The Roles And Mechanism Of Butyric Acid(A Short-chain Fatty Acid) In Cold-induced Hypertension

Background:Cold stimulation induces sympathetic responses and causes activation of the autonomic nervous system,which leads to vasoconstriction and forms acute hypertension.Prolonged cold stimulation is considered as an important environmental factor in the development of hypertension.International studies and previous research of our group have shown that prolonged cold stimulation can promote the development of hypertension,which may be related to the changes in the intestinal flora and short-chain fatty acids（SCFAs）.The role of SCFAs in the increase of blood pressure due to cold exposure and the related mechanisms need to be further studied.Objectives:（1）To investigate the effects of cold exposure on intestinal flora and the role of butyric acid.（2）To investigate the possible mechanism by which butyrate increased brown fat（BAT）thermogenesis,accelerated lipid metabolism,and reduced CIH through activation of SCFAs receptors（GPR43 and GPR109a）.（3）To explore the possible mechanism by which butyrate reduced the oxidative stress response to hypertension induced by cold exposure,modulating the Renin angiotensin system（RAS）,and activating SCFAs receptors,thereby lowering blood pressure.Methods:SPF male SD rats aged 5-6 weeks were purchased in this experiment.The rats were divided into six groups,which were low temperature exposure control group（LT,n=8）,normal temperature control group（NT,n=8）,low temperature medium dose continuous administration group（LMC,n=8）,low temperature high dose continuous administration group（LHC,n=8）,low temperature medium dose administration after six weeks group（LMA,n=8）,and low temperature high dose administration after six weeks group（LHA,n=8）according to the weight.The NT group was kept in a clean-grade animal room（20±2℃）.The LT,LMC,LHC,LMA,and LHA groups were housed in a clean-grade artificial climate simulator（4±1℃）and subjected to continuous cold exposure（20 h/d）.Sodium butyrate（0.5 or 1 g/kg/day）was dissolved in saline and given by intragastric administration daily at regular intervals in the LMC,LHC,LMA,and LHA groups.（1）The rats were exposed in cold for 8 weeks.Food intake（g）and water intake（m L）,body weight（g）,blood pressure（mm Hg）,and heart rate（beats/min）were monitored.（2）At the end of the 8^th week,the rats were sacrificed.The blood was collected and the plasma was separated.Heart,kidney,intestine,brown fat and other tissues were extracted and weighed.The concentrations of triglyceride（TG）and high-density lipoprotein（HDL）were measured.（3）The rat intestinal contents were collected and sequenced for 16S rDNA.The sequencing data were used for Alpha diversity analysis,Beta diversity analysis,and screening differential bacteria between groups,respectively.Metabolites of the intestinal contents were determined by using ultra-performance liquid chromatography coupled with mass spectrometry to screen for differential metabolites.The analysis of metabolic pathways was based on the KEGG database and the differential metabolites.（4）The concentrations of angiotensin Ⅱ（ANG Ⅱ）,endothelial nitric oxide synthase（e NOS）,and nuclear transcription factor（NF-κB）in rat plasma were measured.The concentrations of Renin,nitric oxide（NO）,and reactive oxygen species（ROS）in kidney tissues were measured.The expression levels of SCFAs receptors,angiotensin type 1 receptor（AT₁R）,recombinant and synthetic protein（Nrf2）,and heme oxygenase1（HO-1）in rat kidney tissues were measured by Western Blotting,as well as the mRNA levels of the above proteins.（5）The expression of SCFAs receptors,lysine demethylase（LSD-1）,and uncoupling protein 1（UCP-1）,and the mRNA levels of these proteins were measured in BAT.The concentrations of peroxisome proliferator-activated receptor gamma coactivator 1a（PGC-1a）and fibroblast growth factor 21（FGF-21）were measured in BAT.Adipose histopathological sections were prepared and the ultrastructure was observed.