表没食子儿茶素没食子酸酯可改善高脂饮食大鼠胰腺组织炎症状态
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摘要
目的探讨表没食子儿茶素没食子酸酯(epigallocatechin gallate, EGCG)对高脂饮食大鼠胰腺组织炎症状态的作用及其与胰岛素抵抗的关系。方法将30只SPF级雄性SD大鼠随机分为正常饮食组(the normal diet group as the control,NC组,n=10)和高脂饮食组(high-fat diet group,HFD组,n=20)。喂养16周,当两组大鼠体重出现显著差异后,将HFD组按随机区组原则分为单纯高脂组(high-fat diet group,HFD组,n=10)和EGCG干预组(HFD+0.32%EGCG,EGCG组,n=10);干预16周。留取血清及胰腺组织,检测每组大鼠空腹血糖(fasting blood glucose,FBG)、胰岛素(fasting insulin,FINS)及游离脂肪酸(free fatty acids,FFAs),并计算胰岛素抵抗指数(homeostasis model assessment-insulin resistance index,HOMA-IR);应用免疫组织化学方法检测胰岛中CD68+巨噬细胞数目及TNF-α表达;应用Real-time RT-PCR及Western blot方法检测胰腺组织中CD68及Toll样受体4(Toll-like receptor 4,TLR4)、肿瘤坏死因子受体相关因子6(TNF receptor-associated factor 6,TRAF6)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)及白介素6(interleukin-6,IL-6)等炎症相关因子表达水平。结果 HFD组大鼠体重、FFAs和FINS水平与HOMA-IR指数均明显升高,胰岛中巨噬细胞浸润明显增多,胰腺CD68水平及TLR4、TRAF6、TNF-α和IL-6等炎性因子表达明显上升;EGCG干预的HFD大鼠体重、FFAs和FINS水平、HOMA-IR指数、胰岛巨噬细胞浸润、胰腺CD68水平、胰腺TNF-α和IL-6表达的升高不如HFD大鼠明显,与NC大鼠接近;三组大鼠FBG水平无明显统计学差异;EGCG干预的HFD大鼠TLR4表达水平较HFD组未见明显下降。结论 EGCG可减少高脂饮食大鼠胰岛中巨噬细胞浸润,抑制炎症因子TNF-a及IL-6表达的上调,改善胰岛素敏感性;该作用并非通过TLR4信号通路实现。
Objective To investigate the effects of epigallocatechin gallate(EGCG) on inflammation state of pancreas in rats fed a high-fat diet(HFD) and the relationship with insulin resistance. Methods 30 specific pathogen-free(SPF) male Sprague-Dawley rats were randomly divided into 2 groups: the normal diet group as the control(NC group) and HFD group. When there was a significant difference in body weight between the 2 groups after 16 weeks of feeding, the HFD group was further divided into 2 subgroups: the high-fat diet group(HFD, n=10, 16 weeks) and the EGCG group(HFD + 3.2 g/kg EGCG, n=10, 16 weeks) according to the randomized block principle. After 16 weeks of intervention, the serum and pancreatic tissues were collected. Fasting blood glucose(FBG),fasting insulin(FINS), free fatty acids(FFAs) in each group were measured and homeostasis model assessment-insulin resistance index(HOMA-IR) were calculated. The number of macrophage with biomarkers CD68 protein positive and the expression of tumor necrosis factor-α(TNF-α) in pancreatic tissues were evaluated by immunohistochemistry(IHC) and Western blot analysis. Meanwhile,the mRNA and proteins levels of CD68, Toll-like receptor 4(TLR4), tumor necrosis factor receptor-related factor 6(TRAF6), TNF-αand interleukin 6(IL-6) in pancreatic tissue were also detected by real-time RT-PCR and Western blot. Results There was a significant increase in the body weight, HOMA-IR, the level of serum FFAs and FINS, the number of CD68+ macrophages in pancreas islets, and the expression of CD68, TLR4, TRAF6, TNF-α and IL-6 in pancreas, in the HFD group than those in the NC group. EGCG treatment significantly inhibited the HFD-induced increase in the number of CD68+ macrophages in pancreas islets, body weight, HOMA-IR,the levels of serum FFAs and FINS, the expression of CD68, TNF-α and IL-6 in pancreas, bringing them closer to the NC group, in addition to the expression level of TLR4. And there was no significant difference in the FBG level among the three groups. Conclusion In pancreas islet of rats with high-fat diet, EGCG can reduce macrophage infiltration, inhibit the up-regulation of inflammatory factors such as TNF-a and IL-6, and improve insulin sensitivity, by TLR4-independent signaling pathways.
