【作者】 何旭；

【导师】 施勤；

【作者基本信息】 苏州大学 ， 骨外科学， 2024， 硕士

【摘要】 骨质疏松症是一种随着年龄增长而逐步进展的全身性骨骼疾病,是一种以骨密度降低和骨骼微结构退化为特征的慢性疾病,易致骨骼脆性增加和骨质疏松症骨折风险增加。芹菜素(Apigenin,API)作为一种类黄酮类化合物已被证明对多种疾病具有保护作用。本课题旨在研究在骨质疏松症环境下,API对骨代谢的影响及其作用机制。第一部分芹菜素活化SIRT1/HIF1α信号促进骨质疏松症来源的间充质干细胞成骨分化目的:利用卵巢摘除术(Ovariectomy,OVX)后来源的大鼠骨髓间充质干细胞(Bone mesenchymal stem cells from osteoporotic rats,OP-BMSCs),研究API对OP-BMSCs成骨分化的影响;并探究API对OP-BMSCs成骨分化的具体作用机制。方法:首先分离OVX后来源的OP-BMSCs,并对提取的OP-BMSCs并进行流式细胞术鉴定。并通过细胞增殖和毒性试剂盒(Cell counting kit-8,CCK-8)探究API浓度变化对OP-BMSCs和健康小鼠骨髓来源的巨噬细胞(Bone marrow-derived macrophages,BMMs)增殖情况的影响,以选择适合的API浓度。API干预OP-BMSCs成骨分化,在诱导成骨的第5天,提取细胞总RNA检测成骨相关基因(Ocn、Runx2、Bmp2、Alp)的表达情况。在成骨诱导的第7天提取成骨细胞蛋白,利用蛋白质免疫印迹(Western blot,WB)检测成骨相关蛋白(RUNX2、SP7、OCN)的表达情况。在成骨诱导第7天进行碱性磷酸酶染色(Alkaline phosphatase staining,ALP)和AKP活力值检测。在成骨诱导第14天时进行茜素红染色(Alizarin red staining,ARS)检测各组钙结节的形成情况。然后,我们对API干预7天的成骨细胞进行了RNA测序(RNA sequencing,RNA-seq)和富集分析。在成骨诱导的第7天,通过细胞免疫荧光(Immunocytochemistry,ICC)和WB验证了RNA-seq感兴趣基因SIRT1和HIF1α的表达情况。并利用SIRT1选择性抑制剂(EX527),在成骨诱导第7天和第14天分别进行ALP染色和ARS染色来探究API是否通过SIRT1影响OP-BMSCs成骨分化。同时提取OP-BMSCs的蛋白验证EX527抑制SIRT1后信号通路的变化。此外,在破骨诱导第5天通过抗酒石酸酸性磷酸酶(Tartrate-resistant acid phosphatase,TRAP)染色观察API对BMMs破骨分化的影响。并利用实时荧光定量PCR(Quantitative real-time PCR,q RT-PCR)检测基因的表达。结果:(1)通过显微镜观察和流式细胞术对原代OP-BMSCs进行鉴定,CD44、CD29的阳性率分别为95.5%和97.5%,而CD34、CD45的阳性表达率分别为2.45%和5.52%。CCK-8结果显示在0.005到5μM的范围内,API不会影响OP-BMSCs的增殖,并且5μM对BMMs活力无影响。因此5μM被选作最适浓度进行后续实验。(2)q RT-PCR结果显示,与单纯成骨诱导(OB)组相比,API的干预后显著提高了OP-BMSCs成骨相关基因Ocn、Runx2、Bmp2、Alp的表达。WB结果显示,API的干预使成骨细胞中RUNX2和SP7蛋白的表达增强。(3)在成骨诱导7天后,ALP染色及AKP定量结果显示,相较于OB组而言,API的干预显著提升细胞ALP的表达。在成骨诱导14天后,ARS染色及定量结果显示,API的干预促进了成骨细胞骨矿化的能力。(4)RNA-seq结果表明,API可能通过调控SIRT1/HIF1α通路来促进OP-BMSCs成骨分化。WB和ICC结果显示,与OB组相比,API的干预显著提高了成骨细胞SIRT1和HIF1α的表达水平。(5)利用API干预OP-BMSCs成骨分化,同时加入1μM的EX527抑制SIRT1的活性。在成骨诱导的第7天和第14天分别进行ALP和ARS染色结果显示,与OB组相比,API的干预增加了成骨细胞ALP的表达和矿化钙结节的形成,而同时加入API和EX527后,成骨细胞ALP的表达和矿化钙结节形成的能力下降。(6)WB结果显示,与OB相比,API的加入促进了成骨细胞中SIRT1和HIF1α的表达,而加入EX527后HIF1α的表达水平下降。(7)在破骨分化第5天进行TRAP染色,结果显示,与破骨细胞组相比,API的加入显著抑制BMMs的破骨分化。q RT-PCR结果显示,与破骨细胞组相比,API的加入显著抑制破骨相关基因(Nfatc、Ctsk、Trap)的表达。结论:API促进OP-BMSCs成骨分化,与激活SIRT1/HIF1α信号相关;同时API抑制BMMs破骨分化。第二部分芹菜素缓解卵巢摘除术后小鼠的骨质流失目的:在OVX后,给小鼠腹腔内注射API,分析小鼠的骨量、骨形成和骨吸收变化,研究API对OVX小鼠骨流失的作用。方法:购买20只6周龄的雌性C57BL/6小鼠,10只为假手术(Sham)组,10只为OVX组。适宜环境饲养1周后对小鼠进行双侧卵巢摘除术,以建立骨质疏松症模型,Sham组只暴露卵巢,不对卵巢进行切除。术后1周开始给予小鼠API(100mg/kg,PBS为溶剂)处理,每3天注射1次,持续8周,将Sham组和OVX组各随机分为2组,即Sham+PBS组、Sham+API组、OVX+PBS组和OVX+API组,每组5只。在标准饲养环境下饲养8周后,分别收取小鼠的血清和下肢骨标本。使用小动物三维断层扫描系统(Micro computed tomography,Micro-CT)扫描和分析各组小鼠下肢骨骨参数的变化,并对新鲜小鼠股骨标本进行三点力学试验。通过H&E染色、Masson染色、TARP染色和免疫组织化学染色观察骨组织标本组织学的变化。通过酶联免疫吸附剂测定(Enzyme-linked-immunosorbent-assay,ELISA)检测血清中I型原胶原氨基端原肽(Procollagen-I-n-terminal-propeptide,PINP)和I型胶原交联羧基端肽(Crosslinked-c-telopeptide-of-type-I-collagen,CTXI)的含量。对小鼠股骨组织切片行SIRT1、HIF1α等免疫组织化学和免疫荧光组织化学染色。结果:(1)Micro-CT扫描结果显示,与Sham+PBS组小鼠相比,OVX+PBS组小鼠骨量明显下降。与OVX+PBS组相比,API的干预可延缓OVX导致的松质骨骨流失。另外,三点力学实验结果显示,与OVX+PBS组相比,API的干预明显提高了OVX+API组小鼠股骨的最大负荷和刚度。(2)H&E染色和骨小梁定量结果显示,与Sham+PBS组相比,OVX术后导致OVX+PBS组小鼠的骨小梁更紊乱,骨髓腔间隙变大和生长板下骨小梁数量减少,而API的干预增加了小鼠的骨小梁体积,使骨小梁排列更加紧密和骨小梁网络的丢失减少。Masson染色和定量结果显示,与OVX+PBS组小鼠相比,API的干预增加OVX+API组小鼠股骨生长板下新生骨的形成。(3)小鼠股骨钙黄绿素双标结果表明,OVX+PBS组小鼠的骨矿物质附着能力(Mineral apposition rate,MAR)比Sham+PBS组低了50%,而API的干预后显著增加了OVX+API组小鼠的骨矿物质附着能力。免疫组化染色结果显示,与OVX+PBS组小鼠相比,API的干预使OVX小鼠股骨生长板区域的成骨标志物RUNX2的阳性率和Osterix(SP7)的阳性率明显增加。(4)血清ELISA结果表明,与Sham+PBS相比,OVX术后会显著降低OVX小鼠血清内骨形成指示物PINP水平,而API的干预可以缓解OVX术后导致的PINP水平降低;相反,OVX术后会提高小鼠血清内骨吸收指示物CTXI水平,而API的干预可以显著降低OVX术后导致的CTXI水平升高。(5)此外,TRAP染色结果证实,与OVX+PBS组小鼠相比,API的干预显著减少了小鼠生长板下破骨细胞阳性率(Osteoclast surface/bone surface,Oc.S/Bs)。(6)小鼠股骨切片体内组织染色结果验证,发现API使小鼠生长板下和骨髓中SIRT1和HIF1α的表达升高。结论:API的干预显著增强了OVX小鼠的骨形成能力,并降低了其骨吸收能力,有效延缓了小鼠的骨量流失。第三部分靶向MSCs递送芹菜素缓解卵巢摘除术后小鼠的骨质流失目的:构建E7化脂质体靶向BMSCs以提高API体内生物利用率,研究E7修饰的脂质体递送API对OVX小鼠骨流失的作用。方法:首先利用薄膜水合法制备单纯脂质体(Lipo)、E7修饰的脂质体(Lipo-E7)和负载API的E7-Lipo(API@Lipo-E7)。利用透射电镜、Zetasizer Advance和酶标仪表征脂质体形态、粒径、Zeta电位和包封率。并通过Di O染色法和流式细胞术研究修饰脂质体对OP-BMSCs的靶向能力。选取6周龄雌性C57BL/6小鼠共20只,适宜环境饲养1周后对小鼠进行双侧卵巢摘除术以建立骨质疏松症模型,术后1周,将Sham组和OVX组小鼠分为4组,即Sham组,OVX+PBS组,OVX+API组和OVX+API@Lipo-E7组每组5只,通过尾静脉注射对小鼠进行不同的干预。在标准饲养环境下饲养8周后,分别收取小鼠的血清、内脏和下肢骨标本。利用Micro-CT扫描分析各组小鼠下肢骨通用参数的变化,并对内脏和骨组织标本进行组织学染色。使用ELISA检测血清中PINP和CTXI的含量。结果:(1)透射电镜结果显示,Lipo、Lipo-E7和API@Lipo-E7具有均匀的球形外观。(2)Lipo,Lipo-E7和API@Lipo-E7的平均直径为144.00±7.55nm、142.33±8.08nm和145.00±6.25nm,且具有良好的分散性。此外,Lipo,Lipo-E7和API@Lipo-E7的平均Zeta电位分别为-30.53±0.87 m V、-22.33±1.49 m V和-22.97±1.21 m V。API@Lipo和API@Lipo-E7的包封率分别为77.0±4.9%和74.5±4.8%。(3)通过荧光显微镜和流式细胞术检测BMSCs对脂质体的摄取,结果显示Lipo-E7与API@Lipo-E7的荧光强度显著高于Lipo组。(4)体内生物相容性H&E染色结果显示,Lipo,Lipo-E7和API@Lipo-E7的尾静脉注射对小鼠内脏无明显影响。(5)Micro-CT扫描结果显示,与OVX组相比,API@Lipo-E7的干预可延缓OVX导致的松质骨骨流失。(6)H&E染色和骨小梁定量结果表明,与OVX组相比,API@Lipo-E7的治疗增加了小鼠的骨小梁体积,使骨小梁排列更加紧密和减少了骨小梁网络的丢失。(7)血清ELISA结果表明,与OVX相比,OVX术后会显著降低小鼠血清内骨吸收标志物PINP水平,而API@Lipo-E7的干预可以缓解OVX术后导致的PINP水平降低;相反,OVX术后会提高小鼠血清内骨吸收标志物CTXI水平,而API@Lipo-E7的治疗可以显著降低OVX术后导致的CTXI水平升高。结论:API@Lipo-E7的干预明显提高了API在体内的生物利用率,并改善了OVX引起的小鼠骨量丢失。更多还原

【Abstract】 Osteoporosis,a systemic skeletal disease that progresses gradually with age,is characterized by decreased bone mineral density and deterioration of bone microarchitecture,leading to increased bone brittleness and the heightened risk of osteoporotic fractures.Apigenin(API),a flavonoid,has demonstrated protective effects against various diseases.The objective of this study was to examine the impact of API on bone metabolism and elucidate its mechanism of action in the context of osteoporosis.Part Ⅰ:Apigenin promotes osteogenic differentiation of osteoporotic mesenchymal stem cells through activation of SIRT1/HIF1αsignalingObjective:Bone marrow mesenchymal stem cells from osteoporotic rats(OP-BMSCs)were isolated following ovariectomy(OVX)to examine the impact of API on the osteogenic differentiation of OP-BMSCs.RNA sequencing(RNA-seq)was conducted to explore the precise mechanism underlying the influence of API on the osteogenic differentiation of OP-BMSCs.Methods:The isolated OP-BMSCs were initially characterized using flow cytometry.Subsequently,the impact of various concentrations of API on the proliferation of OP-BMSCs and healthy mouse bone marrow-derived macrophages(BMMs)was assessed using the Cell Counting Kit-8(CCK-8)to ascertain