| 1 Research purpose Through the collection of clinical data for mining and analysis,the law of drug use in the treatment of rheumatoid arthritis(RA)patients with anemia by the method of invigorating the spleen,removing dampness and unblocking collaterals,and the change of red blood cell parameters in RA patients with anemia and the association rules with laboratory indicators were discussed.A random walking model was constructed to evaluate the effect of spleen invigorating unit therapy on immune inflammation in RA patients with anemia.Through clinical in vivo experiments,we studied the correlation between anemia index,immune inflammation index and iron metabolism index,related cytokines,Janus protein tyrosine kinase 2 / signal transducer and activator of transcription 3(JAK2 / STAT3)signal pathway index,and explored the primary mechanism of RA with anemia.The biological mechanism of Jianpi Huashi Tongluo formula Xinfeng Capsule in treating RA with anemia was studied by cell experiment in vitro.2 Research methods2.1 Research on data Mining1)The medical records and oral traditional Chinese medicine data of inpatients with anemia in RA from June 2012 to January 2018 in the Department of Rheumatic Immunology,Anhui Hospital of traditional Chinese Medicine were selected to analyze the changes of patients' laboratory indexes and the menstruation of traditional Chinese medicine involved in the prescription of traditional Chinese medicine,and to explore the medication rule of the prescription for the treatment of RA with anemia.In order to study the relationship between traditional Chinese medicine for invigorating spleen,resolving dampness and dredging collaterals and the changes of laboratory examination indexes,the Apriori module of SPSS Clementine 11.0 software was used to analyze the relationship between traditional Chinese medicine and red blood cell parameters and immune inflammation indexes. 2)Spleen invigorating unit therapy is a characteristic treatment mode for RA accompanied with anemia,that is,internal administration of traditional Chinese medicine,Xinfeng Capsule + external application of Furong cream)treatment mode.In order to study the therapeutic effect of RA anemia patients using traditional Chinese medicine,the experimental group(taking traditional Chinese medicine internally,Xinfeng Capsule + Furong cream externally)and the control group(taking traditional Chinese medicine only internally)were set up.The core prescriptions of traditional Chinese medicine in the two groups were analyzed by using complex network technology,and the changes of laboratory indexes in the two groups were compared to explore the correlation between traditional Chinese medicine and index improvement.The red blood cell(RBC)count,hemoglobin(HB),C-reactive protein(CRP),erythrocyte sedimentation rate(ESR)of RA patients with anemia were evaluated by random walking model.2.2 Clinical research According to the inclusion and exclusion criteria,39 patients with RA anemia were divided into the study group and 36 healthy people into the control group.The changes of anemia index,iron metabolism and immune inflammation index in the two groups were analyzed.The related cytokines and JAK2 / STAT3 signal pathway indexes were detected by enzyme linked immunosorbent assay(ELISA)and immunoturbidimetry.In the study group,the indexes of anemia,immune inflammation and Hepcidin(Hepc),iron metabolism,cytokines,JAK2 / STAT3 signal pathway were analyzed.2.3 Basic experimental research Peripheral blood mononuclear cells(PBMCs)were isolated from 24 RA patients with anemia and 6 healthy people.The experiment was divided into five groups: group I(normal group: PBMCs of healthy subjects),group II(model group: PBMCs of RA patients with anemia),group III(XFC group: PBMCs of RA patients with anemia with the best concentration of XFC),group IV(AG490 group: AG490 intervention of PBMCs of RA patients with anemia),group V(XFC + AG490 group: PBMCs of RA patients with anemia with the best concentration of XFC and AG490 intervention).The effect of XFC on the proliferation of PBMCs was detected by MTT method.The effect of XFC on the expression of JAK2,STAT3 and Hepcidin was detected by immunofluorescence(IF).The effect of XFC on the expression of IL-4,IL-10,IL-6,TNF-? and