川芎嗪抑制NF-κB P65磷酸化对LPS诱导的骨关节炎软骨细胞凋亡和炎症反应的调节作用
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摘要
目的:探讨川芎嗪对LPS诱导的骨关节炎软骨细胞凋亡和炎症反应的影响及机制。方法:LPS诱导骨关节炎模型。软骨细胞分为4个组:对照组;川芎嗪(20μmol/L)组; LPS(100 ng/ml)组;川芎嗪(20μmol/L)+LPS(100 ng/ml)组。Hoechst33258染色检测细胞凋亡。ELISA检测一氧化氮(Nitric oxide,NO)、肿瘤坏死因子-α(TNF-α)和白细胞介素(IL)-6水平。蛋白印迹检测Ⅱ型胶原(collagenⅡ)、聚集蛋白聚糖(aggrecan)和基质金属蛋白酶-13(MMP-13)、NF-κB P65和p-NF-κB P65蛋白水平。结果:川芎嗪(20μmol/L)可改善LPS诱导的骨关节炎软骨细胞形态异常及细胞数量的减少。LPS组细胞凋亡率明显高于对照组(P<0. 05)。川芎嗪可降低LPS诱导的细胞凋亡率的升高(P<0. 05)。与对照组相比,LPS组Ⅱ型胶原和聚集蛋白聚糖表达明显降低,MMP-13表达明显升高(P<0. 05)。川芎嗪可抑制LPS诱导的Ⅱ型胶原和聚集蛋白聚糖表达水平的下降及MMP-13表达水平的上升(P<0. 05)。LPS组NO、TNF-α和IL-6水平明显高于对照组(P<0. 05)。LPS+川芎嗪组NO、TNF-α和IL-6水平显著低于LPS组(P<0. 05)。与对照组相比,LPS组p-P65/P65比值明显升高(P<0. 05)。川芎嗪可减弱LPS诱导的p-P65/P65比值的上升(P<0. 05)。结论:川芎嗪通过抑制NF-κB P65磷酸化减轻LPS诱导的骨关节炎软骨细胞凋亡和炎症反应。
Objective: To explore the effect of ligustrazine on the LPS-induced apoptosis and inflammatory response of osteoarthritis chondrocytes. Methods: Osteoarthritis model was induced by LPS. Chondrocytes were divided into four group: control group,ligustrazine( 20 μmol/L) group,LPS( 100 ng/ml) group and ligustrazine( 20 μmol/L) +LPS( 100 ng/ml) group. Apoptosis was measured by Hoechst33258 staining. The levels of nitric oxide( NO),tumor necrosis factor α( TNF-α) and interleukin( IL)-6 were detected by ELISA. The protein levels of collagenⅡ,aggrecan,matrix metalloproteinase 13( MMP-13),NF-κB P65 and p-NF-κB P65 were tested by Western blot. Results: The LPS-induced abnormal cell morphology and decreased number of cells were ameliorated by ligustrazine( 20 μmol/L). The apoptosis in LPS group was higher than control group( P<0. 05). The LPS-induced enhancive apoptosis was reduced by ligustrazine( P<0. 05). Compared with control group,the expression of collagenⅡ and aggrecan was alleviated with increased expression of MMP-13( P<0. 05). The LPS-induced declined expression of collagenⅡ and aggrecan and elevated expression of MMP-13 was inhibited by ligustrazine( P<0. 05). The levels of NO,TNF-α and IL-6 in LPS group were higher than control group( P<0. 05). The levels of NO,TNF-α and IL-6 in LPS+ligustrazine group were lower than LPS group( P<0. 05).Compared with control group,the rate of pP65/P65 in LPPS group was enhanced( P< 0. 05). The LPS-indiced increased rate of p-P65/P65 was decreased by ligustrazine( P <0. 05). Conclusion: Ligustrazine alleviates the LPS-induced apoptosis and inflammatory response of osteoarthritis chondrocytes via inhibiting phosphorylation of NF-κB P65.
