柚皮苷通过P38 MAPK/NF-κB通路抑制脂多糖致HaCaT细胞炎症反应
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摘要
目的探讨柚皮苷通过P38 MAPK/NF-κB通路对脂多糖(LPS)致HaCaT细胞炎症损伤的抑制作用。方法LPS(0、0.1、1.0、10.0、20.0μg/mL)孵育24 h刺激人永生化角质细胞HaCaT,模拟银屑病模型,MTT法观察细胞存活率变化,Western bloting法检测白介素(IL)-6和NF-κB蛋白表达;MTT法观察柚皮苷(0、5、10、20、40、80、160、320μmol/L)作用24 h对HaCaT存活率的影响;选择LPS、柚皮苷最佳作用浓度。柚皮苷(20和40μmol/L)作用银屑病模型HaCaT细胞24 h,实时荧光定量PCR(qRT-PCR)法检测IL-6、IL-1β、IL-17和肿瘤坏死因子-α(TNF-α)的mRNA表达水平变化;Western blotting法检测细胞核内转录因子NF-κB p65、P38 MAPK磷酸化蛋白水平以及炎症因子IL-6蛋白水平。结果LPS增加HaCaT细胞存活率,并上调NF-κB p65和IL-6蛋白表达,呈剂量相关性,炎症反应在20μg/mL达到高峰;5～160μmol/L柚皮苷对HaCaT细胞无明显毒性作用。与模型组比较,柚皮苷能够显著抑制炎症因子IL-6、IL-1β、IL-17和TNF-α的转录水平(P<0.05);同时能够显著抑制LPS诱导的NF-κB p65和P38 MAPK磷酸化蛋白水平、抑制IL-6蛋白的表达(P<0.05)。结论柚皮苷抑制脂多糖致HaCaT细胞炎症反应,发挥改善银屑病的作用,机制可能与抑制P38 MAPK/NF-κB信号通路有关。
Objective To investigate the inhibitory effects of naringin on HaCaT cells inflammation induced by lipopolysaccharide(LPS) through NF-κB/P38 pathway. Methods LPS(0, 0.1, 1.0, 10.0, 20.0 μg/mL) incubated for 24 hours stimulated human immortalized keratinocytes HaCaT to simulate psoriasis model. MTT method was used to observe the changes of cell survival rate,and Western blotting method was used to detect the expression of IL-6 and NF-κB protein. MTT method was used to observe the effect of naringin(0, 5, 10, 20, 40, 80, 160, 320 μmol/L) on the survival rate of HaCaT cells. The optimal concentration of LPS and naringin was selected. Naringin(20 and 40 μmol/L) acted on HaCaT cells of psoriasis model for 24 hours. Reverse transcription polymerase chain reaction(qRT-PCR) was performed to determine the mRNA expression levels of IL-6, IL-1β, IL-17 and TNF-α.Western blotting were used to detect the phosphorylation levels of P38 MAPK and NF-κB p65 and the inflammatory factor IL-6.Results LPS increased the survival rate of HaCaT cells, and up-regulated the protein expression of NF-κB p65 and IL-6, which was dose-related, and the inflammatory response peaked at 20 μg/mL. 5 ～ 160 μmol/L naringin had no obvious toxic effect on HaCaT cells. Compared with model group, naringin can significantly inhibit the transcription of inflammatory cytokines IL-6, IL-1β, IL-17 and TNF-α(P < 0.05). Meanwhile, naringin can significantly inhibit LPS-induced phosphorylation of P38 MAPK and NF-κB p65,inhibiting the expression of IL-6 protein(P < 0.05). Conclusion Naringin inhibits inflammation of HaCaT cells induced by lipopolysaccharide and plays a role in improving psoriasis. The mechanism may be related to the inhibition of P38 MAPK/NF-κB signaling pathway.
Objective To investigate the inhibitory effects of naringin on HaCaT cells inflammation induced by lipopolysaccharide(LPS) through NF-κB/P38 pathway. Methods LPS(0, 0.1, 1.0, 10.0, 20.0 μg/mL) incubated for 24 hours stimulated human immortalized keratinocytes HaCaT to simulate psoriasis model. MTT method was used to observe the changes of cell survival rate,and Western blotting method was used to detect the expression of IL-6 and NF-κB protein. MTT method was used to observe the effect of naringin(0, 5, 10, 20, 40, 80, 160, 320 μmol/L) on the survival rate of HaCaT cells. The optimal concentration of LPS and naringin was selected. Naringin(20 and 40 μmol/L) acted on HaCaT cells of psoriasis model for 24 hours. Reverse transcription polymerase chain reaction(qRT-PCR) was performed to determine the mRNA expression levels of IL-6, IL-1β, IL-17 and TNF-α.Western blotting were used to detect the phosphorylation levels of P38 MAPK and NF-κB p65 and the inflammatory factor IL-6.Results LPS increased the survival rate of HaCaT cells, and up-regulated the protein expression of NF-κB p65 and IL-6, which was dose-related, and the inflammatory response peaked at 20 μg/mL. 5 ～ 160 μmol/L naringin had no obvious toxic effect on HaCaT cells. Compared with model group, naringin can significantly inhibit the transcription of inflammatory cytokines IL-6, IL-1β, IL-17 and TNF-α(P < 0.05). Meanwhile, naringin can significantly inhibit LPS-induced phosphorylation of P38 MAPK and NF-κB p65,inhibiting the expression of IL-6 protein(P < 0.05). Conclusion Naringin inhibits inflammation of HaCaT cells induced by lipopolysaccharide and plays a role in improving psoriasis. The mechanism may be related to the inhibition of P38 MAPK/NF-κB signaling pathway.
