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. 2017 Aug 23;12(8):e0183688.
doi: 10.1371/journal.pone.0183688. eCollection 2017.

Effect of naringin on gp120-induced injury mediated by P2X7 receptors in rat primary cultured microglia

Qiang Chen  1 , Hui Wu  2 , Jia Tao  2 , Chenglong Liu  1 , Zeyu Deng  1 , Yang Liu  3 , Guoqiao Chen  3 , Baoyun Liu  3 , Changshui Xu  1
Affiliations

Effect of naringin on gp120-induced injury mediated by P2X7 receptors in rat primary cultured microglia

Qiang Chen et al. PLoS One. .

Abstract

Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein 120 has been shown to activate microglia, causing release of inflammatory and toxic factors. The P2X7 receptor, primarily expressed on microglia, is closely associated with inflammation. Naringin, a plant bioflavonoid, has anti-inflammatory and anti-oxidative properties. We hypothesized that P2X7 receptor mediated gp120-induced injury in primary cultured microglia, and that naringin would have a protective effect. We showed that HIV-1 gp120 peptide (V3 loop, fragment 308-331) appeared to induce apoptosis of primary cultured microglia. However, there was a decrease of microglia apoptosis in gp120+naringin group compared with gp120 group. Using qPCR, Western blot, and immunofluorescence, we showed that gp120 stimulated expression of P2X7 mRNA and receptor protein, and this stimulation was inhibited by naringin. Treatment with gp120 increased concentrations of eATP, TNFα and IL-1β, and these effects were inhibited by naringin. Taken together, these results suggested that gp120 contributed to microglial cell injury and neurotoxic activity by up-regulating expression of P2X7, in a naringin-protective manner.

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Conflict of interest statement

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

Figures

Fig 1
Fig 1. Primary cultured microglia purity identification by immunofluorescence.
Blue signal indicates nuclear staining with DAPI; green signal represents CD11b staining with FITC; arrows represent microglia with typical morphology.
Fig 2
Fig 2. Gp120 up-regulated expression of P2X7 mRNA and receptor protein in primary cultured microglia.
A, Relative expression of P2X7 mRNA detected by qPCR. B, SDS-PAGE electrophoresis of P2X7 receptor protein. C, Relative expression of P2X7 receptor protein; **P < 0.01, versus Ctrl group; n = 5.
Fig 3
Fig 3. Primary cultured microglia apoptosis detected by TUNEL.
A, Typical fluorescence image of apoptotic microglia in each group; blue signal indicates nuclear staining with DAPI; green signal represents TUNEL staining of fragmented DNA with FITC; arrows indicate apoptotic microglial cells; a, Ctrl group; b, gp120 group; c, gp120+naringin group; d, gp120+DMSO group. B, The percentage of apoptotic microglial cells in each group; **P < 0.01, versus Ctrl group; ##P < 0.01, versus gp120 group; n = 3.
Fig 4
Fig 4. Concentrations of eATP in the supernatant of primary cultured microglia.
**P < 0.01, versus Ctrl group; ##P < 0.01, versus gp120 group; n = 3.
Fig 5
Fig 5. Expression of P2X7 mRNA in different treatment groups detected by qPCR.
**P < 0.01, versus Ctrl group; ##P < 0.01, versus gp120 group; n = 5.
Fig 6
Fig 6. Expression of P2X7 protein measured by Western blot.
A, SDS-PAGE electrophoresis of P2X7 receptor proteins in primary cultured microglia. B, Relative expression level of P2X7 receptor protein in primary cultured microglia; **P < 0.01, versus Ctrl group; ##P < 0.01, versus gp120 group; n = 5.
Fig 7
Fig 7. Co-expression of CD11b and P2X7 receptor in primary cultured microglia detected by immunofluorescence double labeling.
A, Typical fluorescence image of primary cultured microglia; green signal represents CD11b staining with FITC; red signal indicates P2X7 staining with TRITC; Merge represents the P2X7 and CD11b double staining image; arrows represent microglia with co-expression of P2X7 receptor and CD11b. B, Co-expression values of CD11b and P2X7 receptor in each group; **P < 0.01, versus Ctrl group; ##P < 0.01, versus gp120 group; n = 3.
Fig 8
Fig 8. Concentrations of TNFα and IL-1β in the supernatant of primary cultured microglia.
A, TNFα concentration in each group. B, IL-1β concentration in each group; **P < 0.01, versus Ctrl group; #P < 0.05, ##P < 0.01, versus gp120 group; n = 3.
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Grants and funding

This work was supported by a grant (No. 81260187) from the National Natural Science Foundation of China (URL: http://www.nsfc.gov.cn/), a grant (No. 20153BCB23033) from the Cultivating Foundation of Young Scientists (Star of Jinggang) of Jiangxi Province (URL: http://www.jxstc.gov.cn/); all grants were received by Changshui Xu. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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