Login Register Cart 0
Generic placeholder image

Current Molecular Medicine

Editor-in-Chief

ISSN (Print): 1566-5240
ISSN (Online): 1875-5666

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

siRNA Mediated Downregulation of RhoA Expression Reduces Oxidative Induced Apoptosis in Retinal Ganglion Cells

Author(s): Qian Liu, Changgeng, Liu and Bo Lei*

Volume 24, Issue 5, 2024

Published on: 22 June, 2023

Page: [630 - 636] Pages: 7

DOI: 10.2174/1566524023666230511095628

Price: $65

Abstract

Backgrounds: Glaucoma is the second leading cause of blindness. Apoptosis of retinal ganglion cells (RGCs) is an important mechanism of glaucomatous optic injury. Rho kinase expression is significantly increased in apoptotic RGCs. This study aimed to investigate the role of RhoA, a Rho GTPase, on the survival of RGCs and further to explore its potential therapeutic applications.

Methods: RGCs were treated with siRhoA for 24 hours in vitro. Knockdown of RhoA was confirmed with quantitative RT-PCR. Oxidative stress was induced by treating the RGCs with 200 μM of H2O2 for 1 hour, and apoptosis of RGCs was quantified with TUNEL assay in situ, and with flow cytometry. The mRNA expression levels of RhoA, Nogo receptor, caspase 3 and Bcl-2 were evaluated by quantitative RT-PCR, and the protein levels of RhoA, ROCK1, ROCK2, Nogo receptor, caspase 3 and Bcl-2 were evaluated by Western blot. We found siRhoA treatment efficiently downregulated the expression of RhoA in RGCs and protected against H2O2-induced injury in RGCs in vitro. Apoptosis of RGC cells under oxidative stress was quantified in situ using TUNEL assay and confirmed with flow cytometry (FCM).

Results: With the knockdown of RhoA, the expression of ROCK1, ROCK2, Nogo Receptor, Casepase-3 were decreased, while the expression of Bcl-2 was increased in both mRNA and protein level. Our data indicated that siRhoA prevented H2O2-induced apoptosis in RGC cells by modulating the RhoA/ROCK pathway.

Conclusion: The results suggested that siRhoA may exert potentially effective neuroprotection for RGCs by reducing injury.

