基于雌激素受体研究氯化锂干预成骨细胞的分化及自噬
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摘要
背景:临床应用锂治疗造血系统疾病和双极性疾病已经有数十年,并且发现锂可以直接刺激骨髓间充质干细胞的增殖。目的:研究雌激素受体抑制下氯化锂对大鼠成骨细胞增殖分化及自噬的影响。方法:实验选取10只出生72 h的SPF级SD乳鼠(由黑龙江中医药大学提供)。采用酶消化法和茜素红染色法获得和鉴定成骨细胞。取对数生长期成骨细胞,于培养基中加入不同浓度的氯化锂(0,1,2,5 nmol/L)处理24 h,采用CCK-8法检测各组细胞增殖情况。另取成骨细胞随机分为空白组(正常培养细胞)、抑制剂组(10~(-7) mol/L ICI182780)、治疗组(5 nmol/L氯化锂+10~(-7) mol/L ICI182780)。采用钙化结节染色及碱性磷酸酶活性检测各组成骨细胞分化情况;Western blot法测定各组成骨细胞中Beclin1和Runx2蛋白表达情况。结果与结论:(1)倒置显微镜下观察细胞多为单核、多边形及梭形,局部有细胞密集的细胞团;(2)CCK-8法检测结果显示不同浓度的氯化锂都能对成骨细胞增殖产生促进作用,呈浓度依赖性;(3)茜素红染色结果显示与空白组比较,抑制剂组成骨细胞矿化能力明显降低,与抑制剂组比较,治疗组成骨细胞矿化结节能力明显增加(P <0.05);活性测定结果显示与空白组比较,抑制剂组中碱性磷酸酶活性明显降低,与抑制剂组比较,治疗组中碱性磷酸酶活性明显增加(P <0.05);(4)Western blot结果显示与空白组比较,抑制剂组中Beclin1和Runx2蛋白表达明显降低,与抑制剂组比较,治疗组中Beclin1和Runx2蛋白表达显著增加(P <0.05);(5)结果提示,雌激素受体抑制下氯化锂可以提高大鼠成骨细胞增殖分化及自噬。
BACKGROUND:Lithium has been used in the treatment of hematopoietic diseases and bipolar diseases for decades, and it can directly stimulate the proliferation of bone marrow mesenchymal stem cells.OBJECTIVE:To investigate the effects of lithium chloride on the proliferation, differentiation and autophagy of rat osteoblasts inhibited by estrogen receptor.METHODS:Ten neonatal Sprague-Dawley rats, 72 days old, SPF grade, were provided by the Heilongjiang University of Chinese Medicine.(1) Osteoblasts were isolated by enzymatic digestion and alizarin red staining, and logarithmically growing osteoblasts were then cultured in media containing different concentrations of lithium chloride(0, 1, 2, 5 nmol/L) for 24 hours. Cell proliferation was measured by cell counting kit-8.(2) Osteoblasts at logarithmical growth phase were randomly divided into blank group(normal cultured cells), inhibitor group(10~(-7) mol/L ICI182780), treatment group(5 nmol/L lithium chloride +10~(-7) mol/L ICI 182780). Calcified nodule staining and alkaline phosphatase activity were used to detect the differentiation of osteoblasts. Expression of Beclin1 and Runx2 in osteoblasts were determined by western blot.RESULTS AND CONCLUSION:Under the inverted microscope, the cells were mostly mononuclear, polygonal and fusiform, with local cell-dense cell clusters. Results from the cell counting kit-8 assay showed that different concentrations of lithium chloride promoted the proliferation of osteoblasts in a concentration-dependent manner. Alizarin red staining results showed that the mineralization ability of osteoblasts was significantly reduced in the inhibitor group compared with the blank group, while compared with the inhibitor group, the mineralization ability of osteoblasts was increased significantly in the treatment group(P < 0.05). The activity of alkaline phosphatase was significantly reduced in the inhibitor group compared with the blank group, while compared with the inhibitory group, the activity of alkaline phosphatase increased significantly in the treatment group(P < 0.05). Western blot results showed that the expression of Beclin1 and Runx2 proteins in the inhibitor group decreased significantly compared with blank group, while compared with the inhibitor group, the expression of Beclin1 and Runx2 protein in the treatment group increased significantly(P < 0.05). In conclusion, lithium chloride can increase the proliferation, differentiation and autophagy of rat osteoblasts inhibited by estrogen receptor.
