甘草黄酮对运动大鼠股四头肌病理学变化及FAT/CD36表达的影响
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摘要
背景:研究发现,骨骼肌中的FAT/CD36通常是在运动的刺激作用下诱导骨骼肌收缩向细胞膜外转运,通过转运增加了细胞膜的FAT/CD36含量,从而增加了脂肪酸进入骨骼肌细胞的数量。目的:观察运动前补充甘草黄酮大鼠股四头肌病理改变和脂肪酸转移酶FAT/CD36的影响。方法:SD雄性大鼠30只,随机分为安静对照组、运动对照组、运动+甘草黄酮组。运动+甘草黄酮组按10.0 mL/(kg·d)进行灌服给药,对照组给等量生理盐水;并且运动2组均进行为期6周的训练,速度为19.3 m/min,跑台坡度为5°。苏木精-伊红染色观察股四头肌的病理学变化;免疫组织化学法和Western Blot法测定FAT/CD36的表达。结果与结论:(1)光镜下观察运动对照组大鼠股四头肌纤维有肿胀、破裂和坏死的现象,运动+甘草黄酮组股四头肌纤维细胞排列较紧密,肌纤维边缘完整,与安静对照组比较无明显的差异;(2)免疫组织化学反应显示,运动对照组大鼠股四头肌FAT/CD36表达高于安静对照组(P<0.05),运动+甘草黄酮组大鼠股四头肌FAT/CD36表达略高于安静对照组,但低于运动对照组(P<0.05);(3)Western Blot检测结果与免疫组织化学具有相同的趋势;(4)结果说明,运动诱导增加了脂肪酸转运能量,出现积累的效应;FAT/CD36通过甘草黄酮协同作用下增加了脂肪酸的运转效率,从而维持骨骼肌脂质中的能量代谢稳态在一定的水平上,防止脂质代谢紊乱。
BACKGROUND: Fatty acid translocase/CD36(FAT/CD36) in skeletal muscle has been proved to induce skeletal muscle contraction transport to the outer membrane stimulated by movement, which increases FAT/CD36 content in cellular membrane, further leading to more fatty acids into skeletal muscle cells. OBJECTIVE: To observe the effects of Glycyrrhiza Flavone administration before exercise on the pathological changes of quadriceps and FAT/CD36 in rats. METHODS: Thirty male Sprague-Dawley rats were randomly divided into quiet, exercise and combination groups. The rats in the combination group were administrated with 10 m L/(kg·d) Glycyrrhiza Flavone, while the other two groups were given the same volume of normal saline. The rats in the exercise and combination groups were subjected to 6-week training, 19.3 m/minute at the treadmill slope of 5°. The pathological changes of quadriceps were observed by hematoxylin-eosin staining, and the expression level of FAT/CD36 was detected by immunohistochemistry and Western blot assay. RESULTS AND CONCLUSION: Under light microscope, swollen, ruptured and necrotic muscle fibers in the quadriceps were found in the excise group. In the combination group, muscle fibers tightly arranged in the quadriceps with complete edge, which had no significant difference compared with the quiet group. Immunohistochemistry showed that the expression level of FAT/CD36 in the exercise group was significantly higher than that in the quiet group(P < 0.05); the expression level of FAT/CD36 in the combination group was slightly higher than that in the quiet group, but significnatly lower than that in the exercise group(P < 0.05), all above findings were consistent with the western blot assay. Our results indicate that exercise can induce fatty acid transport and accumulation. Due to the synergistic action of Glycyrrhiza Flavonoids, FAT/CD36 increases the efficiency of fatty acid transport, thereby maintaining lipid metabolism balance in the skeletal muscle, which avoids lipid metabolic disorders.
