| 1.ObjectTo explore the changes of echocardiographic parameters in patients with decreased cardiac function in patients with ankylosing spondylitis(AS)and its correlation with laboratory indexes,the clinical data were collected and analyzed by data mining and analysis,and the medication rule of invigorating spleen,resolving dampness and dredging collaterals was used to treat patients with decreased cardiac function in AS.The association rules were used to evaluate the improvement of immune inflammation index of spleen-invigorating unit therapy in patients with decreased cardiac function in patients with AS.Through in vivo study,to study the correlation of echocardiographic parameters,immuno-inflammatory indexes,related cytokines,micro RNA-23a-3p(miR-23a-3p)and PI3K/AKT/mTOR(phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin pathway)signal pathway,and to explore the preliminary mechanism of cardiac dysfunction in AS.Through the cell experiment in vitro,to study the biological mechanism of Jianpi Huashi Tongluo recipe-Xinfeng capsule in the treatment of cardiac dysfunction in AS.2.Methods2.1 Research on data MiningTo screen the medical records and oral traditional Chinese medicine(TCM)data of AS inpatients in the Department of Rheumatology and Immunology,Anhui Hospital of traditional Chinese Medicine from June 2012 to July 2020.To analyze the changes of patients’laboratory indexes and the menstruation of TCM involved in the prescription,and to explore the medication rule of the prescription for the treatment of AS.In order to study the relationship between the changes of laboratory indexes and TCM for invigorating spleen,resolving dampness and dredging collaterals,the Apriori module of SPSS Clementine 12.0 software was used to evaluate the effect of TCM treatment on immune and inflammatory indexes in patients with decreased cardiac function of AS.Spleen-strengthening unit therapy is a characteristic treatment mode for the treatment of AS,that is,‘internal administration of TCM+Xinfeng capsule’treatment mode.In order to study its therapeutic effect,the AS patients who used TCM were divided into the experimental group(taking TCM+Xinfeng capsule)and the control group(taking TCM only).The core prescriptions of the two groups of TCM were analyzed by complex network technology,and the changes of laboratory indexes of the two groups were compared to explore the relationship between TCM and index improvement.2.2 Experimental study in vivo30 patients with impaired cardiac function in AS were selected as the study group and30 normal controls as the control group.The changes of cardiac function parameters and clinical indexes in the two groups were analyzed.Real-time fluorescence quantitative(real-time fluorescence quantitative PCR,RT-qPCR)was used to detect the expression of m RNA(miR-23a-3p,PI3K,AKT,enzyme-linked immunosorbent method)to detect the changes of cytokines.To analyze the correlation between cardiac function parameters,clinical indexes and miR-23a-3p,PI3K/AKT/mTOR signal pathway and cytokines in patients with impaired cardiac function in AS.2.3 Experimental study in vitro2.3.1 The effect of XFC drug-containing serum on AS-CD4~+T cells co-cultured with AS-FLS in miR-23a-3p Influence of PI3K/AKT/mTOR signal pathwayCD4~+T cells were isolated from peripheral blood of normal persons and AS patients,and co-cultured with AS fibroblast-like synoviocytes(FLS)in transwell chamber.RT-qPCR method was used to detect the expression of miR-23a-3p,PTEN,PI3K,AKT and mTOR in AS-FLS.The expression of IL-1β,IL-6,IL-10 and TNF-αin the supernatant of co-culture compartment was detected by ELISA method.Western blotting(WB)was used to detect the expression of PTEN,PI3K,AKT,p-AKT,mTOR and p-mTOR in AS-FLS.The data were analyzed by SPSS 21.0 statistical software.2.3.2 miR-23a-3p/co-cultured with AS-CD4~+T cells and HCM with XFC drug-containing serum Influence of PI3K/AKT/mTOR signal pathwayCD4~+T cells were isolated from peripheral blood of normal persons and AS patients,and co-cultured with normal cardiomyocytes(HCM)in transwell chamber.ELISA method was used to detect the expression of IL-1βand IL-6,IL-10,TNF-αin the supernatant of transwell co-culture compartment,RT-qPCR method was used to detect the expression of miR-23a-3p,PTEN,PI3K,AKT and mTOR,WB method was used to detect the expression of PTEN,PI3K,AKT,p-AKT,mTOR and p-mTOR protein in the lower compartment HCM.SPSS 21.0 statistical software and Graghpad 8.0 analysis were used.3 Results3.1 Research on data Mining3.1.1 Analysis of the changes of cardiac function parameters of AS and its correlation with immuno-inflammatory indexesAmong the 238 patients,there were 97 cases of abnormal cardiac function parameters,accounting for 40.75%.Correlation analysis showed that cardiac function indexes were correlated with Ig M,hs-CRP and ESR,but not with other items(P>0.05).3.1.2 Analysis of Association rules between Cardiac function parameters and immune inflammation Indexes in patients with AS.It is found that the increase of AODd is strongly related to the increase of hs-CRP,and the decrease of SV is strongly related to the increase of hs-CRP and ESR.The support,confidence and improvement of the above association rules are all more than 30%,50%and 1 respectively.3.1.3 Binary Logistics regression analysis of cardiac function parameters and immuno-inflammatory indexes in patients with AS.IgG and hs-CRP are the risk factors of AODd(OR>1),ESR and the increase of Ig M are the risk factors of the decrease of SV(OR>1).3.1.4 use of traditional Chinese medicine in patients with AS.Among the 1237 prescriptions,the top 20 drugs used were:(a)spleen-invigorating and dampening drugs:tangerine peel,Poria cocos,Coix seed,yam,alisma;(b)anti-wind and dampness medicine:Weilingxian,Zizhao grass,Dicranthes bidentata,Achyranthes bidentata;(c)for promoting blood circulation and removing blood stasis:Salvia miltiorrhiza,safflower,peach kernel,chicken blood vine,Chuanxiong;(d)heat-clearing and detoxifying drugs:dandelion,Hedyotis diffusa,Scutellaria baicalensis,Phellodendron,Rhizoma Anemarrhenae.3.1.5 Correlation Analysis of traditional Chinese Medicine with Cardiac function parameters and immune inflammatory Indexes.Dandelion combined with Salvia miltiorrhiza was associated with the decrease of CRP,and Rhizoma Rehmanniae with yam was associated with the decrease of ESR.Coix seed,Poria cocos,yam,tangerine peel,single live,Rhizoma Alismatis,Rhizoma Corydalis,Radix Salviae Miltiorrhizae,Radix Salviae Miltiorrhizae and Ligusticum chuanxiong were associated with the decrease of Ig A,Coix seed and Eucommia ulmoides,Coix seed and Chinese yam,C3,Rhizoma chuanxiong and alisma,respectively.Poria cocos combined with yam was associated with the decrease of Ig M,while Coix seed combined with Chinese yam was associated with the decrease of IgG.The combination of Coix seed and Eucommia ulmoides was associated with the decrease of SV.3.1.6 Analysis of the relationship between XFC combined with traditional Chinese Medicine and immune inflammatory Indexes and Cardiac function parameters.XFC combined with single Chinese medicine Coix seed has a high correlation with the decrease of hs-CRP(confidence is 80.31%,),XFC,Scutellaria baicalensis,Weilingxian,and ESR decline is high(confidence is 82.45%,83.25%,73.48%,),XFC combined with Coix seed,dandelion,Dan is involved in the decline of Ig M has a high correlation(confidence is 73.32%,72.28%,72.38%).There was a high correlation between XFC combined with safflower and the decrease of AODd(confidence level was 65.29%).3.2 Experimental study in vivo3.2.1 General situation.Among the 30 cases of AS group,there were 26 males and 4 females,the age was 42.50±9.91years old,and 30 cases of(HCs)in the control group,26 males and 4 females,aged 43.50±5.60 years.There was no significant difference in age and sex between the two groups.3.2.2 Comparison of immuno-inflammatory indexes and cardiac function parameters between the two groups.The levels of hs-CRP and ESR in AS group were significantly higher than those in HCs group.There was no significant difference in the indexes of Ig A,IgG and Ig M between the two groups(P>0.05).There was no significant difference in EF,SV and AODd between the two groups,and there was no significant difference in other cardiac function parameters between the two groups(P>0.05).3.2.3 Comparison of related Indexes of miR-23a-3p/PTEN/PI3K/AKT/mTOR Pathway between the two groups.Compared with the normal group,the expression levels of miR-23a-3p,PI3K,AKT and mTOR in AS group were significantly increased(P<0.05).The expression level of PTEN decreased significantly.3.2.4 Comparison of cytokines between the two groups.Compared with the normal group,the expression of IL-1β,IL-6 and TNF-αin AS group was significantly up-regulated,while the expression of IL-10 was significantly down-regulated.3.2.5 Correlation analysis of cardiac function parameters with miR-23a-3p/PI3K/AKT/mTOR pathway and cytokines in patients with AS.AODd was positively correlated with miR-23a-3p and TNF-α,and SV was positively correlated with PI3K.3.3 Experimental study in vitro.3.3.1 Effect of XFC drug-containing serum on miR-23a-3p/PI3K/AKT/mTOR signal pathway in co-culture of AS-CD4~+T cells and AS-FLS(1)The inhibitory effect of XFC-containing serum on AS-CD4~+T cells co-cultured with AS-FLS was detected by CCK-8.Compared with the low dose group in the same period,the cell inhibition rate of middle and high dose XFC groups increased,and the inhibition rate of XFC on AS-CD4~+T cells combined with AS-FLS increased gradually with the extension of action time;the inhibition rate of low dose XFC on AS-CD4~+T cells+AS-FLS was close to 50%after48 hours.Therefore,the middle dose of XFC is the best concentration,and 48h is the best time.(2)The expression of cytokines in each group was detected by ELISA.Compared with control-1 and control-2 groups,the expression of IL-1β,IL-6,TNF-αwas up-regulated and IL-10 was down-regulated in model group,while the expression of IL-1β,IL-6,TNF-αwas down-regulated in XFC group compared with model group,and the expression of anti-inflammatory factor IL-10 was opposite.Compared with model group,the expression of IL-1βand TNF-αin mimics group was significantly up-regulated,while the expression of IL-10 was down-regulated.Compared with mimics group,the expression of IL-1βand TNF-αwas down-regulated and the expression of IL-10 was up-regulated in mimics+XFC group.(3)The expression level of miR-23a-3p/PI3K/AKT/mTOR in each group was detected by RT-qPCR.Compared with control-1 and control-2 groups,the expression of miR-23a-3p,PI3K,AKT and mTOR in model group was up-regulated and the expression of PTEN was down-regulated.Compared with model group,the expression of miR-23a-3p,PI3K,AKT and mTOR in XFC group was significantly up-regulated,while the expression of PTEN was significantly down-regulated.Compared with model group,the expression of miR-23a-3p,PI3K,AKT and mTOR in mimics-miR-23a group was significantly up-regulated,while the expression of PTEN was down-regulated.Compared with mimics group,the expression of miR-23a-3p,PI3K,AKT and mTOR was down-regulated and the expression of PTEN was up-regulated in mimics+XFC group.(4)The expression level of PTEN/PI3K/AKT/mTOR protein in each group was detected by WB.Compared with control-1 and control-2 group,the expression of PI3K,AKT,p-AKT,mTOR,p-mTOR in model group was up-regulated,while the expression of PTEN was down-regulated in XFC group.Compared with model group,the expression of PI3K,AKT,p-AKT,mTOR and p-mTOR was significantly down-regulated,while the expression of PTEN was significantly down-regulated in XFC group.Compared with model group,the expression of PI3K,AKT,p-AKT,mTOR and p-mTOR in mimics group was significantly up-regulated,while the expression of PTEN was down-regulated.Compared with mimic group,the expression of PI3K,AKT,p-AKT,mTOR and p-mTOR in mimics+XFC group was down-regulated,while the expression of PTEN was up-regulated.3.3.2 Effect of XFC drug-containing serum on miR-23a-3p/PTEN/PI3K/AKT/mTOR signal pathway in co-culture of AS-CD4~+T cells and HCM(1)The effect of XFC drug-containing serum on the survival rate of AS-CD4~+T cells co-cultured with HCM.Compared with the negative control group in the same period,there was little difference in cell survival rate in low-dose XFC group.The effect of XFC on the survival rate of AS-CD4~+T cells+HCM cells in each dose group was prolonged.The survival rate of AS-CD4~+T cells+HCM cells treated with medium-dose XFC serum decreased by27.45%after 48 hours(P<0.01).Therefore,the middle dose of XFC is the best concentration,and 48h is the best time.(2)ELISA method was used to detect the effect of serum containing XFC on cytokines.Compared with control-1 and control-2 groups,the expression of IL-1β,IL-6,TNF-αwas up-regulated and the expression of IL-10 was down-regulated in model group,while the expression of IL-1β,IL-6,TNF-αwas down-regulated and the expression of IL-10 was up-regulated in XFC group compared with model group.Compared with model group,the expression of IL-1βand TNF-αin mimics group was significantly up-regulated,while the expression of IL-10 was down-regulated.Compared with mimics group,the expression of IL-1βand TNF-αwas down-regulated and the expression of IL-10 was up-regulated in mimics+XFC group.(3)RT-qPCR method was used to detect the effect of XFC drug-containing serum on miR-23a-3p/PTEN/PI3K/AKT/mTOR.Compared with control-1 and control-2 group,the expression of miR-23a-3p,PI3K,AKT and mTOR was up-regulated and PTEN was down-regulated in model group,while the expression of miR-23a-3p,PI3K,AKT and mTOR was significantly down-regulated and PTEN expression was significantly down-regulated in XFC group compared with model group.Compared with model group,the expression of miR-23a-3p,PI3K,AKT and mTOR in mimics-miR-23a-3p group was significantly up-regulated,while the expression of PTEN was down-regulated.Compared with mimics group,the expression of miR-23a-3p,PI3K,AKT and mTOR was down-regulated and the expression of PTEN was up-regulated in mimics+XFC group.(4)The effect of XFC drug-containing serum on the expression of PTEN/PI3K/AKT/mTOR protein.Compared with control-1 and control-2 group,the expression of PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR was up-regulated and PTEN was down-regulated in model group,while the expression of PI3K,AKT,p-AKT,mTOR and p-mTOR was significantly down-regulated and PTEN expression was significantly down-regulated in XFC group compared with model group.Compared with model group,the expression of PI3K,AKT,p-AKT,mTOR and p-mTOR in mimics group was significantly up-regulated,while the expression of PTEN was down-regulated.Compared with mimic group,the expression of PI3K,AKT,p-AKT,mTOR and p-mTOR in mimics+XFC group was down-regulated,while the expression of PTEN was up-regulated.(5)The effect of XFC-containing serum on the expression of cardiomyocyte markers was detected by immunofluorescence.Under normal conditions,Troponin I and Troponin T are continuously activated and distributed in the whole cell.After drug treatment,Troponin I and Troponin T were abnormally aggregated in the cytoplasm of HCM cells.BNP and Connexin 43 were distributed in the nuclei of myocardium.Among them,the enhanced fluorescence of Troponin I,Troponin T,BNP and Connexin 43 in group F was the strongest,and the distribution in cardiomyocytes became disordered.However,the fluorescence of cardiomyocytes in group An and group B decreased or even disappeared,and the fluorescence spots in group D and group G were significantly lower than those in group C and group F respectively.4 Conclusion4.1 40.75%of the patients with decreased cardiac function of AS were associated with immuno-inflammatory indexes.There are mainly four kinds of drugs for the decline of cardiac function of AS in the rheumatology department of our hospital:drugs for invigorating spleen and removing dampness,heat-clearing and detoxifying drugs,drugs for promoting blood circulation and removing blood stasis,drugs for dispelling wind and dampness and dredging collaterals,with drugs for invigorating spleen and removing dampness as the core.There is a strong correlation between the use of spleen-invigorating and dampness-resolving drugs and the improvement of immune inflammatory indexes in patients.4.2 There were significant differences in cardiac function parameters,miR-23a-3p/PI3K/AKT/mTOR signaling pathway,cytokines and immuno-inflammatory indexes between AS patients and normal patients.The parameters of AS cardiac function and immune inflammation are directly related to the abnormal activation of miR-23a-3p/PTEN/PI3K/AKT/mTOR signal pathway and the imbalance of cytokines.XFC can improve the cardiac function of AS by down-regulating miR-23a-3p,and up-regulating PTEN,to inhibit the expression of PI3K/AKT/mTOR,inhibit the activation of signal pathway,and then down-regulate pro-inflammatory cytokines IL-1β,IL-6,TNF-α,up-regulate IL-10,to improve immuno-inflammatory response,so as to improve cardiac function in patients with AS. |
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