黄芪甲苷对2型糖尿病肾病大鼠肾组织PI3K/Akt/FoxO1信号调控的影响
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摘要
目的:研究黄芪甲苷对2型糖尿病肾病大鼠肾脏的保护作用及其对磷酯酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/叉头框转录因子O亚族1(FoxO1)信号的调控作用,探讨黄芪甲苷保护2型糖尿病肾病的机制。方法:6周高糖高脂饮食后,给予链脲佐菌素(STZ)一次性腹腔注射(35 mg·kg~(-1))建立2型糖尿病大鼠模型,随机分为正常组,模型组,黄芪甲苷(20,40,80 mg·kg~(-1))组及盐酸二甲双胍(阳性药)组。连续给药8周后检测各组大鼠体质量,肾脏指数,血糖,24 h尿蛋白,尿微量白蛋白,尿肌酐,血肌酐及血尿素氮含量的变化;苏木素-伊红(HE)染色观察肾脏组织病理形态学变化;马松(Masson)三色染色观察胶原表达水平;过碘酸六胺银(PASM)染色观察基底膜的变化;免疫组化检测磷酸化FoxO1(p-FoxO1)蛋白表达;蛋白质免疫印迹法(Western blot)分析PI3K/Akt/FoxO1信号蛋白及自噬标记蛋白PI3K,微管相关蛋白1轻链3(LC3)Ⅰ/Ⅱ,B细胞淋巴瘤-2(Bcl-2)/腺病毒E1B 19 k Da相互作用蛋白3(BNIP3),Beclin1,p-Akt,Akt表达水平。结果:与正常组比较,模型组肾小球基底膜增厚,细胞外基质增多,系膜扩张,胶原蛋白显著增加,PI3K/Akt/FoxO1信号增强(P <0. 01),自噬活性减弱(P <0. 01);与模型组比较,黄芪甲苷中、高剂量肾脏组织病变明显改善,明显抑制肾组织PI3K,Akt及FoxO1磷酸化水平(P <0. 05,P <0. 01),同时增强自噬反应,上调BNIP3,LC3Ⅱ/LC3Ⅰ和Beclin1的表达(P <0. 05,P <0. 01)。结论:黄芪甲苷可能通过抑制PI3K/Akt/FoxO1信号增加肾组织细胞自噬活性,减缓了2型糖尿病肾病的发展进程。
Objective: To study the protective effect of astragaloside( AS) Ⅳ on kidney in type 2 diabetic nephropathy rats and its regulation effect on phosphoinositide 3-kinase( PI3K)/protein kinase B( Akt)/forkhead box O1( FoxO1) signaling, and investigate the mechanism of glycosides against type 2 diabetic nephropathy. Method: After 6 weeks of high-glucose and high-fat diet,rat models of type 2 diabetes were established by intraperitoneal injection of streptozotocin( STZ)( 35 mg·kg~(-1)) and randomly divided into model group,AS-Ⅳ( 20,40,80 mg·kg~(-1)) groups and metformin hydrochloride( positive) group. After 8 weeks of continuous administration,changes in body weight,kidney index,blood glucose,24-hour urinary protein,urinary microalbumin,urinary creatinine,serum creatinine,and blood urea nitrogen were measured in each group;hematoxylin-eosin( HE) staining was used to observe the pathological changes of renal tissues; Masson trichrome staining was used to observe the collagen expression level. Periodontium hexaammine silver( PASM) staining was used to observe the changes of basement membrane. Immunohistochemistry assay was used to detect phospho-FoxO1( p-FoxO1) protein expression,and Western blot was used to analyze the expression levels of autophagy marker protein PI3 K, microtubule-associated protein 1 light chain 3( LC3)/Ⅱ, B-cell lymphoma-2( Bcl-2)/adenovirus E1 B19 kDa interacting protein 3( BNIP3),Beclin1,p-Akt,and Akt. Result: As compared with the normal group,the glomerular basement membrane of the model group was thicker; the extracellular matrix was increased; the mesangial was expanded; the collagen was significantly increased; the PI3 K/Akt/FoxO1 signal was increased( P < 0. 01); and the autophagic activity was weakened( P < 0. 01). As compared with the model group,the phosphorylation of PI3 K,Akt,and FoxO1 in renal tissue was significantly inhibited in AS-Ⅳ high and medium dose groups( P < 0. 05,P < 0. 01); autophagy was increased; and the expression of BNIP3,LC3 Ⅱ/LC3 I,and Beclin1 was up-regulated( P < 0. 05,P < 0. 01). Conclusion: AS-Ⅳ may increase the autophagic activity of renal cells by inhibiting PI3K/Akt/FoxO1 signal,slowing down the development of type 2 diabetic nephropathy.
