丹皮酚对大鼠糖尿病视网膜病变的改善作用及其调节miR-802-5p表达的作用机制

doi:10.13481/j.1671-587X.20220111

摘要/Abstract

摘要： 目的

探讨丹皮酚对大鼠糖尿病视网膜病变（DR）的改善作用，并阐明其可能的作用机制。

方法

40只SPF级SD大鼠随机分为对照组、DR模型组、15 mg·kg^－1丹皮酚组和30 mg·kg^－1丹皮酚组，每组10只，除对照组外其余3组大鼠腹腔注射55 mg·kg^－1链脲佐菌素（STZ）构建DR模型。建模成功后，对照组和DR模型组大鼠皮下注射8 mL·kg^－1生理盐水，15和30 mg·kg^－1丹皮酚组大鼠皮下注射15和30 mg·kg^－1丹皮酚磺酸钠注射液，每日1次，共2周。取大鼠视网膜组织用于实时荧光定量PCR（RT-qPCR）、Western blotting和酶联免疫吸附测定（ELISA）检测，HE染色观察各组大鼠视网膜组织病理形态表现。采用高糖（HG，33 mmol·L^－1葡萄糖）培养基培养人视网膜微血管内皮细胞（HRMECs）。采用不同剂量（0、5、25、50、70 和100 mg·L^－1）丹皮酚分别干预HRMECs和HG处理 24 h 后的HRMECs，采用CCK-8法和流式细胞术检测各组细胞活性和凋亡率，以确定后续实验中丹皮酚最适作用剂量。HRMECs进行转染后分为对照组、HG组、HG+NC inhibitor组、HG+miR-802-5p inhibitor组、HG+丹皮酚（25 mg·L^－1）组、HG+丹皮酚+NC mimic组和HG+丹皮酚+miR-802-5p mimic组。采用RT-qPCR法检测各组大鼠视网膜组织和各组细胞中miR-802-5p表达水平，荧光素酶报告基因和RNA免疫共沉淀法检测HRMECs 中miR-802-5p与SIRT6的结合关系，Western blotting法检测各组大鼠视网膜组织和各组细胞中沉默调节蛋白6（SIRT6）、色素上皮衍生因子（PEDF）和血管内皮生长因子（VEGF）蛋白表达水平，ELISA法检测各组大鼠视网膜组织和各组细胞中活性氧（ROS）水平及过氧化氢酶（CAT）和超氧化物歧化酶（SOD）活性。

结果

与对照组比较，DR模型组大鼠视网膜组织中miR-802-5p表达水平和VEGF蛋白表达水平明显升高（P<0.01），SIRT6和PEDF蛋白表达水平明显降低（P<0.01），ROS水平明显升高（P<0.01），CAT和SOD活性明显降低（P<0.01）；与DR模型组比较，不同剂量丹皮酚组大鼠视网膜组织中miR-802-5p表达水平和VEGF蛋白表达水平明显降低（P<0.05），SIRT6和PEDF蛋白表达水平明显升高（P<0.01），ROS水平明显降低（P<0.05或P<0.01），CAT和SOD活性明显升高（P<0.05或P<0.01）。HE染色，对照组大鼠视网膜组织结构清晰，内外核层细胞排列规则，未见新生血管生成；DR模型组视网膜组织结构不清晰，神经纤维层伴水肿，内外核层细胞排列疏松，有新生血管生成；不同剂量丹皮酚组大鼠视网膜组织结构较DR清晰，内外核层细胞排列较整齐，新生血管生成均较DR模型组有明显改善。与对照组比较，HG组细胞凋亡率升高（P<0.01）；与HG组比较，HG+丹皮酚组细胞凋亡率明显降低（P<0.01）。与对照组比较，HG组细胞中miR-802-5p表达水平明显升高（P<0.01）；与HG+NC inhibitor组比较，HG+miR-802-5p inhibitor组细胞中miR-802-5p表达水平明显降低（P<0.01）。荧光素酶报告基因分析和RNA免疫共沉淀检测，SIRT6是miR-802-5p的靶基因。与对照组比较，HG组细胞中SIRT6和PEDF蛋白表达水平及CAT和SOD活性明显降低（P<0.01），VEGF蛋白表达水平和ROS水平明显升高（P<0.01）；与HG组比较，HG+miR-802-5p inhibitor组细胞中VEGF蛋白表达水平明显降低（P<0.01），SIRT6和PEDF蛋白表达水平明显升高（P<0.01），ROS水平明显降低（P<0.05），CAT和SOD活性明显升高（P<0.01）；与HG组比较，HG+丹皮酚组细胞中ROS水平明显降低（P<0.01），CAT和SOD活性明显升高（P<0.01）；与HG+丹皮酚+NC mimic组比较，HG+丹皮酚+ miR-802-5p mimic组细胞中ROS水平明显升高（P<0.05），CAT和SOD活性明显降低（P<0.01）。

