甘草酸二铵脂质配位体对非酒精性脂肪性肝病大鼠肝组织AMPK信号通路的影响
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摘要
目的研究甘草酸二铵脂质配位体(DGLL)对非酒精性脂肪性肝病(NAFLD)大鼠的治疗作用,并探讨其可能的作用机制。方法以高脂饲料喂养建立NAFLD大鼠模型,在造模成功后分别给予DGLL或多烯磷脂酰胆碱(PPC)灌胃16周。采用免疫组化法检测肝组织磷酸化的磷酸腺苷激活蛋白激酶α(P-AMPKα)蛋白表达,采用Real-time PCR法检测肝组织过氧化物酶体增殖物激活受体γ(PPAR-γ)、肉毒碱棕榈酰基转移酶-1(CPT-1)、葡萄糖转运蛋白4(GLUT-4)、酰基辅酶A氧化酶(ACO)、肝脏型脂肪酸结合蛋白(L-FABP)、乙酰辅酶A羧化酶(ACC)m RNA。结果 DGLL组血清瘦素为(3.87±0.38)ng/ml,显著低于模型组【(4.68±0.39)ng/ml,P<0.01】或PPC组【(4.55±0.24)ng/ml,P<0.01】;DGLL组脂联素为(12.27±0.64)μg/ml,显著高于模型组【(10.46±0.28)μg/ml,P<0.01】,但与PPC组【(12.13±0.56)μg/ml,P>0.05】比,无统计学差异;DGLL组大鼠肝组织P-AMPKα蛋白阳性表达面积为(8.38±3.27)%,显著高于模型组【(1.81±0.90)%,P<0.01】或PPC组【(5.50±0.73)%,P<0.05】;DGLL组肝组织PPAR-γ、CPT-1、GLUT-4 m RNA水平分别为(1.07±0.14、0.91±0.05、1.61±0.54),显著高于模型组【分别为(0.26±0.12、0.14±0.01、0.10±0.03,P<0.05】,但ACO m RNA水平(0.23±0.09)与模型组比,无统计学差异【(0.24±0.02),P>0.05】;PPC组PPAR-γ和CPT-1 m RNA水平分别为(0.65±0.16)和(0.22±0.05),显著低于DGLL组(P<0.05),而GLUT-4和ACO m RNA水平(1.32±0.12和0.14±0.05)与DGLL组比,无统计学差异(P>0.05);DGLL组肝组织ACC m RNA水平为(1.67±0.23),显著低于模型组【(3.31±0.02),P<0.05】或PPC组【(2.69±0.14),P<0.05】;DGLL组L-FABP m RNA水平为(1.06±0.04),显著低于模型组【(1.26±0.02),P<0.05】,而与PPC组【(1.06±0.09),P>0.05】比,无统计学差异。结论 DGLL能降低NAFLD大鼠血脂、血糖、胰岛素、HOMA-IR、瘦素水平,升高脂联素,并增加肝组织P-AMPKα蛋白表达,上调PPAR-γ、CPT-1、GLUT-4 m RNA水平、下调L-FABP和ACC m RNA水平,这些作用可能与其激活AMPK通路和改善NAFLD糖脂代谢紊乱有关。
Objective To study the therapeutic effect of diammonium glycyrrhizinate lipid ligand(DGLL)on non-alcoholic fatty liver disease(NAFLD) and to explore its possible mechanism. Methods The model of NAFLD was established in rats by feeding high fat diet,and then the DGLL was given with polyene phosphatidylcholine capsules(PPC) as control. Serum liver function index,blood lipids and glucose,insulin,leptin,adiponectin were detected. The expression of P-AMPKα was immunohistochemically assayed,and PPAR-γ,CPT1,GLUT-4,ACO,L-FABP and ACC m RNA were detected by real time PCR. Results The serum levels of leptin in DGLL group were(3.87±0.38) ng/ml,much lower than in the model group [(4.68 ±0.39) ng/ml,P<0.01] or inPPC group [(4.55 ±0.24) ng/ml,P <0.01];serum adiponectin levels was(12.27 ±0.64)μg/ml,much higher than in the model【(10.46±0.28)μg/ml,P<0.01】,while it was not significantly different from in PPC group 【(12.13 ±0.56)μg/ml,P >0.05】;the P-AMPKα expression area in liver tissue was(8.38±3.27)%,much higher than in the model 【(1.81 ±0.90)%,P<0.01】 or in PPCgroup 【(5.50 ±0.73)%,P <0.05】;the PPAR-γ,CPT-1,GLUT-4 m RNA in liver tissues were(1.07 ±0.14,0.91 ±0.05,1.61±0.54,respectively),much higher than in the model 【(0.26±0.12,0.14±0.01,0.10±0.03,respectivelyt,P<0.05】,while the ACO m RNA was not significantly different from in the model [(0.23±0.09)vs.