薯蓣皂苷对破骨细胞分化及骨吸收功能的调控作用及机制研究

目的 观察薯蓣皂苷对破骨细胞(OC)分化及骨吸收功能的调控作用,并探讨可能的机制.方法 取对数期RAW 264.7细胞,分为对照组、低剂量组、中剂量组、高剂量组(加入薯蓣皂苷0,1,3,9μmol·mL-1)、高剂量+茴香霉素组(薯蓣皂苷9μmol·mL-1、茴香霉素2000 mg·L-1).诱导分化第1,7天进行抗酒...     

淫羊藿苷对破骨细胞的分化及骨吸收功能的影响

目的研究淫羊藿苷对破骨细胞的分化及骨吸收功能的影响,评价淫羊藿苷对骨质疏松的预防及治疗作用。方法采用1,25-(OH)2D3诱导兔骨髓单核细胞形成破骨细胞样细胞以及原代分离的乳兔成熟破骨细胞与骨片共培养的方法,考察淫羊藿苷对破骨细胞的分化及骨吸收功能的影响。结果浓度为100、50μmol.L-1的淫羊藿苷明显抑制1,25-(OH)2D3诱导兔骨髓单核细胞形成破骨细胞样细胞的数目,当其浓度为100、50、10μmol.L-1时,明显抑制破骨细胞的骨吸收功能,具体表现在吸收陷窝数目及表面积均明显减少。结论淫羊藿苷不仅可以明显抑制破骨细胞的分化,同时具有抑制破骨细胞的骨吸收功能的作用,提示淫羊藿苷具有改善骨吸收功能亢进的潜力。     

整合素在破骨细胞介导的骨吸收中的作用

在骨吸收的过程中,破骨细胞是唯一效应细胞。破骨细胞表面高度表达的整合素αVβ3通过与骨基质的黏附可促进破骨细胞的成熟与分化,在骨吸收过程中发挥重要作用。     

女贞子提取物对破骨细胞分化及成骨细胞增殖的影响

目的:探讨女贞子提取物对破骨细胞分化及成骨细胞增殖的影响。方法:以1α,25-二羟基维生素D3诱导兔原代骨髓细胞获取破骨细胞,经女贞子醇提物和水煎物低、中、高剂量(均为2、20、200 mg/L)处理后,采用抗酒石酸酸性磷酸酶(TRACP)活性测定法检测各组细胞裂解液中TRACP活性及细胞中TRACP阳性细胞数,采用甲苯胺蓝染色法检测各组细胞骨吸收陷窝数量及骨陷窝面积百分率。以L-抗坏血酸、β-甘油磷酸钠、地塞米松诱导小鼠颅顶前成骨细胞亚克隆14细胞获取成骨细胞,采用MTT法检测各组细胞的相对增殖率,采用碱性磷酸酶(APK)活性测定法检测各组细胞中APK活性。结果:经不同剂量女贞子提取物处理后,TRACP阳性细胞数、骨吸收陷窝数量及其面积均有不同程度的改变,其中女贞子醇提物各剂量组和水煎物高剂量组TRACP活性及阳性细胞数、骨吸收陷窝数量及面积百分率,以及女贞子水煎物中剂量组TRACP活性及阳性细胞数、骨陷窝面积百分率均显著降低或减少,而水煎物低剂量组细胞的骨吸收陷窝数量显著增多(P<0.05或P<0.01);女贞子提取物低、中剂量组细胞的相对增殖率以及提取物各剂量组细胞的APK活性均显著升高,而女贞子提取物高剂量组细胞的相对增殖率均显著降低(P<0.01)。结论:女贞子提取物可抑制破骨细胞的TRACP活性,改变破骨细胞的骨吸收功能和成骨细胞的增殖行为,并增加成骨细胞的APK活性。     

阿仑磷酸钠对体外破骨细胞性骨吸收影响的研究

目的　研究阿仑磷酸钠片对体外破骨细胞性骨吸收的影响。方法　采用破骨细胞体外培养法、显微摄片、显微光密度仪扫描及计算机图象分析技术。结果　Alen(0 5、5、5 0 μmol·L-1)使体外破骨细胞性骨吸收陷窝数呈剂量依赖性减少、吸收陷窝表面积明显减少。结论　Alen通过抑制破骨细胞的活性 ,而直接抑制破骨细胞性骨吸收作用 ,对骨质疏松可能有一定的治疗作用     

