Inhibition Of ERK/Calpain-2 Pathway On Neuroinflammation And Necroptosis In Brain Injury Of Rats After Cardiopulmonary Resuscitation

Background and objective:Global cerebral ischemia-reperfusion injury(CIRI)after cardiac arrest(CA)and cardiopulmonary resuscitation(CPR)is closely related to the poor neurological prognosis of the patients.The pathophysiological mechanism of CIRI is complex,in which programmed cell death pathways(such as autophagy,necroptosis)play an important role.The necroptosis is mediated by a signal complex called necrosome,which can be initiated by the pro-inflammatory factor tumor necrosis factor-α(TNF-α),and participates in the neuronal death after CIRI.Extracellular signal-regulated kinase(ERK)is a member of the mitogen-activated protein kinase(MAPK)family,which is involved in the regulation of intracellular physiological functions.Calpain is a downstream protein of ERK.Its activity depends on the concentration of calcium and is closely related to CIRI.In previous CPR-induced CIRI studies,it was found that ERK inhibitor PD98059 improved neurological function by inhibiting mitochondrial division and autophagy which mediated by ERK phosphorylation.Whether this protective mechanism is related to calpain and whether it is involved in the regulation of neuroinflammation and necroptosis is still unclear.In this study,we established a CA-CPR rat model to observe the effects of PD98059 on brain calpain-related proteins,and understand pathophysiological changes of calpain involved in CIRI,focusing on the potential role and molecular mechanism of ERK/calpain-2 pathway in cerebral neuroinflammation and necroptosis.Methods:The first part is the effect of ERK inhibitor PD98059 on calpain-related proteins in brain cortex of rats after CA-CPR: Except for the Sham group,the other rats were all induced by esophageal electrical stimulation to induce cardiac arrest and cardiopulmonary resuscitation.Sprague-Dawley rats that successfully restored spontaneous circulation were divided into 3 groups: model control group(NS),8% dimethyl sulfoxide solvent group(DMSO)and the PD98059group(PD,0.3 mg/kg)。All rats were slowly injected with the corresponding drugs through the femoral vein within 30 minutes after resuscitation.Record the CPR duration of model rats and observe the following indicators of all rats 24 h after resuscitation: 1)survival rate and neurological defect score(NDS);2)observation of the pathological changes of cerebral cortex by HE staining and Nissl staining;3)western blotting(WB)was used to detect the expression of phosphorylated-ERK(p-ERK),ERK,calpain-1,calpain-2 and calpain inhibitor protein(calpastatin);4)the co-expression of p-ERK and calpain-2 was detected by double immunofluorescence.The second part is the effect and mechanism of calpain inhibitor MDL28170 on calpain-related proteins in cerebral cortex of rats after CA-CPR:The SD rats that successfully restored spontaneous circulation were divided into4 groups: Sham group,NS group,DMSO group,low-dose MDL28170 group(MDL-l,1.5 mg/kg)and high-dose MDL28170 group(MDL-h,3.0 mg/kg).Record the CPR duration of model rats and observe the following indicators of all rats 24 h after resuscitation: 1)survival rate and NDS after resuscitation;2)HE staining and Nissl staining were used to observe the pathological changes of cerebral cortex;3)WB was used to detect the expression of calpain-1,calpain-2,calpastatin proteins and pro-inflammatory factors Interleukin-1beta(IL-1β)and TNF-α in cerebral cortex.After determining the effective dose of MDL28170,the following indexes were observed in Sham,NS and MDL-h groups: 1)ultrastructure and autophagy of neurons were observed under electron microscope;2)WB was used to detect the expression of P62,Beclin-1 and Lc3 in cerebral cortex;3)double immunofluorescence staining was used to detect the co-expression of Lc3 and calpain-2.The third part is the effect of inhibition of ERK/calpain-2 pathway on cerebral neuroinflammation and necroptosis in rats after CA-CPR: The SD rats that successfully restored spontaneous circulation were divided into 6 groups:Sham group,NS group,DMSO group,MDL group(MDL28170,3.0 mg/kg),PD group(PD98059,0.3 mg/kg)and MP group(3.0mg/kg MDL28170 +0.3mg/kg PD98059).Record the CPR duration of model rats and observe the following indicators of all rats 24 h a fter resuscitation: 1)survival rate and NDS of rats after resuscitation;2)HE staining,Nissl staining and transmission electron microscope were used to observe the morphological damage of neurons;3)WB and immunohistochemical detection of p-ERK,ERK,calpain-2,neuroinflammatory markers [glial fibrillary acidic portein(GFAP),ionized calcium binding adaptorm(Iba1),Interleukin-1beta(IL-1β),TNF-α,tumor necrosis factor receptor 1(TNFR1)] and necroptosis proteins[receptor-interacting serine/threonine-protein