电针对去卵巢大鼠Wnt3a和β-catenin的基因及蛋白表达的影响
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摘要
目的研究电针对去卵巢骨质疏松模型大鼠Wnt3a和β-catenin的蛋白及基因表达的影响。方法选取60只雌性SD大鼠,随机分为空白组、假手术组、模型组、药物组、针刺组,每组12只。空白组不做任何处理,常规饲养;假手术组只暴露双侧卵巢但不切除;其余3组行双侧切卵巢法建立骨质疏松模型大鼠。造模13周后开始治疗,针刺组取"肾俞"、"脾俞"、"三阴交"、"足三里",每次电针刺激15 min(断续波,2 Hz,1.0 mA)并灌服与药物组相同体积的蒸馏水,每日1次,连续治疗5 d,间隔2 d,20 d为1个疗程,共治疗3个疗程;药物组以戊酸雌二醇水溶液(0.02 mg/mL,1 mL/100 g体质量)灌胃,疗程同针刺组;假手术组、模型组灌服与药物组相同体积的蒸馏水,疗程同针刺组;各组大鼠每周称重1次,以此调整给药剂量。治疗结束后麻醉处死大鼠,分离股骨和胫骨,分别用RT-PCR分析法和免疫组化法检测各组大鼠股骨和胫骨Wnt3a,β-catenin蛋白及基因的表达。结果模型组Wnt3a和β-catenin蛋白表达量最低,与空白组和假手术组比较差异有统计学意义(P<0.05);经针刺或药物干预后,Wnt3a和β-catenin蛋白表达显著升高,与模型组比较差异具有统计学意义(P<0.05);模型组Wnt3a和β-catenin mRNA表达量最低,与空白组和假手术组比较差异有统计学意义(P<0.05);经针刺或药物干预后,Wnt3a和β-catenin mRNA表达显著升高,与模型组比较具有统计学意义(P<0.05)。结论针刺防治绝经后骨质疏松症的分子机制可能是通过调控Wnt3a和β-catenin的基因和蛋白表达水平,从而激活Wnt/β-catenin信号通路,以此调节骨重建系统的平衡。
Objective To study the effects of electroacupuncture on gene and protein expression of Wnt3 a and β-catenin in ovariectomized rats. Methods Sixty female SD rats were randomly divided into sham operation group, blank group, model group, electroacupuncture group, and medicine group, with 12 rats in each group. The rats in blank group received no treatment. The bilateral ovaries of rats in the sham operation group were only exposed but not removed, while bilateral ovarian resection was performed in the other groups to establish the osteoporosis model. After 13 weeks of modeling, rats in the electroacupuncture group were stimulated at Shenshu(BL23), Pishu(BL20), Sanyinjiao(SP6), and Zusanli(ST36) points for 15 min(intermittent wave, 2 Hz, 1.0mA) and were fed with the same volume of distilled water to the other groups. The treatment was once a day and two days off after lasting 5 days. Rats in the medicine group received estradiol 0.02 mg/mL, 1 mL/100 g weight by gavage, and electroacupuncture. Rats in the rest groups were fed with an equal volume of distilled water, and the electroacupuncture was the same. The rats in each group were weighed once a week and then the dosage of medicine was adjusted. The rats were killed under anesthesia after the experiment. The femurs and the tibia were isolated. The gene and protein expression of wnt3 a and β-catenin of the femurs and the tibia was detected using RT-PCR analysis and immunohistochemistry. Results The expression of Wnt3 a and β-catenin protein in model group was significantly lower than that in the blank group and Sham operation group(P<0.05). The expression of Wnt3 a and β-catenin protein increased significantly after acupuncture or medicine intervention compared with the model group, with statistical significance(P<0.05). The mRNA expression of Wnt3 a and β-catenin was the lowest in the model group, and it significantly decreased(P<0.05) compared with that in the blank group and Sham operation group. After acupuncture or medicine intervention, the mRNA expression of Wnt3 a and β-catenin significantly elevated, which was statistically significant compared with the model group(P<0.05). Conclusion The mechanism of electroacupuncture in the treatment of postmenopausal osteoporosis may be through regulating the gene and protein expression of wnt3 a and β-catenin, and activating the Wnt/β-catenin signaling pathway, then regulating the balance of bone remodeling.
