姜黄素对脂多糖诱导的巨噬细胞炎症与氧化应激的影响及机制
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摘要
目的探讨姜黄素对脂多糖(LPS)诱导的大鼠肺泡巨噬细胞(NR8383)炎症反应和氧化应激的影响及机制。方法 NR8383细胞分正常细胞组、LPS组(仅LPS刺激)及姜黄素预处理组[用姜黄素(5、10、15、20、25μmol/L)预处理1 h后,再加入0. 5μg/m L LPS刺激NR8383细胞],用细胞计数试剂盒-8(CCK-8)法测细胞活性;舍弃细胞毒性大的姜黄素浓度组,用酶联免疫吸附实验(ELISA)法测培养上清中肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)含量、用流式细胞仪测细胞内活性氧簇(ROS)水平、WST-8法检测超氧化物歧化酶(SOD)活性; 15μmol/L姜黄素预处理细胞,同前LPS刺激细胞,提取细胞总蛋白,蛋白质印迹法(Western blot)法测paralemmin-3(PALM3)蛋白表达。结果与正常细胞组相比,0. 5μg/m L LPS及5~20μmol/L姜黄素+0. 5μg/m L LPS对细胞活性无影响(P> 0. 05); 25μmol/L姜黄素+0. 5μg/m L LPS有细胞毒性(P <0. 05)。5~20μmol/L姜黄素可抑制LPS诱导的TNF-α、IL-1β及ROS的产生,提高胞内SOD活性,并呈剂量依赖性(P <0. 05);其中15μmol/L与20μmol/L姜黄素的抑制效力相似(P>0. 05)。LPS上调NR8383细胞内PALM3蛋白水平,15μmol/L姜黄素抑制LPS诱导的PALM3表达上调(P <0. 05)。结论姜黄素可抑制LPS诱导的大鼠肺泡巨噬细胞炎症反应和氧化应激,抑制LPS诱导的PALM3高表达是可能的分子机制之一。
Objective To investigate the effect of curcumin on the inflammatory response and oxidative stress induced by lipopolysaccharide( LPS) in alveolar macrophages and its mechanism. Methods The rat alveolar macrophages( NR8383 cells) were conventionally cultured,seeded in 96-well plates,and divided into 3 groups,normal cell group,LPS-stimulation group and curcumin-pretreated group. After stimulation with different doses( 5、10、15、20、25 μmol/L) of curcumin and LPS( 0. 5 μg/m L),the cellular survival rates were determined by cell counting kit-8( CCK-8)assay. The levels of tumor necrosis factor-α( TNF-α) and interleukin-1β( IL-1β) in cell culture supernatant were assessed by enzyme-linked immunosorbent assay( ELISA). Meanwhile,the reactive oxygen species( ROS) level and superoxide dismutase( SOD) activity in macrophages were also determined. After stimulation with curcumin( 15 μmol/L)and LPS( 0. 5 μg/m L),the protein expression of paralemmin-3( PALM3) in NR8383 cells was determined by Western blot analysis. Results The results of the CCK8 assay demonstrated that LPS( 0. 5 ng/m L),or curcumin( 5 ~ 20 μmol/L) plus of LPS( 0. 5 ng/m L) did not suppress the cell viability of NR8383 cells compared with the normal NR8383 cells( P > 0. 05). However,curcumin of 25 μmol/L plus LPS( 0. 5 ng/m L) significantly influence the cell viability of NR8383 cells( P < 0. 05). Curcumin( 5 ~ 20 μmol/L) significantly inhibited the production of TNF-α,IL-1β and ROS,and recovered the activity of SOD in a dose-dependent manner in NR8383 cells after LPS-stimulation( P <0. 05). As for the effect of curcumin on,There were no significant difference on inflammation and oxidative stress of curcumin at 15. 0 μmol/L and 20. 0 μmol/L( P > 0. 05). LPS significantly upregulated the protein expression of PALM3,which was inhibited by curcumin( 15. 0 μmol/L). Conclusions Curcumin inhibits the inflammatory response and oxidative stress induced by LPS in alveolar macrophages and its mechanism is partially associated with the inhibition of LPS-induced PALM3 overexpression.
