Salidroside inhibits extracellular matrix deposition and oxidative stress in human trabecular meshwork cells by suppressing the TNF signaling pathway and upregulating MMP3 expression.

BACKGROUND: The functional integrity of human trabecular meshwork cells (HTMCs) is essential for regulating intraocular pressure (IOP). In primary open-angle glaucoma (POAG), pathological stimuli induce oxidative damage and promote excessive extracellular matrix (ECM) deposition in HTMCs, thereby hindering aqueous humor outflow. Salidroside (Sal), a compound with diverse pharmacological activities, has been reported to inhibit oxidative stress and reduce ECM accumulation. This study aimed to investigate whether Sal exerts its antioxidant effects by regulating matrix metalloproteinase 3 (MMP3) expression via the TNF signaling pathway.

METHODS: Potential targets of salidroside and glaucoma were predicted using SwissTargetPrediction and GeneCards online databases, and overlapping targets were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on these intersecting targets. Meanwhile, the glaucoma GEO dataset (GSE27276) was downloaded for differential gene expression analysis, and the results were further compared with the intersecting targets to identify key genes. An oxidative damage model of HTMCs was established using hydrogen peroxide (H₂O₂) induction. Cell viability was assessed using the MTT assay to determine the appropriate H₂O₂ concentration for modeling, as well as the safe dosage and treatment duration of Sal. Oxidative stress markers were measured as follows: superoxide dismutase (SOD) and malondialdehyde (MDA) levels were determined by enzyme-linked immunosorbent assay (ELISA), while reactive oxygen species (ROS) levels were assessed using DCFH-DA staining. Furthermore, the expression of ECM proteins such as collagen type I (COL-I), fibronectin (FN), and laminin (LN), as well as MMP3 and TNF signaling pathway-related proteins, was detected by Western blot and immunofluorescence (IF). Rescue experiments were performed using the TNF inhibitor R7050 and the broad-spectrum MMP inhibitor GM6001. In addition, a rat glaucoma model was established by injecting microbeads into the anterior chamber of SD rats. Intraocular pressure changes were monitored using a TonoLab tonometer. Retinal tissue morphology was observed by hematoxylin-eosin (H&E) staining. Retinal ganglion cell apoptosis was detected by TUNEL assay. The expression of oxidative stress markers and ECM-related proteins in the retina and serum was measured by Western blot and ELISA.

RESULTS: A total of 68 intersecting targets were identified from the database analyses. Enrichment analysis indicated that these targets were primarily involved in biological processes such as oxidative stress and ECM regulation, as well as the TNF signaling pathway. Experimental results further demonstrated that Sal significantly increased the viability of H₂O₂-damaged HTMCs and effectively inhibited intracellular oxidative stress and ECM protein expression. Moreover, the TNF signaling pathway inhibitor R7050 enhanced the inhibitory effects of Sal on oxidative stress and ECM deposition in HTMCs. Screening of the GEO database identified MMP3 as a key gene. Western blot results showed that Sal upregulated MMP3 expression in H₂O₂-treated HTMCs. Furthermore, the MMP3 inhibitor GM6001 reversed the protective effects of Sal against oxidative stress and ECM deposition. Interestingly, in the presence of Sal, co-treatment with R7050 and GM6001 revealed that GM6001 reversed the effects of R7050 on oxidative stress and ECM, suggesting that Sal may act through the TNF/MMP3 axis. In vivo experiments further confirmed that Sal effectively reduced IOP in glaucomatous rats, alleviated retinal pathology, and significantly decreased oxidative stress levels and ECM deposition.

CONCLUSION: Sal, both in vitro and in vivo, promotes MMP3 expression by inhibiting the activation of the TNF signaling pathway, thereby suppressing ECM deposition and mitigating oxidative damage in trabecular meshwork cells.