糖肾平胶囊通过PI3K/Akt信号通路干预高糖+LPS诱导足细胞凋亡的机制
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摘要
目的:观察糖肾平对高糖环境下脂多糖(LPS)对足细胞凋亡的影响,并探讨其作用机制。方法:利用雄性Wistar大鼠,分别用糖肾平小、中、大剂量(0. 525,1. 05,2. 1 g·kg-1),厄贝沙坦(17. 5 mg·kg-1)灌胃给药,制备相应药物血清。采用高糖,LPS体外刺激大鼠足细胞建立模型。并分为正常组(5%正常大鼠血清),高糖组(5%模型大鼠血清+4. 5‰葡萄糖),高糖+LPS组(5%模型大鼠血清+4. 5‰葡萄糖+LPS 1 mg·L-1),厄贝沙坦组(5%厄贝沙坦组大鼠血清+4. 5‰葡萄糖+LPS1 mg·L-1),糖肾平小剂量组(5%糖肾平小剂量大鼠血清+4. 5‰葡萄糖+LPS 1 mg·L-1),中剂量组(5%糖肾平中剂量大鼠血清+4. 5‰葡萄糖+LPS 1 mg·L-1),大剂量组(5%糖肾平大剂量大鼠血清+4. 5‰葡萄糖+LPS 1 mg·L-1)。采用蛋白免疫印迹法(Western blot)及逆转录聚合酶链式反应(RT-PCR)检测足细胞中CD2相关蛋白(CD2AP),nephrin和磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号转导通路中关键因子的蛋白和mRNA的表达水平。结果:与正常组比较,高糖组和高糖+LPS组足细胞nephrin,CD2AP,PI3K,Akt蛋白及mRNA表达明显减少(P <0. 05,P <0. 01); B淋巴细胞瘤-2相关凋亡蛋白(Bad蛋白)及mRNA表达明显增加(P <0. 05,P <0. 01)。与高糖+LPS组比较,厄贝沙坦组足细胞CD2AP,PI3K,Akt蛋白及mRNA表达明显增加(P <0. 05,P <0. 01); nephrin mRNA表达增加; Bad蛋白及mRNA蛋白表达减少。糖肾平各组足细胞CD2AP,PI3K,Akt蛋白及mRNA表达明显增加(P <0. 05,P <0. 01); Bad蛋白及mRNA蛋白表达减少(P <0. 05,P <0. 01)。结论:糖肾平维持足细胞裂孔隔膜蛋白稳定表达,保护肾小球滤过屏障,同时进一步激活PI3K/Akt信号通路,抑制足细胞凋亡;是其多靶点防治糖尿病肾病的作用机制之一。
Objective: To observe effect of Tangshenping capsule on lipopolysaccharide(LPS)-stimulated podocytes apoptosis under high glucose conditions of podocyte in rats with diabetic nephropathy(DN),and discuss possible mechanism. Method: With cultured rat podocytes as object of study,the podocytes model was established with high glucose and LPS,and divided into 7 groups: control group,high glucose group,high glucose plus LPS group, irbesartan group, and low, moderate and high-dose Tangshenping capsule groups.Protein and mRNA expressions of CD2-associated protein(CD2 AP),nephrin, phosphatidylinositol 3-kinase(PI3 K),protein kinase B(Akt) and B-cell lymphoma-2 associated death protein(Bad protein) of podocyte in each group were detected by Western blot and reverse transcription polymerase chain reaction(RT-PCR). Result:Compared with control group, the protein and mRNA expressions of podocyte CD2 AP,nephrin,PI3 K,Akt decreased definitely(P < 0. 05,P < 0. 01),while Bad protein and mRNA expressions increased definitely(P <0. 05,P < 0. 01). Compared with high glucose plus LPS group,the protein and mRNA expressions of podocyte CD2 AP,PI3 K,Akt increased definitely(P < 0. 05,P < 0. 01),whereas Bad protein and mRNA expressions decreased definitely(P < 0. 05,P < 0. 01) in irbesartan group. The protein and mRNA expressions of podocyte nephrin,CD2 AP,PI3 K,Akt increased definitely(P < 0. 05,P < 0. 01),and Bad protein and mRNA expressions decreased definitely(P < 0. 05,P < 0. 01) in three Tangshenping capsule groups. Conclusion: Tangshenping capsule can maintain a stable protein expression of Slit diaphragm(SD) in podocyte,inhibit podocyte apoptosis by activating PI3 K/Akt signaling pathway. This is one of mechanisms for preventing and treating diabetic nephropathy.
