The Effect And Mechanism Of High Temperature Stimulation On LPS Induced Inflammation Of Bovine Mammary Epithelial Cells

In the summer of high temperature,cows are not only easy to produce heat stress,but also have a high incidence of mastitis.Heat stress can promote the occurrence of mastitis,leading to the decline of milk yield and milk quality,and having a great impact on the production and economic benefits of dairy industry.Current studies have shown that heat shock transcription factor(HSF1)is closely related to the occurrence of heat stress.It can induce the expression of heat shock protein(HSP70)during heat stress,thus buffering cell stress.Lipopolysaccharide(LPS)is a component of cell wall of Gram-negative bacteria.It is the main virulence factor that induces inflammatory reaction of cells.Cells respond to inflammatory response through the expression of various inflammatory factors.There are also reports that tight junctions are involved in the heat stress response of cells,and inflammatory factors can regulate tight junctions.Tight junction structure plays an important role in the realization of the mammary barrier function.The mammary barrier provides a stable internal environment for mammary cells and the mammary gland could normally and orderly play its lactation function.The changes of its structure will affect the material exchange and immune function,leading to imbalance of the internal and external environment of the mammary gland.Heat stress related genes,inflammatory factors and tight junction related genes may be involved in the regulation of dairy cow mastitis,but the regulatory mechanism is not very clear.Therefore,in this study,we analyzed the inflammatory factors(IL-6 and IL-8),heat stress related genes(HSF1 and HSP70),tight junction related genes(claudin-4,occludin,ZO-1 and so on)Objective to investigate the effect of high temperature on inflammation of bovine mammary epithelial cells in vitro induced by LPS and its mechanism.The main results are as below:(1)Heat stress related genes(HSF1,HSP70)and tight junction related genes(claudin-4,occludin,ZO-1 and symplekin)were highly expressed in normal bMECs;(2)High temperature stimulation could promote the expression of Hsp70 gene,but inhibit the expression of inflammatory factor IL-8 and tight junction related genes:the expression of HSP70 increased significantly at 0 h and 1 h after heat stimulation(P < 0.001),and the expression of IL-8 decreased significantly at 1 h and 3 h after heat stimulation(P < 0.05),the expression levels of claudin-4 and occludin were significantly lower than the normal levels(P < 0.01);(3)LPS stimulation could induce inflammation of bMECs,but inhibit the expression of heat stress related genes and tight junction related genes: LPS stimulation significantly increased the expression of inflammatory factors(IL-6 and IL-8)in bMECs(P < 0.001),and decreased the expression of heat stress related genes(HSF1 and HSP70)(P < 0.001),the expression levels of tight junction related genes(claudin-4,occludin,ZO-1 and symplekin)were significantly decreased(P < 0.001);(4)High temperature stimulation may promote LPS induced inflammation of bovine bMECs by reducing the expression of inflammatory factors: the results showed that the expression of IL-8 was significantly lower than that of LPS alone at6 h and 12 h of recovery period,which may lead to the decrease of anti-inflammatory ability of cells;(5)High temperature stimulation may affect LPS induced inflammation of bovine bmecs by decreasing the expression of tight junction related genes: High temperature stimulation inhibited the expression of four kinds of tight junction related genes;the expression of claudin-4 was decreased most significantly after the mixed stimulation of high temperature and LPS under three different stimulation;therefore,it is speculated that high temperature stimulation may reduce the expression of tight junction related genes in mammary cells.The damage of tight junction structure leads to more inflammatory reaction.In conclusion,the inflammation of bovine bMECs can be induced by LPS stimulation,and high temperature can promote the inflammation of bMECs: When stimulated by high temperature,on the one hand,it can reduce the expression of inflammatory factors,which may reduce the anti-inflammatory ability of cells;on the other hand,reduce the expression of tight junction related genes,which may lead to the damage of tight junction structure and the cells are more prone to inflammatory reaction;in addition,when stimulated by LPS,the expression of heat stress relatedgenes is lower than the normal level,which makes the cell anti-heat stress ability down.The results provide reference for the study of the relationship between heat stress and mastitis,and the incidence rate of mastitis in summer.The results also provide a reference for the prevention and reduction of dairy cow mastitis in summer.This study needs to further study the effect and mechanism of high temperature on mastitis from protein expression level and individual level of cows.