the optimal concentration of API for further experimentation.API supplementation influenced the osteogenic differentiation process of OP-BMSCs.On day 5 of osteogenesis induction,total cell RNA was extracted to assess the expression of osteogenesis-related genes(Ocn,Runx2,Bmp2,Alp).Total cell protein was extracted on day 7 of osteogenesis induction,and the expression of osteogenesis-related proteins(RUNX2,SP7,OCN)was analyzed via protein immunoblotting(Western blot,WB).Alkaline phosphatase staining(ALP)and AKP viability values were conducted on day 7 of osteogenic induction.Alizarin red staining(ARS)was performed on day 14 of osteogenic induction to evaluate the formation of calcium nodules in each experimental group.Then,RNA-seq and enrichment analysis of osteoblasts were conducted at 7 days after API intervention.The expression of RNA-seq identified genes of interest(SIRT1 and HIF1α)was confirmed through cellular immunocytochemistry(ICC)and Western blot(WB)analysis on day 7 of osteogenic induction.To investigate whether API affects the osteogenic differentiation of OP-BMSCs via SIRT1,ALP and ARS staining were performed on day 7and day 14 of osteogenic induction,respectively,utilizing the SIRT1 selective inhibitor(EX527).Furthermore,proteins extracted from OP-BMSCs were analyzed to verify alterations in signaling pathways following SIRT1 inhibition by EX527.Additionally,the impact of API on the osteogenic differentiation of BMMs was assessed through Tartrate-resistant acid phosphatase(TRAP)staining on day 5 of osteoblast induction.The expression of osteoblast-related genes was evaluated using quantitative real-time fluorescence PCR(q RT-PCR).Results:(1)The identification of primary OP-BMSCs by microscopic observation and flow cytometry showed that the positivity rates of OP-BMSCs for CD44 and CD29 were95.5%and 97.5%,respectively,while the positive expression rates of CD34 and CD45 were2.45%and 5.52%,respectively.CCK-8 assays indicated that API concentrations ranging from 0.005 to 5μM had no effect on OP-BMSCs proliferation,and 5μM had no impact on BMMs viability.Therefore,5μM was selected as the optimal concentration for subsequent experiments.(2)q RT-PCR analysis demonstrated a significant increase in the expression of osteogenesis-related genes(Ocn,Runx2,Bmp2,Alp)in OP-BMSCs after API intervention compared to the osteogenesis-induced(OB)group alone.Additionally,WB results revealed enhanced expression of RUNX2 and SP7 proteins in osteoblasts due to API intervention.(3)After 7 days of osteogenic induction,ALP staining and AKP quantification revealed that API intervention significantly elevated ALP expression in cells compared to the OB group.Following 14 days of osteogenic induction,ARS staining and quantitative analyses demonstrated that API intervention enhanced the ability of osteoblasts to mineralize bone.(4)RNA-seq results suggested that API might enhance osteogenic differentiation of OP-BMSCs by modulating the SIRT1/HIF1αpathway.WB and ICC results revealed that API intervention significantly elevated the expression levels of SIRT1 and HIF1αin osteoblasts compared to the OB group.