| 1 ObjectiveBased on data mining and miR-23a-3p/PTEN/PI3K/AKT/mTOR signaling pathway,the method of invigorating the spleen and removing dampness and clearing heat was discussed from three aspects:clinical data mining,clinical in vivo observations and in vitro cell co-culture experiments to improve patients with gouty arthritis(GA)The scientific connotation of immune metabolism,inflammation and changes in heart function.2 Methods2.1 Data mining research2.1.1 Analysis of association rules between the rule of invigorating the spleen,dissipating dampness and clearing heat and the changes of heart function in patients with gouty arthritisThe inpatient medical record data processing system of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine(Anhui Provincial Hospital of Traditional Chinese Medicine)was used to collect the medical record data and oral Chinese medicine data of GA inpatients from June 2012 to June 2020.Analyze the changes in patients’clinical indicators and the prescriptions involving the nature and flavor of traditional Chinese medicines,and explore the rules of therapeutic medication.And use SPSS Modeler 14.1 to analyze the association rules between the Chinese medicine for invigorating the spleen,dissipating dampness and clearing heat and the changes of cardiac function parameters.2.1.2 Data mining study of Huangqin Qingre Chubi Capsule on improving the changes of heart function in patients with gouty arthritisAccording to whether or not to use Huangqin Qingre Chubi Capsules(HQC),patients were divided into control group and scutellaria group.Laboratory indicators between the two groups were compared,and random walking model was used to evaluate the homocysteine(Hcy),homocysteine(Hcy),HQC of GA patients.The effect of high sensitivity C-reactive protein(hs-CRP).2.2 Clinical researchAccording to the inclusion and exclusion criteria,peripheral blood samples of 30 GA patients and 30 normal healthy persons were collected,and real-time quantitative PCR(RT-qPCR)was used to detect miR-23a-3p RNA,PTEN mRNA,PI3K mRNA,AKT mRNA,The expression of mTOR mRNA was detected by enzyme-linked immunosorbent assay(ELISA)to detect the inflammatory parameters IL-6,IL-1β,IL-10 and TNF-αin 30 GA patients and 30 normal healthy people.Statistical analysis of the correlation between the relative expression of miR-23a-3p,PTEN,PI3K/AKT/mTOR genes and cardiac function parameters,VAS score,SF-36 and other integral values.2.3 In vitro experimental research2.3.1 Co-culture of CD4~+T cells and synovial cells in GA patientsThe peripheral blood of GA patients was collected and CD4~+T cells were extracted.Synovial cells were derived from Shanghai Cybertron Biotechnology Co.,Ltd.Under aseptic conditions,follow the steps to prepare MSU crystal culture solution to further stimulate synovial cells.The CD4~+T cells and synovial cells were co-cultured in a transwell chamber,with CD4~+T cells in the upper chamber and synovial cells in the lower chamber.Use CCK8,Western blot,RT-qPCR,ELISA and other detection methods.2.3.2 Co-culture of CD4~+T cells and cardiomyocytes in GA patientsThe peripheral blood of GA patients was collected and CD4~+T cells were extracted.Cardiomyocytes were obtained from the Scientific Cell Research Laboratory(San Diego,California,United States,lot number:26583).Under aseptic conditions,follow the steps to prepare MSU crystal culture solution to further stimulate cardiomyocytes.CD4~+T cells and cardiomyocytes were placed in a transwell chamber for co-culture,in which CD4~+T cells were in the upper chamber and the cardiomyocytes were in the lower chamber.Use CCK8,IF,Western blot,RT-qPCR,ELISA and other detection methods..3 Results3.1 The results of clinical data mining research on 1500 GA patients3.1.1 Changes in clinical parameters of GA patientsBefore treatment,there were 14 patients with abnormal cardiac function parameters EF,accounting for 19.44%of the total;16 patients with LADs were abnormal,accounting for 22.22%of the total;12 patients with LVDd were abnormal,accounting for 16.67%of the total;7 patients with SV were abnormal.Accounted for 9.72%of the total number;BNP had 11 abnormalities,accounting for 42.31%of the total number;Hcy had 75 abnormalities,accounting for 54.35%of the total number.Compared with after treatment,the differences in BNP,Hcy,and EF were statistically significant(p<0.05).Compared with after treatment,the differences in BNP,Hcy and EF were statistically significant(p<0.05).The differences of hs-CRP,ESR,AAG,WBC,IgA,LgG,IgM,UA,TG were statistically significant(p<0.05).3.1.2 Analysis of the frequency of traditional Chinese medicine for the treatment of GAThe prescriptions for 1500 Chinese medicine patients involved a total of 305 Chinese medicines.The 20 Chinese medicines with the highest frequency were concentrated in four categories:spleen-tonifying and dampening medicines,heat-clearing and detoxifying medicines,dispelling