Study About The Effect And Mechanism Of Artesunate Against Endotoxima Of Heat Stroke

BackgroudHeat stroke is an illness caused by the effect of heat during violent physical activity or working in the high temperature. Along with hot-house effect become make more serious, people always focus on not only the process and mechanism of the injure 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. Recently, it has been found endotoximia of intestinal plays a key role in the process of the disease. According to such theory, using drugs of anti- endotoxin may probably be have the effect positively to the treatment of heat stroke. Abrotani herba is orthodox eliminating summer-heat by cooling drug, beginning of 20 century 70 age, abstract and accredit arteannuin that possess anti-ague active component, miscellaneous invest to arteannuin and it's erivate, as if treat pneumocystis carinii pneumoniaimproveimmunocell, cytoactiveantibiosis and against esogenous endotoxin effect and so on. Then, whether or not artesunate can against endotoxima of heat stroke in the condition of high temperature and hign wet? Now to be short of correlated report still in the world.ObjectiveTo observe the effect of Artesunate 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 efficacy to heatstroke endotoxemia. And to build an injury of macrophage model on the high temperature and LPS composite factors to simulate the course of heat stroke, futher approach medicamentous mechanism through ex vivo cell experiment. Provide experimental evidence for the application of the sort of medicine in the treatment of this disease.MethodsThe experiments consisted of two parts. The first part: The effects of artesunate on heatstroke endotoxemia mice. 32 Jamming mice randomly were divided into four groups including artesunate group,dexamethasone group,NS group and heat stroke group, eight per group. The dosage of medicine and administer ways were made as same as related reference, artesunate group is peritoneal injection according to 60mg/kg, dexamethasone group is peritoneal injection according to 5mg/kg, NS group is iso-volume injection. Anus temperature were measured as basal body temperature half an hour before experiments. Administered by different medicines, after half hour, the mice of four 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 record. 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 every group mice, the paraffin section of 1×1×0.3cm³ part of liver dyed by HE of liver kept by malondialdehyde of five groups were made. The pathologic change of liver using light microscope were observed. The second part: study about the mechanism of medicine. To build an injury of macrophage model on the high temperature and LPS composite factors to simulate the course of heat stroke, futher approach medica -mentous mechanism through ex vivo cell experiment. Used the macrophage line called RAW264.7 which came from mouse as objection while the high temperature and 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 itcan be used for research. 2. cellular damage modeling on the high temperature and LPS composite factors: reference internal and abroad literature, excitation cell add theLPS 1μl according to experimental demand, and then put them into cell culture incubator of 95%O₂, 5%CO₂ (the temperature of the cell culture incubator was40℃). Cell morphology change in Normal group and the high temperature and LPS composite factors group was observed differencely, and accredit normal cell appearance using dyeing.3. bolting medicine concentration: experiment groups including normal control group,high temperature and lps group,artesunate+high temperature and lps group(artesunate concentration is selected to 80,40,20,10,5,2.5,1.25μg/ml), and set up normal control group, detect to change of cell longevity using MTT, observe to medicamentous noxious property, accordingly bolting suitable drug action density use for next experiment. 4. experiment subgroup and determin index:experiment is including high temperature and lps1μg/ml group,artesunate+high temperature and lps group(artesunate concen -tration is selected to20,10,5,2.5,1.25μg/ml )and normal control group, determin index:â‘ the SOD using the method of xanthine oxidase and MDA using TBA of liver homogenate were observed;â‘¡. NF—κB mRNA using RT-PCR of macroph -age and contents of NO;â‘¢. HSP70 mRNA using RT-PCR and proteic express using Western blot;â‘£. the apoptosis circumstance using fluorescent staining of AO/EB, the apoptosis rate using flow cytometer.Results:The first part: The effects of artesunate on heatstroke endotoxemia mice.1. The physiological change of mice The increased rate of anus temperature of Artesunate group is slower than Dexamethasone group,saline group and heat stroke group, the data of four groups are 0.039±0.008℃/min,0.049±0.007℃/min,0.052±0.005℃/min and 0.056±0.009℃/min respectively(P<0.05); the survival time of Artesunate group is longer than any other three groups (P<0.01). These data are 126.3±12.8min,103.3±11.3min,95.8±13.0min and 94.3±7.2min respectively.2. The concentration change of endotoxin in plasma two hours after heat exposure, The concentration change of endotoxin dexamethasone group,saline group and heat stroke group is 0.211±0.037ng/ml,0.239±0.054ng/ml and 0.254±0.030ng/ml, higher insignificantly compared with normal group(0.110±0.010ng/ml) (P<0.01). The concentration change of artesunate group is 0.132±0.006ng/ml, higher a little compared with normal group, but no significant difference(P>0.05).3.The pathologic change of liver tissue liver tissue of dexamethasone group,saline group and heat stroke group are considerable congestion, nuclear swelling, central phlebectasis, it is thus clear that considerable air bladde; but liver tissue of artesunate group is only see sparing congestion and nuclear swelling, not yet air bladder, is close to normal group. The second part: study about the mechanism of medicine.1. detect to change of cell longevity using MTT cell longevity is 55.42±5.18% on the high temperature and LPS composite factors, lower insignificantly compared with normal group (P<0.05). after force-conservancy in different density artesunate(1.25μg/ml,2.5μg/ml,5μg/ml,10μg/ml,20μg/ml,40μg/ml,8μg/ml), cell longevity is: 58.85±5.84%,64.37±6.41%,69.35±8.45%,80.92±11.40%,89.44±10.21%,84.42±9.21 %,74.73±14.37%. It is thus clear that, there is no amelioration also with 1.25μg/ml artesunate, from beginning to 2.5μg/ml, cell persistence is accrescence along with Artesunate concentr to heighten, achieve zenith in 20ug/ml, get close to normal control group. From beginning to 40μg/kg, cell survival is assume downtrend, elucidae, when Artesunate concentr exceed 20μg/kg, there is conspicuous toxic action to macrophage. thus, to select 2.5μg/kg,5μg/kg,10μg/kg and 20μg/kg four effective concentration use in next experiment.2. The expression change of NF—κB mRNA of macrophage The expression of NF—κB mRNA of high temperature and lps group is higher obviously normal control group (P<0.01) , after applicate artesunate, the expression of mRNA is degrade gradually along with medicine saturation higher, have conspicuous quant-potency dependence. 2.5μg/ml artesunate have protection now (P<0.05) , contrast have significance in 10μg/ml (P<0.01), arrive to zenith in 20μg/ml, near to normal control group ,not statistically significant between optimal dose group and normal control group(P>0.05).3. The content change of NO There have conspicuous lure effect to NO on high temperature and lps composite factor, in cell culture clear supernatant liquid the content of nitrite is higher obviously, reach to 56.80±6.57μmol/L; there have distinguished statistical significance compare with normal control group (P<0.01) ; add in invariably concentr artesunate can abaissement obviously the content of NO on high temperature and lps composite factor, along with concentration of artesunate increase (2.5,5,10,20μg/ml) , depressant effect increase to NO, the content of NO is 46.22±8.60μmol/L,25.02±3.51μmol/L,13.85±3.46μmol/L and 7.13±1.83μmol/L, compare to high temperature and lps group, havepatency statistical significance all (P<0.01).4. The expression change of HSP70 mRNA and proteinum of macrophage The expression of HSP70 mRNA is low lever in normal cell, it can induced generous the expression of HSP70 on high temperature and lps composite factor, difference possess significance (P<0.01) ; make use of artesunate, may further induce produce of endogenous cell protector protein HSP70, offer strengthen tendency along with concentrate higher; compare with high temperature and lps group, 5μg/ml artesunate possess statistical significance (P<0.01) , 10,20μg/ml arrived peak, show platform tendency ,between both not statistics contrast (P>0.05). it proteinum express accord with the expression of mRNA.5. The change of MDA and SOD in macrophage The concentration of SOD of normal group cell culture spernate is 14.90±0.652 U/ml, on high temperature and lps composite factor, the concentration of SOD significantlydegrade, it's value 13.07±0.488 U/ml, contrast possess significance compare with normal control group(P<0.01) ; after make use of artesunate, may be dose dependently raise the concentration of SOD, Compare with high temperature and lps, 2.5μg/ml artesunate have statistical significance at once (P<0.05) , 10μg/ml artesunate significance of difference (P<0.01) , in 20μg/ml artesunate the concentration of SOD approach to normal control group, between both notstatistically significant (P>0.05). Likewise, MDA show correspondingtendency also, the concentration of MDA originated high temperature and lps taper along with medicine concentration increaser. The concentration of MDA in noral controlgroup,high temperature and lps group and different concentration artesunate (2.5μg/ml,5μg/ml,10μg/ml,20μg/ml) are 1.75±0.234nmol/ml,3.10±0.156nmol/ml,2.91±0.115nmol/ml,2.90±0.189 nmol/ml,2.11±0.189 nmol/ml and 1.85±0.113 nmol/ml.6. The change of apoptosis of macrophage AO can get into normocellular envelope, show vir or flavor-green fluorescence with DNA,binding with RNA show crocus fluorescence; but EB only can get into impair cell, show samon fluorescence with DNA. Fluorescent staining of AO/EB experiments show macrophage of high temperature and lps group have a great middle-late apoptosis cells and a small die cell, after add into different concentr artesunate, discover apoptosis cell grow downwards along with medicine concentr, 20ug/ml artesunate nucelrs most show green fluorescence, have thimbleful die cell, similar to normal group; flow cytometry show alike tendency with AO/EB.Conclusion:1. Artesunate is an effective medicine against endtoxemia which can decrease the increased rate of anus temperature, reduce the moral rate, and can significant depress the content of LPS in mice blood, at the same time mitigate damage of the important organs such as liver, et al, protect the mice attacked by endotoxin.2. To build an injury of macrophage model on the high temperature and LPS composite factors to simulate the course of heat stroke, even confirm artesunate curative effect of treat heatstroke from in vivo and vitro experiment.3.Artesunate can down regulation the express of NF—κB mRNA that lps signal transduction iter's receptor, abaissement secrete of NO, protect organism function, enhance phagocytosis through increasing energy metabolism of macrophage which can eliminate endtoxemia effectively and restrict indirectly damnification caused by endtoxemia. It is also one of the most important mechanisms of the medicine against endtoxemia of heat stroke, this is important mechanism of bring into full play biological effect.4. Artesunate can further induce to HSP70 express, accordingly increase endogenous protection of damage cell. From a firenew angle approach medicine gene expression changed mechanism on high temperature and lps composite factor, and to include the aspect research lead to penereate.5. Artesunate can abatement cell lipid peroxidation, refrain macrophage apoptosis. The medicine can increase the archaeus of SOD and degrade content of MDA in cell supernate, this can active suppression lipid peroxidation damage to cell cause hige temperature and lps composite factor. Meanwhile, artesunate can restrain macrophage apoptosis, protect macrophage function, strengthen ability to kill lps. So abatemeng cell lipid peroxidation level, refrain cell apoptosis are another important mechanism against heatstroke endotoxemia.6. The trait that Artesunate 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.