润目灵护眼液治疗苯扎氯铵诱导的干眼症及其作用机制研究

目的 探究润目灵护眼液经超声雾化机雾化后对过度蒸发性干眼症的治疗效果及作用机制。方法 16只新西兰兔随机分为空白对照组、模型组、原方组和考察组，每组4只，采用苯扎氯铵滴眼法诱导过度蒸发性兔干眼症模型，经超声雾化机给予润目灵护眼液，通过检测兔眼泪液的分泌量、泪膜的破裂时间和角膜的荧光染色，以评价润目灵组方对兔干眼症模型的干预作用。免疫荧光染色法检测兔眼结膜黏蛋白-5亚型AC(MUC5AC)的表达情况；在光学显微镜下观察兔眼角膜和结膜的病理情况；ELISA法检测兔眼泪腺和房水中TNF-α和IL-6的含量。Western blot法检测兔眼角膜中JNK/P38通路以及MMP-9和MUC5AC蛋白的表达水平，以评价润目灵护眼液对兔干眼症模型的治疗作用。结果 经HPLC测定，原方组中绿原酸、金丝桃苷、木犀草苷的浓度分别为0.123 63、0.017 25、0.095 83 mg·mL^-1。通过优化提取工艺，考察组中绿原酸、金丝桃苷、木犀草苷的含量显著提升，浓度分别为0.225 18、0.042 11、0.183 10 mg·mL^-1。与模型组相比，原方...

Investigation on Treatment of Xerophthalmia Induced by Benzalkonium Chloride with Runmuling Eye-Drops and Its Underlying Mechanism

  OBJECTIVE  To explore the therapeutic effects and mechanism of Runmuling eye-drops on hyperevaporative xerophthalmia after atomization by ultrasonic atomizer.  METHODS  Sixteen New Zealand rabbits were randomly divided into the blank control group, the model group, the original formula group, and the investigation group (n=4), and the rabbit hyperevaporative xerophthalmia model was induced by using benzalkonium chloride (BAC) eye-drops method. After treatment of Runmuling eye-drops by ultrasonic atomizer, the intervention effects of Runmuling formula on the rabbit xerophthalmia model were evaluated by measuring the amount of tear secretion, the rupture time of tear film, and the corneal fluorescence staining. The expression of mucin-5 subtype AC (MUC5AC) in conjunctival membrane was detected by immunofluorescence staining, the pathological changes of cornea and conjunctiva were observed under the light microscope, and the contents of TNF-α and IL-6 in lacrimal gland and aqueous humor were determined by ELISA. Western blot was employed to detect the expression levels of JNK/P38 pathway, MMP-9, and MUC5AC proteins in cornea for evaluating the therapeutic effects of Runmuling eye-drops on rabbit xerophthalmia model.  RESULT  The concentrations of chlorogenic acid, hyperoside, and cynaroside in the original formula group were 0.123 63, 0.017 25 and 0.095 83 mg·mL-1, respectively by the determination of HPLC. By optimizing the extraction process, the contents of chlorogenic acid, hyperoside, and cynaroside in the investigation group significantly increased and the concentrations were 0.225 18, 0.042 11 and 0.183 10 mg·mL-1, respectively. Compared to model group, the volumes of rabbit tear secretion increased (P < 0.05) and the stabilities of rabbit tear film were enhanced (P < 0.05) in the original formula and the investigation groups, and the volume of rabbit tear secretion in the investigation group was higher than that in the original formula group (P < 0.05). Compared to the model group, the expression levels of MUC5AC in the conjunctiva of rabbits increased in the original formula and the investigation groups (P < 0.05), and the conjunctival cells stained with MUC5AC antibody in the investigation group were more than those in the original formula group (P < 0.05). Compared to the model group, the epithelial cells in cornea and conjunctiva of rabbits repaired and the contents of the inflammatory factors in lacrimal gland and aqueous humor of rabbits were decreased in the original formula and the investigation groups (P < 0.01), and the expression level of IL-6 in aqueous humor of the investigation group was lower than that of the original formula group (P < 0.05). Compared to the model group, the expression levels of JNK/P38 pathway-related protein decreased (P < 0.05, P < 0.01), the expression level of MUC5AC increased (P < 0.05, P < 0.01), and the expression level of MMP-9 decreased (P < 0.05, P < 0.01) in cornea of rabbits in the original formula and the investigation groups. At the same time, compared to the original formula group, the improvement effects of P-P38/P38, P-P65/P65, MMP-9, and MUC5AC in cornea of rabbits in the investigation group were more obvious (P < 0.05).  CONCLUSION  The pathogenesis of xerophthalmia might be related to inflammatory pathways. Among them, hyperoside, luteolin, and chlorogenic acid contained in Runmuling formula could increase the amount of tear secretion, maintain the stability of tear film, and effectively ameliorate the injury of ocular surface and inhibit the expression of inflammation on ocular surface, so as to achieve the therapeutic effects of xerophthalmia.