| 1 ObjectiveData mining method was used to analyze the changes of heart function in Rheumatoid arthritis(RA)and its correlation with immune inflammatory indexes,and to explore the effect of strengthening spleen,reducing dampness and dredging collateral on it.Cardiac functional parameters,immunoinflammatory markers and cytokines,microRNA-23A-3P/chromosome 10 deficient phosphatase and tensin homologue genes/phosphatidylinositol 3-kinase/protein kinase/mammal-target rapamycin(miR-23a-3p/PTEN/PI3K/Akt/mTOR)signaling pathway to explore the primary mechanism of cardiac function decline in RA.In vitro cell experiments were conducted to study the biological mechanism of improving cardiac function of RA with Xinfeng Capsule(XFC)and Jianpi,Huashi and Tongluo prescription.2 Methods2.1 Data mining researchThe medical records of RA inpatients in the Department of Rheumatology of the First Affiliated Hospital of Anhui University of Chinese Medicine from September 2016 to July 2020 were studied.Observed the changes of cardiac function parameters in RA patients and the correlation and correlation with immune inflammatory indicators,and analyzed the risk factors of cardiac function decline in RA by binary Logistics regression.The complex network diagram was used to analyze the core prescriptions of patients with cardiac function decline in RA,and the frequency of TCM flavor returning to menstruation was counted.Association rules were used to explore the influence of traditional Chinese medicine on immune inflammation indexes and cardiac function parameters in patients with cardiac function decline in RA.2.2 In vivo study30 patients with cardiac function decline of RA were selected as the study group(RA group)and 30 normal healthy patients as the control group(HCs group).The changes of cardiac function parameters and immune inflammatory indexes in the two groups were analyzed.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expressions of miR-23a-3p,PTEN,PI3K,AKT and mTOR.Enzyme-linked immunosorbent method(ELISA)was used to detect the changes of interleukin-1β(IL-1β),IL-6,IL-10 and tumor necrosis factor(TNF)-α.To analyze the correlation between cardiac function parameters,immune inflammatory indexes and miR-23a-3p/PTEN/PI3K/AKT/mTOR signaling pathway and cytokines in patients with cardiac function decline in RA.2.3 In vitro experimental study2.3.1 Effects of XFC-containing serum on miR-23a-3p/PTEN/PI3K/AKT/mTOR signaling pathway in co-culture of RA-CD4+T cells combined with RA-FLSCD4+T cells were isolated from the peripheral blood of normal persons and RA patients,and cocultured with RA fibroblast-like synoviocytes(FLS)by transwell chamber.The cells were divided into 7 groups,control-1 group:RA-FLS;control-2 group:normal CD4+T and RA-FLS co-culture;model group:RA-CD4+T and RA-FLS co-culture;XFC group:model+containing the optimal concentration of XFC;mimics-NC group:model+mimics-miR-23a-3p+NC;mimics groups:model+mimics-miR-23a-3p;mimics+XFC group:model+mimics-miR-23a-3p+containing the optimal concentration of XFC.The expressions of miR-23a-3p,PTEN,PI3K,AKT and mTOR in lower ventricular cells were detected by RT-qPCR.The expression of cytokines in the supernatant of the upper compartment was detected by ELISA.Western blotting(WB)was used to detect the protein expressions of PTEN,PI3K,AKT,p-AKT,mTOR and p-mTOR in lower ventricular cells.SPSS 21.0 statistical software was used to analyze the data.2.3.2 Effects of XFC-containing serum on miR-23a-3p/PTEN/PI3K/AKT/mTOR signaling pathway in co-culture of RA-CD4+T cells combined with human normal cardiomyocytesCD4+T cells were isolated from the peripheral blood of normal people and RA patients,and were co-cultured with Human Cardiac Myocyte(HCM)in transwell chamber.The cells were divided into 7 groups.Control-1 group:HCM;control-2 group:normal CD4+T cells and HCM co-culture;model group:RA-CD4+T cells and HCM co-culture;XFC