| Objective:This study focuses on the neuroprotective effect of Paeoniflorin on neonantal rat hippocampal slice cultures after oxygen-glucose deprivation and the regulating effect of Paeoniflorin on the expressions of the components of NLRP3 and NLRP1 inflammasome as well as the release of inflammatory cytokines and apoptosis in the downstream. The study therefore provides a new clue to the neuroprotective mechanisms of Paeoniflorin.Methods:(1) Different time of oxygen-glucose deprivation (OGD) on rat hippocampal slices: slices were separated into four groups after organotypic hippocampal slice cultures following the Stoppini method:Control group, OGD 30 min group, OGD 45 min group and OGD 60 min group, the Control group received no treatment. The culture medium were replaced by 1 mL of PBS, the slices were then incubated in the incubator under the conditions of 94%N2, 5%CO2,1%O2 and 37℃ for 30,45 and 60 minutes. After the incubation, PBS were replaced by fresh culture medium and the slices were then cultured under the conditions of 5% CO2 for further 48 hours. Propidium Iodide (PI) staining method was used to determine the damages in the slices and therefore the most optimal length of time of OGD.(2) Neuroprotective effect of Paeoniflorin on hippocampal slices after OGD:slices were grouped as Control group, OGD/R group, Paeoniflorin-Low Concentration (PL,1μM) group, Paeoniflorin-Medium Concentration (PM,10 μM) group and Paeoniflorin-High Concentration (PL,100 μM) group. All groups were treated with OGD except Control group. Respective concentrations of Paeoniflorin were then added to the culture medium for a further culture of 24 hours. PI staining was used to determined the neuro-damages in the slices.(3) Effects of Paeoniflorin on NLRP3 and NLRP1 inflammasome as well as the release of inflammatory cytokines and apoptosis in the downstream in hippocampal slices after OGD: after 24 of drug intervene, ELISA was applied to determine the expressions of IL-18 and IL-1β in the supernate; TUNEL assay was applied to determine the apoptosis ratio; RT-qPCR was applied to determine the gene expressions of NLRP3, NLRP1, ASC and Caspase-1 and Western Blot was applied to determine the protein expressions of NLRP3, NLRP1, ASC, Caspase-1/3, Bax and BcL-2.Result:(1) After the OGD process, OGD 30 min group had no significant change in comparison to Control group (P>0.05); However, OGD 45 and 60 min group showed strong PI uptake intensity comparing with Control group (P<0.01). This result indicates that after 45 minutes of OGD process, the damage on hippocampal slices were significantly increased. Moreover, 60 minutes of OGD process might cause irreversible damage, thus we regard the length of 45 mintues as the best length of time of OGD process.(2) The PI uptake intensity was significantly increased in comparison to Control group (P<0.01), this result indicates that the OGD model was successfully built. After the intervene of different concentrations of Paeoniflorin, the PI uptake intensity was significantly decreased comparing with OGD group following a concentration-dependent manner.(3) The protein and gene expressions of NLRP3, NLRP1, ASC and Caspase-1 were significantly increased in comparison to Control group (P<0.01). The protein expressions of NLRP1, ASC, NLRP3, Cl.Caspase-1 and Pre.Caspase-1 in PH and PM group were significantly reduced comparing with OGD group (P<0.01). The gene expressions of NLRP1, ASC, NLRP3, Caspase-1 in PH, PM and PL group were significantly reduced comparing with OGD group (P<0.01).(4) The protein expressions of IL-18, IL-1p, Bax and Cl.Caspase-3 as well as the apoptosis ratio were significantly increased in comparison to Control group, and the protein expression of BcL-2 was significantly reduced in comparison to Control group (P<0.01). The protein expressions of IL-18, IL-1β,Bax and Cl.Caspase-3 as well as the apoptosis ratio in PH group were significantly reduced in comparison to OGD group, and the protein expression of BcL-2 was significantly increased in comparison to OGD group (P<0.01)Conclusion:Paeoniflorin has the neuroprotective effects on hippocampal slices processed with OGD, the mechanism might relate to the down-regulating effect of Paeoniflorin on the expressions of the components of NLRP3 and NLRP1 inflammasome, thus mediating the release of inflammatory cytokines and apoptosis in the downstream. |
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