BACKGROUND: Fatty acid translocase/CD36(FAT/CD36) in skeletal muscle has been proved to induce skeletal muscle contraction transport to the outer membrane stimulated by movement, which increases FAT/CD36 content in cellular membrane, further leading to more fatty acids into skeletal muscle cells. OBJECTIVE: To observe the effects of Glycyrrhiza Flavone administration before exercise on the pathological changes of quadriceps and FAT/CD36 in rats. METHODS: Thirty male Sprague-Dawley rats were randomly divided into quiet, exercise and combination groups. The rats in the combination group were administrated with 10 m L/(kg·d) Glycyrrhiza Flavone, while the other two groups were given the same volume of normal saline. The rats in the exercise and combination groups were subjected to 6-week training, 19.3 m/minute at the treadmill slope of 5°. The pathological changes of quadriceps were observed by hematoxylin-eosin staining, and the expression level of FAT/CD36 was detected by immunohistochemistry and Western blot assay. RESULTS AND CONCLUSION: Under light microscope, swollen, ruptured and necrotic muscle fibers in the quadriceps were found in the excise group. In the combination group, muscle fibers tightly arranged in the quadriceps with complete edge, which had no significant difference compared with the quiet group. Immunohistochemistry showed that the expression level of FAT/CD36 in the exercise group was significantly higher than that in the quiet group(P < 0.05); the expression level of FAT/CD36 in the combination group was slightly higher than that in the quiet group, but significnatly lower than that in the exercise group(P < 0.05), all above findings were consistent with the western blot assay. Our results indicate that exercise can induce fatty acid transport and accumulation. Due to the synergistic action of Glycyrrhiza Flavonoids, FAT/CD36 increases the efficiency of fatty acid transport, thereby maintaining lipid metabolism balance in the skeletal muscle, which avoids lipid metabolic disorders.
引文
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[2]Tanaka D,Suga T,Tanaka T,et al.Ischemic Preconditioning Enhances Muscle Endurance during Sustained Isometric Exercise.Int J Sports Med.2016;37(8):614-618.
[3]Holloway GP,Bezaire V,Heigenhauser GJ,et al.Mitochondrial long chain fatty acid oxidation,fatty acid translocase/CD36 content and carnitine palmitoyltransferase I activityin human skeletal muscle during aerobic exercise.J Physiol.2006;57I(Pt1):201-210.
[4]Smith BK,Jain SS,Rimbaud S,et al.CD36 is located on the outer mitochondria membrane,upstream of long-chain acyl-Co A synthetase,and regulates palmitate oxidation.Biochem J.2011;437(1):125-134.
[5]Xie YC,Dong XM,Wu XM,et al.Inhibitory effects of flavonoids extracted from licorice on lipopolysaccharide-induced acute pulmonary inflammation in mice.International Immunopharmacology.2009;9(2):194-200.
[6]Tang S,Huang W,Ji S,et al.Prenylated flavonoids from Glycyrrhiza uralensis as promising anti-cancer agents:a preliminary structure-activity study.Journal of Chinese Pharmaceutical Sciences.2016;25(1):23-29.
[7]黄东,卜凯,阳毅.甘草黄酮对运动大鼠体内糖及脂肪组织RBP4m RNA表达的影响[J].中国实验方剂学杂志,2014,20(2):124-128.
[8]黄东,李跃林,卜凯,等.黄酮对运动大鼠脂肪代谢酶与ERRα表达的影响[J].广西师范大学学报:自然科学版,2016,34(1):168-173.
[9]Bedford TG,Tipton CM,Wilson NC,et al.Maximum oxygen consumption of rats and its changes with various experimental procedures.J Appl Physiol.1979;47(6):1278-1283.
[10]邢国秀,李楠,王童,等.甘草中黄酮类化学成分的研究进展[J].中国中药杂志,2003,28(7):593-597.
[11]Bode AM,Dong Z.Chemopreventive Effects of Licorice and Its Components.Current Pharmacology Reports.2015;1(1):60-71.
[12]何薇,宁静,吴敬敬,等.甘草化学成分与细胞色素P450酶间的相互作用研究进展[J].中草药,2016,47(11);1974-1981.
[13]景晶,赵金英,华冰,等.甘草总黄酮抑制硫代乙酰胺诱导肝纤维化大鼠肝组织中TGF-及Caspase-3的表达[J].中国中药杂志,2015,40(15):3034-3040.
