黄嘌呤氧化酶在激素诱导成骨细胞凋亡过程中的作用及机制
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摘要
目的探究黄嘌呤氧化酶在糖皮质激素诱导小鼠胚胎成骨细胞前体细胞(MC3T3-E1)细胞凋亡过程中的作用及机制。方法将细胞分为空白组、模型组、5 mol/L组、10 mol/L组和15 mol/L组,接种完成后培养24 h,确认细胞贴壁后换液。空白组加入10%FBS培养基2 mL,模型组加入1 10-6 mol/L DEX培养基2 mL, 5 mol/L组加入5 mol/L别嘌醇DEX培养基2 mL,10 mol/L组加入10 mol/L别嘌醇DEX培养基2 mL,15 mol/L组加入15 mol/L别嘌醇DEX培养基2 mL。培养72 h后检测Caspase-3、STAT-1、Bax、Bcl-2等蛋白表达情况,检测细胞凋亡情况以及活性氧簇含量。检测细胞内黄嘌呤氧化酶活性、丙二醛(MDA)含量及线粒体膜电位。结果模型组中Caspase-3、STAT-1和Bax蛋白表达量明显升高,Bcl-2蛋白表达量降低,细胞凋亡比例增高,细胞内活性氧簇含量增高,细胞内黄嘌呤氧化酶活性、丙二醛含量增高,而线粒体膜电位降低,与空白组、5 mol/L组、10mol/L组和15 mol/L组相比,差异有统计学意义(P <0.05)。结论黄嘌呤氧化酶通过产生过量的活性氧簇从而导致成骨细胞氧化应激损伤,通过激活STAT-1和Bax蛋白来诱导成骨细胞凋亡,同时诱导线粒体膜电位稳态崩溃触发内源性线粒体凋亡途径。别嘌醇可以较好地抑制黄嘌呤氧化酶的活性,减少活性氧簇的产生,通过减少Caspase-3、STAT-1和Bax蛋白表达,稳定线粒体膜电位,从而减少成骨细胞凋亡。
Objective To investigate the role and mechanism of xanthine oxidase in glucocorticoid-induced apoptosis in MC3 T3-E1 cells.Methods MC3 T3-E1 cells were divided into blank control group,model group,5 mol/L group,10 mol/L group and 15 mol/L group.After the inoculation was completed,the cells were incubated in an incubator for24 hours.After confirming the attachment of the cells.Blank control group was added 2 mL 10% FBS medium.Model group was added 1 10-6 mol/L DEX medium 2 mL.5 mol/L group was added 5 mol/L allopurinol DEX medium 2 mL.10 mol/L group was added 10 mol/L allopurinol DEX medium 2 mL.15 mol/L group was added 15 mol/L allopurinol DEX medium 2 mL.The related indicators,such as Caspase-3,STAT-1,Bax,Bcl-2,apoptosis,reactive oxygen species(ROS) contents,intracellular xanthine oxidase,malondialdehyde and mitochondrial membrane potential were detected after 72 hours of cultivation.Results In model group,the expression of Caspase-3,STAT-1 and Bax was significantly increased while the expression of Bcl-2 protein was decreased.The proportion of apoptotic cells and the content of intracellular reactive oxygen species were also increased.The activity of intracellular xanthine oxidase,malondialdehyde content was also increased.But the mitochondrial membrane potential was significantly decreased.These indicators were significant difference with other groups(P<0.05).Conclusion The xanthine oxidase may generate excessive ROS,which leads to cell oxidative stress injury and induce the apoptosis in MC3 T3-E1 cells through activating STAT-1 and Bax.The endogenous mitochondrial apoptosis pathway was triggered by mitochondrial membrane potential collapse.Allopurinol can inhibit the activity of xanthine oxidase and reduce the production of reactive oxygen species.It can reduce osteoblast apoptosis by decreasing the expression of STAT-1,Caspase-3 and Bax protein and stabilizing the mitochondrial membrane potential.
