去甲斑蝥素对实验性过敏性结膜炎眼部炎症及Th1/Th2平衡的影响及机制

doi:10.3969/j.issn.1006-6233.2022.04.06

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摘要 目的:探究去甲斑蝥素(norcantharidin,NCTD)对实验性过敏性结膜炎(allergic conjunctivitis,AC)大鼠眼部炎症及Th1/Th2平衡的调节作用及其机制。方法:采用卵清蛋白(OVA)法建立SD大鼠AC模型,将大鼠随机分为对照组、模型组、NCTD低剂量组和NCTD高剂量组,除对照组外,其余各组均诱导AC模型,NCTD低、高剂量组分别按0.05mg·kg^-1·d^-1、0.1mg·kg^-1·d^-1给予NCTD灌胃,连续10d;大鼠OVA末次致敏后,观察眼部体征表现并评分,苏木精-伊红(HE)染色和高碘酸‐希夫(PAS)染色分别观察角膜与结膜组织病理学变化,酶联免疫吸附法(ELISA)检测血清中细胞因子IL-2、IL-4、IL-5、IL-10及IFN-γ的含量,流式细胞术检测脾脏淋巴细胞内Th1、Th2细胞比例,实时荧光定量PCR检测结膜组织IL-4、IFN-γ mRNA表达水平,免疫荧光染色检测角膜组织VEGF表达,蛋白质印迹(Western Blot)实验检测结膜组织VEGF、VEGFR2蛋白表达水平。结果:大鼠造模后出现结膜充血红肿,眼裂变小、流泪、分泌物增多等AC病理现象,炎症评分高于对照组(P<0.01),说明AC动物模型构建成功;经过NCTD低剂量组和NCTD高剂量组处理的大鼠,结膜充血红肿的现象明显缓解,流泪、分泌物均减少,炎症评分下降,角膜上皮细胞排列较为紧密,新生血管减少,结膜中杯状细胞数目有所增加,血清中IL-2、IFN-γ含量升高,IL-4、IL-5及IL-10含量降低,脾脏中Th1细胞比例和Th1/Th2比值均增加,Th2细胞比例则减少,结膜组织中IFN-γ mRNA表达上调、IL-4 mRNA表达下调(P均<0.01);同时,角膜组织中VEGF阳性表达率减少,结膜组织中VEGF、VEGFR2蛋白表达均下调(P均<0.01);此外,NCTD高剂量的作用效果优于NCTD低剂量(P均<0.01)。结论:去甲斑蝥素能够缓解实验性过敏性结膜炎大鼠的病变进程,改善Th1/Th2细胞免疫失衡,其机制可能与抑制VEGF途径相关。

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	庄海容
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关键词 ：过敏性结膜炎, 去甲斑蝥素, Th1/Th2平衡, 血管内皮生长因子

Abstract：Objective: To explore the regulating effect and mechanism of norcantharidin (NCTD) on ocular inflammation and Th1/Th2 balance in experimental allergic conjunctivitis (AC) rats. Methods: The SD rat AC model was established by the ovalbumin (OVA) method. The rats were randomly divided into control group, model group, NCTD low-dose group and NCTD high-dose group. Except for the control group, all other groups were induced with AC model, the NCTD low-dose and high-dose groups were given NCTD by gavage at 0. 05 mg·kg^-1·d^-1 and 0.1 mg·kg^-1·d^-1 respectively for 10 days; After the last sensitization of OVA, the rats were observed and scored for ocular signs, hematoxylin-eosin (HE) staining and periodate-Schiff (PAS) staining for histopathological changes in the cornea and conjunctiva, respectively, and enzyme-linked immunosorbent assay (ELISA) for the determination of cytokines IL-2, IL-4, IL-5, IL-10 and IFN-γ in the serum. Flow cytometry was performed to detect the ratio of Th1 and Th2 cells in spleen lymphocytes, real-time fluorescence PCR was performed to detect IL-4 and IFN-γ mRNA expression in conjunctival tissue, immunofluorescence staining was performed to detect VEGF expression in corneal tissue, and western blotting was performed to detect VEGF and VEGFR2 protein expression in conjunctival tissue. Results: After the rats were modeled, conjunctival hyperemia, redness, swelling, ocular fission, tearing, increased secretions and other AC pathological phenomena appeared. The inflammation score was higher than that of the control group (P<0.01), indicating that the AC animal model was successfully constructed. In the rats treated with the NCTD low-dose group and the NCTD high-dose group, the conjunctival congestion and redness were significantly relieved, tearing and secretion were reduced, the inflammation score decreased, the corneal epithelial cells were tightly arranged, neovascularization was reduced, and the numberthe of goblet cells in the conjunctiva increased, the serum levels of IL-2, IFN-γ increase, the levels of IL-4, IL-5 and IL-10 decrease, the ratio of Th1 cells and Th1/Th2 in the spleen both increased, and the ratio of Th2 cells decreaseed. The expression of IFN-γ mRNA in the conjunctival tissue was up-regulated, and the expression of IL-4 mRNA was down-regulated (all P＜0.01); at the same time, the positive expression rate of VEGF in corneal tissue decreased, and the expression of VEGF and VEGFR2 protein in conjunctival tissue were all down-regulated (all P<0.01). In addition, the effect of the high dose of NCTD was better than that of the low dose of NCTD (all P<0.01). Conclusion: Norcantharidin can alleviate the pathological process of experimental allergic conjunctivitis rats and improve Th1/Th2 cell immune imbalance by a mechanism that may be related to the inhibition of the VEGF pathway.

Key words： Allergic conjunctivitis Norcantharidin Th1/Th2 balance Vascular endothelial growth factor

基金资助:海南省卫生健康行业科研项目,(编号:19A200053)

引用本文:

庄海容, 吴子东, 陈雪红, 李成军. 去甲斑蝥素对实验性过敏性结膜炎眼部炎症及Th1/Th2平衡的影响及机制[J]. 河北医学, 2022, 28(4): 554-560.
ZHUANG Hairong, WU Zidong, CHEN Xuehong, et al. A Preliminary Investigation on the Effect of Norcantharidin on Ocular Inflammation and Th1/Th2 Balance in Experimental Allergic Conjunctivitis and Its Mechanism. HeBei Med, 2022, 28(4): 554-560.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2022.04.06 或 http://www.hbyxzzs.cn/CN/Y2022/V28/I4/554

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