| Research 1:Data mining researchObjectiveAnalyzing the clinical characteristics of acute gouty arthritis(AGA)through data mining,and exploring the rules of prescription and medication for clearing heat and removing dampness method in the treatment of AGA.MethodsUsing case series analysis,patients with AGA who were hospitalized in the Department of Endocrinology of Guangdong Provincial Hospital of Traditional Chinese Medicine from January 1,2010 to July 1,2022 were included.Extract and standardize the medical record information,and input the data into the auxiliary platform V2.50 and EXCEL for clinical feature analysis,frequency statistics,rule analysis,entropy clustering analysis and complex network analysis.ResultsA total of 585 medical records were included in this study,including 477 males and 108 females.The incidence ratio of males and females was 4.42:1.The average age of onset was 62.41 ± 16.24 years old.The most cases had a disease course of 6-10 years.The average length of hospitalization was 8.39±3.86 days,and the average number of hospitalization was 2.13 ± 2.57 times.The inducements were listed from high to low in order of frequency:improper diet,eating seafood,drinking alcohol,eating too much meat,getting cold due to weather changes,intense exercise,surgery or trauma,fatigue,uric acid lowering treatment,and long-term poor posture.The first metatarsophalangeal joint and ankle joint were the most common sites.When acute attack of gout occurs,some patients(6.51%)may have fever,accompanied by an increase in respiratory and pulse frequency.More than half of the patients(69.03%)were overweight or obese.The average value of serum uric acid is 540.76 μmol/L,of which 84.12%of patients had elevated serum uric acid level.57.25%,31.30%,44.31%,33.82%,80.72%and 45.98%of patients with abnormal blood glucose,elevated total cholesterol,elevated triglyceride,elevated low density lipoprotein,decreased high density lipoprotein and elevated blood pressure,respectively.The proportion of patients with increased leukocyte count,neutrophil percentage,hypersensitive C-reactive protein,procalcitonin and erythrocyte sedimentation rate was 49.81%,39.67%,89.51%,84.77%and 87.96%,respectively.The symptoms of damp-heat accumulation syndrome that occur frequently include joint pain(97.09%),joint local tenderness(87.86%).joint dysfunction(77.26%),joint skin temperature increase(76.07%),joint swelling(72.99%),sudden onset(70.60%),dry mouth(26.84%),poor sleep(25.64%),bitter mouth(15.21%),constipation(13.50%),fatigue(12.31%),joint swelling(11.11%),and loose stool(10.26%).The largest proportion of tongue coating and pulse was dark red tongue(51.28%),yellow and greasy tongue coating(64.27%),and smooth pulse(34.19%).According to the order of frequency of use,the top 28 high-frequency Chinese herbs are:coix seed,soil poria cocos,achyranthes bidentata,lily,honeysuckle vine,atractylodes lanceolata,plantain seed,fritillaria amurensis,red peony,phellodendron amurense,rhizoma polygoni,tuckahoe,anemarrhena asphodeloides,angelica sinensis,raw rehmannia glutinosa,silkworm excrement,rhubarb,peach kernel,forsythia suspensa,plantain,bixie,tetrandra,Scutellaria baicalensis,pinellia,chuanxiong,wild chrysanthemum,and angelica.The statistical results of efficacy classification showed that the most frequently used herbs were antipyretic herbs,diuretic herbs,and blood-activating herbs.According to the statistical results of the frequency of menstruation of nature and taste,cold herbs account for the largest proportion of herbal properties,followed by warm herbs.Bitter herbs accounted for the largest proportion of the herbal flavor,followed by sweet herbs.The liver channel accounts for the largest proportion of meridians,followed by spleen and stomach channels.Based on the entropy clustering method of complex systems,16 core combinations have been evolved.Based on the unsupervised entropy hierarchical clustering method,a total of 8 new prescriptions for the treatment of this syndrome were proposed.ConclusionThe patients with AGA usually have multiple metabolic disorders such as abnormal blood glucose,abnormal blood lipids,overweight or obesity,and high blood pressure.Damp-heat accumulation syndrome is the core