(0.24±0.02),P>0.05】;the hepatic PPAR-γ and CPT-1 m RNA in PPC group were(0.65±0.16) and(0.22±0.05),much lower than in DGLL group(P<0.05),while the GLUT-4 and ACO m RNA were not significantly different from in the DGLL group(P>0.05);the hepatic ACC m RNA in DGLL group was(1.67±0.23),much lower than in the model【(3.31±0.02),P<0.05】 or in the PPC group【(2.69±0.14),P<0.05】;the hepatic L-FABP m RNA in DGLL group was(1.06±0.04),much lower than in the model 【(1.26 ±0.02),P <0.05】,but not significantly diferent from in PPC group【(1.06 ±0.09),P >0.05】. Conclusion We found DGLL could reduce serum transaminase,blood lipids,blood glucose,insulin,HOMA-IR and leptin,increase hepatic PPAR-γ,CPT-1 and GLUT-4 m RNA and decrease the LFABP and ACC m RNA levels in rats with NAFLD. DGLL could significantly improve liver function and lipid metabolism in NAFLD,which may be related to the activation of AMPK pathway.
Objective To study the therapeutic effect of diammonium glycyrrhizinate lipid ligand(DGLL)on non-alcoholic fatty liver disease(NAFLD) and to explore its possible mechanism. Methods The model of NAFLD was established in rats by feeding high fat diet,and then the DGLL was given with polyene phosphatidylcholine capsules(PPC) as control. Serum liver function index,blood lipids and glucose,insulin,leptin,adiponectin were detected. The expression of P-AMPKα was immunohistochemically assayed,and PPAR-γ,CPT1,GLUT-4,ACO,L-FABP and ACC m RNA were detected by real time PCR. Results The serum levels of leptin in DGLL group were(3.87±0.38) ng/ml,much lower than in the model group [(4.68 ±0.39) ng/ml,P<0.01] or inPPC group [(4.55 ±0.24) ng/ml,P <0.01];serum adiponectin levels was(12.27 ±0.64)μg/ml,much higher than in the model【(10.46±0.28)μg/ml,P<0.01】,while it was not significantly different from in PPC group 【(12.13 ±0.56)μg/ml,P >0.05】;the P-AMPKα expression area in liver tissue was(8.38±3.27)%,much higher than in the model 【(1.81 ±0.90)%,P<0.01】 or in PPCgroup 【(5.50 ±0.73)%,P <0.05】;the PPAR-γ,CPT-1,GLUT-4 m RNA in liver tissues were(1.07 ±0.14,0.91 ±0.05,1.61±0.54,respectively),much higher than in the model 【(0.26±0.12,0.14±0.01,0.10±0.03,respectivelyt,P<0.05】,while the ACO m RNA was not significantly different from in the model [(0.23±0.09)vs.(0.24±0.02),P>0.05】;the hepatic PPAR-γ and CPT-1 m RNA in PPC group were(0.65±0.16) and(0.22±0.05),much lower than in DGLL group(P<0.05),while the GLUT-4 and ACO m RNA were not significantly different from in the DGLL group(P>0.05);the hepatic ACC m RNA in DGLL group was(1.67±0.23),much lower than in the model【(3.31±0.02),P<0.05】 or in the PPC group【(2.69±0.14),P<0.05】;the hepatic L-FABP m RNA in DGLL group was(1.06±0.04),much lower than in the model 【(1.26 ±0.02),P <0.05】,but not significantly diferent from in PPC group【(1.06 ±0.09),P >0.05】. Conclusion We found DGLL could reduce serum transaminase,blood lipids,blood glucose,insulin,HOMA-IR and leptin,increase hepatic PPAR-γ,CPT-1 and GLUT-4 m RNA and decrease the LFABP and ACC m RNA levels in rats with NAFLD. DGLL could significantly improve liver function and lipid metabolism in NAFLD,which may be related to the activation of AMPK pathway.
