七叶皂苷与曲安奈德合用对血—视网膜屏障损伤的保护作用及其机制

发布时间：2018-04-19 19:23

本文选题：七叶皂苷 + 曲安奈德　；参考：《山东大学》2013年博士论文

【摘要】：研究背景血-视网膜屏障(blood-retinal barrier, BRB)包括由视网膜毛细血管内皮细胞及其紧密连接构成的内屏障,和由视网膜色素上皮细胞及其紧密连接构成的外屏障。正常情况下视网膜内屏障可阻止血管内液体和大分子物质渗漏到视网膜；外屏障则阻止脉络膜毛细血管血液渗漏到视网膜。各种原因,如缺血、糖代谢异常、炎性细胞因子的释放、玻璃体以及视网膜前膜的机械牵引等都可以造成血-视网膜屏障的破坏,血管内液体及大分子物质渗漏到细胞外间隙,形成视网膜水肿,发生于黄斑区的水肿称为黄斑水肿。黄斑水肿直接引起患者视力下降,如果长期得不到缓解,可造成视网膜组织不可逆性损伤终致失明。因此,黄斑水肿的早期治疗十分重要。黄斑水肿目前常用的治疗方法包括：①激光治疗,一定程度上缓解黄斑水肿,但对于弥漫性黄斑水肿患者则疗效欠佳,甚至部分患者在治疗后黄斑水肿加重；②玻璃体内注射糖皮质激素(如曲安奈德triamcinolone acetonide,TA)、抗血管内皮生长因子(vascular endothelial growth factor,VEGF)抗体(如雷珠单抗)等取得了较好疗效,但药物半衰期短,需反复多次注射,增加了玻璃体内感染的几率,以及眼内压升高和白内障发生的几率(发生几率高达30%-50%)。研究表明,由occludi、claudin-5、与zonula occludens-1等蛋白构成的细胞间紧密连接是维持血-视网膜屏障生理功能的重要保障。糖皮质激素在体内外能与糖皮质激素受体结合,促进细胞间紧密连接蛋白的表达进而降低受损血-视网膜屏障的通透性,这可能是糖皮质激素治疗黄斑水肿的机制之一七叶皂苷是中药娑罗子的主要成分,具有稳定血管内皮细胞、保护血脑屏障和增加静脉张力等作用,临床常用于治疗创伤及各种手术后水肿、视网膜静脉阻塞以及中心性渗出性脉络膜视网膜炎等。前期研究显示,七叶皂苷可上调糖皮质激素受体的表达,抑制炎症相关因子,在抗炎作用方面与糖皮质激素具有显著协同效应,但无免疫抑制作用。由此我们推测,七叶皂苷有可能通过提高糖皮质激素受体的表达,进而促进糖皮质激素对血-视网膜屏障的保护作用(协同保护作用)。如果假设成立,可为黄斑水肿的治疗提供新的思路,降低糖皮质激素的使用剂量,减少其产生的不良反应。研究目的基于玻璃体内注射糖皮质激素治疗黄斑水肿易导致眼内感染、白内障及眼内压升高等的现状,本研究拟探讨七叶皂苷(escin)与曲安奈德联合应用对血-视网膜屏障损伤的保护作用及其机制,为降低糖皮质激素的使用剂量、减少其不良反应提供实验依据。研究方法本研究分为两部分：第一部分为体内实验,研究七叶皂苷与曲安奈德联合应用对急性高眼压导致的大鼠血-视网膜屏障损伤的保护作用及其机制；第二部分为体外实验,研究七叶皂苷与曲安奈德联合应用对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)及视网膜色素上皮细胞-19(retinal pigment epithelium cells-19, ARPE-19)紧密连接的影响及其机制。 