| Part Ⅰ The role of intestinal flora in the pathogenesis of hypertension caused by high-salt diet1 IntroductionHypertension is one of the most common chronic diseases,and the incidence rate has increased rapidly in recent years.According to the definition of the 2017 American Hypertension Guidelines(systolic blood pressure(SBP)≥130 mmHg,diastolic blood pressure(DBP)≥80 mmHg),45.6%of Americans have hypertension.Even in China,according to loose standards(SBP≥140 mmHg,DBP≥90 mmHg),nearly half of adults(44.7%)are hypertensive patients,compared with 29.6%in 2014.Only about 30%of hundreds of millions of hypertension patients in China have received treatment,but only 7.2%of patients with blood pressure(BP)control standards,and the more hypertension control rate did not exceed 10%.Hypertension has become a topic of global public health and an important independent risk factor for vascular disease,cerebrovascular disease and kidney disease.Controlling the occurrence and development of hypertension is an important part of human health.At present,antihypertensive drugs include diuretics,calcium channel blockers(CCB),beta blockers and angiotensin Ⅱ receptor antagonists,more than 60 categories.But the treatment of hypertension still faces low treatment rates and compliance difficulties,low compliance rate,large adverse drug reactions,and drug resistance.The pathogenesis of hypertension is complicated.It is currently believed that it is mainly related to the excessive activation of the central sympathetic nervous system,the retention of sodium and water in the kidney,the activation of the renin-angiotensin-aldosterone system(RAAS),abnormalities in arterial structure and function,and insulin resistance.According to whether the cause of the disease is clear,hypertension is divided into primary hypertension and secondary hypertension,the secondary hypertension with a clear cause accounts for only 5%,and the primary hypertension with an unknown cause is as high as 95%.Essential hypertension is a multi-factor,multi-link,multi-stage and individual-disease disease,which is the result of the interaction of genetic factors with diet,smoking,mental stress and other environmental factors.Reasonable diet control is an important principle for the treatment of hypertension,and restricting sodium intake is a top priority in diet control.The characteristics of hypertension in China that are high in the north and low in the south are also related to the high-salt diet in the north.The pathogenesis of high-salt hypertension is complicated.It is currently believed that it is mainly caused by abnormal water and sodium retention in the kidney,increased blood volume,and increased peripheral vascular resistance.Some genetic factors(such as GNB3,ADD1 and ACE polymorphisms)also affect the sensitivity to high salt,but the specific mechanism that causes these pathological changes has not yet been fully elucidated.The reported mechanisms include:abnormal activation of the RAAS,the sympathetic nervous system,and the kallikrein-kinin system;the interaction of the endocrine system(including androgen and insulin resistance)with high salt;one Nitric oxide signaling pathway and vascular endothelial glycocalyx layer damage and vascular endothelial dysfunction caused by enhanced oxidative stress.Nevertheless,the exact pathogenesis of high-salt hypertension still needs more scientific experiments for further research.About 1013-1014 bacteria(i.e.intestinal flora)coexist in the human gut,which is an important environmental factor that affects the occurrence of multiple diseases in genetically susceptible individuals.The intestinal flora colonizes the gastrointestinal tract,and the colon becomes the preferred colonization site for the intestinal flora due to its unique anaerobic,high-nutrient environment.The intestinal flora can not only play an important role in the host’s food digestion,but also perform many other important functions and interact with the host.A large number of studies in recent years have shown that intestinal flora is closely related to metabolic diseases,immunological diseases,and nervous system diseases.Early studies showed that BP in sterile rats increased,suggesting that intestinal flora may be involved in BP regulation.Recent studies have found that the diversity and richness of intestinal microflora in hypertensive animals are significantly reduced,and the short-chain fatty acids(SCFAs,such as acetic acid and butyric acid)-producing bacteria are significantly reduced,while lactic acid-producing bacteria,pro-inflammatory bacteria and opportunistic pathogens A significant increase.SBP is positively correlated with the