BACKGROUND:Lithium has been used in the treatment of hematopoietic diseases and bipolar diseases for decades, and it can directly stimulate the proliferation of bone marrow mesenchymal stem cells.OBJECTIVE:To investigate the effects of lithium chloride on the proliferation, differentiation and autophagy of rat osteoblasts inhibited by estrogen receptor.METHODS:Ten neonatal Sprague-Dawley rats, 72 days old, SPF grade, were provided by the Heilongjiang University of Chinese Medicine.(1) Osteoblasts were isolated by enzymatic digestion and alizarin red staining, and logarithmically growing osteoblasts were then cultured in media containing different concentrations of lithium chloride(0, 1, 2, 5 nmol/L) for 24 hours. Cell proliferation was measured by cell counting kit-8.(2) Osteoblasts at logarithmical growth phase were randomly divided into blank group(normal cultured cells), inhibitor group(10~(-7) mol/L ICI182780), treatment group(5 nmol/L lithium chloride +10~(-7) mol/L ICI 182780). Calcified nodule staining and alkaline phosphatase activity were used to detect the differentiation of osteoblasts. Expression of Beclin1 and Runx2 in osteoblasts were determined by western blot.RESULTS AND CONCLUSION:Under the inverted microscope, the cells were mostly mononuclear, polygonal and fusiform, with local cell-dense cell clusters. Results from the cell counting kit-8 assay showed that different concentrations of lithium chloride promoted the proliferation of osteoblasts in a concentration-dependent manner. Alizarin red staining results showed that the mineralization ability of osteoblasts was significantly reduced in the inhibitor group compared with the blank group, while compared with the inhibitor group, the mineralization ability of osteoblasts was increased significantly in the treatment group(P < 0.05). The activity of alkaline phosphatase was significantly reduced in the inhibitor group compared with the blank group, while compared with the inhibitory group, the activity of alkaline phosphatase increased significantly in the treatment group(P < 0.05). Western blot results showed that the expression of Beclin1 and Runx2 proteins in the inhibitor group decreased significantly compared with blank group, while compared with the inhibitor group, the expression of Beclin1 and Runx2 protein in the treatment group increased significantly(P < 0.05). In conclusion, lithium chloride can increase the proliferation, differentiation and autophagy of rat osteoblasts inhibited by estrogen receptor.
引文
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[18]Chen XX,Yang T.Roles of leptin in bone metabolism and bone diseases.J Bone Miner Metab.2015;33(5):474-485.
[19]Hart NH,Nimphius S,Rantalainen T,et al.Mechanical basis of bone strength:influence of bone material,bone structure and muscle action.J Musculoskelet Neuronal Interact.2017;17(3):114-139.
[20]Nguyen HTT,Bo VS,Nguyen TV,et al.Sex hormone levels as determinants of bone mineral density and osteoporosis in Vietnamese women and men.J Bone Miner Metab.2015;33(6):658-665.
[21]Black DM,Rosen CJ.Clinical practice.postmenopausal osteoporosis.N Engl J Med.2005;353(6):595-603.
[22]李继钢.骨质疏松药物治疗研究进展[J].中国处方药,2018,16(4):17-18.
[23]陶亚军.骨质疏松药物治疗的研究进展[J].医学综述,2016,22(9):1715-1718.
[24]Gennari L,Rotatori S,Bianciardi S,et al.Treatment needs and current options for postmenopausal osteoporosis.Expert Opin Pharmacother.2016;17(8):1141-1152.
[25]Horák P,SkácelováM.Current treatment options for postmenopausal osteoporosis.Klinicka Farmakologie AFarmacie.2014;28(3):99-104.
[26]吴智鸿,赵水平.他汀类药物抗骨质疏松作用的研究进展[J].中华老年医学杂志,2005,24(1):64-66.