Objective To investigate the role and mechanism of xanthine oxidase in glucocorticoid-induced apoptosis in MC3 T3-E1 cells.Methods MC3 T3-E1 cells were divided into blank control group,model group,5 mol/L group,10 mol/L group and 15 mol/L group.After the inoculation was completed,the cells were incubated in an incubator for24 hours.After confirming the attachment of the cells.Blank control group was added 2 mL 10% FBS medium.Model group was added 1 10-6 mol/L DEX medium 2 mL.5 mol/L group was added 5 mol/L allopurinol DEX medium 2 mL.10 mol/L group was added 10 mol/L allopurinol DEX medium 2 mL.15 mol/L group was added 15 mol/L allopurinol DEX medium 2 mL.The related indicators,such as Caspase-3,STAT-1,Bax,Bcl-2,apoptosis,reactive oxygen species(ROS) contents,intracellular xanthine oxidase,malondialdehyde and mitochondrial membrane potential were detected after 72 hours of cultivation.Results In model group,the expression of Caspase-3,STAT-1 and Bax was significantly increased while the expression of Bcl-2 protein was decreased.The proportion of apoptotic cells and the content of intracellular reactive oxygen species were also increased.The activity of intracellular xanthine oxidase,malondialdehyde content was also increased.But the mitochondrial membrane potential was significantly decreased.These indicators were significant difference with other groups(P<0.05).Conclusion The xanthine oxidase may generate excessive ROS,which leads to cell oxidative stress injury and induce the apoptosis in MC3 T3-E1 cells through activating STAT-1 and Bax.The endogenous mitochondrial apoptosis pathway was triggered by mitochondrial membrane potential collapse.Allopurinol can inhibit the activity of xanthine oxidase and reduce the production of reactive oxygen species.It can reduce osteoblast apoptosis by decreasing the expression of STAT-1,Caspase-3 and Bax protein and stabilizing the mitochondrial membrane potential.
引文
[1] Laporte DM, Mont MA, Mohan V, et al. Multifocal osteonecrosis[J]. J Rheumatol, 1998, 25(10):1968-1974.
[2] Chernetsky SG, Mont MA, Laporte DM, et al. Pathologic featuresin steroid and nonsteroid associated osteonecrosis[J]. Clin OrthopRelat Res, 1999, 368(368):149-161.
[3] Weinstein RS. Glucocorticoid-induced osteoporosis and osteonecr-osis[J]. Endocrinol Metab Clin North Am, 2012, 41(3):595-611.
[4] Weinstein RS. Glucocorticoid-induced osteonecrosis[J]. Endocri-ne, 2012, 41(2):183-190.
[5] Kang P, Gao H, Pei F, et al. Effects of an anticoagulant and a lipid-lowering agent on the prevention of steroid-induced osteonecrosisin rabbits[J]. Int J Exp Pathol, 2010, 91(3):235-243.
[6] Beckmann R, Shaheen H, Kweider N, et al. Enoxaparin preventssteroid-related avascular necrosis of the femoral head[J]. Scien-tific World Journal, 2014, 2014(3):347813.
[7] Sun Z, Yang S, Ye S, et al. Aberrant CpG islands'hypermethylationof ABCB1 in mesenchymal stem cells of patients with steroid-as-sociated osteonecrosis[J]. J Rheumatol, 2013, 40(11):1913-1920.
[8] Zhang Y, Yin J, Ding H, et al. Vitamin K2 Ameliorates Damage ofBlood Vessels by Glucocorticoid:a Potential Mechanism for ItsProtective Effects in Glucocorticoid-induced Osteonecrosis of theFemoral Head in a Rat Model[J]. Int J Biol Sci, 2016, 12(7):776-785.
[9] Zhang Q, L V J, Jin L. Role of coagulopathy in glucocorticoid-in-duced osteonecrosis of the femoral head[J]. J Int Med Res, 2018,46(6):2141-2148.
[10] Zalavras C, Shah S, Birnbaum MJ, et al. Role of apoptosis in glu-cocorticoid-induced osteoporosis and osteonecrosis[J]. Crit RevEukaryot Gene Expr, 2003, 13(2-4):221-235.
[11] Weinstein RS, Jilka RL, Parfitt AM, et al. Inhibition of oste-oblastogenesis and promotion of apoptosis of osteoblasts and os-teocytes by glucocorticoids. Potential mechanisms of their del-eterious effects on bone[J]. J Clin Invest, 1998, 102(2):274-282.
[12] Weinstein RS, Nicholas RW, Manolagas SC. Apoptosis of osteo-cytes in glucocorticoid-induced osteonecrosis of the hip[J]. J ClinEndocrinol Metab, 2000, 85(8):2907-2912.
[13] Almeida M, Han L, Ambrogini E, et al. Glucocorticoids and tumornecrosis factor alpha increase oxidative stress and suppress Wntprotein signaling in osteoblasts[J]. J Biol Chem, 2011, 286(52):44326-44335.