pathogenesis of AGA.The typical clinical manifestations are joint redness,swelling and pain,sudden onset,dry mouth and bitter mouth,poor sleep,dark red tongue,yellow and greasy fur,and stringy,and slippery pulse.The treatment is mainly based on clearing heat and removing dampness method.The commonly used herbs include coix seed,poria cocos,achyranthes bidentata,lily,honeysuckle vine,atractylodes lanceolata,plantain seed,and fritillaria.This study provides theoretical basis and data support for the treatment of clearing heat and removing dampness method for AGA.Research 2:Meta-analysisObjectiveA meta-analysis was carried out by including the randomized controlled trials(RCTs)on the treatment of AGA with a series of prescriptions for clearing heat and removing dampness to evaluate the effectiveness and safety of clearing heat and removing dampness in the treatment of AGA,and to provide evidence based medical basis for later clinical application.MethodsNine Chinese and English databases were retrieved,including RCTs of the series of prescriptions for clearing heat and removing dampness to treat damp-heat accumulation type of AGA.The retrieval time limit was from the establishment of the database to February 1,2022.According to the evaluation criteria provided by the revised Cochrane tool(RoB 2.0),the quality of the included documents was evaluated,and the data was statistically analyzed using Stata software.ResultsFinally,37 RCTs were included in the meta-analysis.Results showed that the total effective rate of treatment in the test group(heat clearing and dampness removing treatment group)was higher than that in the control group(western medicine group)(RR=1.15,95%CI:1.12-1.18,P<0.001).The treatment group of clearing heat and removing dampness can significantly reduce the levels of IL-1 β,IL-6,IL-8,IL-18 and TNF-α compared with the western medicine group(SMD=-1.02,95%CI:-1.32 to-0.73,P<0.001;SMD=-0.54,95%CI:-0.76 to-0.32,P<0.001;SMD=-1.02,95%CI:-1.45 to-0.60,P<0.001;SMD=-0.68,95%CI:-0.96 to-0.40,P<0.001;SMD=-0.76,95%CI:-1.02 to-0.50,P<0.001).Compared with the control group,the test group can reduce the levels of serum uric acid,C-reactive protein,and erythrocyte sedimentation rate(WMD=-56.80,95%CI:-85,52 to-28.07,P<0.001;WMD=-4.71,95%CI:-6.24 to-3.18,P<0.001;WMD=-7.00,95%CI:-10.58 to-3.41,P<0.001).In addition,compared with the control group,the test group can reduce VAS score,TCM syndrome score,joint pain score,tenderness score,activity limit score,swelling score,pain relief time,time of disappearance of redness and swelling,and the incidence of adverse reactions(WMD=-1,02,95%CI:-1.43 to-0.61,P<0.001;WMD=-2.48,95%CI:-3.87 to-1.09,P<0.001;WMD=-0.72,95%CI:-0.91 to-0.54,P<0.001;WMD=-0.34,95%CI:-0.48 to-0.20,P<0.001;WMD=-0.46,95%CI:-0.59 to-0.33,P<0.001;WMD=-0.40,95%CI:-0.52 to-0.27,P<0.001;WMD=-6.21,95%CI:-9.96 to-2.46,P=0.001;WMD=-9.06,95%CI:-17.14 to-0.99,P=0.028;RR=0.45,95%CI:0.3-0.69,P<0.001).There was no significant difference in the levels of serum creatinine,urea nitrogen,alanine aminotransferase and alanine aminotransferase between the two groups.ConclusionThe clearing heat and removing dampness treatment was effective and safe in the treatment of AGA.Compared with the western medicine group,the heat clearing and dampness removing treatment group can improve the total effective rate,reduce the serum cytokine level,blood uric acid,C-reactive protein,ESR,VAS score,TCM syndrome score,symptom score,joint pain relief time,time of disappearance of joint redness and swelling,and the incidence of adverse reactions.However,due to the limited number of RCTs included and the limitations of many RCTs with low quality,small sample size and short duration,it is necessary to further supplement and verify the efficacy and safety of clearing heat and removing dampness treatment for treating AGA in the future.Research 3:Professor Fan Guanjie’s experience in treating AGAObjectiveThe clinical experience of Professor Fan Guanjie in the prevention and treatment of AGA by using the theory of "dynamic and fixed sequence" was studied theoretically.MethodsTo explore the diagnosis and treatment ideas and experience of AGA under the guidance of Professor Fan Guanjie’s "dynamic and fixed sequence" theory