| Purpose:There are five parts in this paper:clinical observation,data mining,network pharmacology,clinical retrospective research and animal experiment,mainly analyze the pathogenetic characteristics and syndrome elements of metabolism-related fatty liver disease(MAFLD).According to Professor Lu Bingjiu’s clinical experiences,we formulated the decoction of Li qi hua shi,and explored the material basis,clinical efficacy and mechanism of action for the treatment of MAFLD.Material and method:Clinical observation:A total of 329 cases were collected from outpatients with NAFLD treated by Professor Lu Bingjiu from Jan.1,2017 to Dec.31,2021.Then the relevant Western medical testing indicators,the Chinese medicine symptom and questionnaires of the included study cases were collated and statistically analyzed,and statistical analysis of the pathological factors and disease location factors of MAFLD were performed by frequency analysis,frequency statistics and factor analysis.Data mining:A total of 310 cases of MAFLD treated by Professor Lu Bingjiu during the period from 1 Jan.2017 to 31 Dec.2021,with a total of 1,149 consultations were collected,and the traditional Chinese medicine used by Professor Lu Bingjiu in the treatment of MAFLD were statistically analysed by frequency analysis,frequency statistics,correlation analysis and cluster analysis to summarise their properties,taste,meridian tropism and efficacy types.In order to inductive the valuable experience of Professor Lu Bingjiu in the clinical treatment of MAFLD.Network pharmacology : We formulated the decoction of Li qi hua shi based on the experience of Professor Lu Bingjiu in the treatment of MAFLD.The main chemical components and their targets of Citri Reticulatae Pericarpium,Poria,Atractylodis Rhizoma,Alismatis Rhizoma,Psyllium,Arecae Pericarpium,Hawthorn and red yeast rice were retrieved by the TCMSP database and BATMAN-TCM database as well as the related literature review.Then we selected the components that simultaneously satisfy the two ADME property paramaters of OB(oral bioavailability)≥30% and DL(drug-likeness)≥0.18 as the active components and targets of the decoction,and applied the Uniprot Protein data to standardize the target name as the gene symbol.The target of "fatty liver" and "metabolic disease" that satisfied the Relevance score ≥ 5 were retrieved from the Gene Cards database,and their intersection was used as the MAFLD-related disease target.The intersection of decoction of Li qi hua shi-related target genes and MAFLD-related disease target genes was plotted on a Venn Diagram.The cytoscape 3.6.11 software was used to construct the network of herb-active ingredients-therapeutic targets,Network Analyzer was used to analyze the network topological parameters of the network to obtain the components of Li qi hua shi decoction that exerts the primary pharmacological effects and the primary therapeutic targets.A high-confidence interaction network that satisfied the minimum interaction score >0.9000 was filtered with the String database,and the PPI network of targets of Li-qi-hua-shi decoction-MAFLD was constructed by cytoscape 3.6.1,and the potential core sub-networks in the PPI network were obtained using the MCODE.The GO analysis and KEGG signaling pathway analysis were performed by Metascape platform at P < 0.01 to establish the target-signaling pathway network of Li-qi-hua-shi decoction.Clinical research:A total of 340 patients who attended the hepatology clinic of the First Hospital of Liaoning University of Chinese Medicine from Mar.2021 to Dec.2021 and met both the Chinese and Western medical diagnostic standard of MAFLD were selected.They were divided into 83 cases in the Chinese herbal medicine group and 257 cases in the control group according to receiving treatment with the Li-qi-hua-shi decoction and silybin meglumine tablets.Propensity score matching(PSM)method was used to obtain the data on36 groups of comparable cases.The BMI index,transaminase,serum lipid level,liver ultrasound structure and quantitative score of TCM symptom integral meter were compared between the two groups after 4W,8W and 12 W of treatment to evaluate the efficacy and safety of the Li-qi-hua-shi Decoction on MAFLD.Animal experiment:The rats in the Chinese medicine group and the Western medicine group were administered with the Li-qi-hua-shi Decoction and silybin meglumine respectively.After 8 weeks of intervention,the body weight,liver wet weight,liver index,transaminase,serum lipid level,FIN,FBG,IRI,general and pathomorphological changes of liver tissue,serum levels of adipokines and inflammatory factors were compared and