Proteomics And Metabolomics Analysis Of Camel Urine And Its Protective Effect On CCl₄-induced Liver Injury In Mice

The liver is an important organ that metabolizes endogenous and exogenous substances.A variety of stimulating factors(such as viral infection,hepatotoxic substances or liver ischemia reperfusion,etc.)can cause acute hypohepatia,and long-term repeated damage can cause chronic liver damage further leads to liver fibrosis,cirrhosis,and even liver cancer and liver failure.Therefore,preventing and controlling the occurrence and development of liver injury has the great clinical significance for the treatment of liver disease.Camel urine has anti-cancer,antibacterial,anti-platelet and other activities,which can be used to treat and prevent cancer,liver damage,dysentery and other diseases.At present,there are few studies on the mechanism of anti-liver injury activity of camel urine.Therefore,this study firstly comprehensively demonstrated the composition and function of camel urine protein and metabolites through proteomics and metabolomics technology,to provided basic data for studying the mechanism of camel urine against liver injury and screening of anti-liver injury active substances.Secondly,use CCl₄to establish acute and chronic liver injury models in mice,through the use of serum biochemical indicators(ALT,AST and TBA),liver antioxidant stress indicators(MDA,GSH,SOD,CAT and GSH-px)and tissue Pathological analysis,combined with mouse liver transcriptomics to study the effect of camel urine on liver injury and its molecular mechanism.This study not only provides a theoretical basis for further revealing the anti-liver injury mechanism of camel urine,but also lays a foundation for further research and development of anti-liver injury active components and products in camel urine.The main findings are as follows:(1)A total of 827 camel urine proteins were identified according the LC-MS mass spectrometry.GO functional analysis revealed that 357 proteins are involved in the immune system process;27 proteins are involved in the detoxification process;27 proteins have antioxidant activity.The KEGG pathway analysis revealed that these proteins are mainly involved in the complement and coagulation cascade pathways(54 proteins),PI3K-Akt signaling pathway(47 proteins),and lysosomes(43 proteins).Some of these high-abundance proteins may participate in the immune response or have antibacterial activity,thereby ensuring the sterility of urine.At the same time,the 27 proteins involved in the detoxification process are mainly related to glutathione metabolism;in addition,there are some proteins with antioxidant activity such as(SOD,GSH,PRDX,GPX,etc.).The results showed that these proteins with antioxidant and detoxification functions may be contribute to the antioxidant activity of camel urine.(2)a total of 245 highly reliable metabolites were identified in camel urine,through the Non-targeted metabolome detection by LC-MS.These metabolites including amino acids,fatty acids,and small molecular carboxylic acids etc.Camel urine contains some biologically active metabolites,including:protocatechuic acid,resorcinol,cholic acid,dextroibuprofen and so on.According to the research,these compounds have anti-oxidant,anti-bacterial,anti-inflammatory,anti-spasmodic and anti-cancer effects.In addition,protocatechuic acid,betaine,perillyl alcohol and gingerone exhibited different extent of liver protection activity.The results indicate that these active metabolites may play a role in the anti-liver injury activity of camel urine.(3)In the acute liver injury study,through the detection of biochemical indicators and histopathological analysis of mice,it was found that the serum ALT,AST,TBA,inflammatory factors(IL-1?,TNF-?and IL-6)and liver MDA levels were significantly reduced,while liver SOD activity and GSH levels were significantly increased,suggesting that camel urine can inhibit the production of liver lipid peroxides and pro-inflammatory factors.Through transcriptome sequencing,it was found that 1790 differential genes between the camel uine group and the model group were significantly enriched in 41pathways including MAPK signaling pathway,Bile secretion,and glutathione.The m RNA expression of GPX7,Pdia5,Pdia4,Ggt,Anpep,Idh1 and Nat8 in the urine group increased significantly,suggesting that camel urine may promote the synthesis of glutathione and increase the antioxidant capacity of the mouse liver,thereby playing an anti-oxidative stress effect.In addition,the expression of 14 genes related to bile acid secretion was reversed in the camel urine group.It is suggested that camel urine may change the synthesis and transport of bile acids by inhibiting the excessive down-regulation of FXR and SHP and further regulating the expression of transporters,thereby protecting the liver.The results of the study indicate that camel urine may alleviate CCl4-induced liver damage and lipid peroxidation in mice by promoting glutathione synthesis and metabolism,regulating bile secretion and transport.(4)In the study of chronic liver injury,the results of biochemical indicators in mice showed that camel urine can effectively improve the disorder of ALT and AST levels caused by CCl₄,and improve the antioxidant capacity of liver tissue.At the same time,the results of histological analysis showed that the treatment of camel urine significantly reduced collagen deposition and cell apoptosis caused by CCl₄,improved liver histological morphology,reduced liver damage and prevented the formation of liver fibrosis.Transcriptomics analysis showed that 714 differential genes were significantly enriched in 14 pathways including Cell cycle,ECM-receptor interaction,and Glutathione metabolism.Camel urine significantly inhibited the m RNA expression of 8 liver ECM-receptor related genes and cell cycle related genes(cyclins A1,cyclins B1,cyclins B2 and CDK1).The results show that camel urine may inhibit the activation of HSC to block it in the G2/M phase,thereby reducing the accumulation of collagen in the liver tissue,and play a role in preventing and treating liver fibrosis.