[14] Lin H, Gao X, Chen G, et al. Indole-3-carbinol as inhibitors of glu-cocorticoid-induced apoptosis in osteoblastic cells through block-ing ROS-mediated Nrf2 pathway[J]. Biochem Biophys Res Com-mun, 2015, 460(2):422-427.
[15] Sato AY, Tu X, Mcandrews KA, et al. Prevention of glucocorticoidinduced-apoptosis of osteoblasts and osteocytes by protecting ag-ainst endoplasmic reticulum(ER)stress in vitro and in vivo in fe-male mice[J]. Bone, 2015, 73:60-68.
[16]肖骏,佘强,罗开良,等.别嘌呤醇对心肌梗死后大鼠心肌细胞凋亡的抑制作用[J].第三军医大学学报, 2008, 30(8):735-738.
[17] Faria TO, Simoes MR, Vassallo DV, et al. Xanthine Oxidase Acti-vation Modulates the Endothelial(Vascular)Dysfunction Relatedto HgCl2 Exposure Plus Myocardial Infarction in Rats[J]. Card-iovasc Toxicol, 2018, 18(2):161-174.
[18] Akahane H, Gomes RZ, Paludo KS, et al. The influence of allop-urinol and post-conditioning on lung injuries induced by lower-limb ischemia and reperfusion in Wistar rats[J]. Acta Cir Bras,2017, 32(9):746-754.
[19] Mccord JM, Roy RS, Schaffer SW. Free radicals and myocardialischemia. The role of xanthine oxidase[J]. Adv Myocardiol, 1985,5:183-189.
[20] Guo F, Yang F, Zhu YH. Scutellarein from Scutellaria barbata in-duces apoptosis of human colon cancer HCT116 cells through theROS-mediated mitochondria-dependent pathway[J]. Nat ProdRes, 2018, 19:1-4.
[21] Lee MH, Hong SH, Park C, et al. Hwang-Heuk-San induces apop-tosis in HCT116 human colorectal cancer cells through the ROS-mediated activation of caspases and the inactivation of the PI3K/Akt signaling pathway[J]. Oncol Rep, 2016, 36(1):205-214.
[22] Zhong J, Yu H, Huang C, et al. Inhibition of phosphodiesterase 4by FCPR16 protects SH-SY5Y cells against MPP(+)-induced de-cline of mitochondrial membrane potential and oxidative stress[J].Redox Biol, 2018, 16:47-58.
[23] Huang TC, Chiu PR, Chang WT, et al. Epirubicin induces apop-tosis in osteoblasts through death-receptor and mitochondrial pat-hways[J]. Apoptosis, 2018, 23(3-4):226-236.
[24] Li K, Li Y, Mi J, et al. Resveratrol protects against sodium nitrop-russide induced nucleus pulposus cell apoptosis by scavengingROS[J]. Int J Mol Med, 2018, 41(5):2485-2492.
[25] Jacob S, Herndon DN, Hawkins HK, et al. Xanthine oxidase con-tributes to sustained airway epithelial oxidative stress after scaldburn[J]. Int J Burns Trauma, 2017, 7(6):98-106.
[2] Chernetsky SG, Mont MA, Laporte DM, et al. Pathologic featuresin steroid and nonsteroid associated osteonecrosis[J]. Clin OrthopRelat Res, 1999, 368(368):149-161.
[3] Weinstein RS. Glucocorticoid-induced osteoporosis and osteonecr-osis[J]. Endocrinol Metab Clin North Am, 2012, 41(3):595-611.
[4] Weinstein RS. Glucocorticoid-induced osteonecrosis[J]. Endocri-ne, 2012, 41(2):183-190.
[5] Kang P, Gao H, Pei F, et al. Effects of an anticoagulant and a lipid-lowering agent on the prevention of steroid-induced osteonecrosisin rabbits[J]. Int J Exp Pathol, 2010, 91(3):235-243.
[6] Beckmann R, Shaheen H, Kweider N, et al. Enoxaparin preventssteroid-related avascular necrosis of the femoral head[J]. Scien-tific World Journal, 2014, 2014(3):347813.
[7] Sun Z, Yang S, Ye S, et al. Aberrant CpG islands'hypermethylationof ABCB1 in mesenchymal stem cells of patients with steroid-as-sociated osteonecrosis[J]. J Rheumatol, 2013, 40(11):1913-1920.
[8] Zhang Y, Yin J, Ding H, et al. Vitamin K2 Ameliorates Damage ofBlood Vessels by Glucocorticoid:a Potential Mechanism for ItsProtective Effects in Glucocorticoid-induced Osteonecrosis of theFemoral Head in a Rat Model[J]. Int J Biol Sci, 2016, 12(7):776-785.
