Clinical Characteristics And Mechanism Of Lingguizhugan Decoction On Metabolic Syndrome

As an outstanding representative of traditional Chinese medicine prescriptions,classical prescriptions are effective carriers for the inheritance and innovation of traditional Chinese medicine.They are still widely used in clinical practice with unique characteristics and advantages.In the past 10 years,the state has issued a series of incentive policies to support the promotion of research and development of classical prescriptions of traditional Chinese medicine.The core vitality of classical prescriptions lies in clinical efficacy.How to provide support and reference for precise clinical positioning of classical prescriptions by means of modern technology to guide their more rational and effective clinical application is one of the important issues of classical prescriptions research.At present,the methods of literature research,clinical experience mining,modern technology and evidence-based evaluation have been put forward for the clinical application of classical prescriptions,but they are still scattered and have not yet formed a systematic research model.Taking Lingguizhugan Decoction as an example,its bibliometric study shows that Lingguizhugan Decoction has been widely used in the treatment of cardiovascular system,nervous system and digestive system diseases,while following the original records of "Treatise on Febrile and Miscellaneous Diseases".However,there are still a lack of deep and systemical research on the same application field and effective interaction between clinical and basic research results.So it is necessary to carry out further systematic research.In recent years,with the rapid development of multi-omics technology and large data resources,the understanding of organisms and diseases has changed from decomposition and reduction to system integration.Integrative pharmacology is an interdisciplinary subject which studies the law and principle of integration among complex systems from different levels and perspectives.It coincides with the holistic concept of traditional Chinese medicine.The purpose of this study is to provide evidence and support for the precise clinical application and scientific connotation interpretation of Lingguizhugan Decoction,and to provide reference for the relevant research of classical prescriptions,taking the application of Lingguizhugan Decoction in patients with metabolic syndrome of spleen deficiency and dampness as an entry point.This study includes two parts:The first part is the study of clinical effect characteristics.252 patients with metabolic syndrome were enrolled in a multi-center and prospective case series study.After 16 weeks of Lingguizhugan Decoction intervention relevant data were collected comprehensively.Based on the clinical benefit points of metabolic syndrome such asblood sugar,lipid,blood pressure,waist circumference and insulin resistance,The clinical characteristics of Lingguizhugan Decoction in metabolic syndrome were analyzed with logistic regression.At the same time,the clinical decision-making pathway of Lingguizhugan Decoction in metabolic syndrome was constructed with the help of classification tree.These results would provide reference for clinical precise application of Lingguizhugan Decoction.The second part is the study of mechanism of Lingguizhugan Decoction for metabolic syndrome.Based on UPLC-MS detection,network pharmacology,basic experiments,metabolomics and so on,the mechanism of Lingguizhugan Decoction for metabolic syndrome is explained on the overall level of "drug components-pathway prediction-basic level-clinical level".Part I:Clinical characteristics of Lingguizhugan Decoction on metabolic syndrome.OBJECTIVE:To explore the characteristics of clinical effect population of Lingguizhugan Decoction for metabolic syndrome and further to construct the clinical decision-making path based on the clinical benefit points of metabolic syndrome with logistic regression and classification tree data analysis and mining methods so as to provide reference for the precise clinical application of Lingguizhugan Decoction.METHODS:It is a large sample,multi-center and prospective case series study including 252 eligible patients with metabolic syndrome(spleen deficiency and dampness excess syndrome)carrying on more than 10 sites of the country.After 16 weeks of treatment with Lingguizhugan Decoction,the relevant data of each component of metabolic syndrome were collected comprehensively.Data mining methods such as univariate analysis,logistic regression and classification tree were used to analyze the data.Based on the clinical benefit points of metabolic syndrome such as blood sugar,blood lipid,blood pressure,waist circumference,insulin resistance,the characteristics of clinical effect population of Lingguizhugan Decoction for metabolic syndrome were analyzed,and the clinical decision-making pathway of Lingguizhugan Decoction for metabolic syndrome was constructed.RESULT:1.Univariate analysis resultsThe improvement of waist