胰岛素信号通路与骨质疏松症发病机制及中药治疗荟萃分析

发布时间：2018-01-24 13:20

  本文关键词： 2型糖尿病 骨质疏松症 胰岛素信号通路 骨髓脂肪组织 皮质骨 脊柱骨折 中草药　出处：《河北医科大学》2015年博士论文　论文类型：学位论文

【摘要】：2型糖尿病(Type 2 diabetes mellitus,T2DM)和骨质疏松症(Osteoporosis,OP)作为老年人的常见病、多发病,其发病率呈逐年升高趋势,严重影响人们的生活质量。国内外各项研究对1型糖尿病与骨质疏松症的关系已经得出较为肯定的结论,即1型糖尿病是骨质疏松症的高风险因素,并可由此导致病理性骨折,其原因可能与胰岛素分泌绝对不足引发糖类、脂类及蛋白质代谢异常有关,而糖类、脂类、蛋白质代谢异常可导致机体钙磷代谢失衡,从而减低骨量;而2型糖尿病患者骨量变化情况尚未得到较为一致的结论,二者发病机制是否存在共通之处亦不甚明确。近年来研究发现,胰岛素信号通路可能与2型糖尿病和骨质疏松症的发病机制有关。胰岛素信号通路包括:胰岛素受体底物(insulin receptor substrate,IRS)的磷酸化,磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)的活化,Akt(即蛋白激酶B)活化,雷帕霉素靶蛋白(molecular target of rapamycin,m TOR)活化,核糖体蛋白S6激酶(p70 S6 kinase,p70 S6 k)活化等。正常情况下,胰岛素或胰岛素样生长因子(insulin-like growth factor-1,IGF-1)在细胞表面与胰岛素受体结合,诱导胰岛素受体发生自身磷酸化,酪氨酸激酶被激活,活化的酪氨酸激酶使胰岛素受体底物(insulin receptor substrate,IRS)的酪氨酸磷酸化,该底物被激活。活化的IRS在细胞膜处与磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)的p85-p110亚基结合,PI3K被激活,后者生成磷脂酰肌醇(3、4)二磷酸(PIP2)和磷脂酰肌醇(3、4、5)三磷酸(PIP3),并与磷脂酰肌醇依赖性激酶(phosphoinositide dependent kinase,PDK)-1结合,进一步使下游通路中蛋白激酶B(protein kinase B,PKB)丝氨酸残基磷酸化,最终导致葡萄糖转运蛋白-4(glucose transporter 4,GLUT-4)移位到细胞膜而发挥降糖作用。T2DM时,外周组织对胰岛素的敏感性降低,细胞对葡萄糖的摄取能力下降,葡萄糖调节受损,血糖升高。近来研究发现,胰岛素信号通路不仅是糖代谢的关键信号通路之一,其各因子也参与骨代谢的调节。PI3K参与成骨细胞和破骨细胞的分化过程,在骨生成和骨重建方面发挥重要作用。实验发现,PI3K信号通路的活化在成骨前体细胞MC3T3-E1的分化过程中是必需的,阻断该信号通路在成骨细胞中的活化将严重抑制成骨细胞的正常分化。Akt是PI3K信号通路的主要下游分子。在成骨细胞和破骨细胞上,Aktl的表达占绝对优势,敲除小鼠Akt1基因发现其骨量减少,骨组织形态测量显示骨形成和骨吸收活性均明显下降,二次骨化中心形成延迟;Akt1-/-野生型小鼠成骨细胞分化指标(如ALP、I型胶原、骨钙素表达等)均降低。此外,Akt与骨形态发生蛋白2共同调节间充质细胞向成骨细胞的分化。骨质疏松症不仅与体重和身体总脂肪有关,与骨髓脂肪细胞也密切相关。骨髓间充质干细胞(Mesenchymal stem cells,MSCs)具有多向分化潜能。若MSCs向脂肪细胞方向分化增加,会导致其向成骨细胞分化减少,故骨髓成骨细胞与脂肪细胞呈竞争性负相关关系。激素、脂肪因子,PPARγ以及机械性刺激均可影响MSCs向成骨或成脂方向转化。抑制成骨细胞内信号通路可促使MSCs向脂肪细胞方向转化。由骨质疏松症所导致的椎体压缩性骨折(OVCFs)十分常见,严重影响患者的生活质量,给家庭和社会都带来沉重的经济负担。过去的十年中,制药公司已经研发出一系列可提高骨量的骨质疏松治疗药物。这些药物主要在促进骨形成(如特立帕肽,锶盐等)或抑制骨吸收(如双磷酸盐类)方面发挥治疗骨质疏松症的作用。虽然这些药物对骨质疏松症疗效明显,但发热、骨痛加重等副反应也伴之而来。中草药可与已有的药物联合应用于治疗由骨质疏松症所导致的脊柱压缩性骨折。第一部分胰岛素下游信号因子PI3K、Akt1、Akt2及炎症因子NFκB在2型糖尿病骨质疏松大鼠肝、肾及骨骼肌组织中表达目的:探讨胰岛素信号通路下游PI3K、Akt1、Akt2及炎症因子NFκB表达变化是否影响2型糖尿病合并骨质疏松症的发生、发展。方法:选取100只健康雌性Wistar大鼠(合格证书编号905105,2.5-3月龄)适应性喂养1周后随机分为正常对照组(NS组,N=24),单纯去卵巢组(NOVX组,N=26),2型糖尿病(DS组,N=24),2型糖尿病去卵巢组(DOVX组,N=26)。采用高糖高脂饲料喂养联合小剂量STZ腹腔注射法制作T2DM大鼠模型,双侧卵巢切除法制作OP大鼠模型。去卵巢后12周麻醉处死大鼠,分离双侧肝脏、肾脏及右侧股四头肌组织,分别制作各组织石蜡切片和提取RNA和总蛋白,应用免疫组织化学和western blot方法检测NS组、NOVX组、DS组、DOVX组各组织PI3K、Akt1、Akt2、NFκB蛋白表达,反转录聚合酶链反应(RT-PCR)方法检测PI3K、Akt1、Akt2、NFκB m RNA的表达。应用SPSS13.0统计软件处理实验数据,所有实验数据均采用均数±标准差(x±s)表示,计量资料多组间比较采用方差分析,P0.05认为有统计学意义。结果:应用免疫组化方法观察大鼠肝、肾、骨骼肌组织PI3K、Akt1、Akt2、NFκB分别在大鼠肝细胞胞浆、肾小管上皮细胞胞浆及骨骼肌细胞胞浆中表达。各组织免疫组化结果显示,与NS组相比,NOVX、DS和DOVX的PI3K、Akt1和Akt2均降低,以DOVX组降低最明显(P0.05)。NFκB趋势则相反,12周时NOVX、DS和DOVX的NFκB均升高,以DOVX组升高最明显(P0.05)。肝组织RT-PCR结果显示,DOVX组PI3K、Akt1和Akt2降低最为明显(P0.05),NFκB升高最明显(P0.05)。DS组和DOVX组Akt2无明显差别(P0.05),NOVX组、DS组和DOVX组NFκB无明显差别(P0.05)。肾组织RT-PCR结果显示,DOVX组PI3K、Akt1和Akt2降低最为明显(P0.05),NFκB升高最明显(P0.05)。NOVX组和DS组PI3K无明显差别(P0.05),NOVX组、DS组和DOVX组Akt2表达无明显变化(P0.05)。