| Objective: To investigate the effect of P2X4 receptor(P2X4R)overexpression on the expression of interleukin-6(IL-6)in the substantia nigra(SN)of Parkinson’s disease(PD)rats induced by 6-OHDA,and to explore the role of P2X4 R and its mediating inflammatory factor IL-6 in the pathogenesis of PD.Methods: Male Wistar rats were randomly divided into 6 groups: control group,6-OHDA group,lentivirus vectors carrying P2X4 R overexpression target gene(P2X4R-OE)group,vector carrying P2X4 R negative control(P2X4R-NC)group,P2X4R-OE + 6-OHDA group and P2X4R-NC + 6-OHDA group.The treatment methods of each group of rats were as follows: 1.Control group: normal saline was injected into the left SN.2.6-OHDA group: 6-OHDA was injected into the left SN.3.P2X4R-OE group: lentivirus carrying P2X4 R overexpression target gene was injected into the left SN.4.P2X4R-NC group: lentivirus carrying no load RNA was injected into the SN.5.P2X4R-OE+6-OHDA group: lentivirus carrying P2X4 R overexpression target gene was injected into the left SN.The animals were re-injected with 6-OHDA after one week of initial treatment.6.P2X4R-NC + 6-OHDA group: lentivirus carrying no load RNA was injected into the left SN.The animals were re-injected with 6-OHDA after one week of initial treatment.Two weeks after modeling,rats were given intraperitoneal injection of apomorphine for behavioral detection.The numbers of rotations to the right within 30 minutes was recorded.The successful induction of PD was validated with the rotation speed,which was at least ≥7/min.The rats were induced once a week for 3 consecutive weeks.After excluding the animals that failed the test,20 animals were kept in each group.After 4 weeks of modeling,rats in each group were anesthetized by isoflurane,then decapitated.Brain tissues were taken out for different treatment and preservation.The number of TH positive dopaminergic neurons in the SN was detected by immunofluorescence staining,and the expression levels of P2X4 R and IL-6 protein in the SN were detected by Western blot.Results: 1.Immunofluorescence staining: Compared to the control group,the number of TH positive DA neurons in the SN of 6-OHDA group and P2X4R-NC + 6-OHDA group decreased significantly(P < 0.001);compared to P2X4R-NC+ 6-OHDA group,the number of TH positive dopaminergic neurons in the SN of P2X4R-OE + 6-OHDA group decreased significantly(P< 0.001).There was no significant difference in TH positive dopaminergic neurons between control group,P2X4R-NC group and P2X4R-OE group.2.Western blots: a.Detection of P2X4 R expression in the SN: Compared to the control group,the expression of P2X4 R in the SN of rats in 6-OHDA group and P2X4R-OE group was significantly increased(P < 0.001).Compared to the control group,the expression of P2X4 R in the SN of rats in P2X4R-NC group had no significant difference.Compared with P2X4R-NC + 6-OHDA group,the expression of P2X4 R protein in P2X4R-OE + 6-OHDA group was significantly increased(P < 0.001).b.Detection of IL-6 expression in the SN: Compared to the control group,the expression of IL-6 in the SN of 6-OHDA group was significantly increased(P < 0.001).There was no significant difference between P2X4R-NC group and P2X4R-OE group(P > 0.05).The expression of IL-6 in the SN of P2X4R-OE + 6-OHDA group was significantly higher than that of P2X4R-NC + 6-OHDA group,the difference was statistically significant(P < 0.001).Conclusions: The results of this study showed that the injection of lentivirus vector carrying P2X4 R overexpression target gene can reduce TH positive dopaminergic neurons,and upregulate the expression of inflammatory factor IL-6 in the substantia nigra of rats.According to the existing experimental evidence,we conclude that ATP binds to P2X4 R on the surface of microglia,and then activates microglial downstream signaling pathways to produce cytokines and interleukin factors such as IL-6.The released cytokines then bind to their respective receptors on microglial surface to further accelerate the activation of microglia.Our study can provide further theoretical basis for clarifying the etiology and pathogenesis of PD. |
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