Effects of paraquat on proliferation and neurogenesis of neural stem cells by promoting secretion of IL-1β by microglia

Background Paraquat (PQ) is a widely used herbicide that exerts neurotoxicity. The effects of PQ on neural stem cells (NSCs) through microglia mediated neuroinflammation remain limitedly studied.

Objective To investigate the effects of PQ on the proliferation and neurogenesis of NSCs through neuroinflammation mediated by microglia.

Methods Microglial cell lines (BV2 cells) and primary NSCs were used. BV2 cells were exposed to 0, 1, 3.3, 10, 33, and 100 μmol·L⁻¹ of PQ for 6 h followed by viability assessment. The highest PQ concentration that had no effect on cell viability was selected as the final exposure concentration (33 μmol·L⁻¹). To exclude the direct effect of PQ on NSCs, after the BV2 cells were cultured in complete medium containing 33 μmol·L⁻¹ PQ for 6 h, the BV2 culture medium was replaced by NSCs complete medium without PQ for 24 h. The concentration of interleukin-1β (IL-1β) in supernatant was detected by enzyme-linked immune sorbent assay. Besides, in order to detect the effects of IL-1β on NSCs proliferation and neurogenesis, NSCs isolated from hippocampus of adult mice were cultured in the supernatant obtained above and divided into four groups: control supernatant + control antibody, control supernatant + IL-1β neutralizing antibody (10 ng·mL⁻¹), PQ supernatant + control antibody, PQ supernatant + IL-1β neutralizing antibody (10 ng·mL⁻¹). Proportion of Ki67-positive NSCs was detected by flow cytometry (FCS) and immunofluorescence after 24 h culture, and neurogenesis was detected by FCS and immunofluorescence after 3-7 d of culture.

Results The IL-1β concentration in the supernatant of BV2 cells was significantly increased after the 33 μmol·L⁻¹ PQ exposure compared with the control group (t=3.020, P<0.05). After the NSCs were cultured with the supernatant of PQ-treated BV2 cells, the proportion of Ki67-positive NSCs (t=9.129, P<0.01) and the proportion of newborn neurons (t=4.638, P<0.01) were significantly decreased compared to the control group. After neutralizing IL-1β, the proportion of Ki67-positive NSCs (t=22.05, P<0.01) and the proportion of newborn neurons (t=11.09, P<0.01) were significantly higher than those in the un-neutralized group. The results of immunofluorescence detection also showed that after neutralizing IL-1β secreted by 33 μmol·L⁻¹ PQ-treated BV2 cells, the number of Ki67-positive NSCs and the number of newborn neurons were significantly higher than those in the un-neutralized group.

Conclusion The secretion of IL-1β by microglia is increased after PQ treatment, resulting in a decrease in the proliferation and neurogenesis of NSCs. These results suggest that neuroinflammation is involved in NSCs damage caused by PQ.