益气养阴祛瘀方含药血清对干燥综合征人颌下腺细胞AQP5和M3R表达的影响
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摘要
目的观察益气养阴祛瘀方含药血清对干燥综合征(■;gren′s syndrome, SS)人颌下腺细胞(human submandibular gland cells, HSG cells)中水通道蛋白5(aquaporin protein-5, AQP5)和毒蕈碱样胆碱能受体3(type 3 muscarinic acetylcholine receptors, M3R)表达的影响。方法制备益气养阴祛瘀方含药血清,将对数生长期的HSG细胞随机分为空白组(RPMI 1640培养液)、空白血清组(10%的空白血清+RPMI 1640培养液)、含药血清组(10%的益气养阴祛瘀方含药血清+RPMI 1640培养液)、IFN-γ组(IFN-γ+RPMI 1640培养液)、IFN-γ+IFN-γ拮抗剂组(IFN-γ+IFN-γ拮抗剂+RPMI 1640培养液)、IFN-γ+含药血清组(IFN-γ+10%益气养阴祛瘀方含药血清+RPMI 1640培养液),共6组。培养24 h后,Western blot法检测HSG细胞中AQP5和M3R蛋白表达;RT-PCR法检测HSG细胞AQP5 mRNA和M3R mRNA表达;免疫荧光法观察HSG细胞AQP5蛋白通道表达变化。结果与空白血清组比较,含药血清组AQP5、M3R蛋白及AQP5 mRNA和M3R mRNA表达升高(P<0.05)。与IFN-γ组比较,空白组、空白血清组、含药血清组、IFN-γ+IFN-γ拮抗剂组AQP5、M3R蛋白及mRNA表达均升高(P<0.05)。结论益气养阴祛瘀方具有与IFN-γ拮抗剂类似功效,能上调主管水运的AQP5、M3R表达,增加唾液量的分泌。
Objective To observe effect of Yiqi Yangyin Quyu Recipe(YYQR) containing serum on aquaporin protein-5(AQP5) and type 3 muscarinic acetylcholine receptors(M3R) of human submandibular gland cells with ■;gren′s syndrome(SS). Methods YYQR containing serum was prepared. HSG cells in logarithmic growth phase were randomly divided into the blank group(RPMI 1640 medium), the blank serum group(10% blank serum + RPMI 1640 medium), the medicated serum group(10%YYQR containing serum + RPMI 1640 medium), the IFN-γ group(IFN-γ + RPMI 1640 medium), the IFN-γ + IFN-γ antagonist group(IFN-γ + IFN-γ antagonist +RPMI 1640 medium), and the IFN-γ + medicated serum group(IFN-γ + 10%YYQR containing serum + RPMI 1640 medium). After 24 hours of culture, protein and mRNA were extracted. Protein expressions of AQP5 and M3R in HSG cells were detected by Western blot. AQP5 and M3R mRNA expressions in HSG cells were detected by RT-PCR. Protein channel expression changes of AQP5 in HSG cells was observed by laser confocal microscope. Results Compared with the blank serum group, protein and mRNA expressions of AQP5 and M3R in the medicated serum group increased(P<0.05). Compared with the IFN-γ group, protein and mRNA expressions of AQP5 and M3R in the remaining five groups increased(P<0.05). Conclusion YYQR has similar efficacy with IFN-γ antagonist, it can increase the AQP5 and M3R expressions which were in charge of water transportation, and increase the secretion of saliva.
Objective To observe effect of Yiqi Yangyin Quyu Recipe(YYQR) containing serum on aquaporin protein-5(AQP5) and type 3 muscarinic acetylcholine receptors(M3R) of human submandibular gland cells with ■;gren′s syndrome(SS). Methods YYQR containing serum was prepared. HSG cells in logarithmic growth phase were randomly divided into the blank group(RPMI 1640 medium), the blank serum group(10% blank serum + RPMI 1640 medium), the medicated serum group(10%YYQR containing serum + RPMI 1640 medium), the IFN-γ group(IFN-γ + RPMI 1640 medium), the IFN-γ + IFN-γ antagonist group(IFN-γ + IFN-γ antagonist +RPMI 1640 medium), and the IFN-γ + medicated serum group(IFN-γ + 10%YYQR containing serum + RPMI 1640 medium). After 24 hours of culture, protein and mRNA were extracted. Protein expressions of AQP5 and M3R in HSG cells were detected by Western blot. AQP5 and M3R mRNA expressions in HSG cells were detected by RT-PCR. Protein channel expression changes of AQP5 in HSG cells was observed by laser confocal microscope. Results Compared with the blank serum group, protein and mRNA expressions of AQP5 and M3R in the medicated serum group increased(P<0.05). Compared with the IFN-γ group, protein and mRNA expressions of AQP5 and M3R in the remaining five groups increased(P<0.05). Conclusion YYQR has similar efficacy with IFN-γ antagonist, it can increase the AQP5 and M3R expressions which were in charge of water transportation, and increase the secretion of saliva.
