水飞蓟宾通过调控AMPK活性抑制自由脂肪酸诱导的肝细胞炎症因子分泌
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摘要
目的研究水飞蓟宾对自由脂肪酸(FFA)诱导肝细胞炎症因子分泌的抑制作用,并探讨其是否通过调节腺苷单磷酸活化蛋白激酶(AMPK)信号通路实现。方法将HepG2细胞分为对照组、模型组和水飞蓟宾组。水飞蓟宾组(80和160μmol·L-1)预孵育HepG2细胞4h后,用500μmol·L-1的FFA处理HepG2细胞24h。Real-time PCR和Elisa法测定炎症因子IL-1β和TNF-α的mRNA及蛋白表达,油红O染色检测脂质生成情况,Western Blot测定AMPK和p-AMPK的蛋白相对表达量。用AMPK抑制剂或水飞蓟宾孵育细胞后用FFA处理,Elisa法测定炎症因子的生成情况。结果水飞蓟宾组可降低FFA诱导炎症因子生成增加,降低肝细胞中脂质含量的积累。Western Blot测定结果发现,水飞蓟宾可增加AMPK的磷酸化,AMPK抑制剂可逆转水飞蓟宾对炎症因子生成的抑制作用。结论水飞蓟宾组可通过AMPK信号通路抑制FFA诱导肝细胞炎症因子的表达。
Objective To investigate the inhibitory effects of silybin on the inflammatory factor secretion of HepG2 cells induced by free fatty acid(FFA)and to explore whether the underlying mechanism was associated with regulating adenosine monophosphate activated protein kinase(AMPK)signaling pathway.Methods HepG2 cells were divided into 3groups:control group,model group and silybin group.After pre-incubated with silybin(80and 160μmol·L-1),HepG2 cells were treated with FFA(500μmol·L-1)for 24 h.Real-time PCR and Elisa method were used to detect the level of mRNA and protein expression of IL-1βand TNF-α,respectively.The level of lipid accumulation was measured by oil-red O staining.The expression of AMPK and p-AMPK was detected by Western Blot.After incubation with silybin in the absence or presence of AMPK inhibitor,the HepG2 cells treated with FFA and the inflammatory factor levels were determined by Elisa method.Results Silybin can reduce the increased inflammatory cytokines production and reduce the accumulation of lipid content in HepG2 cells induced by FFA.Western Blot results showed that silybin increased the phosphorylation of AMPK in HepG2 cells treated with FFA,and the AMPK inhibitors reversed the inhibitory effect of silybin on the FFA-induced inflammatory factors production.Conclusion Silybin decreased the HepG2 cells inflammatory cytokines expression induced by FFA via inhibiting AMPK activity.
Objective To investigate the inhibitory effects of silybin on the inflammatory factor secretion of HepG2 cells induced by free fatty acid(FFA)and to explore whether the underlying mechanism was associated with regulating adenosine monophosphate activated protein kinase(AMPK)signaling pathway.Methods HepG2 cells were divided into 3groups:control group,model group and silybin group.After pre-incubated with silybin(80and 160μmol·L-1),HepG2 cells were treated with FFA(500μmol·L-1)for 24 h.Real-time PCR and Elisa method were used to detect the level of mRNA and protein expression of IL-1βand TNF-α,respectively.The level of lipid accumulation was measured by oil-red O staining.The expression of AMPK and p-AMPK was detected by Western Blot.After incubation with silybin in the absence or presence of AMPK inhibitor,the HepG2 cells treated with FFA and the inflammatory factor levels were determined by Elisa method.Results Silybin can reduce the increased inflammatory cytokines production and reduce the accumulation of lipid content in HepG2 cells induced by FFA.Western Blot results showed that silybin increased the phosphorylation of AMPK in HepG2 cells treated with FFA,and the AMPK inhibitors reversed the inhibitory effect of silybin on the FFA-induced inflammatory factors production.Conclusion Silybin decreased the HepG2 cells inflammatory cytokines expression induced by FFA via inhibiting AMPK activity.
引文
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[9] Gu M,Zhao P,Huang J,et al.Silymarin ameliorates metabolic dysfunction associated with diet-induced obesity viaactivation of farnesyl X receptor[J].Front Pharmacol,2016,7:345.
[10]Zhang B,Xu D,She L,et al.Silybin inhibits NLRP3inflammasome assembly through the NAD+/SIRT2pathway in mice with nonalcoholic fatty liver disease[J].FASEB J,2018,32:757-767.
[11]Smith B K,Marcinko K,Desjardins E M,et al.Treatment of nonalcoholic fatty liver disease:role of AMPK[J].Am J Physiol Endocrinol Metab,2016,311:E730-E740.
