| Staphylococcus aureus is one of the main pathogenic bacteria causing cow mastitis,and its virulence factor PV-leukocidin(PVL)is the main virulence protein produced by Staphylococcus aureus.In recent years,the detection rate of pvl gene in the milk of cows with mastitis has been increasing,which proves that pvl gene is one of the main causes of mammary epithelial cell injury in cows.Our previous studies showed that rPVL showed strong toxicity and damage to bovine mammary epithelial cells,and baicalin showed protective effect in this damage process.Cytoskeleton is an important,complex and dynamic cell component,which plays an important role in maintaining cell morphology,cell movement and apoptosis.Previous experiments have found that damaged cytoskeleton can induce apoptosis of bovine mammary epithelial cells and change the effect of lactation in bovine.At present,the mechanism of phosphide modification of skeleton protein by rPVL in the process of damaging the cytoskeleton of bovine mammary epithelial cells is not clear,and the molecular targets regulated by baicalin in this process have not been confirmed.Therefore,by clarifying the pathological characteristics of bovine mammary epithelial microfilaments and microtubules rearrangement caused by Staphylococcus aureus ATCC49775,Δpvl 49775,C-Δpvl 49775 and rPVL,this study further explored the effect and molecular mechanism of baicalin on this pathological process,and provided experimental basis for the clinical application of baicalin.Promote the application of basic research results.In this study,cytoskeleton-specific drugs were used to construct cytoskeletal instability models of bovine mammary epithelial cells,and to analyze the effects of damaged cytoskeletal stability on cell apoptosis and lactation function,as well as whether it affected the ability of Staphyoco accus aureus to adhere to and invade bovine mammary epithelial cells.To study the effects of Staphylococcus aureus ATCC49775,Δpvl 49775,C-Δpvl 49775 and rPVL on the microfilaments and microtubules structure of bovine mammary epithelial cells and the classical regulatory pathways of cytoskeletal related protein nuclei,and the molecular mechanism of baicalin’s intervention in the microfilaments and microtubules rearrangement of bovine mammary epithelial cells induced by PVL.The results showed that 5 μg/mL,10μg/mL of cytorelaxation D and 0.25 μg/mL,0.5 μg/mL,1μg/mL of colchicine significantly increased the expression of apoptosis-related proteins in bovine mammary epithelial cells.β-casein expression in bovine mammary epithelial cells was significantly decreased after treatment with 10μg/mL cytorelaxin D and 1μg/mL colchicine.Treatment with 2.5 μg/mL,5 μg/mL,10 μg/mL cytorelaxation D and colchicine inhibited the invasion of Staphylococcus aureus into bovine mammary epithelial cells.After the infection of Staphylococcus aureus ATCC49775,Δpvl 49775,C-Δpvl 49775 and rPVL,the microfilament arrangement was disordered,and the protein expressions of F-actin,cofilin and p-cofilin were significantly increased.Compared with model group,the structure of cell microfilaments was more obvious in baicalin intervention group,the protein expressions of F-actin and p-cofilin were significantly decreased,and the protein expression of cofilin was significantly decreased in baicalin intervention group at 5 μg/mL.The expression of RhoA and LIMK1/2 in baicalin intervention groups decreased significantly at 2.5 μg/mL and 5 μg/mL.The expression of p-LIMK 1/2 protein in 5 μg/mL baicalin intervention group was significantly increased;The expressions of ROCK2 and p-ROCK2 protein were significantly increased.After infecting bovine mammary epithelial cells with Staphylococcus aureus ATCC49775,Δpvl 49775,C-Δpvl 49775 and rPVL,microtubules were spot-like,and the expression of Ace-Tubulin was significantly decreased,while the expressions of Tau,p-Tau,CRMP2 and p-CRMP2 were significantly increased.Compared with model group,Tau protein expression in baicalin intervention groups of 2.5 μg/mL and 5 μg/mL was significantly decreased,and p-Tau protein expression in baicalin intervention groups of 2.5 μg/mL,5 μg/mL and 10 μg/mL was significantly decreased.The protein expressions of CRMP2 and p-CRMP2 in baicalin intervention groups of 2.5 and 5 μg/mL were significantly decreased.The expression of PI3K protein was significantly decreased in baicalin intervention groups of 2.5 μg/mL and 5 μg/mL,the expression of p-AKT protein was significantly decreased in baicalin intervention groups of 2.5 μg/mL and 5 μg/mL,the expression of GSK3β protein was significantly increased in baicalin intervention groups of 5 μg/mL and 10 μg/mL,and the expression of p-GSK3β protein was significantly increased.The results showed that apoptosis of bovine mammary epithelial cells was induced by cytoskeleton injury,and both death receptor pathway and mitochondrial pathway were involved in the process.Cytoskeleton injury can affect the synthesis of milk fat and milk protein in bovine mammary epithelial cells.Cytoskeleton injury did not affect the adhesion of Staphylococcus aureus to bovine mammary epithelial cells,but inhibited the invasion of Staphylococcus aureus into bovine mammary epithelial cells.The microfilaments and microtubules were damaged after Staphylococcus aureus ATCC49775,Apvl 49775,C-Δpvl 49775 and rPVL infected bovine mammary epithelial cells,and the degree of damage was related to Staphylococcus aureus secreted PVL.rPVL regulates the rearrangement of microfilaments and microtubules in bovine mammary epithelial cells by activating RhoA/ROCK and PI3K/AKT signaling pathways.Baicalin inhibits the activation of RhoA/ROCK and PI3K/AKT pathways induced by rPVL,regulates the expression of related proteins,and reduces the over-phosphorylation of cytoskeletal related proteins cofilin,Tau and CRMP2,thus alleviating the rearrangement of microfilaments and microtubules in bovine mammary epithelial cells. |
PDF Full Text Request