神经退行性病
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Figure 1｜The study groups and interventions.
Rats were divided randomly into four treatment groups (n = 20 in each group; Figure 1). Control group: after 1 week of pretreatment with 0.9% physiological saline, we injected 0.02% ascorbic acid into the substantia nigra pars compacta (SNpc) stereotactically; 6-hydroxydopamine (6-OHDA) group: after 1 week of pretreatment with 0.9% physiological saline, we injected 6-OHDA into the SNpc stereotactically to establish PD; 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) group: after 1 week of pretreatment with 5-BDBD (a selective P2X4R antagonist), we injected ascorbic acid into the SNpc stereotactically; and 5-BDBD + 6-OHDA group: after 1 week of pretreatment with 5-BDBD, we injected 6-OHDA into the SNpc stereotactically.
Figure 2｜Effect of 5-BDBD on the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta.
There were significantly fewer TH-positive neurons in the 6-OHDA and 5-BDBD + 6-OHDA groups compared with the 5-BDBD and the control groups (all P < 0.01). Compared with the 6-OHDA group, the quantity of TH-positive neurons increased significantly in the 5-BDBD + 6-OHDA group (P < 0.01). There were no significant differences in the number of TH-positive neurons in the 5-BDBD group compared with the control group (P > 0.05; Figure 2).
Figure 3｜Effect of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) on the protein expression of P2X4R (A), NLRP3 (B), caspase-1 (C), IL-1β (D), and IL-18 (E) in the substantia nigra pars compacta.
We investigated the effect of 5-BDBD on the mRNA and protein expression levels of P2X4, NLRP3, caspase-1, IL-1β, and IL-18 in the SNpc. The mRNA and protein levels of P2X4, NLRP3, caspase-1, IL-1β, and IL-18 were significantly higher in the 6-OHDA and 5-BDBD + 6-OHDA groups than the control and 5-BDBD groups (all P < 0.01). The mRNA and protein levels of P2X4, NLRP3, caspase-1, IL-1β, and IL-18 were significantly lower in the 5-BDBD + 6-OHDA group than the 6-OHDA group (P < 0.01). There were no significant statistical differences in P2X4 mRNA and protein expression between the control and 5-BDBD group (P > 0.05; Figure 3 and Table 1).
Figure 4｜Effect of P2X4R overexpression and silencing on the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta.
Figure 5｜Distribution of the lentivirus in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta in P2X4R-RNA and P2X4R-siRNA groups.
The numbers of TH-positive neurons were significantly lower in the 6-OHDA, P2X4R-NC + 6-OHDA, P2X4R-RNA + 6-OHDA, and P2X4R-siRNA + 6-OHDA groups compared with the control, P2X4R-NC, P2X4R-RNA, and P2X4R siRNA groups (all P < 0.01). Compared with the P2X4R-NC + 6-OHDA group, there were significantly fewer TH-positive neurons in the P2X4R-RNA + 6-OHDA group (P < 0.01) and significantly more TH-positive neurons in the P2X4R-siRNA + 6-OHDA group (P < 0.01). There were no significant differences between the control and P2X4R-NC groups (P > 0.05; Figures 4 and 5).
Figure 6｜Effect of a lentivirus carrying P2X4R on the protein expression levels of P2X4R (A), NLRP3 (B), caspase-1 (C), IL-1β (D), and IL-18 (E) in the substantia nigra pars compacta.
Figure 7｜Effect of the lentivirus carrying siRNA for P2X4R on the protein expression levels of P2X4R (A), NLRP3 (B), caspase-1 (C), IL-1β (D), and IL-18 (E) in the substantia nigra pars compacta.
NLRP3, caspase-1, IL-1β, and IL-18 protein levels were significantly higher in the 6-OHDA, P2X4R-NC + 6-OHDA, P2X4R-RNA + 6-OHDA, and P2X4R-siRNA + 6-OHDA groups compared with the control, P2X4R-NC, P2X4R-RNA, and P2X4R siRNA groups (P < 0.01). NLRP3, caspase-1, IL-1β, and IL-18 protein levels were significantly higher in the P2X4R-RNA + 6-OHDA group (P < 0.01) and significantly lower in the P2X4R-siRNA + 6-OHDA group (P < 0.01). There were no significant differences in the expression of NLRP3, caspase-1, IL-1β, and IL-18 proteins between the control and P2X4R-NC groups (P > 0.05; Figures 6 and 7).