Objective To investigate the effect of curcumin on the inflammatory response and oxidative stress induced by lipopolysaccharide( LPS) in alveolar macrophages and its mechanism. Methods The rat alveolar macrophages( NR8383 cells) were conventionally cultured,seeded in 96-well plates,and divided into 3 groups,normal cell group,LPS-stimulation group and curcumin-pretreated group. After stimulation with different doses( 5、10、15、20、25 μmol/L) of curcumin and LPS( 0. 5 μg/m L),the cellular survival rates were determined by cell counting kit-8( CCK-8)assay. The levels of tumor necrosis factor-α( TNF-α) and interleukin-1β( IL-1β) in cell culture supernatant were assessed by enzyme-linked immunosorbent assay( ELISA). Meanwhile,the reactive oxygen species( ROS) level and superoxide dismutase( SOD) activity in macrophages were also determined. After stimulation with curcumin( 15 μmol/L)and LPS( 0. 5 μg/m L),the protein expression of paralemmin-3( PALM3) in NR8383 cells was determined by Western blot analysis. Results The results of the CCK8 assay demonstrated that LPS( 0. 5 ng/m L),or curcumin( 5 ~ 20 μmol/L) plus of LPS( 0. 5 ng/m L) did not suppress the cell viability of NR8383 cells compared with the normal NR8383 cells( P > 0. 05). However,curcumin of 25 μmol/L plus LPS( 0. 5 ng/m L) significantly influence the cell viability of NR8383 cells( P < 0. 05). Curcumin( 5 ~ 20 μmol/L) significantly inhibited the production of TNF-α,IL-1β and ROS,and recovered the activity of SOD in a dose-dependent manner in NR8383 cells after LPS-stimulation( P <0. 05). As for the effect of curcumin on,There were no significant difference on inflammation and oxidative stress of curcumin at 15. 0 μmol/L and 20. 0 μmol/L( P > 0. 05). LPS significantly upregulated the protein expression of PALM3,which was inhibited by curcumin( 15. 0 μmol/L). Conclusions Curcumin inhibits the inflammatory response and oxidative stress induced by LPS in alveolar macrophages and its mechanism is partially associated with the inhibition of LPS-induced PALM3 overexpression.
引文
[1]Qian L,Zhao Y,Guo L,et al.Activating transcription factor 3(ATF3)protects against lipopolysaccharide-induced acute lung injury via inhibiting the expression of TL1A[J].J Cell Physiol,2017,232(12):3727-3734.
[2]Xiang B,Chen L,Wang X,et al.Transplantation of Menstrual Blood-Derived Mesenchymal Stem Cells Promotes the Repair of LPS-Induced Acute Lung Injury[J].Int J Mol Sci,2017,18(4):pii:E689.
[3]Wu DD,Pan PH,Liu B,et al.Inhibition of Alveolar Macrophage Pyroptosis Reduces Lipopolysaccharide-induced Acute Lung Injury in Mice[J].Chin Med J(Engl),2015,128(19):2638-2645.
[4]Soleimani V,Sahebkar A,Hosseinzadeh H.Turmeric(Curcuma longa)and its major constituent(curcumin)as nontoxic and safe substances:Review[J].Phytother Res,2018,32(6):985-995.
[5]孙慧男,陈旭昕,韩志海.姜黄素对脂多糖诱导肺泡上皮细胞炎性反应及单免疫球蛋白白介素-1相关蛋白表达的影响[J].实用医学杂志,2017,33(7):1070-1073.
[6]Moutinho MSS,Arag2o S,Carmo D,et al.Curcumin and Rutin Down-regulate Cyclooxygenase-2 and Reduce Tumor-associated Inflammation in HPV16-Transgenic Mice[J].Anticancer Res,2018,38(3):1461-1466.
[7]Kim J,Jeong SW,Quan H,et al.Effect of curcumin(Curcuma longa extract)on LPS-induced acute lung injury is mediated by the activation of AMPK[J].J Anesth,2016,30(1):100-108.