Objective To study the effects of electroacupuncture on gene and protein expression of Wnt3 a and β-catenin in ovariectomized rats. Methods Sixty female SD rats were randomly divided into sham operation group, blank group, model group, electroacupuncture group, and medicine group, with 12 rats in each group. The rats in blank group received no treatment. The bilateral ovaries of rats in the sham operation group were only exposed but not removed, while bilateral ovarian resection was performed in the other groups to establish the osteoporosis model. After 13 weeks of modeling, rats in the electroacupuncture group were stimulated at Shenshu(BL23), Pishu(BL20), Sanyinjiao(SP6), and Zusanli(ST36) points for 15 min(intermittent wave, 2 Hz, 1.0mA) and were fed with the same volume of distilled water to the other groups. The treatment was once a day and two days off after lasting 5 days. Rats in the medicine group received estradiol 0.02 mg/mL, 1 mL/100 g weight by gavage, and electroacupuncture. Rats in the rest groups were fed with an equal volume of distilled water, and the electroacupuncture was the same. The rats in each group were weighed once a week and then the dosage of medicine was adjusted. The rats were killed under anesthesia after the experiment. The femurs and the tibia were isolated. The gene and protein expression of wnt3 a and β-catenin of the femurs and the tibia was detected using RT-PCR analysis and immunohistochemistry. Results The expression of Wnt3 a and β-catenin protein in model group was significantly lower than that in the blank group and Sham operation group(P<0.05). The expression of Wnt3 a and β-catenin protein increased significantly after acupuncture or medicine intervention compared with the model group, with statistical significance(P<0.05). The mRNA expression of Wnt3 a and β-catenin was the lowest in the model group, and it significantly decreased(P<0.05) compared with that in the blank group and Sham operation group. After acupuncture or medicine intervention, the mRNA expression of Wnt3 a and β-catenin significantly elevated, which was statistically significant compared with the model group(P<0.05). Conclusion The mechanism of electroacupuncture in the treatment of postmenopausal osteoporosis may be through regulating the gene and protein expression of wnt3 a and β-catenin, and activating the Wnt/β-catenin signaling pathway, then regulating the balance of bone remodeling.
引文
[1] Liu Y Q,Han X F,Liu T, et al. A cell-based model of bone remodeling for identifying activity of icarrin in the treatment of osteoporosis[J]. Biotechnology Letters, 2015, 37(1): 219-226.
[2] Leslie WD, Morin SN. Osteoporosis epidemiology 2013:implications for diagnosis, risk assessment, and treatment. Curr Opin Rheumatol, 2014: 26 (4): 440-446.
[3] 康轶鑫,王道全,刘忠厚.针灸治疗骨质疏松症的实验研究进展[J]. 中国骨质疏松杂志, 2011, 17(1): 73-79.Kang YX, Wang DQ, Liu ZH. The experimental research progress of acupuncture therapy for the treatment of osteoporosis [J]. Chinese Journal of Osteoporosis, 2011, 17(1): 73-79.
[4] 李金艳, 陈颖,唐艳丽,等. 针灸治疗骨质疏松症效应机制研究进展[J]. 中国骨质疏松杂志, 2014, 20(3): 315-321.Li JY, Chen Y, Tang YL, et al. Research Progress on the effect mechanism of acupuncture and moxibustion in the treatment of osteoporosis [J]. Chinese Journal of Osteoporosis, 2014, 20(3): 315-321.
[5] 卢化爱.绝经后骨质疏松发病率调查与预防[J]. 中国妇幼保健, 2007, 22(21): 3033-3034.Lu HA. Investigation and prevention of postmenopausal osteoporosis incidence [J]. China's Maternal and Child Health, 2007, 22(21): 3033-3034.
[6] 刘峰. 骨质疏松流行病学及防治研究进展[J]. 医学理论与实践, 2017, 30(22): 3321-3322.Liu F. Advances in epidemiology and control of osteoporosis [J]. Medical Theory and Practice, 2017, 30(22): 3321-3322.
[7] 申艳,郑清莲.胎肾细胞悬液对去卵巢大鼠骨组织形态计量学参数的影响[J]. 中国中医骨伤科杂志, 2017, 25(12): 4-7.Shen Y, Zheng QL. Effect of fetal renal cell suspension on the morphometric parameters of bone tissue in ovariectomized rats [J]. Chinese Journal of Orthopaedic Orthopedics, 2017, 25(12): 4-7.
[8] 彭娟娟,王冰,王旭东.中医治疗绝经后骨质疏松症研究进展[J]. 河南中医 2016, 36(5): 921-923.Peng JJ, Wang B, Wang XD. Progress in treatment of postmenopausal osteoporosis by traditional Chinese medicine [J]. Henan Traditional Chinese Medicine, 2016, 36(5): 921-923.
[9] 陈克明. 淫羊藿总黄酮的抗骨质疏松作用机制[J]. 中华中医药杂志, 2017, 32(12): 5485-5489.Cheng KM. Anti-osteoporosis mechanism of total flavonoids from epimedium [J]. Chinese Journal of Chinese Medicine, 2017, 32(12): 5485-5489.