[14]张佳莹,魏苗苗,初晓,等.甘草黄酮对小鼠急性肺损伤保护机制的研究[J].中国农学通报,2012,28(8):56-62.
[15]冯亚娟,胡滨青,周建华.甘草黄酮对糖尿病大鼠血糖、血脂水平及抗氧化能力的影响[J].山东医药,2016,56(3):23-25.
[16]张明发,沈雅琴.甘草及其有效成分的抗糖尿病药理作用的研究进展[J].抗感染药学,2015,12(1):1-4.
[17]卢宁清,赵伟鸿,樊紫周,等.宁夏栽培甘草的黄酮提取物降糖作用实验研究[J].宁夏医科大学学报,2013,35(5):510-514.
[18]赵金英,杨卫东,李红兵,等.栽培甘草中甘草黄酮提取物对糖尿病大鼠血糖血脂的调节作用[J].时珍国医国药,2012,23(1):101-103.
[19]阳毅,莫伟彬,李启畅.甘草黄酮对运动大鼠肝组织自由基代谢及P53 m RNA表达的影响[J].中国实验方剂学杂志,2013,19(19):245-249.
[20]杨衍滔.甘草黄酮对运动训练大鼠睾酮、皮质酮及血脂代谢的影响[J].现代预防医学,2015,42(16):2979-2982.
[21]Tao N,Wagner SJ,Lublin DM.CD36 is palmitoylated on both N-and C-terminal cytoplasmic tails.J Biol Chem.1996;271(37):22315-22320.
[22]Wellen KE,Hotamisligil GS.Obesity-induced inflammatory changes in adipose tissue.J Clin Invest.2003;112(12):1785-1785.
[23]Zineb T,Azeddine I.Insight into the mechanism of lipids binding and uptake by CD36 receptor.Bioinformation.2015;11(6):302-306.
[24]Tsuzuki S,Amitsuka T,Okahashi T,et al.A single aldehyde group can serve as a structural element for recognition by transmembrane protein CD36.Biosci Biotechnol Biochem.2016;80(7):1375-1378.
[25]Stewart CR,Stuart LM,Wilkinson K,et al.CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer.Nature Immunology.2010;11(2):155-161.
[26]MoulléVS,Picard A,Le Foll C,et al.Lipid sensing in the brain and regulation of energy balance.Diabetes Metabolism.2014;40(1):29-33.
[27]Harb D,Bujold K,Febbraio M,et al.The role of the scavenger receptor CD36 in regulating mononuclear phagocyte trafficking to atherosclerotic lesions and vascular inflammation.Cardiovasc Res.2009;83(1):42-51.
[28]Tilg H,Moschen AR.Inflammatory mechanisms in the regulation of insulin resistance.Molecular Medicine.2008;14(3-4):222.
[29]张艳艳,赵蕾,谢云霞,等.FAT/CD36在高脂喂养小鼠脂肪组织炎症中的作用[J].中国病理生理杂志,2015,31(3):463-467.
[30]Sundaresan S,Abumrad NA.Dietary Lipids Inform the Gut and Brain about Meal Arrival via CD36-Mediated Signal Transduction.J Nutr.2015;145(10):2195-200.
[31]Liw,Wang D,Chi Y,et al.7-Ketocholesteryl-9-carboxynona--noate enhances the expression of ATP-binding cassette transporter A1 via CD36.Atherosclerosis.2013;226(1):102-109.
[32]Gross B,Pawlak M,Lefebvre P,et al.PPARs in obesity-induced T2DM,dyslipidaemia and NAFLD.Nat Rev Endocrinol.2017;13(1):36-49.
[33]Ahn SB,Jang K,Jun DW,et al.Expression of liver X receptor correlates with intrahepatic inflammation and fibrosis in patients with nonalcoholic fatty liver disease.Dig Dis Sci.2014;59(12):2975-2982.
[34]Xie P,Zhang AT,Wang C,et al.Molecular cloning,characterization,and expression analysis of fatty acid translocase(FAT/CD36)in the pigeon(Columba livia domestica).Poultry Science.2012;91(7):1670-1679.
