Researching On The Expression Of HO-1、nNOS In Hippocampal Neurons Oxidative Stress Injury After Curcumin

Objective: To explore the protection mechanism of curcumin onhippocampal neurons oxidative stress, which would provide some theoreticalbases for the clinical application of curcumin. Methods:Hippocampal neuronsof new-born SD rats(≤24h) were cultured by non-blood serum(Neurobasal+2％B27). The morphology of neuron was observed by inverted phase contrastmicroscope and the cell purity was detected with MTT. We identifiedhippocampal neurons by NeuN and NF-200. The hippocampal neuronscultured for10days were divided randomly into6groups: control group,injured group and four curcumin groups (curcumin concentrations was at2.5μmol/L,5.0μmol/L,10.0μmol/L,20.0μmol/L respectively.). Thehippocampal neurons were exposed to H2O2(1mmol/L) for1hour to make theoxidative stress model. And then four curcumin groups were incubated withcurcumin at2.5μmol/L,5.0μmol/L,10.0μmol/L,20.0μmol/L respectivelyfor6hours, while non-blood serum (300μl) was used in control group andinjured group. As stated above, the morphology of neurons was observed byinverted phase contrast microscope, and the survival rate was detected byMTT, the activity of LDH, the level of MDA were tested by colorimetry andthio-barbituric acid method, the expression of HO-1and nNOS were evaluatedby IHC, IF and RT-PCR. Results:1. The cultured cells grew against the wallof flask after plating3~4hours. The cell prominences grew the1thday and developed quickly in the7~8thday. The prominences interlaced to net,forming typical nerve fiber net in the9~11thday. After20days, cells axonswere disconnecting and shortened, and the neuronal bodies were Halo-free.The cell purity was92%when using above mentioned culture methods.Compared with other time points, the activity of hippocampal neurons in the9~11thday was the highest, which was the proper time to establish oxidativestress model, p <0.05.2. Observing the models in1hour post oxidative stressdamage, we had found that the synaptic adhesion strength was to lower, axonswere rough and fracture, parts of cells floated on the medium and vacuolesappeared in cell bodies.3. Compared with1hour, there were no obviousmorphological changes in6hours in curcumin groups (at5.0μmol/L and10.0μmol/L). The axons were shorten but not fracture in6hours when comparedwith1hour in curcumin groups at2.5μmol/L and20.0μmol/L. Cells axonsand the neuronal bodies were more shortened, fractured, and Halo-free ininjured group than in1hour. Axons and the neuronal bodies were whole,smooth, webbed and Halo in control group in1hour.4. MTT assay Theneurons activity in curcumin groups (at5.0μmol/L,10.0μmol/L) and controlgroups was more stronger than injured group, P <0.05. The activity ofneurons in curcumin treatment groups between at5.0μmol/L,10.0μmol/Land at2.5μmol/L,20.0μmol/L has significant difference, P <0.05. Theactivity of cells in treatment groups at2.5μmol/L and20.0μmol/L was noobviously different from injured group, P>0.05.5. Activity detection of LDH Compared with injured group, the activity of LDH in curcumintreatment groups (5.0μmol/L,10.0μmol/L) and blank control group waslower, P <0.05. The activity of LDH in curcumin treatment groups at5.0μmol/L,10.0μmol/L and at2.5μmol/L,20.0μmol/L has obvious difference,P <0.05. Compared with blank control group, the activity of LDH incurcumin treatment groups (at2.5μmol/L,20.0μmol/L) was higher, P <0.05.6. Concentration detection of MDA Compared with injured group, theconcentration of MDA in curcumin treatment groups (5.0μmol/L,10.0μmol/L) and control group was lower, P <0.05. Compared the concentrationof MDA in curcumin treatment groups between5.0μmol/L,10.0μmol/L and2.5μmol/L,20.0μmol/L, there was significant difference, P <0.05.Compared with blank control group, the concentration of MDA in curcumintreatment groups (2.5μmol/L,20.0μmol/L) was higher, P <0.05.7. Thedetection of IHC, IF and RT-PCR Compared with injured group, theexpression of HO-1was increased and the expression of nNOS was decreasedin curcumin treatment groups (5.0μmol/L,10.0μmol/L) and control group, P<0.05. And when compared in curcumin treatment groups, the expression ofHO-1and nNOS in groups at5.0μmol/L,10.0μmol/L and at2.5μmol/L,20.0μmol/L has significant difference, P <0.05. Compared with injured group, theexpression of HO-1and nNOS in curcumin treatment groups (2.5μmol/L,20.0μmol/L) was no significant difference, P>0.05. Conclusion:1.Thecurcumin solution at5μmol/L and10μmol/L has particularly protective effect on hippocampal neurons oxidative stress injury by increasing the expressionof HO-1and decreasing the expression of nNOS.2. Lipid peroxidation takespart in the hippocampal neurons oxidative stress injury. The curcumin solutionat5μmol/L and10μmol/L plays protective role on neural membrane bydecreasing lipid peroxidation and reducing the release of LDH.