[27]田发明,张柳.他汀类药物抗骨质疏松作用的实验研究进展[J].中国骨与关节杂志,2015,4(5):419-422.
[28]Arioka M,TakahashiYanaga F,Sasaki M,Acceleration of bone regeneration by local application of lithium:Wnt signal-mediated osteoblastogenesis and Wnt signal-independent suppression of osteoclastogenesis.Biochem Pharmacol.2014;90(4):397-405.
[29]Zhu Z,Yin J,Guan J,et al.Lithium stimulates human bone marrow derived mesenchymal stem cell proliferation through GSK‐3β‐dependentβ‐catenin/Wnt pathway activation.FEBS J.2015;281(23):5371-5389.
[30]Pan J,He S,Yin X,et al.Lithium enhances alveolar bone formation during orthodontic retention in rats.Orthod Craniofac Res.2017;20(3):146-151.
[31]陈天钢,勾禹,田发明,等.雌激素及选择性雌激素受体调节剂对骨关节炎作用的研究进展[J].中国临床药理学杂志,2018(1):73-76.
[32]Lee WY,Ji HY,Lee DH,et al.2B24 application of vibration signal processing for gear fault detection(the 12th international conference on motion and vibration control)[c]dynamics and design conference.The Japan Society of Mechanical Engineers.2017:385-385.
[33]Hori YS,Hosoda R,Akiyama Y,et al.Chloroquine potentiates temozolomide cytotoxicity by inhibiting mitochondrial autophagy in glioma cells.J Neurooncol.2015;122(1):11-20.
[34]郭鑫,杨俊.Beclin1调控自噬、凋亡与炎症反应的分子机制[J].中国老年学,2014(18):5289-5291.
[35]唐欢,许海甲,侯煜东,等.Runx2基因对骨代谢调控的研究进展[J].中国骨质疏松杂志,2014(12):1501-1505.
[2]杨宇,凯赛尔江·艾合买提.自噬现象与骨类疾病的关系[J].临床军医杂志,2015,43(2):196-199.
[3]She C,Zhu LQ,Zhen YF,et al.Activation of AMPK protects against hydrogen peroxide-induced osteoblast apoptosis through autophagy induction and NADPH maintenance:new implications for osteonecrosis treatment.Cell Signal.2014;26(1):1-8.
[4]Bonnelye E,Chabadel A,Saltel F,et al.Dual effect of strontium ranelate:stimulation of osteoblast differentiation and inhibition of osteoclast formation and resorption in vitro.Bone.200842(1):129-138.
[5]王伟舟,郭皓,袁勇.自噬在绝经后骨质疏松中作用的研究进展[J].中国骨质疏松杂志,2017(12):1675-1680.
[6]Han SY,Lee KH,Kim YK.Poligoni Multiflori Radix enhances osteoblast formation and reduces osteoclast differentiation.Int J Mol Med.2018;42(1):331-345.
[7]Inokondo A,Hotokezaka H,Kondo T,et al.Lithium chloride reduces orthodontically induced root resorption and affects tooth root movement in rats.Angle Orthod.2018;88(4):474-482.
[8]Lin FX,Zheng GZ,Chang B,et al.Connexin 43 modulates osteogenic differentiation of bone marrow stromal cells through GSK-3beta/Beta-Catenin signaling pathways.Cell Physiol Biochem.2018;47(1):161-175.
[9]Pan J,He S,Yin X,et al.Lithium enhances alveolar bone formation during orthodontic retention in rats.Orthod Craniofac Res.2017;20(3):146-151.
[10]Liu J,Wen J,Kai M,et al.Elevated risk of subsequent endometrial cancer after first primary breast cancer according to estrogen and progesterone receptor status:a SEER analysis.Meeting of the American-Society-Of-Breast-Surgeons.2015:72-73.
[11]王双利,查振刚,刘宁,等.改进酶消化法培养SD大鼠成骨细胞及其鉴定[J].实用医学杂志,2008,24(6):915-918.
[12]李启活.补骨脂素激活MAPK-NF-κB信号通路促进成骨细胞增殖的研究[D].广州:广州中医药大学,2016.