analyzed.Results:No.1 Analysis of the syndrome and pathological elements of 329 cases of MAFLD.1.Results of the general data analysis: the gender distribution of patients was mainly male,with 68.39% and 31.61% of patients of both sexes respectively;the age distribution of patients was mainly in the age groups of 46-60 years old,among which there were significantly more male patients than female patients in the age group below 45 years old,and the gap gradually narrowed above 46 years old;obese patients accounted for 56.23% of the total number of patients,and the proportion of obese patients was the highest among both male and female patients.The proportion of obese patients was the highest among both male and female patients,followed by overweight patients,with only 1.33% of male patients and15.38% of female patients having a BMI within the normal range.2.Laboratory indicators results: Among the MAFLD cases included in this statistical data,70.21% of the total number of patients with abnormal ALT level and 46.2% of the total number of patients with abnormal AST level;in terms of abnormal liver function in patients of different genders,72.89% of male patients and 57.1% of female patients had abnormal ALT level,47.76% of male patients and 39.06% of female patients had abnormal AST level;in terms of abnormal liver function in male patients of different ages,the abnormal liver function in patients of all ages was generally similar.In terms of abnormal liver function in male patients of different age groups,the abnormalities of liver function were generally similar in all age groups,and in terms of abnormal liver function in female patients of different age groups,the highest percentage of abnormal liver function indicators were found in patients under 30 years old.Dyslipidemia was prevalent among the patients with MAFLD,with abnormalities in levels of TC,TG,HDL-C and LDL-C accounting for 69%,83.28%,80.55%and 76.29% respectively.In terms of dyslipidemia in patients of different genders,male patients had a higher rate of dyslipidemia than female patients.The highest rate of abnormal TG level was demonstrated in both male and female patients,and there were no significant differences between all of the age groups.In the cases of this research,the percentage of patients with MAFLD who had abnormal FBG level was 46.2% and 30.09% of those with abnormal SUA level.In terms of the distribution of indicators among patients of different genders,male and female patients with abnormal FBG level accounted for 53.29% and46.71% of the same sex prevalence,respectively,and male and female patients with abnormal SUA level accounted for 31.31% and 68.69% of the same sex prevalence,respectively with the detection rate of abnormal SUA is significantly higher in female patients than in males.3.Comorbid status:The top five highest frequency of comorbid occurring in the MAFLD cases collected in this study were type 2 diabetes(38.3%),hypertension(21.58%),hepatitis B(15.5%),coronary heart disease(10.64%)and chronic renal insufficiency(6.69%).4.The Chinese medical syndrome and pathological elements of MAFLD : The highest frequency of the tongue image is a pale red tongue with the thin fer,and higher pulse condition ais the slippery pulse,followed by the heavy pulse,the stringy pulse and and the thin pulse.The pathological elements involved were yin deficiency,yang deficiency,qi deficiency,dampness,fire,phlegm,blood stasis and the stagnation of qi.The disease location factors include liver,spleen and kidneys.No.2 A data-mining-based exploration of Professor Lu Bingjiu’s medication patterns in the treatment of MAFLDA total of 903 Chinese medicine prescriptions were included in this study,using 292 kinds of Chinese medicine,with a total frequency of 12,962 doses,with a maximum of 30 flavors and a minimum of 1 flavor in a single prescription,as well as an average of 14.35 flavors.1.Types of the medicine: In terms of medication,the more frequent ones were reinforcing drugs(22.33%),diuretic drugs(13.47%),heatclearing drugs(9.40%),Qi regulating drugs(8.20%)and dampness resolving drugs(8.12%);In terms of drug properties,the more frequent ones were warm drugs(47.80%),cold drugs(25.55%)and balanced drugs(21.36%);By medicine taste,the most frequently ones were sweet(34.27%),bitter(30.88%)and pungent(23.80).By meridian,the higher frequencies were in the spleen(22.22%),liver(16.55%)and stomach(14.31%)meridians.2.High frequency drugs : 37 high frequency drugs were identified through medication statistics,including Citri Reticulatae Pericarpium(68.14%),Atractylodes macrocephala(63.72%),Poria(63.16%),Atractylodis Rhizoma(59.73%)and Alismatis