骨骼肌RT-PCR结果显示,DOVX组PI3K、Akt1和Akt2降低最为明显(P0.05),NFκB升高最明显(P0.05)。NOVX组、DS组和DOVX组PI3K、Akt1、NFκB无明显差别(P0.05)。肝组织western blot结果显示,以DOVX组PI3K、Akt1和Akt2降低最为明显(P0.05),NOVX组和DS组PI3K无明显差别(P0.05)。NFκB以DOVX组升高最明显(P0.05),NOVX组和DS组NFκB无明显差别(P0.05)。肾组织western blot结果显示,DOVX组PI3K、Akt1和Akt2降低最明显(P0.05),NFκB升高最明显(P0.05)。NOVX组和DS组PI3K、NFκB无明显差别(P0.05),DS组与DOVX组Akt2无明显差别(P0.05)。骨骼肌western blot结果显示,DOVX组PI3K、Akt1和Akt2降低最明显(P0.05),NFκB升高最明显(P0.05)。NOVX组和DS组Akt1、NFκB表达无明显差别(P0.05)。结论:胰岛素信号通路中PI3K、Akt1、Akt2、NFκB表达受抑制可能与2型糖尿病合并骨质疏松症的发病有关。第二部分绝经后骨质疏松患者骨髓脂肪组织与皮质骨区关系的研究目的:探讨绝经后骨质疏松症患者骨髓脂肪组织与皮质骨区是否存在负相关关系。方法:收集2013年1月-2013年12月在河北医科大学第三医院内分泌二科住院的2型糖尿病女性患者30例。采用核磁共振技术测量患者右侧股骨BMA,右侧股骨CBA,右侧股骨皮下脂肪组织(SAT)及右侧股骨骨骼肌,并将图像用Slice Omatic分析软件进行数据分析。采用SPSS13.0统计分析软件进行统计分析,描述性指标以均数±标准差表示。采用Pearson相关系数计算CBA、BMA、体重、BMI百分比、年龄、SAT和骨骼肌之间的关系。采用多元线性回归进行相关分析。结果:Pearson相关分析显示,右侧股骨BMA和右侧股骨CBA都与体重、BMI、年龄、右侧股骨皮下脂肪组织(SAT)及右侧股骨骨骼肌呈显著正相关(分别为r=0.081,r=-0.215,r=0.175,r=-0.252,r=0.317;r=0.385,r=0.153,r=-0.298,r=-0.087,r=0.650;P0.05)。结论:BMA与CBA之间呈负相关关系。这可能与骨髓间充质干细胞优先向脂肪细胞分化而向成骨细胞分化减少有关,为骨质疏松症提供新的治疗方向。第三部分传统中药治疗骨质疏松性椎体压缩性骨折的系统性回顾研究目的:系统评价中草药治疗在骨质疏松性椎体压缩性骨折的临床效果。方法:计算机检索三个电子数据库(中国国家知识基础设施工程,中国科技期刊数据库(VIP)和万方数据库),检索时间从1999年至2013年2月,搜索的中文关键字是中国草药、草药、骨质疏松性椎体压缩性骨折。本研究还使用了以下关键字的任意组合:一)压缩性骨折、腰椎骨折、胸椎骨折;和b)传统中国医药或者中国传统药物。搜索的英文关键词包括Chinese herb、herbal、osteoporotic vertebral compression fracture、compression fracture、vertebra fracture、lumbar vertebrae fracture、thoracic vertebrae fracture、traditional Chinese medicine、traditional Chinese drug。实验类型为随机对照实验。根据入排标准对入选文章进行严格筛选、评估质量、提取数据,采用Rev Man 5.1软件进行数据分析。结果:第三部分:最终纳入11项随机对照试验,共953例受试者。结果显示,与安慰剂组相比,使用中草药治疗的患者其骨密度明显升高。此外,与安慰剂组相比,联合中药治疗的患者其骨密度提高更明显。敏感性分析证实了这一点。结论:使用中草药治疗可以增加脊柱骨密度。中草药可作为一种创伤小、成本低的治疗骨质疏松症导致的脊柱压缩性骨折的方法。
[Abstract]:Type 2 diabetes mellitus (Type 2 diabetes mellitus, T2DM) and osteoporosis (Osteoporosis, OP) is a common disease, the elderly disease, its incidence is increasing year by year, seriously affecting people's quality of life at home and abroad. The research on the relationship between type 1 diabetes and osteoporosis has drawn more positive conclusion: type 1 diabetes is a high risk factor for osteoporosis, and thus lead to pathological fracture, the reason may be related to insulin secretion absolutely caused by insufficient carbohydrate, lipid and protein metabolism, abnormal lipid and carbohydrate, protein, metabolic abnormalities can lead to imbalance of calcium and phosphorus metabolism, thereby reducing bone mass changes; the situation of bone mass in patients with type 2 diabetes has not been consistent conclusions, the pathogenesis of the existence of two common is not very clear. Recent studies have found that the Shima Sonobu pathway may be related to type 2 diabetes The pathogenesis of diabetes and osteoporosis. The insulin signaling pathway including insulin receptor substrate-1 (insulin receptor, substrate, IRS) phosphorylation of phosphatidylinositol 