| Most lameness diseases are caused by claw lesion in dairy cattle.Laminitis is the most frequent causes of lameness,which is believed to result from highly carbohydrate in diet.However,the exact pathogenesis of laminitis is still unclear.Using acute laminitis model of oligofructose overload,this study was investigated the change of polymorphonuclear neutrophil(PMN)activity and several related protease in order to provide theoretical and experiment basis in future research of dairy cattle laminitis.Twelve clinically healthy and non-lame Holstein cows was used for the experiments and randomly equally divided into two groups: control(CON)and oligofructose(OF).OF cows received 17 g/kg of BW oligofructose dissolved in 2 L/100 kg of BW tap water at time 0 h via an oral-rumen tube.CON cows received 2 L/100 kg of BW tap water.At 0,6,12,18,24,36,48,60 and 72 h after overload,80 m L of blood was collected from jugular vein to collect plasma and PMN.All cows were euthanasia and collect lamellar tissue after 72 h.The activity of matrix metalloproteinases(MMP2 and MMP9)and tissue inhibitors of metalloproteinases(TIMP1 and TIMP2)in plasma and lamellar tissue was measured by zymography and reverse zymography,respectively.Western blot was used to analyze neutrophil elastase(NE)and myeloperoxidase(MPO)protein expression in lamellar tissue.The endogenous and phorbol myristate acetat(PMA)-stimulated reactive oxygen species(ROS)production of PMN was determinate by fluorophotometric method.The genes expression of PMN was analyze by quantitative real-time PCR in each time point,including inflammation markers,adhesion,migration,oxidative stress,metabolism and receptor.The result shows that plasma MMP9 activity in OF cows was significant increased from 36 to 60 h;Plasma TIMP1 activity of OF cows was decreased after 18 h(P < 0.05)and TIMP2 activity was elevated after 48 h(P < 0.01).Plasma MMP9/TIMP1 was also significant elevated after 18 h(P < 0.01).There was no difference in plasma MMP2 activity comparing CON and OF cows.In lamellar tissue,the activity of MMP9 was increased(P < 0.05)and the activity of TIMP1 was decreased(P < 0.05)in OF cows.Lamellar MMP9/TIMP1 in OF cows was also markedly higher than CON cows(P < 0.01),while the activity of MMP2 and TIMP2 was no difference.The result of western blot shows that lamellar NE protein in OF cows expression was significant higher than CON cows(P < 0.01),while lamellar MPO protein was no difference between CON and OF cows(P > 0.05).The result of ROS production of PMN shows that PMA-stimulated ROS production was significantly higher than endogenous ROS production at all time points in CON cows(P < 0.01),while PMA-stimulated ROS production was decreased and no difference from 12 h to 48 h in OF cows(P > 0.05).The result of quantitative real-time PCR shows that inflammation markers(IL1?,IL1?,IL6,IL10,TNF?,STAT3,TLR4,MMP9,and HP)and eicosanoids(ALOX5,ALOX5 AP,and PLA2G4A)was significant upregulated in OF cows,while IL1?,ALOX5 and ALOX5 AP maintain a high level after 72 h.The expression of adhesion and migration(CXCR2,CXCL8,CD62 L,ITGA4,ITGAM,and ITGB2)in OF cows was increased compared with CON cows,while CXCR2 and ITGA4 still maintain a high level after 72 h.The expression of SOD1 and MPO were downregulated and other oxidative stress genes were upregulated.The expression of SOD2 and S100A8 in OF cows were still higher than CON cows after 72 h.In metabolism and receptor genes,the expression of Fas,GR? and INSR was downregulated in OF cows,while LDHA and PANX1 were no difference(P > 0.05).In summary,the activity of MMP9 was increase and the activity of its inhibitor TIMP1 was decrease in plasma and lamellar tissue during acute laminitis model of oligofructose overload in cows,which could contribute to PMN's migration and basement membrane's degradation.In addition,the change of ROS production and genes expression of PMN is involved the inflammatory respond of systemic and local lamellar tissue,which may play an important role in the pathological process of laminitis of dairy cattle. |
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