circumference was more obvious in patients with higher TC level;the improvement of blood sugar was more obvious in patients with obesity(relatively large body weight and BMI),higher TC and better diet control;the improvement of blood pressure was more obvious in patients with better diet and exercise control;the improvement of blood lipid was more obvious in older patients with larger waist circumference,higher HDL-C and better diet control.Youth metabolic syndrome patients with large body weight,waist circumference,low systolic blood pressure and good exercise self-management benefit more obviously from blood sugar,blood pressure and blood lipid.2.Multivariate analysis resultsThe waist circumference of patients with dizziness,white greasy moss improved more obviously after treatment.The blood sugar of patients with TC abnormalities improved more obviously after treatment.Lingguizhugan Decoction improved blood pressure of MS patients with high systolic pressure more obviously,and good diet and exercise self-management also helped improving blood pressure of MS patients.The improvement of blood lipid and insulin resistance was more obvious of patients with higher LDL-C after Lingguizhugan Decoction treatment.The comprehensive benefit of blood glucose,blood pressure and blood lipid was more obvious in young MS patients with better exercise self-management if systolic blood pressure was relatively high and tongue coating was not white and greasy.3.Construction of clinical decision-making pathway(1)Construction of clinical decision-making pathway based on insulin resistance benefits:Lingguizhugan Decoction is more beneficial for improving insulin resistance in metabolic syndrome patients under the following circumstances:Motion score>3.3,TG<2 mmol/L,TC>5.1 mmol/L,and blood pressure<135 mmHg.The exercise score>3.3,TG>2 mmol/L,and blood pressure(systolic pressure)<141 mmHg.The prediction accuracy of the model was 77.7%.(2)Construction of clinical decision-making pathway based on blood glucose benefit:Lingguizhugan Decoction is more beneficial for improving blood glucose in MS patients under the following circumstances:Age>42 years old,waist circumference<91 cm,,TG>1.3 mmol/L,LDL-C>3mmol/L.Age<42 years old,TG<1.9 mmol/L.The prediction accuracy of the model was 68.0%.(3)Construction of clinical decision-making pathway based on blood pressure benefit:Lingguizhugan Decoction is more beneficial for improving blood pressure in metabolic syndrome patients under the following circumstances:Dietary score<3.8 and TG<1.5mmol/L.Diet score>3.8,exercise score<4.8,fasting blood sugar<6.9 mmol/L and glycosylated hemoglobin>6.2%.Diet score>3.8,exercise score>4.8,fasting blood sugar<6.9 mmol/L.The prediction accuracy of the model was 57.3%.(4)Construction of clinical decision-making pathway based on blood lipid benefit:Lingguizhugan Decoction is more beneficial for improving blood lipid in metabolic syndrome patients under the following circumstances:Systolic blood pressure>130 mmHg and diastolic blood pressure<87 mmHg,body weight<86 kg,fasting blood sugar<6.4 mmol/L,tongue coating white and greasy.Systolic blood pressure>130 mmHg,body weight<86 kg,no white greasy tongue coating.Systolic blood pressure>130 mmHg and body weight>86kg with drinking history,age>50 years old.Systolic blood pressure>130mmHg.body weight>86kg,no history of drinking.Systolic blood pressure<130mmHg.The prediction accuracy of the model is 55.3%.(5)Construction of clinical decision-making pathway based on waist circumference benefit:Lingguizhugan Decoction improves waist circumference benefit greatly for MS patients who is dietary score>2.5 points,body weight<71 kg and age<47 years.The prediction accuracy of the model is 92.2%.CONCLUSION:1.Clinical Characteristics and Decision Pathway of Lingguizhugan Decoction Based on Blood Glucose BenefitWhen Lingguizhugan Decoction is used to improve the abnormal blood sugar of metabolic syndrome,attention should be paid to the age and blood lipid of patients.When patients are younger(<42 years old)and complicated with abnormal blood lipid(TG>1.9 mmol/L),the improvement of blood sugar may be more significant after the use of Lingguizhugan Decoction.2.Clinical Characteristics and Decision Pathway of Lingguizhugan Decoction Based on Blood Pressure BenefitGood diet and exercise self-behavior management(diet score>3.8 points,exercise score? 