Objective: To explore the effect of ligustrazine on the LPS-induced apoptosis and inflammatory response of osteoarthritis chondrocytes. Methods: Osteoarthritis model was induced by LPS. Chondrocytes were divided into four group: control group,ligustrazine( 20 μmol/L) group,LPS( 100 ng/ml) group and ligustrazine( 20 μmol/L) +LPS( 100 ng/ml) group. Apoptosis was measured by Hoechst33258 staining. The levels of nitric oxide( NO),tumor necrosis factor α( TNF-α) and interleukin( IL)-6 were detected by ELISA. The protein levels of collagenⅡ,aggrecan,matrix metalloproteinase 13( MMP-13),NF-κB P65 and p-NF-κB P65 were tested by Western blot. Results: The LPS-induced abnormal cell morphology and decreased number of cells were ameliorated by ligustrazine( 20 μmol/L). The apoptosis in LPS group was higher than control group( P<0. 05). The LPS-induced enhancive apoptosis was reduced by ligustrazine( P<0. 05). Compared with control group,the expression of collagenⅡ and aggrecan was alleviated with increased expression of MMP-13( P<0. 05). The LPS-induced declined expression of collagenⅡ and aggrecan and elevated expression of MMP-13 was inhibited by ligustrazine( P<0. 05). The levels of NO,TNF-α and IL-6 in LPS group were higher than control group( P<0. 05). The levels of NO,TNF-α and IL-6 in LPS+ligustrazine group were lower than LPS group( P<0. 05).Compared with control group,the rate of pP65/P65 in LPPS group was enhanced( P< 0. 05). The LPS-indiced increased rate of p-P65/P65 was decreased by ligustrazine( P <0. 05). Conclusion: Ligustrazine alleviates the LPS-induced apoptosis and inflammatory response of osteoarthritis chondrocytes via inhibiting phosphorylation of NF-κB P65.
引文
[1]Cross M,Smith E,Hoy D,et al.The global burden of hip and knee osteoarthritis:estimates from the global burden of disease 2010study[J].Ann Rheum Dis,2014,73(7):1323-1330.
[2]Glyn-Jones S,Palmer AJ,Agricola R,et al.Osteoarthritis[J].Lancet,2015,386(9991):376-387.
[3]Cameron M,Chrubasik S.Oral herbal therapies for treating osteoarthritis[J].Cochrane Database Syst Rev,2014,5(5):Cd002947.
[4]Melainie C,Sigrun C.Topical herbal therapies for treating osteoarthritis[J].Cochrane Database Syst Rev,2013,6(3):Cd010538.
[5]Guo M,Liu Y,Shi D.Cardiovascular actions and therapeutic potential of tetramethylpyrazine(active component isolated from rhizomachuanxiong):roles and mechanisms[J].Biomed Res Int,2016,2016:2430329.
[6]Gao HJ,Liu PF,Li PW,et al.Ligustrazine monomer against cerebral ischemia/reperfusion injury[J].Neural Regen Res,2015,10(5):832-840.
[7]Lu C,Xu W,Zhang F,et al.Ligustrazine prevents alcohol-induced liver injury by attenuating hepatic steatosis and oxidative stress[J].Int Immunopharmacol,2015,29(2):613-621.
[8]Shao H,Zhao L,Chen F,et al.Efficacy of ligustrazine injection as adjunctive therapy for angina pectoris:a systematic review and meta-analysis[J].Med Sci Monit,2015,21:3704-3715.
[9]Cao S,Zhao W,Bu H,et al.Ligustrazine for the treatment of unstable angina:a meta-analysis of 16 randomized controlled trials[J].Evid Based Complement Alternat Med,2016,2016:8617062.
[10]Koushki D,Latifi S,Norouzi Javidan A,et al.Efficacy of some nonconventional herbal medications(sulforaphane,tanshinone IIA,and tetramethylpyrazine)in inducing neuroprotection in comparison with interleukin-10 after spinal cord injury:A meta-analysis[J].J Spinal Cord Med,2015,38(1):13-22.
[11]Tang X,Wang S,Zhan S,et al.The prevalence of symptomatic knee osteoarthritis in china:results from the china health and retirement longitudinal study[J].Arthritis Rheumatol,2016,68(3):648-653.
[12]Taruc-Uy RL,Lynch SA.Diagnosis and treatment of osteoarthritis[J].Prim Care,2013,40(4):821-836.
[13]Newman DJ,Cragg GM.Natural products as sources of new drugs from 1981 to 2014[J].J Nat Prod,2016,79(3):629-661.
