The effect of Xuebijing on the gut microbiota and metabolic of heat 1 1 stroke rat 2

The goal of the present study was to evaluate the fecal microbiome and serum metabolites in 16 Xuebijing (XBJ)-injected rats after heat stroke using 16S rRNA gene sequencing and gas 17 chromatography-mass spectrometry (GC-MS) metabolomics. Eighteen rats were divided into the 18 control group (CON), heat stroke group (HS), and XBJ group. The 16S rRNA gene sequencing results 19 revealed that the abundance of Bacteroidetes was overrepresented in the XBJ group compared to the 20 HS group, while Actinobacteria was underrepresented. Metabolomic profiling showed that the 21 pyrimidine metabolism pathway, pentose phosphate pathway, and glycerophospholipid metabolism 22 pathway were upregulated in the XBJ group compared to the HS group. Taken together, these results demonstrated that heat stroke not only altered the gut microbiome community structure of rats but also greatly affected metabolic functions, leading to gut microbiome toxicity.


Introduction 26
Heat stroke is a life-threatening syndrome induced by environmental or metabolic factors. Heat 27 stroke causes body temperature dysregulation, sweat gland failure and electrolyte disturbances 1 , 28 2 leading to debilitating central nervous system damage and the potential for the subsequent rapid 29 development of cardiovascular dysfunction 2,3 . Body temperature continues to rise, cardiac output is 30 difficult to maintain balance, induce cytotoxicity and inflammation, and ultimately lead to multiple 31 organ failure 4-6 . 32 The gastrointestinal mucosal barrier has the function of preventing harmful substances in the 33 intestinal cavity from entering the blood circulation. When heatstroke occurs, intestinal blood loss, 34 intestinal blood flow decreases, intestinal epithelial cell vitality decreases, and cell wall permeability 35 increases 7 . In addition, the oxides and nitrosamines produced in the intestine can stimulate the cell 36 membrane and destroy the tight junctions between cells. The endotoxins and potential pathogens in 37 the intestine enter the blood, pass through the systemic circulation, and reach the liver. The liver's 38 detoxification ability cannot meet the demand, leading to toxemia. Microbial products in the intestine 39 will also be transferred to the systemic circulation 8 . The gastrointestinal microbiota is a collection of 40 microorganisms that colonize the gastrointestinal tract, which have co-evolved inside humans and are considered 41 to provide several mutually beneficial host functions 9 , including digesting food components, synthesizing vitamins,

Pathway analysis of metabolites 135
KEGG analysis was also used to identify the most crucial signal transduction pathway and 136 biochemical metabolic pathway relevant to differential metabolites. The differential metabolites of the 137 CON and HS groups were primarily enriched in the ascorbate and aldarate metabolic pathways ( Fig.  138 3A, 3B). Different metabolites between the HS_XBJ and HS groups were distributed in the pyrimidine 139 metabolism pathway, pentose phosphate pathway, and glycerophospholipid metabolism pathway (Fig.  140 3C, 3D). 141

