| Study 1:data mining studyObjective:To analyze the characteristics of the syndromes of gouty arthritis and medication rules of Professor Zhu Yuelan in the treatment of gouty arthritis by data mining,and to summarize the core prescription and the academic thinking of my tutor by combing the "damp-stasis" theory of goutMethods:The medical records of patients with gouty arthritis treated in outpatient or inpatient department of rheumatology of Dongfang Hospital of Beijing University of Chinese Medicine and prescribed by Professor Zhu Yuelan were collected from January 2012 to December 2019.After standardized processing,the medical record information was input into the Traditional Chinese Medicine Inheritance Support System V2.5 and Excel for frequency statistics,rule analysis,entropy cluster analysis and complex network analysis.Results:(1)A total of 612 medical records were included in this study,including 580 male patients and 32 female patients,with an average age of 51 years old.There were 41 comorbidities,with hypertension being the most common.(2)Overall data mining results:Drinking was the most common cause of gouty arthritis.There were 126 symptoms,tongue and pulse,with "local skin burning feeling,joint swelling heat pain" and "dark red tongue,yellow greasy fur,taut and smooth pulse"being the most common.There were 5 types of syndromes,with "syndrome of damp-heat accumulation" being the most common.There were 207 kinds of Chinese herbs among 612 prescriptions,with "Huzhang,Tufuling,Gancao" being the most common.Efficacy classification showed that heat-clearing drugs were used most frequently and most of the drugs were natured and cold.Besides,most of the medicinal flavor were sweet,bitter and spicy,and most of the channel tropism were in the liver,spleen and stomach channel.(3)Hierarchical data mining results:? Syndrome of damp-heat accumulation:There were 106 symptoms,tongue and pulse,with "joint swelling heat pain,local skin burning feeling" being the core symptoms and "dark red tongue,yellow greasy fur,taut and smooth pulse" being the most common.There were 166 kinds of Chinese herbs,with 12 Chinese herbs such as Huzhang,Tufuling,Chuanniuxi as the core,and 11 new prescriptions were obtained.? Syndrome of stasis-heat block:There were 72 symptoms,tongue and pulse,with "joint swelling and pain,purple and dark skin" as the main symptoms and "dark red tongue,greasy fur,taut and smooth pulse" being the most common.There were 112 kinds of Chinese herbs,with 9 Chinese herbs such as Tufuling,Huzhang,Chuanniuxi as the core,and 13 new prescriptions were obtained.?Syndrome of insufficiency of the spleen with overabundance of dampness:There were 61 symptoms,tongue and pulse,with "hyperuricemia" as the main symptom and "dark red tongue,white greasy fur,smooth pulse" being the most common.There were 107 kinds of Chinese herbs,with Liujunzi Decoction as the core,and 5 new prescriptions were obtained.?Syndrome of deficiency of liver and kidney:There were 74 symptoms,tongue and pulse,with"joint deformities,repeated outbreak" as the main symptoms and "dark tongue,thin and yellow fur,taut and thready pulse" being the most common.There were 136 kinds of Chinese herbs,with 12 Chinese herbs such as Chuanniuxi,Shudihuang as the core,and 8 new prescriptions were obtained.?Syndrome of phlegm turbidity block:There were 50 symptoms,tongue and pulse,with "joint sore,joint swelling and pain" as the main symptoms and "dark and fat tongue,white greasy fur,taut pulse" being the most common.There were 88 kinds of Chinese herbs,with 10 Chinese herbs such as Gancao,Sharen,Fuling as the core,and 2 new prescriptions were obtained.Conclusion:(1)The syndrome of damp-heat accumulation is the most common in clinic,followed by the syndrome of stasis-heat block.Dampness,stasis and heat may be the main characteristic factors of gout.(2)Based on the theory of "damp-stasis",Professor Zhu Yuelan considers that damp heat and blood stasis is the core pathogenesis of gout.The methods of treatment are clearing heat,removing dampness,promoting blood circulation and stopping pain.Qingre Lishi Prescription is the core representative prescription.Study 2:network pharmacological studyObjective:To explore the main ingredients,targets and pathways of Qingre Lishi Prescription in the treatment of gouty arthritis,and to predict its mechanism by means of network pharmacology,so as to provide ideas and theoretical basis for subsequent experimental study.Methods:The main active ingredients and action targets of Qingre Lishi Prescription were collected from TCMSP database,and the targets of the main active ingredients were predicted from Swiss Target Prediction database.The targets related to gouty arthritis were collected from OMIM,TTD,Disgenet and CTD databases.Through the Venn diagram,the common targets of the disease and the herbs were obtained.The herb-ingredient-target network was constructed by Cytoscape3.7.2 software.The common targets were uploaded to the STRING database to construct protein-protein interaction network,and the key targets were obtained after analysing the network.Finally,gene ontology(GO)function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed for key targets.Results:A total of 140 active ingredients and 358 targets of Qingre Lishi Prescription and 1494 targets related to gouty arthritis were collected and screened out.A total of 93 common targets and 38 key