鹿茸胶原酶解物对地塞米松诱导成骨细胞MC3T3-E1凋亡的保护作用

目的研究鹿茸胶原酶解物(VCH)作为治疗药物对地塞米松(DEX)所致MC3T3-E1细胞损伤的影响,探究VCH对糖皮质激素性骨质疏松的保护作用。方法分别用不同浓度的VCH处理成骨细胞MC3T3-E1,MTT法测定细胞活力,通过AnnexinⅤ-FITC/PI双染流式细胞术检测细胞凋亡率,实时PCR及Western印迹检测MC3T3-E1细胞分化相关因子的m RNA及蛋白表达水平。结果 VCH 0.3 g·L~(-1)作用于MC3T3-E1细胞48 h以后能显著促进MC3T3-E1细胞的增殖;VCH可促进骨细胞的活性,可上调成骨细胞分化的标志蛋白ALP的表达和转录,可明显减少DEX所诱导的MC3T3-E1细胞凋亡(P<0.05)。结论 VCH抑制糖皮质激素引起成骨细胞凋亡,促进骨形成,为骨质疏松症的防治提供理论依据。     

左旋肉毒碱对MC3T3-E1成骨细胞及OPG表达的作用

目的探讨左旋肉毒碱对MC3T3-E1成骨细胞及OPG的表达作用,为临床预防骨质疏松提供参考。方法以小鼠骨细胞MC3T3-E1成骨细胞为研究对象,用左旋肉毒碱对其表达进行干预,最后,以免疫组化法检测左旋肉毒碱干预前后OPG蛋白质表达水平的改变,分析左旋肉毒碱对MC3T3-E1成骨细胞OPG的表达作用。结果 1 mmol/L左旋肉毒碱对小鼠成骨细胞无明显作用,10~100 mmol/L左旋肉毒碱能促进小鼠成骨细胞增殖,显著抑制人成骨细胞凋亡,增加小鼠骨密度。结论左旋肉毒碱可通过促进MC3T3-E1成骨细胞OPG的表达,促进成骨细胞增殖,呈剂量依赖性和时间依赖性,并且左旋肉毒碱可抑制人成骨细胞凋亡,延长其存活时间。     

丹参素对地塞米松诱导MC3T3-E1成骨细胞损伤的保护作用

目的 探讨丹参素对地塞米松诱导MC3T3-E1成骨细胞损伤的保护作用。方法 构建地塞米松1μmol/L诱导MC3T3-E1成骨细胞损伤模型;其中,C、D、E组分别采用丹参素10、20、40μmol/L预处理。另设空白对照组(A组)和地塞米松1μmol/L模型对照组(B组)。MTT法检测细胞存活率,二硝基苯肼法和流式细胞术分别检测乳酸脱氢酶(LDH)和活性氧(ROS)水平,Western blot检测p21、核因子E2相关因子2(Nrf2)和血红素氧合酶1(HO-1)蛋白水平。检测丹参素40μmol/L预处理小干扰RNA沉默p21表达(F组)后地塞米松1μmol/L诱导MC3T3-E1成骨细胞(G组)的细胞存活率以及Nrf2和HO-1蛋白水平。结果 与A组比较,B、C、D、E组细胞存活率降低,LDH和ROS水平升高,且p21、Nrf2和HO-1蛋白表达水平升高(P<0.05)。与B组比较,C、D、E组细胞存活率和p21、Nrf2和HO-1蛋白表达水平呈丹参素浓度依赖性升高,而LDH和ROS水平降低(P<0.05)。G组细胞存活率高于B组,但低于E组(P<0.05)。G组...     