[35]薄俊霞,李敬达,刘庆平.脂肪酸移位酶FAT/CD36转运长链脂肪酸研究进展[J].动物医学进展,2017,38(5):93-97.
[36]Takai M,Kozai Y,Tsuzuki S,et al.Unsaturated long-chain fatty acids inhibit the binding of oxidized low-density lipoproteins to a model CD36[J].Bioscience Biotechnology&Biochemistry.2014;78(2):238-244.
[37]Kozai Y,Tsuzuki S,Takai M,et al.Further validation of unsaturated long-chain fatty acids as inhibitors for oxidized low-density lipoprotein binding to CD36 via assays with synthetic CD36 peptide-cross-linked plates.Biosci Biotechnol Biochem.2014;78(5):839-842.
[38]Demers A,Samami S,Lauzier B,et al.PCSK9 Induces CD36Degradation and Affects Long-Chain Fatty Acid Uptake and Triglyceride Metabolism in Adipocytes and in Mouse Liver Significance.Arterioscler Thromb Vasc Biol.2015;35(12):2517-2525.
[39]Hoshino D,Yoshida Y,Kitaoka Y,et al.High-intensity interval training increases intrinsic rates of mitochondrial fatty acid oxidation in rat red and white skeletal muscle.Applied Physiology Nutrition&Metabolism.2013;38(3):326-333.
[40]Hrometz SL,Ebert JA,Grice KE,et al.Potentiation of Ecstasy-induced hyperthermia and FAT/CD36 expression in chronically exercised animals.Temperature.2016:24(8):1-5.
[41]Talanian JL,Holloway GP,Heigenhauser GJ,etal.Exercise training increases sarcolemmal and mitochondrial fatty acid transport proteins in human skeletal muscle.Ajp Endocrinology Metabolism.2010;299(2):E180-188.
[42]齐洁任,彩玲,傅建,等.运动降低MG53表达及其在缓解高脂膳食大鼠IR中的作用[J].中国运动医学杂志,2016,35(3):240-247.
[43]Yoshida Y,Jain SS,Mcfarlan JT,et al.Exercise-and training-induced upregulation of skeletal muscle fatty acid oxidation are not solely dependent on mitochondrial machinery and biogenesis.J Physiol.2013;591(Pt18):4415-4426.
[44]Jain SS,Chabowski A,Snook LA,et al.Additive effects of insulin and muscle contraction on fatty acid transport and fatty acid transporters,FAT/CD36,FABPpm,FATP1,4 and 6.Febs Letters.2009;583(13):2294-300.
[45]Lee JK,Lee JS,Park H,et al.Effect of l-carnitine supplementation and aerobic training on FABPc content andβ-HAD activity in human skeletal muscle.Eur J Appl Physiol.2007;99(2):193-199.
[2]Tanaka D,Suga T,Tanaka T,et al.Ischemic Preconditioning Enhances Muscle Endurance during Sustained Isometric Exercise.Int J Sports Med.2016;37(8):614-618.
[3]Holloway GP,Bezaire V,Heigenhauser GJ,et al.Mitochondrial long chain fatty acid oxidation,fatty acid translocase/CD36 content and carnitine palmitoyltransferase I activityin human skeletal muscle during aerobic exercise.J Physiol.2006;57I(Pt1):201-210.
[4]Smith BK,Jain SS,Rimbaud S,et al.CD36 is located on the outer mitochondria membrane,upstream of long-chain acyl-Co A synthetase,and regulates palmitate oxidation.Biochem J.2011;437(1):125-134.
[5]Xie YC,Dong XM,Wu XM,et al.Inhibitory effects of flavonoids extracted from licorice on lipopolysaccharide-induced acute pulmonary inflammation in mice.International Immunopharmacology.2009;9(2):194-200.
[6]Tang S,Huang W,Ji S,et al.Prenylated flavonoids from Glycyrrhiza uralensis as promising anti-cancer agents:a preliminary structure-activity study.Journal of Chinese Pharmaceutical Sciences.2016;25(1):23-29.
