Study On Mechanism Of Lingguizhugan Decoction Regulating Gut Microbiota And Its Metabolite TMAO To Improve Chronic Heart Failure

Objective1.In the clinical study,the distribution characteristics and rules of TCM syndromes in patients with chronic heart failure were preliminarily discussed in order to provide a reference for TCM clinical syndrome differentiation and treatment;2.To seek the optimal scheme by observing the modelling of doxorubicin-induced chronic heart failure in rats under different schemes and to assess the changes in the composition and structure of the gut microbiota and the correlation with doxorubicin-induced cardiotoxicity;3.Based on the traditional Chinese medicine theory of "the heart stands in interior-exterior relationship with the small intestine",the rats with chronic heart failure induced by doxorubicin were treated with the low,middle and high doses of Linggui Zhugan Decoction,respectively,to observe its effect on improving myocarditis and antioxidant stress after six weeks of administration,and to clarify its mechanism from two aspects:TMAO-Sirt3-SOD2-mtROS pathway and changes in the composition and structure of gut microbiota,It provides a reasonable reference basis for the follow-up clinical treatment of heart failure.Method1.Clinical study on the syndrome analysis of Traditional Chinese Medicine in patients with chronic heart failureCase data of inpatients with chronic heart failure admitted to the cardiovascular department of Shenzhen Hospital of Guangzhou University of Chinese Medicine（Futian）from January 2019 to January 2022 were collected,and relevant original data such as syndrome differentiation and classification of Traditional Chinese Medicine were obtained.After standardized processing,descriptive statistical analysis was conducted.2.Experimental study of doxorubicin-induced chronic heart failure in ratsNinety-nine SD rats were randomly divided into the model group（DOX group,eighty-one SD rats）and the control group（CON group,eighteen SD rats）,and the intervention was performed once a week for six weeks.There were nine groups in the DOX group,which were divided into the following groups according to the route of doxorubicin administration,the mode of administration and the total cumulative dose:（1）Tail vein group（fixed dose administration,same dose per week）,including:DOX-A group:administered in 2mg/kg body weight for a total cumulative dose of 12mg/kg;DOX-B group:administered in 2.5mg/kg body weight for a cumulative total dose of 15 mg/kg;DOX-C group:administered in 3mg/kg body weight for a total cumulative dose of 18mg/kg;（2）Tail vein group（alternating doses,same dose every other week）,including:DOX-D group:3 mg/kg body weight in the first,third and fifth weeks,and 1 mg/kg body weight in the second,fourth,and sixth weeks,for a total cumulative dose of 12 mg/kg;DOX-E group:3.5 mg/kg body weight in the first,third and fifth weeks,and 1 mg/kg body weight in the second,fourth,and sixth weeks,for a cumulative total of 15 mg/kg;DOX-F group:4mg/kg body weight in the first,third and fifth weeks,and 1 mg/kg body weight in the second,fourth,and sixth weeks,for a total cumulative dose of 18mg/kg;（3）Intraperitoneal injection group（alternate doses administered at the same dose every other week）,including:DOX-G group:3mg/kg body weight in the first,third and fifth weeks,and 1 mg/kg body weight in the second,fourth,and sixth weeks,for a total cumulative dose of 12mg/kg;DOX-H group:3.5 mg/kg body weight in the first,third and fifth weeks,and 1mg/kg body weight in the second,fourth,and sixth weeks,for a cumulative total of 15mg/kg;DOX-I group:4mg/kg body weight in the first,third and fifth weeks,and 2 mg/kg body weight in the second,fourth,and sixth weeks,for a total cumulative dose of 18mg/kg.There were two groups in the CON group,including:（1）CON-J group:tail vein injection of an equal volume of saline.