Effects Of β-sitosterol On Inflammatory Injury And Milk Protein And Fat Syntheses-related Genes Expression In Bovine Mammary Epithelial Cells

As a common dietary nutrient,milk provides energy,high protein,the main minerals and vitamins needed by human body.It has the reputation of "the most perfect food" and "white blood",has a high status in national life.Nowadays,in the era of more pursuit of healthy life,the nutritional elements in milk such as milk protein,milk fat have been widely concerned,and the quality of milk has become a hot topic of national concern.Although the development of China’s milk quality has met international standards,there is still room for improvement.At present,in order to improve dairy farming level,milk quality and market competitiveness,healthy dairy farming and disease-resistant nutrition are needed.Therefore,using green and safe means(development of new feed additives,etc.)to improve milk protein content and milk quality,prevent and alleviate the negative effects of dairy cow stress response is one of the important means to promote the development of dairy industry in China.As one of the phytosterols,β-sitosterol has multiple physiological activities such as anti-inflammatory and growth promotion.At present,β-sitosterol as a new feed additive,has been allowed to be added to the breeding of poultry and growth-finishing pigs.However,there are only a few reports on the use of β-sitosterol in improving milk synthesis in dairy cows.Therefore,this study took bovine mammary epithelial cell line(MAC-T cells)as the experimental object to investigate whether β-sitosterol could alleviate the inflammatory response of bovine mammary epithelial cells induced by Lipopolysaccharide(LPS)and whether could affect the synthesis of milk protein from bovine mammary epithelial cells.In order to investigate the anti-inflammatory effect of β-sitosterol and its functional effect on improving milk quality in MAC-T cell model.1.The cell viability of MAC-T cells treated with β-sitosterol(0,0.01,0.1,1,5,10,20,30,40 μM)was measured by CCK-8 during the growth phase of MAC-T cells.The results showed that the activity of MAC-T cells was not significantly influenced when β-sitosterol concentration was 0.01-20 μM,and β-sitosterol at 30 μM and 40μM significantly decreased cell viability.Therefore,1 μM β-sitosterol combined with1 μg/m L LPS was used to treat MAC-T cell lines in subsequent experiments.Real-time fluorescence quantitative assay(q PCR)was used to detect the expression levels of inflammation-related genes,β-sitosterol significantly down-regulated the expression of LPS-induced inflammatory factors including IL-1β,TNF-α and NF-κB(P65).By q PCR and Western Blotting,β-sitosterol significantly reduced caspase3,bax expression of apoptosis factors.β-sitosterol reduced LPS induced intracellular ROS accumulation,has the function of resisting the inflammatory response of bovine mammary epithelial cells.In addition,β-sitosterol significantly increased m TOR,HIF-1α m RNA expression level(P<0.05).2.Different concentrations of β-sitosterol(0,0.01,0.1,1,5,10,20,30,40 μM)were added to induce MAC-T cells during the differentiation stage to evaluate the regulation of β-sitosterol on milk protein synthesis and related gene expression.The results showed that low concentrations of β-sitosterol(0.1,1,5,10,20 μM)promoted the expression of β-casein m RNA expression,and β-sitosterol(30,40 μM)significantly inhibited β-casein m RNA expression.According to the results ofβ-casein m RNA expression,0.1,1,10 and 30 μM β-sitosterol were selected as the final concentration for subsequent experiments.Consistent with the gene expression results,β-sitosterol significantly increased the protein expression of β-casein.QPCR and Western Blotting were used to detect JAK2-STAT5 and m TOR signaling pathway related gene expression and protein level.β-sitosterol(0.1,1,10 μM)increased m RNA and protein expression levels of signal transduction activator 5(STAT5),mammalian target of rapamycin(m TOR)and ribosomal protein S6 kinase β-1(S6K1)in JAK2/STAT5 and m TOR signaling pathways(P<0.05).3.QPCR and Western Blotting were used to detect the effects of β-sitosterol on the expression of milk fat synthesis related genes during the differentiation stage of MAC-T cells.The results showed that β-sitosterol stimulated milk fat synthesis related factors.The m RNA expression levels of sterol regulatory element binding protein 1(SREBP1),peroxisome-proliferator-activated receptor γ(PPARγ),acetyl-Co A carboxylase(ACC),lipoprotein lipase(LPL)and stearoyl-co A desaturase(SCD)were significantly increased,and the protein expression levels of SREBP1,PPARγ and SCD were also enhanced(P<0.05).4.The lactation function of mammary epithelial cells is also affected by multiple hormones and cytokines,mainly prolactin and insulin-like growth factor.In order to further explore the mechanism of β-sitosterol regulating milk synthesis,we examined the expression of growth hormone/insulin-like growth factor-1(GH/IGF-1)axis,hypoxia-inducible factor-1 α(HIF-1α),and cytokine signaling inhibitor(SOCS)related genes associated with JAK2/STAT5 and m TOR signaling pathways.The results showed that compared with the control group,β-sitosterol treatment group significantly increased the expression of related genes of GH/IGF-1 axis and HIF-1α.It was noted that SOCS2 m RNA and protein expression were inhibited by β-sitosterol at two lower concentrations(0.1 and 1 μM),but SOCS2 m RNA and protein expression were significantly increased at the highest concentration of β-sitosterol(30μM)(P<0.05).In conclusion,β-sitosterol has anti-inflammatory properties,prevent and alleviate LPS-induced inflammation in MAC-T cell.In addition,β-sitosterol may regulate the GH/IGF-1 axis to enhance JAK2/STAT5 and m TOR pathway by affecting the expression of the SOCS family,to promote β-casein synthesis;and improved milk fat synthesis related genes(ACC,LPL)by activating SREBP1,PPARγ to improve milk fat synthesis.The results showed that β-sitosterol could be used as a potential feed additive to improve milk quality.