targets were obtained.A total of 1504 enrichment results were obtained by GO enrichment analysis,and 126 related signaling pathways were obtained by KEGG enrichment analysis.The mechanism mainly involved interleukin(IL)-17 signaling pathway,tumor necrosis factor(TNF)signaling pathway,helper T cell 17 differentiates,toll-like receptors(TLRs)signaling pathway,nucleotide-binding oligomerization domain-like receptors(NLRs)signaling pathway,etcConclusion:The Qingre Lishi Prescription contains a variety of active ingredients and targets,and its mechanism of action is complex.This study predicts the mechanism of Qingre Lishi Prescription in the treatment of gouty arthritis,which may play its role through TLRs signaling pathway,NLRs signaling pathway,TNF signaling pathway,etc.It reflects the multi-ingredient,multi-target and multi-pathway characteristics of Chinese herbs and provides a theoretical basis and direction for further study.Study 3:animal experimental studyObjective:To explore the therapeutic effect and mechanism of Qingre Lishi Prescription in treating acute gouty arthritis rats,based on TLRs signaling pathway and NOD-like receptor protein 3(NLRP3)inflammasome.Methods:Forty-eight healthy male SD rats were randomly divided into control group,model group,diclofenac sodium group and Qingre Lishi Prescription group,with 12 rats in each group.Except for the control group,the model of acute gouty arthritis was made in the other 3 groups,and the same amount of normal saline was injected into the same part of the control group.Two hours after modeling,the control group and the model group were gavaged with distilled water,and the other groups were gavaged with relevant drugs.Gastric gavage was given once a day for 5 days.The general condition of the rats was observed,and the degree of ankle joint swelling,disability index and the ankle joint inflammation index were measured at different time points.Two hours after the last gavage,serum IL-1?,IL-6,IL-8,TNF-? were determined in each group,and the histopathological changes of synovial membrane of ankle joint were observed.And the expression of TLR2,TLR4,nuclear factor-kappa B(NF-?B),NLRP3 protein and mRNA were detected.Results:(1)After modeling for 6 h,the joint disability index of Qingre Lishi Prescription group began to decrease gradually,and 48 h after modeling,the difference was statistically significant compared with model group at each point in time(P<0.05,P<0.01).Compared with diclofenac sodium group,there was no significant difference at each point in time(P>0.05).(2)After modeling for 12 h,the joint inflammation index of Qingre Lishi Prescription group began to decrease gradually,and 72 h after modeling,the difference was statistically significant compared with model group(P<0.05).Compared with diclofenac sodium group,there was no significant difference at each point in time(P>0.05).(3)After modeling for 6 h,the degree of ankle joint swelling of Qingre Lishi Prescription group began to decrease gradually,and 12 h after modeling,the difference was statistically significant compared with model group at each point in time(P<0.05,P<0.01).Compared with diclofenac sodium group,there was no significant difference at each point in time(P>0.05).(4)Compared with model group,serum IL-1?,IL-6,IL-8 and TNF-? of Qingre Lishi Prescription group were significantly decreased(P<0.01).Compared with diclofenac sodium group,serum IL-8 of Qingre Lishi Prescription group was significantly increased(P<0.01),and the differences of serum IL-1?,IL-6,and TNF-? were not statistically significant(P>0.05),though these inflammatory factors showed an increasing trend.(5)Compared with model group,the pathology results of the synovial membrane of ankle joints of Qingre Lishi Prescription group showed that there were only a small amount of inflammatory cell infiltration and some slightly edema and hyperplasia cells,and necrosis and degeneration of cells coule be seen occasionally.Compared with diclofenac sodium group,there was no significant difference.(6)Compared with model group,the TLR2,NF-?B,NLRP3 protein expressions of the synovial membrane of ankle joint of Qingre Lishi Prescription group were significantly decreased(P<0.05),and the TLR4 protein expression was also decreased,but the difference was not statistically significant(P>0.05).Compared with diclofenac sodium group,the protein expressions of TLR2,TLR4,NF-?B and NLRP3 were increased,but the differences were not statistically significant(P>0.05).(7)Compared with model group,the TLR2,TLR4,NF-?B and NLRP3 mRNA expressions of the synovial membrane of ankle joint of Qingre Lishi Prescription group were significantly decreased(P<0.05,P<0.01).Compared with diclofenac sodium group,the TLR2,TLR4 and NF-?B mRNA expressions were significantly increased(P<0.05,P<0.01),and the mRNA expression of NLRP3 was increased,but the difference was not statistically significant(P>0.05).Conclusion:(1)Qingre Lishi Prescription can effectively relieve the inflammatory symptoms of acute gouty arthritis rats and improve the swelling and inflammatory infiltration of the ankle joint.And Qingre Lishi Prescription can significantly reduce the content of serum inflammatory factors of acute gouty arthritis rats.(2)Qingre Lishi Prescription may play an anti-inflammatory role by inhibiting TLR2/NF-?B signaling pathway and NLRP3 inflammasome. |
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