Objective To investigate the effects of epigallocatechin gallate(EGCG) on inflammation state of pancreas in rats fed a high-fat diet(HFD) and the relationship with insulin resistance. Methods 30 specific pathogen-free(SPF) male Sprague-Dawley rats were randomly divided into 2 groups: the normal diet group as the control(NC group) and HFD group. When there was a significant difference in body weight between the 2 groups after 16 weeks of feeding, the HFD group was further divided into 2 subgroups: the high-fat diet group(HFD, n=10, 16 weeks) and the EGCG group(HFD + 3.2 g/kg EGCG, n=10, 16 weeks) according to the randomized block principle. After 16 weeks of intervention, the serum and pancreatic tissues were collected. Fasting blood glucose(FBG),fasting insulin(FINS), free fatty acids(FFAs) in each group were measured and homeostasis model assessment-insulin resistance index(HOMA-IR) were calculated. The number of macrophage with biomarkers CD68 protein positive and the expression of tumor necrosis factor-α(TNF-α) in pancreatic tissues were evaluated by immunohistochemistry(IHC) and Western blot analysis. Meanwhile,the mRNA and proteins levels of CD68, Toll-like receptor 4(TLR4), tumor necrosis factor receptor-related factor 6(TRAF6), TNF-αand interleukin 6(IL-6) in pancreatic tissue were also detected by real-time RT-PCR and Western blot. Results There was a significant increase in the body weight, HOMA-IR, the level of serum FFAs and FINS, the number of CD68+ macrophages in pancreas islets, and the expression of CD68, TLR4, TRAF6, TNF-α and IL-6 in pancreas, in the HFD group than those in the NC group. EGCG treatment significantly inhibited the HFD-induced increase in the number of CD68+ macrophages in pancreas islets, body weight, HOMA-IR,the levels of serum FFAs and FINS, the expression of CD68, TNF-α and IL-6 in pancreas, bringing them closer to the NC group, in addition to the expression level of TLR4. And there was no significant difference in the FBG level among the three groups. Conclusion In pancreas islet of rats with high-fat diet, EGCG can reduce macrophage infiltration, inhibit the up-regulation of inflammatory factors such as TNF-a and IL-6, and improve insulin sensitivity, by TLR4-independent signaling pathways.
引文
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[26]Klaus S,Pültz S,Th?ne-Reineke C,et al.Epigallocatechingallate attenuates diet-induced obesity in mice by decreasingenergy absorption and increasing fat oxidation.Int J Obes(Lond),2005,29(6):615-623.
[27]Chen YK,Cheung C,Reuhl KR,et al.Effects ofgreen tea polyphenol(-)-epigallocatechin-3-gallate on newlydeveloped high-fat/Western-style diet-induced obesity andmetabolic syndrome in mice.J Agric Food Chem,2011,59(21):11862-11871.
[28]Mlinar B,Marc J,Janez A,et al.Molecular mechanisms of insulin resistance and associated diseases.Clin Chim Acta,2007,375(1-2):20-35.
[29]Geloneze B,Tambascia MA.Laboratorial evaluation anddiagnosis of insulin resistance.Arq Bras Endocrinol Metabol,2006,50(2):208-215.
[30]Pavan Kumar P,Radhika G,Rao GV,et al.Interferonγand glycemic status indiabetes associated with chronic pancreatitis.Pancreatology,2012,12(1):65-70.
[31]Zhang Z,Ding Y,Dai X,et al.Epigallocatechin-3-gallate protects pro-inflammatory cytokine induced injuriesin insulin-producing cells through the mitochondrialpathway.Eur JPharmacol,2011,670(1):311-316.
[32]Han MK.Epigallocatechingallate,a constituent of green tea,suppresses cytokine-induced pancreatic beta-cell damage.Exp Mol Med,2003,35(2):136-139.
[33]Fu Z,Zhen W,Yuskavage J,et al.Epigallocatechingallatedelays the onset of type 1 diabetes in spontaneous non-obesediabetic mice.Br J Nutr,2011,105(8):1218-1225.
[34]Poggi M,Bastelica D,Gual P,et al.C3H/HeJ mice carrying a toll-like receptor 4mutation are protected against the development of insulinresistance in white adipose tissue in response to a high-fatdiet.Diabetologia,2007,50(6):1267-1276.
[35]Tsukumo DM,Carvalho-Filho MA,Carvalheira JB,et al.Loss-of-function mutation inToll-like receptor 4 prevents diet-induced obesity and insulinresistance.Diabetes,2007,56(8):1986-1998.