引文
[1]Donati G,Stagni B,Piscaglia F,et al.Increased prevalence of fatty liver in arterial hypertensive patients with normal liver enzymes:role of insulin resistance.Gut,2004,53(7):1020-1023.
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[4]Forbes S,Taylor-Robinson SD,Patel N,et al.Increased prevalence of non-alcoholic fatty liver disease in european women with a history of gestational diabetes.Diabetologia,2011,54(3):641-647.
[5]Yatsuji S,Hashimoto E,Kaneda H,et al.Diagnosing autoimmune hepatitis in nonalcoholic fatty liver disease:s the international autoimmune hepatitis group scoring system useful.J Gastroenterol,2005,40(12):1130-1138.
[6]Marchesini G,Bugianesi E,Forlani G,et al.Nonalcoholic fatty liver,steatohepatitis,and the metabolic syndrome.Hepatology,2003,37(4):917-923.
[7]茹仁萍,吴锡铭.18α甘草酸及其脂质配位体的生物利用度与抗肝损害作用的比较.浙江医学,2001,23(8):18-20.
[8]Xu P,Zhang XG,Li YM,et al.Research on the protection effect of pioglitazone for non-alcoholic fatty liver disease(NAFLD)in rats.J Zhejiang Univ Sci B,2006,7(8):627-633.
[9]Savvidou S,Hytiroglou P,Orfanou-Koumerkeridou H,et al.Low serum adiponectin levels are predictive of advanced hepatic fibrosis in patients with NAFLD.J Clin Gastroenterol,2009,43(8):765-772.
[10]Emoto M,Nishizawa Y,Maekawa K,et al.Homeostasis model assessment as a clinical index of insulin resistance in type 2diabetic patients treated with sulfonylureas.Diabetes Care,1999,22(5):818-822.
[11]Hawley SA,Davison M,Woods A,et al.Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase.J Biol Chem,1996,271(44):27879-27887.
[12]Kemp BE,Stapleton D,Campbell DJ,et al.AMP-activated protein kinase,super metabolic regulator.Biochem Soc Trans,2003,31(Pt 1):162-168.
[13]Rutter GA,Da Silva Xavier G,Leclerc I.Roles of 5'-AMP-activated protein kinase(AMPK)in mammalian glucose homoeostasis.Biochem J,2003,375(Pt 1):1-16.
[14]Mac Lean PS,Zheng D,Jones JP,et al.Exercise-induced transcription of the muscle glucose transporter(GLUT 4)gene.Biochem Biophys Res Commun,2002,292(2):409-414.
[15]Liu Y,Wan Q,Guan Q,et al.High-fat diet feeding impairs both the expression and activity of AMPKa in rats'skeletal muscle.Biochem Biophys Res Commun,2006,339(2):701-707.
[16]Rangwala SM,Lazar MA.Peroxisome proliferator-activated receptor gamma in diabetes and metabolism.Trends Pharmacol Sci,2004,25(6):331-336.
[17]Lazar MA.PPAR gamma,10 years later.Biochimie,2005,87(1):9-13.
[18]Tamori Y,Masugi J,Nishino N,et al.Role of peroxisome proliferator-activated receptor-gamma in maintenance of the characte ristics of mature 3T3-L1 adipocytes.Diabetes,2002,51(7):2045-2055.
