番石榴叶总黄酮对2型糖尿病模型小鼠肝糖异生ERRγ/CREBH信号通路相关因子的影响
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摘要
目的:探讨番石榴叶总黄酮(GLTF)对2型糖尿病(T2DM)模型小鼠肝脏雌激素相关受体γ(ERRγ)/环磷酸腺苷应答元件结合蛋白H(CREBH)通路相关因子的影响,探讨其降血糖作用的具体机制。方法:取健康雄性ICR小鼠,采用高糖高脂饲料喂养联合多次腹腔注射小剂量链脲佐菌素的方法建立T2DM模型。将造模成功的小鼠按血糖值随机分为模型组、二甲双胍组(阳性对照,0.17 g/kg)、消渴降糖胶囊组(阳性对照,0.75 g/kg)和GLTF低、高剂量组(0.047、0.094 g/kg),每组12只;另选12只正常小鼠作为正常组。除正常组和模型组小鼠灌胃等体积水外,其余各组小鼠灌胃相应药物溶液10 mL/kg,qd,连续21 d。给药结束后,检测各组小鼠空腹血糖值、血清胰岛素水平,计算胰岛素敏感指数(ISI);采用苏木素-伊红染色法观察小鼠肝脏和胰腺组织病理学变化;采用免疫印迹法检测小鼠肝组织中ERRγ、CREBH的蛋白表达水平;采用实时定量聚合酶链式反应法检测小鼠肝组织中过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC1α)、CREB调节转录辅激活因子2(TORC2)的mRNA表达水平。结果:与正常组比较,模型组小鼠空腹血糖和血清胰岛素水平均显著升高,ISI值显著降低(P<0.01);肝组织病变明显、可见大量空泡;胰腺组织中胰岛数目减少、体积变小,胰岛细胞呈轻度空泡病变;肝组织中ERRγ、CREBH的蛋白表达水平及PGC1α、TORC2的mRNA表达水平均显著升高(P<0.01)。与模型组比较,GLTF各剂量组小鼠上述血糖、胰岛素指标及病理学变化均明显改善;肝组织中ERRγ、CREBH的蛋白表达水平及PGC1α、TORC2的m RNA表达水平均显著降低(P<0.05或P<0.01)。结论:GLTF对T2DM模型小鼠具有明显降糖及肝、胰腺组织保护作用;其降血糖作用的机制可能与抑制肝脏ERRγ/CREBH信号通路有关。
OBJECTIVE:To investigate the effects of total flavonoids from Psidium guajava leaves(GLTF) on the related factors for estrogen-associated receptor γ(ERRγ)/cyclic adenosine monophosphate responsive element binding protein H(CREBH)pathway in liver of type 2 diabetes mellitus(T2 DM)model mice,and to investigate the specific mechanism of its hypoglycemic effect. METHODS:Healthy male ICR mice were collected. T2 DM models were established by high-sugar and high-fat diet feeding combined with repeated intraperitoneal injection of low dose streptozotocin. According to the blood glucose value,model mice were randomly divided into model group,metformin group(positive control,0.17 g/kg),Xiaoke jiangtang capsule group(positive control,0.75 g/kg),GLTF low-dose and high-dose groups(0.047,0.094 g/kg),with 12 mice in each group. Another 12 normal mice were included in normal group. Except that normal group and model group were given constant volume of water intragastrically,other groups were given relevant solution 10 m L/kg intragastrically,qd,for consecutive 21 d. After administration,fasting blood glucose and serum insulin levels of mice were measured and insulin sensitivity index(ISI)was calculated. Histopathological changes of liver and pancreas were observed by HE staining. The protein expressions of ERRγ and CREBH in liver tissues were detected by immunoblotting. The mRNA expression of peroxisome proliferator-activated receptor-γ coactivator-1α(PGC1α)and CREB protein co-activator 2(TORC2)in liver tissue of mice were measured by RT-PCR. RESULTS:Compared with normal group,the levels of fasting blood glucose and serum insulin in T2 DM mice were significantly increased in model group,while ISI was decreased significantly(P<0.01). The pathological changes of liver tissue were obvious and a large number of vacuoles could be seen. The number and volume of islets in pancreatic tissue decreased,and islet cells showed mild vacuolar lesions. The protein expression of ERRγ and CREBH,mR NA expression of PGC1α and TORC2 in liver tissue were increased significantly(P<0.01).Compared with model group, above blood glucose, insulin indexes and pathological changes were improved significantly in GLTF groups. Protein expression of ERRγ and CREBH,mR NA expression of PGC1α and TORC2 in liver tissue were decreased significantly(P<0.05 or P<0.01).CONCLUSIONS:GLTF showed obvious hypoglycemic effect on T2 DM model mice;its mechanism might be related to inhibiting ERRγ/CREBH signaling pathway.