(5)API was utilized to intervene in the osteogenic differentiation of OP-BMSCs,while 1μM of EX527 was added to inhibit the activity of SIRT1.The results of ALP and ARS staining at day 7 and 14 of osteogenic induction,respectively,showed that the intervention of API increased the expression of ALP and the formation of mineralized calcium nodules in osteoblasts compared to the OB group,while the simultaneous addition of API and EX527 decreased the expression of ALP and the ability of mineralized calcium nodule formation in osteoblasts.(6)WB results indicated that API supplementation enhanced the expression of SIRT1 and HIF1αin osteoblasts compared to OB,whereas the addition of EX527 decreased the expression level of HIF1α.(7)TRAP staining conducted on day 5 of osteoblastic differentiation revealed that API significantly inhibited osteoclast differentiation in BMMs compared to the osteoclast group.q RT-PCR results showed that API addition significantly upregulated the expression of osteoclast-related genes(Nfatc,Ctsk,Trap)compared to the osteoclast group.Conclusion:API promoted osteogenic differentiation of OP-BMSCs,which was associated with activation of SIRT1/HIF1αsignaling;meanwhile API inhibited osteoclast differentiation of BMMs.Part Ⅱ: Apigenin attenuates bone loss in mice after ovariectomyObjective: After OVX,mice received intraperitoneal injections of API to examine alterations in bone mass,bone formation,and bone resorption,as well as to investigate the impact of API on bone loss in OVX mice.Methods: Twenty 6-week-old female C57BL/6 mice were purchased,with 10 allocated to the sham operation(Sham)group and the remaining 10 to the OVX group.The mice were bilaterally ovariectomized after 1 week of rearing in a suitable environment to establish an osteoporosis model,and only the ovaries were exposed in the Sham group without ovariectomy.Mice were treated with API(100 mg/kg,PBS as solvent)starting at 1 week after surgery and injected every 3 days for 8 weeks,and each of the Sham and OVX groups was randomly divided into 2 groups: the Sham+PBS group,the Sham+API group,the OVX+PBS group,and the OVX+API group,with 5 animals in each group.After 8 weeks of acclimation in a standardized environment,serum and lower limb bone samples were collected from the mice.Bone parameters of the lower limb bones in each group were assessed using Micro computed tomography(Micro-CT)scanning and analysis.Additionally,three-point mechanical tests were conducted on fresh femur specimens obtained from mice.Histological alterations in bone tissue specimens were examined using a variety of staining techniques including H&E staining,Masson staining,TARP staining,and immunohistochemical staining.The serum levels of Procollagen I n-terminal propeptide(PINP)and Cross-linked c-telopeptide of type I collagen(CTXI)in serum.Immunohistochemical and immunofluorescent histochemical staining for SIRT1 and HIF1α were performed on mouse femur tissue sections.Results:(1)Micro-CT scanning results showed that bone mass was significantly decreased in the OVX+PBS group compared to mice in the Sham+PBS group.The intervention of API delayed the OVX-induced bone loss in cancellous bone compared with the OVX+PBS group.In addition,the results of three-point mechanical experiments showed that the intervention of API significantly increased the maximum load and stiffness of the femur in mice in the OVX+API group compared with the OVX+PBS group.