补阳还五汤对LPS诱导巨噬细胞活化与自噬的影响
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摘要
目的:探讨磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/m TOR)信号通路在补阳还五汤抗脂多糖(LPS)诱导巨噬细胞活化与自噬中的作用。方法:细胞增殖毒性检测(CCK-8)法筛选诱导RAW264.7巨噬细胞活力值的最适LPS浓度。在最适LPS浓度下,利用PI3K阻断剂3-甲基腺嘌呤(3-MA)(5 mmol·L~(-1)),Akt阻断剂MK2206(5μmol·L~(-1)),m TOR阻断剂Rapamycin(10μmol·L~(-1)),Beclin-1阻断剂Spautin-1(5μmol·L~(-1))及不同剂量的补阳还五汤含药血清(5%,10%,20%)处理RAW264.7巨噬细胞24 h后,酶联免疫吸附测定(ELISA)检测RAW264.7巨噬细胞炎症因子白细胞介素-1β(IL-1β),白细胞介素-6(IL-6),肿瘤坏死因子-α(TNF-α)的浓度,蛋白免疫印迹法(Western blot)测定磷酸化(p-)PI3K,p-Akt,p-mTOR通路蛋白的表达及微管轻链蛋白3(LC3),泛素结合蛋白1(p62),Beclin-1的水平。自噬双标腺病毒转染检测RAW264.7细胞自噬流的变化。结果:CCK-8结果显示10 mg·L~(-1)LPS作用时,细胞活性显著增强。模型组IL-1β,IL-6,TNF-α浓度显著高于空白组(P<0.01),与模型组比较,Rapamycin显著升高IL-6浓度,其他给药组均可降低IL-1β,IL-6,TNF-α的水平(P<0.05,P<0.01)。模型组p-Akt,p-PI3K,p-mTOR蛋白表达量明显低于空白组(P<0.05),LC3,p62蛋白表达量显著高于空白组(P<0.01),与模型组比较,Rapamycin显著降低p-Akt蛋白表达量,不影响p-mTOR表达,补阳还五汤各剂量组与其他各阻断剂组均可明显升高p-Akt,p-PI3K,p-mTOR蛋白表达量(P<0.05,P<0.01),降低LC3,p62蛋白表达量(P<0.05,P<0.01),各组Beclin-1蛋白表达量均无显著性差异。与空白组比较,模型组中有明显的自噬体斑点形成,自噬流顺畅;与模型组比较,补阳还五汤含药血清组,3-MA,Spautin-1组自噬斑点的形成明显减少或消失,MK2202,Rapamycin组自噬体斑点大小明显减小,但自噬活力仍较强。结论:补阳还五汤抗LPS诱导巨噬细胞活化与自噬与抑制巨噬细胞炎症反应,调控PI3K/Akt/m TOR信号通路,抑制自噬的过度发生有关。
Objective:To investigate the effect of Buyang Huanwu Tang in resisting lipopolysaccharide(LPS)-induced macrophage activation and autophagy through phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein(PI3K/Akt/m TOR)signaling pathway.Method:The cell counting kit-8(CCK-8)method was used to screen out the optimal LPS concentration for inducing the activity of RAW264.7macrophages.RAW264.7 macrophages were treated separately with PI3K blocker 3-methyladenine(3-MA)(5 mmol·L~(-1)),Akt blocker MK2206(5μmol·L~(-1)),m TOR blocker Rapamycin(10μmol·L~(-1)),Beclin1blocker Spautin-1(5μmol·L~(-1)),different doses of Buyang Huanwu Tang serum(5%,10%,20%)and the optimum concentration of LPS for 24 h.The concentrations of inflammatory factors interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in RAW264.7 macrophages were detected by enzymelined immunosorbent assay(ELISA).Western blot was used to detect the expression levels of phosphorylated PI3K,phosphorylated Akt,phosphorylated m TOR protein,microtubule light chain protein 3(LC3),ubiquitinbinding protein 1(p62)and Beclin-1.The autophagy flow of RAW264.7 cells was detected by transfection with autophagy double-labeled adenovirus.Result:Results of CCK-8 showed the highest cell viability when10 mg·L~(-1)LPS was applied.The concentrations of IL-1β,IL-6 and TNF-αin the model group were significantly higher than those in the blank group(P<0.01).Compared with the model group,Rapamycin significantly increased IL-6 concentration(P<0.05),and other administration groups could decrease the levels of IL-1β,IL-6and TNF-α(P<0.05,P<0.01).The expression levels of p-Akt,p-PI3K and p-mTOR in the model group were significantly lower(P<0.05),and LC3 and p62 protein expressions were significantly higher than those in the blank group(P<0.01).Compared with the model group,Rapamycin significantly decreased the expression of pAkt protein,with no impact on the expressions of p-mTOR.However,Buyang Huanwu Tang and other blockers significantly increased p-Akt,p-PI3K and p-mTOR(P<0.05,P<0.01),while decreased the protein expressions of LC3 and p62(P<0.05,P<0.01).There was no significant difference in the expression level of Beclin-1 protein in each group.Compared with the blank group,there were obvious