Protective Effect And Mechanism Of Curcumin Against Gp120V3 Loop Damaged Microglias And Cortical Neurons

Object:Our experiment used gp120V3 loop to activate N9 microglia and induce cortical neuron apoptosis, which were similar to the main pathologic changes of HIV associated neurocognitive disorders(HAND).We studied the protective effect and mechanism of curcumin against gp120V3 loop damaged microglias and cortical neurons,which provides experimental basis for the application of curcumin in the prevention and treatment of HAND.Methods:1 MTT assay to screen out the doses of curcumin and gpl20V3 loopN9 microglia was treated with 1,5,10,15 and 20μmol/L curcumin for 24 hours and then we evaluated the survival rate of the cells by MTT assay to screen out the safe dose of curcumin. Microglia was cultured with serum free medium for 24 hours, and then given 0.5,1,2 and 4mg/L gp120V3 loop for 24 hours. We screened out the low-toxic dose of gp120V3 loop by MTT assay.2 Qualitative measuring of the reactive oxygen species (ROS) level within microglia by fluorescence microscopeAccording to the results of MTT assay, the doses of curcumin and gp120V3 loop were chosen in follow-on experiment. On the basis of related literatures,we use 3mmol/L N-acetylcysteine (NAC),2mmol/L 4-aminopyridine (4-AP) and 2mmol/L tetraethylammonium (TEA). The experiment included 10 groups:control group, curcumin group, NAC group,4-AP group, TEA group. gp120V3 loop group, gp120V3 loop plus curcumin group, gp120V3 loop plus NAC group, gp120V3 loop plus 4-AP group and gp120V3 loop plus TEA group. DCFH-DA probe method was adopted. Two hours after curcumin, NAC,4-AP and TEA were added. gp120V3 loop were added and interacted with microglia for one hour. Then the probe was loaded. The fluorescence intensity of different groups was observed with fluorescence microscope.3 Quantitative measuring of the reactive oxygen species (ROS) level within microglia by flow cytometryThe experimental groups and doses of the drugs were the same as the part of fluorescence microscope measuring. Two hours after curcumin, NAC,4-AP and TEA were added, gp120V3 loop were added and interacted with microglia for one hour. At once, the cells were digested with trypsin and collected, centrifugalized. After the probe was loaded, we measured the fluorescence intensity of different groups.4 Measuring tumor necrosis factor-α(TNF-α) and monocyte chemoattractant protein-1 (MCP-1) mRNA level of N9 microglia with fluorescence quantitative PCRThe experimental groups and doses of the drugs were the same as the part of fluorescence microscope measuring. Two hours after curcumin, NAC,4-AP and TEA were added, gp120V3 loop were added and interacted with microglia for three hours. The TNF-αand MCP-1 mRNA level of different groups were tested.5 Detecting primary cortical neuron apoptosis with Hoechst33342We prepared conditioned media from microglia at first. The experiment included control group, curcumin group,4-AP group, TEA group, gp120V3 loop group, gp120V3 loop plus curcumin group, gp120V3 loop plus 4-AP group, gp120V3 loop plus TEA group. Microglia was cultured with serum free medium for 24 hours. Two hours after curcumin, NAC,4-AP and TEA were added, gpl20V3 loop were added and interacted with microglia for 12 hours. The cell supernatant fluid was conditioned media. The groups of Hoechst 33342 assay were the same as above. The above-mentioned conditioned media interacted with cortical neurons of corresponding groups for 24 hours. The cells were dyed with Hoechst33342 to observe the nucleus morphological changes of cortical neurons of different groups with fluorescence microscope.6 Observing effects of curcumin on gp120V3 loop-induced voltage gated potassium channel current changes of primary cortical neurons by whole-cell patch clamp.The experiment included control group, gp120V3 loop group and gp120V3 loop plus curcumin group. The cell was clamped at the potential of -80mV and given stairstep depolarization pulse stimulation from -60mV to +60mV. The step is lOmV. The delayed rectification and transient outward potassium current was recorded through changing bath solutions. The effects of curcumin on gp120V3 loop-induced voltage gated potassium channel current changes were observed.Results:1 MTT assay indicated 1,5,10,15μmol/L curcumin had no influences on