[14]Lu H,Jiang J,Xie G,et al.Effects of an aqueous extract of Eucommia on articular cartilage in a rat model of osteoarthritis of the knee[J].Exp Ther Med,2013,6(3):684-688.
[15]Akhtar N,Khan NM,Ashruf OS,et al.Inhibition of cartilage degradation and suppression of PGE2 and MMPs expression by pomegranate fruit extract in a model of posttraumatic osteoarthritis[J].Nutrition,2017,33:1-13.
[16]Chin KY.The spice for joint inflammation:anti-inflammatory role of curcumin in treating osteoarthritis[J].Drug Des Devel Ther,2016,10:3029-3042.
[17]Wang XY,Ma ZC,Wang YG,et al.Tetramethylpyrazine protects lymphocytes from radiation-induced apoptosis through nuclear factor-kappaB[J].Chin J Nat Med,2014,12(10):730-737.
[18]Zhang H,Li D,Li Z,et al.Effect of Ligustrazine on rat peritoneal mesothelial cells treated with lipopolysaccharide[J].Ren Fail,2016,38(6):961-969.
[19]Lin KH,Kuo WW,Jiang AZ,et al.Tetramethylpyrazine ameliorated hypoxia-induced myocardial cell apoptosis via HIF-1alpha/JNK/p38 and IGFBP3/BNIP3 inhibition to upregulate PI3K/Akt survival signaling[J].Cell PhysiolBiochem,2015,36(1):334-344.
[20]Xia B,Chen D,Zhang J,et al.Osteoarthritis pathogenesis:a review of molecular mechanisms[J].Calcif Tissue Int,2014,95(6):495-505.
[21]Wang X,Zhao X,Tang S.Inhibitory effects of EGb761 on the expression of matrix metalloproteinases(MMPs)and cartilage matrix destruction[J].Cell Stress Chaperones,2015,20(5):781-786.
[22]Kwon HO,Lee M,Kim OK,et al.Effect of Hijikiafusiforme extracts on degenerative osteoarthritis in vitro and in vivo models[J].Nutr Res Pract,2016,10(3):265-273.
[23]Masutani T,Tanaka YT,Kojima H,et al.Cynaropicrin is dual regulator for both degradation factors and synthesis factors in the cartilage metabolism[J].Life Sci,2016,158:70-77.
[24]Liang QQ,Ding DF,Xi ZJ,et al.Protective effect of ligustrazine on lumbar intervertebral disc degeneration of rats induced by prolonged upright posture[J].Evid Based Complement Alternat Med,2014,2014:508461.
[25]Berenbaum F.Osteoarthritis as an inflammatory disease(osteoarthritis is not osteoarthrosis!)[J].Osteoarthritis Cartilage,2013,21(1):16-21.
[26]Wang X,Hunter D,Xu J,et al.Metabolic triggered inflammation in osteoarthritis[J].Osteoarthritis Cartilage,2015,23(1):22-30.
[27]Chen YJ,Tsai KS,Chiu CY,et al.EGb761 inhibits inflammatory responses in human chondrocytes and shows chondroprotection in osteoarthritic rat knee[J].J Orthop Res,2013,31(7):1032-1038.
[28]Jeong JW,Lee HH,Lee KW,et al.Mori folium inhibits interleukin-1beta-induced expression of matrix metalloproteinases and inflammatory mediators by suppressing the activation of NF-kappaB and p38 MAPK in SW1353 human chondrocytes[J].Int JMol Med,2016,37(2):452-460.
[29]Nsir H,Szychlinska MA,Cardile V,et al.RETRACTED:Polar and apolar extra virgin olive oil and leaf extracts as a promising anti-inflammatory natural treatment for osteoarthritis[J].Acta Histochem,2017,119(4):407-416.
[30]Kim M,Kim SO,Lee M,et al.Tetramethylpyrazine,a natural alkaloid,attenuates pro-inflammatory mediators induced by amyloid beta and interferon-gamma in rat brain microglia[J].Eur J Pharmacol,2014,740:504-511.
[31]谈冰,刘健,章平衡,等.基于NF-κB信号通路的变化探讨新风胶囊改善骨关节炎患者高凝状态的机制[J].中国免疫学杂志,2016,32(6):842-848.Tan B,Liu J,Zhang PH,et al.XFC improved mechanism of hypercoagulable state in OA patients based on NF-κB signaling pathway:a mechanism exploration[J].Chin J Immun,2016,32(6):842-848.