autophagosome spots in the model group,and the autophagic flow was smooth.Compared with the model group,the formation of autophagic spots in 3-MA,Spautin-1 group and Buyang Huanwu Tang groups were significantly decreased or disappeared,and the size of autophagosome spots in MK2206 and Rapamycin groups was significantly reduced,but the autophagy activity was still strong.Conclusion:Buyang Huanwu Tang can resist LPS-induced macrophages activation and autophagy,inhibit macrophage inflammatory response,regulate PI3K/Akt/m TOR signaling pathway,and inhibit the excessive occurrence of autophagy.
Objective:To investigate the effect of Buyang Huanwu Tang in resisting lipopolysaccharide(LPS)-induced macrophage activation and autophagy through phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein(PI3K/Akt/m TOR)signaling pathway.Method:The cell counting kit-8(CCK-8)method was used to screen out the optimal LPS concentration for inducing the activity of RAW264.7macrophages.RAW264.7 macrophages were treated separately with PI3K blocker 3-methyladenine(3-MA)(5 mmol·L~(-1)),Akt blocker MK2206(5μmol·L~(-1)),m TOR blocker Rapamycin(10μmol·L~(-1)),Beclin1blocker Spautin-1(5μmol·L~(-1)),different doses of Buyang Huanwu Tang serum(5%,10%,20%)and the optimum concentration of LPS for 24 h.The concentrations of inflammatory factors interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in RAW264.7 macrophages were detected by enzymelined immunosorbent assay(ELISA).Western blot was used to detect the expression levels of phosphorylated PI3K,phosphorylated Akt,phosphorylated m TOR protein,microtubule light chain protein 3(LC3),ubiquitinbinding protein 1(p62)and Beclin-1.The autophagy flow of RAW264.7 cells was detected by transfection with autophagy double-labeled adenovirus.Result:Results of CCK-8 showed the highest cell viability when10 mg·L~(-1)LPS was applied.The concentrations of IL-1β,IL-6 and TNF-αin the model group were significantly higher than those in the blank group(P<0.01).Compared with the model group,Rapamycin significantly increased IL-6 concentration(P<0.05),and other administration groups could decrease the levels of IL-1β,IL-6and TNF-α(P<0.05,P<0.01).The expression levels of p-Akt,p-PI3K and p-mTOR in the model group were significantly lower(P<0.05),and LC3 and p62 protein expressions were significantly higher than those in the blank group(P<0.01).Compared with the model group,Rapamycin significantly decreased the expression of pAkt protein,with no impact on the expressions of p-mTOR.However,Buyang Huanwu Tang and other blockers significantly increased p-Akt,p-PI3K and p-mTOR(P<0.05,P<0.01),while decreased the protein expressions of LC3 and p62(P<0.05,P<0.01).There was no significant difference in the expression level of Beclin-1 protein in each group.Compared with the blank group,there were obvious autophagosome spots in the model group,and the autophagic flow was smooth.Compared with the model group,the formation of autophagic spots in 3-MA,Spautin-1 group and Buyang Huanwu Tang groups were significantly decreased or disappeared,and the size of autophagosome spots in MK2206 and Rapamycin groups was significantly reduced,but the autophagy activity was still strong.Conclusion:Buyang Huanwu Tang can resist LPS-induced macrophages activation and autophagy,inhibit macrophage inflammatory response,regulate PI3K/Akt/m TOR signaling pathway,and inhibit the excessive occurrence of autophagy.