[12]Nati M,Haddad D,Birkenfeld A L,et al.The role of immune cells in metabolism-related liver inflammation and development of non-alcoholic steatohepatitis(NASH)[J].Rev Endocr Metab Disord,2016,17(1):29-39.
[13]Arrese M,Cabrera D,Kalergis A M,et al.Innate immunity and inflammation in NAFLD/NASH[J].Dig Dis Sci,2016,61(5):1294-1303.
[14]Fan S,Li L,Chen S,et al.Silibinin induced-autophagic and apoptotic death is associated with an increase in reactive oxygen and nitrogen species in HeLa cells[J].Free Radic Res,2011,45(11/12):1307-1324.
[15]Marra F,Svegliati-Baroni G.Lipotoxicity and the gutliver axis in NASH pathogenesis[J].J Hepatol,2018,68:280-295.
[16]袁秋贞,郑旭,陈瑞明,等.心肌片通过抑制NF-κB p65信号通路发挥心肌缺血再灌注损伤的保护作用[J].西北药学杂志,2017,32(2):171-174.
[17]Feldstein A E,Werneburg N W,Canbay A,et al.Free fatty acids promote hepatic lipotoxicity by stimulating TNF-αexpression via a lysosomal pathway[J].Hepatology,2004,40(1):185-194.
[2]孙康悦,李颖,李悦琪,等.国内降血脂药物研究进展[J].西北药学杂志,2017,32(1):122-124.
[3] Araujo A R,Rosso N,Bedogni G,et al.Global epidemiology of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis:what we need in the future[J].Liver Int,2018,38(Suppl 1):47-51.
[4] Crespo J,Cayon A,Fernandez-Gil P,et al.Gene expression of tumor necrosis factorαand TNF-receptors,p55and p75,in nonalcoholic steatohepatitis patients[J].Hepatology,2001,34(6):1158-1163.
[5] Del Campo J A,Gallego P,Grande L.Role of inflammatory response in liver diseases:therapeutic strategies[J].World J Hepatol,2018,10(1):1-7.
[6] Li W,Zheng L,Sheng C,et al.Systematic review on the treatment of pentoxifylline in patients with non-alcoholic fatty liver disease[J].Lipids Health Dis,2011,10:49.
[7] Bijak M.Silybin,a major bioactive component of milk thistle(Silybum marianum L.Gaernt.)-chemistry,bioavailability,and metabolism[J].Molecules,2017,22:1942.
[8]王红军,姜媛媛,路平,等.水飞蓟宾的抗肿瘤、抗氧化和免疫调节分子药理学机制研究进展[J].药学学报,2010,45(4):413-421.
[9] Gu M,Zhao P,Huang J,et al.Silymarin ameliorates metabolic dysfunction associated with diet-induced obesity viaactivation of farnesyl X receptor[J].Front Pharmacol,2016,7:345.
[10]Zhang B,Xu D,She L,et al.Silybin inhibits NLRP3inflammasome assembly through the NAD+/SIRT2pathway in mice with nonalcoholic fatty liver disease[J].FASEB J,2018,32:757-767.
[11]Smith B K,Marcinko K,Desjardins E M,et al.Treatment of nonalcoholic fatty liver disease:role of AMPK[J].Am J Physiol Endocrinol Metab,2016,311:E730-E740.
[12]Nati M,Haddad D,Birkenfeld A L,et al.The role of immune cells in metabolism-related liver inflammation and development of non-alcoholic steatohepatitis(NASH)[J].Rev Endocr Metab Disord,2016,17(1):29-39.
[13]Arrese M,Cabrera D,Kalergis A M,et al.Innate immunity and inflammation in NAFLD/NASH[J].Dig Dis Sci,2016,61(5):1294-1303.
[14]Fan S,Li L,Chen S,et al.Silibinin induced-autophagic and apoptotic death is associated with an increase in reactive oxygen and nitrogen species in HeLa cells[J].Free Radic Res,2011,45(11/12):1307-1324.
[15]Marra F,Svegliati-Baroni G.Lipotoxicity and the gutliver axis in NASH pathogenesis[J].J Hepatol,2018,68:280-295.
[16]袁秋贞,郑旭,陈瑞明,等.心肌片通过抑制NF-κB p65信号通路发挥心肌缺血再灌注损伤的保护作用[J].西北药学杂志,2017,32(2):171-174.
[17]Feldstein A E,Werneburg N W,Canbay A,et al.Free fatty acids promote hepatic lipotoxicity by stimulating TNF-αexpression via a lysosomal pathway[J].Hepatology,2004,40(1):185-194.
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