[36]Kim F,Pham M,Luttrell I,et al.Toll-like receptor-4 mediates vascular inflammation and insulinresistance in diet-induced obesity.Circ Res,2007,100(11):1589-1596.
[37]Pal D,Dasgupta S,Kundu R,et al.Fetuin-A acts asan endogenous ligand of TLR4 to promote lipid-inducedinsulin resistance.Nat Med,2012,18(8):1279-1285.
[38]Bao S,Cao Y,Fan C,et al.Epigallocatechingallate improves insulin signaling by decreasingtoll-like receptor 4(TLR4)activity in adipose tissues of high-fat diet rats.Mol Nutr Food Res,2014,58(4):677-686.
[39]Olefsky JM,Glass CK.Macrophages,inflammation,and insulin resistance.Annu Rev Physiol,2010,72:219-246.
[2]Hotamisligil GS.Inflammation and metabolic disorders.Nature,2006,444(7121):860-867.
[3]Schenk S,Saberi M,Olefsky JM.Insulin sensitivity:modulation by nutrients and inflammation.J Clin Invest,2008,118(9):2992-3002.
[4]Grant RW,Dixit VD.Mechanisms of disease:inflammasome activation and the development of type 2 diabetes.Front Immunol,2013,4:50.
[5]Gupta D,Krueger CB,Lastra G.Over-nutrition.Obesity andinsulin resistance in the development ofβ-cell dysfunction.Curr Diabetes Rev,2012,8(2):76-83.
[6]Montane J,Cadavez L,Novials A.Stress and the inflammatory process:a major cause of pancreatic cell death in type 2diabetes.Diabetes Metab Syndr Obes,2014,7:25-34.
[7]Rabinovitch A,Suarez-Pinzon WL.Cytokines and their roles inpancreatic islet beta-cell destruction and insulin-dependentdiabetes mellitus.Biochem Pharmacol,1998,55(8):1139-1149.
[8]Odegaard JI,Chawla A.Connecting type 1 and type 2 diabetesthrough innate immunity.Cold Spring Harb Perspect Med,2012,2(3):a007724.
[9]Kawai T,Akira S.The roles of TLRs,RLRs and NLRs inpathogen recognition.Int Immunol,2009,21(4):317-337.
[10]Shi H,Kokoeva MV,Inouye K,et al.TLR4links innate immunity and fatty acid-induced insulinresistance.J Clin Invest,2006,116(11):3015-3025.
[11]Youssef-Elabd EM,McGee KC,Tripathi G,et al.Acute and chronic saturated fatty acid treatment as a key instigator of the TLR-mediatedinflammatory response in human adipose tissue,in vitro.J Nutr Biochem,2012,23(1):39-50.
[12]Tian YF,He CT,Chen YT,et al.Lipoic acid suppressesportal endotoxemia-induced steatohepatitis and pancreaticinflammation in rats.World J Gastroenterol,2013,19(18):2761-2771.
[13]Eguchi K,Manabe I,Oishi-Tanaka Y,et al.Saturated fatty acid and TLR signaling linkβ-celldysfunction and islet inflammation.Cell Metab,2012,15(4):518-533.
[14]Lee JY,Sohn KH,Rhee SH,et al.Saturated fatty acids,but not unsaturated fatty acids,induce the expression ofcyclooxygenase-2 mediated through Toll-like receptor 4.J Biol Chem,2001,276(20):16683-16689.
[15]Richardson SJ,Willcox A,Bone AJ,et al.Islet-associated macrophages in type 2 diabetes.Diabetologia,2009,52(8):1686-1688.
[16]Mangine GT,Gonzalez AM,Wells AJ,et al.The effect of a dietary supplement(N-oleyl-phosphatidyl-ethanolamine and epigallocatechin gallate)on dietary compliance and body fat loss in adults who are overweight:a double-blind,randomized control trial.Lipids Health Dis,2012,11:127.
[17]Wolfram S,Wang Y,Thielecke F.Anti-obesity effects of green tea:from bedside to bench.Mol Nutr Food Res,2006,50(2):176-187.
[18]Dickinson D,Derossi S,Yu H,et al.Epigallocatechin-3-gallate modulates anti-oxidantdefense enzyme expression in murine submandibular andpancreatic exocrine gland cells and human HSG cells.Autoimmunity,2014,47(3):177-184.
[19]Hara Y,Fujino M,Takeuchi M,et al.Green-tea polyphenol(-)-epigallocatechin-3-gallate provides resistance to apoptosis inisolated islets.J Hepatobiliary Pancreat Surg,2007,14(5):493-497.