OBJECTIVE:To investigate the effects of total flavonoids from Psidium guajava leaves(GLTF) on the related factors for estrogen-associated receptor γ(ERRγ)/cyclic adenosine monophosphate responsive element binding protein H(CREBH)pathway in liver of type 2 diabetes mellitus(T2 DM)model mice,and to investigate the specific mechanism of its hypoglycemic effect. METHODS:Healthy male ICR mice were collected. T2 DM models were established by high-sugar and high-fat diet feeding combined with repeated intraperitoneal injection of low dose streptozotocin. According to the blood glucose value,model mice were randomly divided into model group,metformin group(positive control,0.17 g/kg),Xiaoke jiangtang capsule group(positive control,0.75 g/kg),GLTF low-dose and high-dose groups(0.047,0.094 g/kg),with 12 mice in each group. Another 12 normal mice were included in normal group. Except that normal group and model group were given constant volume of water intragastrically,other groups were given relevant solution 10 m L/kg intragastrically,qd,for consecutive 21 d. After administration,fasting blood glucose and serum insulin levels of mice were measured and insulin sensitivity index(ISI)was calculated. Histopathological changes of liver and pancreas were observed by HE staining. The protein expressions of ERRγ and CREBH in liver tissues were detected by immunoblotting. The mRNA expression of peroxisome proliferator-activated receptor-γ coactivator-1α(PGC1α)and CREB protein co-activator 2(TORC2)in liver tissue of mice were measured by RT-PCR. RESULTS:Compared with normal group,the levels of fasting blood glucose and serum insulin in T2 DM mice were significantly increased in model group,while ISI was decreased significantly(P<0.01). The pathological changes of liver tissue were obvious and a large number of vacuoles could be seen. The number and volume of islets in pancreatic tissue decreased,and islet cells showed mild vacuolar lesions. The protein expression of ERRγ and CREBH,mR NA expression of PGC1α and TORC2 in liver tissue were increased significantly(P<0.01).Compared with model group, above blood glucose, insulin indexes and pathological changes were improved significantly in GLTF groups. Protein expression of ERRγ and CREBH,mR NA expression of PGC1α and TORC2 in liver tissue were decreased significantly(P<0.05 or P<0.01).CONCLUSIONS:GLTF showed obvious hypoglycemic effect on T2 DM model mice;its mechanism might be related to inhibiting ERRγ/CREBH signaling pathway.
引文
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[2]JIAO Y,ZHANG M,WANG S,et al.Consumption of guava may have beneficial effects in type 2 diabetes:a bioactive perspective[J].Int J Biol Macromol,2017.DOI:10.1016/j.ijbiomac.2017.03.130.
[3]JAYACHANDRAN M,VINAYAGAM R,AMBATI RR,et al.Guava leaf extract diminishes hyperglycemia and oxidative stress,preventsβ-cell death,inhibits inflammation,and regulates NF-κB signaling pathway in STZ induced diabetic rats[J].Biomed Res Int,2018.DOI:10.1155/2018/4601649.
[4]郭胜男.番石榴叶总黄酮对糖尿病小鼠肝脏胰岛素信号通路及肝脏葡萄糖异生的调控机制研究[D].天津:天津医科大学,2015.
[5]KUMARI K,ADHYA AK,RATH AK,et al.Estrogen-related receptors alpha,beta and gamma expression and function is associated with transcriptional repressor EZH2 in breast carcinoma[J].BMC Cancer,2018,18(1):690-701.
[6]MISRA J,KIM DK,JUNG YS,et al.O-GlcNAcylation of orphan nuclear receptor estrogen-related receptorγpromotes hepatic gluconeogenesis[J].Diabetes,2016,65(10):2835-2848.