through theoretical analysis.ResultsThe theory of "dynamic and fixed sequence" was founded by Professor Fan Guanjie.It originated from the thinking and exploration of the treatment theory of endocrine diseases.According to the law of disease change,it adopted the combination of syndrome differentiation and disease differentiation,combined with the "three factors" and "dynamic and fixed" transformation treatment principle,and the "sequential" treatment at different stages,breaking through the previous treatment mode of "single to single" and "single to compound".This theory reflects the overall concept of traditional Chinese medicine and the concept of syndrome differentiation of constancy,and can be used in many metabolic diseases such as gout and diabetes.Professor Fan Guanjie summarized many years of clinical practice and believed that the accumulation of dampness and heat was the core pathogenesis of AGA,the root cause of acute gout attack,and the decisive factor of gout onset.During the treatment Professor Fan Guanjie emphasized that the basic treatment of AGA should be clearing heat and removing dampness.Professor Fan Guanjie used seven traditional Chinese medicines composed of Fan’s gout formula(30g of Tuckahoe,10g of Fritillaria,30g of Lily,30g of Plantago seed.30g of Lonicera japonica.30 g of Polygonum cuspidatum,and 30g of wild chrysanthemum)as the core drug chain for the treatment of AGA.The clinical symptoms were added and subtracted according to the individual conditions of patients.Early clinical studies showed that the core Chinese medicine string and clinical treatment of AGA under the guidance of the theory of "dynamic and fixed sequence" has a definite effect,which can improve joint pain,joint tenderness,joint acute redness and swelling,and joint movement limitation,and reduce the level of blood uric acid,C-reactive protein and erythrocyte sedimentation rate.ConclusionProfessor Fan Guanjie believes that the accumulation of dampness and heat is the core pathogenesis of AGA,and the basic treatment is to clear heat and remove dampness.Under the guidance of the theory of "dynamic and fixed sequence",the core drug string has a definite clinical effect in the treatment of AGA.Research 4:Animal experimental research(therapeutic effect part)ObjectiveBased on the NLRP3/ASC/Caspase-1 signal pathway,the effects and mechanism of Fan’s gout formula in the treatment of AGA were discussed.MethodsThirty male SD rats were randomly divided into 6 groups by random number table method,including blank group,model group,colchicine group(positive control group)and high,middle and low dose group of Fan’s gout formula,with 5 rats in each group.Except the blank normal group,the other groups were injected with sodium urate suspension injection to replicate the AGA model,and the rats in the blank group were injected with the same amount of normal saline at the same site.After the establishment of the model,the medicine was administered continuously by gavage for 3 days,twice a day.The experimental group was given high,middle and low dose of Fan’s gout formula,the blank group and the model group were given the same volume of normal saline,and the positive control group was given colchicine.Evaluate the joint symptoms of rats at different time nodes.Measure the inflammatory factor IL-1β and IL-6 in serum and joint fluid.The pathomorphology of ankle joint tissue,and the level of NLRP3,ASC,Caspase-I protein and mRNA in synovial tissue were also measured.Results(1)Joint symptom evaluation:1)Joint dysfunction index:the right ankle dysfunction index of rats in the colchicine group was significantly lower than that in the model group at 8h,48h,and 72h after modeling(P=0.037,P=0.012,P=0.002),and the right ankle dysfunction index of rats in the high dose group of Fan’s gout formula was significantly lower than that in the model group at 48h and 72h after modeling(P=0.012,P=0.002).The dysfunction index of the right ankle joint in the middle dose group of Fan’s gout formula was significantly lower than that in the model group 72 hours after the establishment of the model(P=0.002).At each time point,there was no statistically significant difference in the ankle dysfunction index between