group:model+containing the optimal concentration of XFC;mimics-NC group:model+mimics-miR-23a-3p+NC;mimics groups:model+mimics miR-23a-3p;mimics+XFC group:model+mimics-miR-23a-3p+containing the optimal concentration of XFC.The expressions of miR-23a-3p,PTEN,PI3K,AKT and mTOR in lower ventricular cells were detected by RT-qPCR.The expressions of cytokines in the supernatant of the upper compartment were detected by ELISA.The protein expressions of PTEN,PI3K,AKT,p-AKT,mTOR and p-mTOR in lower ventricular cells were detected by WB method.Immunofluorescence(IF)was used to detect the expression of brain natriuretic peptide(BNP),connexin-43(Cx43),cardiac Troponin I(cTnI),and cardiac Troponin T(cTnT)in lower ventricular cells.SPSS 21.0 statistical software was used to analyze the data.3 Results3.1 Dating mining research results3.1.1 Correlation analysis of cardiac function parameters and immune inflammatory indexes in RAAmong the 1596 patients,there were 1011 cases(63.35%)of changes in cardiac function parameters,among which the stroke volume(SV)decreased in the most 611 cases,accounting for 38.28%.Correlation analysis showed that right ventricular outflowtract diameter(RVOTd),aorta diameter(AODd),left atrium diameter(LADs,)SV were positively correlated with C3,while left ventricular end-diastolic diameter(LVDd)was negatively correlated with C3(P<0.05).AODd,LADs and right ventricular end-diastolic diameter(RVDd)were positively correlated with hs-CRP,while ejection fraction(EF)and fractional shortening(FS)were negatively correlated with hs-CRP,anti-CCP(P<0.05).LVDd was positively correlated with C4(P<0.05).3.1.2 Association rule analysis of cardiac function parameters and immunoin-flammatory markers in RA patientsThe association rules found that the increase of AODd and the decrease of SV were strongly correlated with the increase of anti-CCP,ESR,hs-CRP and RF,with the minimum support of 20%and the minimum confidence of 85%.3.1.3 Binary logistic regression analysis of cardiac function parameters and immune inflammatory indicators in RA patientsThe results showed that C3,ESR,Immunoglobulin M(IgM),RF were the risk factors of cardiac function reduction in RA.3.1.4 Use of traditional Chinese medicine in patients with cardiac function changes of RAAmong the 4,622 prescriptions of 314 traditional Chinese medicines,the top 20 drugs in frequency of use were:a)drugs for promoting blood circulation and removing blood stasis:safflower,salvia miltiorrhiza,peach kernel,caulis spatholobus,ligusticum chuanxiong and angelica;(b)Splene-strengthening and humidifying drugs:poria cocos,coix seed,alisma alisma,malt,tangerine peel,pinellia ginger,licorice,yam;(c)wind-dispelling and dehumidifying drugs:siegesbeckiae,verticillium verticilliae,honeysuckle verticilliae,xu changqing;(d)antipyretic poisons:dandelion and diffusa.Cold and warm medicinal properties,sweet,bitter,spicy and medicinal tastes,liver,lung,spleen,stomach,kidney,heart meridian of traditional Chinese medicine appear and use frequency is high.Commonly used for spleen and wet medicine(tangerine peel,coix seed,tuckahoe)and blood circulation and stasis medicine(safflower,salvia miltiorrhiza),heat antidote(dandelion).3.1.5 Correlation analysis of TCM and immune inflammatory markers in patients with cardiac function changes in RADandelion,safflower seed,tuckahoe,safflower and hs-CRP decline had high correlation,dandelion,tangerine peel,tuckahoe and C4 decline had high correlation,coix seed,safflower,dandelion,tuckahoe and ESR decline had high correlation.Compatibility of red peach kernel,dandelion,dried tangerine or orange peel,coix seed,poria cocos,salvia miltiorrhiza and hs-CRP decreased incidence degree,poria cocos compatibility coix seed,dried tangerine or orange peel and hs-CRP decreased incidence degree,and dandelion compatibility semen coicis and hs-CRP decreased incidence degree,the compatibility of Safflower with Poria cocos and