DISCUSSION 142
After heat stroke, the intestine is the most vulnerable target organ 16 . Intestinal barrier dysfunction 143 is related to the development of multiple organ dysfunction syndrome caused by trauma-hemorrhagic 144 6 shock 17 . Our results showed that the intestinal tract showed oozing blood and that the intestinal barrier 145 was damaged after heat shock, indicating that heat shock is likely to cause trauma-hemorrhagic shock. Bacteroidetes. Prevotella is the primary fiber-degrading genus in the in the phylum Bacteroidetes. The 169 decrease in Prevotella-9 and Prevotella-1 indicated that the fiber digestion ability was weakened in the 170 HS group, which may have affected the energy supply. In addition, members of the phyla 171 Actinobacteria and Proteobacteria were also the primary dominant bacteria in the guts of rats. 172 Compared to the control group, members of both phyla increased after heat shock. The phylum 173 Proteobacteria was previously reported to be the primary source of various genes involved in carbon 174 metabolism and the biosynthesis of unique secretion systems. Moreover, the phylum Proteobacteria 175 includes many pathogens, such as Escherichia coli, Salmonella, Vibrio cholerae, and Helicobacter 176 pylori, which cause intestinal inflammation and damage to the intestines 24 . An increased abundance 7 of Proteobacteria and a decreased abundance of Bacteroidetes was observed in the guts of patients with 178 kwashiorkor 25 . The alterations in Proteobacteria and Bacteroidetes abundances observed in the present 179 study were similar to those observed in the above study, indicating that heat stroke may cause 180 malnutrition in rats. With XBJ injection after heat shock, the proportion of Bacteroidetes was close to 181 that of the CON group, while the proportion of Proteobacteria was close to that of the HS group, 182 indicating that XBJ may promote Bacteroidetes proliferation but may have no such effect on 183 Proteobacteria. The abundance of the Actinobacteria phylum increased in the HS group, but the 184 abundance of Roseburia, a member of the phylum Actinobacteria, decreased. Members of the genus 185 Roseburia can ferment carbohydrates into butyric acid. In a previous study, Roseburia was 186 transplanted into the intestine of apolipoprotein E-deficient mice, and the results showed that systemic 187 inflammation was relieved, the risk of atherogenesis was reduced, fatty acid utilization was accelerated, 188 and glycolysis was slowed 26 . In addition, the removal of butyric acid-producing bacteria often induces 189 the overexpression of nitric oxide synthase type 2 and the production of nitrate, which eventually leads Faecalibaculum can regulate symbiotic probiotics and improve the energy metabolism pathways, such 202 as those associated with short-chain fatty acids, carbohydrates, and amino acids 32 . The increase in 203 Bifidobacterium and Faecalibaculum after heat shock may be related to the self-repair of intestinal 204 damage, but further proof is needed to assess this possibility. 205 Vitamin C (Vc), also called ascorbic acid, is a polyhydroxy compound that easily undergoes 206 oxidative dehydrogenation reactions. Vc participates in the synthesis and decomposition of many 207 important metabolites 33 . In the present study, the ascorbic acid and aldonic acid metabolic pathways 208 were observed to be downregulated after heat shock, indicating that heat shock would cause a decrease 209 in Vc. The results of a previous study showed that Vc is highly effective in treating sepsis patients 210 8 with a body temperature ≥37.1℃ and white blood cell count ≥15.0 1000/mm 3 , indicating that Vc is 211 associated with fever and high inflammation 34 . Vc not only inhibits the expression of pro-212 inflammatory cytokines but also directly eliminates reactive oxygen species, thereby maintaining 213 cortical barrier function, which indicates that intestinal injury in the heat shock group may be related 214 to a decrease in the Vc content 35 . In patients with septic shock, increasing the clearance rate of 215 thiamine and lactic acid can greatly reduce mortality, showing that thiamine and Vc can alleviate the 216 damage caused by oxidative stress 36 . The findings of such studies provide more possibilities for Vc 217 treatment in patients with sepsis 37 . Compared to that observed in the HS group, the metabolite 218 glucuronate was significantly higher in the HS_XBJ group. Glucuronate is related to anticoagulation, 219 indicating that XBJ can increase the production of Vc and promote blood coagulation. It has been 220 reported that an impaired intestinal barrier can lead to interruption of oxygen and nutrient supplies, 221 which is the primary cause of metabolic changes in specific tissues 38 . Therefore, we hypothesize that 222 heat shock may disrupt those changes that are primarily used to compensate for intestinal dysfunction. 223 The results showed that after the injection of XBJ, the pentose phosphate pathway metabolic pathway 224 was significantly upregulated in the intestinal tract of heat shock-affected rats, especially the level of 225 5-phosphate ribose, which increased significantly. Ribose 5-phosphate plays a crucial role in 226 nucleotide and nucleic acid synthesis 39 . In the pentose phosphate pathway, ribose 5-phosphate can be 227 Sphingosine 1-phosphate (S1P) is one of the metabolites of sphingomyelin, a natural lysophosphatidic 236 with important biological activity, which is involved in many physiological processes, such as cell 237 growth, proliferation, migration, cytoskeleton remodeling and lymphocyte transport 44 . S1P is 238 converted from sphingosine through sphingosine kinase and has the effect of preventing oxidative 239 damage 45 . In the present study, the S1P content in the HS group was significantly increased and was 240 significantly reduced in the XBJ group. In addition, phosphatidylcholine is the primary component of 241 low-density lipoprotein and is related to the metabolism of polyunsaturated fatty acids and 242 sphingomyelin 46,47 . After injection with XBJ, the level of phosphatidylcholine decreased, and the 9 content of 1-acyl-sn-glycero-3-phosphocholine increased. Therefore, it is speculated that XBJ can 244 protect intestinal health by inhibiting the sphingolipid metabolism pathway in heat shock-affected rats. 245 In summary, heat shock significantly modified the structure and composition of the gut bacteria in 246 rats. In the present study, we demonstrated heat shock-induced microbial disorders and the 247 dysregulation of microbial metabolites. The results showed that the abundance of Bacteroidetes 248 decreased and the abundance of Actinobacteria increased after heat shock, while the abundance of both 249 phyla in the XBJ injection group was consistent with that of the control group. At the genus level, heat 250 shock inhibits the proliferation of the Prevotella-9 and Prevotella-1 genera, but XBJ can promote their 251 reproduction. Furthermore, we demonstrated the capabilities of a nontargeted GC-MS-based 252 metabolomics approach to successfully identify 170 different metabolites between the CON and HS 253 groups and 37 different metabolites between HS and XBJ groups. 254

Animals and Reagents 256
The experimental procedures were approved by Ethics Committee of the General Hospital of Infinity ultrahigh-performance liquid chromatograph and an Agilent 6538 UHD and Accurate-Mass 331 Q-TOF mass spectrometer with an ACQUITY UPLC®HSS T3 column (2.1×100 mm, 2.5 µm). The 332 original data were transformed using Agilent MassHunter Qualitative Analysis B.04.00 (Agilent 333 Technologies, USA). The processed data set was then entered into the SIMCA-P software package 334 (v13.0, Umetrics, Umeå, Sweden). The normalized data were then used to perform principal 335 component analysis (PCA) and orthogonal partial least squares-discriminate analysis (OPLS-DA) 49 . 336

Statistical analysis 337
Statistical analyses were performed using GraphPad Prism version 8.0. All data are presented as the 338 means ± SEMs. Multiple comparisons were analyzed using one-way ANOVA. Statistical significance 339 was defined at P≤ 0.05.