糖肾清2号提取物调控AMPK信号通路对高糖环境下肾小球系膜细胞炎症因子表达的影响
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摘要
目的:观察糖肾清2号提取物对高糖环境下的小鼠肾小球系膜细胞株AMPK-α1、IκBα、TLR-4mRNA转录和蛋白表达的影响,探讨该方抑制高糖环境下肾小球系膜细胞株免疫炎性病理损伤的分子机制。方法:采用小鼠肾小球系膜细胞株SV40 MES 13,在5%CO_2、37℃恒温培养箱常规培养。实验分组:空白对照组(D-葡萄糖5.6 mmol/L)、高糖组(D-葡萄糖30 mmol/L)、中药低浓度组(高糖+糖肾清2号提取物5μmol/L)、中药中浓度组(高糖+糖肾清2号提取物10μmol/L)、中药高浓度组(高糖+糖肾清2号提取物20μmol/L),共同孵育12、24、36 h进行指标检测。采用RT-PCR技术检测肾小球系膜细胞AMPKα1、IκBα、TLR4 mRNA转录水平;Western-blot技术检测肾小球系膜细胞AMPKα1、IκBα、TLR4的蛋白表达水平。结果:与空白对照组比较,高糖组AMPKα1、IκBαmRNA转录水平及蛋白表达下调(P<0.05),TLR4 mRNA转录水平及蛋白表达水平上调(P<0.01),差异有统计学意义。与高糖组比较,中药各浓度组AMPKα1、IκBα蛋白表达水平上调(P<0.05),中药高、低浓度组AMPKα1、IκBαmRNA转录水平上调(P<0.05)。中药各浓度组TLR4mRNA转录水平下调(P<0.01),中、高浓度组TLR4蛋白表达水平下调(P<0.01)。结论:糖肾清2号抑制高糖环境下肾小球系膜细胞免疫炎性代谢性损伤,此分子机制可能与调节AMPK信号通路相关。
Objective: To observe the effects of Tangshenqing Formula Ⅱ(TSQF Ⅱ)extract on the gene transcription and protein expressions of mouse glomerular mesangial cell line including AMP-activated protein kinase-α1(AMPK-α1)、inhibitor of nuclear factor kappa B-α(IκBα)、toll like receptor-4(TLR-4) mRNA, and to explore its the molecular mechanism of inhibiting the immune-inflammatory pathological injury of mesangial cell lines in high glucose environment. Methods: To culture mouse glomerular mesangial cell line SV40 MES 13 in 5% CO2, 37 ℃ constant temperature incubator. Experimental groups:control group(D-Glucose 5.6/L),high glucose group(D-Glucose 30 mmol/L),high glucose+TSQF Ⅱextract 5 μmol/L group,high glucose+TSQF Ⅱextract 10 μmol/L group,high glucose+TSQF Ⅱextract 20 μmol/L group.Cells in all groups were coculture12 h,24 h,36 h.Rt-PCR was used to detect mRNA transcriptional levels of AMPKα1,IκBα,TLR4 mRNA.Western-blot was used to detect protein expression levels of AMPKα1,IκBα,TLR4.Results: compared with the control group,the mRNA transcriptional and protein expression level of AMPKα1 and IκBα in the high glucose group were down-regulated(P<0.05),the mRNA transcription and protein expression level of TLR4 were up-regulated(P<0.01).Compared with the high glucose group,the expression level of AMPKα1 and IκBα proteins were up-regulated(P<0.05), and the mRNA transcriptional level of AMPKα1 and IκBα were up-regulated in the high and low dose Chinese medicine groups(P<0.05).TLR4 mRNA transcription level was down-regulated in all groups of TSQF Ⅱ extract(P<0.01), and the TLR4 protein expression level was down-regulated in the middle and high dose groups(P<0.01).Conclusion: TSQF Ⅱextract can inhibit immune-inflammatory pathological injury of mesangial cell lines in high glucose environment, and this molecular mechanism may be related to the regulation of AMPK signaling pathway.
