Study About The Effect And Mechanism Of Neoandrographolide Against Endotoxemia

BackgroundHeat stroke is an acute illness caused by the effect of heat during violent physical activity or working in the high temperature.Endotoxemia is an important pathology and physiology process in heatstroke.The prevention and cure is always the hot research.Along with hot-house effect become more serious,people always focus on not only the process and mechanism of the injury but also the treatment strategy and method of it.Hoping to explore further with the problem,people can gain progression in the field of prevention,treatment and rehabilitation of heat stroke.It is important to study the heat stress response caused by heat stroke,which has great meaning in the hygieology and phylaxiology.The pathophysiology mechanism of this disease is not clearly understood presently.Lately,with the research of endotoxin signal transduction pathway in-depth and clear,endotoxemia therapy is gradually turn to the aspect of effecting pivotal receptors' expression in lipopolysaccharide(LPS) signal transduction pathway.Recently,it has been found endotoximia of intestinal plays a key role in the process of the disease.Neoandrographolide has been found recently it's a good medicament to antagonize extrinsic endotoxemia but there was no use of it in heatstroke field in overseas and internal.Therefore,to discuss the effects of Neoandrographolide on heatstroke endotoxemia and LPS signal transduction pathway could clearly know the mechanisms of medicament function.And it would base the foundations of this kind of medicine for applying in heatstroke endotoxemia field.Objective:To observe the effect of Neoandrographolide a novel endotoxin-antagonist made from traditional Chinese medical herbs,in the treatment of endtoxemia of heat stroke through establishing animal model of endtoxemia of heat stroke,to observe it's therapeutic dfficacy to heatstroke endotoxemia.And to build an injury of macrophage model on the LPS to simulate the course of heat stroke, futher approach medicamentous mechanism through ex vivo cell experiment.Provide experimental evidence foe the application of the sort of medicine in the treatment of this disease.Methods:The experiments consisted of two parts.The first part:The effects of Neoandrographolide on heatstroke endotoxemia mice.40 Kunming mice were divided into five groups randomly,including Neoandrographolide group,dexamethasone group,NS group,DMSO group and heat stroke group,eight per group.The dosage of medicine and administer ways were made as same as related reference,Neoangrographolide group is peritoneal injection according to 5mg/kg, dexamethasone group is peritoneal injection according to 5mg/kg,NS group is iso-volume injection.Anus temperature was measured as basal body temperature half an hour before experiments.Administered by different medicines,after haif an hour, the mice of five groups were put into the artificial simulator under circum stances of dry bulb temperature(35±0.5)℃and(65±0.5)%relative humidity which remained certain condition until to die.Measure anus temperature per 30 min,and increased rate of anus temperature and subsistence time of the animals were observed and recorded.Simultaneously,Kunming mice get in the artificial simulator after two hours,every group mice eye globe is culled blood,EDTAK2 anticoagula,to detect the quantity of endotoxin using limuloid method,and to select liver and lung every group mice,the paraffin section of 1×1×0.3cm~3 part of liver and lung dyed by HE kept by malondiadehyde of five groups were made.The pathologic change of liver and lung using light microscope were observed.The second part:study about the mechanism of the medicine.To build an injury of macrophage model on LPS to simulate the course of heat stroke,futher approach meicamentous mechanism through ex vivo cell experiment.Used the macrophage line called RAW264.7 which came from mouse as objection while the LPS was the stimulant in our research.1. RAW264.7 cell culture:RAW264.7 cell was serial subcultivation in the DMEM complete medium.When the cell spread on the bottom of the culture flask nearly,it can be used for research.2.Cellular damage modeling on the LPS:reference internal and abroad literature,excitation cell add the LPS 0.1μg/ml