Functions And Molecular Mechanisms Of TGF-β1 In The Process Of Bovine Mammary Fibrosis

Mastitis and mammary fibrosis are the most common diseases of bovine,and both of them have high incidence rates.They coulddevelop into mastoscirrhus and thus lead to the obsolete of bovine and severe losses of dairy industry.Bovine mammary fibrosis always was developed from chronic uncured mastitis.Different bacteria lead to clinical or subacute mastitis,and both of them may develop to mammary fibrosis in various degrees.All of the hyperplasia of connective tissue,accumulation of extracellular matrix protein,(ECM)and epithelial-mesenchymal transition(EMT)are associated with difference types of organs and tissues fibrosis.Although it is well established that transforming growth factor-β1(TGF-β1)plays a crucial role in chronic inflammation in various tissues and is related to inflammation-caused organ fibrogenesis associated with EMT and the deposition of ECM,the effects of TGF-β1 on bovine mammary epithelial cells(BMECs)with bovine mammary fibrosis and the mechanisms remain unknown.The purpose of this thesis is to study the role of TGF-β1 in bovine mammary fibrosis and its mechanism.Firstly,we analyzed the expression level of TGF-β1 in inflamed mammary tissues by western blot,and the results showedthat TGF-β1 protein expression level in inflamed mammary tissues was higher than that in normal tissues.ELISA and western blot analysis showed TGF-β1 protein expression level and secretion level in lipopolysaccharides(LTA)-induced inflamed BMECs and lipoteichoic acid(LPS)-induced inflamed BMECs were also higher than those in normal BMECs,suggesting that TGF-β1 involve in the inflammation response process of bovine mammary gland.BMECs were treated with TGF-β1,and a series of molecular biological methods were used to evaluate the effect of TGF-β1 on BMECs.Phase contrast microscope observation and F-actin fluorescence staining were used for morphological analysis,and the results showedthat TGF-β1 were able to induce EMT in BMECs.EMT-related gene and protein expression changes were evaluated using real-time quantitativepolymerase chain reaction(RT-qPCR),western blot,and immunofluorescence.Results showed that TGF-β1 up-regulated the expression of α-smooth muscle actin(α-SMA),vimentin,albumin and fibronectin,and down-regulated the E-cadherin expression.In addition,TGF-β1 also increased ECM component expression,such as collagen type I(collagen I),matrix metalloproteinase(MMP)2 and MMP9.The results demonstrated that TGF-β1 was able to induce anobvious EMT and ECM overexpression in BMECs.In the next section,we use western blot and immunofluorescence to explore the molecular mechanisms of the EMT induced by TGF-β1.The results showedthat TGF-β1 activated Smad2 and caused a transfer of Smad4 from cytoplasm to nucleus,indicating that TGF-β1 treatment activated the TGF/Smad signaling pathway in BMECs.We also inhibited the TGF/Smad signaling pathway with its receptor inhibitor,and we found that the ECM deposition and EMT caused by TGF-β1 was inhibited at the same time,suggesting that TGF-β1 induce ECM deposition and EMT of BMECs via TGF/Smad signaling pathway.To ascertain whether TGF-β1 also induces fibrosis in vivo,a mouse model was used.Mammary glands of mice were injected with TGF-β1 through milk duct four times,with one administered every three days.Routine histopathological analysis showed significant fibrosis and inflammatory infiltration in the mammary glands.On the contrary,normal mice mammary glands andmammary glands of mice injected once did not show any abnormity.It declared that sustained stimulation incubation of TGF-β1 within mammary glands can cause mastitis and mammary fibrosis.In conclusion,TGF-β1 expression was up-regulated in mammary tissues with mastitis and inducible inflammatory BMECs,and this aberrant up-regulation of TGF-β1 could cause EMT and ECM accumulation in mammary epithelial cells,whichwas associated with mammary fibrosis caused by chronic mastitis.