Investigation on Treatment of Xerophthalmia Induced by Benzalkonium Chloride with Runmuling Eye-Drops and Its Underlying Mechanism
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摘要:
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，原方组与考察组兔眼泪液的分泌量增加(P < 0.05)，兔眼泪膜的稳定性增强(P < 0.05)，且考察组泪液分泌量高于原方组(P < 0.05)；与模型组相比，原方组与考察组兔眼结膜中的MUC5AC表达水平升高(P < 0.05)，且考察组中被MUC5AC抗体染色的结膜细胞多于原方组(P < 0.05)；与模型组相比，原方组与考察组中兔眼角膜及结膜中的上皮细胞得到修复，兔眼泪腺和房水中的炎症因子含量降低(P < 0.01)，且考察组房水中IL-6的表达水平低于原方组(P < 0.05)；与模型组相比，原方组与考察组兔眼角膜中的JNK/P38通路相关蛋白表达水平降低(P < 0.05，P＜0.01)、MUC5AC表达水平升高(P < 0.05, P < 0.01)以及MMP-9表达水平降低(P < 0.05，P < 0.01)。同时，与原方组相比，考察组兔眼角膜中的P-P38/P38、P-P65/P65、MMP-9以及MUC5AC的改善效果更加明显(P < 0.05)。 结论 干眼症的发病机制可能与炎症通路具有相关性, 其中润目灵方中含有的金丝桃苷、木犀草苷以及绿原酸等成分可以增加兔眼泪液的分泌量, 维持泪膜的稳定性, 并有效改善眼表的损伤和抑制眼表炎症的表达, 从而达到治疗干眼症的效果。 Abstract: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. -
Key words:
- xerophthalmia /
- Runmuling eye-drops /
- inflammatory reaction /
- mucin /
- inflammatory cytokines
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图 1 润目灵组方对兔眼泪液分泌量的影响
注: 与空白对照组相比, ΔP < 0.05;与模型组相比, *P < 0.05, * *P < 0.01;与原方组相比, #P < 0.05, ##P < 0.01。x±s, n=4。
Figure 1. Effects of Runmuling formula on tear secretion in rabbit eyes
图 2 润目灵组方对干眼症模型兔眼泪膜稳定性的影响
注: 与空白对照组相比, ΔP < 0.05;与模型组相比, *P < 0.05。x±s, n=4。
Figure 2. Effects of Runmuling formula on tear film stability in rabbit xerophthalmia model
图 3 造模第14天荧光素钠试纸染色不同程度的眼表损伤
Figure 3. Varying degrees of ocular surface damage after staining with sodium fluorescein test strip at d14 of modeling
图 4 润目灵组方对干眼症模型兔眼角膜荧光染色评分的影响
注: 与空白对照组相比, ΔP < 0.05, ΔΔP < 0.01;与模型组相比, *P < 0.05, * *P < 0.01。x±s, n=4。
Figure 4. Effects of Runmuling formula on scores of corneal fluorescence staining in rabbit xerophthalmia model
图 5 润目灵组方对干眼症模型兔眼结膜中MUC5AC表达水平的影响
注: 与空白对照组相比, ΔΔP < 0.01;与模型组相比, *P < 0.05, * *P < 0.01;与原方组相比, #P < 0.05。x±s, n=4。
Figure 5. Effects of Runmuling Formula on expression levels of conjunctival MUC5AC in rabbit xerophthalmia model
图 6 润目灵组方对干眼症模型兔眼角膜、球结膜和睑结膜的影响
Figure 6. Effects of Runmuling formula on cornea, bulbar conjunctiva and palpebral conjunctiva in rabbit xerophthalmia model
图 7 润目灵组方对干眼症模型兔眼泪腺、房水及哈氏腺中TNF-α及IL-6表达水平的影响
注: 与空白对照组相比，△△P < 0.01；与模型组相比, *P < 0.05, * *P < 0.01;与原方组相比, #P < 0.05。x±s, n=4。
Figure 7. Effects of Runmuling formula on expression levels of TNF-α and IL-6 in lacrimal gland, aqueous humor, and Harderian gland in rabbit xerophthalmia model
图 8 润目灵组方对干眼症模型兔眼角膜中JNK/P38通路相关蛋白表达水平的影响
注: C.空白对照组；M.模型组；Y.原方组；K.考察组；与空白对照组相比, ΔΔΔP < 0.001; 与模型组相比, *P < 0.05, * *P < 0.01;与原方组相比, #P < 0.05。x±s, n=4。
Figure 8. Effects of Runmuling formula on expression levels of JNK/P38 pathway related proteins in cornea in rabbit xerophthalmia model
图 9 润目灵组方对干眼症模型兔眼角膜中MMP-9和MUC5AC蛋白表达水平的影响
注: C.空白对照组；M.模型组；Y.原方组；K.考察组；与空白对照组相比, ΔΔP < 0.01, ΔΔΔP < 0.001; 与模型组相比, *P < 0.05, * *P < 0.01;与原方组相比, #P < 0.05。x±s, n=4。
Figure 9. Effects of Runmuling formula on expression levels of MMP-9 and MUC5AC proteins in cornea in rabbit xerophthalmia model