« Previous Next »
[1]
Kingman S. Glaucoma is second leading cause of blindness globally. Bull World Health Organ 2004; 82(11): 887-8.
[PMID: 15640929]
[2]
Hysi PG, Cheng CY, Springelkamp H, et al. Genome-wide analysis of multi-ancestry cohorts identifies new loci influencing intraocular pressure and susceptibility to glaucoma. Nat Genet 2014; 46(10): 1126-30.
[http://dx.doi.org/10.1038/ng.3087] [PMID: 25173106]
[3]
Guo KX, Wang W, Zhang P, et al. Oxidative stress and mitochondrial dysfunction of retinal ganglion cells injury exposures in long-term blue light. Int J Ophthalmol 2020; 13(12): 1854-63.
[http://dx.doi.org/10.18240/ijo.2020.12.03] [PMID: 33344182]
[4]
Ahmad A, Ahsan H. Biomarkers of inflammation and oxidative stress in ophthalmic disorders. J Immunoassay Immunochem 2020; 41(3): 257-71.
[http://dx.doi.org/10.1080/15321819.2020.1726774] [PMID: 32046582]
[5]
Arfuzir NNN, Lambuk L, Jafri AJA, et al. Protective effect of magnesium acetyltaurate against endothelin-induced retinal and optic nerve injury. Neuroscience 2016; 325: 153-64.
[http://dx.doi.org/10.1016/j.neuroscience.2016.03.041] [PMID: 27012609]
[6]
Kaur C, Foulds WS, Ling EA. Hypoxia-ischemia and retinal ganglion cell damage. Clin Ophthalmol 2008; 2(4): 879-89.
[http://dx.doi.org/10.2147/OPTH.S3361] [PMID: 19668442]
[7]
Lee D, Kim KY, Noh YH, et al. Brimonidine blocks glutamate excitotoxicity-induced oxidative stress and preserves mitochondrial transcription factor a in ischemic retinal injury. PLoS One 2012; 7(10): e47098.
[http://dx.doi.org/10.1371/journal.pone.0047098] [PMID: 23056591]
[8]
Kim SJ, Sung MS, Heo H, Lee JH, Park SW. Mangiferin protects retinal ganglion cells in ischemic mouse retina via SIRT1. Curr Eye Res 2016; 41(6): 844-55.
[http://dx.doi.org/10.3109/02713683.2015.1050736] [PMID: 26200953]
[9]
Chi W, Chen H, Li F, Zhu Y, Yin W, Zhuo Y. HMGB1 promotes the activation of NLRP3 and caspase-8 inflammasomes via NF-κB pathway in acute glaucoma. J Neuroinflammation 2015; 12(1): 137.
[http://dx.doi.org/10.1186/s12974-015-0360-2] [PMID: 26224068]
[10]
Fu Y, Wang Y, Gao X, Li H, Yuan Y. Dynamic expression of HDAC3 in db/db Mouse RGCs and its relationship with apoptosis and autophagy. J Diabetes Res 2020; 2020: 1-8.
[http://dx.doi.org/10.1155/2020/6086780] [PMID: 32190700]
[11]
Tjandra I, Artini W, Siregar NC, Victor AA, Victor AA. Ganglion cells apoptosis in diabetic rats as early prediction of glaucoma: A study of Brn3b gene expression and association with change of quantity of NO, caspase-3, NF-κB, and TNF-α. Int J Ophthalmol 2020; 13(12): 1872-9.
[http://dx.doi.org/10.18240/ijo.2020.12.05] [PMID: 33344184]
[12]
Weishaupt JH, Diem R, Kermer P, Krajewski S, Reed JC, Bähr M. Contribution of caspase-8 to apoptosis of axotomized rat retinal ganglion cells in vivo. Neurobiol Dis 2003; 13(2): 124-35.
[http://dx.doi.org/10.1016/S0969-9961(03)00032-9] [PMID: 12828936]
[13]
Levkovitch-Verbin H, Harris-Cerruti C, Groner Y, Wheeler LA, Schwartz M, Yoles E. RGC death in mice after optic nerve crush injury: Oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci 2000; 41(13): 4169-74.
[PMID: 11095611]
[14]
Rao J, Ye Z, Tang H, et al. The RhoA/ROCK pathway ameliorates adhesion and inflammatory infiltration induced by AGEs in glomerular endothelial cells. Sci Rep 2017; 7(1): 39727.
[http://dx.doi.org/10.1038/srep39727] [PMID: 28054559]
[15]
Issa Bhaloo S, Wu Y, Le Bras A, et al. Binding of Dickkopf-3 to CXCR7 enhances vascular progenitor cell migration and degradable graft regeneration. Circ Res 2018; 123(4): 451-66.
[http://dx.doi.org/10.1161/CIRCRESAHA.118.312945] [PMID: 29980568]
[16]
Schöneborn H, Raudzus F, Coppey M, Neumann S, Heumann R. Perspectives of RAS and RHEB GTPase signaling pathways in regenerating brain neurons. Int J Mol Sci 2018; 19(12): 4052.
[http://dx.doi.org/10.3390/ijms19124052] [PMID: 30558189]
[17]
Shimokawa H, Sunamura S, Satoh K. RhoA/Rho-kinase in the cardiovascular system. Circ Res 2016; 118(2): 352-66.
[http://dx.doi.org/10.1161/CIRCRESAHA.115.306532] [PMID: 26838319]
[18]
Hannan JL, Matsui H, Sopko NA, et al. Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion. Sci Rep 2016; 6(1): 29416.
[http://dx.doi.org/10.1038/srep29416] [PMID: 27388816]
[19]
