distribution.Immunohistochemical staining was used to detect the positive expression of alpha-smooth muscle actin(α-SMA)in liver tissue.External indicators of syndrome of liver yin deficiency,including body weight,24 h food intake,24 h water intake,rectal temperature,tongue surface moisture content,and overall physical sign score(mental state,fur,stool,and urine scores),were assessed.Serum levels of cyclic adenosine monophosphate(cAMP),cyclic guanosine monophosphate(cGMP),triiodothyronine(T3),and thyroxine(T4)were measured,and the cAMP/cGMP ratio was calculated.The correlation analysis was performed between the tongue surface moisture content,rectal temperature and the related indicators of yin deficiency(serum cAMP,cGMP,T3,and T4)in the normal and model groups.Metabolomics and transcriptomics identified differential metabolites,expressed gene changes,and pathways in liver tissue between the normal and model groups,and the combined analysis of omics was performed.The adenosine monophosphate(AMP)content and the mRNA expression of aquaporin 7(Aqp7)in liver tissue were detected in the normal and model groups,and the correlation between them and the related indicators of yin deficiency was analyzed.Results The model group had significantly higher liver organ index and serum levels of ALT,AST,TBIL,TBA,γ-GT,ALP,LN,HA,TGF-β1,cAMP,T3,T4,and cAMP/cGMP ratio,and lower MMP-2 and cGMP levels compared to the normal group(P<0.05).In liver tissue,the contents of IL-10 and GSH and the activities of CAT and T-SOD decreased,whereas TNF-α,IL-1β,IL-6,and MDA levels increased(P<0.05).HE staining showed hepatocyte steatosis,ballooning degeneration,and fibrous tissue hyperplasia.Masson and Sirius red stainings indicated increased blue and red collagen fibers,with a significant rise in collagen and α-SMA area percentages in liver tissue(P<0.05).The 24 h water intake and rectal temperature rose,along with an increase in physical sign,mental state,fur condition,and urine scores;however,body weight,tongue surface moisture content,and stool score decreased(P<0.05).Compared with the model group,the Zhibai Dihuang Pill group reversed these indicators,except for body weight(P<0.05).In the normal and model groups,the tongue surface moisture content was very strongly negatively correlated with cAMP,T3,and T4,and very strongly positively correlated with cGMP(P<0.05);the rectal temperature was very strongly positively correlated with cAMP,T3,T4,and very strongly negatively correlated with cGMP(P<0.05).Liver tissue metabolomic analysis identified 247 significant metabolites primarily associated with the cGMP-protein kinase G and cAMP signaling pathways.Transcriptomic analysis identified 2,568 differentially expressed genes linked to transcriptional pathways,such as cytokine-cytokine receptor and extracellular matrix receptor interactions.Integrated omics analysis revealed key signaling pathways,including lipolysis regulation in adipocytes,ascorbate and aldarate metabolism,and purine metabolism.Experimental verification results showed that compared with the normal group,the AMP content and Aqp7 mRNA expression in the liver tissue of the model group were increased(P<0.05).The AMP content was very strongly positively correlated with cAMP,T3,and T4,and very strongly negatively correlated with cGMP(P<0.05).In contrast,Aqp7 was strongly positively correlated with cAMP,T3,and T4,and strongly negatively correlated with cGMP(P<0.05).Conclusion This study successfully developed an animal model of LF with syndrome of liver yin deficiency.Multi-omics verified that the pathological changes and syndrome manifestations in this model were consistent with clinical findings.Additionally,the study explains the syndrome and pathological mechanisms at metabolite and genetic levels,providing additional biological evidence for using this integrated disease-syndrome animal model.