[32]Haseeb A,Khan NM,Ashruf OS,et al.A polyphenol-rich pomegranate fruit extract suppresses NF-kappa B and IL-6 expression by blocking the activation of IKKbeta and NIK in primary human chondrocytes[J].Phytother Res,2017,31(5):778-782.
[33]Wang Z,Wang Q,Wang C,et al.Tetramethylpyrazine attenuates periorbitalallodynia and neuroinflammation in a model of traumatic brain injury[J].J Inflamm(Lond),2017,14:13.
[34]Wang Y,Fu Q,Zhao W.Tetramethylpyrazine inhibits osteosarcoma cell proliferation via downregulation of NF-kappaBin vitro and in vivo[J].Mol Med Rep,2013,8(4):984-988.
[2]Glyn-Jones S,Palmer AJ,Agricola R,et al.Osteoarthritis[J].Lancet,2015,386(9991):376-387.
[3]Cameron M,Chrubasik S.Oral herbal therapies for treating osteoarthritis[J].Cochrane Database Syst Rev,2014,5(5):Cd002947.
[4]Melainie C,Sigrun C.Topical herbal therapies for treating osteoarthritis[J].Cochrane Database Syst Rev,2013,6(3):Cd010538.
[5]Guo M,Liu Y,Shi D.Cardiovascular actions and therapeutic potential of tetramethylpyrazine(active component isolated from rhizomachuanxiong):roles and mechanisms[J].Biomed Res Int,2016,2016:2430329.
[6]Gao HJ,Liu PF,Li PW,et al.Ligustrazine monomer against cerebral ischemia/reperfusion injury[J].Neural Regen Res,2015,10(5):832-840.
[7]Lu C,Xu W,Zhang F,et al.Ligustrazine prevents alcohol-induced liver injury by attenuating hepatic steatosis and oxidative stress[J].Int Immunopharmacol,2015,29(2):613-621.
[8]Shao H,Zhao L,Chen F,et al.Efficacy of ligustrazine injection as adjunctive therapy for angina pectoris:a systematic review and meta-analysis[J].Med Sci Monit,2015,21:3704-3715.
[9]Cao S,Zhao W,Bu H,et al.Ligustrazine for the treatment of unstable angina:a meta-analysis of 16 randomized controlled trials[J].Evid Based Complement Alternat Med,2016,2016:8617062.
[10]Koushki D,Latifi S,Norouzi Javidan A,et al.Efficacy of some nonconventional herbal medications(sulforaphane,tanshinone IIA,and tetramethylpyrazine)in inducing neuroprotection in comparison with interleukin-10 after spinal cord injury:A meta-analysis[J].J Spinal Cord Med,2015,38(1):13-22.
[11]Tang X,Wang S,Zhan S,et al.The prevalence of symptomatic knee osteoarthritis in china:results from the china health and retirement longitudinal study[J].Arthritis Rheumatol,2016,68(3):648-653.
[12]Taruc-Uy RL,Lynch SA.Diagnosis and treatment of osteoarthritis[J].Prim Care,2013,40(4):821-836.
[13]Newman DJ,Cragg GM.Natural products as sources of new drugs from 1981 to 2014[J].J Nat Prod,2016,79(3):629-661.
[14]Lu H,Jiang J,Xie G,et al.Effects of an aqueous extract of Eucommia on articular cartilage in a rat model of osteoarthritis of the knee[J].Exp Ther Med,2013,6(3):684-688.
[15]Akhtar N,Khan NM,Ashruf OS,et al.Inhibition of cartilage degradation and suppression of PGE2 and MMPs expression by pomegranate fruit extract in a model of posttraumatic osteoarthritis[J].Nutrition,2017,33:1-13.
[16]Chin KY.The spice for joint inflammation:anti-inflammatory role of curcumin in treating osteoarthritis[J].Drug Des Devel Ther,2016,10:3029-3042.
[17]Wang XY,Ma ZC,Wang YG,et al.Tetramethylpyrazine protects lymphocytes from radiation-induced apoptosis through nuclear factor-kappaB[J].Chin J Nat Med,2014,12(10):730-737.