4.8 points)has a positive role in improving blood pressure after medication.When patients with abnormal blood glucose and lipid(fasting blood glucose?6.9 mmol/L and TG?1.5 mmol/L),the effect of Linggui Zhugan Decoction alone may be weak.It is suggested to adjust the treatment plan according to the clinical situation.3.Clinical Characteristics and Decision Pathway of Lingguizhugan Decoction Based on Blood Lipid BenefitWhen the tongue coating of MS patients is white and greasy,the improvement of blood lipid may not be obvious after Lingguizhugan Decoction treatment.In MS patients without hypertension(systolic blood pressure<130mmHg),the improvement of blood lipid may be more obvious.4.Clinical characteristics and decision-making pathway of Lingguizhugan Decoction based on waist circumference benefitWhen Lingguizhugan Decoction is used to improve waist circumference abnormality in MS patients,it may be more suitable for young and middle-aged patients with mild obesity who have controlled diet(diet score>2.5 points,weight<71 kg,age<47 years).Part II:The mechanism of Lingguizhugan Decoction in the treatment of metabolic syndromeSection 1:The mechanism of Lingguizhugan Decoction in treating metabolic syndrome based on metabonomicsThis section includes basic metabonomics research(study I)and clinical metabonomics research(study II).Based on animal models and clinical patients of metabolic syndrome,we explore the metabolomic characteristics of Lingguizhugan Decoction before and after intervention,and try to explain the mechanism of Lingguizhugan Decoction in metabolic syndrome combined with network pharmacological prediction results.Study I Non-Clinical MetabonomicsOBJECTIVE:To explore the metabolomic characteristics of Lingguizhugan Decoction(LGZG)on insulin resistance model rats by using UPLC/Q-TOF-MS combined with non-target metabolic profiling analysis.METHODS:A rat model of insulin resistance induced by olanzapine was established.The treatment group was given low,medium and high doses of Lingguizhugan Decoction for 4 weeks.Blood was taken from abdominal aorta after the last administration.One was used for biochemical detection and the other was used for metabolomics analysis.Metabonomics samples were routinely prepared and detected by UPLC-QTof-MS.The results were exported in raw.file format.PCA and OPLS-DA were analyzed by SIMCA 14.1 software to identify the differential metabolites.Metabo Analysis platform was used to analyze the pathways.RESULT:(1)Compared with the blank group,the weight,fasting blood glucose,blood lipid(TC,LDL-C,VLDL),insulin resistance index of the model group were significantly abnormal(p<0.05),indicating that the model was successful.Compared with the model group,there was no significant difference in the above indicators in each dose group of Lingguizhugan Decoction.And there is a certain trend of benign regulation of TC,TG,VLDL,HSCRP in the low dose group of Lingguizhugan Decoction..(2)Nine different metabolites were identified,including lysophosphatidy-lethanolamine(LysoPE)and phosphatidylethanolamine(LysoPE).(3)Differential metabolites are mainly concentrated in four metabolic pathways,among which Glycerophospholipid metabolism is the main pathway,and others include Linoleic acid metabolism,alpha-Linolenic acid metabolism and Arachidonic acid metabolism.Study II Clinical MetabonomicsOBJECTIVE:To explore the metabolomic characteristics of metabolic syndrome patients with insulin resistance benefit after Lingguizhugan Decoction intervention by UPLC/Q-TOF-MS and non-targeted metabolic profiling analysis.METHODS:The samples were collected from patients with metabolic syndrome in Xiyuan Hospital of CACMs and Chinese Medicine Hospital of Changping District,Beijing.Serum samples of subjects with improved insulin resistance index after intervention with Lingguizhugan Decoction were selected for metabolomics test before and after treatment.The methods of sample preparation,detection and data processing of metabonomics are the same as those of non-clinical metabonomics.RESULTS:Eleven different metabolites were found,including lysophosph-atidylcholine(LysoPC),phosphatidylcholine(PC)and L-Carnitine.Differential metabolites are mainly concentrated in four metabolic pathways,which are the same as the results of non-clinical metabolomics.CONCLUSION OF SECTION 1:Metabonomic analysis suggested that the mechanism of Lingguizhugan Decoction in treating metabolic syndrome might be related to Glycerophospholipid metabolism.By regulating the level of LysoPC and L-carntine in the body through this pathway,it can improve the metabolic abnormalities in patients with metabolic syndrome.Section 2:The mechanism of Lingguizhugan Decoction in metabolic syndrome based on network pharmacologyOBJECTIVE:To predict the potential targets and pathways of Lingguizhugan Decoction for metabolic syndrome based on the main components of Lingguizhugan Decoction detected by UPLC-MS with network pharmacology,and further to explore the mechanism of Lingguizhugan Decoction for metabolic syndrome combined with metabonomic analysis results above.METHODS:Firstly,"metabolic syndrome" or "insulin resistance" or"ICD-9:277.7" were used as keywords to retrieve disease targets related to metabolic syndrome in current commonly used disease gene databases.The interaction network of disease targets was constructed and analyzed with STRING database and Cytoscape 3.5.0 software.Secondly,UPLC-MS technology and UNIFI natural product identification software were used to detect the decoction and granules of Lingguizhugan Decoction.The common components were selected as the main components of Lingguizhugan Decoction,and CTlink software was used to predict the targets of the main components of Lingguizhugan Decoction.Thirdly,by mapping and docking the disease target network of metabolic syndrome with the drug target network of Lingguizhugan Decoction,the drug-disease interaction network was constructed,and the effective components and target of Lingguizhugan Decoction for metabolic syndrome were obtained.Finally,through the interaction network analysis of the effect target of Lingguizhugan Decoction for metabolic syndrome,the key effect target was determined.Through the enrichment analysis of KEGG pathway,the potential mechanism of Lingguizhugan Decoction for metabolic syndrome was explored.RESULT:(1)There are 264 metabolic syndrome disease-related targets,among which INS,AKT1,LEP,PPARG,IL6,TNF,SRC,EGFR and IGF1 are the key targets.