(2)H&E staining and quantification of bone trabeculae indicated that postoperative OVX led to more disorganized trabeculae,enlarged bone marrow luminal spaces,and a decreased number of subgrowth plate trabeculae in mice of the OVX+PBS group compared to those in the Sham+PBS group.Conversely,API intervention increased trabecular volume,resulting in tighter trabecular alignment and reduced loss of the trabecular network.Masson staining and quantitative results demonstrated that the intervention of API increased the formation of new bone under the femoral growth plate in the OVX+API group of mice compared to the OVX+PBS group of mice.(3)The results of mouse femur calcein double labeling results showed that the bone mineral apposition rate(MAR)of mice in the OVX+PBS group was 50% lower than that of the Sham+PBS group,whereas the intervention of API significantly increased the bone mineral apposition rate of OVX+API group mice.Immunohistochemical staining indicated that API intervention significantly increased the positivity of the osteogenic marker RUNX2 and Osterix(SP7)in the region of the femoral growth plate in OVX mice compared to mice in the OVX+PBS group.(4)Serum ELISA results showed that OVX significantly decreased the serum endosteal bone formation indicator PINP levels in OVX mice compared to Sham+PBS group,whereas API intervention could alleviate the decrease in PINP levels caused by OVX;on contrast,OVX increased the serum endosteal bone resorption indicator CTXI levels in mice after OVX surgery,whereas API intervention could significantly decrease the elevated CTXI levels resulting from OVX postoperatively.(5)In addition,TRAP staining results confirmed that the intervention of API significantly reduced the mouse subgrowth plate osteoclast surface/bone surface(Oc.S/Bs),compared to mice in the OVX+PBS group.(6)In vivo histological staining of mouse femur sections verified that API elevated the expression of SIRT1 and HIF1α under the growth plate and in the bone marrow of mice.Conclusion: API intervention significantly enhanced bone formation and reduced bone resorption in OVX mice,effectively delaying bone loss.Part Ⅲ: Targeted delivery of apigenin to MSCs alleviates bone loss in mice after ovariectomyObjective: Constructing E7-liposomes targeting BMSCs to improve API bioavailability in vivo and investigating the role of API delivery on bone loss in OVX mice.Methods: Liposomes(Lipo),E7-modified liposomes(Lipo-E7),and API-loaded E7-modified liposomes(API@Lipo-E7)were initially prepared using the thin film hydration method.Liposome morphology,particle size,zeta potential