The Roles And Mechanisms Of Gut Microbiota And ANG Ⅱ-AT₁R Pathway In Cold-induced Hypertension

Objectives:The rat cold-induced hypertension（CIH）models were established by cold exposure,and the fecal microbiota transplantation（FMT）was conducted to explore the roles and mechanisms of gut microbiota in blood pressure regulation under cold stress.By analyzing the gut microbiota composition using microbial 16S rDNA amplicons sequencing,we further explored the roles of gut microbiota and ANGⅡ-AT₁R pathway involved in the development of CIH.Methods:SPF-grade Sprague Dawley rats were purchased and adaptively fed for one week.Rats were then randomly divided into normal temperature group（NT,n=32）and low temperature group（LT,n=32）according to body weight.Rats in the group of LT were exposed to continuous cold exposure（4±1°C,22 h/day）in a meteorological environment simulation chamber.And rats in NT group were placed in a clean-grade normal temperature animal room（20±2°C）for breeding.Cold exposure lasted for 6weeks to achieve the CIH models.FMT donors were selected from the groups of LT and NT.Feces of all rats were collected once a week.To explore the roles of gut microbiota in blood pressure regulation,FMT was conducted in four groups:the rats in NT group+feces from CIH rats（NL group,n=9）,the rats in NT group+feces from rats in NT（NN group,n=9）,the rats in LT group+feces from rats in NT group（LN group,n=9）and the rats in LT group+feces from CIH rats（LL group,n=9）.The FMT was conducted for three times in two weeks.The biomarkers included and relevant analyses were as follows:（1）Daily food intake and water consumption were monitored.Rat body weight（g）,heart rate（times/min）,anal temperature（°C）and blood pressure（mmHg）,including systolic blood pressure（SBP）,diastolic blood pressure（DBP）and mean blood pressure（MBP）were measured once a week during the modeling.After FMT,the indicators were measured after each FMT.（2）After FMT,rats were sacrificed.Plasma was separated from blood samples of the rats.The brains,hearts,kidneys,and livers of rats were also separated and weighed which were then used to calculate organ coefficient.The blood lipid levels of rats were detected,including total cholesterol（T-CHO）,triglyceride（TG）,low density lipoprotein（LDL）and high density lipoprotein（HDL）.（3）Feces were collected from the six groups respectively and 16S rDNA microbial amplicons were sequenced.The sequencing data were used for OTU analysis,Alpha diversity analysis,Beta diversity analysis,and difference analysis of microbiota.The Spearman correlation analysis between microbiota and SBP was conducted in the levels of family and genus.（4）A series of biomarkers were detected,including AngiotensinⅡ（ANGⅡ）,Nitric oxide（NO）,Superoxide diamutase（SOD）,Inducible nitric oxide synthase（iNOS）and Lactic dehydrogenase（LDH）.The expression levels of AngiotensinⅡreceptors 1（AT₁R）were detected by western blotting.To observe the changes of intestinal structure,ileum and colon tissues were prepared for HE pathological sections.（5）By a semi-quantitative method,mucoprotein 2（MUC 2）and tight junction protein（Occludin,OCLN）were detected by immunohistochemical sections of colon tissue.And the metabolic levels of acetic acid,propionic acid and butyric acid in feces were measured by gas chromatography.Results:（1）During the period of cold exposure,the rats in the LT group consumed more food than those in the NT group（P<0.05）,while the water consumption was only significantly lower than NT group in the first,second,and fifth weeks of modeling（P<0.05）.Compared to the rats in NT group,body weight of LT group was significantly lower after the first week（P<0.05）.The blood pressure of the rats in LT group increased obviously in the first two-week cold exposure.The pressure then decreased and maintained at a high and stable level to some extent from the third week of cold exposure and the difference was statistically significant（P<0.05）.From the third week to the sixth week of cold exposure,the SPB of group were 142.33±10.38mmHg,141.83±12.40 mmHg,139.65±11.51 mmHg,137.99±11.66 mmHg,respectively.After FMT,compared with the NN group,SBP of rats in the LL and LN groups were significantly higher（P<0.05）.Although the difference was not statistically significant（P>0.05）,the SBP in the groups of NL and LL was higher than those in NN and LN groups respectively.The T-CHO,TG,and HDL levels in the LN group were significantly higher than those in the NN and NL groups（P<0.05）.The concentrations of T-CHO and TG were significantly higher in the group of LL than those in the NL group（P<0.05）.And the concentrations of HDL in the LL group were significantly higher than those in the NN and NL groups（P<0.05）.For LDL,there was no statistical difference among the groups（P>0.05）.（2）The results of 16S rDNA amplicon sequencing showed that the top three phylums were Firmicutes,Bacteroidetes and Proteobacteria.After cold exposure and FMT,the gut microbiota composition of the rats in each group had a large variation.The ratio between Firmicutes and Bacteroidetes（F/B ratio）in the LL group increased,but the difference was not statistically significant（P>0.05）.Alpha diversity analysis and Beta diversity analysis showed that cold exposure and FMT resulted in lower gut microbiota diversity.And the differences between groups were greater than the differences within the group.Difference analysis of microbiota showed that there was more enrichment of beneficial bacteria in the NT group and enrichment of pathogenic bacteria or conditional pathogenic bacteria in the LT group.Microbiota composition changed significantly after FMT.Spearman correlation analysis showed that some bacteria were significantly correlated with SBP after FMT.At the levels of family,Acidaminococcaceae was positively correlated with SBP;Lachnospiraceae,Rikenellaceae,Peptococcaceae,Clostridiales vadin BB60 group and Elusimicrobiaceae were negatively correlated with SBP.At the genus level,only Phascolarctobacterium is positively correlated with SBP;Ruminiclostridium 9,Oscillibacter and Rikenellaceae RC9 gut group were negatively correlated with SBP.（3）Compared with NN and NL groups,plasma ANGⅡconcentrations in LL and LN groups significantly increased（P<0.05）;SOD activity in kidney tissue significantly reduced（P<0.05）;AT₁R expressed more in the LN and LL groups（P<0.05）.Plasma NO concentrations in the NL,LL,and LN groups significantly reduced（P<0.05）compared to the NN group.Although LDH activity in rat kidney increased in NL,LL and LN groups,there was no significant difference（P>0.05）.Compared with the NN group,iNOS activity in NL and LN groups increased significantly（P<0.05）;Intestinal pathology showed that the wall thickness decreased,the intestinal villi increased,and the width widened after cold exposure.（4）After a six-week cold exposure,the contents of acetic acid,propionic acid,and butyric acid all decreased,but only the butyric acid content was significantly reduced（P<0.05）in the LT group.Although butyric acid content was still lower than the NT group after FMT,the difference was not statistically significant（P>0.05）.Compared with NN group,the expression levels of MUC2 and OCLN in the LN and LL group significant decreased（P<0.05）.Conclusions:（1）Continuous cold stress could induce the increase of blood pressure and lead to CIH in SD rats.FMT might regulate the blood pressure.（2）CIH rats suffer from the dysbiosis of gut microbiota and pathogenic bacteria or conditioned pathogens are significantly enriched.FMT can change the composition of gut microbiota in CIH rats.（3）The increase of plasma ANGⅡconcentration may induce vasoconstriction of vessels and increase blood pressure through AT₁R.（4）Gut microbiota may regulate the intestinal barrier function,oxidative stress and blood pressure of CIH rats through gut microbiota-related metabolites.（5）The changes of gut microbiota,its metabolites and ANGⅡ-AT₁R pathway caused by cold exposure may lead to the development of CIH.