结论

丹皮酚可能通过miR-802-5p/SIRT6轴对DR起到一定的改善作用。

关键词: 糖尿病视网膜病变, 丹皮酚, 微小RNA-802-5p, 沉默调节蛋白6, 高糖

Abstract:

Objective： To investigate the improvement effect of paeonol on diabetic retinopathy （DR） in the rats， and to elucidate its possible mechanism.

Methods

A total of 40 SPF grade SD rats were randomly divided into control group，DR model group，15 mg·kg^－1 paeonol group and 30 mg·kg^－1 paeonol group；there were 10 rats in each group.Except control group，the rats in other groups were intraperitoneally injected with 55 mg·kg^－1 streptozotocin（STZ） to establish the DR medels.After successeful modeling，the rats in control group and DR model group were subcutaneously injected with normal saline，and the rats in 15 and 30 mg·kg^－1 paeonol groups were subcutaneously injected with 15 and 30 mg·kg^－1 paeonol sodium sulfonate.The rat retina tissue was harvested for real-time fluorescence quantitative PCR （RT-qPCR）， Western blotting， and ELISA assays and HE staining was performed to observe the pathomorphology of retina tissue of the rats in various groups.The human retinal microvascular endothelial cells （HRMECs） were cultured in high glucose （HG，33 mmol·L^-1 glucose medium）. Different doses （0， 5， 25， 50， 70 and 100 mg·L^-1） of paeonol were used to intervene the HRMECs and the HRMECs treated with HG for 24 h， respectively；the cell viabilities and apoptotic rates of cells in various groups were detected by CCK-8 assay and flow cytometry， respectively.The optimum dose used in the follow-up experiment was confirmed.The transfected HRMECs were divided into control group， HG group， HG+NC inhibitor group， HG+miR-802-5p inhibitor group， HG + paeonol （25 mg·L^－1） group，HG+paeonol +NC mimic group， and HG+ paeonol + miR-802-5p mimic group.The expression levels of miR-802-5p in the retina tissue and the cells in various groups were determined by RT-qPCR method，and the association between miR-802-5p and SIRT6 in the HRMECs was determined by luciferase reporter gene assay and RNA co-immunoprecipitation assay；the protein expression levels of silent information regulator 6 （SIRT6）， pigment epithelium derived factor （PEDF） and vascular endothelial growth factor （VEGF） in the retina tissue of the rats and the cells in various groups were determined by Western blotting method. The levels of reactive oxygen species （ROS） and the activities of catalase （CAT） and superoxide dismutase （SOD） in the cells in various groups were measured by ELISA.

