Studies On The Protective Role Of Ethyl Pyruvate In Acute Liver Injury

Objective:To study effects of ethyl pyruvte on expression of the tight junction associated protein-ZO-1 between epithelial cells of intestinal mucosa and on release of cytokins from kupffer cells or from human umbilical vein endothelial cells,in order to explore the protective role and pathogenesis of ethyl pyruvte in.acute liver injury.Materials and Methods:The first part of the experiment:Thirty BW 190～250g male wistar rats were randomly divided into three groups(n=10 for each group),normal contral group,acute liver injury group(D-GalN/LPS,700mg/5ug/kg,i.p.)and EP group(pretreatment with EP,40mg/kg,lhour befor i.p with D-GalN/LPS).48 hours after D-GalN/LPS was administered,the parameters of plasma's ALT and Endotoxin were measured and historical staining of intestinal tissue was evaluated and ZO-1 was observed by immunohistochemistry. The second part of the experiment:Thirty BW 190～250g male wistar rats were randomly divided into three groups.(n=10for each group),normal contral group,acute liver injury group(D-GalN/LPS,700mg/5ug/kg,i.p.)and EP group(treatment with EP,40mg/kg,24 hour after i.p with D-GalN/LPS)48 hours after D-GalN/LPS was administered,the parameters of plasma's ALT,TNF-αand HMGB1 were measured respectively.The third part of the experiment:Neonate umbilical vein endothelial cells were primary cultured and identified in vitro,Firstly,the cells were divided into three groups in every experiments,LPS stimulation group,EP group and normal contral group,then the expression of HMGB1,ICAM-1 between umbilical vein endothelial cells were measured by immunohistochemistry.Secondly,the cells were divided into normal contral group,LPS stimulation group and EP group,on every time points(Ohour,6hour,12hour,24hour,48hour),the level of HMGB1 in the supernatant were measured by Western blot.Thirdly,the cells were divided into three groups,0uM EP group, 5uM EP group and 10uM EP group,and the level of HMGB1 in the supematant were measured by Western blot.Results:The first part of the experiment:The levels of ALT and Endotoxin were markly higher in the acute liver injury group than that in the nomal contral group(P＜0.01).In EP group, the levels of ALT and Endotoxin were still higher than that in the nomal contral group but were obviously lower than that in the acute liver injury group(P＜0.05).The staining of ZO-1 in the control rats was similar to a honeycomb,which reflected a continuous and uniform distribution localized at the apical portion of cell-to-cell contact of the enterocytes.In the acute liver injury group,the signals of ZO-1 were disrupted and irregularly distributed at the outer enterocyte periphery.The content of ZO-1 was obviously lower in the acute liver injury group than that in the control group.Meanwhile,the level of ZO-1 in EP,prevention group was higher than that in the acute liver injury group.The second part of the experiment:The levels of ALT and TNF-αin plasma of contral group had no difference in various times,however,the ALT and TNF-αlevels in the acute liver injury group were much higher than that in nomal group.Pretreat with EP could reduce the release of ALT and TNF-α.We evaluated HMGB1 protein levels by Western blot analysis,and couldn't detect the expression of it in contral group.The HMGB1 levels in the EP group(78.79±8.55ng/ml)were much lower than that in the acute liver injury group(171.75±18.80ng/ml)(P＜0.01).The third part of the experiment:The primary cultured cells were verified as vascular endothelial cells by the examination of the immunohistochemical staining of the factorⅧrelated antigen(vWF).The expression of ICAM-1 and HMGB1 in HUVEC which were analysed by immunohistochemistry was very low without stimulation,however,increased obviously after stimulated with LPS,Pretreat with EP could reduce the release of ICAM-1 and HMGB1.After stimulation HUVEC for 6hour,12hour,24hour and 48hour with 100 ng/ml LPS,HMGB1 in the supernate were increased in a time-dependent manner,and reach the peak at 24hour.When pretreated with EP,the HMGB1 protein levels decreased accordingly.After stimulation HUVEC for 16hour with 100 ng/ml LPS,as compared with 0uM EP group,The HMGB1 levels in the 10uM EP group were much lower than that iri the 5uMEP group.Conclusion:1,Ethyl pyruvate could upregulate the expression of ZO-1 between umbilical vein endothelial cells and inhibit the genesis of intestinal endotoxemia.2,Ethyl pyruvate could reduce the release of TNF-αand HMGB1 from kupffer cells and protect acute liver injury.3,Ethyl pyruvate could reduce the expression of HMGB1 and ICAM-1 from human umbilical vein endothelial cells and ameliorate the hepatic microcirculatory disturbance.