新风胶囊对强直性脊柱炎患者疗效及血清免疫球蛋白亚型、外周血淋巴细胞自噬的影响
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摘要
目的观察新风胶囊(XFC)对强直性脊柱炎(AS)患者的症状体征、血清免疫球蛋白、外周血淋巴细胞自噬蛋白、自噬基因的影响,探讨其作用机制。方法将59例AS患者按随机数字表法分为2组,治疗组(39例)和对照组(20例),治疗组给予XFC治疗,每次3粒,每日3次;对照组给予柳氮磺胺吡啶(SASP)治疗,每次4片,每日2次。治疗3个月。统计Bath强直性脊柱炎疾病活动指数(BASDAI)及Bath强直性脊柱炎功能指数(BASFI);采用ELISA法检测两组血清免疫球蛋白(Ig G1、Ig G2、Ig G3、Ig G4、Ig A、SIg A、Ig M)水平,采用Western blot检测两组的淋巴细胞自噬蛋白Beclin1、LC3-Ⅱ、PI3K、Akt、m TOR水平,采用聚合酶链式反应(Polymerase Chain Reaction,PCR)方法检查血清自噬相关基因Atg1、Atg5、Atg12、Atg13、Atg17表达改变。采用Spearman相关分析方法比较AS患者免疫球蛋白亚型与细胞自噬基因的相关性。结果与治疗前比较,治疗组治疗后BASDAI、Ig G1、Ig G3、Ig A水平下降(P<0.01),PI3K、Akt、m TOR蛋白表达下降(P<0.01),ATG1、ATG12、ATG13、ATG17 mRNA表达下降,ATG5 mRNA表达升高(P<0.01);对照组BASDAI、Ig G1、Ig A水平下降(P<0.05,P<0.01),PI3K、Akt、m TOR蛋白表达下降(P<0.05),ATG1、ATG13mRNA表达下降(P<0.05,P<0.01)。与对照组比较,治疗组BASDAI、Ig G1、Ig A水平下降(P<0.05),PI3K、Akt、m TOR蛋白表达水平下降(P<0.01),ATG12、ATG17 mRNA表达下降,ATG5 mRNA表达升高(P<0.01)。相关分析结果显示,AS患者Ig G1、Ig G2、Ig G3、Ig A、SIg A、Ig M与ATG17呈负相关,Ig G4与ATG17呈正相关(P<0.05,P<0.01)。结论 XFC提高AS患者临床疗效,其增强AS患者细胞自噬的机制可能为作用于PI3K/Akt/m TOR信号,影响自噬基因和自噬蛋白的表达,参与调节B淋巴细胞的增殖和分化,加强体液免疫。
Objective To observe the effect of Xinfeng Capsule( XFC) on ankylosing spondylitis( AS) patients' symptoms and signs,serum immunoglobulin levels,peripheral blood lymphocyte autophagy protein,autophagy gene,and to explore its mechanism. Methods Totally 59 AS patients were assigned to the treatment group( 39 cases) and the control group( 20 cases) according to random digit table. Patients in the treatment group received XFC,0. 5 g each pill,three pills each time,3 times per day,while those in the control group received sulfasalazine( SASP),0. 25 g per tablet,4 tablets each time,twice per day. Three months consisted of one therapeutic course. Bath Ankylosing Spondylitis Disease Activity Index( BASDAI) and Bath Ankylosing Spondylitis Functional Index( BASFI) were statistically calculated. Serum immunoglobulins( Ig G1,Ig G2,Ig G3,Ig G4,Ig A,SIg A,and Ig M) were detected using ELISA. Changes of Beclin1,LC3-Ⅱ,phosphatidylinositol 3-kinase( PI3K),Akt,the mammalian target of rapamycin( m TOR) were detected using Western blot. Serum autophagy related genes such as Atg1,Atg5,Atg12,Atg13,and Atg17 were detected using the polymerase chain reaction( PCR). The correlation between immunoglobulin subtypes and autophagy gene in AS patients using Spearman correlation. Results Compared with before treatment,BASDAI,Ig G1,Ig G3,and Ig A decreased(P < 0. 01); PI3 K,Akt,and m TOR protein expressions decreased(P < 0. 01); ATG1,ATG12,ATG13,and ATG17 mRNA expressions decreased,ATG5 mRNA expression increased(P <0. 01) in the treatment group. But BASDAI,Ig G1,and Ig A levels decreased(P < 0. 05,P < 0. 01); PI3 K,Akt,and m TOR protein expressions decreased(P < 0. 05); ATG1 and ATG13 mRNA expressions decreased(P < 0. 05,P < 0. 01) in the control group. Compared with the control group,BASDAI,Ig G1,and Ig A levels decreased(P < 0. 05); PI3 K,Akt,m TOR protein expressions decreased(P < 0. 01); ATG12 and ATG17 mRNA expression decreased,ATG5 mRNA expression increased(P <0. 