Effects of Salidroside on Trabecular Meshwork Cell Extracellular Matrix Expression and Mouse Intraocular Pressure

Abstract

PURPOSE. Excessive accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) reduces aqueous humor outflow, which likely contributes to elevation of IOP in primary open-angle glaucoma (POAG). Salidroside, a phenolic glycoside isolated from Rhodiola rosea is reported to prevent profibrotic responses by inhibiting Smad signaling pathway activated by TGF-b in liver, lung, and kidney tissues. We tested if salidroside can (1) inhibit TGF-b2induced ECM expression in cultured human TM cells, and (2) lower TGF-b2-induced ocular hypertension in the mouse. METHODS. Cultured human TM cells stimulated with 5 ng/mL TGF-b2 for 48 hours were treated with salidroside for 24 hours. The expressions of fibronectin (FN), collagen type IV (COL-IV), and laminin (LN) were evaluated by quantitative PCR, Western blot, and immunocytochemistry. BALB/cJ mice were injected intravitreally with an adenoviral vector encoding a bioactive mutant of TGF-b2 (Ad.hTGF-b2 226/228) in one eye to induce ocular hypertension, with the uninjected contralateral or Ad.Empty-injected eyes serving as controls. Mice were treated with a daily intraperitoneal injection of 40 mg/kg salidroside. Conscious mouse IOP values were measured using a TonoLab rebound tonometer. RESULTS. In cultured human TM cells, treatment with TGF-b2 increased expressions of FN, COL-IV, and LN, as assessed by quantitative PCR, Western blotting, and immunocytochemistry, all of which were significantly and completely ameliorated by 30 lM salidroside. Daily intraperitoneal injections of salidroside (40 mg/kg), starting either at day 0 (same day as Ad.hTGF-b2 226/228 injection) or at day 14, significantly lowered TGF-b2-induced ocular hypertension in the mouse. In contrast, salidroside did not affect IOP of control eyes. CONCLUSIONS. These results demonstrated that salidroside is capable of minimizing TGF-b2induced ECM expression in cultured human TM cells. It also reduced TGF-b2-induced ocular hypertension in the mouse. These findings indicate that this phenolic glycoside may be useful as a novel treatment for POAG.

Osthole Reduces Mouse IOP Associated With Ameliorating Extracellular Matrix Expression of Trabecular Meshwork Cell

“…Similar to our previous findings, 27 the current study demonstrated that TGF-β2 (5 ng/mL) increased both mRNA and protein levels of fibrosis-related ECM molecules, such as FN, COL-IV, and LN, in cultured HTM cells. In this study, we further demonstrated that the TGF-β2–induced expressions of FN, COL-IV, and LN were decreased by osthole in a concentration-dependent manner between 1 and 10 µM.…”

“…Western blots were performed as previously described. 27 Briefly, 20 µg protein samples were denatured at 100°C for 10 minutes, separated on an 8% or 12% sodium dodecyl sulfate polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane. The polyvinylidene difluoride membrane was washed, blocked with 5% nonfat milk for 1 hour at room temperature, and then incubated overnight at 4°C with primary antibodies, including rabbit polyclonal FN antibody (1:1000; catalog #ab2413; Abcam, Cambridge, MA), rabbit polyclonal COL-IV antibody (1:1000; catalog #ab6586; Abcam), rabbit polyclonal LN antibody (1:1000; catalog #ab11575; Abcam), and rabbit polyclonal GAPDH antibody (1:2000; catalog #ab9485; Abcam).…”

“…Primary HTM cells were isolated from fresh human volunteer donor eyes and characterized as previously described. 27 The cells were cultured at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (Gibco, Halethorpe, MD) supplemented with glutamine, penicillin/streptomycin, and 10% fetal bovine serum (Invitrogen-Gibco, Carlsbad, CA).…”

Long-term and potent IOP-lowering effect of IκBα-siRNA in a nonhuman primate model of chronic ocular hypertension

“…No fluorescence was detected in the negative control (NC) group (secondary antibodies only) both in MCM and MTM cells ( Figure S1 B). Besides, a number of criteria, including the expression of RSPO4 (spreading throughout the TM but not surrounding tissues ( Patel et al., 2020 )), neuron-specific enolase (NSE), fibronectin (FN), and collagen type IV (Col IV) ( Fan et al., 2019 ; Guo et al., 2012 ; Mao et al., 2013 ; Patel et al., 2020 ), as well as Dex-induced increased expression of myocilin (MYOC) ( Clark et al., 2001 ; Fan et al., 2019 ; Fingert et al., 2001 ), were used to characterize isolated MTM cells. We first studied the expression RSPO4, NSE, FN, and Col IV in MTM cells by immunofluorescence.…”

Prior inhibition of AKT phosphorylation by BX795 can define a safer strategy to prevent herpes simplex virus-1 infection of the eye

“…A new tip was loaded into the tonometer for every sample group and calibrated prior to measurement. Each measurement was performed in triplicates to account for variability (43).…”