The Mechanism Of Loureirin B Alleviating Lipopolysaccharide-induced Inflammation And Oxidative Stress In Bovine Mammary Epithelial Cells

Mastitis is one of the most common diseases in dairy cow production,which seriously interferes with the normal lactation and milk quality of dairy cows and brings extremely huge economic losses.In addition,in the modern dairy industry,high-yield dairy cows and lactating dairy cows,due to long-term heavy lactation pressure,their mammary glands often experience oxidative stress,which also brings great interference to the health of dairy cows and the development of the dairy industry.Lipopolysaccharide(LPS)is a component of the outer membrane of gram-negative bacteria,an important pathogen causing mastitis in dairy cows.It is released as the gram-negative bacteria die and disintegrates.When LPS acts on tissues or cells,it will induce inflammation and oxidative stress in tissues or cells.Loureirin B is an active ingredient isolated from the traditional Chinese medicine-Dragon’s Blood.Studies have confirmed that Loureirin B has the functions of anti-inflammatory,promoting blood circulation,eliminating thrombus and removing ROS.However,the regulatory effect and mechanism of Loureirin B on the inflammation and oxidative stress of bovine mammary epithelial cells is still unknown.Therefore,the purpose of this study is to explore the influence and molecular mechanism of Loureirin B on LPS-induced inflammation and oxidative stress in bovine mammary epithelial cells.In this study,we first constructed an inflammation and oxidative stress model of MAC-T cells(bovine mammary epithelial cell line)through LPS treatment.The results of CCK-8 showed that LPS at concentrations of(5,10,25,50,100)μg/m L significantly reduced the viability of MAC-T cells.We then chose 10 μg/m L LPS to establish the inflammation and oxidative stress model of MAC-T cells.The results of q RT-PCR showed that LPS treatment significantly increased the m RNA levels of inflammatory factors(IL-6、IL-8、GM-CSF and CCL5)as well as endoplasmic reticulum stress markers(CHOP and GRP78)in MAC-T cells.Detect the changes of antioxidant substances in MAC-T cells,we found that after LPS treatment,MAC-T cells’ T-AOC(total antioxidant capacity),SOD(superoxide dismutase)activity,CAT(catalase)activity,as well as GSH(glutathione)content are significantly reduced,while MDA(malondialdehyde)content is significantly increased.The results of reactive oxygen detection also found that LPS significantly increased the production of reactive oxygen species(ROS)in MAC-T cells.In addition,after LPS treatment,we also tested the milk fat synthesis ability of MAC-T cells.Our results showed that LPS treatment not only inhibited the expression level of genes related to milk fat synthesis,but also inhibited the accumulation of lipid droplets and the secretion of triglyceride in MAC-T cells,and up-regulated several micro RNAs(mi RNAs)which targeting the PPARγ gene.We further used Loureirin B to alleviate the inflammation and oxidative stress caused by LPS to MAC-T cells.First,MAC-T cells were treated with different concentrations of Loureirin B to determine the safe concentration range.The results showed that Loureirin B at 40 μM and lower concentrations did not cause toxicity to MAC-T cells.We continued to co-treat MAC-T cells with different concentrations of Loureirin B and 10 μg/m L LPS in safe concentrations,and found that Loureirin B at the levels of(10,20,30,40)μM can significantly save cell viability,and 20 μM concentration of loureirin B has the best rescue effect,so 20 μM was selected as the subsequent concentration of loureirin B.We then explored the alleviating effect of Loureirin B on LPS-induced inflammation and oxidative stress in MAC-T cells.Our results found that Loureirin B can significantly inhibit the increase in the levels of inflammatory factors(IL-6,IL-8,GM-CSF and CCL5)caused by LPS,add Loureirin B alone can also significantly reduce the expression levels of some inflammatory factors.At the same time,Loureirin B can also inhibit the increase in TLR4 m RNA levels caused by LPS.Western-blot was used to measure the changes of NF-κB signaling pathway proteins in MAC-T cells,and it was found that Loureirin B can inhibit the increase in phosphorylation of NF-κB p65 caused by LPS.In addition,add Loureirin B alone can also inhibit the phosphorylation of NF-κB p65 in MAC-T cells.In terms of anti-oxidation,Loureirin B can inhibit the increase of endoplasmic reticulum stress makers(CHOP,GRP78)caused by LPS,add Loureirin B alone can also down-regulate the expression of endoplasmic reticulum stress makers.Loureirin B can also rescue the decrease in T-AOC,SOD,CAT and GSH levels caused by LPS treatment,and inhibit the increase in MDA content induced by LPS,add Loureirin B alone can also significantly increase the levels of T-AOC,SOD and GSH in MAC-T cells.Western-blot and q RT-PCR were used to detect the expression of antioxidant genes,we found that Loureirin B can increase the m RNA levels of key antioxidant gene: Nrf2 and its downstream molecules: HO-1 and x CT in the presence or absence of LPS,as well as increase the protein expression of Nrf2.The results of the ROS assay found that Loureirin B can significantly inhibit the increase in ROS levels caused by LPS.In addition,Loureirin B can also effectively repair the inhibition of MAC-T cell milk fat synthesis induced by LPS.In summary,our results show that Loureirin B can alleviate the inflammation and oxidative stress of MAC-T cells induced by LPS,and it can also certain anti-inflammatory and antioxidant effects on MAC-T cells under normal conditions.The findings of our study may provide new theoretical basis for the protection and therapy of mastitis and oxidative stress in dairy cows during production.