Mechanism Of Chlorogenic Acid Relieving Liver Lipid Deposition Induced By High Free Fatty Acids

Due to insufficient intake of dry matter during the early lactation period of perinatal cows,the demand for lactation increases,leading to negative energy balance and a large amount of nonesterified fatty acids entering the liver through the blood circulation.After incomplete oxidation and reesterification,a large amount of free fatty acids(FFA)are formed under the action of acyl Co A synthase.After entering the liver,a large amount of FFA are esterified to form triglycerides(TG),thus forming fatty liver.Orai1 is a porin of SOCE on the cell membrane.Our team found that Orai1 regulates endoplasmic reticulum stress and liver fat De novo synthesis synthesis pathway caused by high FFA in perinatal cows,and alleviates liver fat deposition.Chlorogenic acid(CGA)is mainly present in traditional Chinese medicine such as honeysuckle,hawthorn,Eucommia ulmoides,chrysanthemum,and coffee.Research has shown that CGA,as a substance that scavenges free radicals,has antioxidant and anti-inflammatory effects.However,the protective mechanism of CGA on fatty liver in dairy cows has not been fully studied.Therefore,this study aims to clarify how CGA regulates the protection of perinatal cows from fatty liver invasion.This provides experimental basis for the research and development of Chinese herbal excipients,and also lays the foundation for the development of drugs for treating fatty liver disease in dairy cows.This study used high FFA to treat primary calf liver cells to construct a liver lipid deposition model for research.We first screened the optimal concentration of CGA by measuring ROS in calf liver cells.Treat calf liver cells with 1.2 m M FFA for 12 hours,then add different concentrations(10 μg/m L、20 μg/m L、30 μg/m L CGA)and flow cytometry were used to detect ROS content.It was found that the concentration of CGA was at 20 μg/m L The protective effect is best at,which also indicates that CGA has good antioxidant properties.In order to study the effects of CGA on high FFA induced endoplasmic reticulum stress,mitochondrial autophagy and lipid synthesis in calf hepatocytes,the primary calf hepatocytes were treated with 1.2 m M FFA for 12 hours,and then added 20 μg/m L Detect protein levels of PERK,IRE1,GRP78,ATF6,CHOP,IKK,IKB,P65,and ORAI1 at a concentration of CGA for 12 hours.The results showed that FFA could up regulate the level of ORAI1 gene,and then lead to endoplasmic reticulum stress.CGA alleviates the stress of endoplasmic reticulum induced by high FFA and reduces the level of inflammatory markers.The expression of ORAII is closely related to the up-regulation of transcription factor NF-κB p65,and IKK,IKB,P65,and ORAI1 all showed a downward trend after the addition of CGA,suggesting that CGA inhibits endoplasmic reticulum stress caused by high FFA by mediating NF-κB/ORAI1.In order to investigate the effect of CGA on mitochondrial autophagy and lipid deposition in fatty liver cow liver cells,primary calf liver cells were treated with 1.2 m M FFA for 12 hours before adding 20 μg/m L After 12 hours of treatment with CGA,mitochondrial damage related molecules DRP1,MFN2,VDAC1,Cyp D,FIS1,mitochondrial autophagy indicators P62,Park,LC3,lipid synthesis related indicators SREBP and FFA protein expression,as well as lipid droplet morphology were detected.The results showed that compared with the control group,the protein expression of mitochondrial fission protein FIS1,mitochondrial autophagy indexes P62,Parkin,LC3,and lipid synthesis indexes FAS and SREBP were increased under the effect of high FFA.On the contrary,compared with FFA,the indices of mitochondrial division,mitochondrial autophagy and lipid synthesis showed a downward trend after CGA treatment.This indicates that CGA can effectively alleviate mitochondrial autophagy and lipid deposition in calf hepatocytes with high FFA.In order to study how CGA alleviates mitochondrial autophagy and lipid deposition in calf hepatocytes caused by high FFA,After 12 hours of treatment with 1.2 m M FFA,we added endoplasmic reticulum stress activator TG to act for 6 hours.ORAI1,endoplasmic reticulum stress indicators PERK,IRE1,GRP78,ATF6,CHOP,mitochondrial damage related molecules DRP1,MFN2,VDAC1,Cyp D,FIS1,mitochondrial autophagy indicators P62,Parkin,LC3,The results showed that TG could increase the number of lipid droplets,increase the endoplasmic reticulum stress,mitochondrial division,mitochondrial autophagy,and increase the protein expression of lipid synthesis indicators,on the contrary,CGA could alleviate these indicators compared with the FFA group.This indicates that CGA alleviates mitochondrial autophagy and lipid synthesis by mediating the down-regulation of endoplasmic reticulum stress level.In summary,CGA mediates NF-κB/ORAI1 inhibits endoplasmic reticulum stress caused by high FFA,thereby alleviating mitochondrial autophagy and lipid deposition.