引文
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[9] Varma S R, Sivaprakasam T O, Mishra A, et al.Imiquimod-induced psoriasis-like inflammation in differentiated Human keratinocytes:Its evaluation using curcumin[J]. Eur J Pharmacol, 2017, 813:33-41.
[10] Li S J, Xie R J, Jiang C R, et al. Schizandrin a alleviates LPS-induced injury in human keratinocyte cell Hacat through a MicroRNA-127-dependent regulation[J]. Cell Physiol Biochem, 2018, 49(6):2229-2239.
[11]冯森玲.银屑灵对过度增殖人角质形成细胞STAT3通路干预的影响[D].广州:广州中医药大学, 2013.
[12] Chen P, Yuan Y, Zhang T Y, et al. Pentosan polysulfate ameliorates apoptosis and inflammation by suppressing activation of the p38 MAPK pathway in high glucosetreated HK-2 cells[J]. Int J Mol Med, 2018, 41(2):908-914.
[13] Ren X L, Shi Y L, Zhao D, et al. Naringin protects ultraviolet B-induced skin damage by regulating p38MAPK signal pathway[J]. J Dermatol Sci, 2016, 82(2):106-114.
[14] Jian C Y, Ouyang H B, Xiang X H, et al. Naringin protects myocardial cells from doxorubicininduced apoptosis partially by inhibiting the p38 MAPK pathway[J]. Mol Med Rep, 2017, 16(6):9457-9463.
[15] Chen X, Xiu M, Xing J J, et al. Lanthanum chloride inhibits LPS mediated expressions of pro-inflammatory cytokines and adhesion molecules in HUVECs:involvement of NF-κB-Jmjd3 signaling[J]. Cell Physiol Biochem, 2017, 42(5):1713-1724.
[16]黄翠萍,杨和平,张珍祥,等. p38蛋白激酶对脂多糖诱导肺泡巨噬细胞NF-κB活化的调控[J].华中科技大学学报:医学版, 2007, 36(6):804-807.
[2] Todke P, Shah V H. Psoriasis:implication to disease and therapeutic strategies, with an emphasis on drug delivery approaches[J]. Int J Dermatol, 2018, 57(11):1387-1402.
[3] Talwar H, Bauerfeld C, Bouhamdan M, et al. MKP-1negatively regulates LPS-mediated IL-1βproduction through p38 activation and HIF-1αexpression[J]. Cell Signal, 2017, 34:1-10.
[4] Qu R Z, Chen X M, Hu J, et al. Ghrelin protects against contact dermatitis and psoriasiform skin ininflammation by antagonizing TNF-α/NF-κB signaling pathways[J].Sci Rep, 2019, 9(1):1348.
[5] Alam M A, Subhan N, Rahman M M, et al. Effect of citrus flavonoids, naringin and naringenin, on metabolic syndrome and their mechanisms of action[J]. Adv Nutr,2014, 5(4):404-417.
[6] Bharti S, Rani N, Krishnamurthy B, et al. Preclinical evidence for the pharmacological actions of naringin:a review[J]. Planta Med, 2014, 80(6):437-451.
[7] NilamberLal Das R, Muruhan S, Nagarajan R P, et al.Naringin prevents ultraviolet-B radiation-induced oxidative damage and inflammation through activation of peroxisome proliferator-activated receptorγin mouse embryonic fibroblast(NIH-3T3)cells[J]. J Biochem Mol Toxicol, 2019, 33(3):e22263.
[8]刘辽,贾萍,杨云霞,等.柚皮苷对人皮肤角质细胞分泌趋化因子RANTES及其核转录因子-κB信号通路影响的实验研究[J].中国药房, 2010, 21(1):40-42.
[9] Varma S R, Sivaprakasam T O, Mishra A, et al.Imiquimod-induced psoriasis-like inflammation in differentiated Human keratinocytes:Its evaluation using curcumin[J]. Eur J Pharmacol, 2017, 813:33-41.
[10] Li S J, Xie R J, Jiang C R, et al. Schizandrin a alleviates LPS-induced injury in human keratinocyte cell Hacat through a MicroRNA-127-dependent regulation[J]. Cell Physiol Biochem, 2018, 49(6):2229-2239.
[11]冯森玲.银屑灵对过度增殖人角质形成细胞STAT3通路干预的影响[D].广州:广州中医药大学, 2013.
[12] Chen P, Yuan Y, Zhang T Y, et al. Pentosan polysulfate ameliorates apoptosis and inflammation by suppressing activation of the p38 MAPK pathway in high glucosetreated HK-2 cells[J]. Int J Mol Med, 2018, 41(2):908-914.
[13] Ren X L, Shi Y L, Zhao D, et al. Naringin protects ultraviolet B-induced skin damage by regulating p38MAPK signal pathway[J]. J Dermatol Sci, 2016, 82(2):106-114.
[14] Jian C Y, Ouyang H B, Xiang X H, et al. Naringin protects myocardial cells from doxorubicininduced apoptosis partially by inhibiting the p38 MAPK pathway[J]. Mol Med Rep, 2017, 16(6):9457-9463.
[15] Chen X, Xiu M, Xing J J, et al. Lanthanum chloride inhibits LPS mediated expressions of pro-inflammatory cytokines and adhesion molecules in HUVECs:involvement of NF-κB-Jmjd3 signaling[J]. Cell Physiol Biochem, 2017, 42(5):1713-1724.
[16]黄翠萍,杨和平,张珍祥,等. p38蛋白激酶对脂多糖诱导肺泡巨噬细胞NF-κB活化的调控[J].华中科技大学学报:医学版, 2007, 36(6):804-807.
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