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[1] The definition and classification of dry eye disease: Report of the definition and classification subcommittee of the international dry eye workshop (2007)[J]. Ocular Surf, 2007, 5(2): 75-92. [2] SONG PG, XIA W, WANG ML, et al. Variations of dry eye disease prevalence by age, sex and geographic characteristics in China: A systematic review and meta-analysis[J]. J Glob Heal, 2018, 8(2): 020503. doi: 10.7189/jogh.08.020503 [3] JIE Y, XU L, WU YY, et al. Prevalence of dry eye among adult Chinese in the Beijing Eye Study[J]. Eye, 2009, 23(3): 688-693. doi: 10.1038/sj.eye.6703101 [4] MA BK, ZHOU YF, LIU RJ, et al. Pigment epithelium-derived factor (PEDF) plays anti-inflammatory roles in the pathogenesis of dry eye disease[J]. Ocular Surf, 2021, 20: 70-85. doi: 10.1016/j.jtos.2020.12.007 [5] HUANG JJ. Runmuling granules treatment clinical study of dry eye[D]. Nanjing: Nanjing University of Chinese Medicine, 2010. [6] SHI N, GUO HJ, WANG H, et al. Effects of hyperin on mice with acute inflammatory pain[J]. Drug Eval Res, 2016, 39(5): 768-771. [7] LI HX, ZHANG Q, LIU YN, et al. Comparative study on anti-inflammatory activity of luteolin and luteoloside[J]. J Yantai Univ Nat Sci Eng Ed, 2018, 31(2): 114-120. [8] HAI Y. Research on the synergistic anti-inflammatory effect and dosage optimization of chlorogenic acid and quercetin[D]. Xi'an: Northwest University, 2021. [9] WANG QH, DU TT, ZHANG ZH, et al. Advances in research on the pharmacological effects and mechanism of action of chlorogenic acid[J]. Acta Pharm Sin, 2020, 55(10): 2273-2280. [10] XIONG CJ, CHEN D, LIU JB, et al. A rabbit dry eye model induced by topical medication of a preservative benzalkonium chloride[J]. Invest Ophthalmol Vis Sci, 2008, 49(5): 1850. doi: 10.1167/iovs.07-0720 [11] XU YH. Runmuling atomization treatment clinical study of dry eye[D]. Nanjing: Nanjing University of Chinese Medicine, 2009. [12] LEMP M. Report of the national eye institute/industry workshop on clinical trials in dry eyes[J]. CLAO J, 1995, 21(4): 221-232. [13] MARKOULLI M, HUI A. Emerging targets of inflammation and tear secretion in dry eye disease[J]. Drug Discov Today, 2019, 24(8): 1427-1432. doi: 10.1016/j.drudis.2019.02.006 [14] BAUDOUIN C, ROLANDO M, BENITEZ DEL CASTILLO JM, et al. Reconsidering the central role of mucins in dry eye and ocular surface diseases[J]. Prog Retin Eye Res, 2019, 71: 68-87. doi: 10.1016/j.preteyeres.2018.11.007 [15] LI H. Anti-inflammation and anti-inflammatory/antioxidant linkage mechanism of flavonoids based on NF-κB signaling pathway[D]. Wuhan: Huazhong Agricultural University, 2019. [16] JIN XN, GAO W, FENG X, et al. Therapeutic effect of Hyperoside on mice with collagen-induced arthritis[J]. J Pract Med, 2021, 37(17): 2199-2203. [17] QIU Y. Studies on anti-inflammation effects and molecular mechanism of chlorogenic acid extracted from Lonicera hypoglauca miq[D]. Nanning: Guangxi Medical University, 2009. [18] BARABINO S, CHEN W, DANA MR. Tear film and ocular surface tests in animal models of dry eye: Uses and limitations[J]. Exp Eye Res, 2004, 79(5): 613-621. doi: 10.1016/j.exer.2004.07.002 [19] WARD KW. Superficial punctate fluorescein staining of the ocular surface[J]. Optom Vis Sci, 2008, 85(1): 8-16. doi: 10.1097/OPX.0b013e31815ed756 [20] MA J, RUBIN BK, VOYNOW JA. Mucins, mucus, and goblet cells[J]. Chest, 2018, 154(1): 169-176. -
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