【作者】 逯伟达；

【导师】 高海青； 肖桂山；

【作者基本信息】 山东大学 ， 老年医学， 2012， 博士

【摘要】 研究背景骨质疏松症是以骨量减少、骨的微观结构退化为特征的,致使骨的脆性增加、易于发生骨折的一种全身性骨骼疾病。骨质疏松症常发生在绝经后妇女和老年人中。随着人口老龄化,罹患人数逐年增加。骨质疏松症已成为一个世界性的公共健康问题。骨是代谢活跃器官,正常状态下,骨形成和骨吸收处于一种动态的平衡。在生长发育期,骨的形成率和吸收率均较高,骨的更新代谢活跃；成年以后骨形成率和吸收率明显减少,骨的结构和成分处于稳定状态；随着年龄的增长或妇女在绝经期以后,骨形成不足同时伴有骨吸收增加,骨代谢平衡发生转换,造成骨量的减少和骨质强度的降低,最终导致骨质疏松等骨相关疾病的发生。老年人中骨质疏松性骨折发病率的增高是与皮质骨和松质骨骨量进行性减少相关联的,特别是与骨形成的减少相关。近年来的研究表明,各种原因导致的骨量减少,如糖皮质激素应用、卵巢切除、长期制动等,常伴有骨髓中脂肪组织含量的增加。骨髓中脂肪组织不仅被动地为骨髓的造血功能保留一定空间,参与机体的脂质代谢及作为局部能量储存库,在骨形成和造血支持中发挥重要作用,而且脂肪组织还具有内分泌调节功能,释放出一系列重要的分泌性因子,在调节骨髓间充质干细胞向脂肪细胞分化过程中起关键作用。骨质疏松患者骨髓中脂肪细胞增多,成骨细胞减少,且两者存在此消彼长的关系。因此,骨髓中脂肪组织含量的增多与骨形成能力的降低存在密切联系。骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)是在骨髓中发现的,来源于中胚层的一类具有自我更新及多向分化能力的干细胞。BMSCs在不同诱导条件下,可以分化为成骨细胞、脂肪细胞、软骨细胞及成纤维细胞等多种细胞系。BMSCs作为成骨细胞和脂肪细胞共同的前体细胞源,其向成骨细胞方向和脂肪细胞方向分化的平衡对骨稳态的维持起着非常重要的作用。如果BMSCs过多地向脂肪细胞方向分化,则向成骨细胞方向分化的细胞数量相应减少,就会打破骨形成与骨吸收之间的动态平衡进而引起骨量减少导致骨质疏松的发生。因此,BMSCs的分化方向及其调控机制在骨质疏松症的发病机制中发挥重要作用。BMSCs的分化方向受多种因素、多个信号途径的调节。其中Wnt信号通路通过旁分泌或者自分泌的方式影响细胞的增长和分化。最主要的是Wnt/β-Catenin经典传导通路。当细胞外因子Wnt与低密度脂蛋白受体相关蛋白5或6(LRP5/6)、跨膜受体Frizzled胞外N端富含半胱氨酸的结构结合后,受体复合物作用于胞质内的Dishevelled (Dsh),使其高度磷酸化被激活,活化后的Dsh蛋白切断P-Catenin的降解途径,抑制β-Catenin蛋白降解和磷酸化,使其在胞质内聚集,并进入核内与核内转录因子(TCF/LEF)相互作用,启动下游靶基因的转录和表达,调节成骨细胞的增殖分化,影响骨形成。研究目的本实验旨在研究骨髓间充质干细胞来源的脂肪细胞在成骨细胞发生分化及骨形成过程中的抑制作用机制。研究方法1.细胞共培养：使用三种不用浓度的脂肪细胞诱导剂诱导人骨髓间充质干细胞(hBMSCs)向脂肪细胞方向分化,14天后与骨髓间充质干细胞共培养,再同时向成骨细胞方向诱导分化。共培养有两种模式：脂肪细胞和骨髓间充质干细胞直接接触(Mode A)和非直接接触(Mode B)。Mode A:组ⅰ,组ⅱ,组ⅲ分别使用无脂肪细胞诱导剂、半量脂肪细胞诱导剂和全量脂肪细胞诱导剂诱导hBMSCs向脂肪细胞方向分化,14天后组i、组ⅱ、组iii同时更换为成骨细胞诱导剂加脂肪细胞维持培养液,向成骨细胞方向诱导分化。脂肪细胞和成骨细胞在同一培养皿内直接接触。Mode B:采用Transwell双层细胞培养板,在上层的Netwell inserts中,组i、组ii、组iii分别使用相应浓度的脂肪细胞诱导剂(同上)诱导hBMSCs向脂肪细胞方向分化；7天时,接种hBMSCs到培养板下层的Snapwell中维持培养。诱导脂肪细胞14天后将上层的Netwell inserts和下层的Snapwell合并在一起,更换为成骨细胞诱导剂加脂肪细胞维持培养液,向成骨细胞方向诱导分化。脂肪细胞和成骨细胞非直接接触。2.形态学分析及分化标志物检测：Mode A共培养14天后进行碱性磷酸酶(ALP)染色、油红染色,28天后进行茜素红染色并采用Northern Eclipse图像定量分析软件和ALP活性检测试剂盒分析了ALP阳性面积和ALP活性的变化；Mode B共培养14天后进行ALP染色、同样的方法分析ALP阳性面积和ALP活性的变化,并分别收集Netwell inserts和Snapwell中的细胞,采用Western blot检测成脂分化标志物-Ppary和成骨分化标志物-Runx2的表达变化。3.mRNA芯片分析：分别收集Mode B组i、组ii、组iii Snapwell中的细胞提取纯化RNA后进行芯片分析；并采用RT-PCR验证S100A6的mRNA水平变化。4.双向凝胶电泳和MALDI-TOF/TOF质谱分析：分别收集Mode B组i、组ii、组iii Snapwell中的细胞提取蛋白进行双向凝胶电泳,Sypro-Ruby染色后酶解差异蛋白点,ZipTip纯化样品后点靶进行MALDI-TOF/TOF质谱分析、搜库鉴定及及数据分析；并采用Western Blot验证Calreticuln、Annexin A2和S100A6蛋白水平表达变化。5.人脂肪因子芯片分析：分别收集Mode B共培养4天后组i、组ii、组iii的培养液进行脂肪因子芯片分析。6.活性TGF-β1含量测定：分别收集Mode B共培养4天、7天、10天、14天后组i、组ii、组iii的培养液,ELISA试剂盒检测培养液中活性TGF-β1的含量。7.TGF-β中和实验：在Mode B组iii共培养0天、4天、7天、10天更换培养液时分别加入5μg/ml和10μg/ml TGF-p中和抗体,相同体积的PBS作为阴性对照。处理14天后,进行ALP染色和ALP活性测定,Western blot检测Runx2、(3-Catenin、S100A6和Calreticulin蛋白水平变化。研究结果1.形态学检测分析,在Mode A中,随着脂肪细胞诱导剂量的增加,油红染色示脂肪细胞的数目逐渐增多,而ALP和钙化结节逐渐减少。同样,在Mode B中,随着脂肪细胞诱导剂量的增加,ALP表达逐渐减少。Mode A和Mode B中ALP染色面积和酶活性测定也随着脂肪细胞诱导剂量的增加逐渐减少。同时Western blot结果验证了随着脂肪细胞诱导剂量的升高,骨形成标志物-Runx-2表达水平下降,脂肪细胞形成标志物-Ppary表达水平上升。2. mRNA芯片结果显示,随着脂肪细胞诱导剂量的增加,230个基因表达水平下降,149个基因表达水平上升,其中与成骨细胞形成相关的基因-COL1A1、Notch1、BMP6和S100A6在组ⅱ和组ⅲ中的表达水平与组i相比明显下降。相反,与脂肪细胞形成相关的基因-Calreticulin、Ppary和SMAD6在组ii和组iii中的表达水平与组i相比明显升高。同时采用RT-PCR验证了S100A6在三组中的表达水平,其结论与mRNA芯片分析结果一致。蛋白质组学分析显示有5个与成骨细胞分化相关的蛋白如Annexin A2和S100A6在组ⅱ、组ⅲ中的表达水平与组i相比明显下降；7个与脂肪细胞形成相关的蛋白如Calreticulin在组ii、组iii中的表达水平与组i相比明显上升。采用Western blot验证Annexin A2、S100A6和Calreticulin的表达水平变化,其变化趋势与蛋白质组学结果相一致。其中,S100A6和Calreticulin在基因水平和蛋白水平都被检测出存在显著差异。