（6）The length of the intestinal tract was measured.The pathological sections of the ileum and colon tissues were made to observe the changes in the intestinal tracts.Immunohistochemical sections of colon tissues were prepared to detect the expression levels of mucoprotein 2（MUC 2）and tight junction protein（Zonulaoccludens-1,ZO-1）,and a semi-quantitative analysis was performed.Results:（1）At the end of the 8^th week,the systolic blood pressure（SBP）in the LT and NT groups were 136.31±3.92 mm Hg and 107.43±4.57 mm Hg,respectively.The SBP was significantly higher in the LT group than in the NT group during cold exposure（P<0.05）.The sodium butyrate intervention significantly reduced blood pressure in the LMC and LHC groups（P<0.05）.The SBP,diastolic blood pressure（DBP）,and mean blood pressure（MBP）in the LMA and LHA groups continued to increase till the 6^th week,which was not significantly different from the LT group.After the sodium butyrate intervention,the SBP in the LMA and LHA groups decreased and were 17.73±1.84 mm Hg and 106.46±2.06 mm Hg at the end of week 8.（2）Analysis based on 16S rRNA sequencing showed that the abundance of Lactobacillus decreased in the LT group and increased after sodium butyrate intervention compared to the NT group.The total number of species,abundance,and diversity of microbial communities decreased in the LT group compared to the NT group and increased in the LHC,LMA,and LHA groups after supplementation with sodium butyrate.The samples were composed of similar microbial communities among the five groups.After continuous cold exposure,the differential metabolites were mainly enriched in 20 metabolic pathways,among which the highest metabolite enrichment was in glycerophospholipid metabolism and arachidonic acid metabolism,and the pathways contained more differential metabolites were steroid hormone biosynthesis and purine metabolism.After sodium butyrate intervention,the differential metabolites in the LMC,LHC,LMA,and LHA groups were mainly enriched in 25,41,16,and 31 metabolic pathways,with a significant change in sphingolipid metabolism in the LMC group compared to the LT group（P<0.05）.（3）Compared to the NT group,the concentrations of FGF-21 and PGC-1a in BAT were reduced in the LT group（P<0.05）.The concentrations of FGF-21 and PGC-1a significantly increased in the LMA and LHA groups after supplementation with sodium butyrate（P<0.05）.The protein and mRNA expression of UCP-1 were increased in the LT group.The protein and mRNA expression of GPR43,GPR109a,and LSD-1 were increased in the LMC,LHC,LMA,and LHA groups after supplementation with sodium butyrate.In the LT group,the number of BAT was significantly increased,the number of lipid droplets（LDs）was reduced and the size became smaller.The number of mitochondria in the cytoplasm increased significantly.The mitochondria were round and tightly arranged and the mitochondrial cristae showed clearly.After supplementation with sodium butyrate,the number of LDs increased and became larger,and the number of mitochondria decreased,but the mitochondrial cristae were evident.（4）Compared with the NT group,the concentrations of ANG Ⅱ,Renin,ROS,and NO were increased in the LT group,and after supplementation with sodium butyrate,the concentrations of ROS were significantly reduced in the LMC,LHC,and LMA groups（P<0.05）.The concentrations of ANG Ⅱ,Renin,and NO declined in the LMC,LHC,and LMA groups,but not significantly（P>0.05）,while the protein of Nrf2increased in the LT group（P<0.05）.The protein and mRNA expression of Nrf2 was significantly reduced in the LHC and LMA groups（P<0.05）.（5）After cold exposure,the wall thickness of the ileal was reduced in the LT group compared to the NT group,and intestinal villi were significantly increased in LT,LMC,LHC,LMA,and LHA groups.The ileal wall thickness was restored in the LMC,LHC,LMA,and LHA groups compared to the LT group.The concentrations of HDL,TG,and NF-κB were increased in the LT group,but the differences were not significant（P>0.05）.The concentrations of NF-κB decreased in the LHC and LMA groups（P<0.05）.HDL and TG concentrations were lower in the LMC,LHC,LMA,and LHA groups,but were not statistically significant（P>0.05）.Conclusion:（1）The mechanism of hypertension induced by cold might be that cold reduced the diversity and richness of intestinal flora,increased harmful bacteria,reduced the level of butyrate,and increased the levels of inflammatory factors,blood lipids and ROS by promoting the production of ANG Ⅱ and affecting the intestinal barrier,thus affecting the vasoconstriction,which led to the increase of blood pressure.（2）The possible mechanism of butyric acid lowering blood pressure in CIH might be that butyric acid alleviated the rise of blood pressure caused by cold by promoting the growth of beneficial bacteria,repairing intestinal barrier,promoting the secretion of beneficial derived factors in BAT,and inhibiting ANG Ⅱ and ROS levels.