（2）CON-K group:intraperitoneal injection of an equal volume of saline.The rats were observed for general conditions.The M-mode echocardiograms performed cardiac function evaluation at the end of six weeks of intervention.Pathological changes in the intestine were observed by H&E staining and in the heart by Masson staining.The serum levels of N-terminal pre-B-type natriuretic peptide（NT-proBNP）and cardiac troponin I（cTnI）were measured by ELISA.The composition and characteristics of gut microbiota were analyzed by 16S rRNA gene sequencing.3.The study based on gut microbiota to explore the mechanism of Lingguizhugan decoction in the treatment of chronic heart failureFifty SD rats were randomly divided into the model group（DOX group,fourty SD rats）and the control group（CON group,ten SD rats）.The rats in the model group were given tail vein injection of doxorubicin,according to 4mg/kg body weight in the first,third and fifth weeks,and 2mg/kg body weight in the second,fourth,and sixth weeks,for a total cumulative dose of 18mg/kg,the control group was were given tail vein injection of an equal volume of saline.At the end of the sixth week after modelling,40 surviving SD rats were randomly divided into the heart failure model group（HF group）,the low-dose Linggui Zhugan decoction group（LGZGT-LD group）,the medium-dose Linggui Zhugan decoction group（LGZGT-MD group）and the high-dose Linggui Zhugan decoction group（LGZGT-HD group）.The rats were observed for general conditions.At the end of the sixth week after intervention,the M-mode echocardiograms performed cardiac function evaluation at the end of six weeks of intervention.Pathological changes in the intestine were observed by H&E staining and in the heart by Masson staining.The serum levels of trimethylamine-N-oxide（TMAO）,N-terminal pre-B-type natriuretic peptide（NT-proBNP）,cardiac troponin I（cTnⅠ）,superoxide dismutase（SOD）,interleukin-1β（IL-1β）,tumour necrosis factor-α（TNF-α）and NOD-like receptor protein 3（NLRP3）were measured by ELISA.The ultrastructure of rat myocardium was observed by transmission electron microscopy,and mitochondria were scored in parallel;The expression of silent mating type information regulation 2 homolog 3（SIRT3）and superoxide dismutase 2（SOD2）was measured by Immunohistochemistry and Western blot;The level of cardiomyocyte apoptosis was detected by Tunel method;The myocardial mitochondrial reactive oxygen species（mtROS）was measured by BCA kit;The composition and characteristics of gut microbiota were analyzed by 16S rRNA gene sequencing.Results1.Clinical study on the syndrome analysis of Traditional Chinese Medicine in patients with chronic heart failure（1）A total of 74 cases were included in this study.5 TCM syndrome types were extracted,including 32 cases of Qi and Yin deficiency,17 cases of Yang deficiency and water genericity,15 cases of Qi deficiency and blood stasis,6 cases of phlegm-stasis inter-association,and 4 cases of heart-lung Qi deficiency.（2）Functional classification of the centre of this study:there were 6 patients with GradeⅡ cardiac function,with the highest frequency of syndrome types being the syndrome of Qi and Yin deficiency and the syndrome of Yang deficiency and water genericity;there were 32 patients with grade Ⅲ cardiac function,and Qi and Yin deficiency syndrome had the highest frequency,there were 36 patients with grade Ⅳ heart function,and Qi and Yin deficiency syndrome had the highest frequency.The patients with Qi and Yin deficiency syndrome and phlegm-stasis interjunction syndrome were mainly distributed in grade Ⅲ.In contrast,the patients with the syndrome of Yang deficiency and water genericity and Qi deficiency and blood stasis were mainly distributed in grade Ⅳ.（3）In this study,there were 51 cases of Qi deficiency,32 cases of Yin deficiency,21 cases of blood stasis,17 cases of Yang deficiency,17 cases of water genericity,and 6 cases of phlegm turbidity;Qi-deficiency and Yin deficiency were the main syndrome-related elements in grade Ⅱ and Ⅲ patients with chronic heart failure,while Qi-deficiency and water genericity were the main syndrome-related elements in grade Ⅳ patients with chronic heart failure.（4）In this study,the combination of pathogenic syndromes included 27 cases of five syndromes combination,23 cases of two syndromes combination,12 cases of three syndromes combination and 12 cases of four syndromes combination;（5）In this study,there were 14 cases of simple deficiency syndrome,54 cases of mixed deficiency and excess syndrome,and 6 cases of simple excess syndrome.