Effect Of CAMP/PKA/CREB Signaling Pathway On Hypoxic Preconditioning Reversing Propofol-induce Neurotoxicity In Hippocampus Of Neonatal Rats

Part 1 Hypoxic preconditioning inhibits the neurotoxicity of ropofol in developing brain of neonatal ratsObjective: Establish an hypoxic preconditioning model.To observe the effect of hypoxic preconditioning on propofol-induce neuroapoptosis in the hippocampus of neonatal rats.Methods: Fifty SD rats of 7 days were randomly divided into normal saline group(N group),fat emulsion group(F group),hypoxic preconditioning group(H group),Propofol group(P group),hypoxic preconditioning + propofol injection group(HP group).N group,F group were given saline and fat emulsion respectively,with the same volume of propofol by intraperitoneal injection;H group was given only hypoxic preconditioning to establish the hypoxic preconditioning model,the sealed tank filled with a concentration of 8% O2 +92% N2 mixed gas,the pups were placed in the tank for 10 minutes,and then placed in the air for 10 minutes,the oxygen concentration was detected every 3minutes,the pups were given 5 cycles of hypoxic preconditioning,then were placed in the air for 2 hours;P group,the pups were given propofol(100 mg / kg)by two times intraperitoneal injection(each 50 mg / kg),the first injection(propofol 50 mg / kg),after the rats restore physical activity,then given 50 mg /kg,the total amount was 100 mg / kg;hypoxic preconditioning + propofol group(HP group),given hypoxic preconditioning first(same as H group)and then given intraperitoneal propofol injection(same as the P group).HE staining was used to observe and evaluate the morphology of neurons.The morphology of neurons was observed by transmission electron microscopy.The apoptosis of hippocampal neurons was observed by TUNEL staining.The expressions of Bcl-2,Bax and cleaved-caspase-3 were detected by western blot.The expressions of cleaved-caspase-3 was detected by immunohistochemistry.Results:1.Compared with N group,the results of HE showed that the pyramidal cells were disordered and the cell bodies were reduced and nuclear pyknosis in P group.Electron microscopy showed that chromatin margination,nuclear pyknosis and nuclear membrane invagination in P group;TUNEL assay showed that the number of TUNEL positive cells was higher in P group(P <0.05);The Western blot results showed that the expression of Bcl-2 protein was decreased(P <0.05),while the expression of Bax and cleaved-caspase-3 protein were increased in P group(P<0.05).The results of immunohistochemistry showed that cleaved-caspase-3 protein expression was increased in P group(P <0.05).2.Compared with P group,the results of HE showed that the pyramidal cells arranged neatly,the cytoplasm was richer and the nucleus was more clear in HP group.The electron microscopy showed that a slight decreased chromatin and more uniform chromatin density in HP group,TUNEL results showed thatthe number of TUNEL positive cells was decreased in HP group(P <0.05);western blot results showed that the expression of Bcl-2 protein was increased(P<0.05);while the expression of Bax protein and cleaved-caspase-3 protein were decreased in HP group(P <0.05).The results of immunohistochemistry showed that the expression of cleaved-caspase-3 protein was lower in HP group(P<0.05).Conclusions:1.Propofol has a damaging effect,and induced neuroapoptosis in hippocampal of neonatal rats.2.Hypoxic preconditioning reduced propofol-induce damage and attenuated neuroapoptosis in hippocampal of neonatal rats.Part 2 The effect of c AMP / PKA / CREB signaling pathway n hypoxic preconditioning reversing propofol-induce eurotoxicity in hippocampus of neonatal ratsObjective: To investigate the Effect of c AMP / PKA / CREB signaling pathway on hypoxic preconditioning reversing propofol-induce neurotoxicity in hippocampus of neonatal rats and elucidate the role of c AMP / PKA / CREB signaling pathway in hypoxic preconditioning reversing the toxicity of propofol.Methods: Seventy SD rats of 7 days were randomly divided into normal saline group(N group),Propofol group(P group),hypoxic preconditioning +propofol injection group(HP group),hypoxic preconditioning + propofol+H89injection group(HPH89group),propofol + H89 injection group(PSP group),normal saline intracerebroventricular injection(NI group),DMSO intracerebroventricular injection(DI group).N group was given saline,with the same volume of propofol by intraperitoneal injection;P group,the pups were given propofol(100mg / kg)by two times intraperitoneal injection(each 50 mg /kg),the first injection(propofol 50 mg / kg),after the rats restore physical activity,then given 50 mg / kg,the total amount was 100 mg / kg;HP group was given hypoxic preconditioning to establish the hypoxic preconditioning model,the sealed tank filled with a concentration of 8% O2 + 92% N2 mixed gas,the pups were placed in the tank for 10 minutes,and then placed in the air for 10 minutes,the oxygen concentration was detected every 3 minutes,the pups were given 5 cycles of hypoxic preconditioning,then were placed in the air for 2hours,then given propofol by intraperitoneal injection(same as P group);hypoxic preconditioning + propofol + H89 group(HPH89 group)were given PKA inhibitor H89 intracerebroventricular injection,after 30 min recovery,hypoxic preconditioning and propofol injection(same as HP group);propofol +sp-camp group(PSP group)was given intracerebroventricular