Sopko NA, Bell M, Matsui H, Hannan J, Bivalacqua T. RhoA/ROCK activation in major pelvic ganglion mediates caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury. Neural Regen Res 2017; 12(4): 572-3.
[http://dx.doi.org/10.4103/1673-5374.205091] [PMID: 28553331]
[20]
Liu X, Zuo Z, Liu W, et al. Upregulation of Nogo receptor expression induces apoptosis of retinal ganglion cells in diabetic rats. Neural Regen Res 2014; 9(8): 815-20.
[http://dx.doi.org/10.4103/1673-5374.131597] [PMID: 25206894]
[21]
Katoch R, Thakur N. RNA interference: A promising technique for the improvement of traditional crops. Int J Food Sci Nutr 2013; 64(2): 248-59.
[http://dx.doi.org/10.3109/09637486.2012.713918] [PMID: 22861122]
[22]
Liu Q, Wu K, Qiu X, Yang Y, Lin X, Yu M. siRNA silencing of gene expression in trabecular meshwork: RhoA siRNA reduces IOP in mice. Curr Mol Med 2012; 12(8): 1015-27.
[http://dx.doi.org/10.2174/156652412802480907] [PMID: 22741561]
[23]
Siddiqui AM, Sabljic TF, Koeberle PD, Ball AK. Downregulation of BM88 after optic nerve injury. Invest Ophthalmol Vis Sci 2014; 55(3): 1919-29.
[http://dx.doi.org/10.1167/iovs.13-12986] [PMID: 24526440]
[24]
Biswas S, Wan KH. Review of rodent hypertensive glaucoma models. Acta Ophthalmol 2019; 97(3): e331-40.
[http://dx.doi.org/10.1111/aos.13983] [PMID: 30549197]
[25]
Xin Y, Huang M, Guo WW, Huang Q, Zhang L, Jiang G. Nano-based delivery of RNAi in cancer therapy. Mol Cancer 2017; 16(1): 134.
[http://dx.doi.org/10.1186/s12943-017-0683-y] [PMID: 28754120]
[26]
Ren X, Lin J, Wang X, et al. Photoactivatable RNAi for cancer gene therapy triggered by near-infrared-irradiated single-walled carbon nanotubes. Int J Nanomedicine 2017; 12: 7885-96.
[http://dx.doi.org/10.2147/IJN.S141882] [PMID: 29138556]
[27]
Chen S, Luo M, Kou H, Shang G, Ji Y, Liu H. A review of gene therapy delivery systems for intervertebral disc degeneration. Curr Pharm Biotechnol 2020; 21(3): 194-205.
[http://dx.doi.org/10.2174/1389201020666191024171618] [PMID: 31749423]
[28]
Kang Y, Jia P, Zhao H, Hu C, Yang X. MicroRNA-26a overexpression protects RGC-5 cells against H2O2-induced apoptosis. Biochem Biophys Res Commun 2015; 460(2): 164-9.
[http://dx.doi.org/10.1016/j.bbrc.2015.02.164] [PMID: 25757910]
[29]
Zhou X, Su CF, Zhang Z, et al. Neuroprotective effects of methyl 3,4-dihydroxybenzoate against H₂O₂-induced apoptosis in RGC-5 cells. J Pharmacol Sci 2014; 125(1): 51-8.
[http://dx.doi.org/10.1254/jphs.13055FP] [PMID: 24849190]
[30]
Hong S, Iizuka Y, Lee T, Kim CY, Seong GJ. Neuroprotective and neurite outgrowth effects of maltol on retinal ganglion cells under oxidative stress. Mol Vis 2014; 20: 1456-62.
[PMID: 25352751]
[31]
Li X, Saint-Cyr-Proulx E, Aktories K, Lamarche-Vane N. Rac1 and Cdc42 but not RhoA or Rho kinase activities are required for neurite outgrowth induced by the Netrin-1 receptor DCC (deleted in colorectal cancer) in N1E-115 neuroblastoma cells. J Biol Chem 2002; 277(17): 15207-14.
[http://dx.doi.org/10.1074/jbc.M109913200] [PMID: 11844789]
[32]
Bromberg KD, Iyengar R, He JC. Regulation of neurite outgrowth by G(i/o) signaling pathways. Front Biosci 2008; 13: 4544-57.
[33]
Fischer D, Petkova V, Thanos S, Benowitz LI. Switching mature retinal ganglion cells to a robust growth state in vivo: gene expression and synergy with RhoA inactivation. J Neurosci 2004; 24(40): 8726-40.
[http://dx.doi.org/10.1523/JNEUROSCI.2774-04.2004] [PMID: 15470139]
[34]
Fujita Y, Yamashita T. Axon growth inhibition by RhoA/ROCK in the central nervous system. Front Neurosci 2014; 8: 338.
[http://dx.doi.org/10.3389/fnins.2014.00338] [PMID: 25374504]
[35]
Li Y, Andereggen L, Yuki K, et al. Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration. Proc Natl Acad Sci USA 2017; 114(2): E209-18.
[http://dx.doi.org/10.1073/pnas.1616811114] [PMID: 28049831]
[36]
Kubo T, Yamashita T. Rho-ROCK inhibitors for the treatment of CNS injury. Recent Patents CNS Drug Discov 2007; 2(3): 173-9.
[http://dx.doi.org/10.2174/157488907782411738] [PMID: 18221230]
[37]
Huang Y, Chen J, Yang B, Shen H, Liang JJ, Luo Q. RhoA/ROCK pathway regulates hypoxia-induced myocardial cell apoptosis. Asian Pac J Trop Med 2014; 7(11): 884-8.
[http://dx.doi.org/10.1016/S1995-7645(14)60154-1] [PMID: 25441988]

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy