Experimental Study On The Preventive Effects And Mechanisms Of Glutamine Combined With Vitamin C Against Heat Stress Induced Enterogenous Endotoxemia In Rats

Background and Purpose:Heat illness is a group of acute disease occurring under constant exposure of thermalradiation and high temperature. According to people,s various tolerance toward it, Thesyndrome consists of thermoregulation dysfunction, water, electrolyte metabolic disorders,and a series symptoms caused by central nervous system and circulatory systemdysfunctions. With the development of global warming caused by greenhouse effect, Heatillness has become a common and frequently-occurring disease in summer. The prevalenceand mortality are increasing year by year, especially in those engaged in high-intensityphysical works under hot weather, including the soldier,s daily field trainning. Thus,inrecent years, the army has added the prevention of heat illness to the common regulations.Related research suggests that the elevated body temperature reduced the obstruction ofintestinal epithelial cells and increase the open rate of cell bypass, then enterogenousendotoxin (ET) followed by inflammatory mediators that it mediated poured into the body’sblood circulation, lead to enterogenous endotoxemia. Role of enterogenous endotoxemia inthe process of heat stroke and its prognosis has gradually raised great attention among themedical world. Current study suggests that the oxidative stress response and enterogenousendotoxemia are both important pathophysiological basis of heat stroke and itscomplications. As one of the body’s important immune defense system, Intestinal barrierfunction is very important in the whole stress process during the heat illness.So complicated the Causes of heat illness is, that the prevention and treatment shouldfocus on multiple pathogenesis and a single drug is often too difficult to achieve goodresults,. But combination therapy or multi-ring drug offen works. Glutamine is an efficientantioxidant, which can act directly on the intestinal epithelial cells, and enhance theirvitality. It can promote intestinal epithelial cells to differentiate, proliferate, repair,regenerate and enchance the intestinal immunity. What,s more,it can also reduce the absorption of enterogenous endotoxin and inflammatory mediators into the blood effectively,and plays a positive role in the protection of intestinal mucosa structure and barrier function.Vitamin C is a highly effective antioxidant,which can remove the oxygen free radicals fromthe body. Otherwise,,It can reduce oxidative stress, alleviate the tissue damage effectivelyfollowed by promotting it to repair, improve the barrier defense function of the mucosal anddecreased the occurrence of enterogenous infection, then prevent heat stroke from heatstress effectively. In heat illness,toll receptor plays a leading role in the pathogenesis of thebody’s systemic endotoxemia. As one of the main components of the Toll family,TLR2receptor is of great importance in the inter-cell interactions and is dominant in the signaltransmission. It also acts as a bridge between specific gene expression and extracellularsignal transmission. Therefore, TLR2receptor has been recognized as one of the startgate of the inflammatory reaction and become a hot spot of medical research in this field.As an important transcription factor, HSF1takes effects through the following signaltransductions in heat stress response. To throw light on its molecules mechanism correctlyhas become an important research direction in medical world. In this experiment, We useglutamine combined with vitamin C, which were commonly used in clinical treatment asthe prevention drug, to study the drugs,preventive effect against enterogenous endotoxemiacaused by heat stress in rats. We have further explored the preventive effects and its possiblemechanism of the glutamine combined with vitamin C against the heat stress, and thenprovided a theoretical basis for preventing heat illness effectively with medical medication.This study is based on our early human trials. Now we continue to exploring its mechanismthrough animal experiments.Through detecting the changes of the superoxide dismutase(SOD), malondialdehyde (MDA), content of serum endotoxin, expression of TLR2mRNAfrom the tissue of the rats；survival time observed in each group tested rats by hightemperature lethal experiments, the expression changes of heat shock transcription factor1(HSF1), the ultrastructural changes of small intestine in the experiment of high-temperaturelethal, we further study the preventive effect and its mechanism of glutamine combined withvitamin C against heat stress-induced enterogenous endotoxemia, then provide thetheoretical basis for preventing heat stroke with clinical medication.Methods:1、the test of heat stress endurance of SD rats: We have the160Sprague-Dwley (SD) health male rats (12-14weeks of age, body mass (205±25) g), divided into five groupsaccording to the random number table: the group of glutamine combined with vitamin C,the glutamine group, the vitamin C group, the heat stress group, and the normal controlgroup. To simulate the high-temperature induced heat stroke environment,The first fourgroups have been taken into the artificial climate chamber simultaneously for2hourseveryday, normal control group has been left at room temperature. The simulatedtemperature in the artificial climate chamber:(38±1)℃, relative humidity:(65±10)%. Thegavaged doses of glutamine enteric-coated capsules:216mg/(kg d), the gavaged dose ofVitamin C tablets:135mg/(kg d),1time/day. The rats in the same batch of stress groupswere given medicines at60min before the experiment everyday, and were gavaged by5mlsaline dissolved and diluted. The single treatment group were given by the single drug, thecombined group were given by the combination of two drugs, and the heat stress groupwere given by5ml saline only. The groups were free to eat and drink before the experiment,but were deprived of water and food strictly in the process of the experiment, and were freeto water and food after the experiment. On the1st,3rd,7th,15thday after stressing, theywere anesthetized from abdomen using2%amyl barbital sodium(4mg/Kg), then took thecleaning