Hepcidin was detected by ELISA.The effect of XFC on the expression of JAK2/STAT3/Hepcidin-m RNA was detected by real-time quantitative PCR(RT-q PCR).Western blot(WB)was used to detect the effect of XFC on the expression of JAK2,STAT3,p-JAK2 and p-STAT3 protein.3 Results3.1 Research on data Mining3.1.1 Exploring the rule of using traditional Chinese medicine to improve anemia in RA through association rules4818 RA patients were included in the study,2612 of them were RA with anemia patients,accounting for 54.21% of the total RA patients.Among them,494 were male and 2118 were female,the ratio of male to female was 1:4.29;The age was(52.39±11.09)years old;The length of hospitalization was(17.43±10.76)days.Compared with before treatment,after treatment,the anemia indexes: RBC,Hb,mean corpuscular volume(MCV),mean corpuscular hemoglobin(MCH)were increased(P < 0.01),mean corpuscular hemoglobin concentration(MCHC)was decreased(P < 0.01).Compared with before treatment,the inflammatory indexes ESR,CRP and immune indexes:complement C3(C3),complement C4(C4),anti cyclic citrullinated peptide antibody(anti-CCP),immunoglobulin A(IGA),rheumatoid factor(RF)decreased(P < 0.01),Ig G increased(P < 0.01).Immunoglobulin M(Ig M)decreased after treatment,but the difference was not statistically significant(P>0.05).The study involved 12873 prescriptions of 329 kinds of traditional Chinese medicine.The traditional Chinese medicine used in the study involved 5 aspects: cold,hot,warm,cool and plain.Among them,cold,warm and plain drugs were used most frequently,with the occurrence rate of more than 24% and the use frequency of more than 92%; There are 7 kinds of traditional Chinese medicine,including sour,bitter,sweet,pungent,salty,astringent and light.Among them,bitter,sweet and pungent herbs are used most frequently,with the frequency of occurrence being over 38% and the frequency of use being over 98%;The prescription of traditional Chinese medicine involves 12 channels such as lung channel,large intestine channel,stomach channel,spleen channel and heart channel.The frequency of liver,lung,stomach,spleen,kidney and heart channel is more than 22%,and the frequency of use is more than 96%.The prescription of traditional Chinese medicine is mainly composed of the following five kinds of drugs: spleen invigorating and dampness removing drugs,heat clearing and detoxifying drugs,blood activating and stasis removing drugs,wind dampness removing and collaterals removing drugs,Qi Benefiting and blood nourishing drugs.The preceding item is 1.In the association analysis,Dan participated in the decline of RF with higher support,confidence and promotion(support 85.91%,confidence 86.28%,improvement 1.01).The preceding item is 1.In the association analysis,Dan participates in the association of RF with high support,confidence and promotion(support 85.91%,confidence 86.28%,promotion 1.01);the relationship between Poria cocos and ESR(support 91.65%,confidence 98.04%,promotion 1.04);In the previous 2 association analysis,the correlation between safflower & Poria cocos and RF(the degree of support,confidence and promotion were 76.99%,86.37% and 1.02 respectively);the correlation between tangerine peel & Poria cocos and ESR(the degree of support,confidence and promotion were 81.05%,83.36% and 1.03 respectively).3.1.2 Random Walk Model Analysis of TCM spleen-invigorating Unit Therapy in improving immune inflammatory Indexes of RA anemia A total of 1251 RA patients with anemia met the study requirements.According to whether to use Xinfeng Capsule(XFC)and Furong ointments,they were divided into control group and experimental group.The control group was treated with traditional Chinese medicine only,while the experimental group was treated with traditional Chinese medicine,XFC and Furong ointment for external use.There were 754 cases in the control group,161 males and 693 females,aged 18-88 years,with an average of(55.45±13.91)years;497 cases in the experimental group,109 males and 388 females,aged 29-87 years,with an average of(56.61±13.32)years.There was no significant difference in age and gender between the two groups(P > 0.05).Compared with the group before treatment,RBC and Hb levels increased(P < 0.05 or P < 0.01),CRP,ESR,Ig A,Ig G,RF,anti-CCP,C3 and