(2)A total of 20 common components in Lingguizhugan Decoction decoction and granules were determined,including 10 from Gancao,4 from Guizhi,5 from Fuling and 1 from Baizhu.(3)20 main components of Lingguizhugan Decoction can act on 634 targets,mapping and docking with the target of metabolic syndrome disease shows that 14 components of Lingguizhugan Decoction can act on 38 metabolic syndrome disease-related targets,one of which is the key target of metabolic syndrome disease.(4)Through the analysis of the interaction network of the effect targets of Lingguizhugan Decoction on metabolic syndrome,8 key effect targets were identified,namely PTGS2,TNF,NR3C1,ESR1,PIK3R1,CYP19A1,ACE and IL2.(5)KEGG pathway enrichment analysis of the target of Lingguizhugan Decoction for metabolic syndrome revealed 22 potential pathways.Regulation of lipolysis in adipocytes may be the most closely related to the mechanism of Lingguizhugan Decoction for metabolic syndrome.CONCLUSION:14 main components of Lingguizhugan Decoction can act on 38 metabolic syndrome related targets,among which PTGS2,TNF,NR3C1,ESR1,PIK3R1,CYP19A1,ACE and IL2 are the key targets.Regulation of lipolysis in adipocytes may be the main pathway for Lingguizhugan Decoction for metabolic syndrome.The detailed intervention mechanism may be to participate in the process of lipid demodulation during fasting and eating,and to influence insulin signaling pathway and arachidonic acid inflammation signaling pathway respectively by interfering with PI3K and COX.Based on the results of network pharmacology and metabolomics analysis,it is speculated that the mechanism of Lingguizhugan Decoction for metabolic syndrome is related to the intervention of regulation of lipolysis in adipocytes and arachidonic acid pathway.AdPLA as a common component of the above pathway,may mediate Lingguizhugan Decoction's further involvement in glycerol phospholipid metabolism pathway by regulating the level of LysoPC and L-carnitine in the body to improve metabolic abnormalities in patients with metabolic syndrome.Summary1.The clinical characteristics of Lingguizhugan Decoction for metabolic syndrome are as follows:(1)When Lingguizhugan Decoction is used to improve the abnormal blood sugar of metabolic syndrome,attention should be paid to the age and blood lipid of the patients.When the patients are younger(<42 years old)and complicated with abnormal blood lipid(TG?1.9 mmol/L),the improvement of blood sugar may be more significant after the treatment of Lingguizhugan Decoction.(2)Good diet and exercise self-behavior management(diet score?3.8 points),exercise score?4.8 points)has a positive role in improving blood pressure in MS patients after medication.When patients with abnormal blood glucose and lipid(fasting blood glucose?6.9 mmol/L)and TG?1.5 mmol/L),the effect of Linggui zhugan Decoction alone may be weak.It is suggested to adjust the treatment plan according to the clinical situation.(3)When the tongue of MS patients is white and greasy,the improvement of blood lipid may not be obvious after the treatment of Lingguizhugan Decoction.In MS patients without hypertension(systolic blood pressure<130mmHg),the improvement of blood lipid may be more obvious.(4)When Lingguizhugan Decoction is used to improve waist circumference abnormality in MS patients,it may be more suitable for young and middle-aged patients with mild obesity who have controlled diet(diet score?2.5 points,weight<71 kg,age<47 years).(5)Lingguizhugan Decoction may be more suitable for patients with metabolic syndrome characterized by dyslipidemia,abnormal blood sugar and mild obesity.Lingguizhugan Decoction may not be effective in the treatment of patients with metabolic syndrome characterized by abnormal blood pressure.2.The mechanism of Lingguizhugan Decoction on metabolic syndrome may be related to the regulation of lipid and inflammation-related pathways:Lingguizhugan Decoction can interfere with the regulation of lipolysis in adipocytes in fasting and eating state,and affect the arachidonic acid inflammation signaling pathway by interfering with COX.AdPLA,as a common component of the above pathway,may mediate Lingguizhugan Decoction to further participate in the glycerol phospholipid metabolism pathwayto regulate the levels of LysoPC and L-carnitine in the body of patients with metabolic syndrome.