siRNA沉默FoxO1对STZ诱导的糖尿病大鼠视网膜IL-1β的影响及其作用机制-东南大学学报医学版

siRNA沉默FoxO1对STZ诱导的糖尿病大鼠视网膜IL-1β的影响及其作用机制

单位：1. 第三军医大学大坪医院野战外科研究所眼科, 重庆 400042;
2. 西南医科大学附属医院眼科, 四川泸州 646000

分类号：R-33;R587.2

出版年·卷·期（页码）：2018·37·第二期（183-190）

摘要：

目的：探讨抑制叉头状转录因子O1（FoxO1）表达对糖尿病大鼠视网膜IL-1β的影响及其作用机制。方法：将人视网膜微血管内皮细胞（HRCECs）用不同浓度葡萄糖处理后，采用RT-PCR和Western blot检测细胞FoxO1、IL-1β表达。HRCECs按照处理方式分为空白对照组（Mock组）、阴性对照组（NC-FoxO1组）和si-FoxO1组（si-FoxO1组），RT-PCR检测各组细胞FoxO1、IL-1β mRNA表达，Western blot检测各组细胞FoxO1、IL-1β、p-P38、p-JNK、p-ERK蛋白表达。60只大鼠随机分为对照组（Control组）、DM组、si-FoxO1转染组（LV-si-FoxO1组）和空病毒转染组（LV-NC组），DM组、LV-si-FoxO1组、LV-NC组建立糖尿病大鼠模型，造模成功后向大鼠玻璃体腔注射LV-si-FoxO1或LV-NC慢病毒载体，注射12周后分离视网膜组织，分别采用免疫组织化学染色、RT-PCR和Western blot检测大鼠视网膜组织FoxO1、IL-1β表达。结果：与5 mmol·L^-1葡萄糖浓度组比较，15 mmol·L^-1浓度组FoxO1、IL-1β mRNA和蛋白表达差异无统计学意义（P>0.05），25 mmol·L^-1浓度组FoxO1、IL-1β mRNA和蛋白表达明显高于5 mmol·L^-1浓度组，而35 mmol·L^-1浓度组FoxO1、IL-1β mRNA和蛋白表达增加的更明显，组间比较差异具有统计学意义（P<0.05）。si-FoxO1组IL-1β mRNA和蛋白表达明显低于NC-FoxO1组和Mock组（P<0.05），NC-FoxO1组和Mock组IL-1β mRNA和蛋白表达比较差异无统计学意义（P>0.05）。DM组和LV-NC组FoxO1、IL-1β mRNA和蛋白表达明显高于Control组和LV-si-FoxO1组（P<0.05），LV-NC组与Control组FoxO1、IL-1β表达差异无统计学意义（P>0.05），DM组和LV-NC组FoxO1、IL-1β表达差异无统计学意义（P>0.05）。si-FoxO1组p-P38、p-JNK、p-ERK蛋白表达明显低于NC-FoxO1组和Mock组（P<0.05），NC-FoxO1组和Mock组p-P38、p-JNK、p-ERK蛋白表达比较差异无统计学意义（P>0.05）。结论：高糖能够诱导HRCECs FoxO1、IL-1β表达增加，抑制FoxO1表达可能是通过降低MAPK磷酸化下调HRCECs细胞IL-1β表达实现的。

Objective:To explore the effect of FoxO1 inhibition in the expressions of IL-1β in the diabetic rats retina induced by STZ and its mechanism. Methods:The human retinal capillary endothelial cells (HRCECs) were treated by different concentration of glucose. The expressions of FoxO1, IL-1β were measured by RT-PCR and Western blot. The HRCECs were divided into Mock group, NC-FoxO1 group and si-FoxO1 group, the expressions of FoxO1, IL-1β mRNA were measured by RT-PCR, the expressions of FoxO1, IL-1β, p-P38, p-JNK, p-ERK protein were measured by Western blot. 60 rats were randamly divided into Control group, DM group, LV-si-FoxO1 group and LV-NC group, the diabetic rats models were established in DM group, LV-si-FoxO1 group and LV-NC group. After models were estabilished sucessfully, the LV-si-FoxO1 or LV-NC lentiviral vector was injected in the vitreous cavity of diabetic rats, the retinal tissue was completely separated at the end of 12 weeks, the expressions of FoxO1, IL-1β in retinal tissue were measured by immunohistochemical staining, RT-PCR and Western blot. Results:Compared to the 5 mmol·L^-1 glucose group, the expressions of FoxO1, IL-1β mRNA and protein in 15 mmol·L^-1 glucose group had no significant differences (P>0.05), the expressions of FoxO1, IL-1β mRNA and protein in 25 mmol·L^-1 glucose group were significantly higher than those in 5 mmol·L^-1 glucose group, and FoxO1, IL-1β mRNA and protein in 35 mmol·L^-1 glucose group increased more obviously, the difference between groups was statistically significant (P<0.05). The expressions of IL-1β mRNA and protein in si-FoxO1 group were significantly lower than those in NC-FoxO1 group and Mock group (P<0.05), there were no significant differences of IL-1β mRNA and protein between NC-FoxO1 group and Mock group (P>0.05). The expressions of FoxO1, IL-1β mRNA and protein in DM group and LV-NC group were significantly higher than those in Control group and LV-si-FoxO1 group (P<0.05), there were no significant differences of FoxO1, IL-1β mRNA and protein between LV-NC group and Control group(P>0.05), there were also no significant differences between DM group and LV-NC group (P>0.05). The expressions of p-P38, p-JNK, p-ERK protein in si-FoxO1 group were significantly lower than those in NC-FoxO1 group and Mock group (P<0.05), and the expressions of p-P38, p-JNK, p-ERK protein between NC-FoxO1 group and Mock group had no significant differences (P>0.05). Conclusion:High glucose can promote the expressions of FoxO1, IL-1β in HRCECs, inhibition the expression of FoxO1 may be done by down-reuglation of IL-1β expression through decreasing the MAPK phosphorylation.