01) in the XFC group. Correlation analysis showed AS patients' Ig G1,Ig G2,Ig G3,Ig A,SIg A,Ig M had negative correlation with ATG17; Ig G4 and ATG17 were positively correlated(P < 0. 05,P < 0. 01). Conclusion XFC could elevate clinical efficacy of AS patients and enhance their autophagy,which might be achieved by acting on PI3 K / Akt / m TOR signal,affecting autophagy gene and autophagy protein expression,taking part in the regulation of proliferation and differentiation of lymphocyte B,and strengthen humoral immunity.
Objective To observe the effect of Xinfeng Capsule( XFC) on ankylosing spondylitis( AS) patients' symptoms and signs,serum immunoglobulin levels,peripheral blood lymphocyte autophagy protein,autophagy gene,and to explore its mechanism. Methods Totally 59 AS patients were assigned to the treatment group( 39 cases) and the control group( 20 cases) according to random digit table. Patients in the treatment group received XFC,0. 5 g each pill,three pills each time,3 times per day,while those in the control group received sulfasalazine( SASP),0. 25 g per tablet,4 tablets each time,twice per day. Three months consisted of one therapeutic course. Bath Ankylosing Spondylitis Disease Activity Index( BASDAI) and Bath Ankylosing Spondylitis Functional Index( BASFI) were statistically calculated. Serum immunoglobulins( Ig G1,Ig G2,Ig G3,Ig G4,Ig A,SIg A,and Ig M) were detected using ELISA. Changes of Beclin1,LC3-Ⅱ,phosphatidylinositol 3-kinase( PI3K),Akt,the mammalian target of rapamycin( m TOR) were detected using Western blot. Serum autophagy related genes such as Atg1,Atg5,Atg12,Atg13,and Atg17 were detected using the polymerase chain reaction( PCR). The correlation between immunoglobulin subtypes and autophagy gene in AS patients using Spearman correlation. Results Compared with before treatment,BASDAI,Ig G1,Ig G3,and Ig A decreased(P < 0. 01); PI3 K,Akt,and m TOR protein expressions decreased(P < 0. 01); ATG1,ATG12,ATG13,and ATG17 mRNA expressions decreased,ATG5 mRNA expression increased(P <0. 01) in the treatment group. But BASDAI,Ig G1,and Ig A levels decreased(P < 0. 05,P < 0. 01); PI3 K,Akt,and m TOR protein expressions decreased(P < 0. 05); ATG1 and ATG13 mRNA expressions decreased(P < 0. 05,P < 0. 01) in the control group. Compared with the control group,BASDAI,Ig G1,and Ig A levels decreased(P < 0. 05); PI3 K,Akt,m TOR protein expressions decreased(P < 0. 01); ATG12 and ATG17 mRNA expression decreased,ATG5 mRNA expression increased(P <0. 01) in the XFC group. Correlation analysis showed AS patients' Ig G1,Ig G2,Ig G3,Ig A,SIg A,Ig M had negative correlation with ATG17; Ig G4 and ATG17 were positively correlated(P < 0. 05,P < 0. 01). Conclusion XFC could elevate clinical efficacy of AS patients and enhance their autophagy,which might be achieved by acting on PI3 K / Akt / m TOR signal,affecting autophagy gene and autophagy protein expression,taking part in the regulation of proliferation and differentiation of lymphocyte B,and strengthen humoral immunity.
引文
[1]汪四海,刘健,杨佳,等.102例强直性脊柱炎患者中医证候回顾性研究[J].中医药临床杂志,2011,23(6):522-524.
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[15]孟明,梁红格,方皓,等.PI3K/Akt/m TOR通路与自噬在类风湿关节炎滑膜细胞增生中的意义[J].医学研究与教育,2013,30(5):69-74.
[16]叶青.自噬的分子机制与病理生理意义[J].国际病理科学与临床杂志,2007,27(3):358-362.
[17]李欣志.缺血/再灌注过程中心肌细胞自噬研究进展[J].中国药理学通报,2008,24(2):704-707.
[18]Reedquist KA,Ludikhuize J,Tak PP.Phosphoinositide 3-kinase signaling and Fox O transcription factors in rheumatoid arthritis[J].Biochem Soc Transactions,2006,34(Pt 5):727-730.
[19]Mathew R,Karantza-Wadsworth V,White E.Role of autophagy in cancer[J].Nat Rev Cancer,2007,7(8):961-967.
[20]Lee BY,Han JA,Im JS,et al.Senescence-associatedβ-galactosidase is lysosomalβ-galactosidase[J].Aging Cell,2006,5(2):187-195.
[21]Yokoi T.Troglitazone.Handbook of experimental pharmacology[M].London:Springer,2011:419-435.
[22]陈洪菊,屈艺,母得志.m TOR信号通路的生物学功能[J].生命的化,2010,30(4):555-560.
[23]钱帅伟,漆正堂,丁树哲.哺乳动物雷帕霉素靶蛋白通路与细胞自噬[J].生命科学,2011,23(8):730-734.
[24]王桂珍,刘健,曹云祥,等.新风胶囊联合中药熏蒸治疗强直性脊柱炎临床观察[J].中国临床保健杂志,2014,26(6):581-583.
[25]黄传兵,谌曦,汪元,等.新风胶囊联合来氟米特治疗强直性脊柱炎临床研究[J].中医药临床杂志,2011,5,23(5):405-406.
[26]Liu Y,Zhang W,Wang XJ.Antitumor effect of Kanglaite Injection in human pancreatic cancer xenografts[J].BMC Complement Alternat Med,2014,228(14):2-6.
[27]Tomson AW,Turnquist HR,Raimondi G.Immunoregulatory functions of m TOR inhibition[J].Nat Rev Immunol,2009,9(11):324-337.
[28]Roczynska B,Kaur S,Platanias LC.Growth suppressive cytokines and the AKT/m TOR pathway[J].Cytokine,2009,48(1-2):138-143.
[29]Bnath I,Roundy KM,Weis JJ,et al.Analysis of the regulatory role of BAFF in controlling the expression of CD21 and CD23[J].Mol Immunol,2007,44(9):2388-2399.
[2]赵驻军,颉玉欣,汤慧华,等.强直性脊柱炎的免疫学指标评价[J].颈腰痛杂志,1998,19(4):283-284.
[3]Kim JE,Chen J.Cytoplasmic nuclear shuttling of FKBP12-rapamycin-associated protein is involved in rapamycin 2 sensitive signaling and translation initiation[J].Proc Natl Acad Sci USA,2000,97(4):14340-14345.
[4]刘健,万磊,黄传兵,等.类风湿关节炎患者肺功能变化及中药新风胶囊干预研究[J].中国临床保健杂志,2014,2,17(1):102-106.
[5]Mcpherron AC,Lawler AM,Lee SJ.Regulation of skeletal muscle mass in mice by a new TGFbeta superfamily member[J].Nature,1997,387(4):83-90.
[6]陆再英主编.内科学教材-强直性脊柱炎[M].第7版.2008:867-868.