Tangerine Peel had a higher correlation with the decline of ESR,and the compatibility of Tangerine Peel with Poria cocos had a higher correlation with the decline of C4.3.1.6 Correlation analysis of traditional Chinese medicine and cardiac function parameters in patients with cardiac function changes in RAPeach kernel,poria cocos,yam,liquorice,safflower,red sage root,dried tangerine or orange peel and SV increases high correlation,salvia miltiorrhiza,dried tangerine or orange peel and AODd fall high correlation,salvia miltiorrhiza,safflower and FS increased incidence degree is higher,poria cocos and RVDd drop correlation is higher,Safflower had a high correlation with EF.The compatibility of safflower with poria cocos,peach kernel,salvia miltiorrhiza,tangerine peel and tuckaing with tangerine peel and salvia miltiorrhiza had higher correlation with SV.3.1.7 Correlation analysis of XFC combined with traditional Chinese medicine and immune inflammatory indexes and cardiac function parametersCoicis seed combined with XFC single traditional Chinese medicine had a high correlation with the decline of hs-CRP,a high correlation between XFC combined with Sibeckiae Sibeckiae and dandelion and the decline of ESR,a high correlation between XFC combined with tangerine bark and dandelion and the decline of C3,XFC combined with peach kernel,weilingia,tuckaea,tangerine peel,safflower,coix seed,dandelion and salviorrhiza miltiorrhiza had a high correlation with RF decline.XFC combined with Safflower had a high correlation with RVOTd and AODd.3.2 In vivo experimental study3.2.1 General situationIn RA group,there were 4 males and 26 females,with an age of 58.2(45-72)years and a course of 11.5(0.5-35)years.There were 7 males and 23 females in the HCs group,aged 57.0(45-70)years.There was no significant difference in age and gender between the two groups(P>0.05).3.2.2 Comparison of immune inflammation indexes and cardiac function parameters between the two groupsCompared with HCS group,the levels of hs-CRP,ESR,RF and anti-CCP in RA group were significantly increased(P<0.05).EF,SV and FS were significantly decreased in RA group(P<0.05).3.2.3 Comparison of miR-23a-3p/PTEN/PI3K/AKT/mTOR pathway related indicators between the two groupsCompared with HCs group,the expression levels of miR-23a-3p,PI3K,AKT and mTOR in RA group were significantly increased,and the expression level of PTEN was significantly decreased(P<0.05).3.2.4 Comparison of cytokines between the two groupsCompared with the HCS group,the expression of IL-1β,IL-6 and TNF-α in RA group was up-regulated,while the expression of IL-10 was down-regulated(P<0.05).3.2.5 Correlation analysis of immunoinflammatory indexes with miR-23a-3p/PTEN/PI3K/AKT/mTOR pathway and cytokines in patients with cardiac function decline in RAMiR-23a-3p and IL-6 were positively correlated with ESR,PTEN and IL-10 were negatively correlated with ESR(P<0.05);miR-23a-3p,PI3K,TNF-α and IL-1β were positively correlated with hs-CRP and RF,PTEN and IL-10 were negatively correlated with hs-CRP and RF(P<0.05).3.2.6 Correlation analysis of cardiac function parameters with miR-23a-3p/PTEN/PI3K/AKT/mTOR pathway and cytokines in patients with cardiac function decline in RAMiR-23a-3p was positively correlated with RVOTd,LADs,RVDd and SV,PTEN was negatively correlated with RVOTd,LADs,RVDd and SV,PI3K was positively correlated with RVOTd(P<0.05),there was a positive correlation between AKT and LVDd(P<0.05),TNF-α and IL-1β were positively correlated with RVOTd,LADs,RVDd,LVDD,AODd and SVIL-6 was positively correlated with RVOTd,LADS and AODd,IL-10 was negatively correlated with RVOTd,RVDd and SV(P<0.05).3.3 In vitro experimental study3.3.1 Effects of XFC-containing serum on miR-23a-3p/PTEN/PI3K/AKT/mTOR signaling pathway in co-culture of RA-CD4+T cells combined with RA-FLS3.3.1.1 CCK-8 detected the inhibitory effect of XFC-containing serum on the co-culture of RA-CD4+T