Protective Effect And Mechanism Of Curcumin On ActD/TNF-α-induced Synergistically Apoptosis In PC12 Cells

Objective:To observe the protective effect of curcumin against ActD/TNF-α-induced synergistically apoptosis in PC12 cells, and explore the mechanism, contributing a new experimental support to the application of curcumin in the prevention and treatment of HIV-associated neurocognitive disorders (HAND) and related diseases.Methods:1. Culture PC12 cells in the DMEM high glucose medium that containing 5% fetal bovine serum, the condition of the evidence is 37℃,5%CO2.The cells grow adherent to the wall, when they covered it, digest them with 0.25% pancreatin and subcultures every 2 days.2. MTT method to evaluate the optimal concentration of curcumin which can protect neurons and ActD/TNF-αwhich can induced neuronal insult.The cells were divided into 6 groups:control group,TNF-αgroup,ActD group,curcumin group,ActD/TNF-αgroup curcumin+ActD/TNF-αgroup, then Selected the appropriate concentrations of curcumin antagonist the effect of cytotoxicity on the PC12 cells induced by ActD/TNF-α. And then the level of lactate dehydrogenase (LDH) in the culture solution was determined.3. To observe the effect of curcumin on the ActD/TNF-α-induced apoptosis. The morphological change were detected by Hoechst 33258 staining and the mitochondrial membrane potential (MMP) was detected by by JC-1 as a fluorescent molecular probe.The apoptosis of PC12 cells was analysed by AnnexinⅤ/PI double staining and flow cytometry.4. Record the field-excitatory postsynaptic potential (fEPSP) of CA1 pyramidal layer of rat hippocampal brain slices with extracellular microelectrode recording technique, to observe and calculate the change of long-term potentiation in different groups.5. To explorer the possible neuroprotection mechanisms of curcumin on ActD/TNF-α-induced PC12 cells injury, detected the level of Ca2+ by flow cytometry, analyzed the expression of Bcl-2, Bax and evaluated the activity of caspase-3 by coelosphere reader.Results:1. MTT assay showed that TNF-a at concentrations of 10,25,50μg/mL didn't affect the cell viability after 24h incubation(P>0.05 vs control), TNF-a at concentrations of 100μg/mL has the a certain damage on the PC12 cells after 24 h incubation (P<0.05 vs control).ActD at concentrations of 5,10ng/mL didn't affect the cell viability after 24h(P>0.05 vs control), while ActD at concentration of 20ng/mL,40ng/mL reduced cell viability remarkably (P<0.05 vs control).incubation curcumin at concentrations of 0.5,1,5μmol/L didn't affect the cell viability after 24 h incubation (P>0.05 vs control), while curcumin at concentration of 10μmol/L reduced cell viability remarkably (P<0.05 vs control). ActD at concentration of 10 ng/mL which add the corresponding concentrations of TNF-a in 25,50,100 ng/mL reduced cell viability remarkably (P<0.05 vs control), which cell viability of 10ng/mL ActD add 50 ng/mL TNF-a induced 59.67±2.81% in PC12 cells. So we chose 10ng/mL ActD plus 50 ng/mL group as a model which further observe the neuronal insult by them.1,5μmol/L curcumin can improve the PC12 cells from damage induced by ActD/TNF-a (P<0.05 vs ActD/TNF-a), and the best protective concentration is 5μmol/L.ActD/TNF-a group induced an increase in activity of LDH in the supernatant, which were inhibited by treatment with 5μmol/L curcumin(P<0.05 vs control), respectively. Therefore, the following experiments were observed the protective effect using 5μmol/L curcumin for 24 hours.2. Hoechst 33258 staining showed that the nuclei of normal cells appeared the same size and regular conformation, while in ActD/TNF-αgroup some cells showed pyknosis and karyorrhexis and this phenomenon changed after treatments of curcumin at concentration of 5μmol/L Compared with control group, MMP were reduced remarkably in ActD/TNF-αgroup, while Following treatments of curcumin at concentration of 5μmol/L, MMP increased remarkably compared with ActD/TNF-a. AnnexinV/PI double staining showed that the apoptosis rate were increased significantly in ActD/TNF-αgroup (P<0.05 vs control), and the apoptotic rate were reduced significantly while in curcumin plus ActD/TNF-αgroup (P<0.05 vs ActD/TNF-α).3. ActD/TNF-αcan inhibit the LTP, depress the hippocampal synaptic plasticity (P<0.05 vs control). Curcumin can inverse the effect of ActD/TNF-αon LTP, and improved the synaptic plasticity (P<0.05 vs ActD/TNF-α).4. ActD/TNF-a increased the intracellular Ca2+ concentration in PC 12 cells, inhibited the expression of Bcl-2 and increased the activity of caspase-3, while curcumin can protect the PC 12 cells against ActD/TNF-a induced Calcium overload from decreasing the intracellular Ca2+ concentration, increased the expression of Bcl-2 and decreased the the activity of caspase-3 (P<0.05 vs ActD/TNF-α).Conclusions:1.10 ng/mL ActD and 50μg/mL TNF-αco-incubated caused neuron pyknosis and karyorrhexis,MMP reduced remarkably, and application of curcumin (5μmol/L) could protect neurons against ActD (10 ng/mL) plus TNF-a (50ng/mL)-induced neuronal apoptosis.2. Curcumin could recover the LTP of hippocampal brain slices inhibited by ActD/TNF-a, so that it can maintain the stability of neuronal function.3. Curcumin could prevent the calcium overload of PC 12 cells induced by ActD/TNF-a, maintain the Ca2+ homeostasis and MMP, increase the expression of Bcl-2 and inhibit the activation of caspase-3.In conclusion, curcumin can protect the ActD/TNF-a-induced synergistically neuronal damage, the mechanisms may include depressing the intracellular Ca2+ concentration, raising the mitochondria membrane potential, increasing the expression of Bcl-2 and inhibiting the activation of caspase-3.