Objective: To observe effect of Tangshenping capsule on lipopolysaccharide(LPS)-stimulated podocytes apoptosis under high glucose conditions of podocyte in rats with diabetic nephropathy(DN),and discuss possible mechanism. Method: With cultured rat podocytes as object of study,the podocytes model was established with high glucose and LPS,and divided into 7 groups: control group,high glucose group,high glucose plus LPS group, irbesartan group, and low, moderate and high-dose Tangshenping capsule groups.Protein and mRNA expressions of CD2-associated protein(CD2 AP),nephrin, phosphatidylinositol 3-kinase(PI3 K),protein kinase B(Akt) and B-cell lymphoma-2 associated death protein(Bad protein) of podocyte in each group were detected by Western blot and reverse transcription polymerase chain reaction(RT-PCR). Result:Compared with control group, the protein and mRNA expressions of podocyte CD2 AP,nephrin,PI3 K,Akt decreased definitely(P < 0. 05,P < 0. 01),while Bad protein and mRNA expressions increased definitely(P <0. 05,P < 0. 01). Compared with high glucose plus LPS group,the protein and mRNA expressions of podocyte CD2 AP,PI3 K,Akt increased definitely(P < 0. 05,P < 0. 01),whereas Bad protein and mRNA expressions decreased definitely(P < 0. 05,P < 0. 01) in irbesartan group. The protein and mRNA expressions of podocyte nephrin,CD2 AP,PI3 K,Akt increased definitely(P < 0. 05,P < 0. 01),and Bad protein and mRNA expressions decreased definitely(P < 0. 05,P < 0. 01) in three Tangshenping capsule groups. Conclusion: Tangshenping capsule can maintain a stable protein expression of Slit diaphragm(SD) in podocyte,inhibit podocyte apoptosis by activating PI3 K/Akt signaling pathway. This is one of mechanisms for preventing and treating diabetic nephropathy.
引文
[1] Briasoni L,Schn Paer H W,Kopp J B. Advnaces in the biology and geneties of the podoeytopathies:implications for diangosis and therapy[J]. Arch Pathol Lab Med,2009,133(2):201-216.
[2] CHUANG P Y,HE J C. Signaling in regulation of podocyte phenotypes[J]. Nephron Physiol,2009,111(2):9-15.
[3]李敏州,高彦彬,马鸣飞,等.糖尿病肾病发病机制研究进展[J].中国实验方剂学杂志,2012,18(22):344-349.
[4] Vezrola D,Gandolof M T,Ferrario F,et al. Aopoptosis in the kidneys of patients with typeⅡdiabetic nephropathy[J]. Kidney Int,2007,72(10):1262-1272.
[5]石格,毛志敏,万毅刚,等.糖尿病肾病足细胞损伤的病理机制及中药的干预作用[J].中国中药杂志,2016,41(13):2416-2421.
[6] ZHANG Z,PENG H,CHEN J,et al. MicroRNA-21protects from mesangial cell proliferation induced by diabetic nephropathy in db/db mice[J]. FEBS Lett,2009,583(12):2009-2014.
[7]朱海慧,叶学锋.中医药防治糖尿病肾病足细胞损伤研究进展[J].辽宁中医药大学学报,2014,16(1):109-111.
[8] LI X Z,CHUANG P Y,D'Agati V D,et al. Nephrin preserves podocyte viability and glomerular structure andfunction in adult kidneys[J]. J Am Soc Nephrol,2015,26(10):2361-2377.
[9]赵宗江,王颖超,杜磊,等.健脾益肾化瘀通络方对糖尿病肾病大鼠氧化应激的影响[J].北京中医药大学学报,2013,36(3):161-165,218.
[10]杜磊,赵敬,赵宗江,等.糖肾平对高糖+LPS刺激足细胞TGF-β1/Smad7信号转导通路影响的研究[J].中国中西医结合肾病杂志,2013,14(2):107-110.