C4 levels decreased(P < 0.05 or P < 0.01).There was no significant difference in Ig M between the two groups(P >0.05).Compared with the difference between the two groups before and after treatment,the experimental group was superior to the control group in increasing RBC,Hb,reducing CRP,ESR and anti-CCP(P < 0.01).There was no significant difference in Ig A,Ig G,Ig M,RF,C3,C4 between the two groups(P > 0.05).In the control group,the positive increasing rates of RBC,Hb,CRP and ESR were0.1321,0.2781,0.1545 and 0.4286 respectively,and the comprehensive improvement rates of each positive step were 23.78%,27.81%,52.19% and 22.11%.In the experimental group,the positive increasing rates of RBC,Hb,CRP and ESR comprehensive evaluation were 0.2178,0.2781,0.6012 and 0.4901 respectively,and the comprehensive improvement rates of each positive step were 37.63%,39.39% and33.89% 29.01%.3.2 Clinical research3.2.1 General situation of the object of study39 RA patients with anemia were divided into study group(9 males and 30 females)with an average age of(56.03 ±13.47)years.36 normal healthy persons were selected as the control group,including 9 males and 27 females,with an average age of(57.97±9.16)years.The baseline of the two groups was compared in terms of age and sex(P > 0.05),and the two groups were comparable.3.2.2 Changes of blood routine indexes,iron metabolism and immune inflammation indexes in two groups Compared with the control group,RBC,Hb,serum iron(SI),total iron binding capacity(TIBC)of RA with anemia patients were significantly reduced(P < 0.01),while transferrin,The levels of CRP,ESR,RF,Ig A and anti-CCP in the study group were significantly higher than those in the control group(P < 0.05 or P < 0.01);there was no significant difference in Ig G and Ig M between the two groups(P > 0.05);the levels of IL-6,TNF-?,JAK2 and STAT3 in RA anemia patients were significantly higher(P < 0.01),and the levels of IL-4 and IL-10 were significantly lower than those in the control group(P < 0.01).3.2.3 Correlation analysis of anemia index,immune inflammation index and iron metabolism,signal pathway and cytokine index in RA patients with anemia The correlation analysis between anemia index and iron metabolism,signal pathway and cytokine index showed that: RBC level was negatively correlated with hepcidin(P< 0.05),JAK2(P < 0.01),SI(P < 0.01),but not with other indexes(P > 0.05).Hb level was positively correlated with Hepcidin(P < 0.05),IL-6(P < 0.05),SI(P < 0.01),and Hb was not correlated with other indexes(P > 0.05).The correlation analysis of inflammatory indexes with iron metabolism,pathway indexes and cytokines showed that ESR level was positively correlated with Hepcidin(P < 0.01),IL-10(P < 0.05),and no correlation with other indexes(P > 0.05).CRP level was negatively correlated with TRF and SI(P < 0.05 or P < 0.01),but not with other indexes(P > 0.05).The correlation analysis between immune index and iron metabolism,JAK2/STAT3 pathway index and cytokine showed that the level of Ig A was positively correlated with IL-6 and IL-10(P < 0.05),but not with other indexes(P > 0.05).RF level was positively correlated with IL-6 and SI(P < 0.05),negatively correlated with TNF-?(P< 0.01),and not correlated with other indexes(P > 0.05).3.3 Basic experimental research3.3.1 Effect of XFC on PBMCs in RA patients with anemia detected by MTT Compared with the negative control group in the same period,the OD of low,middle and high dose XFC groups decreased,and the inhibition rate of XFC on PBMC increased gradually with the extension of action time.The inhibition rate of XFC in the middle dose group on RA patients with anemia was close to 50% at 24 hours.Therefore,in this experiment,the dose of XFC is the best concentration,and 24 hours is the best time.3.3.2 The effect of XFC on IL-4,IL-6,IL-10,TNF-?,hepcidin in PBMCs of RA patients with anemia was detected by ELISA Compared with the normal group,IL-6,TNF-? and Hepcidin in the model group increased(P < 0.01),IL-4 and IL-10 decreased(P < 0.01).Compared with the model group,IL-6,TNF-? and Hepcidin decreased in each group after drug intervention(P <0.01),IL-4 and IL-10 increased significantly(P < 0.01).Compared with XFC group,IL-6,TNF-? and Hepcidin decreased in AG490 group(P < 0.01).IL-4 and