3 kinase (phosphatidylinositol, 3-kinase, PI3K) activation, Akt (i.e. protein kinase B) activation, rapamycin (molecular target of rapamycin, m TOR) activation of ribosomal protein S6 kinase (p70 S6 kinase, p70 S6 K) activation. Under normal circumstances, insulin or insulin-like growth factor (insulin-like growth, factor-1, IGF-1) on the cell surface binding with the insulin receptor, insulin receptor induced autophosphorylation and tyrosine kinase activated tyrosine kinase activation to insulin (insulin receptor substrate receptor tyrosine phosphorylation, IRS), the substrate is activated. The activation of IRS kinase and phosphatidylinositol 3- at the cell membrane (phosphatidylinositol 3-kinase, PI3K) p85-p110 subunit binding, PI3K is activated, the generation of phosphatidylinositol (3,4) two phosphoric acid (PIP2) and phosphatidylinositol (3,4,5) three (PIP3), and phosphate dependent kinase and phosphatidylinositol (phosphoinositide dependent, kinase, PDK) -1 binding protein kinase, further downstream pathways B (protein kinase B, PKB) phosphorylated serine residues, resulting in glucose transporter -4 (glucose transporter 4, GLUT-4) shift to the cell membrane and play the hypoglycemic effect of.T2DM, reduce the sensitivity of peripheral tissues to insulin, decreased ability of glucose uptake by cells, impaired glucose regulation, blood glucose increased recently. The study found that insulin signaling pathway is not only one of the most important signal pathway of glucose metabolism, the factors are also involved in the regulation of bone metabolism of.PI3K in osteoblast and osteoclast differentiation in bone formation and bone remodeling. The surface play an important role. The experimental results revealed that the activation of PI3K signaling pathway in the differentiation of osteoblastic progenitor cells in MC3T3-E1 is necessary, to block the signal pathway in activation of bone cells in severely inhibited the normal differentiation of.Akt into osteoblasts is the main downstream PI3K signaling pathway molecules in osteoblasts and. Osteoclasts, the expression of Aktl dominant Akt1 gene knockout mice found reduced bone mass, bone tissue morphometry showed bone formation and bone resorption activity were significantly decreased, two secondary ossification center formation delay; Akt1-/- wild type mouse osteoblastic cell differentiation index (such as ALP, type I collagen, osteocalcin expression etc.) were reduced. In addition, Akt and bone morphogenetic protein 2 co regulation of mesenchymal cells to differentiate into bone cells. Osteoporosis is not only related with body weight and total body fat, and also closely related to bone marrow mesenchymal cells. Mesenchymal stem cells (Mesenchymal stem cells, MSCs) have the potential of multi-directional differentiation. If MSCs differentiation into adipocytes increased, will lead