[11]王颖超,赵宗江,赵敬,等.糖肾平对糖尿病肾病大鼠足细胞nephrin与CD2AP蛋白及其mRNA表达的影响[J].中国中西医结合肾病杂志,2014,15(2):107-109.
[12]王颖超,于眉,杨敏,等.糖肾平对糖尿病肾病大鼠α-actinin-4表达及足细胞数目的影响[J].辽宁中医药大学学报,2018,20(4):31-35.
[13]王婷,赵宗江,张新雪,等.糖肾宁含药血清对高糖刺激肾小管上皮细胞增殖作用及Rho A/ROCK信号通路影响的实验研究[J].世界科学技术—中医药现代化,2017,19(6):1066-1071.
[14] Boddana P,Caskey F,Casula A,et al. UK renal registry 11thannual report(December 2008):chapter14UK renal registry and international comparisons[J].Nephron Clin Pract,2009,111(Supp11):269-276.
[15] Schernthaner G. Kidney disease in diabetology:lessons from 2010[J]. Nephrol Dial Transplant,2011,26(2):454-457.
[16]刘臻,张铭倩,朱虹.栝楼瞿麦汤对糖尿病肾病大鼠脂肪因子和足细胞nephrin的影响[J].中国实验方剂学杂志,2017,23(23):134-139.
[17] Kelly J C. China faces massive wave of diabetes-linked chronic diseases[J]. JAMA,2013,310:916-917,948-958.
[18] Huber T B,Harfleben B,Kim J,et al. Nephrinand CD2AP associate with phosphoinositide 3-OH kinase an dstimulate Akt-dependent signaling[J]. Mol Cell Bio1,2003,23(14):4917-4928.
[19] LIX Z,HE J C. An update:the role of nephrin inside and outside the kidney[J]. Sci China:Life Sci,2015,58(7):649-657.
[20] Beck J T, Ismail A, Tolomeo C. Targeting the phosphatidylinositol 3-kinase(PI3K)/Akt/mammalian target of rapamycin(m TOR)pathway:an emerging treatment strategy for squamous cell lung carcinoma[J]. Cancer Treat Rev,2014,40(8):980-989.
[21] Theodoropoulou M,Stalla G K. Somatostatin receptors:from signaling to clinical practice[J]. Front Neuroendoctrinol,2013,34(3):228-252.
[22] Lopiccolo J,Blumenthal G M,Bernstein W B,et al.Targeting the PI3K/Akt/m TOR pathway:effective combinations and clinical considerations[J]. Drug Resist Update,2007,11(1/2):32-50.
[23] WANG L S,GAI P Z,XU R G,et al. Shikonin protects chondrocytes from interleukin-1 beta-induced apoptosis by regulating PI3K/Akt signaling pathway[J].Int J Clin Exp Pathol,2015,8(1):298-308.
[24] Tokuhira N,Kitagishi Y,Suzuki M,et al. PI3K/Akt/PTEN pathway as a target for Crohn's disease therapy(review)[J]. Int J Mol Med,2015,35(1):10-16.
[25]贾鹏,陈盛业,甘宇,等.中医药治疗糖尿病肾病研究进展[J].辽宁中医药大学学报,2010,12(7):125-126.
[26]赵宗江,豆小妮,张新雪.糖尿病肾病“肾痿”假说探讨[J].中医杂志,2011,52(S1):8-10.
[2] CHUANG P Y,HE J C. Signaling in regulation of podocyte phenotypes[J]. Nephron Physiol,2009,111(2):9-15.
[3]李敏州,高彦彬,马鸣飞,等.糖尿病肾病发病机制研究进展[J].中国实验方剂学杂志,2012,18(22):344-349.
[4] Vezrola D,Gandolof M T,Ferrario F,et al. Aopoptosis in the kidneys of patients with typeⅡdiabetic nephropathy[J]. Kidney Int,2007,72(10):1262-1272.
[5]石格,毛志敏,万毅刚,等.糖尿病肾病足细胞损伤的病理机制及中药的干预作用[J].中国中药杂志,2016,41(13):2416-2421.
[6] ZHANG Z,PENG H,CHEN J,et al. MicroRNA-21protects from mesangial cell proliferation induced by diabetic nephropathy in db/db mice[J]. FEBS Lett,2009,583(12):2009-2014.