intestinal tissue into liquid nitrogen tank for snap-freezing, and blood2-3ml fromthe heart immediately to a special tube of non-heat source, centrifuged10min,4000r/min,then the upper layer serum samples were stored at-20℃in the refrigerator for indexdetection. Before taken the specimens, the rats needed24h fasting and4h water deprivation.Using RT-PCR technique to detect the Toll-like receptor2(TLR2) mRNA expression insmall intestine, then we can explored the role of intestinal barrier in heat stress inducedenterogenous endotoxemia.2、the test of SD rats lethal by high-temperature:40Sprague-Dwley (SD) healthy malerats,11-13weeks of age, body mass (200±15) g, clean grade, were randomly divided intoglutamine combined with vitamin C group, the glutamine group, the vitamin C group, theheat stress group and normal control group (n=8). The first four groups were taken into theartificial climate chamber simultaneously, with constant heat stress, the temperature inartificial climate:(41±0.5)°C, relative humidity:(65±20)%; and the normal control groupwas left at room temperature.Take the small intestine tissue immediately after they weredead in the high-temperature, randomly divided into three,one were stored in liquid nitrogen tanks for detecting the associated index; one were fixed by10%paraformaldehydefor stained with HE to observe of the intestinal tissue structure and detecting the expressionof HSF1by immunohistochemistry; the left were fixed by2.5%glutaraldehyde forobserving the ultrastructural changes of small intestinal epithelial cells by transmissionelectron microscope.Results:1、The results in the test of heat stress endurance of SD rats show:(1) Compared withthe normal control group, at the same time of heat stress, the content of SOD significantlyreduced, and MDA, serum endotoxin and the expression of TLR2mRNA were allsignificantly increased (p <0.05). There was no significant difference in the contents ofSOD and MDA in the three drugs intervention group (p>0.05). There were significantlyincease in the content of serum endotoxin and the expression of TLR2mRNA in thesingle-glutamine group and the single-vitamin C group (p <0.05). There was no significantdifference on the content of serum endotoxin and the expression of TLR2mRNA in theglutamine group combined with vitamin C group(p>0.05).(2) Compared with them in theheat stress group, the contents of SOD in the same time period in the three drugsintervention groups were significantly increased (except on the7th day in the glutaminesingle-group and single-vitamin C group); the contents of MDA, endotoxin and theexpression of TLR2mRNA were significantly reduced (except the expression of TLR2mRNA on the3rd day,15th day in the vitamin C group)(p <0.05).(3) In the single-vitaminC group and single-glutamine group, the content of SOD were similar to in the glutaminecombined with vitamin C group at the same time, but the content of MDA and serumendotoxin, and the expression of TLR2mRNA were decreased significantly (p <0.05).2、The results in the test of SD rats lethal by high-temperature show:(1) Comparedwith the same time high-temperature stress group, the survival time of rats with three drugsin the intervention group was significantly prolonged(p <0.05); the same time periodglutamine combined with vitamin C group rats survived significantly longer than singleglutamine group and vitamin C group(p <0.05).((2) The same time the optical density ofglutamine combined with vitamin C group HSF1mean significantly higher than the normalcontrol group and the heat stress group(p <0.05),single mean optical density of theglutamine group and the vitamin C group HSF1no significant difference compared with the normal cormal control group and high temperature stress group (p>0.05); the same timeperiod the combined treatment group and a single treatment group compared to the opticaldensity of HSF1mean no significant difference (p>0.05).(3) we found that the structure ofsmall intestinal microvilli, nucleus, cytoplasm and endoplasmic reticulum was normal, andhad less damage in the glutamine combined with vitamin C group by transmission electronmicroscopy. We can find homogenization changes of Nucleus in the single-glutamine group.The nucleus and cytoplasm in the single-vitamin C group showed the changes of edema,electron density decreased. The small intestinal cells were heavily damaged in the heatstress group, with their microvilli disordered, the cytoplasm and nucleus dissolved,endoplasmic reticulum and mitochondria significantly swollen. In the same batch ofexperiments, the survival of the SD rats in the drug intervention group and the tolerance toheat stress in high temperature are better than in the control group, and it is most obvious inthe combined treatment group.Conclusion:The research results show:First，As the sources of nutrients, energy and antioxidant to the intestinal epithelialcells, the application of Glutamine combined with vitamin C can not only effectively reducethe damage of small intestinal mucosa barrier caused by heat stress effectively, but alsopromote the repair of the small intestinal mucosa. Meanwhile, It plays a positive role inconserving the function of the small intestine mucosal barrier, and reducing the occurrenceof enterogenous endotoxemia effectively, Thus it can be used to prevent heat sroke andenterogenous endotoxemia resulted from heat stress.Second, the prevention mechanism of glutamine combined with vitamin C against theenterogenous endotoxemia and heat stroke may be:1.They can not only increase the content of superoxide dismutase (SOD) of theintestinal tissue after heat stress, but also reduce the content of malondialdehyde (MDA),and TLR2mRNA. They can inhibit the oxygen free radicals effectively, suppress oxidativestress, inhibit the production of cytokines, thus alleviate the damages of intestinal tissue andcells in heat stress.2. It an alleviate the damage of the pathological structure as well as ultrastructure ofthe small intestinal tissue and enhance the expression of the HSF1protein. Besides, it can also reduce intestinal absorption of endotoxin produced by heat stress and effectively reducethe accumulation of toxins in vivo. Consequently, it prevent the occurrence of MODSefficiently.