The Effect And Mechanism Of Histamine Regulating The Adhesion Function Of Neutrophils In Dairy Cows With SARA

Systemic inflammatory response is the bond of subacute rumen acidosis(SARA),the secondary inflammatory diseases,and the impaired production performance.It is a key process between SARA itself and other harms.Neutrophils(PMN)have a powerful function of regulating inflammatory amplification.The high concentration of blood lipopolysaccharide(LPS)and histamine may mediate the occurrence of systemic inflammatory response by regulating PMN adhesion in dairy cows with SARA,but the underlying mechanism is still unclear.The present research compared the metabolism and immune indicators in the peripheral blood of SARA(N=15)and healthy cows(N=15).The results showed that the levels of LPS and histamine in the serum of dairy cows with SARA ware significantly increased,the levels of inflammatory cytokines including tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β),as well as the levels of acute phase reactive protein including lipopolysaccharide binding protein(LBP),haptoglobin(Hp),and serum amyloid A(SAA)increased to varying degrees.These results indicate that SARA cows have a systemic inflammatory response,which may be related to the high blood concentrations of LPS and histamine.Accordingly,the following experiment was carried out.In vivo,the PMNs were separated from the peripheral blood of healthy(N=15)and SARA cows(N=15),and PMNs adhesion ability or the expression level of adhesion-related protein were detected by adhesion or western blotting experiment respectively,the results showed that the adhesion ability of PMNs and the expression of the main membrane protein adhesion molecules CD11 a and CD11 b increased markedly in dairy cow with SARA.In vitro experiments,the PMNs ware separated from healthy cows,then treated by pathological concentration of LPS(4EU/m L)or histamine(7μmo L/L)for 4 hours,the results showed that the pathological concentration of histamine(but not LPS)caused the increasing adhesion of PMNs and the increase of main adhesion molecules on the cell membrane.However,there was no significant change in the m RNA level of these adhesion molecules,the results indicate that high blood histamine concentration of cows with SARA can promote the adhesion of PMNs and the accumulation of adhesion molecules,suggesting that the increased level of PMN adhesion molecules may be due to the inhibition of its degradation.Autophagy plays an important role in regulating the circulation of adhesion molecules.In vivo,the autophagy degradation of PMN was hindered in dairy cows with SARA comparing with healthy cows under transmission electron microscopy,and there were more autophagic vesicles in dairy cows with SARA,the results of western blotting showed that the main autophagy-related proteins of PMN in dairy cows with SARA ware accumulated,including microtubule-associated protein 1 light chain 3-II(LC3-II)and autophagy degradation substrate protein P62.In addition,compared with healthy cows,LC3-II and p62 showed no difference in m RNA level in PMNs of cows with SARA,indicating that the autophagy process of PMN of cows with SARA was damaged.In vitro,the results showed that the pathological concentration of histamine significantly increased the levels of autophagy-related proteins LC3-II and P62 and the main adhesion molecules of PMNs.By immunofluorescence experiments,the pathological concentration of histamine significantly increased the accumulation of LC3-II and the main adhesion molecule CD11 b in the cell or on the cell membrane,which effect was similar to that of the autophagy inhibitor chloroquine on PMNs,indicating that the pathological concentration of histamine in vitro can mediate the enhancement of PMNs adhesion by impairing the autophagy flux.All of the results above confirmed that the peripheral blood histamine concentration of dairy cows with SARA is elevated,and the pathological concentration of histamine can mediate the enhancement of PMNs adhesion by impairing the autophagic flux of PMN,which provides a theoretical basis for the clarification of the cause and the regulatory mechanism of systemic inflammatory response in dairy cows with SARA.