Inhibitory effect of SIS3 on trabecular meshwork fibrosis in mice with glucocorticoid-induced ocular hypertension and its mechanism

Objective  To explore the effect of specific inhibitor of Smad3 (SIS3) on glucocorticoid-induced ocular hypertension in mice and its possible mechanism.

Methods  Fifty-one eight-week-old female C57BL/6J mice were randomly divided into control group, dexamethasone group and SIS3 group by the random number table method, with 17 mice in each group.Mice in the control group were injected with 20 μl 2 % polyvinyl alcohol into the conjunctival fornix every week for 4 weeks.Mice in the dexamethasone group and SIS3 group were injected with 20 μl 10 mg/ml dexamethasone acetate every week and SIS3 group was treated with additional 100 μg/ml SIS3 nanomicelle eye drops 3 times daily for up to 4 weeks.Intraocular pressure (IOP) was measured weekly using Icare rebound tonometer.Mice were sacrificed 4 weeks after treatment, and the eyeballs were removed.Morphology of trabecular meshwork (TM) tissues were detected by hematoxylin-eosin (HE) staining.The collagen deposition area in TM tissues were examined by Masson staining.Fibronectin (FN) and collagen type Ⅰ (Col-1) in the extracellular matrix of TM tissue were detected by immunofluorescence staining.TM tissues were obtained from donated patients, and primary human trabecular meshwork cells (HTMCs) were obtained by culture.The expression level of myocilin in dexamethasone-induced HTMCs was detected by immunofluorescence and Western blot for cell identification.Primary HTMCs were divided into normal control group, dexamethasone group and SIS3 group cultured with normal culture medium, medium containing 400 nmol/L dexamethasone, medium containing 400 nmol/L dexamethasone+ 10 μmol/L SIS3 for 48 hours, respectively.The expression levels of FN, Col-1 and p-Smad3/Smad3 proteins were measured by Western blot.The use and care of animals complied with the ARVO statement.This study protocol was approved by the Animal Ethics Committee of Zhengzhou University (No.ZZU-LA20220729).The collection of TM tissue specimens complied with the Declaration of Helsinki and was approved by the Medical Ethics Committee of Henan Provincial Eye Hospital (No.HNEECKY-2022[18]).The patients knew the purpose of the experiment and signed the informed consent forms.

Results  There was a significant overall difference in IOP among the three groups at different time points after administration ( F _group=72.94, P<0.001; F _time=33.19, P<0.001).Compared with baseline, IOP was increased in the dexamethasone group at each time point after administration, and the differences were statistically significant (all P<0.001).The IOP of the control and SIS3 groups at weeks 1, 2, 3, 4 were significantly lower than that of the dexamethasone group (all P<0.001).HE staining showed that the iridocorneal angles of all groups were open with similar morphology of the TM structure.Masson staining showed that the positive expression area of collagen in the control group, dexamethasone group and SIS3 group was (9.57±2.91)%, (27.75±5.88)% and (11.67±3.78)%, respectively, with a statistically significant difference among the three groups ( F=25.91, P<0.001), and the positive expression area of collagen was significantly lower in the control group and SIS3 group than in the dexamethasone group (all P<0.001).The fluorescence expression level of FN in the control group, dexamethasone group and SIS3 group was 8.00±1.92, 14.01±2.74 and 7.85±0.64, respectively, and the fluorescence expression level of Col-1 was 6.90±1.16, 14.36±3.19 and 4.90±0.88, respectively, with statistically significant differences among the three groups ( F=15.93, 30.29; both P<0.001), and the fluorescence expression levels of FN and Col-1 were significantly lower in the control group and SIS3 group than in the dexamethasone group (all P<0.01).Immunofluorescence staining and Western blot showed that the cultured primary cells expressed myocilin and the expression level of myocilin was significantly increased after dexamethasone induction, which was identified as HTMCs.There were statistically significant differences in the relative expression levels of FN, Col-1, and p-Smad3/Smad3 proteins among different groups of cells ( F=8.22, 23.08, 8.78; all P<0.05), and the relative expression levels of FN, Col-1, and p-Smad3/Smad3 proteins were significantly lower in the control group and SIS3 group than in the dexamethasone group (all P<0.05).

Conclusions  SIS3 reduces IOP by inhibiting p-Smad3, reducing extracellular matrix deposition in TM, and reducing fibrosis in the TM tissue.

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