Protective And Inflammation-related Factors Regulation Research Of Tibetan Medicine Jikan Mingmu Drops On Diabetic Dry Eye Syndrome In Mice

ObjectiveJikan Mingmu drops(JMD),a Tibetan medicine recorded in the Tibetan medical classics Four Medical Tantras,can improve eye inflammation and vision related symptoms.It is commonly used in Tibetan medicine to treat diabetes mellitus(DM)-induced dry eye syndrome(DES).However,the protective mechanism and molecular mechanism of JMD on DM-DES are still unclear.Therefore,the aim of this study was to investigate whether JMD can ameliorate DM-DES by inhibiting inflammatory related factors and regulating mitogen-activated protein kinase(MAPK)signaling pathway.MethodsFifty SPF-grade 8-week-old db/db mice were male.The mice were randomly fed in separate cages and fed water freely for one week.The activity and drinking water intake of the mice were recorded.One week later,DES was induced in 40 of the mice by topical administration of 5 ?L of 0.2% benzalkonium chloride to both eyes twice a day for 7 days.The behavioral activities,water intake and fundus changes of each mouse were observed.Tear produce and corneal fluorescein sodium staining score(? 12)were used to determine whether the DM-DES model was successfully established.Based on the clinical evaluations(described below),the mice with dry eye were then randomly divided into five groups(n=8 per group),namely,a model group,a dextran and hypromellose eye drops(DHED)group,and low-,middle-,and high-dose JMD(0.25,0.5,and 1.0 g/mL,respectively)groups.The latter three groups were treated with 5 ?L of JMD three times daily for another 7 d.The other eight db/db mice were used as the control group and the model group received the identical volume of vehicle instead of JMD.Two hours after the last administration,mice were executed by necking and eye tissues were collected.1.Measurement of Tear Volume: Tear production of mice was measured using the phenol red thread tear test with cotton threads at a similar time(5 p.m.)on days 1,3,5,and 7.2.Fluorescein Staining: Corneal fluorescein staining scores were measured using slit-lamp biomicroscopy with a cobalt blue filter on day 7 of administration of JMD 3.Conjunctival histopathological examination: Hematoxylin and eosin staining(HE)was used to detect conjunctival tissue damage in mice;periodic acid-Schiff staining(PAS)was used to detect the morphological and quantitative changes of goblet cells in conjunctival tissue of mice.4.Detection of inflammatory factors in conjunctival tissue: Biochemical method(Elisa)was used to detect the levels of interleukin(IL)-6,IL-17?,IL-1?,tumor necrosis factor-alpha(TNF-?)and vascular endothelial growth factor(VEGF)in conjunctiva of mice.5.The levels of MAPK signaling pathway proteins in conjunctiva: Western Blot performed to assess the levels of p38,p-p38,ERK,p-ERK,JNK and p-JNK proteins associated with the MAPK signaling pathway in conjunctiva.6.Preliminary study on the material basis of JMD: The main chemical constituents of JMD were analyzed by high-performance liquid chromatography and gas chromatography–mass spectrometry.Results 1.A total of 40 mice,8 in each group,met the clinical criteria for dry eye.2.The tear volume of JMD low-dose group and middle-dose group increased on the 3rd day after administration,but the data were not statistically significant(P > 0.05)compared with the model group.The JMD high-dose group had statistical significance(p < 0.05).After 7 days of administration,the tear production of JMD low-,middle-and high-dose group increased significantly,with statistical significance(p < 0.05).3.The corneal fluorescent staining scores of low-,middle-and high-dose JMD groups decreased significantly(p < 0.05)after 7 days of JMD administration compared with the model group.4.The mucosal epithelial cells were arranged in an orderly manner in a single layer with uniform polarity in the control group.The cell morphology and structure were normal.At the same time,the submucosal tissue was neatly arranged,the structure was dense,and no inflammatory cell infiltration was observed.In contrast,the mucosal layer was thicker and the number of epithelial cells was slightly increased in the model group.Moreover,the number of submucosal cells was increased and they exhibited an irregular arrangement,and the submucosal tissue was wider and the nuclei were constricted with some degree of cell necrosis.All of the histopathological changes of the conjunctival epithelial cells observed in the model group as described above were significantly restored in the JMD treatment groups.In addition,PAS staining showed that the number of conjunctival goblet cells increased in JMD middle-and high-dose groups.5.JMD groups could significantly reduce the expression levels of IL-6,IL-17?,IL-1?,TNF-?,and VEGF inflammatory factors in conjunctiva(p < 0.05)compared with the model group.6.Compared with model group,JMD(0.5,and 1.0 g/mL)could significantly reduce the expression levels of p-p38 and p-JNK(p < 0.05 or p < 0.01);Additionally,JMD groups could decrease the expression levels of p-ERK(p > 0.05).7.The concentrations of gallic acid,hydroxysafflor yellow A,magnoflorine,ellagic acid,jatrorrhizine hydrochloride,palmatine hydrochloride,berberine hydrochloride,and tauroursodeoxycholic acid in JMD were determined by HPLC;A total of five compounds in JMD were detected by GC-MS,namely,camphor,isoborneol,borneol,trans-cinnamic acid,and muscone.Conclusions 1.A stable DM-DES model was established in db/db mice by topical administration of 0.2% benzalkonium chloride for 7 days.2.Treatment efficacy of JMD on DES in DM mice showed in a dose-dependent manner.3.JMD treatment could ameliorate the inflammatory response in DES in mice with diabetes mellitus and the mechanism may involve inhibition of the MAPK signaling pathway.