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.
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