[1]李蓉,姚国敏,周凌霄,等.ghrelin-GHSR-1a对高糖环境下视网膜血管内皮细胞的保护作用[J].眼科新进展,2022,42(3):205-209.[doi:10.13389/j.cnki.rao.2022.0041]
　LI Rong,YAO Guomin,ZHOU Lingxiao,et al.Protective effects of ghrelin and growth hormone secretin receptor 1a on retinal microvascular endothelial cells under high glucose conditions[J].Recent Advances in Ophthalmology,2022,42(3):205-209.[doi:10.13389/j.cnki.rao.2022.0041]
点击复制

ghrelin-GHSR-1a对高糖环境下视网膜血管内皮细胞的保护作用/HTML
分享到：

《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
42卷
期数:
2022年3期
页码:
205-209
栏目:
实验研究
出版日期:
2022-03-05

文章信息/Info

Title:
Protective effects of ghrelin and growth hormone secretin receptor 1a on retinal microvascular endothelial cells under high glucose conditions
作者:
李蓉; 姚国敏; 周凌霄; 张敏; 闫瑾
710077　陕西省西安市，西安医学院第一附属医院眼科（李蓉，姚国敏，周凌霄）；710077　西安医学院第一附属医院内分泌科（张敏）；710021　陕西省西安市，西安医学院医学技术学院眼视光教研室（闫瑾）
Author(s):
LI Rong1; YAO Guomin1; ZHOU Lingxiao1; ZHANG Min2; YAN Jin3
1.Department of Ophthalmology,the First Affiliated Hospital of Xi’an Medical University,Xi’an 710077,Shaanxi Province,China
2.Department of Endocrinology,the First Affiliated Hospital of Xi’an Medical University,Xi’an 710077,Shaanxi Province,China
3.Department of Optometry Teaching and Research Section,College of Medical Technology,Xi’an Medical University,Xi’an 710021,Shaanxi Province,China
关键词:
ghrelin; 生长激素分泌素受体1a; 糖尿病视网膜病变; 高糖; 视网膜血管内皮细胞
Keywords:
ghrelin;  growth hormone secretin receptor 1a;  diabetic retinopathy;  high glucose;  retinal microvascular endothelial cells
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2022.0041
文献标志码:
A
摘要:
目的　观察ghrelin及其受体生长激素分泌素受体1a（GHSR-1a）在高糖环境下对视网膜血管内皮细胞的保护作用。方法　将体外培养的人视网膜血管内皮细胞（HRMECs）分为对照组和高浓度葡萄糖组。对照组细胞在含5.5 mmol·L-1葡萄糖的M199培养基中培养48 h，高浓度葡萄糖组细胞在含30.0 mmol·L-1葡萄糖的M199培养基中培养48 h，采用免疫荧光染色法检测两组细胞中GHSR-1a的表达。然后另取HRMECs随机分为5组：正常对照（NC）组、高糖（HG）组、HG+ghrelin组、HG+ghrelin+siGHSR-1a组和HG+ghrelin+NC-siGHSR-1a组，孵育48 h。NC组处理同对照组，HG组处理同高浓度葡萄糖组，HG+ghrelin组细胞在含30.0 mmol·L-1葡萄糖、10.0 nmol·L-1 ghrelin的M199培养基中培养，HG+ghrelin+siGHSR-1a组细胞先转染GHSR-1a-特异性siRNA，24 h后在含30.0 mmol·L-1葡萄糖和10.0 nmol·L-1 ghrelin的M199培养基中培养，HG+ghrelin+NC-siGHSR-1a组细胞先转染非特异性序列，24 h后在含30.0 mmol·L-1葡萄糖和10.0 nmol·L-1 ghrelin的M199培养基中培养。分别采用CCK-8和Annexin-V/FITC-PI凋亡检测试剂盒检测各组HRMECs细胞活力和细胞凋亡情况。Western blot检测凋亡相关蛋白Bax和Bcl-2的表达。结果　高浓度葡萄糖组的GHSR-1a表达低于对照组（P<0.05）。