| BackgroundBlood-retinal barrier (BRB) includes inner BRB (iBRB) constituted by retinal capillary endothelial cells with their tight junction, and ouer BRB (oBRB) constituted by retinal pigment epithelial cells and their tight junctions. In normal circumstances, BRB can prevent intravascular fluid and macromolecular substances into the retina. However, some reasons, such as ischemia, abnormal glucose metabolism, the release of inflammatory cytokines, mechanical traction of the vitreous and epiretinal membrane, can lead to BRB destruction. Thus intravascular fluid and macromolecules leak into the extracellular gap formed retinal edema, including macular edema. Macular edema can cause vision loss even irreversible damage in patients. Therefore, the early treatment of macular edema is very important.The common treatments of macular edema include:①Laser treatment can alleviate ME to some extent, but not very good for diffuse ME in patients, even more serious in some patients after treatment;②Intravitreal injection of hormones (such as triamcinolone acetonide) and anti-VEGF drugs (such as ranibizumab) show good effects, but the effects are different in patients. Furthermore, repeated injections increase intravitreal risk of infection, as well as increased intraocular pressure (the occurrence probability up to30-50%).The study showed that the close connections between cells composed by occludin, claudin-5, VE-Cad and zonula occludens-1and so on, maintain the BRB physiological function. Glucocorticoids combined with the glucocorticoid receptor, can reduce the permeability of the BRB by promoting the protein expression of tight junctions in vivo and vitro, which may be one of the mechanisms of the treatment of ME by glucocorticoid.Escin, the main ingredient of Chinese medicine Aesculi, can stabilize vascular endothelial cells, protect the blood-brain barrier and increase venous tone. In clinic, it is commonly used in the treatment of trauma and post-surgical edema, retinal vein occlusion and central exudative chorioretinopathy. The previous studies showed that escin can upregulate the expression of the glucocorticoid receptor, inhibiting of inflammation-related factors. It was also showed that escin combined with glucocorticoid had synergistic anti-inflammatory effects, but without immunosuppressive effects. Thus, we speculate that escin may amplify the protective effects of glucocorticoid on BRB (synergistic protective effect) by increasing the expression of the glucocorticoid receptor. If the hypothesis is true, the study can provide new ideas for the treatment of macular edema, including reducing the dose of glucocorticoid and adverse reactions.ObjectiveBased on the side effects of infection and increased intraocular pressure after intravitreal injection of glucocorticoid to treat macular edema, the study aimed to investigate the protective effects of triamcinolone acetonide combined with escin on BRB damage and its mechanism. The results will provide new ideas for the treatment of macular edema, including reducing the dose of glucocorticoid and adverse reactions.MethodsThe study is divided into two parts. The first part of the study:the protective effects of triamcinolone acetonide combined with escin on BRB damage caused by increasing intraocular pressure in rats and its mechanism. The second part of the study: the protective effects of triamcinolone acetonide combined with escin on BRB damage in human umbilical vein endothelial cells (HUVECs) and retinal pigment epithelial cells (ARPE-19) and its mechanism. 1. The protective effects of triamcinolone acetonide combined with escin on BRB damage caused by increasing intraocular pressure in rats and its mechanism.First of all, the BRB damage model of rats was made by increasing intraocular pressure as a model of ischemia. Then the animals were divided randomly into the following groups:the model group, triamcinolone acetonide (2μl and5μl) treatment groups, escin (0.9mg/kg and1.8mg/kg) treatment groups, and an escin (0.9mg/kg) and triamcinolone acetonide (2μl) combined treatment group. After induction of generalized anesthesia, pupils were dilated with0.5%tropicamide. The rats were treated with escin through the caudal vein or with triamcinolone acetonide through the vitreous body. A group of rats received the operation without increased intraocular pressure to serve as a sham group. Twenty-four hours after operation, the BRB permeability was assayed by intravenous injection of Evans blue (45mg/kg). The thickness of the retina was measured by Metamorph/BX51image analysis software after