[10] 王亚军,鞠大宏. 针灸治疗妇女绝经后骨质疏松的实验研究进展述评[J]. 按摩与康复医学, 2012, 3(3): 57-59.Wang YJ, Ju DH. Review of the experimental research progress of Acupunture therapy in postmenopausal osteoporosis [J]. Chinese Manipulation & Rehabilitation Medicine, 2012, 3(3): 57-59.
[11] 徐亚莉,金建军,徐登玉. 针灸治疗骨质疏松症研究进展[J].甘肃中医学院学报, 2006, 23(2): 53-56.Xu YL, Jin JJ, Xu DY. The experimental research progress of acupuncture therapy for the treatment of osteoporosis [J]. Journal of Gansu College of Traditional Chinese Medicine, 2006, 23(2): 53-56.
[12] 鞠大宏,于福禄,张丽坤,等. 滋阴补肾法对卵巢切除所致骨质疏松大鼠成骨细胞 COX-2 蛋白和mRNA表达的影响[J]. 中国中医基础医学杂志, 2006, 12(12): 918-920.Ju DH, Yu FL, Zhang LK, et al. The effect of the method of nourishing the kidney-yin on the expression of the osteoblast COX-2 and mRNA in the ovariectomy-induced osteoporosis rats [J]. Chinese Journal of Basic Medicine in Traditional Chinese Medicine, 2006, 12(12): 918-920.
[13] 郭义. 实验针灸学[M]. 北京: 中国中医药出版社, 2008: 1.Guo Y. Experimental acupuncture[M].Beijing: China Press of Traditional Chinese Medicine, 2008: 1.
[14] 欧阳刚,唐曦,莫非,等. 针刺干预对去卵巢大鼠血清胃饥饿素和骨密度的影响[J]. 针刺研究, 2013, 38(4): 277-280.Ou YG, Tang X, Mo F, et al. Influence of electroacupuncture intervention on serum ghrelin content and bone mineral density in ovariectomized rats [J]. Acupuncture Research, 2013, 38(4): 277-280.
[15] 鲍圣涌,张少君,林婉娟, 等. 电针对去卵巢骨质疏松大鼠骨代谢生化指标及肿瘤坏死因子-α和骨胶原代谢生化指标的影响[J]. 中国针灸, 2012, 32(12): 1108-1112.Bao SY, Zhang SJ, Lin WJ, et al. Effect of electroacupuncture on the biochemical indices of bone and bone collagen metabolism and TNF-αin osteoporosis model rats without ovaries [J]. Chinese Acupuncture & Moxibustion, 2012, 32(12): 1108-1112.
[16] 张瑶,朱旭,刘菊, 等. 二仙汤对去卵巢骨质疏松模型大鼠血清 I 型胶原氨基端延长肽影响的实验研究[J]. 广西医学, 2014, 36(11): 1593-1595.Zhang Y, Zhu X, Liu J, et al. Experimental study the effect of erxian decoction on serum amino terminal extension peptide of type I collagen in osteoporosis model rats without ovaries [J]. GuangXi Medical Journa, 2014,36(11):1593-1595.
[17] 杨路昕,蔡辉. 原发性骨质疏松症的中医病因病机研究进展[J]. 河北中医, 2009, 31(3): 467-469.Yang LX, Cai H. The research progress of TCM etiology and pathogenesis on primary osteoporosis [J]. Hebei Journal of Traditional Chinese Medicine, 2009, 31(3): 467-469.
[18] 苑成发,闫秀中,房经武,等.杜仲补肾健骨颗粒联合温针灸治疗肾虚精亏型骨质疏松症临床研究[J].河南中医, 2017,37(11):1981-1986.Yuan CF, Yan XZ, Fang JW, et al. The clinical study on the treatment of kidney deficiency and bone loss by the combination of eucommia and kidney tonic granules [J]. Henan traditional Chinese medicine, 2011,37 (11):1981-1986.
[19] 王亚军,刘梅洁,鞠大宏,等. 针刺对去卵巢骨质疏松模型大鼠骨密度、骨形态计量学的影响[J]. 中国中医基础医学杂志, 2015, 21(7): 857-859,871.Wang YJ, Liu MJ, Ju DH, et al. Influence of acupuncture intervention on bone mineral density and bone histomorphometry in ovariectomized rats. Chinese Journal of Basic Medicine in Traditional Chinese Medicine, 2015, 21(7): 857-859,871.
[20] 马界,罗永芬,蓝肇熙,等. 针灸结合骨立对去势大鼠骨质疏松症骨密度及生物力学影响的研究[J]. 四川中医, 2008, 26(8): 9-11.Ma J, Luo YF, Lan ZX, et al. Study on the effects of acupuncture and Moxibustion combined with guli method on the mineral density and biomechanics of overiectomized rats with osteoporosis. Journal of Sichuan of Traditional Chinese Medicine, 2008, 26(8): 9-11.
[21] 罗成斌,徐金龙,杨增荣,等. 整体调节针法治疗原发性骨质疏松症的临床研究[J].中国骨质疏松杂志, 2016, 22(11): 1459-1465.Luo CB, Xu JL, Yang ZR, et al. Clinical study on the treatment of primary osteoporosis by holistic regulation of Acupuncture [J]. Chinese Journal of Osteoporosis, 2016,22(11):1459-1465.
[22] Amerongen R V, Nusse R. Towards an integrated view of Wnt signaling in development[J]. Development, 2009, 136(19):3205-3214.
[2] Leslie WD, Morin SN. Osteoporosis epidemiology 2013:implications for diagnosis, risk assessment, and treatment. Curr Opin Rheumatol, 2014: 26 (4): 440-446.
[3] 康轶鑫,王道全,刘忠厚.针灸治疗骨质疏松症的实验研究进展[J]. 中国骨质疏松杂志, 2011, 17(1): 73-79.Kang YX, Wang DQ, Liu ZH. The experimental research progress of acupuncture therapy for the treatment of osteoporosis [J]. Chinese Journal of Osteoporosis, 2011, 17(1): 73-79.
[4] 李金艳, 陈颖,唐艳丽,等. 针灸治疗骨质疏松症效应机制研究进展[J]. 中国骨质疏松杂志, 2014, 20(3): 315-321.Li JY, Chen Y, Tang YL, et al. Research Progress on the effect mechanism of acupuncture and moxibustion in the treatment of osteoporosis [J]. Chinese Journal of Osteoporosis, 2014, 20(3): 315-321.
[5] 卢化爱.绝经后骨质疏松发病率调查与预防[J]. 中国妇幼保健, 2007, 22(21): 3033-3034.Lu HA. Investigation and prevention of postmenopausal osteoporosis incidence [J]. China's Maternal and Child Health, 2007, 22(21): 3033-3034.
[6] 刘峰. 骨质疏松流行病学及防治研究进展[J]. 医学理论与实践, 2017, 30(22): 3321-3322.Liu F. Advances in epidemiology and control of osteoporosis [J]. Medical Theory and Practice, 2017, 30(22): 3321-3322.
[7] 申艳,郑清莲.胎肾细胞悬液对去卵巢大鼠骨组织形态计量学参数的影响[J]. 中国中医骨伤科杂志, 2017, 25(12): 4-7.Shen Y, Zheng QL. Effect of fetal renal cell suspension on the morphometric parameters of bone tissue in ovariectomized rats [J]. Chinese Journal of Orthopaedic Orthopedics, 2017, 25(12): 4-7.
[8] 彭娟娟,王冰,王旭东.中医治疗绝经后骨质疏松症研究进展[J]. 河南中医 2016, 36(5): 921-923.Peng JJ, Wang B, Wang XD. Progress in treatment of postmenopausal osteoporosis by traditional Chinese medicine [J]. Henan Traditional Chinese Medicine, 2016, 36(5): 921-923.
[9] 陈克明. 淫羊藿总黄酮的抗骨质疏松作用机制[J]. 中华中医药杂志, 2017, 32(12): 5485-5489.Cheng KM. Anti-osteoporosis mechanism of total flavonoids from epimedium [J]. Chinese Journal of Chinese Medicine, 2017, 32(12): 5485-5489.
[10] 王亚军,鞠大宏. 针灸治疗妇女绝经后骨质疏松的实验研究进展述评[J]. 按摩与康复医学, 2012, 3(3): 57-59.Wang YJ, Ju DH. Review of the experimental research progress of Acupunture therapy in postmenopausal osteoporosis [J]. Chinese Manipulation & Rehabilitation Medicine, 2012, 3(3): 57-59.
[11] 徐亚莉,金建军,徐登玉. 针灸治疗骨质疏松症研究进展[J].甘肃中医学院学报, 2006, 23(2): 53-56.Xu YL, Jin JJ, Xu DY. The experimental research progress of acupuncture therapy for the treatment of osteoporosis [J]. Journal of Gansu College of Traditional Chinese Medicine, 2006, 23(2): 53-56.