abundance of lactic acid-producing Lactobacillus,but negatively correlated with the abundance of SCFAs-producing Odoribacter and Holdemania.Transplanting intestinal flora of hypertensive animals can significantly increase the BP of normal animals,and vice versa lower the BP of hypertensive animals.More than 50%of primary hypertension in the world is salt-sensitive hypertension,but there is still little research on its relationship with intestinal flora.Mell et al.found that the composition of the cecum flora of Dahl salt-sensitive rats and Dahl salt-resistant rats were significantly different;the intestinal flora of the transplanted former did not cause changes in the BP of salt-resistant rats,but the transplantation of intestinal flora to the former will increase the BP and shorten the survival time of the former;and antibiotic treatment can not reduce the BP of the former.This is very different from other studies,and may be related to the large difference in the genetic background of DahI salt-sensitive and-resistant rats.A recent study by Wilck et al.found that a high-salt diet can increase BP and reduce the diversity of intestinal flora in FVB/N mice,which significantly reduced bacteria such as Rothia,Clostridium ⅩⅣa and Lactobacillus,while Parasutterella increased.In particular,Lactobacillus murinus,whose growth is inhibited by high salt in vitro,disappears during HSD in mice and humans,and returns to normal when it is eaten normally.Oral administration of L.murinus can reduce high salt-induced hypertension in mice.The mechanism is that Lactobacillus can metabolize the tryptophan in the diet to produce indole,thereby inhibiting the differentiation of T lymphocytes into TH17 cells and reducing immune inflammation.It is worth noting that the previous studies on intestinal flora and high salt-induced hypertension have found that the differences in intestinal bacteria and intestinal metabolites induced by high-salt diets are not exactly the same,and may be the same as the animal strains and feeding used in the research.The time,feed formula,analysis platform and method are inconsistent.This also shows that the pathogenesis of high salt-induced hypertension is complicated.Whether other bacteria and metabolites still play a role in the development of high salt-induced hypertension still needs further scientific research to clarify.Therefore,in this study,Wistar rats were used as the research object,and a high-salt diet was given for 4 weeks,and the causal relationship between intestinal flora changes and BP changes was clarified through intestinal flora transplantation.Peripheral blood and stool were collected,16S rRNA sequencing was used to determine the abnormal structure of the flora,and non-targeted metabolomics analysis was used to determine the abnormal metabolism in the intestine.Clarify the role and mechanism of intestinal flora in the development of high-salt hypertension.2 Objectives(1)High-salt diet induces an increase in BP in Wistar rats,and the causal relationship between the increase in BP and the change of microflora is clarified through bacterial transplantation;(2)Using 16s rRNA sequencing and non-targeted metabolomics and other technologies to clearly reveal the role and mechanism of intestinal flora in high-salt hypertension.3 Methods3.1 Establish animal models and give drug treatmentHigh-salt diet induced hypertension in rats:4-week-old normal Wistar male rats were selected.The experimental group was fed with high-salt(8%NaCl)feed to induce hypertension,and the normal control group was fed with low-salt(0.49%NaCl)feed.The BP of rats was measured every week,and the SBP>130 mmHg or DBP>80 mmHg was used as the criterion of hypertension.The results of our previous studies showed that the induction success rate exceeded 95%(58/60).Random grouping is as follows:NSD(group A,control(n=32):divided into A1(n=16),A2(n=16)HSD(group B,HSD(n＝32):divided into B1(n=16),B2(n=16))3.2 Fecal Microbiota Transplants(1)Transplantation plan● Group E:Group A1 receives the intestinal flora of Group B1● Group F:Group B2 receives the intestinal flora of Group A2(2)Clear the original intestinal floraA quadruple antibiotic mixture(1 g/L ampicillin,500 mg/L vancomycin,1 g/L neomycin sulfate,1 g/L metronidazole)was given by continuous gavage for 3 weeks to clear the intestinal microflora of the recipient rats,and the dose of tube feeding liquid is 2.5 ml/kg.Mix the antibiotic mixture solution thoroughly before each tube feeding.The drug was stopped 48 hours before fecal microbiota transplants.