【作者】 周晓莉；

【导师】 王华；

【作者基本信息】 湖北中医药大学 ， 针灸推拿学， 2011， 博士

【摘要】 目的观察“双固一通”针法对去卵巢绝经后骨质疏松症(postmenopausal osteoporosis, PMO)模型大鼠调节骨吸收平衡的相关细胞因子系统——护骨素(osteoprotegerin, OPG)/核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)/核因子κB受体活化因子(receptor activator of nuclear factor-κB, RANK)系统的影响,进一步探讨“双固一通”针法治疗本病的作用机制,为针灸治疗PMO提供新的思路和方法,为临床预防和治疗PMO提供实验依据。方法依随机原则将50只SD大鼠分为对照组、模型组、常规取穴针刺组(简称常规组)、“双固一通”针刺组(简称双固一通组)和中成药治疗组(简称中成药组),每组10只。模型组、常规组、双固一通组和中成药组切除双侧卵巢后常规饲养,造成实验性PMO动物模型。90天后常规组、双固一通组采用电针治疗,中成药组早晚予仙灵骨葆胶囊灌胃治疗(早晚以0.4g/次灌胃,药液量以生理盐水溶解,调整至3m1),对照组、模型组早晚予生理盐水3m1灌胃,用药期间常规饲养。疗程均为8周。电针治疗(采用四川恒明科技开发有限公司生产HM-6805型经穴治疗仪,疏密波8～80Hz,疏密波转换14次/分钟,电压1.5V,强度1mA左右,通电15min),每天1次,每次15分钟,留针10分钟,连续治疗6天后休息1天,共治疗8周。常规组取穴：脾俞、胃俞、肾俞,气海俞,均为双侧；双固一通组取关元、后三里(双)、肾俞(双)、膈俞(双)、大杼(双)。用全自动生化仪测定血清钙(calcium, Ca)、磷(phosphorus,P)、碱性磷酸酶(alkaline phosphatase, ALP)和尿钙、尿磷、尿肌酐；用放射免疫法测定血骨钙素(Osteocalcin, BGP);用酶联免疫法(Enzyme-Linked Immunosorbnent Assay, ELISA)测定尿脱氧吡啶啉(Deoxypyridinoline, DPD)的含量；用化学发光免疫分析法检测雌二醇(estradiol,E2); ELISA法检测白细胞介素(interletukin, IL)-6、巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)、OPG、RANKL、RANK;双能x线骨密度仪检测骨密度(bone mass density, BMD); HE染色观察大鼠骨组织形态学的变化；反转录聚合酶链式反应(Polymerase chain reaction, RT-PCR)法检测M-CSF、OPG、RANKL-mRNA表达及OPG/RANKL。大鼠因造模手术、麻醉、灌胃死亡,取材时各组大鼠数量如下：对照组8只,模型组6只,中成药组6只,常规组6只,双固一通组7只。结果1模型组血Ca值明显降低、血P值明显升高、血ALP值明显升高、BGP明显升高,有非常显著性差异(P<0.01)。与模型组比较,双固一通组和中成药组血Ca明显升高、血P明显降低、血ALP明显降低,BGP明显升高有非常显著性差异(P<0.01)；双固一通组优于常规组(P<0.01)。模型组尿Ca/Cr、P/Cr比值明显升高,有非常显著性差异(P<0.01)；与模型组比较,各组Ca/Cr、P/Cr比值均降低,常规组有非常显著性差异(P<0.01),双固一通组Ca/Cr有非常显著性差异(P<0.01),P/Cr有显著性差异(P<0.05)；双固一通组与中成药组和常规组比,无显著性差异(P>0.05)。模型组尿DPD明显升高,有显著性差异(P<0.05)。与模型组比较,双固一通组尿DPD含量明显降低,有非常显著性差异(P<0.01)；双固一通组与中成药组和常规组比,无显著性差异(P>0.05)。模型组血Ca明显降低、而血P、ALP值明显升高、血BGP明显升高(P<0.01)；尿Ca/Cr、P/Cr明显升高(P<0.01),尿DPD明显升高(P<0.05)。说明去卵巢骨质疏松症模型大鼠呈现高骨转化率,骨吸收增加,伴随骨形成代偿性增加,骨量减少,预示骨密度降低,与临床常见的PMO女性的骨高转换率极相似,提示造模成功。而“双固一通”针灸治疗后,BGP显著升高,提示成骨细胞(OB)活性增强,骨形成增加。双固一通组、中成药组均可升高血钙,降低血磷,降低尿Ca/Cr、尿P/Cr、DPD；常规组可升高血钙,降低尿Ca/Cr、尿P/Cr、DPD,对于血P无明显的降低作用。说明中医药治疗可以调节Ca、P代谢,促进骨形成,减缓骨吸收。2模型组腰椎BMD明显降低,有非常显著性差异(P<0.01)。与模型组比较,各治疗组腰椎BMD均明显升高,有非常显著性差异(P<0.01)。与双固一通组比较,有显著性差异(P<0.05)。与对照组比较,模型组股骨BMD明显降低,有非常显著性差异(P<0.01)。与模型组比较,各治疗组股骨BMD均明显升高,有显著性差异(P<0.05)；与双固一通组比较,各组无显著性差异(P>0.05)。3光镜观察结果显示：模型组骨小梁稀疏、细、中断点多、间距宽,部分区域出现较大的空白区域。常规组、中成药组、双固一通组的骨小梁增多、增粗、间隙变窄,中断点减少,三组间的区别不明显。双固一通的骨小梁排列密集,相互之间的连接明显增多,宽度增宽,体积增大,间隙变窄。4模型组血清IL-6明显升高,E2明显降低,有非常显著性差异(P<0.01)。与模型组比较,各治疗组血清IL-6均降低,E2明显升高,有非常显著性差异(P<0.01)。与中成药组比较,双固一通组和常规组血E2无显著性差异(P>0.05)。双固一通组优于常规组和中成药组(P<0.01)。在本实验中模型组E2水平降低,IL-6含量增高,与既往的研究结果一致。“双固一通”针灸治疗后,大鼠血E2显著升高、IL-6显著下降(P<0.05),双固一通组优于常规组和中成药组。表明“双固一通”针法治疗能够升高雌激素,抑制IL-6分泌,抑制OC的活性,促进骨的形成。