Effects Of Xylazine On CaMK/CREB/BDNF Signaling Pathway In Nervous System Of Rat Brain

Xylazine is one of the most important components of multiple anesthetics.It is very important to study the mechanism of anesthesia for the study of high-efficiency and low side effects of compound anesthetics.At present,there are few studies on the neural signaling pathways of xylazine anesthesia.In this experiment,the anesthesia mechanism of xylazine was investigated at the molecular level,which provided a reasonable molecular theoretical basis for future clinical anesthesia.In this experiment,adult Wistar rats and PC12 neurons were studied.In vivo experiments were performed with intraperitoneal injection of xylazine at 70 mg/kg.Experimental groups were determined by continuously observing the behavioral changes in rats,and further examine the index changes in different brain regions of rats during different anesthesia periods,RT-PCR was used to detect mRNA expression of NR1,NR2 A,NR2B,CaMKII,CaMKIV,CREB,BDNF,c-jun,c-fos,and fos-b.Western Blot was used to detect protein expression of NR1,NR2 A,NR2B,CaMKII,p-CaMKII,CaMKIV,p-CaMKIV,CREB,p-CREB,and BDNF.In vitro cell experiments by adding xylazine to the cultured PC12 nerve cells and targeting inhibition of CaMKII and CaMKIV genes by siRNA technology,and further explore investigate the effects of CaMKII and CaMKIV on downstream CREB/BDNF pathways and IEGs(c-jun,c-fos and fos-b),RT-PCR was used to detect mRNA expression of CaMKII,CaMKIV,CREB,BDNF,c-jun,c-fos,and fos-b.Western Blot was used to detect protein expression of CaMKII,p-CaMKII,CaMKIV,p-CaMKIV,CREB,p-CREB,and BDNF.The results:1.The expression of NR1 protein was decreased in cerebellum,hippocampus and thalamus,but increased in brain stem.NR1 mRNA expression was not changed in five brain regions compared with control group.The expression of NR2 A protein was decreased in brain,cerebellum,hippocampus and thalamus.NR2 A mRNA expression was only decreased in brain.NR2 B mRNA expression was decreased in all five brain regions.NR2 B mRNA expression was only decreased in brain.2.CaMKII protein expression was decreased in cerebellum,thalamus and brainstem.CaMKII mRNA expression was only decreased in brainstem.p-CaMKII protein expression was decreased in all five brain regions.CaMKIV protein expression was decreased in five brain area,CaMKIV mRNA expression was decreased in the thalamus and brainstem,and p-CaMKIV protein expression was decreased in all five brain regions.3.CREB protein expression was decreased in cerebellum,hippocampus,thalamus and brainstem.CREB mRNA expression decreased in thalamus and brainstem.p-CREB protein expression was decreased in all five brain regions.BDNF protein expression was decreased in all five brain regions,and BDNF mRNA expression was decreased in all five brain regions.The expression of c-jun,c-fos and fos-b mRNA were decreased in all five brain regions.4.Xylazine Significantly inhibited CaMKII,CaMKIV,CREB,BDNF,c-jun,c-fos,and fos-b mRNA expression in PC12 nerve cells,as well as CaMKII,p-CaMKII,CaMKIV,p-CaMKIV,CREB,p-CREB,and BDNF protein expression significantly;The mRNA expression of CaMKII,CREB,BDNF,c-jun,c-fos,and fos-b in the cells treated with siRNA-CaMKII were significantly decreased,and the protein expression of CaMKII,p-CaMKII,CREB,p-CREB,and BDNF were significantly decreased;The mRNA expression of CaMKIV,CREB,BDNF,c-jun,c-fos,and fos-b in the cells treated with siRNA-CaMKIV were significantly decreased,and the protein expression of CaMKII,CaMKIV,p-CaMKIV,CREB and BDNF were significantly decreased.The results of the study showed that xylazine induced anaesthesia in rats by influencing the expression of the above-mentioned related factors and the cascade reaction between them,and by inhibiting the CaMKII,CaMKIV/CREB/BDNF pathway.