克老素抑制地塞米松诱导的MC3T3-E1成骨细胞凋亡

目的探究体外转染克老素(Klotho,KL)基因对地塞米松(dexamethason,DEX)诱导的MC3T3-E1成骨细胞凋亡的影响。方法用携带KL基因的重组腺病毒(Ad-KL)及携带绿色荧光蛋白基因的重组腺病毒(Ad-GFP)感染细胞,并建立DEX诱导凋亡的细胞模型。运用荧光倒置显微镜观察重组腺病毒的转染效果,qPCR与Western blot法分析KL mRNA和蛋白表达情况;CCK-8法检测不同浓度DEX作用于MC3T3-E1细胞后的存活率及各研究组细胞的生存情况;流式细胞仪分析各研究组细胞的凋亡率;qPCR与Western blot分析凋亡标志物的mRNA、蛋白表达;免疫荧光分析caspase-9的荧光蛋白表达情况。结果重组腺病毒成功感染MC3T3-E1成骨细胞,KL组和KL+DEX组的KL mRNA与蛋白表达量较非转染组明显升高。CCK-8法检测出DEX的适宜干预浓度为2.0 mmol·L~(-1)。DEX组、GFP+DEX组较其余组细胞存活率明显降低、凋亡率明显增加,Bax、Bcl-2的mRNA与蛋白表达分别表现出增加和降低趋势;KL组细胞存活率、凋亡率,以及Bcl-2、Bax mRNA与蛋白表达较DEX组及KL+DEX组均明显改善。结论大剂量DEX的运用能够诱发MC3T3-E1成骨细胞凋亡,上调KL表达具有抵抗DEX诱导成骨细胞凋亡的作用,提示上调KL表达可改善糖皮质激素诱导性骨质疏松,为大剂量使用糖皮质激素所致医源性骨质疏松的治疗提供新的靶点。     

白藜芦醇对脂多糖诱导小鼠急性肺损伤的保护作用

目的研究白藜芦醇对脂多糖(LPS)致小鼠急性肺损伤(ALI)的保护作用,探讨其可能的作用机制。方法以小鼠气道滴注LPS制备急性肺损伤模型,检测气道吸气阻力(Ri)、气道呼气阻力(Re)和动态肺顺应性(Cdyn)的变化,测定支气管肺泡灌洗液(BALF)中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的含量,检测肺湿/干比值和毛细血管通透性,观察组织病理学变化。结果白藜芦醇能明显抑制Ri、Re增长和Cdyn降低,降低BALF中IL-1βI、L-6、TNF-α的含量,降低肺湿/干比值和渗透性,减轻肺组织病理学的损伤。结论白藜芦醇对LPS诱导的ALI具有保护作用,作用机制可能与抑制炎症因子的合成与释放有关。     

芫花素对于MC3T3-E1细胞向成骨细胞分化的影响

目的探索芫花素对于MC3T3-E1细胞向成骨细胞分化的影响。方法采用芫花素1、5、10、15μmol/L处理MC3T3-E1细胞14 d,采用MTS法检测芫花素对MC3T3-E1细胞增殖的抑制作用;实时定量PCR和Western blotting法检测测芫花素对MC3T3-E1细胞中ALP、HDAC1和Runx2 m RNA和蛋白表达的影响。结果芫花素5、10、15μmol/L可以显著促进MC3T3-E1细胞中ALP、Runx2 m RNA和蛋白表达水平的提高(P0.05),HDAC1 m RNA和蛋白表达水平的下调(P0.05),表明芫花素可以促进MC3T3-E1向成骨细胞分化。结论芫花素具有促进MC3T3-E1细胞可以促进成骨细胞分化,对成骨细胞的分化过程有一定的调节作用。     