5模型组血M-CSF明显升高、OPG明显降低、RANKL明显降低、RANK值明显降低,有非常显著性差异(P<0.01)；与模型组比较,双固一通组、常规组和中成药组M-CSF明显降低、OPG值明显升高、RANK明显升高,有非常显著性差异(P<0.01)；与模型组比较,中成药组血清RANKL升高,常规组血清RANKL有降低趋势,但均无显著性差异。双固一通组优于常规组和中成药组。说明针刺可以通过提高血OPG、RANKL、RANK的含量,降低M-CSF,从而抑制破骨细胞(osteoclast,OC)的骨吸收来减少骨量的丢失,且“双固一通,,针法优于其它方法。6模型组骨M-CSFmRNA表达较对照组明显升高,有非常显著性差异(P<0.01)。与模型组比较,各治疗组M-CSFmRNA表达均降低,有非常显著性差异(P<0.01)。与双固一通组比较,常规组M-CSFmRNA表达升高,有显著性差异(P<0.05),中成药组有非常显著性差异(P<0.01)。中成药组、常规组、双固一通组均有效,但双固一通组效最好。模型组骨OPGmRNA表达较对照组明显降低,有非常显著性差异(P<0.01)。与模型组比较,各治疗组OPGmRNA表达均升高,有非常显著性差异(P<0.01)。与中成药组和常规组比较,双固一通组有非常显著性差异(P<0.01),与对照组比较,无显著性差异(P>0.05)。中成药组、常规组、双固一通组均有效,但双固一通组效最好。模型组骨RANKLmRNA表达较对照组明显降低,有非常显著性差异(P<0.01)。与模型组比较,双固一通组骨RANKLmRNA表达升高,有非常显著性差异(P<0.01),中成药组和常规组RANKLmRNA表达与模型组比无显著性差异(P>0.05)。双固一通组与中成药组比有显著性差异(P<0.05)。双固一通组有效,中成药组和常规组均无效。模型组骨OPG/RANKL比值较对照组显著降低,有显著性差异(P<0.05)。与模型组比,双固一通组骨OPG/RANKL比值升高,有非常显著性差异(P<0.01),中成药组与模型组比有显著性差异(P<0.05),常规组与模型组比无显著性差异(P>0.05)。中成药组与双固一通组均有效,但双固一通组效更好。本研究结果表明：模型组骨M-CSFmRNA表达明显升高(P<0.01),提示OC生成增加；各治疗组M-CSFmRNA表达均降低(P<0.01),表明中医药治疗尤其是“双固一通”针法可以抑制OC数量,或降低OC活性,减缓骨吸收。模型组骨OPG、RANKLmRNA表达显著降低(P<0.01),提示OPG蛋白表达降低,破骨吸收增加；RANKL蛋白表达降低,OPG与RANKL结合有一定程度的阻滞OC生成的作用,而综合作用仍表现为骨吸收的增加。OPG/RANKL比值能反映成骨细胞(osteoblast, OB)的形成,比值越大,OB介导的能力越强；englixibaoweiia促进骨吸收,限制骨矿化的速度。模型组骨OPG/RANKL比值表达较对照组明显降低,提示骨形成降低。双固一通组骨OPG、RANKLmRNA表达明显升高(P<0.01)、RANKLmRNA表达降低(P<0.01),OPG/RANKL比值明显升高(P<0.01),提示“双固一通”针法可能通过促进OPG蛋白表达,提高OB活性,通过与RANK竞争和RANKL的结合,阻止OC的生成,阻止骨吸收。结论通过本课题的研究,我们认为：“双固一通”针法对OPG/RANKL /RANK的调节是多环节、多途径的：1“双固一通”针灸治疗后,BGP显著升高,提示成骨细胞活性增强,骨形成增加；血钙升高,血磷降低,尿Ca/Cr、尿P/Cr、DPD降低,提示“双固一通”针灸治疗可以通过增加胃肠吸收功能,促进对钙、磷等各种营养物质的吸收和利用,调节Ca、P代谢,降低尿Ⅰ型胶原分解产物DPD浓度,提高BGP浓度,从而抑制骨吸收、促进骨形成,最终使骨矿含量增加,维持甚至提高骨密度。2“双固一通”针灸治疗能通过提高去卵巢骨质疏松症模型大鼠E2的水平,减少IL-6含量,加强雌激素对IL-6的抑制作用,减少OC的前体细胞形成,抑制骨吸收,表现为BMD的升高、模型大鼠骨组织形态结构的改善。3“双固一通”针灸治疗能升高去卵巢骨质疏松症模型大鼠血清OPG、RANKL、RANK含量和降低M-CSF水平,以抑制OC的活性而减少骨吸收,减少骨量的丢失。4“双固一通”针灸治疗能升高去卵巢骨质疏松症模型大鼠血清OPG、RANKL、RANK含量和降低M-CSF,以抑制OC的活性而减少骨吸收,减少骨量的丢失；“双固一通”针灸治疗能通过降低去卵巢骨质疏松症模型大鼠骨M-CSFmRNA表达来抑制OC数量,或降低OC活性,减缓骨吸收；通过升高骨OPGmRNA表达,升高RANKLmRNA的表达,提高OB活性,通过与RANK竞争和RANKL的结合,阻止OC的生成,阻止骨吸收；通过升高OPG/RANKL比值,调节OPG/RANKL/RANK,能抑制OC活性,拮抗骨吸收。更多还原

【Abstract】 ObjectiveTo observe the effect of ’shuang gu yi tong’ acupuncture therapy on OPG/RANKL/RANK in the rats of postmenopausal osteoporosis (PMO), investigate the method and working mechanism, and obtain the new way of acupuncture therapy and the Experiment basis.Methods:Experiment methods:Fifty SD rats were randomly divided into five groups:control group, model group, common acupuncture group (abbreviated as common group),’shuang gu yi tong’ acupuncture group (abbreviated as shuang gu yi tong group), Herb group. The rats of model group, common group, shuang gu yi tong group, Herb group were ovariectomized to be the PMO Animal model. After 90 days, common groupand