Effects of scutellarin on PKCbig gamma in PC12 cell injury induced by oxygen and glucose deprivation
Abstract
Aim: To evaluate the neuroprotective effect and mechanisms of scutellarin (Scu) against PC12 cell injury after oxygen and glucose deprivation followed by reperfusion (OGD-Rep).
Methods: Undifferentiated rat pheochromocytoma PC12 cells, exposed to oxygen and glucose deprivation followed by reperfusion (OGD-Rep), used as an in vitro model of ischemia/reperfusion. Cell survival was evaluated by diphenyltetrazolium bromide (MTT) assay and the amount of LDH release was determined using assay kits. [Ca2+]i was monitored using a fluorescent Ca2+-sensitive dye Fura-2 acetoxymethyl ester. Cell apoptosis was detected by a DNA ladder and by flow cytometric detection. The expression of protein kinase C (PKC)gamma was determined using both RT-PCR and Western blotting. The translocation of PKCgamma was assayed by subcellular fractionation and Western blotting.
Results: OGD-Rep injury significantly elevated the level of LDH release, [Ca2+]i, mRNA expression and the translocation of PKCgamma compared in the PC12 cells with those of the normal group. Scu (10-100 mumol/L) exerted a protective effect against OGD-Rep injury by reducing LDH release, [Ca2+]i, the percent of apoptosis, and the translocation of PKCgamma.
Conclusion: Scu inhibits the increase of [Ca2+]i and the activation of PKCgamma, exerting protective effects against PC12 cell injury induced by OGD-Rep.
Keywords:
Methods: Undifferentiated rat pheochromocytoma PC12 cells, exposed to oxygen and glucose deprivation followed by reperfusion (OGD-Rep), used as an in vitro model of ischemia/reperfusion. Cell survival was evaluated by diphenyltetrazolium bromide (MTT) assay and the amount of LDH release was determined using assay kits. [Ca2+]i was monitored using a fluorescent Ca2+-sensitive dye Fura-2 acetoxymethyl ester. Cell apoptosis was detected by a DNA ladder and by flow cytometric detection. The expression of protein kinase C (PKC)gamma was determined using both RT-PCR and Western blotting. The translocation of PKCgamma was assayed by subcellular fractionation and Western blotting.
Results: OGD-Rep injury significantly elevated the level of LDH release, [Ca2+]i, mRNA expression and the translocation of PKCgamma compared in the PC12 cells with those of the normal group. Scu (10-100 mumol/L) exerted a protective effect against OGD-Rep injury by reducing LDH release, [Ca2+]i, the percent of apoptosis, and the translocation of PKCgamma.
Conclusion: Scu inhibits the increase of [Ca2+]i and the activation of PKCgamma, exerting protective effects against PC12 cell injury induced by OGD-Rep.