kinase 1(RIPK1),RIPK3,mixed series protein kinase-like domains(MLKL)and phosphorylate-MLKL(p-MLKL)] in cerebral cortex.4)Use multiple immunofluorescence staining methods to detect the co-expression of p-ERK,calpain-2 and RIPK3 in neurons;as well as the multiple fluorescence of GFAP,Iba1 and RIPK3,observe RIPK3 in astrocytes and microglia The level of expression in the cell.Results:The first part: there was no significant difference in the duration of cardiopulmonary resuscitation and 24 h survival rate of experimental rats among the groups of Sham,NS,DMSO and PD(P>0.05).The NDS of NS and DMSO groups was significantly lower than that of the Sham group(all P>0.05),while the NDS of PD group was significantly higher than that of NS group(P <0.05).The pathological damage of the brain tissue in PD group was significantly improved compared with the NS and DMSO groups;the WB results showed that the p-ERK/ERK ratio and calpain-2 levels in the PD group were significantly lower than those in the NS and DMSO groups(P<0.05),but there was no significant difference in the expression of calpain-1 and calpastatin proteins(P>0.05).Double immunofluorescence staining showed that p-ERK and calpain-2 were co-expressed in the cytoplasm and nucleus,and the co-expression rate of p-ERK and calpain-2 in the PD group was significantly lower than that of the NS and DMSO groups(P <0.05).The second part: there was no significant difference in CPR duration time and 24 h survival rate among the groups of Sham,NS,DMSO,MDL-l and MDL-h(P>0.05).Compared with Sham group,the NDS of the other groups was decreased significantly,and the NDS of MDL-h group was significantly higher than that of NS and DMSO groups(P<0.05).After using MDL28170,the pathological damage of brain tissue in MDL-l and MDL-h groups was improved than that in NS and DMSO groups,and the MDL-h group improved the most(P<0.05).The results of WB showed that there was no significant difference in the levels of calpain-1 and calpastatin proteins in each group(P>0.05).The levels of calpain-2,IL-1βp17(mature IL-1β)and TNF-α in cerebral cortex of NS and DMSO groups were significantly up-regulated compared with the Sham group(P<0.05),and the expression levels of these proteins were significantly down-regulated in MDL-h group compared with NS and DMSO groups(P<0.05),while the MDL-l group did not show similar effects.Therefore,comparing the MDL-h group with the Sham and NS groups,it was found that the electron microscope showed that the neurons in the Sham group were normal,the NS group had obvious changes in cell morphology,the nucleus was shrank and deformed,the organelles were damaged and degenerated,and there were autophagy at various stages;while the neuronal damage was significantly improved and the autophagosomes decreased in the MDL-h group.WB showed that the expression of P62 in the cerebral cortex of the NS group was decreased,while the expression of Beclin-1 and the ratio of Lc3 II / Lc3Ⅰwas significantly up-regulated(P <0.05).Compared with the NS group,Beclin-1 and the ratio of Lc3 II / Lc3Ⅰdecreased,while the P62 level increased in MDL-h group(P<0.05).The third part: there was no significant difference in CPR duration time and24 h survival rate among Sham,NS,DMSO,MDL,PD and MP groups(P>0.05).In NS and DMSO groups,morphological changes of injured brain tissues and neurons were observed,NDS decreased,ERK/calpain-2 pathway was activated,and the expressions of neuroinflammatory markers(GFAP,Iba1,IL-1β,TNF-α)and necroptosis proteins(TNFR1,RIPK1,RIPK3 and the ratio of p-MLKL/MLKL)were significantly up-regulated(P<0.05).After the treatment with PD98059 or MDL28170,or the combination of these two drugs,compared with the NS and DMSO groups,the brain function was improved,and the ratio of p-ERK/ERK and calpain-2 levels were down-regulated(P<0.05).After the combination of the two inhibitors,ERK and calpain-2 were blocked at the same time,while the expression of neuroinflammatory and necroptosis-related proteins was significantly down-regulated(P<0.05).Conclusion:This study found that ERK and calpain-2 jointly participated in CIRI after establishing a CA-CPR rat model.PD98059 inhibited the expression of calpain-2 while inhibiting the phosphorylation of ERK,and MDL28170 played a role of neuroprotection by blocking calpain-2 and its anti-inflammatory and anti-autophagy.PD98059 and MDL28170 were used to connect the ERK/calpain-2 pathway,by inhibiting the activation of neuroimmune cells(astrocytes and microglia)and reducing the release of pro-inflammatory cytokines,thereby inhibiting TNF-α-mediated necroptosis occurs,which ultimately reduced neurological damage.Therefore,ERK/calpain-2 can be used as a new target for CIRI treatment,and provides a new idea for the treatment of patients with CA-CPR combined with neurological injury in the future.