wind and dampness medicines,and blood stasis medicines.3.1.3 Association rules between the top 20 traditional Chinese medicines and cardiac function parametersApplication of association rule mining to detect the association between the patient’s drugs and indicators of cardiac function parameters.According to the analysis of the April module,LADs are significantly associated with Huangbai and Chuanniuxi;EF is significantly associated with Huangbai and Chuanniuxi;LVDd is related to Huangbai and Chuanniuxi and Smilax glabra are obviously related.Hcy is obviously related to plantain and Coicis Semen;BNP is obviously related to Chuanniuxi and Coicis Semen.3.2 HQC pre-treatment clinical research resultsA total of 2373 patients met the requirements of this study,including 1586(66.83%)in the control group and 787(33.16%)in the Scutellaria baicalensis group.The laboratory indicators of the two groups changed before and after treatment.The differences in TC,BNP and Hcy in the control group were statistically significant(p<0.05);the differences in TC,HDL-C,TG,BNP and Hcy in the scutellaria baicalensis group were statistically significant(p<0.01).The comparison of laboratory indicators between the two groups after treatment showed statistically significant differences in HDL-C,IgG,TC,TG and Hcy(p<0.05).Random walking model evaluates that Hcy and hs-CRP in the Scutellaria baicalensis group are significantly improved compared with the control group.3.3 Correlation between miR-23a-3p,PTEN,PI3K/AKT/mTOR and related indicators in GA patients3.3.1 Changes in other laboratory indicators before and after treatment in the two groupsThe paired t-test results showed that compared with before treatment,the expression of clinical indicators of GA patients except for HDL-C was significantly reduced,and the differences in TG,IgG,TC and Hcy were statistically significant(p<0.05).3.3.2 Changes in the expression of miR-23a-3p RNA,PTEN mRNA,PI3K mRNA,AKT mRNA and mTOR mRNA in peripheral blood of GA patientsRT-qPCR was used to detect miR-23a-3p RNA,PTEN mRNA,PI3K mRNA,AKT mRNA,mTOR mRNA in 30 GA patients and 30 normal healthy people.The results showed that the GA group(except PTEN)was significantly higher than the normal group,PTEN was lower than the normal group,the difference was statistically significant.3.3.3 Correlation analysis of miR-23a-3p/PTEN/PI3K/AKT/mTOR and cardiac function parametersAfter correlation analysis,miR-23a-3p was positively correlated with EF,SV and IVSTd(p<0.01);PTEN was negatively correlated with IVSTd(p<0.01);AKT was positively correlated with LADs(p<0.01);mTOR was correlated with IVSTd,LVDd It is positively correlated(p<0.01).3.3.4 Correlation analysis between miR-23a-3p/PTEN/PI3K/AKT/mTOR and patient perception parametersAfter correlation analysis,miR-23a-3p was positively correlated with joint pain VAS,patients’assessment of disease VAS,GH,RE,MH(p<0.01);PTEN was negatively correlated with SF,RE(p<0.01);PI3K was correlated with PF,GH,VT,SF,RE,MH are positively correlated(p<0.01);AKT is positively correlated with PF,SF,RE and(p<0.01).3.4 In vitro experimental research results3.4.1 HQC intervention CD4~+T cell and synovial cell co-culture3.4.1.1 HQC regulates the protein levels of PTEN,PI3K,AKT,p-AKT,mTOR and p-mTOR in co-cultured cellsHuangqin Qingre Chubi Capsule has the most obvious inhibitory effect on cell viability when the volume fraction of medicated serum is 20%.The levels of PI3K,AKT,and mTOR in the model group were significantly higher than those in the control and normal groups,while the PTEN level was significantly decreased.HQC reduces the protein levels of PI3K,AKT,p-AKT,mTOR and p-mTOR in co-cultured cells,and increases the protein levels of PTEN.After overexpression of miR-23a-3p,HQC can also reduce the protein levels of PI3K,AKT,p-AKT,mTOR and p-mTOR to varying degrees,and increase the protein level of PTEN.3.4.1.2 HQC regulates the levels of miR-23a-3p RNA,PTEN mRNA,PI3K mRNA,AKT mRNA and mTOR mRNA in co-cultured cellsRT-qPCR results showed that the levels of miR-23a-3p RNA,PI3K mRNA,AKT mRNA and mTOR mRNA in the model group were significantly higher than those in the control and normal groups,and the PTEN mRNA level was significantly decreased(p<0.01).HQC reduced the levels of miR-23a-3p RNA,PI3K mRNA,AKT mRNA and mTOR mRNA in co-cultured cells,and increased the level of PTEN mRNA(p<0.01).After overexpression of miR-23a-3p,HQC can also reduce the levels of miR-23a-3p RNA,PI3K mRNA,AKT mRNA and mTOR mRNA to varying degrees,and increase the level of PTEN mRNA(p<0.01).3.4.1.3 HQC reduces cytokine levels in co-cultured cellsELISA results showed that the levels of IL-1β,IL-6 and TNF-αin the model group were significantly higher than those in the control group and the normal group,and the IL-10 levels were significantly decreased(p<0.01).Under the intervention of HQC,IL-1β,IL-6 and TNF-αdecreased significantly,while the expression of