1.七叶皂苷与曲安奈德联合应用对急性高眼压导致的大鼠血-视网膜屏障损伤的保护作用及其机制。首先通过大鼠眼前房灌注乳酸钠林格氏液升高眼内压制备缺血-再灌注模型造成血-视网膜屏障损伤。然后将模型大鼠随机分为模型组、七叶皂苷小剂量组(0.9mg/kg)、七叶皂苷大剂量组(1.8mg/kg)、曲安奈德小剂量组(2μ1,40mg/ml)、曲安奈德大剂量组(5μl,40mg/ml)以及小剂量七叶皂苷和曲安奈德联合用药组,外加一假手术组作为对照。造模结束后立即给药,其中七叶皂苷尾静脉注射给药,曲安奈德玻璃体内注射给药。假手术组大鼠麻醉后,仅眼前房插针注入乳酸钠林格氏液维持正常眼压,维持时间与大鼠急性高眼压模型前后总时间一致,不做药物治疗。给药后24h,随机取6只大鼠6只眼睛,用尾静脉注射伊文思蓝(45mg/kg)的方法定量检测药物对血-视网膜屏障通透性的影响,同时取鼠眼,甲醛固定,H-E染色,用Metamorph/BX51图像分析软件测量视网膜厚度。进一步通过免疫组化及Western blot技术检测七叶皂苷与曲安奈德合用对血-视网膜屏障损伤大鼠紧密连接蛋白occludin表达的影响。 2.七叶皂苷与曲安奈德联合应用对人脐静脉内皮细胞和视网膜色素上皮细胞-19紧密连接的影响及其机制。首先在Transwell24孔培养板接种人脐静脉内皮细胞和视网膜色素上皮细胞-19,用含10%胎牛血清的DMEM培养液培养,通过细胞电阻仪定期检测电阻,判断细胞间紧密连接的形成情况。细胞紧密连接建立后,加入重组人VEGF2μl(终浓度：视网膜色素上皮细胞-1910ng/ml;人脐静脉内皮细胞100ng/ml)破坏紧密连接,同时分别给予七叶皂苷(终浓度O.1、1、10μg/ml)、曲安奈德(0.01、0.1、1μmol/L)以及七叶皂苷(0.1μg/ml)+曲安奈德(0.01μmol/L),每个浓度设3个平行孔,另设3孔(不加VEGF和药物)作为空白对照,再另设3孔(只加VEGF不加药物)作模型对照孔。加药完成后,振荡混匀,置于C02培养箱中继续培养,分别在不同时间点检测电阻,判断药物对细胞间紧密连接的影响。进一步在体外细胞屏障模型,通过Western blot技术检测七叶皂苷与曲安奈德合用对细胞紧密连接蛋白occludin、zonula occludens-1(ZO-1)以及糖皮质激素受体表达的影响。研究结果 1.七叶皂苷与曲安奈德联合应用对急性高眼压导致的大鼠血-视网膜屏障损伤的保护作用。与假手术组比较,模型组大鼠血-视网膜屏障通透性及视网膜厚度显著增加p0.01)。与模型组比较,高剂量七叶皂苷(1.8mg/kg)i曲安奈德(5μ1)均能显著抑制大鼠血-视网膜屏障通透性p0.05,p0.01)及减轻视网膜厚度p0.05),而低剂量七叶皂苷(0.9mg/kg)与曲安奈德(2μl)对大鼠血-视网膜屏障通透性和厚度未见显著影响。进一步将单用无显著效果的七叶皂苷(0.9mg/kg)与曲安奈德(2μl)联合应用,结果对模型大鼠血-视网膜屏障通透性有显著抑制作用(p0.05),显示出协同作用。 2.七叶皂苷与曲安奈德联合应用对血-视网膜屏障损伤大鼠紧密连接蛋白occludin表达的影响。与假手术组比较,模型组大鼠视网膜occludin蛋白表达显著减少(p0.01)。