according to experimental demand,and then put them into cell culture incubator of 95%02,5%CO2,and the temperature of the cell culture incubator was 37℃.Cell morphology change in Normal group and the LPS group were observed differently,and accredit normal cell appearance using dyeing.3.Bolting medicine concentration:experiment groups including normal control group,LPS group,Neoandrographolide+LPS group (Neoandrographolide concentration is selected from 1.5,3.0,4.5,7.5,11.25,15,30μmol/L),and set up normal control group,detect to change of cell longevity using MTT to observe medicamentous noxious property,accordingly bolting suitable drug action density used for next experiment.4.Experiment subgroup and detected index:experiment is including normal control group,LPS 0.1μg/ml group,Neoandrographolide+LPS(Neoandrographolide concentration is selected from 3.0,4.5,7.5,11.25,15,30μmol/L).Detected index:①.The expression change of TLR4,NF-ΚB(P50) mRNA using RT-PCR of macrophage;②.The contents change of TNF-αusing ELISA assay;③.The expression change of NF-ΚB(P65) using Western blot;④.The apoptosis circumstance using fluorescent staining of AO/EB and Hoechst,the apoptosis rate using flow cytometer.Result:The first part:The effects of Neoandrographolide on heatstroke endotoxemia mice.1.The physiological change of mice①The increased rate of anus temperature of Neoandrographolide group(5mg/kg) and Androprapholide group(5mg/kg) is slower than DMSO control group,Dexamethasone group,NS group and high temperature control group.And the data of six groups are 0.044±0.001℃/min,0.043±0.001℃/min,0.066±0.004℃/min,0.057±0.003℃/min,0.052±0.001℃/min,0.055±0.003℃/min respectively and there is statistical difference among them(P＜0.05).②The change of survival time of different groups:the survival time of Neoandrographolide(5mg/kg) group and Andrographolide(5mg/kg) group is longer than DMSO control group,Dexamethasone group,NS group and high temperature group significantly(P＜0.05),and the data of six groups are 120.0±3.71min,112.5±4.28min,87.8±3.89min,92.1±6.10min,95.8±4.59min,97±3.22min; and the survival time of Neoandroprapholide(5mg/kg) group is longer than Andrographolide(5mg/kg),there is statistical difference between them.2.The concentration change of endotoxin in plasma Two hours after heat exposure,the concentration of endotoxin in plasma of Dexamethasone group,NS group and high temperature control group is 0.210±0.008ng/ml 0.234±0.013ng/ml and 0.259±0.014ng/ml,and is higher than normal control group(0.104±0.004ng/ml) significantly(P＜0.05).The concentration of endotoxin of Neoandrographolide group is 0.132±0.003ng/ml,is higher than the normal group slightly,there is no statistical difference between them.3.The pathologic change of liver and lung tissue Liver tissue of dexamethasone group,NS group and high temperature control group are consider congestion nuclear swelling,central phlebectasis,it is thus clear that considerable air bladde;but liver tissue of Neoangrographolide group is only see sparing congestion and nuclear swelling,not find air bladder,is familiar with normal group.The second part:study about the mechanism of medicine.1.Detect change of cell longevity using MTT method①The RAW264.7 cells is cultured with LPS(concentration is 0.1,1,10,20,40μg/ml respectively) for 24 hours.The longevity of LPS 0.1μg/ml group is 33.57±7.17%,the other groups is 37.23±8.24%,42.39±10.5%,43.234±5.25%,51.20±15.7%.The longevity of LPS 0.1μg/ml is lower than the other groups,and there is statistical difference between them.②The RAW264.7 cells is cultured with Neoandrographolide(concentration is 3.0,4.5,7.5,11.25,15,30,60μmol/L respectively) for 24 hours.The longevity of these groups is 112.5±6.55%,125.1±7.21%,132.9±8.89%,143.6±5.75%,1134±4.44%,102.7±4.77%,42.66±8.936%respectively.It is thus clear that,from 3.0μmol/L to 11.25μmol/L,cell survival is arising.From 11.25μmol/L to 30μmol/L, cell survival is assumed downtrend.When Neoandrographolide concentration exceeds 60μmol/L,there is conspicuous toxic action to macrophage.Thus,we select 3.0,4.5,7.5,11.25,15,30μmol/L for further research.2.The expression change of TLR4 mRNA of macrophage①After cultured in medium(the concentration of LPS is 0,0.1,1,10μg/ml respectively) for 24 hours, the expression of these groups is different.The expression of LPS 0.1μg/ml group is higher than the other groups significantly(P＜0.05).②After cultured in medium(the concentration of Neoandrographolide is 0,3.0,7.5,11.25,15,30μmol/L respectively) for 24 hours,the expression of these groups is different.The expression of Neoandrographolide 11.25μmol/L group is lower than the other groups significantly(P＜0.05).③After cultured in the medium(concentration of LPS is 0.1μg/ml) for 4 hours,we add the Neoandrographolide(the concentration of it is 3.0,7.5,11.25,15,30μmol/L) in the medium.After cultured for 24 hours,the expression of TLR4 mRNA is different among these groups.The expression of TLR4 mRNA of Neoandrographolide 11.25μmol/L group is lower than the LPS 0.1μg/ml group significantly(P＜0.05).3.The expression change of NF-ΚB(p50) mRNA of macrophage①After cultured in medium(the concentration of LPS is 0,0.1,1,10μg/ml respectively) for 24 hours,the expression of these groups is different.The expression of LPS 0.1μg/ml group is higher than the other groups significantly(P＜0.05).②After cultured in medium(the concentration of Neoandrographolide is 0,3.0,7.5,11.25,15,30μmol/L respectively) for 24 hours,the expression of these groups is different. The expression of Neoandrographolide 11.25μmol/L group is lower than the other groups significantly(P＜0.05).③After cultured in the medium(concentration of LPS is 0.1μg/ml) for 4 hours,we add the Neoandrographolide(the concentration of it is 3.0,7.5,11.25,15,30μmol/L) in the medium.After cultured for 24 hours,the expression of TLR4 mRNA is different among these groups.The expression of TLR4 mRNA of Neoandrographolide 11.25μmol/L group is lower than the LPS 0.1μg/ml group significantly(P＜0.05).4.The expression change of TNF-αAfter cultured in the medium(concentration of LPS is 0.1μg/ml) for 4 hours,we add the Neoandrographolide(the concentration of it is 3.0,7.5,11.25,15,30μmol/L) in the medium.After cultured for 24 hours,the expression of TNF-αis different among these groups.The expression of TNF-αof Neoandrographolide 11.25μmol/L group is lower than the LPS 0.1μg/ml group significantly(P＜0.05). 5.The change of apoptosis of macrophage AO can get into normocellular envelope,show vir or flavor-green fluorescence with DNA,binding with DNA show crocus fluorescence;but EB only can get into impair cell,show fluorescence with DNA.Fluorescent staining of AO/EB experiments show macrophage of LPS group have a great middle-late apoptosis cells and a small die cell.After add into different concentration Neoandrographolide,discovering apoptosis cell grow downwards along with medicine concentration.11.25μmol/L Neoandrographolide group nucleus most show green fluorescence,have thimbleful die cell,similar to normal group;flow cytometry show the similar tendency with AO/EB.Conclusion:1.Neoandrographolide is an effective medicine against endtoxemia which can decrease the increased rate of anus temperature,reduce the moral rate,and can significantly depress the content of LPS in mice blood,at the same time mitigate damage of the important organs such as liver and lung,et al,protect the mice attacked by endotoxin.2.The expression of TLR4 and NK-κB mRNA Induced by different LPS concentration,the expression of TLR4 and NK-κB mRNA of RAW264.7 cells is different,and all of them is higher than the normal control group.But aider using Neoandrographolide,the expression of them is decrease rapidly.It is the important mechanism of this medicine.3.The expression of TNF-αInduced by different LPS concentration,the expression of TNF-αof RAW264.7 cells is different,and all of them is higher than the normal control group.But after using Neoandrographolide,the expression of them is decrease rapidly.It is because Neoandrographolide inhibit the expression of TLR4 and NK-κB mRNA.And it is also the important mechanism of this medicine.4.Study the apoptosis of RAW264.7 cell by AO/EB double fluorescence staining,Hoechst fluorescence staining and flow cytometry.The results of these three methods show that Neoandrographolide can decrease the apoptosis of RAW264.7 caused by LPS.5.The trait that Neoandrographolide against endtoxemia from multiplied ways, courses and targets can provide theory fundament for the clinical application of the medicine which caves a new trail for the treatment of heat stroke.