IL-10 increased significantly(p<0.01).After overexpression of miR-23a-3p,HQC can also reduce IL-1β,IL-6 and TNF-αto varying degrees,and increase IL-10 levels(p<0.01).3.4.2 HQC intervention CD4~+T cell and cardiomyocyte co-culture3.4.2.1 HQC improves myocardial specific markers of cardiomyocytesDifferent volume fractions(5%,10%,20%)of Huangqin Qingre Chubi Capsules containing serum after 24 hours,48 hours,72 hours of effect on cardiomyocytes,compared with the control group,the volume fraction of Huangqin Qingre Chubi Capsules containing serum Its inhibitory effect on cell viability is most obvious when it is 20%.The blue fluorescence reflects the cell nucleus,and the green dotted or massive fluorescent spots are BNP,Connexin 43,Tn I,and Tn T.From Merge,it can be seen that BNP,Connexin 43,Tn I,and Tn T are evenly distributed in the nucleus of cardiomyocytes.Among them,it can be seen that the fluorescence of the BNP,Connexin 43,Tn I,and Tn T fluorescent spots of cardiomyocytes in group F has the strongest increase in fluorescence,and the distribution in the cardiomyocytes becomes disordered.The cardiomyocytes of group A and group B had the phenomenon of reduced or missing fluorescence.The fluorescence of group D and group G was significantly reduced compared with group C and group F respectively.3.4.2.2 HQC regulates the protein levels of PTEN,PI3K,AKT,p-AKT,mTOR and p-mTOR in co-cultured cellsHuangqin Qingre Chubi Capsule has the most obvious inhibitory effect on cell viability when the volume fraction of medicated serum is 20%.HQC reduces the protein levels of PI3K,AKT,p-AKT,mTOR,and p-mTOR in co-cultured cells,and increases the protein levels of PTEN.After overexpression of miR-23a-3p,HQC can also reduce the protein levels of PI3K,AKT,p-AKT,mTOR and p-mTOR to varying degrees,and increase the protein level of PTEN.3.4.2.3 HQC regulates the levels of miR-23a-3p RNA,PTEN mRNA,PI3K mRNA,AKT mRNA and mTOR mRNA in co-cultured cellsRT-qPCR results showed that the levels of miR-23a-3p RNA,PI3K mRNA,AKT mRNA and mTOR mRNA in the model group were significantly higher than those in the control and normal groups,and the PTEN mRNA level was significantly decreased(p<0.01).HQC reduced the levels of miR-23a-3p RNA,PI3K mRNA,AKT mRNA and mTOR mRNA in co-cultured cells,and increased the level of PTEN mRNA(p<0.01).After overexpression of miR-23a-3p,HQC can also reduce the levels of miR-23a-3p RNA,PI3K mRNA,AKT mRNA and mTOR mRNA to varying degrees,and increase the level of PTEN mRNA(p<0.01).3.4.2.4 HQC reduces cytokine levels in co-cultured cellsELISA results showed that the levels of IL-1β,IL-6 and TNF-αin the model group were significantly higher than those in the control group and the normal group,and the IL-10 levels were significantly decreased(p<0.01).Under the intervention of HQC,IL-1β,IL-6 and TNF-αdecreased significantly,while the expression of IL-10 increased significantly(p<0.01).After overexpression of miR-23a-3p,HQC can also reduce IL-1β,IL-6,TNF-αand increase IL-10 levels(p<0.01).4 Conclusion4.1 GA patients have disorders of immune,metabolic,and inflammatory reactions and heart function changes.The method of strengthening the spleen and removing dampness and clearing heat has a good effect on it.4.2 While improving the clinical symptoms of GA patients,HQC can also significantly improve the state of cardiac function changes in GA patients,and is superior to the control group in terms of clinical efficacy and improvement of cardiac function changes.4.3 There are significant differences between GA patients’cytokines,cardiac function parameters,miR-23a-3p/PTEN/PI3K/AKT/mTOR signaling pathway and normal healthy people.The changes of abnormal cardiac function parameters in GA patients are directly related to the immune inflammatory metabolic response,cytokine imbalance,and abnormal activation of miR-23a-3p/PTEN/PI3K/AKT/mTOR signaling pathway.4.4 The mechanism by which HQC improves the inflammatory response state and heart function changes in GA patients is as follows:4.4.1HQC controls the inflammatory response and restores the balance of the body by up-regulating anti-inflammatory cytokines(such as IL-10)and down-regulating pro-inflammatory cytokines(such as IL-1β,IL-6,TNF-α),thereby improving clinical symptoms.4.4.2HQC stimulates the down-regulation of miR-23a-3p,then regulates the up-regulation of PTEN,down-regulates the expression of PI3K/AKT/mTOR signaling pathway,and improves the inflammatory state of patients,so as to achieve the effect of improving the state of changes in joint inflammation.4.4.3HQC stimulates the down-regulation of miR-23a-3p,then regulates the up-regulation of PTEN,down-regulates the expression of PI3K/AKT/mTOR signaling pathway,and improves the inflammatory state of cardiomyocytes,so as to achieve the effect of improving the state of cardiac function changes in patients. |
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