Study On The Modulation Of Modified Yiguanjian On Sheep Hepatocyte Injury And Blood Metabolomics Analysis Of Sheep With Pregnancy Toxemia

Pregnancy toxemia(PT)is a common nutritional and metabolic disease in sheep at the end of pregnancy,characterized by ketoneemia and keturia.The disease mainly occurs in ewes with multiple lambs,causing huge economic losses to the sheep industry.However,the mechanism of sheep PT has not been clarified.Many studies have pointed out β-hydroxybutyric acid(BHBA)and non-esterified fatty acids(NEFAs),which are the main components of ketone bodies,play an important role in the occurrence of PT in sheep.The mechanism of action of high concentrations of BHBA and NEFAs on sheep hepatocytes is still unclear.In addition,many studies have shown that Traditional Chinese Medicine has various pharmacological effects such as improving immunity,regulating body metabolism,and resisting oxidative stress.Therefore,in this study,sheep primary hepatocytes were isolated and cultured,and the effects of BHBA and NEFAs on hepatocyte lipid metabolism,oxidative stress and inflammation pathways were studied respectively,and establish a cell injury model by stimulating sheep primary hepatocytes with BHBA and NEFAs to study the effect and mechanism of Modified Yiguanjian against BHBA and NEFAs-induced hepatocellular damage.Finally,high-resolution non-targeted metabolomics technology was used to detect the blood of PT sheep and normal sheep to obtain relevant biomarkers and explore the blood metabolism characteristics and potential pathogenesis of sheep PT.The results are as follows:1.Using collagenase two-step perfusion method to successfully isolate and culture sheep primary hepatocytes.The morphology of hepatocytes was complete and adhered well,with an average survival rate of 92%.PAS and CK-18 fluorescence staining were positive,and the cell purity was more than 95%,which can be used for further study.2.Sheep primary hepatocytes were treated with 0,0.6,1.2,1.8,2.4,4.8 mmol/L BHBA and 0,0.4,0.8,1.2,1.6,3.2 mmol/L NEFAs,respectively.The results showed that the viability of sheep hepatocytes was time-dose-dependent with the concentration of BHBA and NEFAs.As the concentrations of BHBA and NEFAs increased,the viability of sheep hepatocytes was significantly inhibited.Low concentrations of BHBA and NEFAs can activate AMPK pathway and Nrf2 pathway,while high concentrations of BHBA and NEFAs significantly increase the levels of VLDL,TG,T-CHOL,enhance the activities of ALT and AST,inhibit AMPK pathway and Nrf2 pathway,and activate NF-κB pathway.The above results indicate that low concentrations of BHBA and NEFAs can inhibit cellular lipid synthesis,promote lipid oxidation,and improve the antioxidant stress ability of hepatocytes by activating AMPK pathway and Nrf2 pathway.High concentrations of BHBA and NEFAs inhibit AMPK pathway,Nrf2 pathway and activate NF-κB pathway to promote lipid synthesis,inhibit lipid oxidation,and cause cellular inflammatory response.In addition,BHBA has a limited effect on hepatocyte lipolysis,but low concentrations of NEFAs can improve the lipolysis ability of hepatocytes,while high concentrations of NEFAs inhibit hepatocyte lipolysis,which ultimately leads to hepatocyte lipid metabolism disorders and damage.3.According to the diagnostic indicators of sheep PT and the effects of BHBA and NEFAs on sheep hepatocytes,it was determined that 1.8 mmol/L BHBA and 1.6 mmol/L NEFAs were used for the screening of hepatocyte injury models for 24 h.The control group,the BHBA treatment group,the NEFAs treatment group,and the BHBA+NEFAs co-treatment group were set up respectively,and the sheep hepatocytes were treated according to the above groups for 24 h.The results showed that BHBA and NEFAs could aggravate the damage to sheep hepatocytes,and was more suitable for simulating the internal environment of sheep PT hepatocytes.4.In the sheep hepatocyte injury model induced by the combination of BHBA and NEFAs,to assess the efficacy and mechanism of Modified Yiguanjian on cell injury model.The results showed that the safe drug concentration of Modified Yiguanjian on primary hepatocytes of sheep was within 2000 μg/m L.Compared with the model group,400,800,1200 μg/m L Modified Yiguanjian could reduce the levels of VLDL,TG,T-CHOL,inhibit the activities of ALT and AST,and activate AMPK pathway and Nrf2 pathway,but had no effect on the NF-κB pathway.The above results indicate that Modified Yiguanjian can inhibit the hepatocyte injury response caused by BHBA and NEFAs by regulating AMPK pathway and Nrf2 pathway,promote lipid decomposition,inhibit lipid synthesis,and improve the ability to resist oxidative stress.5.Collect the blood of PT sheep and normal sheep,5 samples in each group,use high-resolution non-targeted metabolomics technology to identify the differential metabolites of PT sheep and normal sheep blood,the differential metabolites of PT sheep were screened through univariate statistical and multivariate statistical such as PCA,PLS-DA and OPLS-DA for bioinformatics analysis.The results showed 25 significantly different metabolites,including linoleyl carnitine,glycine,betaine,3-hydroxybutyric acid,palmitic acid,and mannosterol,were obtained,of which 9 were up-regulated and16 were down-regulated.These substances involve fatty acids,amino acids,etc.KEGG enrichment analysis and pathway annotation analysis found that these significantly different metabolites are mainly involved in the adenosine triphosphate binding cassette transporter,2-oxycarbonyl acid metabolism and other metabolic pathways,which are located in metabolism and genetic information processing.Based on results of the above research,BHBA and NEFAs can regulate the lipid metabolism,oxidative stress and inflammation of sheep hepatocytes through AMPK pathway,NF-κB pathway and Nrf2 pathway.Modified Yiguanjian can promote lipid decomposition,inhibit lipid synthesis and enhances the ability of antioxidant stress by regulating AMPK pathway and Nrf2 pathway,so as to alleviate the damage of sheep hepatocytes induced by the combination of BHBA and NEFAs.Metabolomics reveals that the occurrence of sheep PT is mainly through the regulation of adenosine triphosphate binding cassette transporter,2-oxocarbonyl acid and other metabolic pathways,which affect the metabolic state and the functions of multiple biological systems.