[19]王兆君,叶平,张秀锦.衰老对大鼠肝脏组织酰基辅酶A氧化酶水平的影响.中国动脉硬化杂志,2005,13(1):10-12.
[20]王乐,张霞.非酒精性脂肪肝分子机制的研究进展.现代预防医学,2010,37(11):2027-2030.
[2]Diehl AM.Fatty liver,hypertension,and the metabolic syndrome.Gut,2004,53(7):923-924.
[3]常珊珊,徐济良.非酒精性脂肪肝发病相关因子的机制研究新进展.南通大学学报(医学版),2013,33(1):56-60.
[4]Forbes S,Taylor-Robinson SD,Patel N,et al.Increased prevalence of non-alcoholic fatty liver disease in european women with a history of gestational diabetes.Diabetologia,2011,54(3):641-647.
[5]Yatsuji S,Hashimoto E,Kaneda H,et al.Diagnosing autoimmune hepatitis in nonalcoholic fatty liver disease:s the international autoimmune hepatitis group scoring system useful.J Gastroenterol,2005,40(12):1130-1138.
[6]Marchesini G,Bugianesi E,Forlani G,et al.Nonalcoholic fatty liver,steatohepatitis,and the metabolic syndrome.Hepatology,2003,37(4):917-923.
[7]茹仁萍,吴锡铭.18α甘草酸及其脂质配位体的生物利用度与抗肝损害作用的比较.浙江医学,2001,23(8):18-20.
[8]Xu P,Zhang XG,Li YM,et al.Research on the protection effect of pioglitazone for non-alcoholic fatty liver disease(NAFLD)in rats.J Zhejiang Univ Sci B,2006,7(8):627-633.
[9]Savvidou S,Hytiroglou P,Orfanou-Koumerkeridou H,et al.Low serum adiponectin levels are predictive of advanced hepatic fibrosis in patients with NAFLD.J Clin Gastroenterol,2009,43(8):765-772.
[10]Emoto M,Nishizawa Y,Maekawa K,et al.Homeostasis model assessment as a clinical index of insulin resistance in type 2diabetic patients treated with sulfonylureas.Diabetes Care,1999,22(5):818-822.
[11]Hawley SA,Davison M,Woods A,et al.Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase.J Biol Chem,1996,271(44):27879-27887.
[12]Kemp BE,Stapleton D,Campbell DJ,et al.AMP-activated protein kinase,super metabolic regulator.Biochem Soc Trans,2003,31(Pt 1):162-168.
[13]Rutter GA,Da Silva Xavier G,Leclerc I.Roles of 5'-AMP-activated protein kinase(AMPK)in mammalian glucose homoeostasis.Biochem J,2003,375(Pt 1):1-16.
[14]Mac Lean PS,Zheng D,Jones JP,et al.Exercise-induced transcription of the muscle glucose transporter(GLUT 4)gene.Biochem Biophys Res Commun,2002,292(2):409-414.
[15]Liu Y,Wan Q,Guan Q,et al.High-fat diet feeding impairs both the expression and activity of AMPKa in rats'skeletal muscle.Biochem Biophys Res Commun,2006,339(2):701-707.
[16]Rangwala SM,Lazar MA.Peroxisome proliferator-activated receptor gamma in diabetes and metabolism.Trends Pharmacol Sci,2004,25(6):331-336.
[17]Lazar MA.PPAR gamma,10 years later.Biochimie,2005,87(1):9-13.
[18]Tamori Y,Masugi J,Nishino N,et al.Role of peroxisome proliferator-activated receptor-gamma in maintenance of the characte ristics of mature 3T3-L1 adipocytes.Diabetes,2002,51(7):2045-2055.
[19]王兆君,叶平,张秀锦.衰老对大鼠肝脏组织酰基辅酶A氧化酶水平的影响.中国动脉硬化杂志,2005,13(1):10-12.
[20]王乐,张霞.非酒精性脂肪肝分子机制的研究进展.现代预防医学,2010,37(11):2027-2030.