shuang gu yi tong group were treated with Electroacupuncture(Qd), Herb group was treated with Xianlinggubao (0.4g, Bid). Control group and model group were treated wi th NS. The course of treatment was 8 weeks. Electroacupuncture was treated everyday for 15 minutes. Pishu, Weishu, Shenshu, Qihaishu (double) were treated for common group; Guanyuan, Housanli, Shenshu, Geshu, Dazhu were treated for Shuang gu yi tong group. After the experiment, we detected ALP, E2, IL-6, BGP, Ca, P, M-CSF, OPG, RANKL, RANK in the blood and Ca, P, Ca/Cr, DPD in the urine; We detected BMD; We detected M-CSF, OPG, RANKL-mRNA and OPG/RANKL with RT-PCR.Results1 The level of Ca in the blood of model group was decreased, P, ALP and BGP was increased. There was significant difference between model group and control group (P<0.01). The level of Ca in the blood of Shuang gu yi tong group was increased, P, ALP and BGP was decreased. Shuang gu yi tong group was better than common group.The level of Ca/Cr, P/Cr in the urine of model group was increased. There was significant difference between model group and control group(P<0.01). The level of Ca/Cr, P/Cr was decreased. There were no significant difference between Shuang gu yi tong group, herb group and common group (P>0.05).The level of DPD in the urine of model group was increased. There was significant difference between model group and control group (P<0.05). The level of DPD of was decreased. There was significant difference between model group and Shuang gu yi tong group (P<0.01).There was no significant difference between Shuang gu yi tong group, herb group and common group (P>0.05).2 The BMD of lumbar and femur was decreased in model group. There was significant difference between model group and all treatment groups (P<0.01, P<0.05). The BMD in all treatment groups was increased. There was significant difference between Shuang gu yi tong group and other treatment groups on lumbar (P<0.05). There was no significant difference between Shuang gu yi tong group and other treatment groups on femur (P> 0.05) 3 To observe the morphological structure with light microscope. The bone trabe cula of model group was raritas, trifling. There was many breakpoint.The dist was wide. The bone trabe cula of all treatment groups was mult, thickening. The breakpoint and blank was improved. Shuang gu yi tong group was better than common group and herb group.4 The level of IL-6 was increased, E2 was decreased in model group. There was significant difference between model group and control group (P<0.01). The level of IL-6 in all treatment groups was decreased, E2 was increased. There was significant difference between model group