Rhizoma(44.03%).3.Drug correlation analysis: Analysis of the association rules between drug groups showed that the combinations of Chinese herbal medicines commonly used by Prof.Lu Bingjiu included : Hawthorn+Citri Reticulatae Pericarpium→Alismatis Rhizoma,Radix pseudostellariae+Psyllium→ Atractylodes,Arecae Pericarpium+Poria→Citri Reticulatae Pericarpiumi,White Cardamom+Atractylodis Rhizoma,Cassia Twig+Alismatis Rhizoma→Poria,etc.4.Cluster analysis: The types of drugs commonly used by Professor Lu Bingjiu for the treatment of MAFLD as obtained by systematic clustering,were as follows : the first category: Alismatis Rhizoma,Hawthorn,Red yeast rice,Semen Cassiae,Nelumbo nucifera Gaertn,White Cardamom,Atractylodis Rhizoma,Psyllium,Citri Reticulatae Pericarpium,Arecae Pericarpium,Poria;the second category : Gizzard Pepsin,Cuttlebone,Bambusae Caulisin Taenias,Coptidis Rhizoma,Magnoliae Officinalis Cortex,Rhizoma Pinelliae,Radix Glycyrrhizae preparata,Scutellariae Radix,Fructus Aurantii Immaturus;the third category : Astragalus membranaceus,Cassia Twig,Angelica Sinensis Radix,Paeonia Lactiflora;the forth category : Paeoniae Radix Rubra,Rehmanniae Radix,Chuanxiong Rhizoma,Carthamus tinctorius L,Achyranthes bidentata,Radix Rehmanniae Preparata,Dioscoreae Rhizoma,Radix Aconiti Lateralis Praeparata;the fifth category:Atractylodes,Pseudostellariae Radix,Poria cocos(Schw.)Wolf,Salviae Miltiorrhizae Radix et Rhizoma.No.3 Network pharmacology-based analysis of the mechanism of Li-qi-hua-shi Decoction for the treatment of MAFLD1.Filtering of components and targets:After searching and screening,a total of 55 active components were obtained from 8 herbs,including 5 components from Citri Reticulatae Pericarpium,15 components from Poria,9 components from Atractylodis Rhizoma,10 components from Alismatis Rhizoma,9 components from Psyllium,2 components from Arecae Pericarpium,8 components from Hawthorn,2 components from Red yeast rice,and different herbs can contain the same components.There are 254 targets of action,of which 69 targets from Citri Reticulatae Pericarpium,26 targets from Poria,60 targets from Atractylodis Rhizoma,5 targets from Alismatis Rhizoma,146 targets from Psyllium,13 targets from Arecae Pericarpium,233 targets from Hawthorn,48 targets from Red yeast rice.One component may correspond to different targets and one target may be acted upon by different components.2.Screening disease targets of MAFLD:A total of 1643 metabolism-related fatty liver disease targets were obtained.3.The herb-component-target network of Li-qi-hua-shi Decoction for the treatment of MAFLD: The network contains 32 active components corresponding to 160 targets,among which different herbal medicines contain the same active component and different active components act on the same targets.Analysis of the network topological parameters showed that quercetin was the main active ingredient in the treatment of MAFLD with Li-qi-hua-shi Decoction,followed by naringenin,kaempferol,wogonin and nobiletin.The main target of the RQD formula for the treatment of MAFLD was PTGS2,followed by PTGS1、HSP90AA1、PRKACA、PGR、PIK3CG、PPARG、DPP4,etc.4.GO enrichment analysis : The main biological processes involved in GO enrichment analysis are response to lipopolysaccharide,apoptotci signaling pathway,reactive oxygen species metabolic process,regulation of cellular response to stress and response to inorganic substance,etc;the biological molecular functions involved include protein domain specific bindin,DNA-binding transcription factor binding,nuclear receptor activity,oxidoreductase activity and steroid binding,etc.Cellular composition enrichment analysis showed that the active ingredients mainly act on the membrane raft,vesicle lumen and endoplasmic reticulum lumen,etc.5.KEGG enrichment analysis : The signalling pathways obtained by KEGG enrichment analysis covered a wide range of environmental information processing,cellular processes,organismal systems and human diseases,including Non-alcoholic fatty liver disease(NAFLD)),HIF-1 signaling pathway,Epstein-Barr virus infection,PI3K-Akt signaling pathway,AGE-RAGE signaling pathway in diabetic complications and Pathways in cancer,etc.No.4 Evaluation of the clinical efficacy by the method of PSM for the treatment of MAFLD with Li-qi-hua-shi DecoctionA total of 36 groups of comparable case data were obtained after calculation by propensity score matching method.There was a large bias in BMI,ALT,AST,TG and HDL-C between the two groups before matching,and the groups were not comparable;after matching,there were no significant differences in gender,age,BMI,liver ultrasound results,ALT,AST,TG,TC,HDL-C,LDL-C,presence