Mechanism Of Mitofusion 2 On Regulating Mitochondrial Dysfunction And Oxidative Stress In Mammary Gland Of Ketotic Dairy Cows

Ketosis is a common metabolic disease in dairy cows during perinatal period,could cause decrease of milk yield and milk quality.Due to the insufficient energy intake and the increased energy demand,the perinatal cows are in a state of negative energy balance(NEB).NEB can lead to fat mobilization in dairy cows,increase the concentration of NEFA in the blood,and produce ketone body in the liver,causing the occurrence of ketosis in dairy cows.About half of the NEFA was absorbed by mammary epithelial cells.The high concentration of NEFA has lipotoxicity,which could lead to oxidative stress and damage of mitochondrial function in mammary epithelial cells,causes reduction of milk yield and quality.Mitofusion 2(MFN2)is a protein located on mitochondrial outer membrane,and plays an important role on regulating mitochondrial function.But its rule in mammary tissue mitochondrial dysfunction and oxidative stress is not clear.Therefore,the purpose of this experiment is to explore the effect of high concentration of NEFA on mitochondrial function and oxidative stress in mammary gland,and the regulatory mechanism of MFN2 in mitochondrial function and oxidative stress.In this study,we selected healthy cows(n = 15)and clinically ketotic cows(n =15)and collected blood and milk samples.The blood oxidative indicators and antioxidant indicators were measured and the milk composition was analyzed.Compared with healthy cows,ketotic cows have higher serum concentration of NEFA and BHBA,higher levels of MDA content and lower activities of SOD,GSH-Px and CAT.Milk composition shows decreased milk protein and increased milk fat in ketotic cows.The results indicate that clinical ketosis cows exhibited reduced lactation capacity and systemic oxidative stress.For in vitro study,MAC-T cells were cultured and treated with 0,0.6 and 1.2 mmol/L NEFA.The results showed that with the increase of NEFA concentration,the protein and gene expression of MFN2 decreased,the protein expression of mitochondrial function-related protein PGC-1αand TFAM decreased,the level of MDA and ROS increased,and the activity of antioxidant enzymes decreased.The results showed that high concentration of NEFA could inhibit the expression of MFN2 in mammary epithelial cells,damage mitochondrial function and induce oxidative stress.Silencing the expression of MFN2 in MAC-T cells,and added 1.2 mmol/L NEFA.The results showed that silencing MFN2 inhibited the expression of mitochondrial function-related protein,increased oxidative index and decreased the activity of antioxidant enzymes.The results showed that silencing MFN2 could inhibit mitochondrial function and aggravate cellular oxidative stress.Overexpressed MFN2 in MAC-T cells,and added 1.2 mmol/L NEFA.The results showed that overexpression of MFN2 could promote the expression of mitochondrial function-related protein,reduce the MDA content and increase the activity of antioxidant enzymes.The results showed that high expression of MFN2 repaired mitochondrial function and alleviate oxidative stress.MAC-T cells were treated with 1.2 mmol/L NEFA and 10 μmol/L α-tocopherol.The results showed that after treated with α-tocopherol,the MDA content was lower,and the activities of antioxidant enzymes increased.In the meantime,α-tocopherol could alleviate the decrease of β-casein protein abundance caused by NEFA,restore the phosphorylation levels of mTOR and S6K1,and reduce the protein expression of SREBP1 and TG content.The results indicated that vitamin E analogs could reduce the oxidative stress and alleviate the disorder of milk fat and milk protein synthesis caused by high levels of NEFA in mammary epithelial cells.In conclusion,high concentration of NEFA could cause mitochondrial dysfunction and induce oxidative stress by inhibiting MFN2 expression in mammary epithelial cells of ketotic cows;vitamin E could correct disorders in milk fat and protein synthesis induced by high concentration of NEFA by suppressing oxidative stress.This study revealed the regulatory role of MFN2 in mitochondrial dysfunction and oxidative stress in the mammary gland of ketosis cows,demonstrated that vitamin E analogs could prevent and treat oxidative stress and decreased lactation performance in ketosis cows.This study provides data support for preventing and treating oxidative damage to mammary in perinatal cows,improving milk yield and quality.