(3)Preparation and transplantation of donor mouse intestinal flora suspensionThe donor rats were sacrificed after anesthesia,and the colon contents were collected immediately in a sterile operating table.Sterile physiological saline(about 20 ml in total)was added in small amounts and repeatedly.The contents were ground with a sterile glass rod to make it as soluble as possible in physiological saline.Filter the suspension through a 200 mesh screen to remove food debris.The filtrate was centrifuged at 6000g at 4℃ for 20 min.The precipitate was collected and resuspended in 12ml of 10%sterile glycerol solution to prepare a suspension,which was stored in a refrigerator at-80℃ until use.The above operations are carried out under sterile conditions.The corresponding recipient rats were gavaged once every 2 days with a colon content suspension(200 mg/ml)from donor rats,1 ml each time,3 times.Monitor BP twice a week.3.3 Blood pressure measurement(1)tail-cuff methodRats in each group were measured by tail-cuff method before the high-salt(or normal salt)diet,after the high-salt(or normal salt)diet,after antibiotics,and after transplantation.The adaptive measurement was performed 5 days in advance,and each measurement was repeated three times,with an interval of 30s each time,and the average of the three measurement data was used as the BP result of this measurement.(2)Blood pressure measurement by telemetry● Operation of blood pressure implant Animals need to be fasted and provide water for 24 hours before surgery.Intraperitoneal anesthesia with pentobarbital sodium is used to induce complete anesthesia.Prepare 2%glutaraldehyde aqueous solution with distilled water,soak the implant completely,cover and soak for 12-24 hours;take it out aseptically,rinse it with sterilized water and use it.Write down the implant number.According to the principle of aseptic operation,insert the implant into the abdominal aorta,quickly cover the cellulose membrane and add a small amount of adhesive to ensure that there is no bleeding in the puncture.Transfer the animal to the receiving board,enter the correction value in the software and view the waveform.After the waveform is normal,remove the gauze strip and the pull line,clean the abdominal cavity,fix the implant to the abdominal muscle layer,and close the wound.Keep the animal warm after the operation until the animal is awake.4-6 hours after the operation,wait for the animal to defecate before feeding,to prevent postoperative gastrointestinal dysfunction caused by death.● Collect dataConnect the components of the device according to the instructions,place the animal with the implant on the receiving board,and the indicator light is on to start collecting data.3.4 Collection and processing of venous blood and stool samples(1)Collect and process venous bloodUsing a slipknot,fix the rat in a "cross" shape,supine position on a fixed plate(head up,two forelimbs in a straight line and perpendicular to the trunk),and wipe with alcohol cotton balls in the same direction.Draw a horizontal line along the lower edge of the upper extremity and a vertical line along the outer side of the neck base.The intersection point is the needle insertion point.Use a 2ml syringe with a 0.6-gauge blue needle to puncture,and insert the needle at a 30° level with the torso.Once the needle enters the subcutaneous tissue,immediately pull out the piston shaft slightly to form a negative pressure state in the tube.Sting downward at an angle,when blood enters the syringe,fix the syringe and maintain negative pressure until the required amount of blood(1 ml)is collected in the syringe.After blood collection,pull out the puncture needle and gently press the puncture site with a cotton swab for 1-2 minutes to stop bleeding.The rats were awake during the whole process.If the blood was not taken successfully within 5 minutes,the rats should be untied and put back into the cage,and try again later.The blood sample was quickly transferred to an anticoagulation tube,and the supernatant was collected by centrifugation at 3000 rpm and 15 min,and stored at-80℃ until use.