[7]黄东,卜凯,阳毅.甘草黄酮对运动大鼠体内糖及脂肪组织RBP4m RNA表达的影响[J].中国实验方剂学杂志,2014,20(2):124-128.
[8]黄东,李跃林,卜凯,等.黄酮对运动大鼠脂肪代谢酶与ERRα表达的影响[J].广西师范大学学报:自然科学版,2016,34(1):168-173.
[9]Bedford TG,Tipton CM,Wilson NC,et al.Maximum oxygen consumption of rats and its changes with various experimental procedures.J Appl Physiol.1979;47(6):1278-1283.
[10]邢国秀,李楠,王童,等.甘草中黄酮类化学成分的研究进展[J].中国中药杂志,2003,28(7):593-597.
[11]Bode AM,Dong Z.Chemopreventive Effects of Licorice and Its Components.Current Pharmacology Reports.2015;1(1):60-71.
[12]何薇,宁静,吴敬敬,等.甘草化学成分与细胞色素P450酶间的相互作用研究进展[J].中草药,2016,47(11);1974-1981.
[13]景晶,赵金英,华冰,等.甘草总黄酮抑制硫代乙酰胺诱导肝纤维化大鼠肝组织中TGF-及Caspase-3的表达[J].中国中药杂志,2015,40(15):3034-3040.
[14]张佳莹,魏苗苗,初晓,等.甘草黄酮对小鼠急性肺损伤保护机制的研究[J].中国农学通报,2012,28(8):56-62.
[15]冯亚娟,胡滨青,周建华.甘草黄酮对糖尿病大鼠血糖、血脂水平及抗氧化能力的影响[J].山东医药,2016,56(3):23-25.
[16]张明发,沈雅琴.甘草及其有效成分的抗糖尿病药理作用的研究进展[J].抗感染药学,2015,12(1):1-4.
[17]卢宁清,赵伟鸿,樊紫周,等.宁夏栽培甘草的黄酮提取物降糖作用实验研究[J].宁夏医科大学学报,2013,35(5):510-514.
[18]赵金英,杨卫东,李红兵,等.栽培甘草中甘草黄酮提取物对糖尿病大鼠血糖血脂的调节作用[J].时珍国医国药,2012,23(1):101-103.
[19]阳毅,莫伟彬,李启畅.甘草黄酮对运动大鼠肝组织自由基代谢及P53 m RNA表达的影响[J].中国实验方剂学杂志,2013,19(19):245-249.
[20]杨衍滔.甘草黄酮对运动训练大鼠睾酮、皮质酮及血脂代谢的影响[J].现代预防医学,2015,42(16):2979-2982.
[21]Tao N,Wagner SJ,Lublin DM.CD36 is palmitoylated on both N-and C-terminal cytoplasmic tails.J Biol Chem.1996;271(37):22315-22320.
[22]Wellen KE,Hotamisligil GS.Obesity-induced inflammatory changes in adipose tissue.J Clin Invest.2003;112(12):1785-1785.
[23]Zineb T,Azeddine I.Insight into the mechanism of lipids binding and uptake by CD36 receptor.Bioinformation.2015;11(6):302-306.
[24]Tsuzuki S,Amitsuka T,Okahashi T,et al.A single aldehyde group can serve as a structural element for recognition by transmembrane protein CD36.Biosci Biotechnol Biochem.2016;80(7):1375-1378.
[25]Stewart CR,Stuart LM,Wilkinson K,et al.CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer.Nature Immunology.2010;11(2):155-161.
[26]MoulléVS,Picard A,Le Foll C,et al.Lipid sensing in the brain and regulation of energy balance.Diabetes Metabolism.2014;40(1):29-33.
[27]Harb D,Bujold K,Febbraio M,et al.The role of the scavenger receptor CD36 in regulating mononuclear phagocyte trafficking to atherosclerotic lesions and vascular inflammation.Cardiovasc Res.2009;83(1):42-51.
[28]Tilg H,Moschen AR.Inflammatory mechanisms in the regulation of insulin resistance.Molecular Medicine.2008;14(3-4):222.
