Study On The Mechanism Of The Effect Of Different Drinking Water On The Intestinal Microbiota Of Rats Under High Temperature Environment

Objective:Under the high temperature environment,the body’s demand for water will increase significantly,and the impact of drinking water on health will be more prominent at this time.Studies have revealed that the intestinal flora of the organism has a deep relationship with health,while the status of intestinal flora is related to factors such as gender,race,environment,food and water.Currently for different drinking water on the impact of high temperature environment of the body’s intestinal flora research is still relatively rare.The purpose of this study was to investigate the effect and mechanism of different drinking water on the intestinal flora of rats with short-term dehydrated heat exposure and long-term heat exposure.Provide data support for the public to choose suitable healthy drinking water in high temperature environment.Methods:1.Effects of different drinking water on intestinal microflora in rats with short-term thermal dehydration:The 6-week-old SD male rats were randomly divided into five groups:pure water group,natural water group,mineral water group,tap water group,and tap water control group.The first 4 groups were dehydrated for 2 hours per day in a small animal incubator at a temperature of（35±1）℃and humidity of 65%±10%with no food and water,so that the rats were moderately dehydration（dehydration weight loss of 4%or more）,and then the rats were allowed to drink purified water,natural water,mineral water and tap water freely for 2 hours,respectively;for 7 days.The rats were allowed to drink tap water for the rest of the time.Samples of blood,tissues,and blind contents were collected from rats on days 1 and 7,respectively,for the detection and analysis of blood biochemical indexes,organs and intestinal flora.2.Study on the mechanism of the effect of different drinking water on the intestinal flora of rats under long-term high temperature environment:Six-week-old SD male rats were randomly divided into six groups:purified water heat exposure group,natural water heat exposure group,mineral water heat exposure group,purified water control group,natural water control group,and mineral water cotronl group.The purified water heat exposure group,the natural water heat exposure group,and the mineral water heat exposure group were exposed to heat at a temperature of（35±1）℃and relative humidity of 65%±10%for 2 hours per day,while the purified water control group,the natural water control group,and the mineral water control group were placed at room temperature（25±3）℃and the same humidity for 2 hours per day for28 days.The blood,tissue and fecal samples of rats in each group were collected for serum electrolyte,blood lipid,liver and kidney function,neuroendocrine,16S r DNA sequencing and metabonomic analysis.Fecal bacteria transplantation experiment:The 6-week-old SD rats were randomly divided into transplanted pure water heat exposure group,transplanted natural water heat exposure group,transplanted mineral water heat exposure group,and control group.The intestinal flora of rats in the three groups after long-term heat exposure to drinking water were transplanted to the transplanted purified water heat exposure group,the transplanted natural water heat exposure group,and the transplanted mineral water heat exposure group using the fecal bacteria transplantation technique,in order to make the flora colo-nized,and then the heat resistance experiment was conducted.The rat fecal samples were collected for 16S r DNA sequencing analysis during the experiment.Results:1.The rate of weight loss in each drinking water group on days 1 and 7 of thermal dehydration was above 4.99%,indicating that the rat model of moderate dehydration was successfully established.On day 1,serum adrenocorticotropin-releasing hormone（CRH）,adrenocorticotropic hormone（ACTH）and cortisol（Cor）concentrations were signifi-cantly lower in the purified water group than in the tap water group,serum total choles-terol（CHO）and hypothalamic noradrenaline（NA）were significantly lower in the puri-fied water group than in the mineral water group,and serum thyroxine（T4）was signifi-cantly higher than in the tap water and mineral water groups;serum CRH concentration in the mineral water group was significantly lower than that in the tap water group.On day 7,liver T-AOC and serum creatinine（CRE）and NA concentrations were significantly lower in the tap water group compared with the tap water control group;serum ALD,hypothalamic 5-hydroxytryptamine（5-HT）and dopamine（DA）concentrations were sig-nificantly higher in the purified water group than in the tap water group,and serum so-dium ion（Na⁺）and chloride ion（Cl^-）were significantly higher than in the mineral water group;serum magnesium ion(Mg²⁺)concentration and creatinine（CRE）concentration in the mineral water group were