[8]何爱文,陈寿权,冷巧云,等.百草枯中毒大鼠急性肺损伤机制及姜黄素的干预研究[J].中华危重症医学杂志:电子版,2016,9(1):14-19.
[9]Zhang Y,Liang D,Dong L,et al.Anti-inflammatory effects of novel curcumin analogs in experimental acute lung injury[J].Respir Res,2015,16:43.
[10]Chen X,Tang L,Feng J,et al.Downregulation of Paralemmin-3 Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Rats by Regulating Inflammatory Response and Inhibiting Formation of TLR4/My D88 and TLR4/TRIF Complexes[J].Inflammation,2017,40(6):1983-1999.
[11]Chen XX,Tang L,Fu YM,et al.Paralemmin-3 contributes to lipopolysaccharide-induced inflammatory response and is involved in lipopolysaccharide-Toll-like receptor-4 signaling in alveolar macrophages[J].Int J Mol Med,2017,40(6):1921-1931.
[12]Luo M,Hu L,Li D,et al.MD-2 regulates LPS-induced NL-RP3 inflammasome activation and IL-1beta secretion by a My D88/NF-κB-dependent pathway in alveolar macrophages cell line[J].Mol Immunol,2017,90:1-10.
[13]Wang Y,Mao M,Xu JC.Cell-surface nucleolin is involved in lipopolysaccharide internalization and signalling in alveolar macrophages[J].Cell Biol Int,2011,35(7):677-685.
[14]Sun K,He SB,Qu JG,et al.IRF5 regulates lung macrophages M2 polarization during severe acute pancreatitis in vitro[J].World J Gastroenterol,2016,22(42):9368-9377.
[15]Soni S,Wilson MR,O'Dea KP,et al.Alveolar macrophage-derived microvesicles mediate acute lung injury[J].Thorax,2016,71(11):1020-1029.
[16]陈旭昕,孟激光,韩志海,等.干扰paralemmin-3表达抑制脂多糖诱导的肺泡上皮细胞的炎性反应[J].西部医学,2015,27(7):964-966,970.
[17]陈旭昕,孟激光,张燕,等.干扰PALM3表达对小鼠内毒素性急性肺损伤的影响[J].广东医学,2015,36(20):3101-3104.
[18]Li S,Guo L,Zhao Y,et al.Silencing of Paralemmin-3 Protects Mice from lipopolysaccharide-induced acute lung injury[J].Peptides,2016,76:65-72.
[19]Chen X,Wu X,Zhao Y,et al.A novel binding protein of single immunoglobulin IL-1 receptor-related molecule:Paralemmin-3[J].Biochem Biophys Res Commun,2011,404(4):1029-1033.
[20]Bae H,Jeong CH,Cheng WN,et al.Oxidative stress-induced inflammatory responses and effects of N-acetylcysteine in bovine mammary alveolar cells[J].J Dairy Res,2017,84(4):418-425.
[21]Kumar S,Gupta E,Kaushik S,et al.Evaluation of oxidative stress and antioxidant status:Correlation with the severity of sepsis[J].Scand J Immunol,2018,87(4):e12653.
[22]Jung KI,Pyo CW,Choi SY.Influenza A virus-induced autophagy contributes to enhancement of virus infectivity by SOD1 downregulation in alveolar epithelial cells[J].Biochem Biophys Res Commun,2018,498(4):960-966.
[2]Xiang B,Chen L,Wang X,et al.Transplantation of Menstrual Blood-Derived Mesenchymal Stem Cells Promotes the Repair of LPS-Induced Acute Lung Injury[J].Int J Mol Sci,2017,18(4):pii:E689.
[3]Wu DD,Pan PH,Liu B,et al.Inhibition of Alveolar Macrophage Pyroptosis Reduces Lipopolysaccharide-induced Acute Lung Injury in Mice[J].Chin Med J(Engl),2015,128(19):2638-2645.
[4]Soleimani V,Sahebkar A,Hosseinzadeh H.Turmeric(Curcuma longa)and its major constituent(curcumin)as nontoxic and safe substances:Review[J].Phytother Res,2018,32(6):985-995.
