| Research Background and Objectives:Cerebral ischemia-reperfusion injury after cardiac arrest is the key pathological process of cerebral resuscitation after cardiopulmonary resuscitation.Recent studies show that neuronal autophagy is a key mechanism of ischemic neuronal damage.In particular,a large number of circular RNAs(ciRS-7)exist in the nervous system and lead neuron to autophagy by affecting expression of miRNA-7 and then to interfere expression of mTOR and it’s downstream target p70S6 K.Our previous studies find that Shenfu injection can block the neurological function of brain injury after cardiopulmonary resuscitation by blocking Nogo1-Ng R,suggests that Shenfu injection can promote the repair function of neuronal cells.No reports about neuronal autophagy on the mechanism of brain injury after cardiopulmonary resuscitation have been reported.Based on these,we hypothesize that Shenfu injection may protect brain cells by regulating the ciRS-7 / miR-7 /mTOR / p70S6 K signaling pathway to mediate neuronal autophagy after CPR.In this study,the ciRS-7 /miR-7/mTOR/p70S6 K signaling pathway willbedetected by immunohistochemistry,weston-blot,electron microscopy and immunofluorescence confocal microscopy on duplicated rat models of cardiopulmonary resuscitation(CPR),to explore the mechanism of neuronal autophagy after cardiopulmonary resuscitation and the intervention effect of Shenfu injection,reveal the effective therapeutic target of Shenfu injection on cerebral ischemia-reperfusion injury after cardiopulmonary resuscitation,and provide a new objective basis for the treatment of cerebral injury after resuscitation by Shenfu Injection.Methods SD male mice were randomly divided into 5 groups: sham operation group,routine model group,antagonist group(cirs-7-KD),Shenfu injection group and Shenfu + antagonist group(cirs-7-KD).Except for the sham operation group,the other 4 groups of mice were induced to cardiac arrest by potassium chloride chloride,and the model of cardiac arrest/cardiopulmonary resuscitation(CA/CPR)mice was established.After successful resuscitation,the mice were given the same volume of drugs through the femoral vein 1-2min.The sham group did not use drugs.3 h,24 h,3 d and 7 d after resuscitation were used as observation points to compare the changes of related indexes in each group.The experiment was mainly carried out from two aspects: 1.Animal in vivo experiment: firstly,the effects of Shenfu injection on survival rate and neurological function of resuscitation mice were observed at the animal level.Then,the neuronal autophagy structure and neuronal apoptosis in the cortex and hippocampus of resuscitation mice were observed at the tissue and molecular levels,and the expressions of cirs-7 /miR-7/mTOR/p70S6 K related nucleic acid and protein indexes were determined.2.In vitro experiment,the regulation of this pathway was confirmed by cell experiment.Specific indicators include the following:1.CIRS-7 antagonist was prepared by packaging and in vivo validation test of CDR1 as gene sh RNAAAV9 adeno-associated virus.2.Survival rate was calculated and neurological impairment score was used to evaluate the survival rate and neurological impairment in each group.3.The expressions of cirs-7,miR-7,mTOR and p70S6 K in each group were detected at each time point in the hippocampus of mice after resuscitation by q PCR4.Autophagy and apoptosis of neuronal cells after resuscitation were observed by TUNEL and transmission electron microscopy5.The effects of Shenfu injection on cirs-7 /miR-7/mTOR/p70S6 K pathway and on autophagy and apoptosis of nerve cells were detected by q PCR,immunohistochemistry,TUNEL detection,Nissl staining and transmission electron microscopy in brain tissue of resuscitation mice.6.The effects of Shenfu injection on autophagy and apoptosis of oxygen and sugar deprivation(OGD/R)PC12 cells were detected.Results1.Mice were infected with different doses of CDR1 as interference virus.According to the expression results of CDR1 as detected by q PCR,2 l viral amount was selected as the cirs-7 antagonist for intracerebral injection and the next experiment.2.Survival rate and neurological function score of CA/CPR resuscitation mice:(1)Survival rate: Compared with the sham operation group,the survival rate at24 and 48 hours after resuscitated mice in the antagonist and conventional model groups was significantly decreased(P < 0.05).Compared with the conventional treatment,the survival rate at 24 h and 48 h after resuscitation was increased in the Shenfu group and Shenfu + antagonist group(P < 0.05).(2)the nerve function score: compared with the conventional model group,the antagonist group and neural function of mice and attached + antagonist in 3 and7 days have increased significantly(P < 0.05),and join with groups of mice 24 hours after successful recovery,3 days,7 days nerve function score significantly decreased(P < 0.05,P < 0.01).3.The expression of cirs-7,miR-7,mTOR,and p70S6 K at each time point in the hippocampus of mice after resuscitation.