3.脂肪因子芯片分析结果显示有23个细胞因子,如TGF-β,在组ii、组iii中的表达水平与组i相比明显上升；同时有25个细胞因子,如MCP-3,在组ii、组iii中的表达水平与组i相比明显下降。采用ELISA试剂盒检测培养液中活性TGF-β1的含量,结果显示组ii和组iii中活性TGF-β1的含量与组i相比明显升高,并且这种变化趋势从4天一直持续到14天。为了明确TGF-β1在骨形成中的作用机制,加入TGF-p中和抗体后,形态学检测发现成骨细胞数量、ALP染色阳性面积和酶活性都明显升高。Western blot分析发现与骨形成相关的蛋白-Runx2、β-Catenin和S100A6的表达水平显著升高,而Calreticulin的表达水平明显下降。研究结论及意义随着脂肪细胞诱导剂量的升高,成骨细胞的分化及骨形成逐渐减少。其中在mRNA水平和蛋白表达水平均有显著差异变化的两个关键基因-S100A6和Calreticulin通过作用于β-Catenin,调控Wnt/β-Catenin信号传导通路来影响成骨细胞的分化和骨形成。重要的分泌性细胞因子TGF-β可通过S100A6和/或Calreticulin间接作用于β-Catenin,通过Wnt/β-Catenin信号传导通路干预成骨细胞的分化。这些结果揭示了骨质疏松患者骨量和骨形成减少的部分原因,可为以后进一步探索脂肪细胞在骨形成过程中的抑制作用机制提供新的线索。同时为骨质疏松的预防和治疗提供新的靶点,开拓新的视野。研究背景糖尿病是一种慢性全身代谢性疾病,不仅以持续性的高血糖为其特点,更重要的是高血糖所带来的诸如糖尿病心脑血管疾病、糖尿病肝病、糖尿病肾病、糖尿病视网膜病变等并发症的损害。其中,2型糖尿病发病机制最为复杂、患病率最高。胰岛素抵抗和胰岛β细胞分泌障碍是引起2型糖尿病发生的主要因素。2型糖尿病多合并脂代谢异常,脂质可在包括肝脏在内的多个器官异常沉积。机体内持续的高血糖和血脂异常不仅损伤胰岛β细胞功能,影响胰岛素信号传导途径,导致胰岛素抵抗；还能引起机体氧化应激反应和自由基的释放,引起或加重糖尿病相关并发症的发生和进展。因此,在积极控制血糖的同时,加强对高脂血症的治疗和对机体氧化应激反应的控制,能有效减轻胰岛素抵抗和改善胰岛β细胞分泌功能,从而提高2型糖尿病的综合治疗水平。根皮苷(Phlonzin)是根皮素(Phloretin)的2’-β-D葡萄糖苷,存在于多种苹果属植物中。根皮苷具有多种生物活性,如抗氧化,其抗氧化能力高于维生素C和维生素E；平衡雌激素；改善记忆力；保护心脏等。根皮苷通过竞争性地抑制钠离子葡萄糖共转运载体(SGLT)对葡萄糖的运输,减少肠道对葡萄糖的摄取和肾脏对葡萄糖的重吸收。根皮苷作为从苹果中提取的纯天然物质,有着良好的安全记录,副作用小。随着人类基因组工作框架图的完成,生命科学的研究进入了后基因组时代。蛋白质组学被认为是后基因组研究中最主要的部分。为满足蛋白质组学研究的需要,一种功能强大的可同时对4种或8种样品进行相对和绝对定量研究的方法-同位素标记相对和绝对定量(iTRAQ)技术发展迅速。iTRAQ技术与液相色谱-质谱技术的结合应用为研究不同状态下蛋白质的差异表达,揭示生物体内复杂的病理生理过程提供了一种有效方式。目前,国内外尚无采用iTRAQ标记及相关分析技术来研究根皮苷对2型糖尿病肝脏损伤保护机制的报导。研究目的本实验旨在研究根皮苷对2型糖尿病并发症,特别是肝脏损伤的保护机制。研究方法1.实验动物分组及给药：雄性C57BLKS/J db/db小鼠16只及同窝的db/m小鼠8只,鼠龄7周。8只db/m小鼠为对照组。db/db糖尿病小鼠随机分配到两个实验组：根皮苷治疗组(DMT组)8只和糖尿病组(DM组)8只。DMT组小鼠进行根皮苷溶液灌胃,每天20mg/kg,给药10周。对照组和DM组小鼠每天用相同体积的生理盐水灌胃,给药10周。整个实验过程中,所有小鼠不进行任何降糖治疗。小鼠每周测量体重。给药结束后,禁食不禁水12小时后腹主动脉取血处死。全血3000rpm离心15min,小心吸取上层血清,分装后-80℃冰箱保存。切取肝脏组织,一份置于10%甲醛溶液中,HE染色备用；另一份用冰生理盐水冲洗后,分装-80℃冰箱保存备用。2.血清学指标检测方法：采用德国拜耳公司DVI-1650全自动生化分析仪测定空腹血糖(FBG)、血清总胆固醇(TC)和甘油三酯(TG)水平。3.肝脏HE染色：取出固定于10%甲醛溶液中的肝脏组织依次进行脱水、透明、浸蜡、包埋、切片后进行HE染色,封固标本后光镜下观察,拍照。4. iTRAQ蛋白质组学分析：每组取4只小鼠的肝脏组织约50mg混合后共同加入液氮研磨提取蛋白。采用FASP酶解蛋白成肽段后,iTRAQ试剂标记相应样品(114标记对照组,116标记DMT组,117标记DM组)。将标记后样品混合进行强阳离子(SCX)色谱分级后采用毛细管高效液相色谱和电喷雾质谱检测。每次全扫描(full scan)后采集5个碎片图谱(MS2scan)获取MS/MS质谱数据。采用Turbo SEQUEST程序自动进行非冗余International Protein Index (IPI)小鼠蛋白数据库(版本3.72)搜索鉴定多肽分子,并用iTRAQ Result Multiple FileDistiller分析定量数据。定量方法采用ProteomicsTools软件。最后,采用Identified Protein iTRAQ Statistic Builder将鉴定数据及定量数据进行合并处理,得到鉴定和定量结果。5.生物信息学分析：利用IPA软件(Ingenuity Pathway Analysis www.ingenuity.com)对差异蛋白进行亚细胞定位、功能及相互作用网络分析。6.通过Western blot检测CAT、ACACA、HMGCS2和NSDHL4个蛋白的表达水平验证蛋白质组学结果的可靠性。研究结果1.小鼠一般情况：DM组小鼠的初始体重、最终体重及体重增长都明显高于对照组小鼠。自给予根皮苷治疗第二周开始到处理结束,DMT组小鼠的体重明显低于DM组小鼠体重。DM组小鼠的空腹血糖(FBG)、甘油三酯(TG)和胆固醇(TC)水平明显高于对照组。根皮苷治疗10周后,DMT组小鼠的FBG、TG和TC水平明显低于DM组。2.组织学检查：根皮苷治疗10周后,肝细胞肿胀坏死,肝细胞索状结构消失,肝细胞质内大量圆形脂滴、脂肪变性等肝细胞严重损伤症状得到明显改善。3.经LC-LTQ质谱检测和数据分析,共鉴定出1821个蛋白。以114作为对照,116标记峰的峰面积与117峰面积的比值大于1.5或小于0.67的蛋白即为在根皮苷治疗过程中表达有显著差异的蛋白,共261个。215个蛋白在DM组的表达水平与对照组相比升高,经根皮苷治疗后表达水平明显回落。46个蛋白在DM组的表达水平与对照组相比下降,经根皮苷治疗后表达水平明显上升。其中,涉及糖酵解和丙酮酸氧化脱羧过程的关键酶与对照组相比在DM组表达水平上升,经根皮苷治疗后表达水平明显回落,同时三羧酸循环中的限速酶经根皮苷治疗后表达水平与DM组相比明显升高。可见,根皮苷治疗后用于脂质合成的原料-乙酰辅酶A的数量减少。涉及脂酸合成和胆固醇合成的关键酶与对照组相比在DM组的表达水平上调,经根皮苷治疗后表达水平明显降低,而脂酸p-氧化的限速酶经根皮苷治疗后被激活,表达水平与DM组相比明显升高。同样与氧化应激过程相关的抗氧化酶在DMT组也被激活,表达水平与DM组相比明显升高,而与产生自由基相关的酶在DMT的表达水平与DM组相比明显下降。4.使用IPA软件对261个差异蛋白进行定位分析显示大多数差异蛋白为胞浆蛋白,主要涉及脂质代谢、碳水化合物代谢和自由基清除等生物过程。得分最高的两个蛋白相互作用网络也主要与脂质代谢、能量代谢、小分子生物化学等过程相关,这与根皮苷的作用过程相一致。5. Western blot结果显示ACACA、HMGCS2和NSDHL在DMT组的表达水平与DM组相比明显降低,CAT在DMT组的表达水平与DM组相比明显升高,变化趋势与iTRAQ结果相一致。因此,iTRAQ的实验结果真实可靠。研究结论及意义根皮苷能有效减轻糖尿病小鼠体重,控制血糖、甘油三酯和总胆固醇等生化指标。肝脏差异蛋白质组学研究结果表明根皮苷通过影响碳水化合物代谢、脂酸合成和p-氧化、胆固醇合成及自由基清除等生物过程中的关键蛋白来达到改善脂质代谢和减轻氧化应激等对肝脏细胞造成的损伤,从而起到保护糖尿病小鼠肝脏的作用。目前,临床上常用的口服降糖药虽对糖尿病治疗有一定效果,但同时也存在一定的副作用。而根皮苷作为从苹果属中提取的纯天然物质,有着良好的安全记录副作用小,且又具有抗氧化等多种功效。因此,根皮苷作为一种新型的功能成分,将为糖尿病预防和治疗提供新的思路和手段。更多还原

【Abstract】 IntroductionOsteoporosis is a systemic skeletal disease, with the characteristics of reduction in bone mass, deterioration in bone microstructure, increase in brittleness, and inclination for bone fracture. Osteoporosis frequently occurs in elder people, especially postmenopausal women.Bone is a dynamic tissue that undergoes continuous remodeling/renewal to maintain skeletal appearance and structural integrity. In adults, bone destruction and formation are kept in a dynamic balance, and bone mass is maintained in a steady state, which is determined by mechanical usage and largely by homeostatic factors. Starting from the fifth decade of life or menopause for women, bone destruction begins to exceed bone formation, resulting in local or systemic bone loss and bone fragility, vertebrae collapses, fractures, and ultimately disabling the mobility of individuals having bone-related disorders such as osteoporosis.The increasing risk of osteoporotic fracture in seniors is associated with progressive loss of cancellous and cortical bone, partially caused by reduced osteoblastic bone formation. Recently, several studies have unveiled a link between accumulation of bone marrow fat and age-related bone loss, as in the cases of glucocorticoid treatment, immobilization, and ovariectomy. Marrow fat not only exerts a passive role by simply occupying the space, but also participates in lipid metabolism and functions as a localized energy reservoir, thus supporting blood cell lineage and osteoblasts in bone formation. Adipocytes may influence bone formation through the secretion of fatty acids and adipokines that regulate the differentiation of marrow precursors into osteoblasts. There is an inverse correlation between accumulation of adipocyte in bone marrow tissue and bone volume in osteoporotic patients. An increasing body of evidence supports a reciprocal relationship between MSC-derived adipocytes and MSC-derived osteoblasts. Bone marrow mesenchymal stem cells (BMSCs) have the capacity for renewal and the potential to differentiate into several cell types including osteoblasts, chondrocytes, adipocytes, astrocytes, myocytes, oligodendrocytes, and neurons. Differentiation of BMSCs into osteoblast and adipocyte lineages is particularly related to the maintenance of normal bone homeostasis.Therefore, the underlying mechanism, which regulates the differential direction of BMSCs is crucial for the study of etiology of osteoporosis. Particularly, Wnt signal transduction pathway regulates cell differentiation and growth via paracrine or autocrine. The canonical Wnt signaling pathway is activated when Wnts interact with Lrp/Fzd receptor complexes. Receptor engagement activates an unknown kinase(s)(K) that phosphorylates the cytoplasmic tail of Lrp5/6. These phosphorylated residues (P) serve as docking sites for Axin, APC, Dsh, and β-Catenin complex. The interaction results in the inhibition of GSK-3activity, causing the prevention of β-Catenin degradation and accumulation of β-Catenin in the cytoplasm. Upon reaching a certain concentration level, β-Catenin translocates to the nucleus where it associates with the Tcf/Lef family of transcription factors to regulate the expression of the target genes and bone formation.ObjectivesTo demonstrate that adipocytes have an inhibitory effect on osteoblastogenesis and elucidate the underlying molecular mechanisms during adipocyte-regulated osteoblastogenesis.Methods1. hBMSCs were cultured in two types of co-culture modes. In each mode, three groups were designated according to the quantity of adipocytes as follows:Group i, hBMSCs were treated without adipogenic induction; Group ii, hBMSCs were stimulated under an adipogenic condition using a half dose of stimulators; Group iii, hBMSCs were stimulated under a standard adipogenic condition. Mode A, co-culture with intercellular contact (direct regulation):osteoblasts and adipocytes were present in the same plate. Briefly, human hBMSCs were induced into adipocytes according to the above grouping for14days, followed by a uniform osteogenic stimulation in adipocyte maintenance medium for another14days.Mode B, co-culture without intercellular contact (indirect regulation):osteoblasts and adipocytes were plated on separate surfaces within the same well of transwell plates. Briefly, human hBMSCs were induced into adipocytes according to the above grouping for14days within the netwell inserts. At the same time, human hBMSCs were cultured on the lower surfaces of the Snapwell. These two parts were later assembled and cells were maintained in adipocyte maintenance medium using uniform osteogenic stimulation as above for another14days.2. To analyze the activity of the alkaline phosphatase (ALP) on day14, we performed ALP staining and quantitative ALP activity assay using the Alkaline Phosphatase Activity Assay Kit in Mode A and B. To analyze mineralization on day28, Alizarin Red S staining was performed in Mode A. To examine adipogenesis on day14, we performed oil red staining in Mode A. Proteins were separately extracted from the insert wells and snap wells in Mode B to determine the expression levels of osteoblastogenesis marker-Runx2and adipogenesis marker-Ppary in Mode B.3. To understand the influence of MSC-derived adipocytes on osteoblast differentiation, mRNA expression profile in osteoblasts from Snapwells in Groups i, ii, and iii in Mode B were analyzed using mRNA microarray. The expression level of S100A6mRNA was validated by RT-PCR.4. To study the mechanism underlying the influence of adipocytes on osteoblastogenesis during MSC differentiation, we studied changes in protein expression during adipocyte-regulated osteoblastogenesis. Total proteins from cultured cells in Mode B were extracted. The proteins were separated using immobilized pH gradients2-DE and visualized by Sypro-Ruby staining. The differential protein spots were cut from the gels and subjected to in-gel digestion with trypsin. The peptides were desalted by using C18ziptips, and then subjected to identification by MALDI-TOF/TOF MS. The expression levels of Runx2, β-Catenin, S100A6and Calreticulin were confirmed using Western blot analysis.5. To further understand the mechanism underlying inhibition on osteoblastogenesis by adipocytes, medium proteins from adipocyte maintenance medium from Mode B on day4after osteogenic stimulation were analyzed using a human adipokine array.6. Using the ELISA Development kit, we determined the amount of active TGF-β1in the co-culture media for MSCs from Group i to iii in Mode B after4,7,10, and14days.7. Anti-human TGF-P neutralizing antibodies were added to both the upper and lower chambers in group iii, at5μg/ml (treatment2, Trt2) and10μg/ml (treatment3, Trt3) in Mode B at different time points,0day,4days,7days and10days of the culture. Equal volume of PBS was applied in negative control (treatment1, Trt1). After14days of co-culture, cells were collected for ALP staining, analysis of ALP activity and Western blot.Results1. In mode A, the quantity of adipocytes, as shown in oil red staining, increased significantly from Group i to Group iii, while the number of osteoblasts reduced progressively from Group i to Group iii as shown in ALP-positive area and calcified area. The inverse correlation of MSC-derived adipocytes and osteoblasts was also shown in the indirect co-culture Mode B. The results of Western blot analysis showed that Runx2was significantly down-regulated in Groups ii and iii compared to Group i. Conversely, the levels of Ppary were low in Group i but significantly higher in Groups ii and iii.2. The results from mRNA microarray revealed that247genes were significantly down-regulated, and163genes were substantially up-regulated when comparing Group i to Groups ii and iii. The genes related to osteoblastogenesis, COL1A1, Notchl and S100A6, were over-expressed in Group i, and significantly down-regulated in the other groups; yet the genes related to adipogenesis, Calreticulin, Ppary and SMAD6were low in Group i but up-regulated in Groups ii and iii. The expression of S100A6was validated by RT-PCR. Among the differentially expressed proteins, proteins related to osteoblast functions, such as Annexin A2and S100A6, were down-regulated significantly in Groups ii and iii compared to Group i. In contrast, the level of Calreticulin was low in Group i, but up-regulated remarkably in Groups ii and iii. The differentially expressed proteins were further verified by Western blot analysis. S100A6and Calreticulin appeared in both mRNA and protein expression profiles.3. Among the differential expressions of secreted adipokines, there were23adipokines, such as TGF-β, which were significantly up-regulated in Groups ii and iii as compared to Group i; on the other hand, there were25adipokines, such as MCP-3, which were remarkably down-regulated in Group ii and iii as compared to Group i. ELISA assay shows that the levels of active TGF-P were much higher in the media of Group ii and iii. The difference was detected starting from day4and lasted for14days. To illustrate TGF-P regulation of osteoblastogenesis, we neutralized the TGF-β produced by adipocytes in Group iii of the co-culture. The percentage of osteoblasts and the ALP activity after TGF-P neutralizing were increased in Trt2and Trt3compared to Trt1. The expression levels of bone formation markers such as Runx2, S100A6and β-Catenin were significantly up-regulated in Trt2and Trt3compared to Trt1. Conversely, the level of Calreticulin was high in Trt1but significantly reduced in Trts2and3by Western blot analysis.ConclusionIn summary, the present study demonstrates that adipocytes regulate osteoblast differentiation indirectly through secreted factors. The increase in TGF-P in the medium, in accord with the increase in adipocyte numbers, could affect the expressions of calreticulin and S100A6to suppress canonical Wnt signaling activity and osteoblast differentiation. Our study elucidates the mechanism by which adipocytes regulate osteoblast differentiation, thus providing clues for how TGF-P inhibits osteoblast differentiation in later phases. These results revealed partial reasons for reduction in bone mass and bone formation in osteoporotic patients, offering new clues for further study on the inhibitory mechanism of adipocytes on bone formation in the future. Meanwhile, our study discovered new target proteins for the prevention and treatment of osteoporosis. IntroductionType2diabetes is one of the most common endocrine diseases in developed countries, which is caused by absolute or relative deficiencies in insulin secretion or insulin action. Chronic hyperglycemia is the major biochemical alteration in type2diabetes. Moreover, individuals with type2diabetes usually display a marked disruption of lipid metabolism, with an abnormal accumulation of fat in various tissues including the liver. Hyperglycemia and hyperlipidemia not only impair β-cell function and increase insulin resistance in peripheral tissues, such as the muscle, liver and adipose tissue, but also induce oxidative stress reactions, which cause initiation and progression of diabetes-related diseases such as diabetic cardiovascular disease, diabetic nephropathy and diabetic retinopathy. Consequently, regulation of dyslipidemia and reduction in oxidative stress have been regarded as important treatment methods for alleviating diabetes and its complications.Phlorizin(glucose,1-[2-(p.D-glucopyranosyloxy)-4,6-dihydroxyphenyl]-3-(4-hyd roxyphenyl)-1-propanone) is a member of the chalcone class of organic compounds and is mainly distributed in the plants of the genus Malus. Phlorizin has multiple pharmacological activities, such as anti-oxidative, estrogenic and anti-estrogenic activities, memory improvement and cardioprotective activities. Reports show that phlorizin inhibits intestinal glucose uptake and renal glucose reabsorption by inhibiting the sodium D-glucose cotransporter. Phlorizin has been reported to normalize the effects of insulin on glucose metabolism in the liver and other peripheral tissues. However, little is known regarding the effect of phlorizin on hepatic damage associated with type2diabetes. With the completion of human genome sequencing project, life science has entered post-genomic era. Proteomics is regarded as the most important part in post-genomic studies. The rapidly emerging field of quantitative proteomics provides a powerful technique-isobaric tag for relative and absolute quantitation (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the identification and characterization of protein profiles. This technique can lessen variation, enhance throughput and enable quantitative analysis.ObjectiveTo investigate the protective mechanism of phlorizin on hepatic damage in diabetic db/db mice through profiling the global protein expression changes.Methods1. Male C57BLKS/J db/db and age-matched db/m mice (n=24,7weeks of age) were kept under observation for one week prior to the start of the experiments. C57BLKS/J db/m mice were selected as the control group (n=8). The db/db mice were randomly divided into two groups:the vehicle-treated diabetic group (DM, n=8) administered normal saline solution and the other diabetic group treated with phlorizin at a dosage of20mg/kg (DMT, n=8). Phlorizin was administered in normal saline solution by intragastric administration for10weeks. Each group of mice was observed without any administration of other hypoglycemic therapy throughout the experiment. At the end of the intervention, all mice were fasted overnight and then sacrificed. Fasting blood was collected, and the liver tissue was dissected. The tissues and sera were kept at-80℃until further analysis.2. Animals were weighed every week. The levels of fasting blood glucose (FBG), blood triglycerides (TG) and blood total cholesterol (TC) were determined using DVI-1650Automatic Biochemistry and Analysis Instrument (Bayer, Germany) at the end of the treatment.3. In order to ascertain whether phlorizin has any beneficial effect on hepatocellular damage in db/db mice, the excised parts of the livers were immediately fixed in10%paraformaldehyde and embedded in paraffin. After solidification,5-μm sections were cut from the blocks. After hematoxylin and eosin (H&E) staining, the sections were examined using light microscopy.4. To investigate the protective mechanism of phlorizin on hepatic damage in diabetic db/db mice, we studied the global protein expression changes in the liver using iTRAQ approach. Total proteins were extracted from the pooled liver tissue from each of four mice from each group. After the proteins were digested using filter aided proteome preparation (FASP), the peptides of each group was labeled with iTRAQ reagents (114for the peptides of the control group,116for the peptides of the DMT group, and117for the peptides of the DM group). The labeled samples were separated by strong cation exchange (SCX) chromatography. Mass spectrometric analysis was performed using a micro-liquid chromatography system and a LTQ-Velos ion trap mass spectrometer. For protein identification and statistical validation, the acquired MS/MS spectra were automatically searched against the non-redundant International Protein Index (IPI) mouse protein database (version3.72) using the Turbo SEQUEST program in the Bio WorksTM3.1software suite. Data filtering parameters were chosen to generate false-positive protein identification rates of<1%.5. The differentially expressed genes were analyzed through the use of Ingenuity Pathway Analysis.6. The expression levels of CAT、ACACA、HMGCS2and NSDHL were confirmed using Western blot analysis.Results1. The initial, final, and gain in body weight in the DM group were significantly higher than those values in the control group. However, the body weight gain was significantly inhibited from the second week after phlorizin administration in the DMT group compared to the DM group.The levels of FBG, TG and TC in the DM group were markedly elevated compared with the levels in the control group. Moreover, these three parameters were significantly decreased by10weeks of phlorizin administration in the DMT group when compared with the DM group.2. The degree of hepatocellular damage was higher in the livers of the DM group than that of the control group. Yet, phlorizin treatment significantly ameliorated the hepatocellular damage in DMT group.3. Protein profiling was analyzed using iTRAQ approach. A total of1821proteins were identified. A strict cutoff value of a1.5-fold change was used for identification of differential proteins. Two hundred and fifteen proteins were elevated in the DM group compared with the control group and were then inhibited by phlorizin treatment. Forty-six proteins were inhibited in the DM group compared with control group and then restored by phlorizin treatment. The combination of enhancement in glycolysis and suppression in pyruvate oxidation leads to accumulation of mitochondrial acetyl-CoA, which can be transported into the cytosol and utilized for de novo biosynthesis of fatty acids and cholesterol. The key-limiting enzymes in glycolysis and oxidation of pyruvate were down-regulated in the DMT group compared to the DM group. Meanwhile, the key enzymes involved in the TCA cycle were up-regulated in the DMT group, indicating the reduction in the amount of acetyl-CoA. The rate-limiting enzymes for fatty acid biosynthesis and cholesterol biosynthesis were down-regulated in the DMT group compared to the DM group. However, in the DM group, several enzymes involved in fatty acid β-oxidation were down-regulated. Part of the inhibition was reversed by phlorizin administration. Meanwhile, the key anti-oxidative enzymes appeared to be down-regulated in the DM group but were stimulated again by phlorizin treatment. The enzymes involved in generation of superoxide and hydrogen peroxide was decreased in the DMT group.4. Ingenuity Pathway Analysis with all261differentially expressed proteins showed that most of the proteins were from the cytoplasm and the top-ranked biological functions included lipid metabolism and free radical scavenging. The top two protein networks generated by the pathway analysis consisted of proteins involved in lipid metabolism, energy production and small molecule biochemistry, which was consistent with the biological function of phlorizin.5. Four candidate proteins were validated using western blot analysis. ACACA, HMGCS2and NSDHL were found to be inhibited whereas CAT was enhanced in the DMT group compared to the DM group. This result verified the reliability of the iTRAQ results.ConclusionFor the first time, we established quantitative iTRAQ profiles of global liver proteins in a db/db diabetic mouse model treated with or without phlorizin. Phlorizin ameliorated hyperlipidemia and oxidative stress by affecting the expression of a set of proteins which were involved in carbohydrate metabolism, fatty acid synthesis and (3-oxidation, cholesterol biosynthesis, and free radical scavenging in the mouse liver. At the same time, phlorizin successfully decreased body weight, blood glucose, blood TG, and blood TC. Our study provides novel information in regards to the mechanisms of phlorizin on type2diabetic hepatic damage. Phlorizin, as a natural product, may become an effective therapeutic agent in the treatment of diabetes mellitus, particularly for complications in the liver.更多还原