of type 2 diabetes and TCM syndrome score between the two groups,and the groups were comparable as there was no significant difference between the groups.1.BMI index:The BMI of patients in the Chinese medicine group was seen to decrease significantly after 12W(P<0.05),while the BMI of the control group did not change significantly after 4W,8W and 12W(P>0.05),and the BMI of patients in the control group increased after 12 W compared with that at 8W;the BMI of patients in both groups did not differ significantly after 4W,8W and 12W(P> 0.05).This result indicates that the control group was not effective in improving the BMI,while the BMI of the patients in the Chinese medicine group showed a decreasing trend throughout but only showed significant improvement after 12 W.2.Liver function levels: the ALT levels of patients in the Chinese medicine group decreased significantly after 4W(P<0.05)and those of patients in the control group were seen to decrease significantly after 8W(P<0.05);the efficacy of improving ALT levels in the Chinese medicine group was significantly better than that in the control group after 8W of intervention in both groups(P<0.05),but with the extension of treatment time,there was no significant(P>0.05).Similarly,the AST levels of patients in the Chinese medicine group decreased significantly after 4W(P<0.05),while the ALT levels of patients in the control group changed after 8W;a significant difference in efficacy was seen between the two groups after 4W of treatment(P<0.05),and there was no significant difference between the two groups after 8W of treatment(P>0.05),but a significant difference was seen between the two groups after 12 W of treatment as the intervention time was extended(P<0.05).3.Lipid levels: TG levels of patients in both the Chinese medicine group and the control group showed a significant decrease after 4W(P<0.05),with no significant difference in efficacy between the two groups after 4W and 8W of treatment(P>0.05),and a significant difference after 12 W of treatment(P<0.01).The TG levels of patients in the TCM group decreased significantly after 4W(P>0.05),and the TG levels of patients in the control group decreased significantly after 8W as well as the TCM group(P<0.01);there was no significant difference in the efficacy between the two groups after 4W,8W and 12 W of treatment(P>0.05).HDL levels in the TCM group increased significantly after 8W(P<0.05),while HDL-C levels in the control group increased significantly after 12W(P<0.05);no significant difference was seen between the two groups after 8W of treatment(P>0.05).In the Chinese medicine group,LDL-C levels decreased significantly after 4W of treatment(P<0.05),while in the control group,a significant decrease was seen after 8W of treatment(P<0.05);no significant difference was seen between the two groups in HDL-C levels after 4W,8W and 12 W of treatment(P>0.05).4.Liver imaging efficacy: In terms of liver ultrasound results,there was no significant difference between the Chinese medicine group and the control group after 4W,8W and 12 W of treatment(p>0.05).5.The efficacy of the Chinese medical evidence: In terms of the efficacy of symptoms and signs,there was no significant difference between the Chinese medicine group and the control group after 4W of treatment(P>0.05),and a significant difference was seen after 8W of treatment(P<0.05),followed by a significant difference after 12 W of treatment(P<0.01),suggesting that the interventions in the Chinese medicine group improved the patients’ symptoms and signs better than those in the treatment group.6.Evaluation of comprehensive efficacy: From the evaluation of comprehensive efficacy,there was no significant difference between the Chinese medicine group and the control group after 4W and 8W of treatment(P>0.05),with the extension of treatment time,the efficacy of the two groups was significantly different after 12W(P<0.05),suggesting that the comprehensive efficacy of interventions in the Chinese medicine group was better than that in the control group.7.Safety analysis: Patients in the Chinese medicine group and the control group showed no significant abnormalities in blood routine and renal function and other safety indicators before and after treatment.No.5 The experimental research of the hepatic protection and regulation of glucolipid metabolism on MAFLD modal by Li-qi-hua-shi Decoction1.Body weight,liver wet weight and liver index: the body weight of rats in the model group was significantly higher than that of the normal group(P<0.01);the body weight of rats in the Chinese medicine group was significantly lower than that