引文
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[14]Zaffagnini G,Savova A,Danieli A,et al. p62 filaments capture and present ubiquitinated cargos for autophagy[J]. EMBO J,2018,37(5):e98308.
[15]许继文,李金霞,张华敏.中医药调控心血管疾病自噬研究进展[J].中国医药导报,2018,15(10):38-41.
[16]吕仕超,李艳阳,张军平.中医药调控自噬防治心血管疾病的研究[J].时珍国医国药,2016,27(9):2249-2250.
[2]王和峰,翟纯刚,庞文会,等. PI3K/Akt/m TOR信号通路在巨噬细胞自噬及动脉粥样硬化斑块不稳定中的作用[J].中国病理生理杂志,2013,29(3):390-397.
[3]董雯,李保玉,屈园利,等.补阳还五汤治疗脑梗死恢复期气虚血瘀证的Meta分析[J].中西医结合心脑血管病杂志,2016,14(6):580-585.
[4]刘玉晖,侯贝贝,游宇,等.补阳还五汤稳定Apo E-/-小鼠动脉粥样硬化易损斑块的作用机制[J].中国实验方剂学杂志,2018,24(15):112-119.
[5]杨琳,卢斌,庞晓丽,等.基于TGF-β1/Smad4信号探讨缺氧条件下补阳还五汤促进新生血管成熟的机制[J].中国实验方剂学杂志,2018,24(3):114-119.
[6]刘志勇,游宇,刘玉晖,等.补阳还五汤通过调节MAPK信号通路抗内皮细胞损伤的作用[J].中国临床药理学杂志,2016,32(22):2098-2103.
[7]游宇,李林,侯贝贝,等.补阳还五汤抗Apo E-/-小鼠动脉粥样硬化作用与调控巨噬细胞自噬的机制研究[J].中药药理与临床,2018,34(4):2-6.
[8]吴伟烽,李翔,刁胜朋,等.全反式维甲酸对兔颈动脉粥样硬化斑块中TGF-β1、IL-10、MMP-2和MMP-9表达的影响[J].药物评价研究,2018,41(9):1606-1610.
[9]郑冠琳.巨噬细胞在Irisin抗动脉粥样硬化中的作用及相关机制研究[D].济南:山东大学,2018.
[10]密泗宇,周影,王静巧,等.巨噬细胞自噬在动脉粥样硬化中作用的研究进展[J].基础医学与临床,2018,38(7):1020-1024.
[11]Meijer A J,Codogno P. Regulation and role of autophagy in mammalian cells[J]. Int J Biochem Cell Biol,2004,36(12):2445-2462.
[12]Mathew R,Karp C M,Beaudoin B,et al. Autophagy suppresses tumorigenesis through elimination of p62[J]. Cell,2009,137(6):1062-1075.
[13]Jacquin E, Stéphanie L M, Judon C, et al.Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation[J]. Autophagy,2017,13(5):854-867.
[14]Zaffagnini G,Savova A,Danieli A,et al. p62 filaments capture and present ubiquitinated cargos for autophagy[J]. EMBO J,2018,37(5):e98308.
[15]许继文,李金霞,张华敏.中医药调控心血管疾病自噬研究进展[J].中国医药导报,2018,15(10):38-41.
[16]吕仕超,李艳阳,张军平.中医药调控自噬防治心血管疾病的研究[J].时珍国医国药,2016,27(9):2249-2250.