NC组、HG组、HG+ghrelin组、HG+ghrelin+siGHSR-1a组和HG+ghrelin+NC-siGHSR-1a组的细胞活力分别为1.00、69.87%±0.68%、92.31%±3.62%、75.98%±4.67%和90.87%±1.95%，总体比较差异有统计学意义（P<0.001）；两两比较显示，除HG+ghrelin组与HG+ghrelin+NC-siGHSR-1a组的细胞活力差异无统计学意义（P>0.05）外，其余各组细胞活力比较差异均有统计学意义（均为P<0.05）。NC组、HG组、HG+ghrelin组、HG+ghrelin+siGHSR-1a组和HG+ghrelin+NC-siGHSR-1a组的细胞凋亡率分别为5.76%±0.36%、20.53%±0.38%、10.84%±0.52%、14.93%±0.18%和11.03%±0.62%，总体比较差异有统计学意义（F=468.88，P<0.001）；两两比较显示，除HG+ghrelin组与HG+ghrelin+NC-siGHSR-1a组细胞凋亡率差异无统计学意义（P>0.05）外，其余各组细胞凋亡率比较差异均有统计学意义（均为P<0.05）。NC组、HG组、HG+ghrelin组、HG+ghrelin+siGHSR-1a组和HG+ghrelin+NC-siGHSR-1a组细胞的Bax蛋白和Bcl-2蛋白相对表达量分别为0.17±0.03和0.64±0.05，0.75±0.04和0.14±0.02，0.38±0.04和0.49±0.08，0.56±0.04和0.33±0.07，0.40±0.06和0.46±0.09，总体比较差异均有统计学意义（均为P<0.001）；两两比较显示，除HG+ghrelin组与HG+ghrelin+NC-siGHSR-1a组Bax蛋白和Bcl-2蛋白表达差异均无统计学意义（均为P>0.05）外，其余各组Bax蛋白和Bcl-2蛋白表达比较差异均有统计学意义（均为P<0.05）。结论　ghrelin-GHSR-1a系统对高糖环境下的HRMECs功能具有保护作用，并抑制高糖诱导的HRMECs凋亡。
Abstract:
Objective　To investigate the protective effects of ghrelin and its receptor, the growth hormone secretin receptor 1a (GHSR-1a), on retinal microvascular endothelial cells under high glucose conditions.Methods　Human retinal microvascular endothelial cells (HRMECs) cultured in vitro were divided into the control and high glucose groups. Cells in the control group were cultured in M199 medium containing 5.5 mmol·L-1 glucose for 48 h, while cells in the high glucose group were cultured in M199 medium containing 30.0 mmol·L-1 glucose for 48 h. The expression of GHSR-1a in each group was detected by immunofluorescence staining. Additional HRMECs were randomly divided into five groups and incubated for 48 h: normal control (NC) group, high glucose (HG) group, HG+ghrelin group, HG+ghrelin+siGHSR-1a group, and HG+ghrelin+NC-siGHSR-1a group. Cells in the NC group were treated the same as the control group, and cells in the HG group were treated the same as the high glucose group. Cells in the HG+ghrelin group were cultured in M199 medium containing 30.0 mmol·L-1 glucose and 10.0 nmol·L-1 ghrelin. Cells in the HG+ghrelin+siGHSR-1a group were first transfected with GHSR-1a-specific siRNA, and 24 h later, cultured in M199 medium containing 30.0 mmol·L-1 glucose and 10.0 nmol·L-1 ghrelin. Cells in the HG+ghrelin+NC-siGHSR-1a group were transfected with non-specific sequence, and 24 h later, cultured in M199 