cells combined with RA-FLSThe cell inhibition rate increased gradually with time and concentration increasing.The inhibitory rate of 10%XFC containing serum on cells was close to 50%after 48 hours of treatment.Therefore,10%XFC containing serum was the best concentration and 48h was the best time of action.3.3.1.2 The expression level of miR-23a-3p/PTEN/PI3K/AKT/mTOR in each group was detected by RT-qPCRCompared with the control-1 and control-2 groups,the expressions of miR-23a-3p,PI3K,AKT and mTOR in the model group were up-regulated,while the expression of PTEN was down-regulated(P<0.05);Compared with the model group,the expressions of miR-23a-3p,PI3K,AKT and mTOR in the XFC group were down-regulated,while the expression of PTEN was up-regulated(P<0.05).Compared with the mimics-NC group,miR-23a-3p expression was up-regulated,PI3K,AKT,mTOR expression was up-regulated,and PTEN expression was down-regulated in the mimics group(P<0.05).Compared with the mimics group,the expressions of miR-23a-3p,PI3K,AKT and mTOR were down-regulated and the expressions of PTEN were up-regulated in the mimics+XFC group(P<0.05).3.3.1.3 ELISA was used to detect the expression levels of cytokines in each groupCompared with the control-1 and control-2 groups,the expression of IL-1β,IL-6 and TNF-α in the model group was up-regulated,and the expression of IL-10 was down-regulated(P<0.05);Compared with the model group,the expression of IL-1β,IL-6 and TNF-α in XFC group was down-regulated,and the expression of IL-10 was up-regulated(P<0.05).Compared with the mimics-NC group,the expression of IL-1β and TNF-α in the mimics group was up-regulated,and the expression of IL-10 was down-regulated(P<0.05).Compared with the mimics group,the expressions of IL-1β,IL-6,TNF-α and IL-10 in the mimics+XFC group were down-regulated and the expression of IL-10 was up-regulated(P<0.05)3.3.1.4 WB was used to detect the expression levels of PTEN/PI3K/AKT/mTOR protein in each groupCompared with the control-1 and control-2 groups,the expression of PI3K,AKT,p-AKT,mTOR,p-mTOR in the model group was up-regulated,and the expression of PTEN was down-regulated(P<0.05);Compared with the model group,the expression of PI3K,AKT,p-AKT,mTOR,p-mTOR in XFC group was down-regulated,while the expression of PTEN was up-regulated(P<0.05).Compared with the mimics-NC group,the expression of PI3K,AKT,p-AKT,mTOR,p-mTOR were up-regulated and PTEN was down-regulated in the mimics group(P<0.05).Compared with mimics group,the expressions of PI3K,AKT,p-AKT,mTOR,p-mTOR in mimics+XFC group were down-regulated,and the expressions of PTEN were up-regulated(P<0.05).3.3.2 Effects of XFC-containing serum on miR-23a-3p/PTEN/PI3K/AKT/mTOR signaling pathway in co-culture of RA-CD4+T cells combined with HCM3.3.2.1 CCK-8 was used to detect the inhibitory effect of XFC-containing serum on the co-culture of RA-CD4+T cells combined with HCMWith the increase of drug concentration,the cell inhibition rate increased gradually.The inhibitory rate of 10%XFC containing serum on cells was close to 50%after 48 hours of treatment.Therefore,10%XFC containing serum was the best concentration and 48h was the best time of action.3.3.2.2 Expression levels of miR-23a-3p/PTEN/PI3K/AKT/mTOR in each group were detected by RT-qPCRCompared with the control-1 and control-2 groups,the expressions of miR-23a-3p,PI3K,AKT and mTOR in the model group were up-regulated,while the expression of PTEN was down-regulated(P<0.01);Compared with the model group,the expression levels of miR-23a-3p,PI3K,AKT and mTOR in the XFC group were down-regulated,while the expression of PTEN was down-regulated(P<0.01).Compared with the mimics-NC group,the expression of miR-23a-3p in the mimics group was up-regulated,the expression of PI3K,AKT and mTOR were up-regulated,and the expression of PTEN was down-regulated(P<0.01).Compared with the mimics group,the expressions of miR-23a-3p,PI3K,AKT and