formalin-fixing and HE staining.Furthermore, the Occludin expression after treatment by triamcinolone acetonide combined with escin was also detected by immunohistochemistry and Western blot.2. The protective effects of triamcinolone acetonide combined with escin on BRB damage in human umbilical vein endothelial cells (HUVECs) and retinal pigment epithelial cells (ARPE-19) and its mechanism.Firstly, HUVECs and ARPE-19cells were cultured in a transwell24-well plate with DMEM medium containing10%fetal bovine serum. The formation of tight junctions between cells was detected through the cell resistance meter. When cell tight connection was established,2μl rhVEGF (final concentration:ARPE-19cells10ng/ml; HUVECs100ng/ml) was added. At the same time, escin (a final concentration0.1,1,10μg/ml), triamcinolone acetonide (0.01,0.1,1μmol/L), triamcinolone acetonide combined with escin were added too. Atl2h, the effects of drugs on cell tight connection were detected by the cell resistance meter.Furthermore, the occludin, zonula occludens-1and glucocorticoid receptor expression after treatment by triamcinolone acetonide combined with escin was also detected by Western blot. Results1. The protective effects of triamcinolone acetonide combined with escin on BRB damage caused by increasing intraocular pressure in rats.Compared with the sham rats, the model rats showed more retinal Evans Blue leakage, and the retinal thickness increased significantly (p<0.01). High doses of escin (1.8mg/kg) and triamcinolone acetonide (5μl) significantly inhibited retinal Evans Blue leakage compared with the model group (p<0.05and p<0.01, respectively). Low doses of escin (0.9mg/kg) or triamcinolone acetonide (2μl) alone did not significantly affect retinal Evans Blue leakage compared with the model group, but administered together they significantly inhibited retinal Evans Blue leakage (p<0.05).2. The effects of triamcinolone acetonide combined with escin on the occludin, ZO-1expression of retina in rats with BRB damage.Compared with the sham group, the occludin, zonula occludens-1protein expression in retinal of model rats significantly reduced (p<0.01). Low dose escin or triamcinolone acetonide alone did not enhance the occludin and zonula occludens-1expression compared with the model rats. However, when escin and triamcinolone acetonide were administered together, occludin and zonula occludens-1expression in the ganglion cell layer increased significantly in the immunohistochemistry results (p <0.05). The western blot assay also showed the parallel results.3. The protective effects of triamcinolone acetonide combined with escin on BRB damage in human umbilical vein endothelial cells (HUVECs) and retinal pigment epithelial cells (ARPE-19).Compared with the control group, rhVEGF can significantly increase the permeability of tight junctions of HUVECs and ARPE-19cells (p<0.05). Escin (1,10μg/ml) and triamcinolone acetonide (0.1,1μM) can significantly decrease the permeability (p<0.05, p<0.01); however, escin (0.1μg/ml) and triamcinolone acetonide (0.01μmol/L) had no significant impact on the permeability of tight junctions, but administered together they significantly decreased the permeability of cell tight junction (p<0.05). 4. The effects of triamcinolone acetonide combined with escin on the occludin, zonula occludens-1expression of human umbilical vein endothelial cells (HUVECs).Compared with the control group, the occludin, zonula occludens-1protein expression treated with rhVEGF in HUVECs significantly reduced (p<0.05). Low dose escin or triamcinolone acetonide alone did not enhance the occludin and ZO-1expression compared with the rhVEGF group. However, when escin and triamcinolone acetonide were administered together, occludin and zonula occludens-1expression increased significantly (p<0.05).5. The effects of triamcinolone acetonide combined with escin on glucocorticoid receptor expression of human umbilical vein endothelial cells (HUVECs).Low dose escin or triamcinolone acetonide alone did not enhance the glucocorticoid receptor expression compared with the rhVEGF group. However, when escin and triamcinolone acetonide were administered together, glucocorticoid receptor expression increased significantly (p<0.05).ConclusionTriamcinolone acetonide combined with escin shows synergistic protective effects on the BRB damage, and the mechanism may attribute to increasing glucocorticoid receptor expression, then increasing the expression of tight junction protein including both occludin and zonula occludens-1. |
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