[7]朱海慧,叶学锋.中医药防治糖尿病肾病足细胞损伤研究进展[J].辽宁中医药大学学报,2014,16(1):109-111.
[8] LI X Z,CHUANG P Y,D'Agati V D,et al. Nephrin preserves podocyte viability and glomerular structure andfunction in adult kidneys[J]. J Am Soc Nephrol,2015,26(10):2361-2377.
[9]赵宗江,王颖超,杜磊,等.健脾益肾化瘀通络方对糖尿病肾病大鼠氧化应激的影响[J].北京中医药大学学报,2013,36(3):161-165,218.
[10]杜磊,赵敬,赵宗江,等.糖肾平对高糖+LPS刺激足细胞TGF-β1/Smad7信号转导通路影响的研究[J].中国中西医结合肾病杂志,2013,14(2):107-110.
[11]王颖超,赵宗江,赵敬,等.糖肾平对糖尿病肾病大鼠足细胞nephrin与CD2AP蛋白及其mRNA表达的影响[J].中国中西医结合肾病杂志,2014,15(2):107-109.
[12]王颖超,于眉,杨敏,等.糖肾平对糖尿病肾病大鼠α-actinin-4表达及足细胞数目的影响[J].辽宁中医药大学学报,2018,20(4):31-35.
[13]王婷,赵宗江,张新雪,等.糖肾宁含药血清对高糖刺激肾小管上皮细胞增殖作用及Rho A/ROCK信号通路影响的实验研究[J].世界科学技术—中医药现代化,2017,19(6):1066-1071.
[14] Boddana P,Caskey F,Casula A,et al. UK renal registry 11thannual report(December 2008):chapter14UK renal registry and international comparisons[J].Nephron Clin Pract,2009,111(Supp11):269-276.
[15] Schernthaner G. Kidney disease in diabetology:lessons from 2010[J]. Nephrol Dial Transplant,2011,26(2):454-457.
[16]刘臻,张铭倩,朱虹.栝楼瞿麦汤对糖尿病肾病大鼠脂肪因子和足细胞nephrin的影响[J].中国实验方剂学杂志,2017,23(23):134-139.
[17] Kelly J C. China faces massive wave of diabetes-linked chronic diseases[J]. JAMA,2013,310:916-917,948-958.
[18] Huber T B,Harfleben B,Kim J,et al. Nephrinand CD2AP associate with phosphoinositide 3-OH kinase an dstimulate Akt-dependent signaling[J]. Mol Cell Bio1,2003,23(14):4917-4928.
[19] LIX Z,HE J C. An update:the role of nephrin inside and outside the kidney[J]. Sci China:Life Sci,2015,58(7):649-657.
[20] Beck J T, Ismail A, Tolomeo C. Targeting the phosphatidylinositol 3-kinase(PI3K)/Akt/mammalian target of rapamycin(m TOR)pathway:an emerging treatment strategy for squamous cell lung carcinoma[J]. Cancer Treat Rev,2014,40(8):980-989.
[21] Theodoropoulou M,Stalla G K. Somatostatin receptors:from signaling to clinical practice[J]. Front Neuroendoctrinol,2013,34(3):228-252.
[22] Lopiccolo J,Blumenthal G M,Bernstein W B,et al.Targeting the PI3K/Akt/m TOR pathway:effective combinations and clinical considerations[J]. Drug Resist Update,2007,11(1/2):32-50.
[23] WANG L S,GAI P Z,XU R G,et al. Shikonin protects chondrocytes from interleukin-1 beta-induced apoptosis by regulating PI3K/Akt signaling pathway[J].Int J Clin Exp Pathol,2015,8(1):298-308.
[24] Tokuhira N,Kitagishi Y,Suzuki M,et al. PI3K/Akt/PTEN pathway as a target for Crohn's disease therapy(review)[J]. Int J Mol Med,2015,35(1):10-16.
[25]贾鹏,陈盛业,甘宇,等.中医药治疗糖尿病肾病研究进展[J].辽宁中医药大学学报,2010,12(7):125-126.
[26]赵宗江,豆小妮,张新雪.糖尿病肾病“肾痿”假说探讨[J].中医杂志,2011,52(S1):8-10.