(2)Collect and process stool samplesThe experimenter wears protective gloves to fix the rat with one hand,disinfects the skin near the anus with an alcohol cotton ball,promotes bowel movement by rectal massage,and collects fecal particles directly from the anus with a sterile tissue cry otube(2-3 capsules each time)stool).The cryotube containing the fecal particles was immediately put into liquid nitrogen for quick freezing and transferred to the-80℃refrigerator as soon as possible for storage.During the whole process,care should be taken to avoid contact between the nozzle and the skin of the anus,and to avoid contamination by bacteria.3.5 16s rRNA gene sequencing and analysisUsing the gDNA of each group of intestinal flora as a template,the v3-v4 region of the 16S rRNA gene was amplified by PCR.After purification,the linker sequence was added by PCR.After quantification,the sequence was sequenced.The sequencing volume was>200,000 reads/sample.The raw date is processed to remove the joints and low-quality bases to generate clean data for subsequent analysis.Combine tools such as QIIME2,USearch,and Calypso to determine amplicon sequence variants(AVS)and perform annotation and difference analysis to reveal the compositional characteristics of intestinal flora in each group.Then use clustering,PCA and PcoA feature extraction tools to compare the diversity,types and richness of intestinal flora before and after nifedipine.3.6 Non-targeted metabolomics analysisApproximately 100 mg of feces was extracted with 1 mL of methanol,and 60 μL of 2-chloro-L-phenylalanine and pimelic acid were added as internal standards.After vortexing,sonication and centrifugation,the supernatant was transferred to a new tube and dried.Add 60 pL of 15 mg/mL methoxypyridine and N,O-bis(trimethylsilyl)trifluoroacetamide reagent in sequence,and incubate at 37℃.After centrifugation,the ACQUITY UPLC system(Waters Corporation,Milford,USA)in combination with the AB SCIEX Triple TOF 6600 system(AB SCIEX,Framingham,MA)was used in positive and negative ion modes for analysis according to the manufacturer’s instructions.All metabolite ions were normalized and further analyzed by SIMCA 14.0(Umetrics,Umea,Sweden)and XCMS 1.50 software(RSD%<30%).Orthogonal partial least squares discriminant analysis(OPLS-DA)and its verification are carried out after the average centering and unit variance scaling by SIMCA.Based on the combination of the projection(VIP)value(>1)obtained from the OPLS-DA model and the p-value(<0.05)of the Wilcoxon rank sum test for the normalized peak area,the difference peak feature was selected.Retain at least one set of peak features with non-zero values exceeding 50%,and use them to identify differential metabolites through reference material databases established by HMDB,METLIN and Dalian Chemical Data Solutions Information Technology Co.,Ltd.The metabolic cloud map was generated using XCMS online(https://xcmsonline.scripps.edu/).3.7 Multi-omics integration analysisIntegrate 16s rRNA sequencing of each group of rats,omics data of metabolome,BP and other index data into a two-dimensional expression matrix,and then use WGCNA,feature selection and factor analysis and other bioinformatics methods and R multivariate analysis tools to integrate analysis Based on the above multi-dimensional data,intestinal bacteria and corresponding metabolites related to high-salt hypertension are found.3.8 Enzyme-linked immunosorbent assay(ELISA)ELISA is used to detect the level of inflammatory factors in serum.All experiments were performed according to the instructions.The intra-assay and inter-assay coefficients of variation were controlled within 10%and 15%,respectively.All samples of the ELISA were run in duplicate.3.9 Real-time quantitative PCR to verify the level of intestinal bacteriaAs mentioned previously,the CTAB method was used to extract gDNA from stool samples.Then,quantitative PCR was performed on the CFX96 real-time PCR system(Bio-Rad)using TB Green Premix Ex Taq(TaKaRa)and specific primers.3.10 Statistical AnalysisData were presented as mean ± SEM or median(minimum to maximum).The Shapiro-Wilk test was used to evaluate data normality.For normally distributed data,t-test and one-way ANOVA test with post hoc test(Turky or Dunnett)for multiple testing correction were used for comparison between two groups and multi-group comparison,respectively.For data not distributed normally,Wilcoxon rank sum test and Kruskal-Wallis test with post hoc test(Dunn)for multiple testing correction were used for comparison between two groups and multi-group comparison,respectively.For certain multiple comparison or statistical analysis of omic data including metabolomic and 16S rRNA gene sequencing data,t-test or Wilcoxon rank sum test adjusted by Benjamini-Hochberg method with FDR<5%was used.Statistical analysis was performed using GraphPad Prism v8 and R packages.The p<0.05 or adjusted p value<0.05 was considered statistically significant All experiments were repeated independently at least thrice.4 Results4.1 HSD can significantly induce hypertension in Wistar ratsNormal Wistar male rats were fed high salt(8%NaCl)feed to induce hypertension(HSD group),and normal feed was used as the control group.During the feeding period,rats in both groups gained weight,but there was no significant difference between the