The Role And Mechanism Of Nrf2 In TGF-β1-induced Mammary Fibrosis In Mice

Cow mammary fibrosis is a chronic,pathological process of sexual dysfunction,the pathological process will shorten the lactating years of dairy cows,milk production significantly decreased,milk quality deteriorates,severe can develop mammary sclerosis,and eventually lead to the elimination of dairy cows,seriously restrict the development of dairy industry.At present,the research progress of cow mammary fibrosis is slow and there is a lack of effective treatment.Therefore,it is of great research significance and value to effectively alleviate mammary fibrosis of cows and deeply explore its potential pathogenesis.Nuclear factor erythroid 2 related factor 2（Nrf2）is the main regulator of adaptive anti-oxidative stress response in cells,which has multiple biological functions such as relieving inflammation,anti-oxidation and regulating metabolism.However,the role and mechanism of Nrf2 in mammary fibrosis have not been reported.Therefore,based on the Epithelial mesenchymal transformation（EMT）process,systematically investigate the effects and its mechanisms of Nrf2 on TGF-β1-induced mice mammary fibrosis,in order to provide theoretical basis for the prevention and treatment of mammary fibrosis in cows by targeting Nrf2.First,in order to clarify the effect of Nrf2 on mice mammary fibrosis,the study explored the effect of Nrf2 gene deletion on related indicators of mice mammary fibrosis by constructing a mice mammary fibrosis model induced by TGF-β1 and using histopathology,Western blot and q RT-PCR.The results showed that compared with wild-type mice,collagen fiber deposition and fibrosis-related protein and m RNA levels in mammary tissue of Nrf2 knockout mice were significantly increased.Subsequently,we added the activator（t BHQ）and inhibitor（RA）of Nrf2 into m MECs,and further clarified the influence of Nrf2 on mammary fibrosis based on the EMT process induced by TGF-β1.The results showed that,compared with the normal control group,fibrosis-related protein and m RNA levels increased significantly after the addition of RA.t BHQ significantly inhibited the levels of fibrosis-related proteins and m RNA.These results indicate that the activation of Nrf2 inhibit the increase of fibrosis phenotypic indexes and thus alleviate the development of mammary fibrosis in mice.In order to explore the mechanism of Nrf2 in mice mammary fibrosis,the activation of TGF/Smad signaling pathway was detected by Western blot experimental methods in vivo and in vitro.The results showed that,compared with the control group,inhibition or knockout of Nrf2 significantly activated TGF/Smad signaling pathway,while activation of Nrf2 significantly inhibited the activation of TGF/Smad signaling pathway.Subsequently,mitochondrial damage and mitochondrial autophagy were detected in this study,and the results showed that compared with the WT-TGF-β1 group,mitochondrial damage was more serious in Nrf2^-/--TGF-β1 group,and the expression of mitochondrial autophagy related proteins was significantly decreased,and the production of ROS was increased.The results of in vitro study were consistent with those in vivo.Finally,in order to further explore whether the effect of Nrf2 on mammary fibrosis in mice is mediated by ROS,we detected the expression of fibrosis-related proteins after adding ROS scavher（NAC）respectively in vitro and in vivo.The results showed that NAC significantly alleviated the increase of fibrosis-related protein expression induced by TGF-β1 in vitro and in vivo.Further mechanism studies revealed that excessive accumulation of ROS significantly activated TGF/Smad signaling pathway and promoted the expression of fibrosis-related proteins,while the addition of NAC significantly inhibited the activation of TGF/Smad signaling pathway and thus alleviated the development of mammary fibrosis in mice.These results indicate that the activation of Nrf2 alleviate the development of TGF-β1-induced mammary fibrosis in mice,which affects ROS generation mainly through the regulation of mitochondrial autophagy and mitochondrial damage,and then mediates the mammary fibrosis process in mice through the TGF/Smad signaling pathway.Therefore,Nrf2 is expected to be a potential target for alleviating mammary fibrosis in dairy cows.