[7]中华医学会风湿病学分会.强直性脊柱炎诊断及治疗指南[S].中华风湿病学杂志,2010,14(8):557-559.
[8]黄泽伟,黄玉香.800例免疫球蛋白与补体检测结果分析[J].吉林医学,2012,33(6):1262-1263.
[9]李培培.BAFFBAFF-R介导PI3K-Akt-m TOR信号通路上调胶原性关节炎大鼠B淋巴细胞的功能及芍药苷的调节作用[D].合肥:安徽医科大学,2012.
[10]王和峰.PI3K/Akt/m T0R信号通路在巨噬细胞自噬及动脉粥样硬化斑块不稳定中的作用[D].济南:山东大学,2013.
[11]陈惠黎.生物大分子的结构和功能[M].上海:上海医科大学出版社,1999:1.
[12]刘大舟.免疫学基础[M].北京:中国协和医科大学出版社,2009:225.
[13]Bohensky J,Shapiro IM,Leshinsky S,et al.HIF-1 regulation of chondrocyte apoptosis:induction of the autophagic pathway[J].Autophagy,2007,3(3):207-214.
[14]Settembre C,Artea Solis E,Mc Kee MD,et al.Proteoglycan defalcation determines the deficiency of chondrocyte autophagy and the extent of FGF signaling during endochondral ossification[J].Genes Dev,2008(19):2645-2650.
[15]孟明,梁红格,方皓,等.PI3K/Akt/m TOR通路与自噬在类风湿关节炎滑膜细胞增生中的意义[J].医学研究与教育,2013,30(5):69-74.
[16]叶青.自噬的分子机制与病理生理意义[J].国际病理科学与临床杂志,2007,27(3):358-362.
[17]李欣志.缺血/再灌注过程中心肌细胞自噬研究进展[J].中国药理学通报,2008,24(2):704-707.
[18]Reedquist KA,Ludikhuize J,Tak PP.Phosphoinositide 3-kinase signaling and Fox O transcription factors in rheumatoid arthritis[J].Biochem Soc Transactions,2006,34(Pt 5):727-730.
[19]Mathew R,Karantza-Wadsworth V,White E.Role of autophagy in cancer[J].Nat Rev Cancer,2007,7(8):961-967.
[20]Lee BY,Han JA,Im JS,et al.Senescence-associatedβ-galactosidase is lysosomalβ-galactosidase[J].Aging Cell,2006,5(2):187-195.
[21]Yokoi T.Troglitazone.Handbook of experimental pharmacology[M].London:Springer,2011:419-435.
[22]陈洪菊,屈艺,母得志.m TOR信号通路的生物学功能[J].生命的化,2010,30(4):555-560.
[23]钱帅伟,漆正堂,丁树哲.哺乳动物雷帕霉素靶蛋白通路与细胞自噬[J].生命科学,2011,23(8):730-734.
[24]王桂珍,刘健,曹云祥,等.新风胶囊联合中药熏蒸治疗强直性脊柱炎临床观察[J].中国临床保健杂志,2014,26(6):581-583.
[25]黄传兵,谌曦,汪元,等.新风胶囊联合来氟米特治疗强直性脊柱炎临床研究[J].中医药临床杂志,2011,5,23(5):405-406.
[26]Liu Y,Zhang W,Wang XJ.Antitumor effect of Kanglaite Injection in human pancreatic cancer xenografts[J].BMC Complement Alternat Med,2014,228(14):2-6.
[27]Tomson AW,Turnquist HR,Raimondi G.Immunoregulatory functions of m TOR inhibition[J].Nat Rev Immunol,2009,9(11):324-337.
[28]Roczynska B,Kaur S,Platanias LC.Growth suppressive cytokines and the AKT/m TOR pathway[J].Cytokine,2009,48(1-2):138-143.
[29]Bnath I,Roundy KM,Weis JJ,et al.Analysis of the regulatory role of BAFF in controlling the expression of CD21 and CD23[J].Mol Immunol,2007,44(9):2388-2399.