significantly higher than those in the tap water group.Different drinking water alters the intestinal flora of short-term dehydrated heat-ex-posed rats.On day 1,PD＿whole＿tree and Chao1 index were elevated in the tap water group compared with the tap water control group;Shannon index and ace index were higher in the purified water group than in the tap water group,and PD＿whole＿tree,Chao1,and ace index were significantly higher in the mineral water group than in the tap water group.There were differences inβ-diversity among the purified water group,tap water group and mineral water group.On day 7,PD＿whole＿tree was significantly lower in the pure water and natural water groups than in the mineral water group.Further through Metastat analysis,it was found that Lactobacillus significantly decreased in the purified water group compared to the tap water group,and Bacteroides,NK4A214＿group,Chris-tensenellaceae＿R-7＿group,Oscillibacter,and Colidextribacter significantly increased;mineral water group compared to tap water group[Eubacterium]＿siraeum＿group and Akkermansia significantly increased,Akkermansia was associated with improved metab-olism.The joint analysis revealed that serum Mg²⁺concentration in the mineral water group was positively correlated with Akkermansia.2.After 28 days of heat exposure,serum total cholesterol（CHO）and high-density lipoprotein cholesterol（HDL-C）concentrations of rats in the purified water heat-exposed group showed a decrease and were significantly lower than those in the mineral water heat-exposed group.Liver malondialdehyde（MDA）,superoxide dismutase（SOD）and total antioxidant capacity（T-AOC）concentrations were significantly higher in the purified water heat-exposed group than in the mineral water heat exposed group.The concentrations of serum ACTH and CRH in the mineral water heat exposure group were significantly lower than those in the mineral water control group.Using Metastat to analyze the differential flora of different drinking water heat exposure groups,it was found that the relative abundance of the harmful flora Prevotellaceae＿Ga6A1＿group and Desulfovibrio in the mineral water heat exposure group was significantly reduced,while the relative abundance of butyrate-producing bacteria Ruminococcus and Blautia increased significantly in mineral water heat exposure group.Non-targeted metabonomic analysis showed that some beneficial metabolites increased significantly in mineral water heat exposure group,such as 4-phenylbutyric acid,L-（+）-arabinose,N-acetyl-D-glucosamine,carnosine,N6-acetyl L-lysine,DL-hydrastine,L-cysteine,goose deoxycholic acid and citrulline.Rats in the transplanted purified water heat exposed group,the transplanted natural water heat-exposed group,and transplanted mineral water heat-exposed group all showed a significant increase in heat tolerance after colonization with long-term heat exposed intestinal flora.The relative abundance of Rikenellaceae＿RC9＿gut＿group was signifi-cantly increased and the relative abundance of Lactobacillus was significantly decreased in all three groups compared with the control group.Transplanted purified hydrothermal exposure group and transplanted natural hydrothermal exposure group showed a signifi-cant decrease in beneficial bacteria genus Clostridium＿sensu＿stricto＿1 and a significant increase in harmful bacteria genus Helicobacter compared to the control group.Conclusion:1.Short-term dehydrating heat exposure followed by consumption of purified water is not conducive to recovery of serum electrolytes,inhibition of HPA axis activation,reduction of metabolic heat production in the body,and can lead to hypothalamic damage and a significant increase in pathogenic bacteria in the intestinal flora.Mineral water is rich in minerals,which can help restore the electrolyte balance of rats caused by heat-exposed dehydration,improving metabolism,accelerating the body’s recovery and improving intestinal flora disorders.2.Long-term heat exposure to mineral water can reduce oxidative damage to the liver,regulate lipid metabolism,and protect the nervous system in heat-exposed rats.The mechanism of action may be that mineral water rich in minerals（calcium,magnesium,lithium,strontium ions）is involved in the organic metabolism,nervous system regulation,promoting the increase of beneficial intestinal flora and the decrease of harmful flora,increasing beneficial fecal metabolites,thus promoting the health of the organism.When the fecal intestinal flora of three kinds of drinking water long-term heat-exposed rats were colonized to normal rats,the heat resistance of the three transplanted groups was signifi-cantly increased,among which the longest heat resistance time was transplanted to the heat-exposed group of mineral water rats.It was shown that long-term heat exposure by drinking different drinking water could regulate the intestinal flora of rats and affect the heat resistance and health status of rats.