Objective: To study the protective effect of astragaloside( AS) Ⅳ on kidney in type 2 diabetic nephropathy rats and its regulation effect on phosphoinositide 3-kinase( PI3K)/protein kinase B( Akt)/forkhead box O1( FoxO1) signaling, and investigate the mechanism of glycosides against type 2 diabetic nephropathy. Method: After 6 weeks of high-glucose and high-fat diet,rat models of type 2 diabetes were established by intraperitoneal injection of streptozotocin( STZ)( 35 mg·kg~(-1)) and randomly divided into model group,AS-Ⅳ( 20,40,80 mg·kg~(-1)) groups and metformin hydrochloride( positive) group. After 8 weeks of continuous administration,changes in body weight,kidney index,blood glucose,24-hour urinary protein,urinary microalbumin,urinary creatinine,serum creatinine,and blood urea nitrogen were measured in each group;hematoxylin-eosin( HE) staining was used to observe the pathological changes of renal tissues; Masson trichrome staining was used to observe the collagen expression level. Periodontium hexaammine silver( PASM) staining was used to observe the changes of basement membrane. Immunohistochemistry assay was used to detect phospho-FoxO1( p-FoxO1) protein expression,and Western blot was used to analyze the expression levels of autophagy marker protein PI3 K, microtubule-associated protein 1 light chain 3( LC3)/Ⅱ, B-cell lymphoma-2( Bcl-2)/adenovirus E1 B19 kDa interacting protein 3( BNIP3),Beclin1,p-Akt,and Akt. Result: As compared with the normal group,the glomerular basement membrane of the model group was thicker; the extracellular matrix was increased; the mesangial was expanded; the collagen was significantly increased; the PI3 K/Akt/FoxO1 signal was increased( P < 0. 01); and the autophagic activity was weakened( P < 0. 01). As compared with the model group,the phosphorylation of PI3 K,Akt,and FoxO1 in renal tissue was significantly inhibited in AS-Ⅳ high and medium dose groups( P < 0. 05,P < 0. 01); autophagy was increased; and the expression of BNIP3,LC3 Ⅱ/LC3 I,and Beclin1 was up-regulated( P < 0. 05,P < 0. 01). Conclusion: AS-Ⅳ may increase the autophagic activity of renal cells by inhibiting PI3K/Akt/FoxO1 signal,slowing down the development of type 2 diabetic nephropathy.
引文
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[15] Masuko K,Maki I,Hirokazu F,et al. Cancer genetics and ge-nomics of human FOX family genes[J]. Cancer Letters,2013,328(2):198-206.
[16]孙燕.中医中药在肿瘤综合治疗中的应用[J].中国中西医结合杂志,1997,17(6):323-324.
[17] Brezniceanu M L,Lau C J,Godin N,et al. Reactive oxygen species promote Caspase-12 expression and tubular apoptosis in diabetic nephropathy[J]. J Am Soc Nephrol,2010,21(6):943-954.
[18] Lemasters J J. Selective mitochondrial autophagy,or mitophagy,as a targeted defense against oxidative stress,mitochondrial dysfunction,and aging[J]. Rejuvenation Res,2005,8(1):3-5.
[19] Takahashi A,Kimura T,Takabatake Y,et al. Autophagy guards against cisplatin-induced acute kidney injury[J]. Am J Pathol,2012,180(2):517-525.
[20] Pallet N,Bouvier N,Legendre C,et al. Autophagy protects renal tubular cells against cyclosporine toxicity[J]. Autophagy,2008,4(6):783-791.
[21] Chargui A,Zekri S,Jacqullet G,et al. Cadmium-induced autophagy in rat kidney:an early biomarker of subtoxic exposure[J]. Toxicol Sci,2011,121(1):31-42.
[22] HUANG C,LIN M Z,CHENG D,et al. Thioredoxininteracting protein mediates dysfunction of tubular autophagy in diabetic kidneys through inhibiting autophagic flux[J]. Lab Invest,2014,94(3):309-320.
