Mitigation Of Heat Stress-induced Intestinal Damage In Mice By Lactobacillus Rhamnosus

BackgroundIn a high temperature environment,when the body produces more heat than dissipating,it can lead to a continuous increase in core temperature,heat stress（HS）reaction further develop into heatstroke,which is mainly characterized by hyperpyrexia（usually>40.5 ℃）,central nervous system dysfunction,systemic inflammation and multiple organ function damage.Long-term heat exposure can damage cardiovascular function,leading to syncope,myocardial ischemia and circulatory failure.The insufficient gastrointestinal perfusion leads to local ischemia,coupled with the cytotoxicity of heat stress,which not only increases intestinal permeability and inflammatory response,but also makes intestinal bacteria and endotoxins enter the blood circulation,leading to systemic inflammatory response.After endotoxin enters the bloodstream,innate and adaptive immune systems can sense and respond to endotoxin through toll-like receptors,which leads to the secretion of a variety of pro-inflammatory and anti-inflammatory cytokines.The inflammatory pathway mediated by systemic inflammatory response is not completely clear.The NLRP3 inflammasome,which can recognize exogenous pathogenic microorganisms and endogenous stimulation signals,mediating the release of inflammatory factors such as IL-1β and IL-18,play an important role in systemic chronic inflammatory diseases,but the role of NLRP3 in systemic inflammatory response caused by heat stress is not identified.One of the important causes of systemic inflammation caused by heat stress is the damage of intestinal mucosal barrier,and the mechanical damage of intestinal barrier and inflammatory response are closely related to the imbalance of intestinal flora.Therefore,protecting intestinal mucosal barrier and regulating intestinal flora is the key link in reducing heat stress.Studies have found that probiotics can protect intestinal mucosal damage by increasing the secretion of tight junction proteins between cells,and regulating intestinal flora at the same time.The role of probiotics in alleviating systemic inflammatory response caused by heat stress and regulating intestinal flora is not clear.Therefore,the main purpose of this study is to explore the role of activation of NLRP3 inflammasome in intestinal injury and the mechanism of Lactobacillus rhamnoues in alleviating intestinal injury caused by heat stress.This could provide effective strategies for the prevention and treatment of heatstroke.Methods1.Wild type C57BL/6 mice were used to establish a heat stress model,which was divided into room temperature control（CON）group and heat stress（HS）group.Heat stress group of NLRP3 knockout mice（Nlrp3-/-HS）group and Lactobacillus rhamnosus pretreatment in room temperature（LR）group and in heat stress condition,（LR-HS）group were compared with those group under the same conditions.2.The concentrations of serum IL-1 β and TNF-α were detected by ELISA kit,and the level of endotoxin was detected by endotoxin kit.3.The pathological changes of liver,kidney,spleen,brain and intestine were detected by HE staining.4.Western blot,qPCR and immunohistochemistry（IHC）were used to detect the expression of intestinal tight junction protein（ZO-1 and Occludin）,NLRP3,Caspase-1,IL-1β and TLR4-Myd88-NF-κB signaling pathway in each group.5.16S rDNA sequencing was used to confirm the changes of intestinal flora.6.Pretreatment Caco-2 intestinal epithelial cell with Lactobacillus rhamnosus supernatant was used to detect the changes of cell permeability and permeability after heat stress and the lever of LDH/ROS.Results1.HS model was successfully established:in HS group,the core temperature increased sharply,the anal temperature exceeded 42℃ with convulsions and coma.However,the body temperature in Nlrp3-/-HS group and LR-HS group increased slowly,and the anal temperature and weight loss of HS group were lower than those in HS group.2.The pathological changes in HS group were more severe than those in Nlrp3-/-HS group and LR-HS group.3.The serum contents of TNF-α,IL-1β and endotoxin in HS group increased,only the release of IL-1 β decreased in Nlrp3-/-HS group,but the release of inflammatory factors and endotoxin all decreased in LR-HS group.4.The expression of ZO-1 and Occludin in intestinal tissue decreased in HS group,while the expression of ZO-1 and Occludin in Nlrp3-/-HS group and LR-HS group was higher than that in HS group.5.The expressions of NLRP3,Caspase-1 and IL-1β in intestinal tissue in HS group were increased,while the expressions of NLRP3,Caspase-1 and IL-1β in Nlrp3-/-HS group and LR-HS group was lower.The protein changes of TLR4-Myd88-NF-κB pathway were consistent with NLRP3 inflammasome.6.Analysis of the difference of intestinal flora.（1）Comparing with CON group,the richness radio of intestinal flora in HS group decreased,while the richness radio in LR-HS group was higher than that in HS group.（2）At the phylum level,the richness ratio of Bacteroidetes,Tenericutes and Verrucomicrobia in HS group was significantly lower than that in CON group.On the level of genus,the proportion of intestinal beneficial bacteria such as Akkermansia,Parabacteroides,Parasutterella and Ruminococcus₁ genus in HS group was decreased compared with CON group,while the proportion of common intestinal pathogenic bacteria such as Escherichia-Shigella,Enterococcus and Lachnospiraceae_UCG-001 increased in HS group.But,the level of Lentisphaerae and Verrucomicrobia phylum increased in LR-HS group,along with Akkermansia,Parabacteroides,Parasutterella and Ruminococcus₁ genus was increased,and Escherichia-Shigella,Enterococcus and Lachnospiraceae_UCG-001 genus decreased comparing to HS group.7.Pretreatment with Lactobacillus rhamnosus supernatant alleviated the negative effect of HS on the integrity and permeability of Caco-2 monolayer cell model and reduced production of LDH and ROS.ConclusionThe intestinal injury induced by HS is related to the activation of NLRP3 inflammasome.Lactobacillus rhamnosus can alleviate the intestinal injury caused by HS,which may be related to reducing the activation of NLRP3 inflammasome and regulating intestinal flora.