Study Of The Mechanism Of Systemic Lupus Erythematosus Thrombocytopenia By Qihuang Jianpi Zishen Granule Based On LncRNA GAS5 Regulating PI3K/AKT/Bax Pathway

1 ObjectiveTo observe the effect of Qihuang Jianpi Zishen Granule(QJZG)on clinical efficacy and platelet-related indexes in patients with systemic lupus erythematosus(SLE),and explore its possible mechanism.Based on the regulation of PI3K/AKT/Bax cell apoptosis pathway by lncRNA GAS5,the possible mechanism of QJZG in improving SLE thrombocytopenia was studied in animal experiments.2 Methods2.1 Clinical study ⅠSixty-four female patients with SLE(35 cases in the outpatient clinic and 29 cases in the inpatient department)from October 2020 to October 2022 at the Department of Rheumatology,The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine were selected as the study subjects.Another 30 cases of healthy women from the physical examination center were selected as the healthy group.The expression levels of platelet-related indexes,immunoinflammatory indexes,antibody levels,and lncRNA GAS5/PI3K/AKT/Bax signaling axis-related factors were detected.PLT was correlated with other indexes using Spearman or Pearson correlation analysis.2.2 Clinical study ⅡSixty-four selected female patients with SLE were divided into a control group and an observation group according to the random number table method,with 32 cases in each group.In the control group,prednisone acetate tablets and hydroxychloroquine sulfate tablets were administered orally,while in the observation group,Qihuang Jianpi Zishen Granule was added on the basis of the control group.The duration of treatment in both groups was 12 weeks.The platelet count(PLT),platelet pressure product(PCT),mean platelet volume(MPV),thrombopoietin(TPO),anti-dsDNA antibody,renal function indexes[blood urea nitrogen(BUN),blood creatinine(Scr),24 h urine protein quantification(24hPro)],SLE disease activity(SLEDAI),inflammation indexes[blood sedimentation(ESR),C-reactive protein(CRP)],TCM symptom score,immunoglobulin G(IgG),immunoglobulin A(IgA),immunoglobulin M(IgM),complement 3(C3),complement 4(C4),serum inflammatory factors[interleukin 10(IL-10),interleukin 17(IL-17),interleukin 23(IL-23),interferon gamma(IFN-γ)],platelet surface antibodies(PAIgG,PAIgA,PAIgM)changes.2.3 Experimental studyTwenty-four MRL/lpr lupus mice were randomly divided into model group(MC),Qihuang Jianpi Zhishen granule group(QJZG),prednisone acetate group(PRED)and PI3K inhibitor group(LY294002),with 6 mice in each group.Six C57BL/6 mice were used as blank control group(NC).NC group and MC group were given l0ml·kg-1·d-1 normal saline intragastric administration,once a day;QJZG group:12.6g·kg-1·d-1 QJZG solution was given by gavage,once a day;PRED group:5mg·kg-1·d-1 prednisone acetate solution was given intragastric administration,once a day;LY294002 group:10ml·kg-1·d-1 normal saline was injected into the stomach,and 1mg/kg LY294002 solution was injected intraperitoneally,twice/w.Each group received continuous medication for 8w.The levels of PLT,anti-DS-DNA antibody,ANA antibody and C3 in peripheral blood of mice were detected.mRNA expression levels of GAS5,(PI3K,AKT,Bax,Bcl-2)in platelet suspension were detected by RT-qPCR.The expression levels of PI3K/AKT/Bax axis-related proteins in platelet suspension were detected by Western Blot.Platelet cell apoptosis rate was determined by flow cytometry.3 Results3.1 Results of clinical study3.1.1 Comparison of PLT,ESR,CRP,IgG and C3 between SLE group and healthy groupCompared with the healthy group,PLT and C3 in SLE group were significantly decreased(P<0.01 or P<0.05),while ESR,CRP and IgG levels were significantly increased(P<0.01).3.1.2 Comparison of GAS5 expression and PI3K/AKT/Bax pathway protein in platelet suspension