成骨生长肽对MC3T3-E1成骨细胞增殖的影响及其可能机制

目的探讨成骨生长肽(osteogenic growth peptide,OGP)对MC3T3-E1成骨细胞的增殖的影响及其机制。方法常规培养小鼠成骨细胞MC3T3-E1,分为OGP高、中、低剂量组(1×10-8、1×10-10、1×10-12mol/L)以及对照组,利用MTT法检测OGP作用24 h、48 h以及72 h对MC3T3-E1成骨细胞增殖作用,利用免疫化学染色法检测成骨细胞I型胶原蛋白水平,ELISA法检测骨桥蛋白(osteopontin,OPN)含量。结果在24 h时,各剂量OGP对MC3T3-E1的增殖无影响;作用48、72 h,OGP 10-10mol/L以及OGP 1×10-8mol/L显著促进MC3T3-E1细胞增殖(P<0.05),其中OGP 1×10-8mol/L 48 h促进MC3T3-E1细胞增殖作用最强。作用24 h,各组成骨细胞I型胶原蛋白IOD值差异均无统计学意义;作用48 h,OGP 1×10-8mol/L组细胞I型胶原蛋白IOD值显著高于对照组(P<0.05);作用72 h,OGP 1×10-10mol/L组以及OGP 1×10-8mol/L组细胞I型胶原蛋白IOD值均显著高于对照组(P<0.05)。作用24 h,各组成骨细胞OPN OD值差异均无统计学意义;作用48 h、72 h,OGP 1×10-10mol/L组以及OGP1×10-8mol/L组细胞OPN OD值显著高于对照组(P<0.05)。结论 OGP能够明显促进MC3T3-E1成骨细胞的增殖,其可能的作用机制是增加成骨细胞I型胶原蛋白的表达以及OPN的表达,且其作用与浓度密切相关,在1×10-8mol/L时,其促成骨细胞增殖作用最强。     

高糖环境中吡格列酮对MC3T3-E1成骨细胞的作用及其机制

目的观察高糖环境下吡格列酮对MC3T3-E1成骨细胞的作用并探讨其可能机制。方法高糖(22.5 mmol·L~(-1))环境培养MC3T3-E1细胞,分为对照组,吡格列酮2.5、5、10μmol·L~(-1)组,干预24、48 h。检测细胞增殖活性、凋亡率、骨钙素和碱性磷酸酶(ALP)分泌水平,以及过氧化物酶体增殖物激活受体γ(PPARγ)、成骨因子runt相关基因2(Runx2)和骨形态蛋白2(BMP-2)mRNA的表达水平。并分析PPARγ、Runx2的表达与骨钙素、ALP、BMP-2的相关性。结果在相同干预时限,吡格列酮各组MC3T3-E1成骨细胞增殖活性、骨钙素和ALP的分泌水平、Runx2 mRNA和BMP-2 mRNA的表达均低于对照组,凋亡率和PPARγmRNA的表达高于对照组(P<0.05)。随吡格列酮浓度增加,细胞增殖活性、骨钙素和ALP的分泌、Runx2 mRNA和BMP-2 mRNA的表达均降低,而细胞凋亡率和PPARγmRNA的表达增高(P<0.05)。与干预24 h相比,干预48 h时相同浓度吡格列酮组细胞增殖活性,骨钙素和ALP的分泌水平,PPARγ、Runx2、BMP-2 mRNA的表达或无变化或略增加。结论高糖环境下吡格列酮对成骨细胞有损害作用,促进PPARγ表达、抑制Runx2的表达可能为其作用机制之一。     

重组成骨蛋白-1在钛合金微粒环境下对MC3T3-E1成骨细胞增殖、分化、矿化的影响

目的在钛-6铝-4钒(Ti-6Al-4V)合金微粒环境下观察重组成骨蛋白-1(recombinant OP-1,r OP-1)对成骨细胞的影响,为防治关节假体无菌性松动提供新的治疗途径。方法根据小鼠颅顶骨前成骨细胞亚克隆14(MC3T3-E1)中是否加入Ti-6Al-4V微粒和r OP-1,分为微粒组(5、10、15μg/m L Ti-6Al-4V)、处理组(微粒组加入200 ng/m L r OP-1)、阳性组(加入200ng/m L r OP-1)和对照组,检测各组24、72、120 h MC3T3-E1细胞增殖能力、72 h碱性磷酸酶(akaline phosphatase,AKP)、骨钙素(osteocalcin,OCN)和骨桥蛋白(osteopontin,OPN)mRNA的表达,及120 h成骨细胞的矿化能力。结果 1r OP-1无促进Ti-6Al-4V微粒环境下成骨细胞增殖能力,与微粒组比较,差异无统计学意义(P>0.05);2r OP-1可提高成骨细胞分化,与对照组比较差异有统计学意义(P<0.05);同时逆转Ti-6Al-4V微粒抑制成骨细胞分化,与微粒组比较差异有统计学意义(P<0.05);3茜素红S染色后Ti-6Al-4V微粒钙结节数量随着浓度增加逐渐降低,和微粒组比较,加入r OP-1后钙结节数量呈增多趋势。结论 Ti-6Al-4V微粒环境下,r OP-1无提高成骨细胞增殖能力,能提高细胞分化矿化能力,r OP-1可以作为潜在治疗关节假体无菌性松动一种方法。     