groups.With the increase of high salt feeding time,the BP of rats in HSD group gradually increased,and SBP reached 167.9±13mmHg and DBP 125.4±17mmHg at the fourth week,which was significantly higher than that of control rats(101.7)with normal BP at the same age ±10/76.5±9 mmHg),meeting hypertension standards.4.2 HSD significantly dysregulated the intestinal flora of ratsThe principal component analysis and principal axis analysis of the 16s data found that the intestinal flora of the control and HSD groups was clearly separated,indicating that the intestinal flora characteristics of the two groups of rats were significantly different.Compared with the control group of rats,the F/B ratio of the intestinal flora of the HSD group was significantly doubled,which indicates that the high salt hypertensive rats have developed serious intestinal flora dysbiosis.In addition to the obvious changes in the Firmicutes and Bacteroidetes,the proportion of spirochaete in the intestine of hSIH rats increased significantly,while Verrucomicrobia significantly decreased.4.3 HSD significantly changed the symbiotic relationship between the intestinal flora of ratsThere are complex symbiotic relationships and interactions between intestinal bacteria.The results of the common network analysis showed that the symbiotic relationship between the 4-week high-salt-feeding bacteria,high salt intake caused the interaction between certain probiotics to be broken.Many of these bacteria with significantly altered abundance are closely related to increased BP.4.4 Lactobacillus and Bacteroides are significantly reduced in the intestinal flora of hypertensive rats induced by HSDAfter a high-salt diet,the abundance of 31 fungi or species in the rat intestine changed significantly,and the abundance of 22 out clusters decreased significantly.Spearman correlation analysis of bacterial abundance and BP also found that in the differential AVS,all the differential bacteria in the Bacteroridetes,Lactobacillus in the Firmicutes,and SBP and DBP were significantly negative correlation.And among the top ten different intestinal bacteria with the highest abundance,the abundance of Bacteroides is much higher than that of other bacteria,which suggests that it may play a more important role in the pathogenesis of hSIH.4.5 HSD significantly changes the metabolic profile of intestinal floraOrthogonal partial least squares discriminant analysis(OPLS-DA)indicates that the metabolic data clusters of the control group and the HSD group are separated from each other.In the ES+and ES-modes,the peak intensities of 955 and 641 peak features changed significantly.These results indicate that HSD significantly changes the metabolic profile of intestinal flora.4.6 Multiple metabolites are significantly associated with blood pressure and differential bacteriaThere was a significant correlation between intestinal differential bacterial abundance,BP and the levels of many metabolites(|r|>0.5,p<0.05 after FDR adjustment).For example,dihydro tachysterol and acetylcholine were significantly negatively correlated with SBP and DBP,and significantly positively and negatively correlated with Odoribacter and Flexispira,respectively.Prostaglandin E2 and leukotriene B4 were strongly positively correlated with BP,and were significantly negatively and positively correlated with Distasonis and Psychrobacter respectively.It is worth noting that the concentration of intestinal corticosterone in the HSD group increased by 4.4 times.Corticosterone is significantly negatively correlated with certain highly abundant bacteria(such as Bacteroides,Lactobacillus,and Alice Bass).In addition,it is also significantly positively correlated with SBP and DBP.4.7 Hypertension induced by HSD can be transmitted through intestinal flora transplantationThe elevated SBP and DBP in the HSD group decreased significantly after antibiotic treatment,and after further transplantation of the intestinal flora of the control group of rats with normal BP and normal diet,their BP.further decreased to nearly normal.In contrast,the control group of rats treated with antibiotics only increased their SBP SBP significantly,but did not significantly increase DBP.However,after further transplantation of intestinal flora of hypertensive HSD rats,their SBP and DBP increased significantly.These results indicate that the abnormality of the rat intestinal microflora caused by the high-salt diet is one of the causes and mechanisms of high salt-induced hypertension,or is it just a concomitant phenomenon in this process.5 Conclusion(1)High salt intake increases BP in wistar rats,which can be transmitted via FMT;(2)High salt