IL-10increased(P < 0.01 or P < 0.05).Compared with AG490 group,IL-6,TNF-? and hepcidin in XFC + AG490 group decreased(P < 0.01),IL-4 and IL-10 increased(P <0.01).3.3.3 Detection of JAK2 / STAT3 / Hepcidin m RNA in PBMCs of RA patients with anemia by RT-q PCR Compared with the normal group,JAK2 m RNA,STAT3 m RNA and Hepcidin-m RNA in the model group increased(P<0.01);Compared with the model group,JAK2 m RNA,STAT3 m RNA and Hepcidin-m RNA in the model group decreased(P< 0.01);Compared with the XFC group,JAK2 m RNA,STAT3 m RNA and Hepcidin-m RNA in the AG490 group decreased(P < 0.01)Compared with AG490 group,JAK2 m RNA,STAT3 m RNA and Hepcidin m RNA decreased in XFC + AG490 group(P < 0.01).3.3.4 Effect of XFC on JAK2,STAT3 and Hepcidin of PBMCs in RA patients with anemia detected by immunofluorescence The positive expression of JAK2,STAT3 and hepcidin showed red fluorescence.Compared with the normal group,the fluorescent intensity of the model group was significantly enhanced(P < 0.01).Compared with the model group,the fluorescence intensity of each group decreased after drug intervention(P < 0.01).Compared with the XFC group,the proportion of red fluorescent cells in the AG490 group was decreased and the intensity was decreased(P < 0.01).Compared with AG490 group,the expression of red fluorescence in AG490 + XFC group decreased and the intensity decreased(P < 0.01 or P < 0.05).In XFC + AG490 group,the number of red fluorescent cells was the lowest and the intensity was the weakest.3.3.5 Western blot analysis of the effect of XFC on JAK2,p-JAK2,STAT3 and p-STAT3 in PBMCs of RA patients with anemia Compared with the normal group,the protein levels of JAK2,p-JAK2,STAT3 and p-STAT3 in the model group were higher than those in the normal group(P < 0.01).Compared with the model group,the levels of JAK2,p-JAK2,STAT3 and p-STAT3 in each group decreased after drug intervention(P < 0.01).Compared with XFC group,JAK2 and p-STAT3 in AG490 group decreased(P < 0.01),while p-JAK2 and STAT3 decreased,but there was no significant difference(P ? 0.05).Compared with AG490 group,JAK2,p-JAK2,STAT3 and p-STAT3 decreased in AG490+XFC group(P <0.01).4 Conclusion4.1 The rheumatology department of our hospital treats RA with anemia mainly with the following five kinds of drugs: spleen invigorating and dampness removing drugs,heat clearing and detoxification drugs,blood activating and stasis removing drugs,wind dampness removing and collaterals removing drugs,Qi Benefiting and blood nourishing drugs,with spleen invigorating and dampness removing drugs as the core.There is a strong correlation between the use of spleen invigorating and dampness eliminating drugs and the improvement of patients' immune and inflammatory indexes.4.2 There is a long-term relationship between the treatment mode of "spleen strengthening unit therapy" and the comprehensive evaluation indexes of patients.The improvement of the comprehensive evaluation indexes of RBC,Hb,ESR and CRP of patients treated with unit therapy is better than that of patients treated with traditional Chinese medicine alone,which reflects the exact role of the treatment mode in improving anemia and reducing immune inflammation.4.3 There were significant differences in iron metabolism,JAK2 / STAT3 signaling pathway,immune inflammation index between RA patients with anemia and normal healthy patients.The high level of immune inflammation in RA patients with anemia is directly related to the disorder of iron metabolism,the imbalance of cytokines and the abnormal activation of JAK2 / STAT3 signaling pathway related to Hepcidin.4.4 XFC can treat RA and anemia by the following mechanisms: 1)XFC can improve the immune inflammatory response of patients by down-regulating pro-inflammatory factors IL-6 and TNF-?,up-regulating anti-inflammatory factors IL-4 and IL-10,regulating JAK2/STAT3 signal pathway.2)XFC has a AG490-like blocking effect.XFC down-regulates the level of IL-6,inhibits the expression of total JAK2 protein,reduces the activity of p-JAK2,further down-regulates the levels of STAT3 and p-STAT3,and reduces the number of p-STAT3 that binds to the promoter of Hepcidin gene,resulting in a decrease in Hepcidin synthesis and improvement of anemia. |
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