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Shravan K. Chintala, Balabharathi Burugula, BhagyaLaxmi S. Ganesh; Curcumin Attenuates Protease-mediated Death of Retinal Ganglion Cells Both in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5356.
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Staurosporine (SS), a protein kinase inhibitor, was found to cause retinal ganglion cell (RGC) death in vivo, but the mechanisms underlying SS-induced cell death were unclear. Since our previous studies on cultured RGC-5 cells indicate that SS induces cell death due to elevated levels of proteases, the purpose of this was to investigate whether: (a) SS induces RGC loss in vivo by elevating protease levels and (b) Curcumin, an activator of cell protective NF-kB pathway, prevents protease-mediated death of RGCs in vitro and in vivo.
Transformed mouse RGC-5 cells were cultured in serum-free Dulbecco’s Modified Eagle’s medium (DMEM) and treated them with 2.0 uM SS to induce cell death. To investigate the effect of Curcumin, RGC-5 cells were treated with 2.0 uM SS and various doses of Curcumin. Two optimal doses of SS (12.5 and 100 nM) were injected into the vitreous humor of C57BL/6 mice to induce death of RGCs along with two doses of Curcumin (2.5 and 10 uM) to investigate Curcumin’s effect. Proteolytic activities of matrix metalloproteinase -9 (MMP-9), tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA) were assessed by zymography assays. RGC loss in vivo was assessed by immunostaining of flat-mounted retinas with Brn3a and cell viability of RGC-5 cells in vitro was assessed by MTT (dimethyl sulfoxide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) assays. Frozen retinal cross sections prepared from SS-injected animals were used for the immunostaining of Nuclear factor (NF-kB).
Staurosporine induced uPA and tPA levels in RGC-5 cells in vitro, MMP-9, uPA, and tPA levels in vivo, and promoted the death of RGCs both in vitro and in vivo. In contrast, Curcumin attenuated the loss of RGCs both in vitro and in vivo despite the presence of elevated levels of proteases observed in retinal proteins extracted from CB7BL/6 animals or conditioned medium collected from cultured RGC-5 cells. A NF-kB inhibitory peptide, but not a control peptide, reversed curcumin-mediated protective effect in vitro, but did not inhibit elevated levels of proteases. In vivo results indicate that Curcumin does not inhibit elevated levels of proteases, but attenuates RGC loss, in part, by restoring Staurosporine-mediated down-regulation of NF-kB expression in RGCs.
The results presented in this study show that Curcumin attenuates RGC death despite elevated levels of proteases and raises the possibility that Curcumin may be used as a plausible adjuvant therapeutic agent to prevent the death of RGCs.
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