（6）In this study,the top 9 clinical symptoms were shortness of breath,chest tightness,mental fatigue,fatigue,oedema of limbs,loss of appetite,abdominal distension,poor sleep and cough.2.Experimental study of doxorubicin-induced chronic heart failure in rats（1）From the first to third week after modelling,rats in each group showed no abnormalities in their diet and bowel movements.They were accessible and sensitive to activity response and in good mental condition,and their fur was soft and smooth;However,the body weight of rats in each group continued to increase,from the second week onwards,compared with the CON group,the increase in body weight in each DOX group was reduced to different degrees.The difference was significant（P<0.05）.From the fourth to sixth week after modelling,rats in the CON group did not show any apparent abnormalities;rats in the DOX group showed drug intoxication effects,including inactive mental state,reduced activity and muted response,increased eyelid secretions,decreased eating and drinking,tarnishes of fur and dull yellow fur,occasional hair shedding,accelerated respiration,some rats showed symptoms such as eye congestion,diarrhoea and loose stools,etc.Compared with the rats in the CON group,the body weight of rats in groups DOX-A group to DOX-F group was decreased to varying degrees,and the body weight of rats in groups DOX-G group to DOX-I group was increased to different degrees,the difference was significant（P<0.05）.（2）At the end of the sixth week after modelling,compared with the CON groups,the heart weight of all DOX groups except the DOX-G group decreased,and the difference was significant（P<0.05）;（3）At the end of the sixth week after modelling,eight rats survived in the DOX-B group,seven in each of the DOX-F and DOX-I groups,and nine in the other remaining groups;（4）At the end of the sixth week after modelling,compared with the CON groups,the differences in the interventricular septal thickness at end-diastole and the left ventricular internal diameter at end-diastole between the DOX groups were not significant（P>0.05）;the DOX-E group to DOX-I group all had different degrees of decrease in the interventricular septal thickness at end-systole,and the differences were significant（P<0.05）,while the differences between the remaining DOX groups were not significant（P>0.05）;between the DOX groups,the left ventricular internal diameter at end-systole increased to different degrees,and the left ventricular ejection fraction decreased to different degrees,with significant differences（P<0.05）;（5）At the end of the sixth week after modelling,compared with the CON groups,serum NT-proBNP levels and cTnI levels were increased to different degrees in all DOX groups,and the difference was significant（P<0.05）;（6）At the end of the sixth week after modelling,compared with the CON groups,the intestinal structure was relatively intact in the DOX-A group to DOX-G group,with partial loss of intestinal mucosal integrity,colonic mucosal epithelium oedema,a few glands atrophy,and partial inflammatory cell infiltration in the lamina propria.In the DOX-G group to DOX-I group,the intestinal tissue structure was incomplete,with complete loss of intestinal mucosal integrity and most glands atrophy;The pathological changes in the intestinal tissues were most severe in the DOX-I group,with thinning of the lamina propria and separation of the villi from the basement membrane;（7）At the end of the sixth week after modelling,compared with the CON groups,there was no significant cardiomyocyte collagen deposition in the DOX-A group,DOX-D group and DOX-G group.Focal myocardial collagen deposition was observed in the DOX-B group,DOX-E group,DOX-H group and DOX-I group.In the DOX-C group and DOX-F group,there was diffuse collagen deposition of cardiomyocytes,thickened interstitial fibres,disordered and stacked distribution,widened myocardial space,hypertrophy and swelling of cardiomyocytes,among which the myocardial fibrosis in DOX-F group was the most severe;（8）At the end of the sixth week after modelling,the relative abundance of gut microbiota at the phylum level was dominated by the top 3 species in each group:Bacteroidetes,Firmicutes and Proteobacteria in the DOX-A group to DOX-F group,Bacteroidetes,Firmicutes and Actinobacteria in the DOX-G group,Bacteroidetes,Firmicutes and Actinobacteria in