mTOR were down-regulated and the expressions of PTEN were up-regulated in the mimics+XFC group(P<0.01).3.3.2.3 Expression levels of cytokines in each group were detected by ELISACompared with the control-1 and control-2 groups,the expression of IL-1β,IL-6 and TNF-α in the model group was up-regulated,and the expression of IL-10 was down-regulated(P<0.01);Compared with the model group,the expression of IL-1β,IL-6 and TNF-α in XFC group was down-regulated,and the expression of IL-10 was up-regulated(P<0.01).Compared with the mimics-NC group,the expressions of IL-1β and TNF-α in the mimics group were up-regulated,while the expression of IL-10 was down-regulated(P<0.01).Compared with the mimics group,the expression of IL-1β,IL-6 and TNF-α in the mimics+XFC group was down-regulated,and the expression of IL-10 was up-regulated(P<0.01).3.3.2.4 WB was used to detect the expression levels of PTEN/PI3K/AKT/mTOR protein in each groupCompared with the control-1 and control-2 groups,the expression of PI3K,AKT,p-AKT,mTOR,p-mTOR in the model group was up-regulated,and the expression of PTEN was down-regulated(P<0.01);Compared with the model group,the expression levels of PI3K,AKT,p-AKT,mTOR and p-mTOR in XFC group were down-regulated,while the expression of PTEN was up-regulated(P<0.01).Compared with the mimics-NC group,the expressions of PI3K,AKT,p-AKT,mTOR and p-mTOR in the mimics group were up-regulated(P<0.01),while the expression of PTEN was down-regulated(P<0.05).Compared with mimics group,the expressions of PI3K,AKT,p-AKT,mTOR,p-mTOR in mimics+XFC group were down-regulated,and the expressions of PTEN were up-regulated(P<0.01).3.3.2.5 IF was used to detect the influence of myocardial marker expression in each groupCompared with control-1 and control-2 groups,the positive expressions of BNP,Cx43,cTnI and cTnT in model group were significantly increased(P<0.05);Compared with the model group,the expressions of BNP,Cx43,cTnI and cTnT in mimics-NC group,XFC group and mimics+XFC group were decreased(P<0.05).The expressions of BNP,Cx43,cTnI and cTnT in the mimics+XFC group were decreased compared with the mimics group(P<0.05).4 Conclusion4.1 There were 63.35%of RA patients with abnormal cardiac function parameters,mainly SV decreased;In addition,there was strongly correlated with immune inflammatory indicators.In the treatment of cardiac function decline in RA in the Department of Rheumatology of our hospital,there are mainly the following four types of drugs:drugs for strengthening spleen and dampness,antidotes for clearing heat,drugs for promoting blood circulation and removing blood stasis,and drugs for dispelling wind dampness and dredging collusion,with drugs for strengthening spleen and dampness as the core.The use of invigorating spleen and humidifying drugs was strongly correlated with the improvement of immune inflammatory indexes and cardiac function parameters of patients,and the improvement effect of traditional Chinese medicine combined with XFC was more obvious.4.2 There were significant differences in cardiac function parameters of RA,miR-23 a-3p/PTEN/PI3K/AKT/mTOR signaling pathway,cytokines and immune inflammatory markers between RA and normal healthy.RA cardiac function parameters and immunoinflammatory indicators were directly related to abnormal activation of miR-23a-3p/PTEN/PI3K/AKT/mTOR signaling pathway and imbalance of cytokines.4.3 XFC can improve cardiac function in RA patients.The mechanism of action includes:XFC can down-regulate miR-23a-3p,up-regulate PTEN,inhibit PI3K/AKT/mTOR activation,further down-regulated the expressions of pro-inflammatory factors IL-1β,IL-6,TNF-α,up-regulated the expression of anti-inflammatory factors IL-10,and decreased the expressions of BNP,CX43,cTnI,and cTnT.Improve the immune inflammatory response of patients,thereby improving the cardiac function of RA patients. |
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