[23] Ishihara M,Urushido M,Hamada K,et al. Sestrin-2 and BNIP3 regulate autophagy and mitophagy in renal tubular cells in acute kidney injury[J]. Am J Physiol Renal Physiol,2013,305(4):F495-F509.
[2] Gnudi L,Coward R J,Long D A. Diabetic nephropathy:perspective on novel molecular mechanisms[J]. Trends Endocrinol Metab,2016,27(11):820-830.
[3]石宛鑫,李娜,隋满姝,等.叉头框转录因子O在肾脏疾病中作用研究进展[J].中华实用诊断与治疗杂志,2018,32(2):205-208.
[4] DU M,WANG Q,LI W,et al. Overexpression of Fox O1ameliorates the podocyte epithelial-mesenchymal transition induced by high glucose in vitro and in vivo[J]. Biochem Biophys Res Commun,2016,471(4):416-422.
[5] GUO F,ZHANG Y,WANG Q,et al. Effects of Fox O1 on podocyte injury in diabetic rats[J]. Biochem Biophys Res Commun,2015,466(2):260-266.
[6]陈倩,季旭明,阚东方,等.黄芪甲苷对复合水饮内停大鼠心脏功能的影响[J].中国实验方剂学杂志,2017,23(14):100-105.
[7]田春雨,薄海美,林飞武,等.双益方改善2型糖尿病模型大鼠胰岛素抵抗的药效及作用机制[J].中国实验方剂学杂志,2017,23(22):137-142.
[8]段立军,孙博航.黄芪甲苷的研究进展[J].沈阳药科大学学报,2011,28(5):410-415.
[9]尤良震,林逸轩,方朝晖,等.黄芪甲苷治疗糖尿病及其并发症药理作用研究进展[J].中国中药杂志,2017,42(24):4700-4706.
[10]田春雨,薄海美,林飞武,等.双益方改善2型糖尿病模型大鼠胰岛素抵抗的药效及作用机制[J].中国实验方剂学杂志,2017,23(22):137-142.
[11]鲁旭亮.糖尿病肾病患者血清脂联素和血管内皮生长因子水平与氧化应激的相关性[J].中华实用诊断与治疗杂志,2013,27(2):147-149.
[12]邹万忠.糖尿病肾病的病理与分型[J].中华糖尿病杂志,2009,17(6):423-425.
[13] HUANG H,Tindall D J. Dynamic Fox O transcription factors[J]. Cell Sci,2007,120(15):2479-2487.
[14] ZHAO Y,Tindall D J,HUANG H. Modulation of androgen receptor by FOXA1 and FOXO1 factors in prostate cancer[J]. Int J Biol Sci,2014,10(6):614-619.
[15] Masuko K,Maki I,Hirokazu F,et al. Cancer genetics and ge-nomics of human FOX family genes[J]. Cancer Letters,2013,328(2):198-206.
[16]孙燕.中医中药在肿瘤综合治疗中的应用[J].中国中西医结合杂志,1997,17(6):323-324.
[17] Brezniceanu M L,Lau C J,Godin N,et al. Reactive oxygen species promote Caspase-12 expression and tubular apoptosis in diabetic nephropathy[J]. J Am Soc Nephrol,2010,21(6):943-954.
[18] Lemasters J J. Selective mitochondrial autophagy,or mitophagy,as a targeted defense against oxidative stress,mitochondrial dysfunction,and aging[J]. Rejuvenation Res,2005,8(1):3-5.
[19] Takahashi A,Kimura T,Takabatake Y,et al. Autophagy guards against cisplatin-induced acute kidney injury[J]. Am J Pathol,2012,180(2):517-525.
[20] Pallet N,Bouvier N,Legendre C,et al. Autophagy protects renal tubular cells against cyclosporine toxicity[J]. Autophagy,2008,4(6):783-791.
[21] Chargui A,Zekri S,Jacqullet G,et al. Cadmium-induced autophagy in rat kidney:an early biomarker of subtoxic exposure[J]. Toxicol Sci,2011,121(1):31-42.
[22] HUANG C,LIN M Z,CHENG D,et al. Thioredoxininteracting protein mediates dysfunction of tubular autophagy in diabetic kidneys through inhibiting autophagic flux[J]. Lab Invest,2014,94(3):309-320.
[23] Ishihara M,Urushido M,Hamada K,et al. Sestrin-2 and BNIP3 regulate autophagy and mitophagy in renal tubular cells in acute kidney injury[J]. Am J Physiol Renal Physiol,2013,305(4):F495-F509.