intake significantly changes the intestinal flora of high-salt hypertensive rats and reduces the level of intestinal bacteria;(3)High salt intake significantly changes the metabolism of intestinal flora in high-salt hypertensive rats,and reduces intestinal and serum corticosterone levels.Paper Ⅱ Role and mechanism of enteric corticosterone regulated by intestinal flora in high-salt hypertension1 IntroductionHypertension is one of the most common chronic diseases.In China,nearly half of adults(44.7%)are hypertensive patients(systolic blood pressure(SBP)≥140 mmHg,diastolic blood pressure(DBP)≥90 mmHg),and this proportion was only 29.6%in 2014.Hypertension can cause disorders in the structure and function of vital tissues such as the heart,brain,kidneys and blood vessels.It is the most important risk factor for malignant cardiovascular and cerebrovascular events such as stroke,myocardial infarction and heart failure.It causes at least 45%of the global each year heart disease deaths and 51%of stroke deaths have become one of the world’s most serious public health problems.The pathogenesis of hypertension is complex,and reported mechanisms include abnormal activation of the renin-angiotensin-aldosterone system(RAAS),sympathetic and kallikrein-kinin systems,and vascular endothelial dysfunction.Among these mechanisms,corticosterone and mineralocorticoid receptor(MR)dysfunction are essential for the development of hypertension,especially salt-sensitive hypertension.Glucocorticoids(cortisol in humans,corticosterone in rodents)play an important role in a variety of physiological and pathological processes.The adrenal cortex is the main site for the synthesis and release of corticosterone and cortisol.However,many key enzymes used for sterol synthesis are also expressed in the intestinal epithelium,thymus,brain,and other tissues capable of synthesizing bioactive glucocorticoids.Under physiological conditions,the intestinal production of corticosterone is very low,but it increases significantly in an inflammatory environment.Gut-derived corticosterone can even restore plasma corticosterone levels to normal levels in mice that have undergone adrenal intestinal bacterial depletion.Corticosterone,cortisol,and aldosterone are major regulators of sodium and potassium homeostasis,hemodynamics,and blood pressure(BP)in the body.All of these hormones can activate MR,leading to sodium sodium retention and elevated BP.Therefore,the abnormal rise of these hormones is closely related to the occurrence of hypertension.The intestinal flora is closely related to many diseases.Recent studies have shown significant changes in the gut flora of patients and animals with hypertension,including reduced bacteria that produce short-chain fatty acids(SCFAs)and increased pro-inflammatory bacteria and opportunistic pathogens.By transplanting the gut flora of patients or animals with hypertension,BP in healthy animals can be significantly increased.Some probiotics or prebiotics can also lower BP in patients or animals with hypertension.However,little research has been done on the mechanism of the relationship between intestinal flora and hypertension.Recently,Wilck et al.Have found that HSD can significantly reduce intestinal Lactobacillus murinus of hypertensive FVB/N mice,and Lactobacillus murinus can produce indole through tryptophan metabolism in the diet,thereby inhibiting T lymphocyte differentiation to Th17 cells,it reduces inflammation and lowers BP.Our previous research results also found that High salt-induced hypertension(hSIH)can be transferred by fecal flora transplantation(FMT),suggesting that the intestinal flora plays a key role in hSIH development.HSD reduced the levels of Bacteroides in the intestine and increased the concentration of fecal corticosterone,while the levels of Bacteroides and corticosterone and BP were significantly related to each other.However,whether the corticosterone in Bacteroides and feces really play an important role in the development of hypertension and the specific mechanism needs further research to clarify.2 Objectives(1)To study the role of Bacteroides and intestinal corticosterone in hypertension,and to reveal the changes of Bacteroides and corticosterone in hypertensive rats and patients;(2)To study the specific relationship between Bacteroides and corticosterone,and to reveal how Bacteroides regulate corticosterone and the specific mechanism of the two in the development of hypertension.3 methods3.1 Establishment of animal models and drug treatmentHigh-salt diet induced hypertension in rats:Normal Wistar male rats aged 4 weeks were selected.The experimental group was fed with high-salt(8%NaCl)feed