[7]KIM DK,RYU D,KOH M,et al.Orphan nuclear receptor estrogen-related receptorγ(ERRγ)is key regulator of hepatic gluconeogenesis[J].J Biol Chem,2012,287(26):21628-21639.
[8]LEE J,SALAZAR HERNáNDEZ MA,AUEN T,et al.PGC-1αfunctions as a co-suppressor of XBP1s to regulate glucose metabolism[J].Mol Metab,2018.DOI:10.1016/j.molmet.2017.10.010.
[9]KIM H,ZHENG Z,WALKER PD,et al.CREBH maintains circadian glucose homeostasis by regulating hepatic glycogenolysis and gluconeogenesis[J].Mol Cell Biol,2017.DOI:10.1128/MCB.00048-17.
[10]NAKAGAWA Y,SHIMANO H.CREBH regulates systemic glucose and lipid metabolism[J].Int J Mol Sci,2018,19(5):1396-1411.
[11]JIANG SJ,DONG H,LI JB,et al.Berberine inhibits hepatic gluconeogenesis via the LKB1-AMPK-TORC2 signaling pathway in streptozotocin-induced diabetic rats[J].World J Gastroenterol,2015,21(25):7777-7785.
[12]傅予,王宏,张岩,等.Box-Behnken设计-响应面法优化番石榴叶总黄酮提取工艺及总黄酮对糖尿病小鼠糖耐量的影响研究[J].中国药房,2018,29(1):49-53.
[13]李仪奎.中药药理实验方法学[M].2版.上海:上海科学技术出版社,2006:603-606.
[14]SCHMITTGEN TD,LIVAK KJ.Analyzing real-time PCRdata by the comparative C(T)method[J].Nat Protoc,2008,3(6):1101-1108.
[15]张琼阁,王超群,薛嵩,等.单用胰岛素控制不佳的2型糖尿病患者联用阿卡波糖或二甲双胍的疗效及安全性比较:一项随机、开放、平行分组对照研究[J].中华内分泌代谢杂志,2018,34(9):755-760.
[16]赵莼,张巧,徐淑静,等.贵阳城区40岁以上中老年人群不同糖代谢状态与心血管疾病风险相关性研究[J].中华内分泌代谢杂志,2018,34(8):643-648.
[17]陈亚琼,王鹏飞,刘浥.基于肝糖异生的降血糖药物研发进展[J].药学进展,2017,41(10):733-741.
[18]潘小康.二甲双胍降糖机制研究进展[J].中国糖尿病杂志,2016,24(7):665-668.
[19]HE L,SABET A,DJEDJOS S,et al.二甲双胍和胰岛素通过CREB结合蛋白的磷酸化作用抑制肝脏糖异生[J].药品评价,2010,7(3):47.
[20]李英,王战建,旋志刚,等.消渴降糖胶囊治疗非胰岛素依赖型糖尿病临床观察[J].河北中医,1999,21(2):79-81.
[21]MISRA J,CHANDA D,KIM DK,et al.Orphan nuclear receptor ERRγinduces c-reactive protein gene expression through induction of ER-bound Bzip transmembrane transcription factor CREBH[J].PLoS One,2014.DOI:10.1371/journal.pone.0086342.
[22]KIM TH,KIM MK,CHEONG YH,et al.Hepatic role in an early glucose-lowering effect by a novel dipeptidyl peptidase 4 inhibitor,evogliptin,in a rodent model of type 2diabetes[J].Eur J Pharmacol,2016.DOI:10.1016/j.ejphar.2015.11.029.
[2]JIAO Y,ZHANG M,WANG S,et al.Consumption of guava may have beneficial effects in type 2 diabetes:a bioactive perspective[J].Int J Biol Macromol,2017.DOI:10.1016/j.ijbiomac.2017.03.130.
[3]JAYACHANDRAN M,VINAYAGAM R,AMBATI RR,et al.Guava leaf extract diminishes hyperglycemia and oxidative stress,preventsβ-cell death,inhibits inflammation,and regulates NF-κB signaling pathway in STZ induced diabetic rats[J].Biomed Res Int,2018.DOI:10.1155/2018/4601649.