[12] 鞠大宏,于福禄,张丽坤,等. 滋阴补肾法对卵巢切除所致骨质疏松大鼠成骨细胞 COX-2 蛋白和mRNA表达的影响[J]. 中国中医基础医学杂志, 2006, 12(12): 918-920.Ju DH, Yu FL, Zhang LK, et al. The effect of the method of nourishing the kidney-yin on the expression of the osteoblast COX-2 and mRNA in the ovariectomy-induced osteoporosis rats [J]. Chinese Journal of Basic Medicine in Traditional Chinese Medicine, 2006, 12(12): 918-920.
[13] 郭义. 实验针灸学[M]. 北京: 中国中医药出版社, 2008: 1.Guo Y. Experimental acupuncture[M].Beijing: China Press of Traditional Chinese Medicine, 2008: 1.
[14] 欧阳刚,唐曦,莫非,等. 针刺干预对去卵巢大鼠血清胃饥饿素和骨密度的影响[J]. 针刺研究, 2013, 38(4): 277-280.Ou YG, Tang X, Mo F, et al. Influence of electroacupuncture intervention on serum ghrelin content and bone mineral density in ovariectomized rats [J]. Acupuncture Research, 2013, 38(4): 277-280.
[15] 鲍圣涌,张少君,林婉娟, 等. 电针对去卵巢骨质疏松大鼠骨代谢生化指标及肿瘤坏死因子-α和骨胶原代谢生化指标的影响[J]. 中国针灸, 2012, 32(12): 1108-1112.Bao SY, Zhang SJ, Lin WJ, et al. Effect of electroacupuncture on the biochemical indices of bone and bone collagen metabolism and TNF-αin osteoporosis model rats without ovaries [J]. Chinese Acupuncture & Moxibustion, 2012, 32(12): 1108-1112.
[16] 张瑶,朱旭,刘菊, 等. 二仙汤对去卵巢骨质疏松模型大鼠血清 I 型胶原氨基端延长肽影响的实验研究[J]. 广西医学, 2014, 36(11): 1593-1595.Zhang Y, Zhu X, Liu J, et al. Experimental study the effect of erxian decoction on serum amino terminal extension peptide of type I collagen in osteoporosis model rats without ovaries [J]. GuangXi Medical Journa, 2014,36(11):1593-1595.
[17] 杨路昕,蔡辉. 原发性骨质疏松症的中医病因病机研究进展[J]. 河北中医, 2009, 31(3): 467-469.Yang LX, Cai H. The research progress of TCM etiology and pathogenesis on primary osteoporosis [J]. Hebei Journal of Traditional Chinese Medicine, 2009, 31(3): 467-469.
[18] 苑成发,闫秀中,房经武,等.杜仲补肾健骨颗粒联合温针灸治疗肾虚精亏型骨质疏松症临床研究[J].河南中医, 2017,37(11):1981-1986.Yuan CF, Yan XZ, Fang JW, et al. The clinical study on the treatment of kidney deficiency and bone loss by the combination of eucommia and kidney tonic granules [J]. Henan traditional Chinese medicine, 2011,37 (11):1981-1986.
[19] 王亚军,刘梅洁,鞠大宏,等. 针刺对去卵巢骨质疏松模型大鼠骨密度、骨形态计量学的影响[J]. 中国中医基础医学杂志, 2015, 21(7): 857-859,871.Wang YJ, Liu MJ, Ju DH, et al. Influence of acupuncture intervention on bone mineral density and bone histomorphometry in ovariectomized rats. Chinese Journal of Basic Medicine in Traditional Chinese Medicine, 2015, 21(7): 857-859,871.
[20] 马界,罗永芬,蓝肇熙,等. 针灸结合骨立对去势大鼠骨质疏松症骨密度及生物力学影响的研究[J]. 四川中医, 2008, 26(8): 9-11.Ma J, Luo YF, Lan ZX, et al. Study on the effects of acupuncture and Moxibustion combined with guli method on the mineral density and biomechanics of overiectomized rats with osteoporosis. Journal of Sichuan of Traditional Chinese Medicine, 2008, 26(8): 9-11.
[21] 罗成斌,徐金龙,杨增荣,等. 整体调节针法治疗原发性骨质疏松症的临床研究[J].中国骨质疏松杂志, 2016, 22(11): 1459-1465.Luo CB, Xu JL, Yang ZR, et al. Clinical study on the treatment of primary osteoporosis by holistic regulation of Acupuncture [J]. Chinese Journal of Osteoporosis, 2016,22(11):1459-1465.
[22] Amerongen R V, Nusse R. Towards an integrated view of Wnt signaling in development[J]. Development, 2009, 136(19):3205-3214.