Study On The Pathway Of Salvia Miltiorrhiza Polysaccharides In Alleviating Liver Injury Induced By Florfenicol In Chicks Based On Transcription And Proteomics

Florfenicol(FFC)is a common antibiotic for the treatment of animal bacterial diseases.Its use in broiler breeding has increased year by year,which can cause liver damage in chickens.Salvia miltiorrhiza polysaccharides(SMPs)is active ingredients with high content in Salvia miltiorrhiza,which has obvious liver protection effect and high clinical application value.Based on previous studies,this study planned to carefully screen and deeply dig the transcriptomic and proteomic data of chicken livers treated with FFC and SMPs.The differential genes and proteins were classified by functional enrichment to explore the mechanism of FFC induced liver injury and the way of SMPs to alleviate the liver toxicity of FFC.A total of 120 1-day-old healthy broilers were randomly divided into control group,FFC group and FFC+SMPs group,with 40 broilers in each group.Chicks in the control group were given tap water,and chicks in the FFC group were given the tap water containing 0.15 g/L FFC(the dosage recommended in the FFC solution instruction),and those in the FFC+SMPs group were given the tap water containing 0.15 g/L FFC and 5 g/L SMPs(the optimal dose was selected by the preliminary test).All three groups were given standard feed.The drugs were administered continuously for 5 days from the first day of the experiment.On the 6th day,the drugs were stopped,and 10 chicks was randomly selected from each group.The blood was collected from the heart and serum and liver were collected.Three chicks were randomly selected from each group and killed.Fresh liver tissues were aseptically extracted and placed in enzyme-free tube for transcriptome and proteome sequencing.Save the remaining sample and test the experimental index.QPCR was used to detect mRNA relative expression levels of differential genes in relevant signaling pathways screened from high-throughput sequencing data.PRM was used to detect the expression level of differential proteins in selected signaling pathways.Spectrophotometric was used to detect the levels of GSH-PX,GST,H2O2,TG,TC and ANDP in chick livers.Microplate was used to detect the levels of GSH in chick livers.TBA was used to detect the levels of MDA in chick livers.ELISA was used to detect the levels of CYP1A1,CYP2E1,NADPH,ROS,IL-2,IL-1β,TNF-α and IFN-y in chick livers.Immunohistochemistry was used to detect the expression levels of COX-2 and iNOS in chick livers.QPCR was used to detect the mRNA relative expression levels of MyD88 and NF-κB in chick livers.The results showed that(1)The hepatocytes in control group had normal morphology,complete structure and regular arrangement.In the FFC group,the hepatocytes were swollen,the cytoplasm was turbid,the cell boundary was blurred,and there were inflammatory cell infiltration and patchy necrosis areas.Compared with FFC group,The tissue and cell morphology and structure of tended to be complete and the number of inflammatory cells and necrotic areas decreased significantly in FFC+SMPs group.(2)Compared with control group,FFC exposure significantly increased AST and ALT levels in serum of chicks(P<0.01).Compared with FFC group,the levels of AST and ALT in the serum of chicks were significantly decreased in FFC+SMPs group(P<0.01).(3)Compared with the control group,1989 genes and 917 proteins with significantly differential expression were screened from the FFC group,of which 495 genes and 429 proteins were up-regulated and 1494 genes and 488 proteins were down regulated.At the same time,compared with FFC group,380 genes and 190 proteins significantly differentially expression were screened from FFC+SMPs group,of which 165 genes and 105 proteins were up-regulated and 215 genes and 85 proteins were down regulated.(4)According to GO function clustering of transcriptome and proteome,FFC and SMPs can regulate metabolic reaction,oxidative stress and inflammatory reaction in the livers of chicks.(5)The enrichment analysis of KEGG pathway showed that FFC and SMPs significantly changed the key factors in PPAR signaling pathway,drug metabolism-cytochrome P450 signaling pathway,glycine serine threonine metabolism signaling pathway,glutathione metabolism signaling pathway and phagosome signaling pathway.To sum up,by constructing transcriptome and proteome databases,digging sequencing data,and then using qPCR and PRM to verify the screened differential genes and proteins,we found that SMPs ameliorated FFC-induced lipid,drug and amino acid metabolism disorders in the liver of chicks by regulating PPAR signaling pathway,drug metabolismcytochrome P450 signaling pathway and glycine serine threonine metabolism signaling pathway.SMPs also alleviated oxidative stress in the liver of chicks induced by FFC by regulating glutathione signaling pathway.And SMPs alleviated inflammation and oxidative stress in the liver of chicks induced by FFC by regulating phagosome signaling pathway.The results of this experiment provided new ideas for the development of new drugs that could reduce the hepatotoxicity of antibiotics,which was of great significance to ensure the safety of animal food and increase animal welfare,and was expected to provide a theoretical basis for the clinical application of safe and effective Chinese herbal extracts.