[20]Cha KH,Song DG,Kim SM,et al.Inhibition ofgastrointestinal lipolysis by green tea,coffee,and gomchui(Ligularia fischeri)tea polyphenols during simulated digestion.J Agric Food Chem,2012,60(29):7152-7157.
[21]Sang S,Yang I,Buckley B,et al.Autoxidativequinone formation in vitro and metabolite formation in vivofrom tea polyphenol(-)-epigallocatechin-3-gallate:studied byreal-time mass spectrometry combined with tandem massion mapping.Free Radic Biol Med,2007,43(3):362-371.
[22]Kida K,Suzuki M,Matsumoto N,et al.Identificationof biliary metabolites of(-)-epigallocatechin gallate in rats.J Agric Food Chem,2000,48(9):4151-4155.
[23]Wang S,Moustaid-Moussa N,Chen L,et al.Novel insights of dietary polyphenols and obesity.J Nutr Biochem,2014,25(1):1-18.
[24]Maki KC,Reeves MS,Farmer M,et al.Green tea catechin consumption enhancesexercise-induced abdominal fat loss in overweight and obese adults.J Nutr,2009,139(2):264-270.
[25]Bogdanski P,Suliburska J,Szulinska M,et al.Green tea extract reduces bloodpressure,inflammatory biomarkers,and oxidative stress andimproves parameters associated with insulin resistance in obese,hypertensive patients.Nutr Res,2012,32(6):421-427.
[26]Klaus S,Pültz S,Th?ne-Reineke C,et al.Epigallocatechingallate attenuates diet-induced obesity in mice by decreasingenergy absorption and increasing fat oxidation.Int J Obes(Lond),2005,29(6):615-623.
[27]Chen YK,Cheung C,Reuhl KR,et al.Effects ofgreen tea polyphenol(-)-epigallocatechin-3-gallate on newlydeveloped high-fat/Western-style diet-induced obesity andmetabolic syndrome in mice.J Agric Food Chem,2011,59(21):11862-11871.
[28]Mlinar B,Marc J,Janez A,et al.Molecular mechanisms of insulin resistance and associated diseases.Clin Chim Acta,2007,375(1-2):20-35.
[29]Geloneze B,Tambascia MA.Laboratorial evaluation anddiagnosis of insulin resistance.Arq Bras Endocrinol Metabol,2006,50(2):208-215.
[30]Pavan Kumar P,Radhika G,Rao GV,et al.Interferonγand glycemic status indiabetes associated with chronic pancreatitis.Pancreatology,2012,12(1):65-70.
[31]Zhang Z,Ding Y,Dai X,et al.Epigallocatechin-3-gallate protects pro-inflammatory cytokine induced injuriesin insulin-producing cells through the mitochondrialpathway.Eur JPharmacol,2011,670(1):311-316.
[32]Han MK.Epigallocatechingallate,a constituent of green tea,suppresses cytokine-induced pancreatic beta-cell damage.Exp Mol Med,2003,35(2):136-139.
[33]Fu Z,Zhen W,Yuskavage J,et al.Epigallocatechingallatedelays the onset of type 1 diabetes in spontaneous non-obesediabetic mice.Br J Nutr,2011,105(8):1218-1225.
[34]Poggi M,Bastelica D,Gual P,et al.C3H/HeJ mice carrying a toll-like receptor 4mutation are protected against the development of insulinresistance in white adipose tissue in response to a high-fatdiet.Diabetologia,2007,50(6):1267-1276.
[35]Tsukumo DM,Carvalho-Filho MA,Carvalheira JB,et al.Loss-of-function mutation inToll-like receptor 4 prevents diet-induced obesity and insulinresistance.Diabetes,2007,56(8):1986-1998.
[36]Kim F,Pham M,Luttrell I,et al.Toll-like receptor-4 mediates vascular inflammation and insulinresistance in diet-induced obesity.Circ Res,2007,100(11):1589-1596.
[37]Pal D,Dasgupta S,Kundu R,et al.Fetuin-A acts asan endogenous ligand of TLR4 to promote lipid-inducedinsulin resistance.Nat Med,2012,18(8):1279-1285.
[38]Bao S,Cao Y,Fan C,et al.Epigallocatechingallate improves insulin signaling by decreasingtoll-like receptor 4(TLR4)activity in adipose tissues of high-fat diet rats.Mol Nutr Food Res,2014,58(4):677-686.
[39]Olefsky JM,Glass CK.Macrophages,inflammation,and insulin resistance.Annu Rev Physiol,2010,72:219-246.
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