[4]郭胜男.番石榴叶总黄酮对糖尿病小鼠肝脏胰岛素信号通路及肝脏葡萄糖异生的调控机制研究[D].天津:天津医科大学,2015.
[5]KUMARI K,ADHYA AK,RATH AK,et al.Estrogen-related receptors alpha,beta and gamma expression and function is associated with transcriptional repressor EZH2 in breast carcinoma[J].BMC Cancer,2018,18(1):690-701.
[6]MISRA J,KIM DK,JUNG YS,et al.O-GlcNAcylation of orphan nuclear receptor estrogen-related receptorγpromotes hepatic gluconeogenesis[J].Diabetes,2016,65(10):2835-2848.
[7]KIM DK,RYU D,KOH M,et al.Orphan nuclear receptor estrogen-related receptorγ(ERRγ)is key regulator of hepatic gluconeogenesis[J].J Biol Chem,2012,287(26):21628-21639.
[8]LEE J,SALAZAR HERNáNDEZ MA,AUEN T,et al.PGC-1αfunctions as a co-suppressor of XBP1s to regulate glucose metabolism[J].Mol Metab,2018.DOI:10.1016/j.molmet.2017.10.010.
[9]KIM H,ZHENG Z,WALKER PD,et al.CREBH maintains circadian glucose homeostasis by regulating hepatic glycogenolysis and gluconeogenesis[J].Mol Cell Biol,2017.DOI:10.1128/MCB.00048-17.
[10]NAKAGAWA Y,SHIMANO H.CREBH regulates systemic glucose and lipid metabolism[J].Int J Mol Sci,2018,19(5):1396-1411.
[11]JIANG SJ,DONG H,LI JB,et al.Berberine inhibits hepatic gluconeogenesis via the LKB1-AMPK-TORC2 signaling pathway in streptozotocin-induced diabetic rats[J].World J Gastroenterol,2015,21(25):7777-7785.
[12]傅予,王宏,张岩,等.Box-Behnken设计-响应面法优化番石榴叶总黄酮提取工艺及总黄酮对糖尿病小鼠糖耐量的影响研究[J].中国药房,2018,29(1):49-53.
[13]李仪奎.中药药理实验方法学[M].2版.上海:上海科学技术出版社,2006:603-606.
[14]SCHMITTGEN TD,LIVAK KJ.Analyzing real-time PCRdata by the comparative C(T)method[J].Nat Protoc,2008,3(6):1101-1108.
[15]张琼阁,王超群,薛嵩,等.单用胰岛素控制不佳的2型糖尿病患者联用阿卡波糖或二甲双胍的疗效及安全性比较:一项随机、开放、平行分组对照研究[J].中华内分泌代谢杂志,2018,34(9):755-760.
[16]赵莼,张巧,徐淑静,等.贵阳城区40岁以上中老年人群不同糖代谢状态与心血管疾病风险相关性研究[J].中华内分泌代谢杂志,2018,34(8):643-648.
[17]陈亚琼,王鹏飞,刘浥.基于肝糖异生的降血糖药物研发进展[J].药学进展,2017,41(10):733-741.
[18]潘小康.二甲双胍降糖机制研究进展[J].中国糖尿病杂志,2016,24(7):665-668.
[19]HE L,SABET A,DJEDJOS S,et al.二甲双胍和胰岛素通过CREB结合蛋白的磷酸化作用抑制肝脏糖异生[J].药品评价,2010,7(3):47.
[20]李英,王战建,旋志刚,等.消渴降糖胶囊治疗非胰岛素依赖型糖尿病临床观察[J].河北中医,1999,21(2):79-81.
[21]MISRA J,CHANDA D,KIM DK,et al.Orphan nuclear receptor ERRγinduces c-reactive protein gene expression through induction of ER-bound Bzip transmembrane transcription factor CREBH[J].PLoS One,2014.DOI:10.1371/journal.pone.0086342.
[22]KIM TH,KIM MK,CHEONG YH,et al.Hepatic role in an early glucose-lowering effect by a novel dipeptidyl peptidase 4 inhibitor,evogliptin,in a rodent model of type 2diabetes[J].Eur J Pharmacol,2016.DOI:10.1016/j.ejphar.2015.11.029.