[9] Zhang Q, L V J, Jin L. Role of coagulopathy in glucocorticoid-in-duced osteonecrosis of the femoral head[J]. J Int Med Res, 2018,46(6):2141-2148.
[10] Zalavras C, Shah S, Birnbaum MJ, et al. Role of apoptosis in glu-cocorticoid-induced osteoporosis and osteonecrosis[J]. Crit RevEukaryot Gene Expr, 2003, 13(2-4):221-235.
[11] Weinstein RS, Jilka RL, Parfitt AM, et al. Inhibition of oste-oblastogenesis and promotion of apoptosis of osteoblasts and os-teocytes by glucocorticoids. Potential mechanisms of their del-eterious effects on bone[J]. J Clin Invest, 1998, 102(2):274-282.
[12] Weinstein RS, Nicholas RW, Manolagas SC. Apoptosis of osteo-cytes in glucocorticoid-induced osteonecrosis of the hip[J]. J ClinEndocrinol Metab, 2000, 85(8):2907-2912.
[13] Almeida M, Han L, Ambrogini E, et al. Glucocorticoids and tumornecrosis factor alpha increase oxidative stress and suppress Wntprotein signaling in osteoblasts[J]. J Biol Chem, 2011, 286(52):44326-44335.
[14] Lin H, Gao X, Chen G, et al. Indole-3-carbinol as inhibitors of glu-cocorticoid-induced apoptosis in osteoblastic cells through block-ing ROS-mediated Nrf2 pathway[J]. Biochem Biophys Res Com-mun, 2015, 460(2):422-427.
[15] Sato AY, Tu X, Mcandrews KA, et al. Prevention of glucocorticoidinduced-apoptosis of osteoblasts and osteocytes by protecting ag-ainst endoplasmic reticulum(ER)stress in vitro and in vivo in fe-male mice[J]. Bone, 2015, 73:60-68.
[16]肖骏,佘强,罗开良,等.别嘌呤醇对心肌梗死后大鼠心肌细胞凋亡的抑制作用[J].第三军医大学学报, 2008, 30(8):735-738.
[17] Faria TO, Simoes MR, Vassallo DV, et al. Xanthine Oxidase Acti-vation Modulates the Endothelial(Vascular)Dysfunction Relatedto HgCl2 Exposure Plus Myocardial Infarction in Rats[J]. Card-iovasc Toxicol, 2018, 18(2):161-174.
[18] Akahane H, Gomes RZ, Paludo KS, et al. The influence of allop-urinol and post-conditioning on lung injuries induced by lower-limb ischemia and reperfusion in Wistar rats[J]. Acta Cir Bras,2017, 32(9):746-754.
[19] Mccord JM, Roy RS, Schaffer SW. Free radicals and myocardialischemia. The role of xanthine oxidase[J]. Adv Myocardiol, 1985,5:183-189.
[20] Guo F, Yang F, Zhu YH. Scutellarein from Scutellaria barbata in-duces apoptosis of human colon cancer HCT116 cells through theROS-mediated mitochondria-dependent pathway[J]. Nat ProdRes, 2018, 19:1-4.
[21] Lee MH, Hong SH, Park C, et al. Hwang-Heuk-San induces apop-tosis in HCT116 human colorectal cancer cells through the ROS-mediated activation of caspases and the inactivation of the PI3K/Akt signaling pathway[J]. Oncol Rep, 2016, 36(1):205-214.
[22] Zhong J, Yu H, Huang C, et al. Inhibition of phosphodiesterase 4by FCPR16 protects SH-SY5Y cells against MPP(+)-induced de-cline of mitochondrial membrane potential and oxidative stress[J].Redox Biol, 2018, 16:47-58.
[23] Huang TC, Chiu PR, Chang WT, et al. Epirubicin induces apop-tosis in osteoblasts through death-receptor and mitochondrial pat-hways[J]. Apoptosis, 2018, 23(3-4):226-236.
[24] Li K, Li Y, Mi J, et al. Resveratrol protects against sodium nitrop-russide induced nucleus pulposus cell apoptosis by scavengingROS[J]. Int J Mol Med, 2018, 41(5):2485-2492.
[25] Jacob S, Herndon DN, Hawkins HK, et al. Xanthine oxidase con-tributes to sustained airway epithelial oxidative stress after scaldburn[J]. Int J Burns Trauma, 2017, 7(6):98-106.