与模型组比较,单独使用七叶皂苷(0.9mg/kg)与曲安奈德(2μ1)对大鼠视网膜occludin蛋白表达未见显著影响,将七叶皂苷(0.9mg/kg)与曲安奈德(2μ1)联合应用,免疫组化和Western blot结果显示神经节细胞层occludin蛋白表达显著增加(p0.05),显示出协同作用。 3.七叶皂苷与曲安奈德联合应用对人脐静脉内皮细胞和视网膜色素上皮细胞-19紧密连接电阻的影响。与空白对照组比较,VEGF能显著降低人脐静脉内皮细胞和视网膜色素上皮细胞-19紧密连接的电阻(p0.05)。与模型对照组(VEGF)(?)匕较,七叶皂苷(1、10μg/ml)与曲安奈德(0.1、1μmol/L)均能显著增加人脐静脉内皮细胞和视网膜色素上皮细胞-19紧密连接的电阻(p0.05或p0.01),而七叶皂苷(0.1μg/ml)与曲安奈德(0.01μmol/L)对人脐静脉内皮细胞和视网膜色素上皮细胞-19紧密连接的电阻未见显著影响。进一步将单用无显著效果的七叶皂苷(0.1μg/ml)与曲安奈德(0.01μmol/L)联合应用,结果显示能显著增大细胞紧密连接电阻(p0.05),显示出协同作用。 4.七叶皂苷与曲安奈德联合应用对人脐静脉内皮细胞紧密连接蛋白occludin、ZO-1表达的影响。与空白对照组比较,VEGF能显著抑制人脐静脉内皮细胞紧密连接蛋白的表达(p0.05)。与模型对照组(VEGF)比较,七叶皂苷(0.1μg/ml)与曲安奈德(0.01μmol/L)对细胞紧密连接蛋白表达未见显著影响,但七叶皂苷(0.1μg/ml)与曲安奈德(0.01μmol/L)联合应用,结果能显著提高人脐静脉内皮细胞紧密连接蛋白的表达(p0.05),显示出协同作用。 5.七叶皂苷与曲安奈德联合应用对人脐静脉内皮细胞糖皮质激素受体表达的影响。与模型对照组(VEGF)比较,七叶皂苷(0.1μg/ml)与曲安奈德(0.01μmol/L)单独用对人脐静脉内皮细胞糖皮质激素受体蛋白表达未见显著影响,但将七叶皂苷(0.1μg/ml)与曲安奈德(0.01μmol/L)联合应用,结果能显著提高人脐静脉内皮细胞糖皮质激素受体蛋白的表达(p0.05)。结论 1.在大鼠高眼压造成缺血再灌注模型,静脉注射大剂量七叶皂苷能显著抑制大鼠血-视网膜屏障通透性增加,减轻缺血导致的水肿视网膜厚度。在体外细胞培养,VEGF造成人脐静脉内皮细胞和视网膜色素上皮细胞紧密连接的破坏,大剂量七叶皂苷能显著增加紧密连接电阻。表明七叶皂苷对血-视网膜屏障有保护作用,早期给予七叶皂苷治疗,可有效防止缺血缺氧性视网膜疾病引起的视网膜水肿和视力下降。 2.在缺血再灌注引起的视网膜水肿大鼠,小剂量七叶皂苷与小剂量曲安奈德单独使用对大鼠血-视网膜屏障通透性和水肿视网膜厚度没有显著影响；但当二者联合应用时则对大鼠血-视网膜屏障通透性有显著抑制作用,显示出明显的协同作用。七叶皂苷可通过放大糖皮质激素的生物效应减少糖皮质激素的用量。 3.七叶皂苷与曲安奈德联合应用对血-视网膜屏障损伤的保护具有协同作用,其机制与提高糖皮质激素受体表达,进而促进紧密连接蛋白occludin、ZO-1表达有关。
[Abstract]:Background of the study