the three groups of Fan’s gout formula and the colchicine group(P>0.05).2)Arthritis index:The arthritis index of the colchicine group was significantly lower than that of the model group at 8h,48h and 72h after modeling(P=0.032,P=0.003,P=0.002).The arthritis index of the high dose group of Fan‘s gout formula was significantly lower than that of the model group at 48h and 72h after modeling(P=0.012,P=0.017).The arthritis index of the middle dose group of Fan’s gout formula was significantly lower than that of the model group at 72h after modeling(P=0.017).At each time point,there was no statistically significant difference in the arthritis index between the high and middle dose group of Fan’s gout formula and the colchicine group(P>0.05).3)The degree of skin temperature rise:At each time point,the number of joint skin temperature rise in the colchicine group was significantly lower than that in the model group(P<0.05).Four hours after modeling,the degree of ankle skin temperature rise in the high and middle dose group of Fan’s gout formula was significantly lower than that in the model group(P=0.005,P=0.029).At 8h,24h,48h and 72h after the establishment of the model,the number of joint skin temperature rise in the high,middle and low dose group of Fan’s gout formula was significantly lower than that in the model group(P<0.05).At each time point,there was no significant difference in the degree of skin temperature rise of ankle joint between the high and middle dose group of Fan’s gout prescription and the colchicine group(P>0.05).4)Joint swelling degree:At each time point,the swelling degree of the right ankle joint of rats in the colchicine group and the high and middle dose group of Fan’s gout formula were significantly lower than that in the model group(P<0.05).At each time point,there was no significant difference in the degree of ankle swelling between the high and middle dose group of Fan’s gout formula and the colchicine group(P>0.05).(2)IL-1β and IL-6 concentration in joint fluid and serum:1)IL-1β concentration in joint fluid:72h after modeling,the concentration of IL-1β in the joint fluid of the colchicine group and the high,middle and low dose group of Fan’s gout formula was lower than that of the model group(P<0.05).The concentration of IL-1β in the joint fluid of the high dose group of Fan’s gout formula did not differ than that of the colchicine group(P=0.224).2)IL-1β concentration in serum:24h and 72h after modeling,the serum IL-1βin the colchicine group and the high and middle dose group of Fan’s gout formula was reduced than that in the model group(P<0.05).The difference of IL-1β concentration was not statistically significant between high and middle dose of Fan’s gout formula groups and the colchicine group(P>0.05).3)IL-6 concentration in synovial fluid:72h after modeling,the concentration of IL-6 in the synovial fluid of the colchicine group and the high,middle and low dose groups of Fan’s gout formula were significantly lower than that of the model group(P<0.05),the concentration of IL-6 in the synovial fluid of the high dose group of Fan’s gout formula was significantly lower than that of the colchicine group(P=0.048),and the concentration of IL-6 in the synovial fluid of the middle and low dose group of Fan’s gout formula was not significantly different from that of the colchicine group(P>0.05).4)Serum IL-6 concentration:At 24h and 72h after modeling,the serum IL-6 concentration in the colchicine group and the high and middle dose group of Fan’s gout formula was significantly lower than that in the model group(P<0.05).There was no significant difference in serum IL-6 concentration between the high and middle dose group of Fan’s gout formula and the colchicine group(P>0.05).(3)Synovial histopathological changes:Compared with the model group,the synovial epithelium of the colchicine group and the high,middle and low dose groups of Fan’s gout formula were relatively intact,synovial cell proliferation and inflammatory cell infiltration was reduced,and neovascularization was reduced.The expression of ankle inflammation in the high and middle dose groups of Fan’s gout formula was similar to that in the colchicine group.