引文
[1] Baldini C, Talarico R, Tzioufas AG, et al. Classification criteria for Sj?gren′s syndrome: A critical review[J]. J Autoimmun, 2012, 39(1-2): 9-14.
[2] Mavragani CP, Nezos A, Moutsopoulos HM. New advances in the classification, pathogenesis and treatment of Sj?gren′s syndrome[J]. Curr Opin Rheumatol, 2013, 25(5): 623-629.
[3] 王新昌, 谢志军, 温成平, 等. 益气养阴祛瘀法治疗原发性干燥综合征的疗效观察[J]. 中国基层医药, 2009, 16(1): 92-93.
[4] 王新昌, 谢志军, 温成平, 等. 益气养阴祛瘀药对干燥综合征患者性激素水平影响的研究[J]. 浙江中医药大学学报, 2009, 33(1): 48-49.
[5] 王新昌, 范永升, 冯健, 等. 益气养阴祛瘀中药对干燥综合征自发性模型非肥胖糖尿病小鼠的治疗作用及机制[J]. 浙江中医药大学学报, 2012, 36(2): 171-174.
[6] 秦源, 林崇泽, 孙晗, 等. 益气养阴祛瘀方对干燥综合征颌下腺细胞AQP5及M3R表达的影响[J]. 中国中医急症, 2017, 26(1): 5-8.
[7] Moore M, Ma T, Yang B, et al. Tear secretion by lacrimal glands in transgenic mice lacking water channels AQP1, AQP3, AQP4 and AQP5[J]. Exp Eye Res, 2000, 70(5): 557-562.
[8] Gresz V, Kwon TH, Hurley PT, et al. Identification and localization of aquaporin water channels in human salivary glands[J]. Am J Physiol Gastrointest Liver Physiol, 2001, 281(1): G247-G254.
[9] Soyfoo MS, Konno A, Bolaky N, et al. Link between inflammation and aquaporin‐5 distribution in submandibular gland in Sj?gren′s syndrome?[J]. Oral Dis, 2012, 18(6): 568-574.
[10] Dawson L, Tobin A, Smith P, et al. Antimuscarinic antibodies in Sj?gren′s syndrome: where are we, and where are we going?[J]. Arthritis Rheum, 2010, 52(10): 2984-2995.
[11] Cavill D, Waterman SA, Gordon TP. Antibodies raised against the second extracellular loop of the human muscarinic M3 receptor mimic functional autoantibodies in Sj?gren′s syndrome[J]. Scand J Immunol, 2004, 59(3): 261-266.
[12] 陆妍, 陈祎, 王亚南, 等. 半夏芩连汤对干燥综合征模型NOD小鼠Th17/IL-17免疫炎性途径的影响[J]. 中国中西医结合杂志, 2015, 35(5): 612-617.
[13] Wang D, Yuan Z, Inoue N, et al. Abnormal subcellular localization of AQP5 and downregulated AQP5 protein in parotid glands of streptozotocin-induced diabetic rats[J]. Biochim Biophys Acta, 2011, 1810(5): 543-554.
[14] 方祯, 华海清. 干扰素在胆系肿瘤治疗中的作用及其研究进展[J]. 临床肿瘤学杂志, 2014, 19(2): 180-185.
[15] Cha S, Brayer J, Gao J, et al. A dual role for interferon-gamma in the pathogenesis of Sj?gren′s syndrome-like autoimmune exocrinopathy in the nonobese diabetic mouse[J]. Scand J Immunol, 2004, 60(6): 552-565.
[16] Cha S, Nagashima H, Brown VB, et al. Two NOD Idd‐associated intervals contribute synergistically to the development of autoimmune exocrinopathy (Sj?gren′s syndrome) on a healthy murine background[J]. Arthritis Rheum, 2002, 46(5): 1390-1398.
[17] 葛琳, 张华东, 姜泉, 等. 路氏滋燥汤对干燥综合征小鼠颌下腺细胞因子IFN-γ、IL-10的影响[J]. 世界中西医结合杂志, 2011, 6(1): 28-30.