[5]孙慧男,陈旭昕,韩志海.姜黄素对脂多糖诱导肺泡上皮细胞炎性反应及单免疫球蛋白白介素-1相关蛋白表达的影响[J].实用医学杂志,2017,33(7):1070-1073.
[6]Moutinho MSS,Arag2o S,Carmo D,et al.Curcumin and Rutin Down-regulate Cyclooxygenase-2 and Reduce Tumor-associated Inflammation in HPV16-Transgenic Mice[J].Anticancer Res,2018,38(3):1461-1466.
[7]Kim J,Jeong SW,Quan H,et al.Effect of curcumin(Curcuma longa extract)on LPS-induced acute lung injury is mediated by the activation of AMPK[J].J Anesth,2016,30(1):100-108.
[8]何爱文,陈寿权,冷巧云,等.百草枯中毒大鼠急性肺损伤机制及姜黄素的干预研究[J].中华危重症医学杂志:电子版,2016,9(1):14-19.
[9]Zhang Y,Liang D,Dong L,et al.Anti-inflammatory effects of novel curcumin analogs in experimental acute lung injury[J].Respir Res,2015,16:43.
[10]Chen X,Tang L,Feng J,et al.Downregulation of Paralemmin-3 Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Rats by Regulating Inflammatory Response and Inhibiting Formation of TLR4/My D88 and TLR4/TRIF Complexes[J].Inflammation,2017,40(6):1983-1999.
[11]Chen XX,Tang L,Fu YM,et al.Paralemmin-3 contributes to lipopolysaccharide-induced inflammatory response and is involved in lipopolysaccharide-Toll-like receptor-4 signaling in alveolar macrophages[J].Int J Mol Med,2017,40(6):1921-1931.
[12]Luo M,Hu L,Li D,et al.MD-2 regulates LPS-induced NL-RP3 inflammasome activation and IL-1beta secretion by a My D88/NF-κB-dependent pathway in alveolar macrophages cell line[J].Mol Immunol,2017,90:1-10.
[13]Wang Y,Mao M,Xu JC.Cell-surface nucleolin is involved in lipopolysaccharide internalization and signalling in alveolar macrophages[J].Cell Biol Int,2011,35(7):677-685.
[14]Sun K,He SB,Qu JG,et al.IRF5 regulates lung macrophages M2 polarization during severe acute pancreatitis in vitro[J].World J Gastroenterol,2016,22(42):9368-9377.
[15]Soni S,Wilson MR,O'Dea KP,et al.Alveolar macrophage-derived microvesicles mediate acute lung injury[J].Thorax,2016,71(11):1020-1029.
[16]陈旭昕,孟激光,韩志海,等.干扰paralemmin-3表达抑制脂多糖诱导的肺泡上皮细胞的炎性反应[J].西部医学,2015,27(7):964-966,970.
[17]陈旭昕,孟激光,张燕,等.干扰PALM3表达对小鼠内毒素性急性肺损伤的影响[J].广东医学,2015,36(20):3101-3104.
[18]Li S,Guo L,Zhao Y,et al.Silencing of Paralemmin-3 Protects Mice from lipopolysaccharide-induced acute lung injury[J].Peptides,2016,76:65-72.
[19]Chen X,Wu X,Zhao Y,et al.A novel binding protein of single immunoglobulin IL-1 receptor-related molecule:Paralemmin-3[J].Biochem Biophys Res Commun,2011,404(4):1029-1033.
[20]Bae H,Jeong CH,Cheng WN,et al.Oxidative stress-induced inflammatory responses and effects of N-acetylcysteine in bovine mammary alveolar cells[J].J Dairy Res,2017,84(4):418-425.
[21]Kumar S,Gupta E,Kaushik S,et al.Evaluation of oxidative stress and antioxidant status:Correlation with the severity of sepsis[J].Scand J Immunol,2018,87(4):e12653.
[22]Jung KI,Pyo CW,Choi SY.Influenza A virus-induced autophagy contributes to enhancement of virus infectivity by SOD1 downregulation in alveolar epithelial cells[J].Biochem Biophys Res Commun,2018,498(4):960-966.