[13]隋国良,彭依群,翟木绪,等.17β-雌二醇下调人成骨细胞CTGF表达的信号转导机制研究[J].中国骨质疏松杂志,2006,12(5):443-446.
[14]包倪荣,赵建宁,李芳秋,等.小鼠颅骨成骨样细胞体外分离方法的改进[J].医学研究生学报,2003,16(4):260-261.
[15]闫娟丽,陈克明,周建,等.脉冲电磁场与正弦交变电磁场对成骨细胞增殖与成熟矿化的比较研究[J].解放军医药杂志,2015,27(3):6-10.
[16]余帮兴,梁文斌,王文.Western blot在大鼠心肌缺血炎性因子研究中的应用[J].实验动物科学,2018,35(2):69-73.
[17]Tanaka S.Development of osteoporosis drugs-the past,the present and the future-.Clin Calcium.2017;27(1):137-141.
[18]Chen XX,Yang T.Roles of leptin in bone metabolism and bone diseases.J Bone Miner Metab.2015;33(5):474-485.
[19]Hart NH,Nimphius S,Rantalainen T,et al.Mechanical basis of bone strength:influence of bone material,bone structure and muscle action.J Musculoskelet Neuronal Interact.2017;17(3):114-139.
[20]Nguyen HTT,Bo VS,Nguyen TV,et al.Sex hormone levels as determinants of bone mineral density and osteoporosis in Vietnamese women and men.J Bone Miner Metab.2015;33(6):658-665.
[21]Black DM,Rosen CJ.Clinical practice.postmenopausal osteoporosis.N Engl J Med.2005;353(6):595-603.
[22]李继钢.骨质疏松药物治疗研究进展[J].中国处方药,2018,16(4):17-18.
[23]陶亚军.骨质疏松药物治疗的研究进展[J].医学综述,2016,22(9):1715-1718.
[24]Gennari L,Rotatori S,Bianciardi S,et al.Treatment needs and current options for postmenopausal osteoporosis.Expert Opin Pharmacother.2016;17(8):1141-1152.
[25]Horák P,SkácelováM.Current treatment options for postmenopausal osteoporosis.Klinicka Farmakologie AFarmacie.2014;28(3):99-104.
[26]吴智鸿,赵水平.他汀类药物抗骨质疏松作用的研究进展[J].中华老年医学杂志,2005,24(1):64-66.
[27]田发明,张柳.他汀类药物抗骨质疏松作用的实验研究进展[J].中国骨与关节杂志,2015,4(5):419-422.
[28]Arioka M,TakahashiYanaga F,Sasaki M,Acceleration of bone regeneration by local application of lithium:Wnt signal-mediated osteoblastogenesis and Wnt signal-independent suppression of osteoclastogenesis.Biochem Pharmacol.2014;90(4):397-405.
[29]Zhu Z,Yin J,Guan J,et al.Lithium stimulates human bone marrow derived mesenchymal stem cell proliferation through GSK‐3β‐dependentβ‐catenin/Wnt pathway activation.FEBS J.2015;281(23):5371-5389.
[30]Pan J,He S,Yin X,et al.Lithium enhances alveolar bone formation during orthodontic retention in rats.Orthod Craniofac Res.2017;20(3):146-151.
[31]陈天钢,勾禹,田发明,等.雌激素及选择性雌激素受体调节剂对骨关节炎作用的研究进展[J].中国临床药理学杂志,2018(1):73-76.
[32]Lee WY,Ji HY,Lee DH,et al.2B24 application of vibration signal processing for gear fault detection(the 12th international conference on motion and vibration control)[c]dynamics and design conference.The Japan Society of Mechanical Engineers.2017:385-385.
[33]Hori YS,Hosoda R,Akiyama Y,et al.Chloroquine potentiates temozolomide cytotoxicity by inhibiting mitochondrial autophagy in glioma cells.J Neurooncol.2015;122(1):11-20.
[34]郭鑫,杨俊.Beclin1调控自噬、凋亡与炎症反应的分子机制[J].中国老年学,2014(18):5289-5291.
[35]唐欢,许海甲,侯煜东,等.Runx2基因对骨代谢调控的研究进展[J].中国骨质疏松杂志,2014(12):1501-1505.