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. 2013 Aug 6;8(8):e70565.
doi: 10.1371/journal.pone.0070565. Print 2013.

Curcumin Protects Microglia and Primary Rat Cortical Neurons Against HIV-1 gp120-mediated Inflammation and Apoptosis

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Free PMC article

Curcumin Protects Microglia and Primary Rat Cortical Neurons Against HIV-1 gp120-mediated Inflammation and Apoptosis

Luyan Guo et al. PLoS One. .
Free PMC article

Abstract

Curcumin is a molecule found in turmeric root that has anti-inflammatory, antioxidant, and anti-tumor properties and has been widely used as both an herbal drug and a food additive to treat or prevent neurodegenerative diseases. To explore whether curcumin is able to ameliorate HIV-1-associated neurotoxicity, we treated a murine microglial cell line (N9) and primary rat cortical neurons with curcumin in the presence or absence of neurotoxic HIV-1 gp120 (V3 loop) protein. We found that HIV-1 gp120 profoundly induced N9 cells to produce reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1). HIV-1 gp120 also induced apoptosis of primary rat cortical neurons. Curcumin exerted a powerful inhibitory effect against HIV-1 gp120-induced neuronal damage, reducing the production of ROS, TNF-α and MCP-1 by N9 cells and inhibiting apoptosis of primary rat cortical neurons. Curcumin may exert its biological activities through inhibition of the delayed rectification and transient outward potassium (K(+)) current, as curcumin effectively reduced HIV-1 gp120-mediated elevation of the delayed rectification and transient outward K(+) channel current in neurons. We conclude that HIV-1 gp120 increases ROS, TNF-α and MCP-1 production in microglia, and induces cortical neuron apoptosis by affecting the delayed rectification and transient outward K(+) channel current. Curcumin reduces production of ROS and inflammatory mediators in HIV-1-gp120-stimulated microglia, and protects cortical neurons against HIV-1-mediated apoptosis, most likely through inhibition of HIV-1 gp120-induced elevation of the delayed rectification and transient outward K(+) current.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effects of HIV-1 gp120 V3 loop and curcumin on microglial viability.
Microglial cells (mouse N9 cell line) were treated with HIV-1 gp120 V3 loop at various concentrations (0.5, 1, 2, and 4 µg/mL), curcumin at various concentrations (1, 5, 10, 15 and 20 µM), or 4 µg/mL of heat-inactivated HIV-1 gp120 V3 loop for 24 h. Cells were subjected to the MTT assay for viability analysis. A) and B) show the effects of HIV-1 gp120 V3 loop and curcumin on microglial viability, respectively. *, p<0.05: experimental group versus medium control. Experiments were repeated 9 times. Inactivated: 4 µg/mL of heat-inactivated HIV-1 gp120 V3 loop.
Figure 2
Figure 2. Curcumin, NAC, TEA and 4-AP alleviated HIV-1 gp120 V3 loop-induced ROS increase in microglia.
After pre-incubation of microglia with curcumin (15 µM), NAC (3 mM), TEA (2 mM) or 4-AP (2 mM) for 2 h, cells were treated with HIV-1 gp120 V3 loop (1 µg/mL) for 1 h, and then subjected to ROS measurement using fluorescence microscope and FACS. A) and B): ROS production in microglia was determined using fluorescence microscope. The arrows indicated ROS-positive microglial cells. C) and D): ROS production in microglia was determined using FACS. Six independent experiments were performed. Each bar graph shows changes of ROS MFI calculating from comparison of experimental group versus medium control. *, P<0.05 vs. control, n = 6; #, P<0.05 vs. HIV-1 gp120 V3 loop alone, n = 6. Cur, curcumin.
Figure 3
Figure 3. Curcumin, NAC, 4-AP and TEA reduced HIV-1 gp120 V3 loop-induced TNF-α and MCP-1 mRNA expression in microglia.
After pre-incubation of microglia with curcumin (15 µM), NAC (3 mM), TEA (2 mM) or 4-AP (2 mM) for 2 h, cells were treated with HIV-1 gp120 V3 loop (1 µg/mL) for 3 h. Cells were then subjected to RNA extraction for measuring TNF-α and MCP-1 mRNA expression using a qPCR assay. A) and B): TNF-α mRNA and MCP-1 mRNA expression, respectively. *, p<0.05 vs. control, n = 6; #, p<0.05 vs. HIV-1 gp120 V3 loop alone. Experiments were repeated 6 times (n = 6). Cur, curcumin.
Figure 4
Figure 4. Curcumin, 4-AP and TEA reduced HIV-1 gp120 V3 loop- induced apoptosis of primary rat cortical neurons.
Primary rat cortical neurons that had been cultured for 7 days were treated with corresponding conditioned media for 24 h and then subjected to apoptosis analysis. A) and B): Apoptosis of neurons was determined by Hoechst 33342 staining. Each bar shows % of MFI of Hoechst 33342-stained cortical neuron nuclei versus medium control or gp120 treatment alone. C) and D): Apoptosis of neurons was determined by western blotting analysis of cleaved caspase-3 levels. Each bar shows % of cleaved caspase-3 level vs medium control or gp120 treatment alone. *, p<0.05 vs. control, n = 6; #, p<0.05 vs. HIV-1 gp120 loop alone, n = 6. The arrows indicate the neuronal nuclei in each group. Cur, curcumin.
Figure 5
Figure 5. Curcumin attenuated HIV-1 gp120 V3 loop-induced increase in the delayed rectifier K+ current and the transient outward K+ current.
Whole-cell patch clamp recordings of primary rat cortical neuronal bioactivity in response to gp120 (5 µg/mL) treatment with or without curcumin (15 µM) were performed in 35-mm culture dishes on an inverted Olympus microscope stage using an Axopatch 200B amplifier. Whole-cell delayed rectifier K+ current and transient outward K+ current were induced through voltage steps ranging from −60 mV to +60 mV in 10-mV increments. A): Schematic representation of the pulse stimulation setting for both recordings of delayed rectifier K+ current and transient outward K+ current. B): A recording of delayed rectifier potassium current in medium (control), cells treated with gp120 (5 µg/mL), curcumin (15 µM),gp120 (5 µg/mL) plus curcumin (15 µM), or post-washing. C): I-V curves of delayed rectifier K+ current densities in response to gp120 (5 µg/mL) treatment with or without curcumin (15 µM). D): Pooled whole-cell recording data of the delayed rectifier K+ current from 6 separated experiments. E): A recording of transient outward K+ current in medium (control), cells treated with gp120 (5 µg/mL), curcumin (15 µM),gp120 (5 µg/mL) plus curcumin (15 µM), or post-washing. F: I–V curves of transient outward K+ current densities in response to gp120 (5 µg/mL) treatment with or without curcumin (15 µM). G: Pooled whole-cell recording data of the transient outward K+ current from 6 separated experiments. Each value represents the mean ± SEM. #, p<0.05 vs. control; *, p<0.05 vs. HIV-1 gp120 V3 loop alone; n = 6.

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Grant support

This work was supported by National Natural Science Foundation of China, number 81171134; National Basic Research Program of China C973 program, number 2011CB707500; Science and Technology Foundation of Guangzhou, number 2010Y1-C291; Science and Technology Foundation of Guangdong, number 2010B030700017; The Key laboratory’s Open Fund of Jinan University, number 2011ZD003. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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