microglia survival rate.However,20μmol/L curcumin decreased the survival rate to (59.64±4.05)%(p<0.05), which was toxic to microglia.15μmol/L curcumin was adopted in the experiment.0.5mg/L gp120V3 loop had no influence on microglia survival rate. 1,2,4mg/L gp120V3 loop decreased the survival rate to (82.08±2.95)%, (69.53±1.07)% and (60.48±0.59)% separatedly (p<0.05), which were all toxic to microglia.We use the low toxic dose 1mg/L.2 By means of fluorescence microscope observation, lmg/L gp120V3 loop increased the ROS fluorenscence intensity within microglia obviously compared to control group. However, curcumin, NAC,4-AP and TEA can decrease the ROS fluorenscence enhancement which was induced by gp120V3 loop.3 By means of flow cytometry measuring, the ROS relative level of microglia was increased in the gp120V3 loop group. The ROS relative level of microglia was decreased in the gp120V3 loop plus curcumin, gp120V3 loop plus NAC, gp120V3 loop plus 4-AP and gp120V3 loop plus TEA group. The differences were significant statistically compared with the gp120V3 loop group (p<0.05).4 Measuring TNF-a and MCP-1 mRNA level with fluorescence quantitative PCR: The result indicated that the expression of TNF-a and MCP-1 mRNA increased in gp120 V3 loop group, which was significant statistically compared with control group (p<0.05). The expression of TNF-a and MCP-1 mRNA decreased in curcumin, NAC,4-AP and TEA group, which was significant statistically compared with control group (p<0.05).The expression of TNF-a and MCP-1 mRNA decreased in gp120V3 loop plus curcumin, gp120V3 loop plus NAC, gp120V3 loop plus 4-AP and gp120V3 loop plus TEA group, which was significant statistically compared with gp120V3 loop group (p<0.05). 5 Observing effects of curcumin on gpl20V3 loop induced nuclear morphological changes of rat cortical neurons with hoechst33342 stainingThe neuron nucleuses were equally distributed light blue in control group. Many nucleuses were highlighted blue in gp120V3 loop group, indicating that the cells were in a apoptosis condition. The highlighted blue cells decreased obviously in gp120V3 loop plus curcumin, gp120V3 loop plus 4-AP and gp120V3 loop plus TEA compared with the gp120V3 loop group.6 Observing effects of curcumin on gp120V3 loop-induced delayed rectification and transient outward potassium current changes of rat cortical neurons by whole-cell patch clamp.After the gp120V3 loop was added into bath solution, peak delayed rectification and transient outward potassium current increased at different clamped voltages. There were statistical differences compared with control group (p<0.05). After curcumin was added into bath solution, peak delayed rectification and transient outward potassium current decreased at different clamped voltages. There were statistical differences compared with gp120V3 loop group (p<0.05).Conclution:1 Gp120V3 loop increased the ROS level, TNF-a and MCP-1 gene expression within microglia.4-AP, TEA and NAC can decrease above-mentioned rises, indicating that gp120V3 loop increased ROS level through influencing the voltage gated potassium channel. ROS increased the expression of inflammatory cytokines further. This maybe mechanism of gp120V3 loop activating microglia and increasing inflammatory cytokines expression. Curcumin obviously decreased the rises of ROS, TNF-a and MCP-1 gene expression induced by gp120V3 loop within microglia. Curcumin has protective effects on microglia activation induced by gp120V3 loop.2 Gpl20V3 loop induced cortical neuron apoptosis.Curcumin,4-AP and TEA can alleviate the apoptosis induced by gp120V3 loop. Gp120V3 loop enhanced delayed rectification and transient outward potassium current of cortical neuron.Curcumin can alleviate the potassium current increasement. It indicated that gp120V3 loop induced neuron apoptosis through enhancing voltage gated potassium current. Curcumin protect neuron from apoptosis through reducing the potassium currentIn conclusion, gp120V3 loop increased ROS level, TNF-amRNA, MCP-1 mRNA and induced apoptosis of cortical neurons by influencing voltage gated potassium channel.Curcumin had protective effects on microglia activation and cortical neuron apoptosis.The mechanism of curcumin protecting neurons from apoptosis maybe reducing the voltage gated potassium current enhance induced by gp120V3 loop.