injection of PKA agonist sp-camp,after 30 min recovery,propofol was injected(same as P group);saline + intracerebroventricular injection of NS and DMSO in the same volume was performed with intracerebroventricular injection(NI group and DI group).HE staining was used to observe and evaluate the morphology of neurons.The morphology of neurons was observed by transmission electron microscopy.The content of c AMP was detected by ELSIA,The expressions of PKA,p CREB,Bcl-2,Bax and cleaved-caspase-3 were detected by western blot.The expressions of PKAc and p CREB was detected by immunohistochemistry.Results:1.Compared with N group,the results of HE showed that the pyramidalcells were disordered and the cell bodies were reduced and nuclear pyknosis in P group.Electron microscopy showed that chromatin margination,nuclear pyknosis and nuclear membrane invagination in P group;ELSIA results showed that the c AMP content decreased in P group;the western blot results showed that the expression of Bcl-2,PKA,p CREB protein was decreased(P <0.05),while the expression of Bax and cleaved-caspase-3 protein were increased(P<0.05);The results of immunohistochemistry showed that the expression of PKA and p CREB were decreased in P group(P <0.05).2.Compared with P group,the results of HE showed that the pyramidal cells arranged neatly,the cytoplasm was richer and the nucleus was more clear in HP group and PSP group;The electron microscopy showed that a slight decreased chromatin and more uniform chromatin density in HP group and PSP group;Western blot results showed that the expression of Bcl-2,PKA and p CREB protein was increased(P <0.05);while the expression of cleaved-caspase-3 protein was decreased in HP group and PSP group(P <0.05).The results of immunohistochemistry showed that the expression of PKA and p CREB protein was higher than the P group(P <0.05).Conclusions:1.Propofol had a damaging effect,and induced neuroapoptosis in hippocampal of neonatal rats by down-regulation of c AMP / PKA / CREB signal pathway.2.Hypoxic preconditioning reduced propofol-induce damage and attenuated neuroapoptosis by reducing the down-regulation of c AMP / PKA /CREB signal pathway in hippocampal of neonatal rats.Part 3 The expression of GLUT1,GLUT3,Drp1 and MFN2 n hypoxia preconditioning attenuating propofol neurotoxicityObjective: To investigate the expression of GLUT1,GLUT3,Drp1 and MFN2 in hypoxia preconditioning reversing propofol neurotoxicity,and explore the relationship between hypoxic preconditioning reversing propofol neurotoxicity and the mechanism.Methods: Fifty SD rats of 7 days were randomly divided into normal saline group(N group),fat emulsion group(F group),hypoxic preconditioning group(H group),propofol group(P group),hypoxic preconditioning + propofol injection group(HP group).N group,F group were given saline and fat emulsion respectively,with the same volume of propofol by intraperitoneal injection;H group was given only hypoxic preconditioning to establish the hypoxic preconditioning model,the sealed tank filled with a concentration of 8% O2 +92% N2 mixed gas,the pups were placed in the tank for 10 minutes,and then placed in the air for 10 minutes,the oxygen concentration was detected every 3minutes,the pups were given 5 cycles of hypoxic preconditioning,then were placed in the air for 2 hours;P group,the pups were given propofol(100mg / kg)by two times intraperitoneal injection(each 50 mg / kg),the first injection(propofol 50 mg / kg),after the rats restore physical activity,then given 50 mg /kg,the total amount was 100 mg / kg;hypoxic preconditioning + propofol group(HP group),given hypoxic preconditioning first(same as H group)and then given intraperitoneal propofol injection(same as the P group).The morphology of neuronal mitochondrial morphology was observed by transmission electron microscopy.The content of ATP was detected by ATP test.The expressions of GLUT1?GLUT3?p Drp1?Drp1and MFN2 were detected by western blot.Theexpressions of GLUT1 and GLUT3 was detected by immunohistochemistry.Results:1.Compared with N group,the electron microscopy showed that mitochondria swollen,part of the mitochondrial inner membrane rupture,cristae loose dissolution,and even cristae fracture,matrix density decreased in P group;ATP assay showed that the content of ATP was decreased in P group(P <0.05);The western blot results showed that the expression of GLUT1 and GLUT3 protein was increased in H group and HP group(P <0.05),while the expression of Bax and cleaved-caspase-3 protein were increased(P<0.05).The results of immunohistochemistry showed that cleaved-caspase-3 protein expression was increased in P group(P <0.05).2.Compared with P group,the electron microscopy showed that mitochondria slightly swollen,more complete mitochondrial cristae,matrix density more uniform in HP group.ATP assay showed that the content of ATP was increased in HP group(P <0.05);Western blot results showed that the expression of p Drp1,GLUT1,GLUT3 protein was increased in HP group(P<0.05);The results of immunohistochemistry showed that the expression of GLUT3 protein was higher than the P group(P <0.05).Conclusions:1.Propofol had a damaging effect in hippocampal of neonatal rats.The mechanism may be related to the excessive mitochondrial fission and the decrease of GLUT3.2.Hypoxic preconditioning reduced propofol-induce damage and attenuated the excessive mitochondrial fission and the decrease of GLUT3.