The Neuroprotective Mechanisms Of Curcumin In Cerebral Ischemia Reperfusion Injury

Objective: Cerebral ischemia-reperfusion injury is one of the most common diseases in the central nervous system.It is a more serious secondary pathological injury caused by reperfusion of cerebral blood flow after ischemic stroke,which has the characteristics of high morbidity,high fatality rate and high disability rate.At present,effective therapeutic drugs for the ischemic cascade reaction caused by blood flow reperfusion are still not available.Therefore,it is important to further study the pathogenesis of cerebral ischemia-reperfusion injury and find effective drugs in protecting damaged neurons.Moreover,it also has a great clinical significance for increasing the survival rate and improving the prognosis of stroke patients.Curcumin is a liposoluble phenolic pigment with anti-inflammatory and antioxidative effects,which can protect many organs from oxidative stress.It has been reported that curcumin has significant neuroprotective effects,which can reduce the infarct volume of cortex after cerebral ischemia and decrease the apoptosis and death of neuronal cells in ischemic injury.Autophagy is a normal physiological metabolic process of eukaryotic cells and generally continues at a lower level to provide cells with the essential raw materials needed for survival.Many studies have shown that autophagy of neuronal cells is activated during ischemia-reperfusion injury,and high levels of autophagy are closely related to ischemic neuronal apoptosis and death levels.Hypoxia inducible factor-1?(HIF-1?)is an important regulator of cerebral ischemia-reperfusion,and its expression will be increased in damaged brain tissue.HIF-1? plays a key role in the transcription process of apoptosis,energy metabolism and inflammatory responses.The study was aimed to determine whether the neuroprotective effects of curcumin during cerebral ischemia-reperfusion injury are related to the regulation of autophagy and HIF-1? levels and the role of autophagy and HIF-1? in this process.Methods: Oxygen glucose deprivation reperfusion(OGD/R)injury model was established using rat adrenal pheochromocytoma cell PC12 to mimic neuronal ischemia-reperfusion injury.The PC12 cells was treated with different concentrations of curcumin under normal conditions and OGD/R conditions.The cell viability was detected by MTS,and the cell apoptosis was detected by flow cytometry.Western blot was used to detect the expression of autophagy-related proteins LC3 II,LC3 I,P62 and HIF-1? in normal control group,OGD/R control group and(OGD/R+curcumin)group.The number of autophagosomes in each group was detected by fluorescence microscopy.The autophagy inhibitor,3-methyladenine(3-MA),was used to inhibit the activation of autophagy,and the HIF-1? expression in cells was silenced by small interfering RNA(siRNA)transfection.MTS assay was used to detect the cell viability of normal control group,OGD/R control group,(OGD/R+curcumin)group,(OGD/R+3-MA)group and(OGD/R+HIF-1?-siRNA)group,flow cytometry was used to detect apoptosis,autophagy-related protein and HIF-1? was detected by western blot,the number of autophagosomes in each group was detected by fluorescence microscopy.Results: 1.MTS results showed that the cell viability under OGD8h/R24 h was decreased to approximately 50% compared with normal cells(P<0.001).The protective effects of curcumin on injuried PC12 cells were dose-dependent.Compared with OGD/R control group,the maximal cell viability was achieved in 5 ?M curcumin treatment group(P<0.001).However,cellular survival and death were not affected by curcumin under normal conditions.Flow cytometry results showed that compared with OGD/R control group,the apoptosis was the lowest in 5 ?M curcumin treatment group(P<0.001).2.Western blot results showed that compared with the normal control group,OGD/R up-regulated the expression levels of LC3 II / I(P=0.002)and HIF-1?(P=0.024)in neuronal PC12 cells,and down-regulated P62 expression levels(P = 0.012).Curcumin inhibited the high levels of LC3 II / I(P = 0.006)and HIF-1?(P = 0.045)induced by OGD/R and promoted the expression of P62(P = 0.005).Fluorescence microscopy showed that curcumin can reduce the number of autophagosomes in cells under OGD/R conditions.3.MTS results showed that the survival level of neuronal PC12 cells after 3-MA pretreatment(P=0.001)or transfection of HIF-1? siRNA(P<0.001)was significantly higher than that of OGD/R control group.Flow cytometry results showed that 3-MA pretreatment(P=0.007)or transfection of HIF-1? siRNA(P=0.003)significantly reduced the apoptosis level of damaged cells compared with the OGD/R control group.4.Western blot results showed that compared with the OGD/R control group,the expression level of HIF-1? was significantly decreased in the(OGD/R+3-MA)group(P=0.002),the level of LC3 II / I in the(OGD/R+HIF-1? siRNA)group was significantly decreased(P = 0.004),and the expression level of P62 was significantly increased(P = 0.010).Fluorescence microscopy showed that the number of autophagosomes was significantly reduced in PC12 cells transfected with HIF-1? siRNA compared to the OGD/R control group.Conclusion: 1.Curcumin has a protective effect on PC12 cells from OGD/R injury.2.The neuroprotective effect of curcumin is closely related to the inhibition of autophagy and HIF-1? levels in cells under OGD/R conditions.3.Under OGD/R conditions,autophagy in a reciprocal manner to regulate HIF-1? expression levels.