Results

Compared with control group， the expression levels of miR-802-5p and VEGF protein in the retina tissue of the rats in DR model group were significantly increased （P<0.01）， and the protein expression levels of SIRT6 and PEDF were decreased （P<0.05）， the ROS level was increased （P<0.01）， and the activities of CAT and SOD were decreased （P<0.05）.Compared with DR model group，the expression levels of miR-802-5p and the expression levels of VEGF protein in retinal tissue of the rats in different dose of paeonal groups were significantly decreased（P<0.05），the expression levels of SIRT6 and PEDF were signifricantly increased（P<0.01），the ROS levels were significantly decreased （P<0.05 or P<0.01），and the activities of CAT and SOD were signifricantly increased（P<0.05 or P<0.01）.The HE staining results showed that the retinal structure of the rats in control group was clear， the cells in the inner and outer nuclear layers were arranged regularly， and no neovascularization was observed； in DR model group， the retinal structure was unclear， the nerve fiber layer was edematous， the inner and outer nuclear layer cells were arranged loosely， and neovascularization was observed； the retinal structures were clear and the cells in the inner and outer nuclear layers were arranged in an orderly manner in different doses of paeonol groups， and the neoangiogenesis was significantly improved compared with that in model group. Compared with control group，the apoptotic rate of cells in HG group was increased（P<0.01）；compared with HG group，the apoptotic rate of cells in HG+paeonol group was decreased（P<0.01）.Compared with control group， the expression level of miR-802-5p in the cells in HG group was significantly increased （P<0.01）；compared with HG+NC inhibitor group，the expression level of miR-802-5p in the cells in HG+miR-802-5p inhibitor group was significantly decreased（P<0.01）.The results of luciferase reporter gene assay and RNA co-immunoprecipitation assay showed that SIRT6 was a target gene of miR-802-5p.Compared with control group，the expression levels of SIRT6 and PEDF proteins and the activities of CAT and SOD in the cells in HG group were significantly decreased（P<0.01），and the expression level of VEGF protein and the ROS level were significantly increased（P<0.01）；compared with HG group， the expression level of VEGF protein in the cells in HG+miR-802-5p inhibitor group was decreased （P<0.01），the expression levels of SIRT6 and PEDF proteins were increased （P<0.05），the ROS level was decreased （P<0.05）， and the activities of CAT and SOD were increased significantly （P<0.01）.Compared with HG group，the ROS level in the cells in HG+paeonol group was decreased（P<0.01），and the activities of CAT and SOD were increased（P<0.01）；compared with HG + paeonol + NC mimic group， the ROS level in the cells in HG + paeonol + miR-802-5p mimic group was increased （P<0.05）， and the activities of CAT and SOD were significantly decreased （P<0.01）.

Conclusion

Paeonol may improve the DR progression through the miR-802-5p/SIRT6 axis.

Key words: Diabetic retinopathy, Paeonol, microRNA-802-5p, Recombinant sirtuin 6, High glucose

中图分类号:

R587.1

孙俊波,高达,赵逸菲,许华,邱帆,赵璐. 丹皮酚对大鼠糖尿病视网膜病变的改善作用及其调节miR-802-5p表达的作用机制[J]. 吉林大学学报(医学版), 2022, 48(1): 82-93.

Junbo SUN,Da GAO,Yifei ZHAO,Hua XU,Fan QIU,Lu ZHAO. Improvement effect of paeonol on diabetic retinopathy in rats and its mechanism of regulating miR-802-5p expression[J]. Journal of Jilin University(Medicine Edition), 2022, 48(1): 82-93.

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	*河南中医药大学龙子湖校区2017级中医专业 △河南中医药大学东明校区2020级中医专业

Group	ROS [m_B/(mmol·g^－1)]	CAT [λ_B/(U·mg^－1)]	SOD [λ_B/(U·mg^－1)]
Control	0.81±0.12	2.75±0.31	44.89±5.02
DR model	8.37±0.19^*	0. 72±0.11^*	21.35±3.38^*
15 mg·kg^－1 paeonol	4.32±0.15^△	1.33±0.10^△	30.72±3.81^△
30 mg·kg^－1 paeonol	1.95±0.13^△△	2.14±0.17^△△	37.08±4.72^△△
F	334.3	231.3	297.9
P	<0.01	<0.01	<0.01

Group	ROS [m_B/(mmol?g^－1)]	CAT [λ_B/(U?mg^－1)]	SOD [λ_B/(U?mg^－1)]
Control	0.11±0.03	0.26±0.09	4.87±0.23
HG	0.86±0.07^*	0.12±0.01^*	2.01±0.11^*
HG+NC inhibitor	0.83±0.08	0.11±0.03	2.04±0.13
HG+miR-802-5p inhibitor	0.25±0.06^△	0.22±0.04^△	4.15±0.15^△
HG+paeonol	0.25±0.04^#	0.23±0.05^#	4.26±0.14^#
HG+paeonol+NC mimic	0.22±0.04	0.26±0.02	4.19±0.13
HG+paeonol+miR-802-5p mimic	0. 91±0.06^○	0.14±0.02^○	2.15±0.16^○
P	<0.01	<0.01	<0.01
F	63.18	11.29	416.71

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