of the model group after the intervention(P<0.05),and the body weight of rats in the western medicine group was not significantly different from that of the model group(P>0.05);the body weight ratio of rats in the Chinese medicine group was significantly lower compared with that of rats in the western medicine group(P<0.05).The liver wet weight of the rats in the model group was significantly higher than that of the normal group(P<0.01);after the intervention,the liver wet weight of the rats in both the Chinese medicine and western medicine groups was significantly lower than that of the model group(P<0.05);there was no significant difference between the liver wet weight of the rats in the Chinese medicine and western medicine groups(P>0.05).The liver index of rats in the model group was significantly higher than that of the normal group(P<0.01);the liver index of rats in both the Chinese medicine and western medicine groups showed a significant decrease after the intervention(P<0.01);there was no significant difference between the liver index of rats in the Chinese medicine and western medicine groups(P>0.05).2.Liver function levels: The serum ALT and AST levels of the rats in the model group were significantly higher than those in the normal group(P<0.01);the serum ALT and AST levels of the rats in the Chinese medicine and western medicine groups were significantly lower than those in the model group after the intervention(P<0.01),and there was no significant difference between the serum ALT and AST of the rats in the Chinese medicine and western medicine groups(P>0.05).3.Lipid levels: The serum TG,TC and LDL-C levels in the model group were significantly higher than those in the normal group(P<0.01),and HDL-C levels were significantly lower than those in the normal group(P<0.01);TG and LDL-C levels in the Chinese medicine group were significantly lower than those in the model group(P<0.01),TC levels were significantly lower than those in the model group(P<0.05),and HDL-C levels were significantly higher than those in the model group(P<0.05).The TG and LDL-C levels in the Western medicine group were significantly lower than those in the model group(P<0.01),and the TC and HDL-C levels were not significantly different from those in the model group(P>0.05);compared with the Western medicine group,the serum TG,TC and LDL-C levels in the Chinese medicine group were significantly lower(P<0.05)and the HDL-C levels were significantly higher(P<0.05).4.Serum FBG,FINS levels and IRI indexes:The serum FBG levels of rats in the model group were significantly higher than those in the normal group(P<0.01);after intervention,the serum FBG levels of rats in the Chinese medicine group were significantly lower than those in the model group(P<0.01),and the serum FBG levels of rats in the western medicine group were not significantly different from those in the model group(P>0.05);comparing the serum FBG levels of rats in the Chinese medicine group and the western medicine group,the The serum FBG levels of rats in the Chinese medicine group were significantly lower than those in the western medicine group(P<0.01).The serum levels of FINS in the model group were significantly higher than those in the normal group(P<0.01);after the intervention,the serum levels of FINS in both the Chinese and Western medicine groups were significantly lower than those in the model group(P<0.01);there was no significant difference between the serum levels of FINS in the Chinese and Western medicine groups(P>0.05).The IRI levels of rats in the model group were significantly higher than those in the normal group(P<0.01);after the intervention,the IRI levels of rats in the TCM group were significantly better than those in the model group(P<0.01);the IRI levels of rats in the TCM group were significantly lower than those in the Western medicine group(P<0.01).5.Observation of liver pathology: The liver of the rats in the model group was yellowish,obscure,with thick and blunt edges and larger size;the rats in the Chinese medicine and western medicine groups were slightly yellowish,with slightly blunt and thick edges and slightly larger size.Under the light microscope,the hepatocytes in the model group were enlarged and disorganized with poorly defined borders,with diffuse fatty changes,nuclei close to the edges,lipid droplets of different sizes deposited in the cytoplasm,ballooning of cells,loose cytoplasm with poorly defined edges,and inflammatory cell infiltration and necrotic foci in the lobules and confluent areas of the liver.Compared with the model group,the liver tissues of the rats in the Chinese herbal medicine and western