[18]Zhang H,Li D,Li Z,et al.Effect of Ligustrazine on rat peritoneal mesothelial cells treated with lipopolysaccharide[J].Ren Fail,2016,38(6):961-969.
[19]Lin KH,Kuo WW,Jiang AZ,et al.Tetramethylpyrazine ameliorated hypoxia-induced myocardial cell apoptosis via HIF-1alpha/JNK/p38 and IGFBP3/BNIP3 inhibition to upregulate PI3K/Akt survival signaling[J].Cell PhysiolBiochem,2015,36(1):334-344.
[20]Xia B,Chen D,Zhang J,et al.Osteoarthritis pathogenesis:a review of molecular mechanisms[J].Calcif Tissue Int,2014,95(6):495-505.
[21]Wang X,Zhao X,Tang S.Inhibitory effects of EGb761 on the expression of matrix metalloproteinases(MMPs)and cartilage matrix destruction[J].Cell Stress Chaperones,2015,20(5):781-786.
[22]Kwon HO,Lee M,Kim OK,et al.Effect of Hijikiafusiforme extracts on degenerative osteoarthritis in vitro and in vivo models[J].Nutr Res Pract,2016,10(3):265-273.
[23]Masutani T,Tanaka YT,Kojima H,et al.Cynaropicrin is dual regulator for both degradation factors and synthesis factors in the cartilage metabolism[J].Life Sci,2016,158:70-77.
[24]Liang QQ,Ding DF,Xi ZJ,et al.Protective effect of ligustrazine on lumbar intervertebral disc degeneration of rats induced by prolonged upright posture[J].Evid Based Complement Alternat Med,2014,2014:508461.
[25]Berenbaum F.Osteoarthritis as an inflammatory disease(osteoarthritis is not osteoarthrosis!)[J].Osteoarthritis Cartilage,2013,21(1):16-21.
[26]Wang X,Hunter D,Xu J,et al.Metabolic triggered inflammation in osteoarthritis[J].Osteoarthritis Cartilage,2015,23(1):22-30.
[27]Chen YJ,Tsai KS,Chiu CY,et al.EGb761 inhibits inflammatory responses in human chondrocytes and shows chondroprotection in osteoarthritic rat knee[J].J Orthop Res,2013,31(7):1032-1038.
[28]Jeong JW,Lee HH,Lee KW,et al.Mori folium inhibits interleukin-1beta-induced expression of matrix metalloproteinases and inflammatory mediators by suppressing the activation of NF-kappaB and p38 MAPK in SW1353 human chondrocytes[J].Int JMol Med,2016,37(2):452-460.
[29]Nsir H,Szychlinska MA,Cardile V,et al.RETRACTED:Polar and apolar extra virgin olive oil and leaf extracts as a promising anti-inflammatory natural treatment for osteoarthritis[J].Acta Histochem,2017,119(4):407-416.
[30]Kim M,Kim SO,Lee M,et al.Tetramethylpyrazine,a natural alkaloid,attenuates pro-inflammatory mediators induced by amyloid beta and interferon-gamma in rat brain microglia[J].Eur J Pharmacol,2014,740:504-511.
[31]谈冰,刘健,章平衡,等.基于NF-κB信号通路的变化探讨新风胶囊改善骨关节炎患者高凝状态的机制[J].中国免疫学杂志,2016,32(6):842-848.Tan B,Liu J,Zhang PH,et al.XFC improved mechanism of hypercoagulable state in OA patients based on NF-κB signaling pathway:a mechanism exploration[J].Chin J Immun,2016,32(6):842-848.
[32]Haseeb A,Khan NM,Ashruf OS,et al.A polyphenol-rich pomegranate fruit extract suppresses NF-kappa B and IL-6 expression by blocking the activation of IKKbeta and NIK in primary human chondrocytes[J].Phytother Res,2017,31(5):778-782.
[33]Wang Z,Wang Q,Wang C,et al.Tetramethylpyrazine attenuates periorbitalallodynia and neuroinflammation in a model of traumatic brain injury[J].J Inflamm(Lond),2017,14:13.
[34]Wang Y,Fu Q,Zhao W.Tetramethylpyrazine inhibits osteosarcoma cell proliferation via downregulation of NF-kappaBin vitro and in vivo[J].Mol Med Rep,2013,8(4):984-988.