Blood - retinal barrier ( BRB ) includes an inner barrier consisting of retinal capillary endothelial cells and their tight junctions , and an outer barrier consisting of retinal pigment epithelial cells and their tight junctions .
Because of ischemia , abnormal glucose metabolism , the release of inflammatory cytokines , vitreous humor and mechanical traction of anterior retinal membrane can cause damage to the blood - retina barrier , the edema of retinal edema and edema in macular region are known as macular edema .

The treatment methods commonly used in macular edema include : ( 1 ) laser treatment to some extent to alleviate macular edema , but in patients with diffuse macular edema , the efficacy is poor , even in some patients after treatment of macular edema ;
( 2 ) The injection of glucocorticoid ( such as Triannamedusa , TA ) and anti - vascular endothelial growth factor ( VEGF ) antibody ( such as anti - vascular endothelial growth factor , VEGF ) in the glass body has achieved good curative effect , but the half - life of the drug is short , repeated multiple injections are needed , the probability of infection in the glass body is increased , and the incidence of intraocular pressure elevation and cataract occurrence is increased ( the probability of occurrence is as high as 30 % -50 % ) .

The study shows that the tight junction between cells composed of protein di , claudin - 5 , zonula and dens - 1 is an important guarantee for maintaining the physiological function of blood - retinal barrier . The glucocorticoid receptor can be combined with glucocorticoid receptor in vivo to promote the expression of intercellular adhesion protein and reduce the permeability of damaged blood - retinal barrier , which may be one of the mechanisms of glucocorticoid in the treatment of macular edema .

Aescin is the main component of Chinese chestnut , and has the functions of stabilizing vascular endothelial cells , protecting blood brain barrier and increasing vein tension , etc . It is commonly used in the treatment of trauma and post - operative edema , retinal vein occlusion , and central inflammatory choroiditis , etc . In the previous studies , it is speculated that aescinat can increase the expression of glucocorticoid receptor and inhibit the protective effect of glucocorticoid on blood - retinal barrier ( synergistic protection ) . If it is established , it can provide new idea for the treatment of macular edema , reduce the dosage of glucocorticoid and reduce the adverse reaction .

Purpose of study

In this study , the protective effect of escin ( escin ) on blood - retinal barrier injury and its mechanism are discussed in this study , which can reduce the dosage of glucocorticoid and reduce its adverse reaction .

Research Methods

This study is divided into two parts : the first part is in vivo experiments , and the protective effect and mechanism of the combination of aescinat and qu annel on the damage of blood - retinal barrier in rats caused by acute ocular hypertension are studied .
The second part of this study was to investigate the effects of aescinat on human umbilical vein endothelial cells ( HUVEC ) and retinal pigment epithelial cells - 19 ( ARPE - 19 ) in vitro .

1 . The protective effect of aescinat combined with Quonneid on the damage of blood - retinal barrier in rats caused by acute ocular hypertension and its mechanism .

The rats were randomly divided into model group , 7 - leaf saponin low - dose group ( 0.9 mg / kg ) , high - dose group ( 1 . 8 mg / kg ) , 7 - leaf saponin group ( 1 . 8 mg / kg ) , small - dose aescinat group ( 1 . 8 mg / kg ) , small - dose aescinat group ( 5 渭l , 40 mg / ml ) , small - dose aescinat group ( 5 渭l , 40 mg / ml ) , small - dose aescinat group ( 5 渭l , 40 mg / ml ) and small - dose aescinat group ( 5 渭l , 40 mg / ml ) .

Furthermore , the effects of aescinat on the expression of closely linked proteins in rats with blood - retinal barrier injury were investigated by immunohistochemistry and Western blot .

2 . The effect and mechanism of combination of aescinat on human umbilical vein endothelial cells and retinal pigment epithelial cells - 19 in human umbilical vein endothelial cells .

Human umbilical vein endothelial cells and retinal pigment epithelial cells - 19 were inoculated in a well 24 - well culture plate . The cells were cultured in DMEM culture medium containing 10 % fetal bovine serum . After the cell - tight connection was established , the recombinant human VEGF2 渭l ( final concentration : retinal pigment epithelial cells - 1910ng / ml ; human umbilical vein endothelial cells 100ng / ml ) were added as control wells .

Further in vitro cell barrier model , the effects of aescinat ( aescinat ) on the expression of closely linked protein ( Zdin ) , zonula concerndens - 1 ( ZO - 1 ) and glucocorticoid receptor were detected by Western blot .

Results of the study

1 . The protective effect of aescinat on the damage of blood - retinal barrier in rats caused by acute ocular hypertension .