Objective: To observe the effects of Tangshenqing Formula Ⅱ(TSQF Ⅱ)extract on the gene transcription and protein expressions of mouse glomerular mesangial cell line including AMP-activated protein kinase-α1(AMPK-α1)、inhibitor of nuclear factor kappa B-α(IκBα)、toll like receptor-4(TLR-4) mRNA, and to explore its the molecular mechanism of inhibiting the immune-inflammatory pathological injury of mesangial cell lines in high glucose environment. Methods: To culture mouse glomerular mesangial cell line SV40 MES 13 in 5% CO2, 37 ℃ constant temperature incubator. Experimental groups:control group(D-Glucose 5.6/L),high glucose group(D-Glucose 30 mmol/L),high glucose+TSQF Ⅱextract 5 μmol/L group,high glucose+TSQF Ⅱextract 10 μmol/L group,high glucose+TSQF Ⅱextract 20 μmol/L group.Cells in all groups were coculture12 h,24 h,36 h.Rt-PCR was used to detect mRNA transcriptional levels of AMPKα1,IκBα,TLR4 mRNA.Western-blot was used to detect protein expression levels of AMPKα1,IκBα,TLR4.Results: compared with the control group,the mRNA transcriptional and protein expression level of AMPKα1 and IκBα in the high glucose group were down-regulated(P<0.05),the mRNA transcription and protein expression level of TLR4 were up-regulated(P<0.01).Compared with the high glucose group,the expression level of AMPKα1 and IκBα proteins were up-regulated(P<0.05), and the mRNA transcriptional level of AMPKα1 and IκBα were up-regulated in the high and low dose Chinese medicine groups(P<0.05).TLR4 mRNA transcription level was down-regulated in all groups of TSQF Ⅱ extract(P<0.01), and the TLR4 protein expression level was down-regulated in the middle and high dose groups(P<0.01).Conclusion: TSQF Ⅱextract can inhibit immune-inflammatory pathological injury of mesangial cell lines in high glucose environment, and this molecular mechanism may be related to the regulation of AMPK signaling pathway.
引文
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[22] Matsui T. Inflammatory cytokines and hypoxia contribute to 18F-FDG uptake by cells involved in pannus formation in rheumatoid arthritis[J].Nucl Med, 2009(50):920-926.
[23] 李景,赵进喜,王世东,等. 中医药综合治疗方案全程干预对糖尿病肾病终点事件的影响[J].中医杂志,2012,53(7):568-571.
[24] Devaraj S,Dasu MR,Jialal I.Diabetes is a proinflammatory state:a translational perspective[J]. Expert Rev Endocrinol Metab,2010,5(1):19-28.
[25] Vaarala 0, Yki-Jrvinen H. Diabetes:Should we treat infection or inflammation to prevent T2DM?[J].Nat Rev Endocrinol.2012,8(6):323-325.
[26] 杜晓燕.miR-101靶向调节AMPK表达抑制高糖环境下肾小球系膜细胞自噬及促进其增殖[D].广州:南方医科大学,2016.
[27] Wilfred Lieberthal, Leiqing Zhang, Vimal A Patel,et al.AMPK protects proximal tubular cells from stress-induced apoptosis by an ATP-independent mechanism: potential role of Akt activation[J]. Am J Physiol Renal Physiol, 2011, 301(6): F1177-F1192.
[28] Houtmann S,Roche R,Hoareau L,et al. Presence of functional TLR3 and TLR4 on human adipocytes[J].Histochemistry and Cell Biology,2007:131-137.
[29] Li M,Yu L,She T,et al.Astragaloside IV attenuates Toll-like receptor 4 expression via NF-κB pathway under high glucose condition in mesenchymal stem cells[J].Eur J Pharmacol,2012,696(1-3):203-209.
[30] 张艳霞,王质刚.人类Toll样受体信号转导途径及其介导的免疫作用[J].国际免疫学杂志,2010,33(2):121-123.
[2] Williams ME.Diabetic nehropathy:the proteinuria hypothesis[J]. Am J Nephrol, 2005, 25 (2) :77-94.
[3] Tuttle KR, Bakris GL, Bilous RW, et al.Diabetic kidney disease:a report from an ADA Consensus Conference[J].Am J Kidney Dis, 2014, 64(4):510-533.
[4] Armstrong C.ADA updates standards of medical care for patients with diabetes mellitus[J].Am Fam Physician, 2017, 95(1):40-43.
[5] 杜月光,钱俊文,宋光明,等.糖肾颗粒对2型糖尿病大鼠肾脏的保护作用及对炎症的影响[J].中华中医药学刊,2013,31(10):2217-2220,2347.
[6] Mooradian AD.Dysli Pidemia in type 2 diabets mellitus[J].Nat Clin Praet Endoeri-nol Metab,2009,5(3):150-159.
[7] 中华医学会内分泌学会.中国成人糖尿病肾脏病临床诊断专家共识[J].中华内分泌代谢杂志,2015,31(5):379-384.