蛇床子素对体外培养破骨细胞骨吸收及细胞凋亡的影响

研究蛇床子素对破骨细胞骨吸收的影响及其分子机制。采用体外分离、培养兔破骨细胞, 与盖玻片及骨磨片共同培养, 使用1×10⁻⁵ mol·L⁻¹蛇床子素刺激破骨细胞, 观察活体细胞并依据HE、TRAP、骨陷窝甲苯胺蓝染色鉴定破骨细胞; 进行骨吸收陷窝和面积定量分析, 吖啶橙染色统计凋亡细胞; real time PCR及Western blotting法检测相关基因和蛋白。与空白对照组比较, 1×10⁻⁵ mol·L⁻¹蛇床子素能够明显提高破骨细胞凋亡率并通过抑制RANKL和TRAP等相关基因及JNK1/2磷酸化水平抑制其骨吸收。结果提示, 蛇床子素可以通过RANK+RANKL/ TRAF6/Mkk/JNK途径刺激破骨细胞凋亡并抑制骨吸收。     

葡萄糖对大鼠骨髓破骨细胞分化的影响

目的观察葡萄糖对大鼠骨髓破骨细胞(OC)分化的影响,探讨糖尿病骨质疏松的发病机制。方法用巨噬细胞克隆刺激因子(M-CSF)、RANKL诱导大鼠骨髓单个核细胞分化为OC,同时给予不同浓度的葡萄糖(0、5.5、15、25mmol/L)干预,通过观察抗酒石酸酸性磷酸酶(TRAP)染色阳性OC数、TRAP活性测定、OC膜表面RANKmRNA表达量来分析葡萄糖对OC分化的影响。结果高糖(25mmol/L)组培养7d时TRAP染色阳性OC数及TRAP活性测定均高于对照组(0mmol/L)、葡萄糖5.5mmol/L组(P<0.01);与对照组相比高糖组培养3d时TRAP染色阳性OC数明显增多(P<0.01);葡萄糖呈浓度依赖性上调OC膜表面RANKmRNA的表达,其中高糖组培养的各时间点与其他三组相比差异有显著性(P<0.01,P<0.05)。结论高糖可促进OC的分化,其促进作用始于诱导分化的早期;骨髓微环境中高糖可引起OC分化增多,可能是糖尿病骨质疏松的发病机制之一。     

国产帕屈磷酸钠对体外破骨细胞性骨吸收影响的研究

研究国产帕屈磷酸钠（ＡＰＤ）对破骨细胞性骨吸收的作用。采用破骨细胞的体外培养法和显微摄片、显微光密度仪扫描及计算机图像分析技术。结果表明：ＡＰＤ（５，５０与５００μｍｏｌ·Ｌ－１）使体外破骨细胞性骨吸收陷窝数日呈剂量依赖性减少，低浓度（５μｍｏｌ·Ｌ－１）ＡＰＤ的抑制作用明显高于同类进口药骨磷（ＧＬ２ＭＤＰ）；两药物可使吸收陷窝表面积明显降低。提示ＡＰＤ具有较强的直接抑制破骨细胞性骨吸收的作用，对骨质疏松症可能有一定的疗效。     

加减青娥方体外对RAW264.7细胞向破骨细胞分化的影响

目的 探讨加减青娥方抑制骨质疏松的作用机制。方法 在体外培养的小鼠破骨前体RAW264.7细胞中加入不同浓度的加减青娥方提取物,利用抗酒石酸酸性磷酸酶(TRAP)染色法检测核因子κ B受体活化因子配体(RANKL)诱导的RAW264.7细胞分化为破骨细胞的数量及其活性的影响;利用荧光定量PCR(RT-PCR)法检测RANKL诱导的RAW264.7细胞分化为破骨细胞雌激素受体(ER)mRNA表达。结果 加减青娥方可抑制RANKL诱导的RAW264.7细胞分化为破骨细胞,该作用可能是通过调控ERα mRNA的表达实现的。结论 加减青娥方可通过调控ERα基因的表达,抑制破骨细胞的分化、增殖,从而实现增加骨密度、防治骨质疏松的作用。     

Quercitrin and Taxifolin stimulate osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in RAW 264.7 cells

Flavonoids are natural antioxidants that positively influence bone metabolism. The present study screened among different flavonoids to identify biomolecules for potential use in bone regeneration. For this purpose, we used MC3T3-E1 and RAW264.7 cells to evaluate their effect on cell viability and cell differentiation. First, different doses of chrysin, diosmetin, galangin, quercitrin and taxifolin were analyzed to determine the optimum concentration to induce osteoblast differentiation. After 48 h of treatment, doses ≥100 μM of diosmetin and galangin and also 500 μM taxifolin revealed a toxic effect on cells. The same effect was observed in cells treated with doses ≥100 μM of chrysin after 14 days of treatment. However, the safe doses of quercitrin (200 and 500 μM) and taxifolin (100 and 200 μM) induced bone sialoprotein and osteocalcin mRNA expression. Also higher osteocalcin secreted levels were determined in 100 μM taxifolin osteoblast treated samples when compared with the control ones. On the other hand, quercitrin and taxifolin decreased Rankl gene expression in osteoblasts, suggesting an inhibition of osteoclast formation. Indeed, osteoclastogenesis suppression by quercitrin and taxifolin treatment was observed in RAW264.7 cells. Based on these findings, the present study demonstrates that quercitrin and taxifolin promote osteoblast differentiation in MC3T3-E1 cells and also inhibit osteoclastogenesis in RAW264.7 cells, showing a positive effect of these flavonoids on bone metabolism.     