Stimulatory effect of Daidzein in osteoblastic MC3T3-E1 cells

Daidzein is a natural isoflavone found in Leguminosae. The effect of daidzein on osteoblastic MC3T3-E1 cells was investigated. Cells were cultured in a serum-free medium for 48 hr in the presence of daidzein (10(-7)-10(-5) M). Daidzein (10(-6) and 10(-5) M) caused a significant elevation of protein content, alkaline phosphatase activity, and DNA content in cells; those increases were about 1.4-, 1.5-, and 2.0-fold, respectively. The ability of daidzein (10(-5) M) to increase protein content, alkaline phosphatase activity, and DNA content in cells was prevented completely by the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis, indicating that the isoflavone has a stimulatory effect on cellular protein synthesis. The anabolic effect of daidzein (10(-6) or 10(-5) M) in cells was not distinguishable from that of genistein (10(-6) or 10(-5) M). Meanwhile, cell protein showed an additive effect of 17beta-estradiol and daidzein, but their effects on alkaline phosphatase were not additive. Moreover, the effect of daidzein (10(-5) M) in elevating cellular protein content and alkaline phosphatase activity was inhibited completely by the presence of tamoxifen (10(-6) M), suggesting that the effect of the isoflavone is mediated partly through estrogen action. This study demonstrates that daidzein has an anabolic effect in osteoblastic MC3T3-E1 cells.     

Inhibitory effects of a tryptamine derivative on ultraviolet radiation-induced apoptosis in MC3T3-E1 mouse osteoblasts

The benzo[b]furan derivative MU314 inhibits in vitro bone resorption as potently as β-estradiol (E(2)). Here, we examined the point of action on the anti-osteoporotic effects of MU314. MU314 (10 nM) suppressed lacunae formation by osteoclastic cells and ICI-182,780, a pure E(2) antagonist, inhibited this effect. Specifically, we ovariectomized (OVX) Wistar female rats and subcutaneously injected them with either MU314 (30 or 100 μg/kg) or E(2) (100 μg/kg) over an 8-week period. Bone mineral content (BMC) in the proximal end of the tibia was significantly decreased (14%) in OVX rats, and MU314 (100 μg/kg) and E(2) significantly suppressed the decline in BMC. OVX rats exhibited decreased cancellous bone in the proximal end of the tibia and induced destruction of its trabecular structure. MU314 suppressed these changes. OVX also reduced the mechanical strength of the femoral neck, which was also recovered by MU314 and E(2). E(2) completely protected against OVX-induced uterine atrophy, but MU314 had no effect. These results strongly indicate that MU314 acts as a selective estrogen receptor modulator.     

Protective effect of apocynin on antimycin A-induced cell damage in osteoblastic MC3T3-E1 cells

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH-oxidase. In the present study, we investigated the protective effects of apocynin on antimycin A (AMA)-induced toxicicy in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to AMA caused significant cell viability loss, as well as mitochondrial membrane potential (MMP) dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca2+]i) elevation and oxidative stress. Pretreatment with apocynin prior to AMA exposure significantly reduced AMA-induced cell damage by preventing MMP dissipation, complex IV inactivation, ATP loss, [Ca2+]i elevation and oxidative stress. These results suggest that apocynin has a protective effect against AMA-induced cell damage by its antioxidant effects and the attenuation of mitochondrial dysfunction. Apocynin also induced the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by AMA. All these data indicate that apocynin may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders.     

