Regulation Of Orai1 On High Fatty Acid-Induced Oxidative Stress And ER Stress In Calf Hepatocytes

Fatty liver disease is an important nutritional metabolic disease with high incidence in perinatal dairy cows,which reduces the production performance of dairy cows.Due to the rapid decrease in dry matter intake(DMI)of postpartum dairy cows,lactating energy expenditure increases.As a result,cows enter a state of negative energy balance.In order to meet the increased energy requirements of pregnancy and lactation,fat is mobilized to provide a large amount of non-esterified fatty acids(NEFAs),resulting in hyper NEFA bloodemia.Fatty liver is caused by a large amount of NEFA entering the liver and being esterified to form triacylglycerol(TAG),which accumulates in the liver.The links between plasma NEFA,fatty liver,and lipid peroxidation are all related to metabolic stress.The homeostasis of Ca2+ is the premise and basis for cells to maintain normal structure and function.Calcium ion entry(SOCE)induced by the calcium pool is the main channel for regulating the intracellular calcium ion concentration Ca2+.Orai1 is a porin of SOCE on the cell membrane.Orai1 regulates the production of liver fat in non-ruminant by mediating Ca2+ homeostasis.At present,it is unclear whether Orai1 mediates the formation of oxidative stress and endoplasmic reticulum stress in calf liver cells caused by negative energy balance.Therefore,this study aimed to determine whether fatty acids activate oxidative stress through Orai1,thereby inducing mitochondrial dysfunction and endoplasmic reticulum stress.In this study,a model of liver lipid deposition constructed by high NEFA stimulation of primary calf liver cells was used for the study.In this experiment,1.2 m M NEFA was used to stimulate primary hepatocytes for 0.5,1,3,6,9,and 12 h,respectively.It was found that NEFA significantly increased the expression level of Orai1 in hepatocytes at 0.5 h and significantly increased Ca2+ concentration at 1 h,oxidative stress-related indicators reduced glutathione(GSH)and superoxide dismutase(SOD)decreased significantly at 1 h,and malondialdehyde(MDA)and hydrogen peroxide(H2O2)increased significantly at 1 h.Endoplasmic reticulum stress-related molecules PERK,IRE1,GRP78,ATF6,and CHOP increased significantly at 6 h.It shows that NEFA activates Orai1 expression,increases intracellular Ca2+ concentration,and promotes oxidative stress and endoplasmic reticulum stress.After adding oxidative stress inhibitor NAC to hepatocytes,NEFA was added.The results showed that the expression of Orai1 and major ER stress proteins caused by NEFA could be blocked by NAC.It shows that endoplasmic reticulum stress can be caused by oxidative stress,and reducing oxidative stress can relieve endoplasmic reticulum stress.To study the role of Orai1 in oxidative stress,mitochondrial dysfunction and endoplasmic reticulum stress.Adding 1.2 m M NEFA to hepatocytes for 6 h,the results showed that NEFA could up-regulate Orai1 expression and the abundance of mitochondrial damage-related molecules VDAC1,CLPP and Cyp D.NEFA induces the transient opening of the mitochondrial permeability transition pore(m PTP)and increases the mitochondrial membrane potential.These changes are highly correlated with changes in mitochondrial morphology.In addition,NEFA induced the decrease of oxidative stress related indexes GSH and SOD,and the increase of MDA,H2O2 and ROS,increased the expression of ER stress related molecules PERK,IRE1,GRP78,ATF6 and CHOP.The calf hepatocytes were subsequently silenced or inhibited by Orai1,SOCE activator thapsigargin(Thapsigargin)or calcium ionophore Ionomycin(Ionomycin)to determine ORAI regulation.After 12 hours of silencing or inhibition of Orai1,NEFA was added to stimulate for 6 hours.It was found that Orai1 silencing or inhibition can eliminate oxidative stress,including increased GSH and SOD content,reduced MDA and H2O2 content,and reduced ROS production.The abundance of ER stress protein is down-regulated and mitochondrial function is restored.In addition,Thapsigargin or Ionomycin induced mitochondrial dysfunction and caused an increase in Orai1 expression.It showed that Orai1 can regulate oxidative stress,mitochondrial dysfunction and endoplasmic reticulum stress,the expression of Orai1 can be activated by NEFA,and then induce the occurrence of mitochondrial dysfunction,oxidative stress and endoplasmic reticulum stress.In summary,the high concentration of NEFA induced by negative energy balance stimulates liver cells,through Ca2+ signal transduction and Orai1 activation of oxidative stress and mitochondrial damage leading to endoplasmic reticulum stress.