Effects Of Methylene-blue On Intestinesin Damage In Endotoxic Shock Rats

Objective: To establish the endotoxic shock rat model,determin DAO and iNOS activity in serum ,observe the changes of intestinal pathological and iNOS expression,as well as,to explore the effects of Methylene-blue (MB) in different dosages on intestinesin damage in endotoxic shock rats model in the early stage and to approve the dose effect of MB. Methods:The first step:To establish the endotoxic shock rat model and determin DAO activity in serum.Thirty-two health Wistar rats were randomly divided into 4 groups:â‘ Normal control group: record heart rate (HR) and mean arterial pressure (MAP) under anesthesia.The samples were harvested when 6h later.â‘¡Septic shock(LPS)for 2h group.â‘¢LPS 4h group.â‘£LPS 6h group.The 3 LPS group were all received LPS(0111:B4) 15mg/kg,at 2h,4h,6h obtain the samples respectively.Each group have 8 rats. In the application of Immunohistochemical(IHC) to observe changes of iNOS expression in rat intestine,to analysis the iNOS activity in intestine and iNOS, DAO activity in serum by UV spectrophotometry.Observe morphological changes of intestinal under Light microscope.The second step:Effects of MB in different dosages on endotoxic shock ratsThe time point of highest expression of iNOS and DAO was chosen respectively for interventional study according to the results of step one experiment. Fourty health Wistar rats were randomly divided into 5 groups: Normal group, LPS group, the low-dose,midlle-dose,high-dose group of MB.Each rats from the four groups received LPS 15mg/kg to induce endotoxic shock except normal group.Each rats in MB groups were infused with MB 5mg/kg,10mg/kg,20mg/kg respectively, meanwhile, other rats were infused with the same-dose balanced solution. Each rats were put to death after infused with LPS for 6 hours and obtain the samples.Targets are same to step one experiment.Results: (1)The MAP decreased obviously after injection of LPS, then recovered to normal, and decreased slowly till death.The HR become faster and faster after injection of LPS,after about 5h,decreased sharply till death. LPS increased DAO activity and iNOS activity obviously; the pathology score of intestine was higher than normal control group.(2) MB can be maintained MAP and HR in the normal range. MB groups decreased the increasing level of iNOS and DAO,and compared to LPS group, there was a statistical significant differenc. With the MB dose increased, iNOS activity decreased; DAO activity in midlle-dose group was lowest;the pathology score of intestinal in the middle and low-dose group was low, but there was no significant difference between high-dose group and LPS group.Conclusion: (1) LPS is major components of endotoxin, 15mg/kg of LPS could establish endotoxic shock rat model quickly and easily,and induce intestinal injury.This model appered intestinal swelling, bleeding, exudation and serum DAO activity increased.(2)MB could improve hemodynamic status and rise MAP by inhibiting the abnormal release of iNOS. MB inhibited iNOS formation in dose-depended way. Middle and low-dose MB could reduced the changes of intestinal damage induced by LPS, but large doses of MB would aggratate intestinal injury,which might be associated with high-dose MB inhibit the expression of iNOS strongly induce to decreased NO significantly,so refrain the beneficial effect.