to the reduction of osteoblastic differentiation, the bone marrow cells and fat cells showed competitive negative correlation. The fat factor hormone, PPAR, gamma stimulation can affect the osteogenic or MSCs adipogenic transformation and Mechanical properties. The inhibition of osteoblasts in signaling pathway can promote the conversion of MSCs into adipocytes. Caused by osteoporosis vertebral compression fracture (OVCFs) is very common, seriously affecting the quality of life of patients has brought heavy economic burden to family and society over the past ten years. In the pharmaceutical company has developed a series of osteoporosis drugs can increase bone mass. These drugs mainly in promoting bone formation (such as teriparatide, strontium salts) or inhibit bone resorption (such as bisphosphonates) play therapy Treatment of osteoporosis. Although these drugs significantly, the curative effect of osteoporosis but fever, pain exacerbated side reactions are coming. Chinese herbal medicine can be used together with existing drugs in the treatment of osteoporosis caused by vertebral compression fractures. The first part of the insulin downstream signal factor PI3K, Akt1 Akt2 NF and inflammatory factor kappa B in type 2 diabetic osteoporosis rat liver, kidney and skeletal muscle tissue expression objective: To explore the insulin signaling pathway downstream of PI3K, Akt1, Akt2 and the expression of inflammatory cytokines NF kappa B whether changes in type 2 diabetic patients with osteoporosis. Methods: 100 healthy female Wistar rats (certificate number 905105,2.5-3 months) after 1 weeks of feeding were randomly divided into normal control group (group NS, N=24), ovariectomized group (NOVX group, N=26), type 2 diabetes (DS group, N=24), ovariectomized group (type 2 diabetes D Group OVX, N=26). The T2DM rat model by high-fat diet and low dose STZ intraperitoneal injection, OP rats were bilaterally ovariectomized. Rats were sacrificed 12 weeks after ovariectomy, anesthesia, bilateral liver, kidney and muscle tissues of the right femoral head four, paraffin section and RNA extraction and the total protein of tissue were made, using immunohistochemical method to detect the NS and western of blot group, NOVX group, DS group, DOVX group PI3K, Akt1, Akt2, expression of NF kappa B protein, reverse transcription polymerase chain reaction (RT-PCR) method for detection of PI3K, Akt1, Akt2, m RNA expression of NF kappa B to deal with the experimental data. The application of SPSS13.0 statistical software, all experimental data were used to mean standard deviation (x + s) said that the measurement data were compared by analysis of variance, P0.05 was considered statistically significant. Results: immunohistochemistry of rat liver, kidney, skeletal muscle PI3K, Akt1, Akt2 ,NF魏B鍒嗗埆鍦ㄥぇ榧犺倽缁嗚優鑳炴祮,鑲惧皬绠′笂鐨�缁嗚優鑳炴祮鍙婇�ㄩ�艰倢缁嗚優鑳炴祮涓�琛ㄨ揪.鍚勭粍缁囧厤鐤�缁勫寲缁撴灉鏄剧ぃ�

本文编号：1460139