| Objective:Through the establishment of PCPA rat insomnia model,intervening with SZRD,to observe the effect of SZRD on expression of GFAP,P2X7R protein and gene mRNA in astrocytes of cerebral cortex of insomnia rats.Intervening with P2X7R antagonist and its agonist,to observe the changes of P3 8MARK gene mRNA expression in the cerebral cortex of insomnia rats after P2X7R antagonist and its agonist acted on P2X7R respectively.To further explore the possible mechanism of PCPA-induced insomnia and the mechanism of SZRD treatment of insomnia.Method:1.The healthy and clean grade SD rats were randomly divided into control group,model group,traditional Chinese medicine treatment group and traditional Chinese medicine control group.The rat insomnia model was established by intraperitoneal injection of PCPA for three days.On the 4th to 10th day,the traditional Chinese medicine treatment group and the traditional Chinese medicine control group were given SZRD gavage.The experimental groups were obtained the prefrontal cortex at the 4th,5th,6th,8th and 10th day.The expression of GFAP and P2X7R protein in the cerebral cortex of rats at different time was detected by immunofluorescence double staining.The expression of P2X7R mRNA in the cerebral cortex was measured by RT-qPCR method.2.Rats were randomly divided into normal control group,operation control group,model group,P2X receptor antagonist group,P2X7 receptor antagonist group and P2X7 receptor agonist group.Apart from the normal control group,the other groups of animals were required to carry out lateral ventricle intubation,and the animals were required to recover for 7 days after the operation.The rat insomnia model was established by intraperitoneal injection of PCPA for three days.Immediately after intraperitoneal injection of each rat,intraventricular injection was performed.P2X receptor antagonist group was given intraventricular injection of OxATP,P2X7 receptor antagonist group was given A-438079 and P2X7 receptor agonist group was given BzATP.P38MARK gene mRNA expression in cortex was detected by RT-qPCR method.Result:1.The results of immunofluorescence double staining showed that the expression of GFAP in the cerebral cortex of PCPA-induced insomnia model was significantly higher than that of control group(P<0.001).GFAP expression in the treatment group was lower than that in the model group on the 5th day(P<0.05).GFAP expression in the treatment group was significantly lower than that in the model group on the 6th day and the 8th day(P<0.01).GFAP expression in the treatment group was significantly lower than that in the model group on the 10th day(P<0.001).The expression of GFAP was decreased in the treatment group from the 4th to 10th.There was no significant difference in GFAP expression between the control group and the traditional Chinese medicine control group.P2X7R expression in the cerebral cortex of the model group was significantly higher than that of the control group at the 4th,5th,6th and 8th day(P<0.001).P2X7R expression in the treatment group was lower than that in the model group on the 6th day(P<0.05).P2X7R expression in the treatment group was significantly lower than that in the model group on the 8th day and the 10th day(P<0.001).The expression of P2X7R was decreased from the 4th to 10th day.There was no significant difference in the expression of P2X7R between the control group and the traditional Chinese medicine control group.RT-qPCR results showed that the relative expression of P2X7R mRNA in the cerebral cortex of rats with insomnia induced by PCPA was significantly higher than that of the control group(P<0.001).The relative expression of P2X7R mRNA in the cerebral cortex of the traditional Chinese medicine group was down-regulated compared with the model group on the 8th day(P<0.05).P2X7R mRNA expression in the cerebral cortex was significantly lower than that in the model group on the 10th day(P<0.001).The expression of P2X7R mRNA in the treatment group was decreased from the 5th to 10th day.There was no significant difference in P2X7R mRNA expression between control group and traditional Chinese medicine control group.2.P38MARK mRNA expression in the cerebral cortex of rats with insomnia induced by PCPA was significantly higher than that of the control group(P<0.001).The expression of P38MARK mRNA in P2X receptor antagonist group and P2X7 receptor antagonist group was significantly lower than that in model group(P<0.05).P38MARK mRNA expression in P2X7 receptor agonist group was higher than that in model group(P<0.05).There was no significant difference in P38MARK mRNA expression between normal control group and operation control group.Conclusion:1.The expression of GFAP and P2X7R protein in the cerebral cortex of PCPA-induced insomnia rats was significantly enhanced.The mechanism of SZRD treatment of insomnia may be related to the decrease of protein expression of GFAP and P2X7R in the cerebral cortex.2.The expression of P2X7R mRNA in the cerebral cortex of the rats with insomnia induced by PCPA was significantly up-regulated.The mechanism of SZRD treatment of insomnia may be related to down-regulation of P2X7R mRNA expression in the cerebral cortex.3.The expression of P38MARK mRNA in cortex of PCPA insomnia rats was significantly up-regulated.P2X7R antagonist can down-regulate the expression of P38MARK mRNA,while P2X7R agonist can up-regulate the expression of P38MARK mRNA,indicating that PCPA-induced insomnia can effectively activate P2X7R and cause P38MARK activation. |
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