Platinum nanoparticles suppress osteoclastogenesis through scavenging of reactive oxygen species produced in RAW264.7 cells

Recent research has shown that platinum nanoparticles (nano-Pt) efficiently quench reactive oxygen species (ROS) as a reducing catalyst. ROS have been suggested to regulate receptor activator of NF-κB ligand (RANKL)-stimulated osteoclast differentiation. In the present study, we examined the direct effects of platinum nano-Pt on RANKL-induced osteoclast differentiation of murine pre-osteoclastic RAW 264.7 cells. The effect of the nano-Pt on the number of osteoclasts was measured and their effect on the mRNA expression for osteoclast differentiation was assayed using real-time PCR. Nano-Pt appeared to have a ROS-scavenging activity. Nano-Pt decreased the number of osteoclasts (2+ nuclei) and large osteoclasts (8+ nuclei) in a dose-dependent manner without affecting cell viability. In addition, this agent significantly blocked RANKL-induced mRNA expression of osteoclastic differentiation genes such as c-fms, NFATc1, NFATc2, and DC-STAMP as well as that of osteoclast-specific marker genes including MMP-9, Cath-K, CLC7, ATP6i, CTR, and TRAP. Although nano-Pt attenuated expression of the ROS-producing NOX-family oxidases, Nox1 and Nox4, they up-regulated expression of Nox2, the major Nox enzyme in macrophages. These findings suggest that the nano-Pt inhibit RANKL-stimulated osteoclast differentiation via their ROS scavenging property. The use of nano-Pt as scavengers of ROS that is generated by RANKL may be a novel and innovative therapy for bone diseases.     

Effects of alkylphenols on bone metabolism in vivo and in vitro

Alkylphenols are endocrine disruptors that show estrogen-like effects in various wildlife species. However, little information is available about the action of these chemicals on bone metabolism. We investigated the effects of alkylphenols, such as nonylphenol (NP) and octylphenol (OP), on the formation of bone using several culture systems for osteoclasts and osteoblasts, as well as in vivo experiments. NP and OP dose-dependently inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) in cocultures of mouse spleen cells or mouse bone marrow cells with ST2 cells. However, beta-estradiol at 10(-9)M to 10(-6)M did not affect this process. In contrast, neither compound affected the proliferation and differentiation of rat calvarial osteoblast-like cells (ROB cells). When NP or OP (0.1mg/kg body weight) was administered subcutaneously to pregnant mice at 10 days, 12 days and 14 days post-coitus, fetuses at 17.5 days post-coitus showed stimulation of sternebrae bone calcification. Our findings suggest that alkylphenols have critical effects on the formation of bone by non-estrogenic effects.     

Hexane-Soluble Fraction of the Common Fig, Ficus carica, Inhibits Osteoclast Differentiation in Murine Bone Marrow-Derived Macrophages and RAW 264.7 Cells

Osteoclasts, derived from multipotent myeloid progenitor cells, play homeostatic roles in skeletal modeling and remodeling, but may also destroy bone in pathological conditions such as osteoporosis and rheumatoid arthritis. Osteoclast development depends critically on a differentiation factor, the receptor activator of NF-κB ligand (RANKL). In this study, we found that the hexane soluble fraction of the common fig Ficus carica (HF6-FC) is a potent inhibitor of osteoclastogenesis in RANKL-stimulated RAW264.7 cells and in bone marrow-derived macrophages (BMMs). HF6-FC exerts its inhibitory effects by suppression of p38 and NF-κB but activation of ERK. In addition, HF6-FC significantly decreased the expression of NFATc1 and c-Fos, the master regulator of osteoclast differentiation. The data indicate that components of HF6-FC may have therapeutic effects on bone-destructive processes such as osteoporosis, rheumatoid arthritis, and periodontal bone resorption.     