(4)Western blot results:Compared with the model group,the content of NLRP3,ASC and Caspase-1 protein in the colchicine group and the high and middle dose group of Fan’s gout formula decreased significantly(P<0.05),while the content of ASC protein in the low dose group of Fan’s gout formula decreased significantly(P<0.05).Compared with the colchicine group,there was no significant difference in the content of NLRP3,ASC and Caspase-1 protein in the high,middle and low dose groups of Fan’s gout formula(P>0.05).(5)PCR results:Compared with the model group,the mRNA expression of NLRP3,ASC and Caspase-1 in the colchicine group and the high and middle dose group of Fan’s gout formula were significantly decreased(P<0.01).Compared with the colchicine group,the mRNA expression of NLRP3,ASC and Caspase-1 in the high dose group of Fan’s gout formula had no significant difference(P>0.05).ConclusionThis study preliminarily showed that Fan’s gout formula may improve the ankle joint dysfunction index,inflammatory index,skin temperature and swelling degree of AGA rats by interfering with NLRP3/ASC/Casase-1 signal pathway,and reduce IL-1β and IL-6 concentration in joint fluid and serum,and then play a role in the treatment of AGA.Research 5:Animal experimental research(preventive effect part)ObjectiveBased on the NLRP3/ASC/Caspase-1 signal pathway,the efficacy and mechanism of Fan’s gout formula in preventing AGA were discussed.MethodsThirty male SD rats were randomly divided into 6 groups by random number table method,including blank group,model group,colchicine group(positive control group),high,middle and low dose group of Fan’s gout formula,with 5 rats in each group.For 14 days,twice a day,the experimental group was given high,middle and low dose of Fan’s gout formula solution,the blank group and the model group were given the same volume of normal saline,and the positive drug group was given colchicine solution.One hour after the 14th day of gavage,except for the blank normal group,the other groups were injected with MSU suspension injection to replicate the rat model of AGA,and the observation continued for 3 days after the establishment of the model.Evaluate the joint symptoms of rats at different time nodes.Measure the inflammatory factor IL-1 in serum and joint fluidβ And IL-6 content,the pathomorphology of ankle joint tissue,and the level of NLRP3,ASC,Caspase-1 protein and mRNA in synovial tissue.Results(1)Evaluation of joint symptoms:1)Joint dysfunction index:The dysfunction index of the right ankle joint in the colchicine group was significantly lower than that in the model group at 8h,24h,and 48h after modeling(P=0.003,P=0.037,P=0.023).The joint dysfunction index in the high dose group of Fan’s gout formula was significantly lower than that in the model group at 4h,8h,24h,48h,and 72h after modeling(P=0.035,P=0.003,P=0.037,P=0.001,P=0.012).The joint dysfunction index in the colchicine group was significantly lower than that in the low dose group of Fan’s gout formula at 8 and 24 hours after modeling(P=0.017,P=0.037).At each time point,there was no statistically significant difference in the joint dysfunction index between the high and middle dose group of Fan’s gout formula and the colchicine group(P>0.05).2)Arthritis index:The inflammation index of the right ankle in the colchicine group was significantly lower than that in the model group at 8 and 48 hours after modeling(P=0.002,P=0.047).Compared with the model group,the joint inflammation index in the high dose group of Fan’s gout formula significantly decreased at 8h,24h,and 48h after modeling(P<0.001,P=0.011,P=0.047).The arthritis index in the middle dose group of Fan’s gout formula was significantly lower than that in the model group at 8 hours after modeling(P=0.017).The arthritis index of the colchicine group was significantly lower than that of the low dose group of Fan’s gout formula at 8 hours after modeling(P=0.002).At each time point,there was no statistically significant difference in the arthritis index between the high and middle dose group of Fan’s gout formula and the colchicine group(P>0.05).3)Degree of skin temperature rise:At each time point,the degree of skin temperature rise in the right ankle joint of rats in the high-dose group of Fan’s gout formula and the colchicine group were significantly lower than that in the model group(P<0.05).At 4h and 8h after modeling,the degree of joint skin temperature rise in the middle-dose group of Fan’s gout formula was significantly lower than that in the model group(P<0.05).At each time point,there was no significant difference in the degree of skin temperature rise of ankle joint between the Fan’s gout formula(high and middle doses)groups and the colchicine group(P>0.05).4)The degree of joint swelling:At 24h,48h and 72h after modeling,the degree of swelling of the right ankle joint of rats in the high-dose group of Fan’s gout formula was significantly lower than that in the model group(P<0.05).At each time point,there was no statistically significant difference in ankle swelling between the three groups of Fan’s gout prescription and the colchicine group(P>0.05).(2)IL-1β and IL-6 concentration in joint fluid and serum:1)IL-1β concentration in joint fluid:72h after model establishment,IL-1β in the joint fluid of three doses of Fan’s gout formula groups and the colchicine group than that of the model group did not differ significantly(P>0.05).The IL-1β in the joint fluid of three Fan’s gout formula groups than that of the colchicine group did not differ significantly(P>0.05).2)Serum IL-1β:Compared with the model group,24h after modeling,the serum IL-1β in the colchicine group and the high and middle doses group of Fan’s gout formula were significantly decreased(P<0.05).The serum IL-1β concentration in the high,middle,and low dose group of Fan’s gout formula was not statistically significant than that in the colchicine group(P>0.05).3)IL-6 concentration in synovial fluid:72h after modeling,the IL-6 concentration in synovial fluid of the high and low dose group of Fan’s gout formula and the colchicine group were significantly lower than that of the model group(P<0.05).There was no significant difference in the IL-6 concentration in synovial fluid between Fan’s gout formula groups(high and low doses)and the colchicine group(P>0.05).4)Serum IL-6:72h after modeling,the serum IL-6 concentration in the high-dose group of Fan’s gout formula and the colchicine group was significantly lower than that in the model group(P<0.05).There was no significant difference in the serum IL-6 concentration between three Fan’s gout formula groups and the colchicine group(P>0.05).(3)Synovial histopathological changes:Compared with the model group,the synovial epithelium of the high-dose group of Fan’s gout formula was relatively intact,the proliferation of synovial cells and infiltration of inflammatory cells were reduced,and the neovascularization was reduced.The degree of inflammation in the colchicine group was between the model group and the high dose group of Fan’s gout formula.Synovial epithelium was incomplete,synovial cell proliferation and inflammatory cell infiltration were obvious.The inflammatory reaction in the middle and low dose groups of Fan’s gout formula was obvious,with synovial cell proliferation,accompanied by a large number of inflammatory cell infiltration and vascular proliferation.(4)Western blot results:Compared with the model group,the content of NLRP3,ASC and Caspase-1 protein in the colchicine group and the high dose group of Fan’s gout formula decreased significantly(P<0.05).Compared with the colchicine group,there was no significant difference in the content of NLRP3,ASC and Caspase-1 protein in the high,middle and low dose groups of Fan’s gout formula(P>0.05).(5)PCR results:Compared with the model group,the mRNA expression of NLRP3,ASC and Caspase-1 in the colchicine group and the high dose group of Fan’s gout formula were significantly decreased(P<0.05).Compared with the colchicine group,the mRNA expression of NLRP3,ASC and Caspase-1 in the high,middle and low dose group of Fan’s gout formula had no significant difference(P>0.05).ConclusionFan’s gout formula may play a role in preventing AGA by interfering with NLRP3/ASC/Casase-1 signal pathway,improving ankle joint dysfunction index,inflammatory index,skin temperature and swelling degree,and reducing IL-1β and IL-6 concentration in joint fluid and serum. |
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