Compared with the sham operation group , the permeability of the blood - retinal barrier and the retinal thickness of the model group increased significantly ( p < 0.01 ) . Compared with the model group , the high - dose aescinat ( 1.8mg / kg ) i - ( 5 & mu ; 1 ) could significantly inhibit the rat blood - retinal barrier permeability ( p0.01 ) and reduce the retinal thickness ( p . 05 ) , while the low - dose aescinat ( 0.9mg / kg ) had no significant effect on the permeability and thickness of the rat ' s blood - retinal barrier .

The synergistic effect of aescinat ( 0.9mg / kg ) on blood - retinal barrier permeability of model rats was significantly inhibited ( p < 0.05 ) .

2 . The effect of aescinat on the expression of tight connexin protein in rats with blood - retinal barrier injury was studied .

Compared with the sham - operated group , the expression of protein in the retina of the model group was significantly decreased ( p < 0.01 ) . Compared with the model group , there was no significant effect on the expression of the retinal ganglion din protein in the rat retina by using aescinat ( 0.9mg / kg ) alone .

3 . The effect of combination of aescinat on human umbilical vein endothelial cells and retinal pigment epithelial cells - 19 in human umbilical vein endothelial cells was studied .

Compared with blank control group , VEGF could significantly decrease the resistance of human umbilical vein endothelial cells and retinal pigment epithelial cells - 19 ( p < 0.05 ) . Compared with model control group ( VEGF ) ( ? ) , aescinat ( 1 , 10 渭g / ml ) and Triannel ( 0.1 , 1 渭mol / L ) could significantly increase the resistance of human umbilical vein endothelial cells and retinal pigment epithelial cells - 19 .

The results showed that the combination of aescinat ( 0.1 渭g / ml ) with no remarkable effect was used in combination with Triad ( 0.01 渭mol / L ) . The results showed that the cell ' s tight junction resistance ( p . 05 ) could be significantly increased , and the synergistic effect was shown .

4 . The effect of combination of aescinat and batroxamine on the expression of human umbilical vein endothelial cells ( HUVEC ) in human umbilical vein endothelial cells ( HUVEC ) was studied .

Compared with the control group , VEGF could significantly inhibit the expression of closely linked protein in human umbilical vein endothelial cells ( p < 0.05 ) . Compared with the model control group ( VEGF ) , aescinat ( 0.1 渭g / ml ) had no significant effect on the expression of tight connexin ( 0.01 渭mol / L ) .

5 . Effects of combination of aescinat and batroxamine on glucocorticoid receptor expression in human umbilical vein endothelial cells .

Compared with the model control group ( VEGF ) , the expression of glucocorticoid receptor protein in human umbilical vein endothelial cells was not significantly affected by aescinat ( 0.1 渭g / ml ) alone or in combination with Triannel ( 0.01 渭mol / L ) , but the expression of glucocorticoid receptor protein in human umbilical vein endothelial cells was significantly improved ( p < 0.05 ) .

Conclusion

1 . In rats with high intraocular pressure resulting in ischemia - reperfusion model , intravenous bolus of aescinat can significantly inhibit the increase of permeability of rat ' s blood - retinal barrier and reduce the retinal thickness caused by ischemia . In vitro cell culture , VEGF results in a close connection between human umbilical vein endothelial cells and retinal pigment epithelial cells .

2 . In rats with retinal edema induced by ischemia / reperfusion , there was no significant effect on the permeability of rat blood - retinal barrier and the thickness of edema retina alone .
However , when they are used in combination , there is a significant inhibitory effect on the permeability of rat ' s blood - retinal barrier , showing a significant synergistic effect . Aescin can reduce the amount of glucocorticoid by amplifying the biological effect of glucocorticoid .

3 . The combination of aescinat and qu ' annel has a synergistic effect on the protection of blood - retinal barrier injury , and its mechanism is related to the improvement of the expression of glucocorticoid receptor in order to promote the expression of compact connexin and ZO - 1 .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774

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