Fluoride-induced c-Fos expression in MC3T3-E1 osteoblastic cells

Excessive systemic exposure to fluoride leads to disturbances of bone homeostasis. c-Fos is known to be essential in bone development by affecting osteoblast and osteoclast differentiation. In this study, we examined the effects of fluoride exposure on c-Fos expression and its regulatory signaling pathways in MC3T3-E1 mouse osteoblast cell line. c-fos mRNA level, c-Fos protein level and c-Fos DNA-binding activity were markedly increased, with a peak at 2 or 4?h, in MC3T3-E1 cells exposed to sodium fluoride (NaF). Fra-1 protein, another member of Fos family, was also elevated, whereas FosB and Fra-2 proteins remained unchanged. NaF further induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), ERK5, c-Jun NH₂-terminal kinase and p38. NaF-induced expression of c-Fos protein was markedly suppressed with U0126, the inhibitor of both activated and non-activated forms of MAPK/ERK kinase 1/2 (MEK1/2) and BIX02189, the MEK5 inhibitor, but partially with SP600125, the JNK inhibitor and SB203580, the p38 inhibitor. Therefore, ERK1/2 and ERK5 signal transduction pathways are important for accumulating c-Fos. siRNA targeting against the mouse c-fos gene further enhanced NaF-induced up-regulation of osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, suggesting that c-Fos might negatively regulate OPG expression induced by fluoride in osteoblastic cells.     

Kaempferol protects MC3T3-E1 cells through antioxidant effect and regulation of mitochondrial function

Kaempferol, a natural flavonoid present in fruits, vegetables, and teas, provides beneficial effects for human health. In this study, we investigated the protective effects of kaempferol on antimycin A (AMA)-induced toxicity in osteoblast-like MC3T3-E1 cells. Exposure of MC3T3-E1 cells to AMA caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, intracellular calcium ([Ca²⁺]_i) elevation, and reactive oxygen species (ROS) production. Pretreatment with kaempferol prior to AMA exposure significantly reduced AMA-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, [Ca²⁺]_i elevation, and ROS production. Kaempferol also induced the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B), and CREB (cAMP-response element-binding protein) inhibited by AMA, which result demonstrates that kaempferol utilizes the PI3K/Akt/CREB pathway to augment metabolic activity inhibited by AMA. All these data indicate that kaempferol may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders.     

Costunolide stimulates the function of osteoblastic MC3T3-E1 cells

The effect of costunolide on the function of osteoblastic MC3T3-E1 cells was studied. Costunolide significantly increased the growth of MC3T3-E1 cells and caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and mineralization in the cells (P<0.05). The effect of costunolide in increasing cell growth was completely prevented by the presence of ICI182780, LY294002, PD98059, rotlerin, or glibenclamide, suggesting that the effect of costunolide might be partly mediated from estrogen receptor (ER), PI3K, ERK, protein kinase C (PKC) and mitochondrial ATP-sensitive K(+) channel. The effect of costunolide in increasing ALP activity was prevented by the presence of ICI182780, PD98059, SB203580, or rotrelin, suggesting that the effect of costunolide on ALP activity might be mediated from ER, ERK, p38, and PKC. The effect of costunolide in increasing collagen content was prevented by the presence of LY294002, PD98059, SB203580, SP600125, or rotrelin, suggesting that the effect of costunolide on collagen synthesis might be mediated from PI3K, ERK, p38, JNK, and PKC. Moreover, cotreatment of ICI182780 or LY294002 inhibited costunolide-mediated upregulation of mineralization, suggesting that the induction of mineralization by costunolide is associated with increased activation of ER and PI3K. Our data indicate that the enhancement of osteoblast function by costunolide may result in the prevention for osteoporosis.