the DOX-H group,Actinobacteria,Bacteroidetes and Firmicutes in the DOX-I group,and Firmicutes,Bacteroidetes and Proteobacteria in the CON group;Compared with the CON-J group,the DOX-A group to DOX-F group showed different decreases in the abundance of Firmicutes,increases in the abundance of Bacteroidetes,and decreases in the ratio of Firmicutes to Bacteroidetes（F/B）;Compared with the CON-K group,the ratio of Firmicutes:Bacteroidetes（F/B）was lower in both the DOX-G group and DOX-I group,while the ratio of Firmicutes:Bacteroidetes（F/B）was higher in the DOX-H group;The relative abundance of gut microbiota at the genus level increased for Escherichia/Shigella in all DOX groups compared with all CON groups,with the most pronounced increase in the DOX-F group;the abundance of Lactobacillus in the DOX-B group,DOX-F group,DOX-G group to DOX-I group;The abundance of Bifidobacterium in DOX-A group,DOX-C group and DOX-I group increased,with the most significant increase in DOX-I group;the abundance of Bacteroides in DOX-A group to DOX-G,and DOX-I group decreased,with the most significant decrease in DOX-C group;（9）At the end of the sixth week after modelling,compared with the CON groups,the Shannon index in the DOX-B group,DOX-C group,DOX-E group and DOX-F group,and the Chao1 index in the DOX-B group,DOX-C group and DOX-F group were reduced,and the difference was significant（P<0.05,P<0.01）;compared with the CON-K group,the Shannon index and Simpson index in the DOX-G group to DOX-I group were reduced,and the difference was significant（P<0.05,P<0.01）,Chao1 index in the DOX-H group was increased,and the difference was significant（P<0.05）;（10）At the end of the sixth week after modelling,Tax4Fun functional prediction analysis showed that,（i）genes related to the Cellular Processes,the gene function of transport and catabolism was enriched in DOX-A group to DOX-D group,DOX-F group and DOX-G group;（ii）genes related to the Genetic Information Processing,the gene function of folding,sorting and degradation was enriched in groups DOX-Agroup to DOX-I group;the gene function of translation was enriched in groups DOX-A group to DOX-C group,DOX-F group,DOX-H group and DOX-I group;the gene function of Replication and repair was enriched in DOX-I group;（iii）genes related to the Metabolism,the gene function of Metabolism of other amino acids was enriched in DOX-A group to DOX-C group,and DOX-E group to DOX-H group;the gene function of Glycan biosynthesis and metabolism was enriched in DOX-A group,DOX-B group,DOX-E group to DOX-G group;the gene function of Enzyme families was enriched in DOX-B group to DOX-D group,DOX-F group and DOX-H group;the gene function of Biosynthesis of other secondary metabolism was enriched in DOX-A group,DOX-B group,DOX-D group and DOX-F group;the gene function of Metabolism of terpenoids and polyketides was enriched in DOX-E group,DOX-F group,DOX-H group and DOX-I group;the gene function of lipid metabolism was enriched in DOX-A group,DOX-B group and DOX-D group;the gene function of Metabolism of cofactors and vitamins was enriched in DOX-A and DOX-B groups;the gene function of Carbohydrate metabolism and Nucleotide metabolism were enriched in DOX-H group and DOX-I group;（iiii）genes related to the Organismal Systems,the gene function of Nervous system was enriched in DOX-B group,DOX-D group and DOX-F group;the gene function of Aging was enriched in DOX-I group;（iiiii）genes related to the Human Diseases,the gene function of Cancers was enriched in DOX-A group to DOX-F group;the gene function of Drug resistance was enriched in DOX-G group to DOX-I group;the gene function of Endocrine and metabolic diseases was enriched in DOX-I group;the gene function of Cardiovascular diseases was enriched in the DOX-A group to DOX-D group,and DOX-F group.3.The study based on gut microbiota to explore the mechanism of Lingguizhugan decoction in the treatment of chronic heart failure（1）At the end of the sixth week after intervention,rats in the CON group had no obvious abnormality in diet and bowel movement,they were accessible and sensitive to activity response and in good mental condition,their fur was soft and smooth,and their weight continued to rise.Compared with the CON group,the rats in the HF group were depressed