between SLE group and healthy groupCompared with healthy group,GAS5 expression level in platelet suspension of SLE group was significantly decreased(P<0.01),and protein levels of PI3K,AKT and Bax were significantly increased(P<0.01).3.1.3 Correlation analysis of SLEDAI,ESR,CRP,TPO and PLT in SLE groupIn SLE group,PLT was negatively correlated with SLEDAI,ESR,CRP and TPO levels(P<0.01).3.1.4 Correlation analysis of IgG,IgA,IgM,C3,C4,anti-DSDNA antibody and PLT in SLE groupIn SLE group,PLT was negatively correlated with IgG,IgA,IgM and dsDNA antibodies(P<0.05 or P<0.01).PLT was positively correlated with C3 and C4 levels(P<0.05).3.1.5 Correlation analysis of GAS5,PI3K,AKT,Bax,Bcl-2,Caspase-3 and PLT in SLE groupGAS5 was positively correlated with PLT in SLE group(P<0.05),PI3K and AKT were not correlated with PLT(P>0.05),Bax and Caspase-3 were negatively correlated with PLT(P<0.01 or P<0.05),and Bcl-2 was positively correlated with PLT(P<0.05).3.2 Results of clinical study Ⅱ3.2.1 Comparison of clinical efficacy between the two groupsAfter treatment,the total effective rate of the observation group(87.50%)was significantly higher than that of the control group(68.79%)(Z=-2.288,P=0.022).The clinical effect of the observation group was better than that of the control group.3.2.2 Comparison of PLT,PCT and MPV before and after treatment between the two groupsAfter treatment,PLT,PCT and MPV were significantly higher than before treatment(P<0.01).The PLT of observation group after treatment was significantly higher than that of control group(P<0.01).PCT and MPV were not significantly increased(P>0.05).3.2.3 Comparison of TPO and anti-DSDNA antibody before and after treatment between the two groupsAfter treatment,TPO and anti-DSDNA antibody were decreased significantly in both groups(P<0.01).TPO and anti-DSDNA antibody in the observation group were significantly lower than those in the control group after treatment(P<0.05 or P<0.01).3.2.4 Comparison of BUN,Scr and 24hPro before and after treatment between the two groupsAfter treatment,BUN,Scr and 24hPro were significantly decreased in both groups after treatment(P<0.01),and BUN,Scr and 24hPro in the observation group were significantly lower than those in the control group after treatment(P<0.05 or P<0.01).3.2.5 Comparison of SLEDAI,ESR and CRP before and after treatment between the two groupsAfter treatment,SLEDAI,ESR and CRP in both groups were significantly lower than before treatment(P<0.01),and after treatment,SLEDAI,ESR and CRP in the observation group were significantly lower than that in the control group(P<0.01).3.2.6 Comparison of TCM symptom scores before and after treatment between the two groupsAfter treatment,the scores of burnout,fatigue,erythema and alopecia were significantly decreased(P<0.05 or P<0.01).After treatment,the scores of burnout,fatigue,alopecia,waist and knee tenderness,fear of cold and preference for warm,diet and deficiency in the observation group were significantly lower than those in the control group(P<0.05 or P<0.01).3.2.7 Comparison of IgG,IgA,IgM,C3 and C4 levels between the two groups before and after treatmentAfter treatment,IgG,IgA and IgM were decreased significantly(P<0.01),while C3 and C4 were increased significantly(P<0.01).After treatment,IgG and IgA in observation group were significantly lower than those in control group(P<0.05 or P<0.01),and C3 was significantly higher than that in control group(P<0.01).3.2.8 Comparison of serum inflammatory factors before and after treatment between the two groupsAfter treatment,IL-10 was significantly increased(P<0.01),while IL-17,IL-23 and IFN-γ were significantly decreased(P<0.01).IL-10 in observation group was significantly higher than that in control group after treatment(P<0.05).IL-17,IL-23 