| This study focuses on diabetic coronary heart disease(DCHD).Diabetes can exacerbate cardiovascular disease,resulting in a poor prognosis for DCHD patients,but its specific pathogenesis was still unspecified.Blood-stasis syndrome is one of the core pathogenesis of coronary heart disease in traditional Chinese medicine.Playing a critical role in the dialectics of blood-stasis syndrome,tongue features are closely related to oral(tongue)microbiota.We try to explore and clarify the relevant mechanism of blood-stasis syndrome promoting DCHD aggravated by diabetes and blood-activating and blood-stasis-removing from the perspective of the human oral cavity and intestinal microbial changes.The study clarifies oral and gut microbiota features of blood-stasis syndrome in DCHD by establishing a clinical discovery cohort and a verifying cohort.The study also explores the disease mechanism of diabetes promoting Myocardial Ischemia-Reperfusion Injury(MIRI)by regulating oral and gut microbiota and the preventing and therapeutic mechanism of the blood circulation drug Salvia Miltiorrhiza-Ligusticum Chuanxiong through in vivo experiments.This PhD thesis is divided into five main parts as follows.Part Ⅰ:Characteristics of Oral and Gut Microbiota in Patients with Diabetic Coronary Heart Disease-Related Blood-stasis SyndromeObjective:To establish expression profiles of oral and gut microbiota in patients with DCHD related blood-stasis syndrome and analyze its microbiome characteristics.Methods:114 consecutive patients with coronary heart disease was recruited from Xiyuan Hospital of China academy of Chinese medical Sciences(CACMS)to establish the discovery cohort.Metagenomic sequencing was conducted on the oral microbiota(tongue coating)and fecal microbiota.According to the disease-syndrome model,patients were classified into DCHD(DCHD)/simple coronary heart disease(CHD)groups(n=57/n=57)and severe blood-stasis syndrome(Z)/mild blood-stasis syndrome(Q)groups(n=68/n=46).Diversity analysis,species differential analysis,and functional annotation methods were used to identify characteristic oral and gut microbiota in DCHD and coronary heart disease with blood-stasis syndrome.The characteristic oral and gut microbiota in DCHD/CHD and blood-stasis syndrome severe/mild were compared using a Venn diagram.Spearman correlation analysis was used to investigate associations between the oral-gut microbiota and disease characteristics by analyzing their correlation with clinical indicators.Results:(1)Compared to patients in group CHD,group DCHD showed an increased abundance of 12 bacterial species(P<0.05)and a decreased abundance of 5 bacterial species(P<0.05)in the oral microbiota.Patients with severe blood-stasis syndrome had an increased abundance of 11 bacterial species(P<0.05)and a decreased abundance of 18 bacterial species(P<0.05)in the oral microbiota,compared to those with mild blood-stasis syndrome.The abundance of Actinomyces naeslundii、Corynebacterium durum、Rothia mucilaginosa、Streptococcus australis were decreased(P<0.05)and that of Fusobacterium nucleatum were increased(P<0.05)in both DCHD and severe blood-stasis syndrome patients.(2)In the gut microbiota,the abundance of 3 bacterial species,including Latcobacillus,were increased(P<0.05),while the abundance of 24 bacterial species,including Eubacterium,were decreased(P<0.05)in DCHD patients.In patients with severe blood-stasis syndrome,the abundance of 2 bacterial species were increased(P<0.05)and that of 5 bacterial species including Actinomyces sp_ICM47 were decreased(P<0.05).There was no overlap of characteristic gut microbiota between DCHD and severe blood-stasis syndrome patients.(3)Functional annotation of the microbiota revealed changes in carbohydrate metabolism and short-chain fatty acid metabolism in the oral and gut microbiota of DCHD patients with blood-stasis syndrome.(4)The abundance of Actinomyces sp ICM47 was decreased in the oral-gut microbiota of patients with severe blood-stasis syndrome.Various oral bacterial species were correlated with Lactobacillus abundance,and there was a correlation between the oral-gut microbiota and clinical indicators such as glycosylated hemoglobin(HBA1c)in DCHD patients.Conclusion:Actinomyces naeslundii,Corynebacterium durum,Rothia mucilaginosa,Streptococcus australis,and Fusobacterium nucleatum are potential oral microbiota biomarkers for DCHD patients with blood-stasis syndrome,while Latcobacillus,Actinomyces,and Eubacterium are potential gut microbiota biomarkers for DCHD patients.Additionally,Bifidobacterium pseudocatenulatum and Actinomyces are potential gut microbiota biomarkers for patients with blood-stasis syndrome.Furthermore,the findings indicate a strong association between oral and gut microbiota and the severity of DCHD and blood-stasis syndrome.Part Ⅱ:Validation of Oral and Gut microbiota Biomarkers in DCHD Patients with Blood-stasis SyndromeObjective:To validate oral and gut microbiota markers in DCHD patients with blood-stasis syndromeMethods:Ten bacterial species identified as characteristic oral and gut microbiota in DCHD blood-stasis syndrome patients in Part I were validated using qPCR.A validation cohort was established with 68 coronary heart disease patients from Xiyuan Hospital of CACMS and absolute quantification sequencing was conducted for oral(tongue coating)and fecal microbiota.The DCHD/CHD group(n=35/n=33)was compared with the severe blood-stasis syndrome/mild blood-stasis group(n=37/n=31)to screen for characteristic oral and gut microbiota in each group,and Spearman correlation analysis was performed on the characteristic oral and gut microbiota and clinical indicators.The characteristic oral-gut microbiota of DCHD with severe bloodstasis syndrome and their clinical functions were validated.Results:DCHD patients had a significantly higher abundance of Fusobacterium nucleatum in the mouth and Latcobacillus in the gut compared to simple CHD patients(P<0.05).Similarly,patients with severe blood-stasis syndrome in CHD had increased abundance of Fusobacterium nucleatum in both the mouth and gut,as well as Latcobacillus in the gut(P<0.05)compared to patients with mild blood-stasis syndrome.A positive correlation was observed between the abundance of Fusobacterium nucleatum in the mouth and Latcobacillus in the gut in DCHD patients(r=0.455,P<0.05).In patients with,the abundance of Fusobacterium nucleatum in the gut was positively correlated with glycated hemoglobin levels(r=0.354,P<0.05).Conclusion:Fusobacterium nucleatum in the mouth and Latcobacillus in the gut are specific bacterial communities in patients with DCHD blood-stasis syndrome.Furthermore,diabetes appears to increase the correlation between Fusobacterium nucleatum in the mouth and Latcobacillus in the gut,suggesting that diabetes may influence the development of CHD by regulating the oral-gut microbiota.Part Ⅲ:Study on the Effect of Diabetes Regulated Oral-Gut microbiota on Exacerbating Myocardial Ischemia-Reperfusion InjuryObjective:To investigate the potential role of dysbiosis of oral-gut microbiota,regulated by diabetes,in exacerbating myocardial ischemia-reperfusion injury(MIRI)in mice.Methods:C57BL/6 mice(n=26)were randomly divided into two groups:SHAM(n=10)and MIRI(n=16).The remaining C57BL/6J mice(n=32)were injected with streptozotocin(STZ)to induce diabetes.The diabetic mice were randomly divided into two groups:diabetes MIRI(DMIRI,n=16)and diabetes MIRI with antibiotics(DMIRI+ABX,n=16).The DMIRI+ABX group received antibiotics by gavage while the other groups received water by gavage for 6 weeks.All mice underwent MIRI surgery,involving left anterior descending coronary artery ligation for 30 minutes followed by reperfusion for 24 hours.Fasting blood glucose(FBG)was monitored dynamically,and TTC staining was used to observe and calculate the area of myocardial infarction.HE staining was used to observe pathological changes in myocardial injury,and Tunel staining was used to calculate the apoptosis of myocardial cells.ELISA was used to measure serum levels of cardiac troponin Ⅰ(cTnI),and qPCR was used to measure the absolute abundance of oral microbiota Fusobacterium nucleatum and gut microbiota Latcobacillus.Results:(1)The FBG levels did not differ significantly between the DMIRI+ABX group and the DMIRI group at various time points(P>0.05).(2)Compared to the SHAM group,the MIRI group showed a significant increase in myocardial infarct area as observed through TTC staining(P<0.05),myocardial injury on HE staining,increased levels of serum cTnI(P<0.05),and increased myocardial apoptosis as observed through Tunel staining(P<0.05).The abundance of Fusobacterium nucleatum in the oral cavity and Latcobacillus in the gut did not show any significant difference(P>0.05).(3)Compared to the MIRI group,the DMIRI group showed a significant increase in myocardial infarct area as observed through TTC staining(P<0.05),more severe myocardial injury on HE staining,increased levels of serum cTnI(P<0.05),and increased myocardial apoptosis as observed through Tunel staining(P<0.05).The abundance of Fusobacterium nucleatum in the oral cavity of DMIRI group increased(P<0.05),while the abundance of Latcobacillus in the gut did not show any significant difference(P>0.05).(4)The DMIRIA+ABX group showed a decrease in myocardial infarct area as observed through TTC staining compared to the DMIRI group(P<0.05),and HE staining showed an improvement in myocardial inflammatory infiltration and cell necrosis.The Tunel staining showed a decrease in myocardial apoptosis(P<0.05).The abundance of Latcobacillus in the gut of DMIRIA+ABX group was significantly reduced(P<0.05),while there was no significant difference in the abundance of Fusobacterium nucleatum in the oral cavity compared to the DMIRI group(P>0.05).Conclusion:Hyperglycemia can promote dysbiosis of the oral microbiota,increase the level of oral Fusobacterium nucleatum,and exacerbate MIRI,leading to increased cardiomyocyte apoptosis during MIRI.The use of broad-spectrum antibiotics can reduce the abundance of gut bacteria such as Latcobacillus and alleviate diabetic myocardial ischemia-reperfusion injury through non-hypoglycemic pathways,reducing cardiomyocyte apoptosis during MIRI.Part Ⅳ:Study on the Mechanism of Fusobacterium nucleatum Regulating Gut microbiota in Aggravating MIRIObjective:To investigate the impact of oral Fusobacterium nucleatum on gut microbiota and understand the mechanisms underlying the dysbiosis of oral-gut microbiota in exacerbating MIRI.Methods:C57BL/6 mice were randomly divided into five groups:SHAM(n=10),model(MOD,n=16),pseudo-germ-free(ABX,n=16),Fusobacterium nucleatum transplanted(F.n.,n=16),and fecal microbiota transplantation from patients with DCHD(FMT,n=16).After six weeks of continuous gavage,mice underwent MIRI surgery(left anterior descending coronary artery ligation for 30 minutes followed by 24 hours of reperfusion).The mice were observed for general conditions such as FBG,body weight,and heart weight index.TTC staining was used to observe and calculate the area of myocardial infarction,HE staining was used to observe pathological changes of myocardial injury,Tunel staining was used to observe and calculate myocardial cell apoptosis,serum cTnI level was measured by ELISA;myocardial PTEN protein expression was measured by WB,miRNA-21 expression levels in the heart and colon was measured by RT-qPCR,and the abundance of oral Fusobacterium nucleatum and gut Latcobacillus was measured by qPCR.Results:(1)There were no significant differences in body weight,heart weight index,and FBG among the groups(P>0.05);(2)Compared with the ABX group,the F.n.group showed a significant increase in myocardial infarct size(P<0.05),elevated serum cTnI levels(P<0.05),disordered arrangement of myocardial fibers detected by HE staining,pyknotic cell nuclei and karyolysis and aggravated myocardial injury,and increased myocardial apoptosis indicated by Tunel staining(P<0.05).The FMT group had an increase in serum cTnI levels(P<0.05)and aggravated myocardial injury showed by HE staining,but there was no statistically significant difference in myocardial infarct size and myocardial apoptosis detected by Tunel staining(P>0.05).(3)Compared to the ABX group,the F.n.group had an increased myocardial miRNA21 level(P<0.05)and decreased myocardial PTEN protein expression(P<0.05),while there was no significant difference in colon miRNA-21 expression level(P>0.05).The FMT group showed no significant differences in myocardial miRNA-21,colon miRNA-21,and myocardial PTEN protein expression levels compared to the ABX group(P>0.05).(4)Compared with the ABX group,the F.n.group showed a significant increase in Fusobacterium nucleatum and Latcobacillus abundance in the gut(P<0.05),while there was no significant difference in Fusobacterium nucleatum abundance in the oral cavity(P>0.05).Compared with the ABX group,the FMT group showed a significant increase in Fusobacterium nucleatum abundance in the gut(P<0.05),but no significant difference in Fusobacterium nucleatum abundance in the oral cavity and Latcobacillus abundance in the gut(P>0.05).Conclusion:(1)Transplantation of Fusobacterium nucleatum can significantly aggravate MIRI and promote myocardial apoptosis,which is more significant than FMT of DCHD patients and not related to hypoglycemia.(2)Transplantation of Fusobacterium nucleatum can induce a dysbiosis of the gut microbiota mainly characterized by increased Latcobacillus abundance,causing oral-gut dysbiosis.(3)Transplantation of Fusobacterium nucleatum aggravates MIRI,and its mechanism involves the activation of myocardial miRNA-21 and the inhibition of myocardial PTEN protein expression.Part V:Study on the Mechanism of Activating Blood Circulation Herbs(Salvia Miltiorrhiza-Ligusticum Chuanxiong)Preventing DMIRI by Regulating Oral-Gut MicrobiotaObjective:To observe the effects of the blood circulation drug Salvia Miltiorrhiza-Ligusticum Chuanxiong herb pairs on oral and gut microbiota and myocardial apoptosis-related protein PTEN in diabetic mice,thus further exploring the therapeutic mechanism of Salvia Miltiorrhiza-Ligusticum Chuanxiong preventing and treating DMIRI.Method:C57BL/6 mice(n=26)were randomly divided into two groups:sham operation group(SHAM,n=10),and Myocardial Ischemia-Reperfusion Injury group(MIRI,n=16);the remaining C57BL/6J mice(n=64)were established a diabetic model after intraperitoneal injection of STZ.The diabetic mice then were randomly divided into four groups:Diabetic Myocardial Ischemia-Reperfusion Injury group(DMIRI,n=16),Dapagliflozin group(Dapa,n=16),Salvia Miltiorrhiza-Ligusticum Chuanxiong high dose group(HGXN,n=16),and Salvia Miltiorrhiza-Ligusticum Chuanxiong low dose group(LGXN,n=16).After six weeks of continuous intervention,the MIRI model has been established by operating on mice(the anterior descending branch of each mouse is ligated for 30 minutes and then perfused for another 24 hours).The mice were observed for general conditions including FBG,body weight,and cardiac mass index;the area of myocardial infarction was observed and calculated by TTC staining;pathological changes of myocardial injury were observed by HE staining;myocardial apoptosis was observed and calculated by Tunal Staining;serum cTnI level was measured by ELISA;myocardial PTEN protein expression was measured by WB;abundance of oral cavity microbiota Fusobacterium nucleatum and gut microbiota Latcobacillus were measured by qPCR method.Results:(1)Compared with the SHAM group,the mice in the MIRI group have a significant