in mental state,had reduced activity and muted response,increased eyelid secretions,decreased eating and drinking,tarnishes of fur and dull yellow fur,occasional hair shedding,accelerated respiration,some rats showed symptoms such as eye congestion,diarrhoea and loose stools;Compared with the HF group,the LGZGT-LD group,the LGZGT-MD group and the LGZGT-HD group have different degrees of improvement in the above conditions,of which LGZGT-HD group has the most apparent improvement;（2）At the end of the sixth week after intervention,compared with the HF group,the differences in the interventricular septal thickness at end-diastole in the LGZGT-LD group,LGZGT-MD group and LGZGT-HD group were not significant（P>0.05）;the interventricular septal thickness at end-systole in the LGZGT-HD group was significantly increased（P<0.05）,and the left ventricular internal diameter at end-diastole was significantly decreased（P<0.05）;The left ventricular internal diameter at end-systole in the LGZGT-MD group and LGZGT-HD group were significantly decreased（P<0.05）;the left ventricular ejection fraction in the LGZGT-LD group,LGZGT-MD group and LGZGT-HD group were significantly increased（P<0.05）;（3）At the end of the sixth week after intervention,compared with the HF group,serum TMAO levels,IL-1β levels,NLRP3 levels,TNF-α levels,ROS levels,cTnI levels and T-proBNP levels were significantly lower（P<0.05）and SOD levels were significantly higher（P<0.05）in the LGZGT-LD group,LGZGT-MD group and LGZGT-HD group;（4）At the end of the sixth week after intervention,compared with the CON group,the HF group showed incomplete intestinal tissue structure,complete loss of intestinal mucosal integrity,atrophy of most glands,thinning of the lamina propria and separation of the villi from the basement membrane;compared with the HF group,the LGZGT-LD group,LGZGT-MD group and LGZGT-HD group showed different degrees of improvement in intestinal mucosal integrity and degree of glandular atrophy,with the LGZGT-HD group showing the most significant changes;（5）At the end of the sixth week after intervention,compared with the CON group,the myocardial interstitial and perivascular collagen fiber expression increased significantly in the HF group,with thickened interstitial fibers,disorganized and piled-up distribution,widened myocardial gaps,and hypertrophy and swelling of myocardial cells;compared with the HF group,the myocardial fiber arrangement and collagen deposition in the LGZGT-LD group,LGZGT-MD group and LGZGT-HD group were all reduced to different degrees,with the most obvious changes in the LGZGT-HD group;（6）At the end of the sixth week after intervention,compared with the CON group,the myofibrils of myocardial myogenic fibers in the HF group were partially dissolved,myofilament bundles were broken,the arrangement was loose and disorganized,the mitochondria were swollen and vacuolated,the matrix was transparent and the granules were lost,the mitochondrial cristae were partially broken,the mitochondrial inner and outer membranes were intact,the borders were blurred and fused;compared with the HF group,the myocardial fiber arrangement and mitochondrial changes in the LGZGT-LD group,LGZGT-MD group and LGZGT-HD group were reduced to different degrees,with the most obvious changes in the LGZGT-HD group;（7）At the end of the sixth week after intervention,compared with the HF group,the apoptotic rate of cardiomyocytes was significantly lower in the LGZGT-MD group and LGZGT-HD group（P<0.05）;（8）At the end of the sixth week after intervention,compared with the HF group,the levels of SOD2 positive expression and Sirt3 positive expression in the LGZGT-LD group,LGZGT-MD group and LGZGT-HD group were significantly higher（P<0.05）;（9）At the end of the sixth week after intervention,compared with the HF group,the expression levels of Sirt3 protein and SOD2 protein were significantly higher in the LGZGT-LD group,LGZGT-MD group and LGZGT-HD group（P<0.05）;（10）At the end of the sixth week after intervention,the relative abundance of gut microbiota at the phylum level,the top 4 dominant species in the CON group,HF group,LGZGT-MD group and LGZGT-HD group were Firmicutes,Bacteroidetes,Proteobacteria and Actinobacteria,in the LGZGT-LD group was Firmicutes,Bacteroidetes