medicine groups showed significant improvement in cell volume,morphology and arrangement,and the deposition of lipid droplets in the cytoplasm was reduced.6.NAS score evaluation: the NAS score of rats in the model group was significantly higher than that of the normal group(P<0.01);after the intervention,the volume of hepatocytes and intrahepatic lipid droplets became smaller,and the inflammation and ballooning of hepatocytes in the lobules were significantly improved in the liver tissue sections of rats in both the Chinese medicine and western medicine groups,and the NAS score was significantly lower than that of the model group(P<0.05);the NAS score of rat liver tissue was not significantly different between the Chinese medicine and western medicine groups.There was no significant difference in the NAS scores between the Chinese medicine and western medicine groups(P>0.05).7.Serum adipokine levels: serum LEP concentration in the model group was significantly higher than that in the normal group,and ADPN concentration was significantly lower than that in the normal group,(P<0.01);rats in both the Chinese medicine and western medicine groups showed significantly lower LEP concentration and higher ADPN concentration after the intervention,(P<0.01);there was no significant difference between the serum LEP and ADPN concentrations in the Chinese medicine and western medicine groups compared with each other(P>0.05).There was no significant difference between the serum LEP and ADPN concentrations in the Chinese medicine group and the western medicine group(P>0.05).8.Serum inflammatory factor levels: serum concentrations of NF-κB,TNF-α and IL-1β in the model group were significantly higher than those in the normal group(P<0.01);the serum concentrations of the three inflammatory factors in the rats in the Chinese medicine and western medicine groups were significantly lower after the intervention compared with those in the model group(P<0.01);compared between the Chinese medicine and western medicine groups,the serum concentrations of NF-κB in the rats in the western medicine group were lower than those in the Chinese medicine group(P<0.05),while the serum concentrations of TNF-α and IL-1β in the rats in the western medicine group were not significantly different(P>0.05).The serum concentrations of TNF-αand IL-1βwere not significantly different between the two groups(P>0.05).Conclusion:1.The prevalence of MAFLD is greater in males than in females,with a high prevalence in the 46-60 age group,and its onset is closely related to BMI;abnormal liver function,lipid levels and T2 DM are risk factors for MAFLD.The TCM pathological elements involved in MAFLD are Yin deficiency,Yang deficiency,Qi deficiency,Dampness,Fire,Phlegm,Blood stasis and stagnation of Qi,and the pathological location factors include liver,kidneys and spleen.2.Professor Lu Bingjiu considered that the pathogenesis of MAFLD is stagnation of liver Qi,deficiency of spleen,ut to stagnation of Qi,phlegm,dampness and stagnation of blood in the liver,then for a long time the illness will reach to kidneys.3.The active ingredients in Li-qi-hua-shi Decoction such as quercetin,naringenin,kaempferol,wogonin and nobiletin could regulate biological processes such as response to lipopolysaccharide,apoptotci,reactive oxygen species metabolic,regulation of cellular response to stress and response to inorganic substance through their effects on PTGS,HSP90AA1,PRKACA,PGR,PIK3 CG,PPARG and DPP4 targets.It also plays a role in the regulation of signalling pathways such as Non-alcoholic fatty liver disease(NAFLD)),HIF-1 signaling pathway,Epstein-Barr virus infection,PI3K-Akt signaling pathway,AGE-RAGE signaling pathway in diabetic complications and Pathways in cancer,etc.,to treat MAFLD by a multiple component-multiple target-multiple approaches synergistic mechanism.4.The efficacy of combining Silybin meglumine with Li-qi-hua-shi Decoction in the treatment of MAFLD is faster and more effective than using Silybin meglumine alone.5.Li-qi-hua-shi Decoction has clinical value in treating MAFLD by improving patients’ BMI,liver function,lipid levels,hepatic morpfology and pathological symptoms,with no adverse effects.6.Li-qi-hua-shi Decoction can reduce body weight,liver wet weight and liver index,lower liver enzymatic levels,regulate dyslipidemia,improve insulin resistance and thus regulate abnormal glucolipid metabolism,as well as regulate abnormal concentrations of adipokines and inflammatory factors in MAFLD model,which may be the main mechanism of action of the decoction in the treatment of MAFLD. |
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