and control group (P<0.01). Shuang gu yi tong group was better than common group and herb group.5 The level of M-CSF in the blood was increased, OPG was decreased, RANKL was decreased in model group. There was significant difference between model group and control group (P<0.01). The level of M-CSF in all treatment groups was decreased, OPG was increased, RANK was increased. There were significant difference between herb group, common group and model group(P<0.01). Shuang gu yi tong group was better than common group and herb group.6 The expression of M-CSFmRNA in the bone of model group was increased (P<0.01). The expression of M-CSFmRNA in all treatment groups was decreased. There was significant difference between common group and Shuang gu yi tong group (P<0.05). There was significant difference between herb group and Shuang gu yi tong group (P<0.01). Shuang gu yi tong group was better than common group and herb group.The expression of OPGmRNA in the bone of model group was decreased. There was significant difference between model group and control group (P<0.01). The expression of OPGmRNA in all treatment groups was increased. There were significant difference between herb group, common group and model group (P<0.01). Shuang gu yi tong group was better than common group and herb group.The expression of RANKLmRNA in the bone of model group was decreased. There was significant difference between model group and control group (P<0.01).The expression of RANKLmRNA of Shuang gu yi tong group was increased. There was significant difference between model group and Shuang gu yi tong group (P<0.01). Shuang gu yi tong group was better than common group and Herb group.The expression of OPG/RANKL in the bone of model group was decreased. There was significant difference between model group and control group (P<0.05).The expression of RANKLmRNA of Shuang gu yi tong group was increased. There was significant difference between model group and Shuang gu yi tong group (P<0.01). Shuang gu yi tong group was better than common group and herb group.Conclusion:1 Shuang gu yi tong acupuncture therapy could increase BGP, Ca, increase P, Ca/Cr, P/Cr, DPD in the Urine to regulate Ca, P metabolism.2 Shuang gu yi tong acupuncture therapy could increase E2, decrease IL-6 to increase BMD, to improve the morphological structure.3 Shuang gu yi tong acupuncture therapy could increase OPG, RANKL, RANK, decrease M-CSF to inhibit the activity of OC, to reduce the loss of bone mass.4 Shuang gu yi tong acupuncture therapy could increase the expression of OPGmRNA, RANKLmRNA, OPG/RANKL, decrease M-CSFmRNA, increase the activity of osteoblasts, prevent bone resorption.更多还原