to induce hypertension,and the normal control group was fed with low-salt(0.5%NaCl)feed.BP was measured weekly in rats,with SBP>130mmHg or DBP>80 mmHg as the criterion for hypertension.Our previous research results showed that the induction success rate was more than 95%(58/60).The random grouping is as follows:Low-salt control group(control(n=32))High-salt hypertension group(HSD(n=32))3.2 Tail-cuff methodTail-cuff method was used to measure BP of rats in each group before high salt(or normal salt)diet,after high salt(or normal salt)diet,after antibiotics,and after transplantation.Sex measurement,each measurement is measured three times,with an interval of 5 minutes,and the average of the three measurements is used as the BP result of this measurement.3.3 Sample collection and processing(1)Collection of clinical samples● Inclusion and exclusion criteria for clinical casesAccording to the diagnostic guidelines for hypertension guidelines(new ACC/AHA hypertension guidelines,2017),we recruited 11 untreated hypertensive patients(SBP≥130 mm Hg,or DBP≥80 mm Hg)from Qilu Hospital of Shandong University and 12 healthy people.The exclusion criteria were as follows:pregnancy,diarrhea;use of the following drugs within 3 months:antibiotics,probiotics,prebiotics,laxatives,Chinese herbs and gastrointestinal surgery.The two groups of subjects were matched for age,gender,and body mass index(BMI).● Collect and process venous blood samplesAfter fasting overnight(≥8 h),5 mL of peripheral blood was collected in the morning,and the blood sample was quickly transferred to an anticoagulation tube.The supernatant was collected by centrifugation at 3000 rpm,15 min,and stored at-80℃for later use.● Collection and processing of stool samplesAll subjects were equipped with a toilet specimen collection kit for fecal collection.About 2 g of fresh feces were collected from each subject,placed in liquid nitrogen for quick freezing,and quickly transferred to-80℃ for storage.Use the QIAamp DNA Stool Mini Kit to purify the intestinal flora gDNA in 180 mg stool samples,dissolve the other 180 mg stool samples in PBS,freeze and thaw repeatedly and homogenize 16000 g,and centrifuge at 4℃ for 10 minutes.Use 16000g each time,centrifuge for 10 minutes at 4℃,take the supernatant,and filter through a 0.2um filter.Finally,combine the supernatants from each centrifugation and store at-80℃ for metabolite analysis.(2)Rat sample collection● Collect and process venous blood of ratsUse a knot to fix the rat in a "cross" shape and supine position on a fixed plate(head up,two forelimbs in a straight line and perpendicular to the trunk)to ensure that the binding line is tight and adequate and fully exposes the sternum,neck,etc.Wipe with alcohol cotton ball in the same direction,jugular jugular sinuses on both sides can be seen,with slight beating.Draw a horizontal line along the lower edge of the upper limb and a vertical line along the outer side of the neck root.The intersection point is the point of needle insertion.A 2ml syringe with a 0.6-gauge blue needle was used for puncture,and the needle was inserted horizontally at 300 with the trunk.Once the needle entered the subcutaneous tissue,the piston shaft was slightly pulled out immediately to form a negative pressure state in the tube,which reached approximately 30 degrees above the collarbone.Puncture down at an angle,as blood enters the syringe,secure the syringe and maintain negative pressure until the required amount of blood(1 ml)is collected in the syringe.If no blood flows into the syringe,stop it.Then slowly withdraw the syringe and maintain negative pressure.In most cases,blood will enter the syringe when it is withdrawn.After blood collection,remove the puncture needle and gently press the puncture site with a cotton swab for 1-2 minutes to stop bleeding.During the whole process,the rats are awake.If blood is not taken within 5 minutes,the rats should be untied and returned to the squirrel,and try again later.The blood sample was quickly transferred to an anticoagulation tube,centrifuged at 3000 rpm,15 min to collect the supernatant,and stored at-80℃ for future use.●Collecting and processing rat stool samplesThe experimenter took protective gloves to fix the rat with one hand,disinfected the skin near the anus with an alcohol cotton ball,urged the rat to defecate by rectal massage,and collected the fecal particles directly in the anus with a sterile tissue cryopreservation tube(2-3 capsules each time stool).The cryopreservation tube containing fecal particles was immediately put into liquid nitrogen for quick freezing a... |
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