[18] Lee BH, Gauna AE, Perez G, et al. Autoantibodies against muscarinic type 3 receptor in Sj?gren′s syndrome inhibit aquaporin 5 trafficking[J]. PLoS One, 2013, 8(1): e53113.
[19] Delporte C, Steinfeld S. Distribution and roles of aquaporins in salivary glands[J]. Biochim Biophys Acta, 2006, 1758(8): 1061-1070.
[2] Mavragani CP, Nezos A, Moutsopoulos HM. New advances in the classification, pathogenesis and treatment of Sj?gren′s syndrome[J]. Curr Opin Rheumatol, 2013, 25(5): 623-629.
[3] 王新昌, 谢志军, 温成平, 等. 益气养阴祛瘀法治疗原发性干燥综合征的疗效观察[J]. 中国基层医药, 2009, 16(1): 92-93.
[4] 王新昌, 谢志军, 温成平, 等. 益气养阴祛瘀药对干燥综合征患者性激素水平影响的研究[J]. 浙江中医药大学学报, 2009, 33(1): 48-49.
[5] 王新昌, 范永升, 冯健, 等. 益气养阴祛瘀中药对干燥综合征自发性模型非肥胖糖尿病小鼠的治疗作用及机制[J]. 浙江中医药大学学报, 2012, 36(2): 171-174.
[6] 秦源, 林崇泽, 孙晗, 等. 益气养阴祛瘀方对干燥综合征颌下腺细胞AQP5及M3R表达的影响[J]. 中国中医急症, 2017, 26(1): 5-8.
[7] Moore M, Ma T, Yang B, et al. Tear secretion by lacrimal glands in transgenic mice lacking water channels AQP1, AQP3, AQP4 and AQP5[J]. Exp Eye Res, 2000, 70(5): 557-562.
[8] Gresz V, Kwon TH, Hurley PT, et al. Identification and localization of aquaporin water channels in human salivary glands[J]. Am J Physiol Gastrointest Liver Physiol, 2001, 281(1): G247-G254.
[9] Soyfoo MS, Konno A, Bolaky N, et al. Link between inflammation and aquaporin‐5 distribution in submandibular gland in Sj?gren′s syndrome?[J]. Oral Dis, 2012, 18(6): 568-574.
[10] Dawson L, Tobin A, Smith P, et al. Antimuscarinic antibodies in Sj?gren′s syndrome: where are we, and where are we going?[J]. Arthritis Rheum, 2010, 52(10): 2984-2995.
[11] Cavill D, Waterman SA, Gordon TP. Antibodies raised against the second extracellular loop of the human muscarinic M3 receptor mimic functional autoantibodies in Sj?gren′s syndrome[J]. Scand J Immunol, 2004, 59(3): 261-266.
[12] 陆妍, 陈祎, 王亚南, 等. 半夏芩连汤对干燥综合征模型NOD小鼠Th17/IL-17免疫炎性途径的影响[J]. 中国中西医结合杂志, 2015, 35(5): 612-617.
[13] Wang D, Yuan Z, Inoue N, et al. Abnormal subcellular localization of AQP5 and downregulated AQP5 protein in parotid glands of streptozotocin-induced diabetic rats[J]. Biochim Biophys Acta, 2011, 1810(5): 543-554.
[14] 方祯, 华海清. 干扰素在胆系肿瘤治疗中的作用及其研究进展[J]. 临床肿瘤学杂志, 2014, 19(2): 180-185.
[15] Cha S, Brayer J, Gao J, et al. A dual role for interferon-gamma in the pathogenesis of Sj?gren′s syndrome-like autoimmune exocrinopathy in the nonobese diabetic mouse[J]. Scand J Immunol, 2004, 60(6): 552-565.
[16] Cha S, Nagashima H, Brown VB, et al. Two NOD Idd‐associated intervals contribute synergistically to the development of autoimmune exocrinopathy (Sj?gren′s syndrome) on a healthy murine background[J]. Arthritis Rheum, 2002, 46(5): 1390-1398.
[17] 葛琳, 张华东, 姜泉, 等. 路氏滋燥汤对干燥综合征小鼠颌下腺细胞因子IFN-γ、IL-10的影响[J]. 世界中西医结合杂志, 2011, 6(1): 28-30.
[18] Lee BH, Gauna AE, Perez G, et al. Autoantibodies against muscarinic type 3 receptor in Sj?gren′s syndrome inhibit aquaporin 5 trafficking[J]. PLoS One, 2013, 8(1): e53113.
[19] Delporte C, Steinfeld S. Distribution and roles of aquaporins in salivary glands[J]. Biochim Biophys Acta, 2006, 1758(8): 1061-1070.