decrease in body weight(P<0.05)and no significant differences in blood glucose and cardiac mass index(P>0.05).Meanwhile,compared with the SHAM group,the mice in the MIRI group have a significant increase in the area of myocardial infarction(P<0.05),have an increase in serum cTnI level(P<0.05),were aggravated in myocardial damages like the sparse and disordered myocardial arrangement and disappearance of nucleus lysis as HE staining showed,have a significant increase in myocardial apoptosis as Tunel staining showed(P<0.05),have no significant differences in the abundance of oral cavity microbiota Fusobacterium nucleatum and gut microbiota Fusobacterium nucleatum and Latcobacillus(P>0.05)and myocardial PTEN protein expression(P>0.05).Compared with the MIRI group,the mice in the DMIRI group have a significant increase in blood glucose(P<0.05)and area of myocardial infarction(P<0.05),have no significant differences in body weight and cardiac mass index(P>0.05)and serum cTnI level(P>0.05),were aggravated in myocardial damage as HE staining showed,have no statistical significance in myocardial apoptosis differences as Tunel staining showed(P>0.05),have an increase in abundance of oral cavity microbiota Fusobacterium nucleatum(P<0.05),no significant differences in the abundance of gut microbiota Fusobacterium nucleatum and Latcobacillus(P>0.05)and in myocardial PTEN protein expression(P>0.05);(2)Compared with the DMIRI group,the mice in the Dapa,HGXN,and LGXN groups have no statistical significance in differences in body weight,cardiac mass index,and blood glucose(P>0.05);(3)Compared with the DMIRI group,the mice in HGXN,LGXN,and Dapa groups have a significant decrease in the area of myocardial infarction(P<0.05),have a decrease in Serum cTnI level(P<0.05),were reduced in damages like myocardial inflammatory infiltration and disordered myocardial arrangement as HE staining showed and in myocardial apoptosis as Tunel staining showed(P<0.05);comparing the MIRI degree of HGXN,LGXN,and Dapa groups,the mice in Dapa group have more reduced in Serum cTnI level than the mice in LGXN group(P<0.05),and the mice in HGXN,LGXN,and Dapa groups have no significant differences in other indicators of myocardial injury.(4)Compared with the SHAM group,the mice in the DMIRI group have a significant decrease in myocardial PTEN expression(P<0.05).Compared with the DMIRI group,the mice in the HGXN group have a significant increase in myocardial PTEN protein expression(P<0.05),and the mice in Dapa and LGXN groups have no statistical significance in myocardial PTEN changes(P>0.05);(5)Compared with the DMIRI group,the mice in Dapa,HGXN,and LGXN groups have a significant decrease in the abundance of Fusobacterium nucleatum in their oral cavity(P<0.05).Compared with the DMIRI group,the mice in the HGXN group have a significant decrease in the abundance of Latcobacillus in their gut(P<0.05).Compared with the DMIRI group,the mice in the HGXN group have a significant decrease in the abundance of Fusobacterium nucleatum in their gut(P<0.05).Different doses of salvia Miltiorrhiza-Ligusticum chuanxiong herb pairs have different effects on the regulation of gut microbiota.Conclusion:(1)The Salvia Miltiorrhiza-Ligusticum Chuanxiong with high and low doses can significantly alleviate MIRI in diabetic mice.Pre-ad ministration can reduce diabetic MIRI,with similar efficacy to the positive drug Dapagliflozin but no significant dose-effect relationship;(2)Hyperglycemia can cause the reduction of myocardial PTEN protein expression during MIRI.The mechanism of the Salvia Miltiorrhiza-Ligusticum Chuanxiong reducing diabetic MIRI is related to that herb pairs can reduce the abundance of Fusobacterium nucleatum in the oral cavity and Latcobacillus in gut and reduce myocardial apoptosis by increasing myocardial PTEN protein expression. |
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