The effects of trans-resveratrol on transforming growth factor-β2 signalling pathways via jnk and p38 on fibronectin deposition in steroid-treated human trabecular meshwork cells

Glaucoma is the leading cause of irreversible blindness. Currently available treatments mainly target the reduction of intraocular pressure (IOP). Increased extracellular matrix (ECM) deposition especially fibronectin (FN) in trabecular meshwork (TM) contributes to aqueous humour (AH) outflow resistance, causing elevated IOP. Transforming growth factor-beta (TGF-β) is a cytokine implicated in the pathophysiology of glaucoma and its TGF-β2 isoform has a more significant association. It enhances ECM deposition in the TM tissue via connective tissue growth factor (CTGF) and the signalling may involve canonical, the SMAD-dependent and/or non-canonical pathways, via mitogen-activated protein kinase (MAPK) such as JNK and p38. Steroid- induced glaucoma, a common type of secondary glaucoma, is also associated with elevated IOP and TM changes similar to those observed in the most common glaucoma subtype, primary open angle glaucoma (POAG). Trans-resveratrol (TR) is a polyphenolic compound that has previously been shown to lower IOP in steroid- induced ocular hypertensive rat model. TR has been shown to minimally affect the canonical TGF-β2 signalling pathway in primary human trabecular meshwork cells (HTMCs). Therefore, this study aims to explore the involvement of non-canonical TGF- β2 signalling via JNK and p38 MAPKs leading to reduction of FN deposition by TR in dexamethasone-treated primary HTMCs. Primary HTMCs were incubated with dexamethasone (100 nM) with and without TR (12.5 µM). The gene and protein expressions of TGF-β2, JNK1, JNK2, p38, CTGF and FN were determined using RT- qPCR and ELISA after 3- and 7-days of treatment, respectively. The active TGF-β2, phosphorylated JNK1 and phosphorylated p38 were also measured using ELISA after 7 days. It was observed that TR downregulates dexamethasone-induced increase in the expression of TGF-β2 gene, and both total and active TGF-β2 proteins. Treatment with dexamethasone-only increased the phosphorylated JNK1 and p38, however it had insignificant effect on the gene and protein expressions of JNK1, JNK2 and p38. TR alone and TR co-treatment with dexamethasone had insignificant effect on JNK1 gene and protein expression and JNK2 gene expression when compared to both dexamethasone-only treated group and control groups. TR however when given alone and with dexamethasone reduced the JNK2 protein expression significantly when compared to dexamethasone-only group and control groups. TR co-treatment with dexamethasone increased JNK1 phosphorylation when compared to the control group and reduced the p38 expression and phosphorylation in comparison to dexamethasone- only group. TR co-treatment with dexamethasone also suppressed both CTGF and FN expression induced by dexamethasone treatment. This current study demonstrated that TR suppressed TGF-β2 pathway leading to reduction in CTGF and FN levels with involvement of p38 MAPK but not JNK. Further investigations are warranted to fully understand the mechanism of action of TR underlying reduction of ECM deposition and its potential use as an antiglaucoma agent.