The Regulatory Mechanism Of PINK1-mediated Mitophagy On Mammary Gland Inflammatory Injury In Ketotic Cows

Ketosis is one of the common nutritional and metabolic diseases of high-yielding dairy cows in the transition period,which causes inflammatory injury to the mammary gland of dairy cows,resulting in decreased milk production and milk quality and causing significant economic losses to the dairy farming industry.Due to severe metabolic stress in ketotic cows,the mitochondria of mammary epithelial cells are damaged,which in turn produces excess mitochondrial reactive oxygen species(Mito-ROS)and activates NOD-like receptor protein 3(NLRP3)-mediated inflammatory signaling pathway,causing Inflammatory injury to mammary gland tissue.PTEN-mediated putative kinase 1(PINK1)is a protein kinase that monitors mitochondrial status,and its mitophagy-mediated mitophagy controls mitochondrial mass and Mito-ROS production by degrading damaged mitochondria,thereby maintaining mitochondrial homeostasis.Mitophagy is negatively regulated by inflammatory response,but the mechanism of mitophagy in mammary gland inflammatory injury in ketotic cows is still unclear.Therefore,this study aimed to clarify the regulatory mechanism of PINK1-mediated mitophagy on mammary gland inflammatory injury in ketotic cows.In vivo,the mammary gland tissue samples of healthy cows,cows with subclinical and clinical ketosis were collected to detect the expression changes of NLRP3 inflammatory signaling pathway,mitochondrial function and mitophagyrelated proteins in mammary gland tissue.The results showed that the serum concentrations of non-esterified fatty acids(NEFA)and β-hydroxybutyric acid(BHBA)of ketotic cows were significantly higher than those of healthy cows,while the concentration of Glucose(GLU)was significantly lower than that of healthy cows,indicating that the ketotic cows have severe metabolic stress.Compared with healthy cows,the NLRP3 inflammatory signaling pathway-related molecules in the mammary gland tissue of cows with ketosis: the protein expression of NLRP3 and CleavedCaspase-1 was significantly increased;the protein and m RNA expression levels of interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were significantly increased,indicating that inflammatory responses and hyperactivation of the NLRP3 inflammasome occur in mammary gland tissue of SCK and CK cows.Compared with healthy cows,mitochondrial oxidative phosphorylation(OXPHOS)complexes in mammary gland tissue of SCK and CK cows: except for the insignificant increase of COⅢ,the protein expressions of COⅠ,COⅡ,COⅢ,COⅣand COⅤ were significantly increased in the mammary gland tissue of SCK cows,indicating that the mitochondrial function in the mammary gland tissue of SCK cows was enhanced;while the protein expressions of COⅠ,COⅡ,COⅢ,COⅣ and COV in the mammary gland tissue of CK cows were significantly decreased,suggesting that mitochondrial dysfunction occurred in the mammary gland tissue of CK cows.Compared with healthy cows,the protein expressions of mitophagy-related molecules: PINK1,Parkin and LC3-II in mammary gland tissue of ketosis cows were significantly increased,while the protein expression of P62 was significantly decreased.In this study,the bovine mammary epithelial cell MAC-T was cultured in vitro,and 1 μg/m L lipopolysaccharide(LPS)was used to stimulate MAC-T cells to construct a mammary epithelial cell inflammation model.After 0 h,3 h,6 h,9 h and12 h of LPS treatment,the results showed that the protein expression levels of mitochondrial oxidative phosphorylation complexes COⅠ,COⅡ,COⅢ,COⅣ and COⅤ in dairy cow mammary epithelial cells were significantly decreased in the 6 h,9 h and 12 h groups.The production of Mito-ROS increased after 9 h of LPS treatment.After LPS treatment,the protein expressions of NLRP3 inflammatory signaling pathway-related molecules: NLRP3 and Caspase-1,as well as the protein and m RNA expression levels of pro-inflammatory factors IL-1β,IL-6 and TNF-α were significantly increased in the 6 h,9 h and 12 h groups.The immunofluorescence intensity of NLRP3 significantly increased after LPS treatment for 9 h.After 0 h,3h,6 h,9 h and 12 h of LPS treatment,the protein expression level of mitophagyrelated molecules PINK1,Parkin and LC3-Ⅱ increased significantly,while the protein expression of P62 decreased significantly.The fluorescence intensity of PINK1/Parkin co-localized with mitochondrial outer membrane translocase 20(TOMM20)increased after LPS treatment for 9 h.The numbers of autophagosomes(yellow puncta)and autophagolysosomes(red puncta)increased after transfection with recombinant adenovirus MRFP-GFP-LC3 for 48 h and adding LPS for 9 h.Overall,these data demonstrated that LPS induces mitochondrial dysfunction in dairy cow mammary epithelial cells,which in turn leads to an inflammatory response and activates mitophagy.To investigate the regulatory mechanism of PINK1-mediated mitophagy on the inflammatory response of bovine mammary epithelial cells.In this study,MAC-T cells were transfected with PINK1 interfering RNA or added with autophagy inhibitor3-MA.The results showed that silencing PINK1 or adding 3-MA inhibited LPSinduced mitophagy by reducing the protein expression of PINK1,Parkin and LC3-II,increasing the protein expression of P62,and reducing the number of autophagosomes and autolysosomes;aggravated LPS-induced mitochondrial dysfunction by reducing the protein expression of COⅠ,COⅡ,COⅢ,COⅣ and COⅤ and increasing the production of Mito-ROS;aggravated the LPS-induced inflammatory response by increasing the protein expression of NLRP3,Caspase-1 and increasing the protein and m RNA expression levels of IL-1β,IL-6 and TNF-α.After transfection of PINK1-overexpressing adenovirus,the results showed that PINK1 overexpression enhanced LPS-induced mitophagy by increasing the protein expression of PINK1,Parkin,LC3-II and reducing the protein expression of P62.In addition,PINK1 overexpression alleviated LPS-induced mitochondrial dysfunction by increasing the expression of COI,COII,COIII,COIV and COV and reducing the production of Mito-ROS;attenuated LPS-induced inflammatory response by reducing the protein expression of NLRP3,Caspase-1 and the protein and m RNA expression levels of IL-1β,IL-6 and TNF-α.These results confirmed that PINK1 alleviated LPS-induced mitochondrial dysfunction and inflammatory response by regulating mitophagy in bovine mammary epithelial cells.In conclusion,when mitochondrial dysfunction occurs in mammary epithelial cells of cows with ketosis,the NLRP3 inflammatory signaling pathway is activated to trigger an inflammatory response,while PINK1-mediated mitophagy negatively regulates NLRP3-mediated inflammatory response.