and encapsulation rate were characterized using transmission electron microscopy,Zetasizer Advance and enzyme markers.The targeting ability of modified liposomes on OP-BMSCs was assessed using Di O staining and flow cytometry.A total of 20 6-week-old female C57BL/6 mice were selected and randomly divided,with 5 mice in the Sham group and 15 mice in the OVX group.After 1 week of acclimation in a suitable environment,mice underwent bilateral ovariectomy to establish an osteoporosis model.One-week post-operation,mice in both the Sham and OVX groups were subdivided into four groups,each comprising 5 mice: Sham group,OVX + PBS group,OVX + API group,and OVX + API@Lipo-E7 group.Subsequently,mice received various interventions via tail vein injections.After 8 weeks of acclimation in a standardized environment,serum,viscera,and lower limb bone specimens were collected separately from the mice.Micro-CT scanning was employed to analyze changes in generic parameters of lower limb bones in each group of mice,while histological staining was conducted on visceral and bone tissue specimens.Additionally,serum levels of PINP and CTXI were measured using ELISA.Results:(1)Transmission electron microscopy results revealed that Lipo,Lipo-E7 and API@Lipo-E7 have a uniform spherical appearance.(2)The average particle sizes of Lipo,Lipo-E7 and API@Lipo-E7 are 144.00 ± 7.55 nm,142.33 ± 8.08 nm,and 145.00 ± 6.25 nm,respectively,and have good dispersibility.The average zeta potential of Lipo,Lipo-E7 and API@Lipo-E7 are-30.53±0.87 m V,-22.33±1.49 m V,and-22.97±1.21 m V.The encapsulation rates of API@Lipo and API@Lipo-E7 were 77.0 ± 4.9% and 74.5 ± 4.8%,respectively.(3)The uptake of liposomes by BMSCs was detected by fluorescence microscopy and flow cytometry,which showed that the fluorescence intensity of Lipo-E7 with API@Lipo-E7 was significantly higher than that of the Lipo group.(4)In vivo biocompatibility evaluation through H&E staining revealed that tail vein injection of Lipo,Lipo-E7,and API@Lipo-E7 did not induce any significant effects on the internal organs of mice.(5)Micro-CT scanning results showed that intervention with API@Lipo-E7 delayed OVX-induced bone loss in cancellous bone compared to the OVX group.(6)H&E staining and trabecular quantification analysis revealed that treatment with API@Lipo-E7 increased trabecular volume,resulting in tighter trabecular alignment and reduced loss of the trabecular network in mice compared to the OVX group.(7)The results of serum ELISA showed that OVX significantly decreased the serum endosteal bone resorption marker PINP levels in mice compared with OVX,and the intervention of API@Lipo-E7 could alleviate the decrease of PINP levels caused by OVX.On the contrary,OVX increased the serum endosteal bone resorption marker CTXI levels in mice after OVX,and the treatment of API@Lipo-E7 significantly reduced the elevated CTXI levels caused by OVX postoperatively.Conclusion: API@Lipo-E7 intervention significantly increased API bioavailability in vivo and ameliorated OVX-induced bone loss in mice.更多还原