medium containing 30.0 mmol·L-1 glucose and 10.0 nmol·L-1 ghrelin. The viability and apoptosis of HRMECs were detected by CCK-8 and Annexin-V/FITC-PI kit, respectively. Western blot was used to detect the expression of apoptosis-related proteins Bax and Bcl-2.Results　The expression of GHSR-1a in the high glucose group was lower than that in the control group (P<0.05). The cell viability in the NC, HG, HG+ghrelin, HG+ghrelin+siGHSR-1a, and HG+ghrelin+NC-siGHSR-1a groups were 1.00, (69.87±0.68) %, (92.31±3.62) %, (75.98±4.67) %, and (90.87±1.95) %, respectively, and the overall difference was statistically significant (P<0.001). The cell viability showed no statistical difference between the HG+ghrelin group and HG+ghrelin+NC-siGHSR-1a group (P>0.05), but in the remaining groups, the cell viability showed significant statistical difference (all P<0.05). The cell apoptosis in the NC, HG, HG+ghrelin, HG+ghrelin+siGHSR-1a, and HG+ghrelin+NC-siGHSR-1a groups were (5.76±0.36)%, (20.53±0.38) %, (10.84±0.52) %, (14.93±0.18) %, and (11.03±0.62) %, respectively, and the overall difference was statistically significant (F=468.88, P<0.001). The cell apoptosis showed no statistical difference between the HG+ghrelin group and HG+ghrelin+NC-siGHSR-1a group (P>0.05), but in the remaining groups, the cell apoptosis showed significant statistical difference (all P<0.05). The relative expression levels of Bax in the NC, HG, HG+ghrelin, HG+ghrelin+siGHSR-1a, and HG+ghrelin+NC-siGHSR-1a groups were 0.17±0.03, 0.75±0.04, 0.38±0.04, 0.56±0.04, and 0.40±0.06, respectively, while the relative expression levels of Bcl-2 in the NC, HG, HG+ghrelin, HG+ghrelin+siGHSR-1a, and HG+ghrelin+NC-siGHSR-1a groups were 0.64±0.05, 0.14±0.02, 0.49±0.08, 0.33±0.07, and 0.46±0.09, respectively, and the overall difference was statistically significant (both P<0.001). The expression levels of Bax protein and Bcl-2 protein showed no statistical difference between the HG+ghrelin group and HG+ghrelin+NC-siGHSR-1a group (both P>0.05), but in the remaining groups, the expression levels of Bax protein and Bcl-2 protein showed significant statistical difference (all P<0.05).Conclusion　Ghrelin-GHSR-1a system can protect the function of HRMECs under high glucose conditions and inhibit their apoptosis induced by high glucose.