西归含药血清对脂多糖诱导的小鼠巨噬细胞RAW264.7炎症因子的影响研究

目的 研究西归含药血清对脂多糖诱导的巨噬细胞炎症因子的影响。方法 SD大鼠灌胃给予西归乙醇提取物以制备西归含药血清,采用噻唑蓝（MTT）法观察给予不同浓度的西归含药血清对RAW 264.7增殖的影响;采用1 μg·mL^-1的脂多糖（LPS）建立RAW 264.7 细胞体外炎症模型,酶联免疫吸附测定(ELISA)法检测细胞上清液中肿瘤坏死因子α(TNF-α) 、白细胞介素6(IL-6) 、白细胞介素2(IL-2)的含量。结果 脂多糖刺激细胞后,给药组均引起肿瘤坏死因子α、白细胞介素6、白细胞介素2的高表达;而西归含药血清干预后,能显著抑制肿瘤坏死因子α和白细胞介素6的高表达（P<0.05或P<0.01）;但对白细胞介素2没有显著影响。结论 西归具有抗炎的作用,其抗炎作用可能是通过抑制脂多糖诱导的巨噬细胞炎症反应来发挥的。     

壮骨颗粒对去卵巢大鼠骨质疏松症的影响

目的探讨壮骨颗粒对原发性骨质疏松症的作用机制,为壮骨颗粒治疗骨质疏松症提供实验依据。方法建立6个月龄雌性SD大鼠卵巢摘除骨质疏松模型,模型成立后随机分为A组（假手术对照组）、B组（去卵巢对照组）、C组（钙尔奇-D组）、D组（壮骨颗粒组）,A、B组喂服生理盐水、C组喂服钙尔奇-D、D组喂服壮骨颗粒。喂药12周后处死取材,检测大鼠离体股骨骨密度和血清钙、磷、碱性磷酸酶（ALP）、骨钙素（BGP）、雌二醇（E2）等的浓度。结果 A组大鼠毛皮颜色光泽,饮食、活动均正常;B组大鼠毛皮颜色无光泽,饮食量减少,活动次数降低,体重高于A组（P〈0.01）,与发生骨质疏松症时出现的脾肾阳虚标准相符;C、D组大鼠毛皮颜色光亮,饮食量增加,活动次数增多,体重高于A组（P〈0.05）。B组股骨骨密度及血清E2水平均显著低于A组（P〈0.05）,C、D组双侧骨密度水平显著高于B组（P〈0.05）。4组间的血清钙、磷水平差异无统计学意义（P〉0.05）;B组血清BGP测定结果显著高于A组（P〈0.05）,C、D组也明显高于A组（P〈0.01）,而与B组差异无统计学意义（P〉0.05）;C、D组血清ALP较A组明显升高（P〈0.01）。结论壮骨颗粒可以提高骨质疏松大鼠的骨密度及BGP、E2水平,增强成骨细胞活性,促进骨形成,抑制骨吸收。     

骨质疏松症破骨细胞的形成与骨吸收活性的研究

目的 分析骨质疏松症骨髓单核细胞向破骨细胞转化及其骨吸收活性的变化。了解骨质疏松骨量丢失的发生机理。方法 分离、诱导培养骨质疏松症骨髓单核细胞，观察细胞的生长与分化状况，测定培养早期的细胞分泌细胞因子的水平，以抗酒石酸酸性磷酸酶（TRAP）染色和噬骨试验检测破骨细胞的形成率及其骨吸收活性，结果 在地塞米松和维生素-D3（1，25-OH2D3）的诱导下，骨髓单核细胞可向多核巨细胞转化，并呈现TRAP阳性反应及明显的噬骨性，骨质疏松症组单核细胞早期可高水平分泌白细胞介素1（IL-1β），白细胞介素6（IL-6），随之发生的TRAP阳性细胞转化率和骨吸收活性高于非骨质疏松对照组。结论 骨髓单核细胞向破骨细胞转化及其骨吸收活性的增强，是骨质疏松骨量丢失的原因之一，因单核细胞异常高水平产生的IL-1β和IL-6在其中起介导作用。     

Effects of Echinococcus multilocularis miR-71 mimics on murine macrophage RAW264.7 cells

The microRNAs (miRNAs) are a class of small regulatory non-coding RNA that contributes to the activation of host-pathogen cross-talk during infection. In helminthes, miR-71 is highly conserved and it has recently been detected in nematode exosomes, as well as in the sera and/or fluids of infected humans and mice. However, the role of miR-71 during infection remains poorly characterized. Herein, we show that Ago1 and Ago4, which encode key components of the small RNA-induced silencing complex (RISC), were up-regulated in murine macrophage RAW264.7 cells transfected by Echinococcus multilocularis miR-71 (emu-miR-71) mimics. Using a miRNA PCR array, none of the 84 miRNAs involved in inflammation or autoimmunity were significantly up- or down-regulated in the transfected cells (p > 0.05). Although it did not influence IL-10 production by the treated cells (p > 0.05), the mimics significantly repressed the production of NO 12 h after treatment with LPS and IFN-γ (p < 0.01), identifying another potential mechanism whereby parasites can carefully regulate host levels of NO. These findings indicate that the release of parasite-derived miR-71 into hosts can affect the functions of macrophages, and possibly represents an exciting direction for studies of the interplay between parasites and hosts.