and Spirochaetota.Actinobacteria,while in the LGZGT-LD group was Firmicutes,Bacteroidetes,Spirochaetota and Proteobacteria;compared with the HF group,the abundance of Bacteroidetes and Spirochaetota in the LGZGT-LD group was significantly higher,while the abundance of Proteobacteria and Actinobacteria in the LGZGT-MD group and LGZGT-HD group were significantly lower;the abundance of Firmicutes,Proteobacteria and Actinobacteria were significantly higher in the LGZGT-MD group and LGZGT-HD group,and the ratio of Firmicutes to Bacteroidetes（F/B）was significantly higher in the LGZGT-HD group,with the most pronounced of these changes in the LGZGT-HD group;The relative abundance of gut microbiota at the genus level was significantly higher for Clostridium_sensu_stricto₁ and Lactobacillus in the LGZGT-HD group compared with the HF group,and for the unclassified genera Muribaculaceae,Dubosiella Dubosiella,and Bacteroides,with the above changes being most pronounced in the LGZGT-HD group;（11）At the end of the sixth week after intervention,compared with the HF group,the Simpson index was significantly higher in the LGZGT-LD group（P<0.05）and significantly lower in the LGZGT-MD group and LGZGT-HD groups（P<0.05）;（12）At the end of the sixth week after intervention,Tax4Fun functional prediction analysis showed that,genes related to the Metabolism,the gene function of Carbohydrate metabolism pathway,Glycan biosynthesis and metabolism pathway,Metabolism of other amino acids,Energy pathway,Metabolism of cofactors and vitamins,Amino acid metabolism,and genes related to the Environmental Information Processing.the gene function of signal transduction pathway was enriched in the LGZGT-LD group;genes related to the Environmental Information Processing,the gene function of membrane transport pathway,translation pathway,replication and repair pathway,and genes related to the Metabolism,the gene function of nucleotide metabolism pathway were enriched in the CON group and LGZGT-HD group.Conclusions1.Clinical study on the syndrome analysis of Traditional Chinese Medicine in patients with chronic heart failureThe deficiency in origin and enrichment in symptoms mainly characterize the pathogenesis of chronic heart failure.The syndromes are mainly characterized by Qi deficiency and blood stasis,Yang deficiency and water genericity.The syndromes are mainly characterized by Qi deficiency,blood stasis and water genericity.It preliminarily reflects the distribution law of common witness type and chronic heart failure syndrome elements.2.Experimental study of doxorubicin-induced chronic heart failure in rats（1）There are differences in the degree of myocardial injury in doxorubicin-induced heart failure rats under different schemes;（2）The composition and structure of the gut microbiota of rats with heart failure induced by doxorubicin differed under different schemes;（3）The experimental results showed that the model of heart failure established by tail vein injection of doxorubicin,administered at 4mg/kg body weight at weeks 1,3 and 5,and at 2mg/kg body weight at weeks 2,4 and 6,with a cumulative total dose of 18mg/kg,was more stable.The degree of myocardial injury and changes in the gut microbiota was the closest to the clinical manifestations of heart failure patients.It is a better protocol to study the correlation between heart failure and gut microbiota.3.The study based on gut microbiota to explore the mechanism of Lingguizhugan decoction in the treatment of chronic heart failure（1）Linggui Zhugan Decoction can effectively improve cardiac function and myocardial injury in rats with chronic heart failure,and the effects were dose-dependent,with the best effect in the high-dose group;（2）Linggui Zhugan Decoctioncan effectively improve the intestinal barrier function in rats with chronic heart failure,with the changes in intestinal flora in the high-dose group being more similar to those in the blank group;（3）The formula of warming yang and dissolving drink,Linggui Zhugan Decoction,can protect the myocardium by down-regulating the expression of TMAO,the metabolite of gut microbiota,up-regulating the expression of SIRT3 and SOD2,and down-regulating the expression of mtROS in the myocardial tissue of rats with chronic heart failure.