参考文献：

[1] WELIKALA R A,DEHMESHKI J,HOPPE A,et al.Automated detection of proliferative diabetic retinopathy using a modified line operator and dual classification[J].Comput Methods Programs Biomed,2014,114(3):247-261.
[2] MCANANY J J,WANEK J,ZELKHA R,et al.Neural constraints on visual acuity in proliferative diabetic retinopathy[J].Optom Vis Sci,2014,91(2):194-199.
[3] SCUDERI S,D'AMICO A G,FEDERICO C,et al.Different retinal expression patterns of IL-1α,IL-1β,and their receptors in a rat model of type 1 STZ-induced diabetes[J].J Mol Neurosci,2015,56(2):431-439.
[4] BEHL Y,KROTHAPALLI P,DESTA T,et al.FOXO1 plays an important role in enhanced microvascular cell apoptosis and microvascular cell loss in type 1 and type 2 diabetic rats[J].Diabetes,2009,58(4):917-925.
[5] 刘晓静,朱弼珺,邹海东,等.氨甲酰促红细胞生成素防治糖尿病视网膜病变的作用及机制[J].中华实验眼科杂志,2014,32(11):980-988.
[6] 韩改玲.2型糖尿病患者CTGF及25-羟维生素D与视网膜病变的相关性[J].现代医学,2016,44(8):1064-1067.
[7] DAY T E,RAVI N,XIAN H,et al.Sensitivity of diabetic retinopathy associated vision loss to screening interval in an agent-based/discrete event simulation model[J].Comput Biol Med,2014,47:7-12.
[8] 曹瑞琪,卢新兰,任牡丹,等.盐酸青藤碱诱导人肝癌细胞凋亡及其对IL-1β,TNF及C/EBPβ水平的影响[J].西安交通大学学报:医学版,2017,38(2):290-294.
[9] RIVERA J C,SITARAS N,NOUEIHED B,et al.Microglia and interleukin-1β in ischemic retinopathy elicit microvascular degeneration through neuronal semaphorin-3A[J].Arterioscler Thromb Vasc Biol,2013,33(8):1881-1891.
[10] BATTIPROLU P K,HOJAYEV B,JIANG N,et al.Metabolic stress-induced activation of FoxO1 triggers diabetic cardiomyopathy in mice[J].J Clin Invest,2012,122(3):1109-1118.
[11] 周英旎,王庆祝,秦贵军,等.FoxO1过表达对大鼠糖尿病肾病的影响及机制研究[J].中华内分泌代谢杂志,2015(2):155-161.
[12] WANG Q,WANG N,DONG M,et al.GdCl3 reduces hyperglycaemia through Akt/FoxO1-induced suppression of hepatic gluconeogenesis in type 2 diabetic mice[J].Clin Sci,2014,127(2):91-100.
[13] DOBIERZEWSKA A,SHI L,KARAKASHIAN A A,et al.Interleukin 1β regulation of FoxO1 protein content and localization:evidence for a novel ceramide-dependent mechanism[J].J Biol Chem,2012,287(53):44749-44760.
[14] 李娜娜,任寿安.慢性间歇低氧对大鼠肝细胞IRS-2、FoxO1表达的影响[J].国际内分泌代谢杂志,2015,35(1):1-5.
[15] HUANG W,YU J,JIA X,et al.Zhenqing recipe improves glucose metabolism and insulin sensitivity by repressing hepatic FOXO1 in type 2 diabetic rats[J].Am J Chin Med,2012,40(4):721-733.
[16] XU F,OTHMAN B,LIM J,et al.Foxo1 inhibits diabetic mucosal wound healing but enhances healing of normoglycemic wounds[J].Diabetes,2015,64(1):243-256.
[17] CHOU M Y,KAO C T,HUNG C J,et al.Role of the P38 pathway in calcium silicate cement-induced cell viability and angiogenesis-related proteins of human dental pulp cell in vitro[J].J Endod,2014,40(6):818-824.
[18] 李永浩,吕林,陈凌燕,等.P38丝裂素活化蛋白激酶信号通路阻断对糖尿病鼠早期血视网膜屏障和视网膜节细胞的保护作用[J].中华眼底病杂志,2010,26(2):139-142.
[19] 刁雪红,申鍔,张跃力,等.抑制Rac1通过降低磷酸化p38 MAPK提高1型糖尿病小鼠的心脏功能[J].中国病理生理杂志,2010,26(7):1285-1289.
[20] KHAN S,JENA G B.Protective role of sodium butyrate,a HDAC inhibitor on beta-cell proliferation,function and glucose homeostasis through modulation of p38/ERK MAPK and apoptotic pathways:Study in juvenile diabetic rat[J].Chem Biol Interact,2014,213:1-12.

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录