参考文献/References:

［1］　SAKATA I,TAKEMI S.Ghrelin-cell physiology and role in the gastrointestinal tract［J］.Curr Opin Endocrinol Diabetes Obes,2021,28(2):238-242.
［2］　AKALU Y,MOLLA M D,DESSIE G,AYELIGN B.Physiological effect of ghrelin on body systems［J］.Int J Endocrinol,2020,2020:1385138.
［3］　LIANG Y,YIN W,YIN Y,ZHANG W.Ghrelin based therapy of metabolic diseases［J］.Curr Med Chem,2021,28(13):2565-2576.
［4］　王睿琦,白洁.Ghrelin在眼部作用的研究进展［J］.解放军医药杂志,2021,33(9):113-116.
WANG R Q,BAI J.Research progress of ghrelin in eye［J］.Med ＆ Pharm J Chin PLA,2021,33(9):113-116.
［5］　GUPTA D,OGDEN S B,SHANKAR K,VARSHNEY S,ZIGMAN J M.A LEAP 2 conclusions?Targeting the ghrelin system to treat obesity and diabetes［J］.Mol Metab,2021,46:101128.
［6］　KOJIMA M,HOSODA H,DATE Y,NAKAZATO M,MATSUO H,KANGAWA K.Ghrelin is a growth-hormone-releasing acylated peptide from stomach［J］.Nature,1999,402 (6762):656-660.
［7］　GNANAPAVAN S,KOLA B,BUSTIN S A,MORRIS D G,MCGEE P,FAIRCLOUGH P,et al.The tissue distribution of the mRNA of ghrelin and subtypes of its receptor,GHS-R,in humans［J］.J Clin Endocrinol Metab,2002,87(6):2988.
［8］　BEDNAREK M A,FEIGHNER S D,PONE S S,MCKEE K K,HRENIUK D L,SILVA M V,et al.Structure-function studies on the new growth hormone-releasing peptide,ghrelin:minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a［J］.J Med Chem,2000,43(23):4370-4376.
［9］　DI FONSO A,GHINASSI B,IZZICUPO P,ZAPPACOSTA R,LIBERATORE M,GALLENGA C E,et al.Novel evidence of ghrelin and growth hormone segretagogue receptor expression by human ocular tissues［J］.Regul Pept,2014,190-191:18-24.
［10］　KATSANOS A,DASTIRIDOU A,GEORGOULIAS P,CHOLEVAS P,KOTOULA M,TSIRONI E E.Plasma and aqueous humour levels of ghrelin in open-angle glaucoma patients［J］.Clin Exp Ophthalmol,2011,39(4):324-329.
［11］　ZANIOLO K,SAPIEHA P,SHAO Z,STAHL A,ZHU T,TREMBLAY S,et al.Ghrelin modulates physiologic and pathologic retinal angiogenesis through GHSR-1a［J］.Invest Ophthalmol Vis Sci,2011，52(8):5376-5386.
［12］　BAI J,YANG F,DONG L,ZHENG Y.Ghrelin protects human lens epithelial cells against oxidative stress-induced damage［J］.Oxid Med Cell Longev,2017,2017:1910450.
［13］　WANG L,CHEN Q,LI G,KE D.Ghrelin stimulates angiogenesis via GHSR1a-dependent MEK/ERK and PI3K/Akt signal pathways in rat cardiac microvascular endothelial cells［J］.Peptides,2012,33(1):92-100.
［14］　LI A,CHENG G,ZHU G H,TARNAWSKI A S.Ghrelin stimulates angiogenesis in human microvascular endothelial cells:implications beyond GH release［J］.Biochem Biophys Res Commun,2007,353(2):238-243.
［15］　WANG X,YANG L,CHEN Y,ZHANG L,FEI H.Ghrelin promotes angiogenesis by activating the Jagged1/Notch2/VEGF pathway in preeclampsia［J］.J Obstet Gynaecol Res,2021,47(2):486-494.
［16］　CONCONI M T,NICO B,GUIDOLIN D,BAIGUERA S,SPINAZZI R,REBUFFAT P,et al.Ghrelin inhibits FGF-2-mediated angiogenesis in vitro and in vivo［J］.Peptides,2004,25(12):2179-2185.
［17］　BAIGUERA S,CONCONI M T,GUIDOLIN D,MAZZOCCHI G,MALENDOWICZ L K,PARNIGOTTO P P,et al.Ghrelin inhibits in vitro angiogenic activity of rat brain microvascular endothelial cells［J］.Int J Mol Med,2004,14(5):849-854.
［18］　ZHANG R.Ghrelin suppresses inflammation in HUVECs by inhibiting ubiquitin-mediated uncoupling protein 2 degradation［J］.Int J Mol Med,2017,39(6):1421-1427.
［19］　NEALE J P H,PEARSON J T,THOMAS K N,TSUCHIMOCHI H,HOSODA H,KOJIMA M,et al.Dysregulation of ghrelin in diabetes impairs the vascular reparative response to hindlimb ischemia in a mouse model; clinical relevance to peripheral artery disease［J］.Sci Rep,2020,10(1):13651.

备注/Memo

备注/Memo:
西安市科技局医学研究项目（编号：21YXYJ0127）
更新日期/Last Update: 2022-03-05