[8] 王三清,黄通,张春惠.灯盏花素及复方丹参滴丸联合缬沙坦治疗糖尿病肾病临床疗效分析[J].临床和实验医学杂志,2009,8(8):27-28.
[9] Tervaert TW,Mooyaart AL, Amann K, et al.Pathologic classification of diabetic nephropathy[J].J Am Soc Nephrol,2010,21(4):556-563.
[10] 郭丛丛,姚金铭,孙洁,等.赤芍治疗糖尿病肾病的网络药理学研究[J].中华中医药学刊,2018,36(10):2420-2423.
[11] 潘永梅,陈志强,贾蕊,等.活血化瘀通络中药对糖尿病肾病大鼠肾小管间质纤维化的影响[J].中华中医药学刊,2017,35(8):2105-2108.
[12] 张继胜,商学征,白羽,等.糖肾清2号方对2型糖尿病模型小鼠肾功能的影响[J].北京中医药大学学报,2016,39(1):16-20.
[13] 张建萍,张红红,卜祥伟,等.糖肾清2号对糖尿病肾病模型小鼠免疫炎性分子的调节作用[J/OL].中国中西医结合杂志,2018:1-6.
[14] Ying C, Chen L, Wang S, et al.Zeaxanthin ameliorates high glucose-induced mesangial cell apoptosis through inhibiting oxidative stress via activating AKT signalling-pathway[J].Biomed Pharmacother, 2017, 90:796-805.
[15] 吴云皓,李伟,陈玉萍,等.山茱萸、生地有效部位及配伍组合抑制糖基化终末产物致肾小球系膜细胞增殖的研究[J].中药药理与临床,2015,31(4):71-75.
[16] Fulco M,Cen Y,Zhao P, et al.Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of Nampt[J].Developmental cell,2008,14(5):661-673.
[17] Canto C, Gerhart-Hines Z,Feige JN,et al.AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity[J]. Nature,2009,458(7241):1056-1060.
[18] Schug TT, Xu Q, Gao H, et al.Myeloid deletion of SIRT1 induces inflammatory signaling in response to environmental stress[J]. Molecular and cellular biology,2010,30(19):4712-4721.
[19] Lin L,Hron JD,Peng SL.Regulation of NF-kappaB,Thactivation,and autoinflammation by the forkhead transcription factor Foxo3a[J]. Immunity,2004,21(2):203-213.
[20] 童楠,张宁.自噬与糖尿病肾病的研究进展[J].世界中西医结合杂志,2017,12(5):601-605.
[21] Connie M Krawczyk, Thomas Holowka, Jie Sun, et al. Toll-like receptor-induced changes in glycolytic metabolism regulate dendritic cell activation[J].Blood, 2010,115(23):912-914.
[22] Matsui T. Inflammatory cytokines and hypoxia contribute to 18F-FDG uptake by cells involved in pannus formation in rheumatoid arthritis[J].Nucl Med, 2009(50):920-926.
[23] 李景,赵进喜,王世东,等. 中医药综合治疗方案全程干预对糖尿病肾病终点事件的影响[J].中医杂志,2012,53(7):568-571.
[24] Devaraj S,Dasu MR,Jialal I.Diabetes is a proinflammatory state:a translational perspective[J]. Expert Rev Endocrinol Metab,2010,5(1):19-28.
[25] Vaarala 0, Yki-Jrvinen H. Diabetes:Should we treat infection or inflammation to prevent T2DM?[J].Nat Rev Endocrinol.2012,8(6):323-325.
[26] 杜晓燕.miR-101靶向调节AMPK表达抑制高糖环境下肾小球系膜细胞自噬及促进其增殖[D].广州:南方医科大学,2016.
[27] Wilfred Lieberthal, Leiqing Zhang, Vimal A Patel,et al.AMPK protects proximal tubular cells from stress-induced apoptosis by an ATP-independent mechanism: potential role of Akt activation[J]. Am J Physiol Renal Physiol, 2011, 301(6): F1177-F1192.
[28] Houtmann S,Roche R,Hoareau L,et al. Presence of functional TLR3 and TLR4 on human adipocytes[J].Histochemistry and Cell Biology,2007:131-137.
[29] Li M,Yu L,She T,et al.Astragaloside IV attenuates Toll-like receptor 4 expression via NF-κB pathway under high glucose condition in mesenchymal stem cells[J].Eur J Pharmacol,2012,696(1-3):203-209.
[30] 张艳霞,王质刚.人类Toll样受体信号转导途径及其介导的免疫作用[J].国际免疫学杂志,2010,33(2):121-123.