[29]张艳艳,赵蕾,谢云霞,等.FAT/CD36在高脂喂养小鼠脂肪组织炎症中的作用[J].中国病理生理杂志,2015,31(3):463-467.
[30]Sundaresan S,Abumrad NA.Dietary Lipids Inform the Gut and Brain about Meal Arrival via CD36-Mediated Signal Transduction.J Nutr.2015;145(10):2195-200.
[31]Liw,Wang D,Chi Y,et al.7-Ketocholesteryl-9-carboxynona--noate enhances the expression of ATP-binding cassette transporter A1 via CD36.Atherosclerosis.2013;226(1):102-109.
[32]Gross B,Pawlak M,Lefebvre P,et al.PPARs in obesity-induced T2DM,dyslipidaemia and NAFLD.Nat Rev Endocrinol.2017;13(1):36-49.
[33]Ahn SB,Jang K,Jun DW,et al.Expression of liver X receptor correlates with intrahepatic inflammation and fibrosis in patients with nonalcoholic fatty liver disease.Dig Dis Sci.2014;59(12):2975-2982.
[34]Xie P,Zhang AT,Wang C,et al.Molecular cloning,characterization,and expression analysis of fatty acid translocase(FAT/CD36)in the pigeon(Columba livia domestica).Poultry Science.2012;91(7):1670-1679.
[35]薄俊霞,李敬达,刘庆平.脂肪酸移位酶FAT/CD36转运长链脂肪酸研究进展[J].动物医学进展,2017,38(5):93-97.
[36]Takai M,Kozai Y,Tsuzuki S,et al.Unsaturated long-chain fatty acids inhibit the binding of oxidized low-density lipoproteins to a model CD36[J].Bioscience Biotechnology&Biochemistry.2014;78(2):238-244.
[37]Kozai Y,Tsuzuki S,Takai M,et al.Further validation of unsaturated long-chain fatty acids as inhibitors for oxidized low-density lipoprotein binding to CD36 via assays with synthetic CD36 peptide-cross-linked plates.Biosci Biotechnol Biochem.2014;78(5):839-842.
[38]Demers A,Samami S,Lauzier B,et al.PCSK9 Induces CD36Degradation and Affects Long-Chain Fatty Acid Uptake and Triglyceride Metabolism in Adipocytes and in Mouse Liver Significance.Arterioscler Thromb Vasc Biol.2015;35(12):2517-2525.
[39]Hoshino D,Yoshida Y,Kitaoka Y,et al.High-intensity interval training increases intrinsic rates of mitochondrial fatty acid oxidation in rat red and white skeletal muscle.Applied Physiology Nutrition&Metabolism.2013;38(3):326-333.
[40]Hrometz SL,Ebert JA,Grice KE,et al.Potentiation of Ecstasy-induced hyperthermia and FAT/CD36 expression in chronically exercised animals.Temperature.2016:24(8):1-5.
[41]Talanian JL,Holloway GP,Heigenhauser GJ,etal.Exercise training increases sarcolemmal and mitochondrial fatty acid transport proteins in human skeletal muscle.Ajp Endocrinology Metabolism.2010;299(2):E180-188.
[42]齐洁任,彩玲,傅建,等.运动降低MG53表达及其在缓解高脂膳食大鼠IR中的作用[J].中国运动医学杂志,2016,35(3):240-247.
[43]Yoshida Y,Jain SS,Mcfarlan JT,et al.Exercise-and training-induced upregulation of skeletal muscle fatty acid oxidation are not solely dependent on mitochondrial machinery and biogenesis.J Physiol.2013;591(Pt18):4415-4426.
[44]Jain SS,Chabowski A,Snook LA,et al.Additive effects of insulin and muscle contraction on fatty acid transport and fatty acid transporters,FAT/CD36,FABPpm,FATP1,4 and 6.Febs Letters.2009;583(13):2294-300.
[45]Lee JK,Lee JS,Park H,et al.Effect of l-carnitine supplementation and aerobic training on FABPc content andβ-HAD activity in human skeletal muscle.Eur J Appl Physiol.2007;99(2):193-199.