and IFN-γ were significantly lower than those in the control group(P<0.05 or P<0.01).3.2.9 Comparison of PAIgG,PAIgA and PAIgM before and after treatment between the two groupsAfter treatment,PAIgG and PAIgA were significantly decreased in both groups(P<0.01),and PAIgG,PAIgA and PAIgM in the observation group were significantly lower than before treatment(P<0.01).3.3 Experimental research results3.3.1 Effects of QJZG on PLT,anti-dsDNA,ANA and C3 in peripheral blood of MRL/lpr Lupus miceCompared with blank group,PLT and C3 levels in model group were significantly decreased(P<0.01).The levels of anti-dsDNA and ANA were significantly increased(P<0.01).Compared with model group,PLT and C3 levels in QJZG,PRED and LY294002 groups were significantly increased(P<0.01 or P<0.05),while anti-dsDNA and ANA levels were significantly decreased(P<0.05).3.3.2 Effects of QJZG on mRNA expression levels of GAS5,(PI3K,AKT,Bax,Bcl-2)in MRL/lpr Lupus miceCompared with the blank group,the relative expression level of GAS5 in the model group was significantly decreased(P<0.01),the relative expression level of(PI3K,AKT,Bax)mRNA was significantly increased(P<0.01),and the relative expression level of Bcl-2 mRNA was significantly decreased(P<0.01).Compared with the model group,the relative expression level of GAS5 in the QJZG group,PRED group,and LY294002 group was significantly increased(P<0.01),and the increase was more obvious in the QJZG group.The mRNA relative expression level of PI3K,AKT,and Bax in the QJZG group,PRED group,and LY294002 group was significantly decreased(P<0.01),while the mRNA relative expression level of Bcl-2 was significantly increased(P<0.01).3.3.3 Effects of QJZG on PI3K/AKT/Bax pathway related proteins in MRL/lpr Lupus miceCompared with the blank group,the protein levels of PI3K and AKT in the model group were not significantly different(P>0.05),while the protein levels of p-PI3K,p-Akt,Bax and Caspase-3 were significantly increased(P<0.01),while the protein level of Bcl-2 was significantly decreased(P<0.01).Compared with the model group,the protein levels of PI3K and AKT in the QJZG group,PRED group,and LY294002 group were not significantly changed(P>0.05),the protein levels of p-PI3K,p-Akt,Bax and Caspase-3 were significantly decreased(P<0.01),and the protein levels of Bcl-2 were significantly increased(P<0.01).3.3.4 Effect of QJZG on the apoptosis rate of platelet cells in MRL/lpr lupus miceCompared with the blank group,the apoptosis rate of platelet cells in the model group was significantly increased(P<0.01).Compared with the model group,the apoptosis rate of platelet cells in the QJZG group,PRED group,and LY294002 group was significantly decreased(P<0.01).The apoptosis of platelet cells in the PRED group was the most obvious.4 Conclusion4.1 In SLE thrombocytopenia patients,PLT was negatively correlated with disease activity and immunoinflammatory markers.PLT was significantly correlated with GAS 5 expression and PI3 K/AKT/B ax apopto sis-related proteins,suggesting that platelet apoptosis in SLE may be involved in the mechanism of thrombocytopenia.4.2 After the intervention of SLE thrombocytopenia,QJZG can significantly increase PLT,reduce inflammation in vivo,regulate immune function,improve renal function and platelet surface antibodies,and the clinical efficacy is higher than that of the control group,with high safety.4.3 QJZG can significantly increase C3 and PLT,reduce anti-dsDNA and ANA levels,effectively improve immune function and increase PLT in lupus mice.QJZG may regulate the signaling axis of PI3K/AKT/B ax apoptotic pathway through lncRNA GAS5,down-regulate pro-apoptotic factor Bax and Caspase-3,up-regulate anti-apoptotic factor Bcl-2,inhibit platelet cell apoptosis,and thus improve thrombocytopenia.