(1)The expression of cirs-7 in the brain tissues of mice in the conventional model group and the antagonist group(cirs-7-KD)decreased in a time-dependent manner at 3h,24 h,3 and 7 days after the recovery of spontaneous circulation,and the expression of cirs-7 was the lowest at 3 days(P< 0.01).(2)The expression of miR-7 in the brain tissues of mice in the conventional model group and the antagonist group(cirs-7-KD)increased in a time dependent manner at 3h,24 h,and 3 days after the recovery of voluntary circulation,and the expression of miR-7 in the brain tissues of mice in the cirs-7-KD group increased in a time dependent manner,with the highest expression at 7d(P <0.05).(3)The expression of mTOR in the brain tissues of the conventional model group and the antagonist group(cirs-7-KD)was significantly higher than that in the sham operation group on the third day(P < 0.05).(4)Compared with the sham operation group,the expression of P70S6 K in the conventional model group and the antagonist group(cirs-7-KD)was increased in a time-dependent manner at 3h,24 h,3 and 7 days after the recovery of spontaneous circulation,and the antagonist group(cirs-7-KD)was significantly increased(P < 0.05).4.Autophagy and apoptosis of neurons after resuscitation in mice(1)Compared with the sham operation group,the apoptosis of brain cells in the conventional model group increased on the 3rd and 7th days after resuscitation by TUNEL detection;Moreover,apoptosis was significantly increased in the cirs-7-KD group compared with the model group.(2)By transmission electron microscopy,compared with the sham operation group,the autophagy of brain cells in the conventional model group increased on the 3rd and 7th days after resuscitation.Moreover,autophagy increased in the cirs-7-KD group compared with the model group.5.Regulatory effect of Shenfu injection on cirs-7 /miR-7/mTOR/p70S6 K signaling pathway(1)The expression of cirs-7 in Shenfu injection group at 24 h,3 days and 7 days was significantly higher than that in model group at corresponding time(P <0.01);The expression of cir S-7 in brain tissues of Shenfu injection + cir S-7-KD group was decreased in a time dependent manner(P < 0.01),and the lowest expression was found at 7 d.(2)Compared with the model group at 24 h,3 d and 7 d,the expression of miR-7in Shenfu injection group was significantly decreased(P < 0.01);The expression of miR-7 in brain tissues of Shenfu injection + cirs-7-KD group was increased in a time-dependent manner(P < 0.05),and the highest expression was found at day 7.(3)The expression of mTOR in Shenfu injection group at 24 h,3 days and 7 days was significantly lower than that in model group at the corresponding time(P <0.01).The expression of mTOR in brain tissues of Shenfu injection + cirs-7-KD group was increased in a time dependent manner(P < 0.05),and the highest expression was observed at day 3.(4)The expression of P70S6 K in Shenfu injection group at 24 h,3 days and 7days was significantly lower than that in model group at the corresponding time.The expression of P70S6 K in the brain tissues of Shenfu injection +cirs-7-KD group was increased in a time-dependent manner(P < 0.05),and the highest expression was found on day 7.6.Effects of Shenfu Injection on apoptosis and autophagy of hippocampal neurons after resuscitation(1)Compared with the sham operation group,the apoptosis of brain cells in the model group was increased by TUNEL detection;Apoptosis in Shenfu injection group was lower than that in model group.CIRS-7-KD promoted the apoptosis of brain cells in Shenfu injection group.(2)By transmission electron microscopy,the autophagy of brain cells in the model group was increased compared with that in the sham group.Autophagy decreased in Shenfu injection group compared with model group.Cirs-7-KD promoted autophagy of brain cells in Shenfu injection group.7.The protein expression of mTOR and p70S6 K in the hippocampus of mice after resuscitated: Western blot detection showed that the protein expression of mTOR and p70S6 K in the cerebral cortex of each group was significantly increased at 24 h,and the contents of both in the antagonistic group and the rhizome + antagonistic group were significantly increased,especially the content of cirs-7-kd in the antagonistic group was significantly higher than that in the sham operation group(P<0.05).However,mTOR and P70S6 K proteins in the control group were not significantly increased compared with the sham group(P<0.05;P<;0.01).8.Expression of autophagy-related proteins and apoptosis in nerve cells(1)On the 24 th hour after resuscitation,immunohistochemical staining showed that compared with the sham operation group,LC3 and Beclin-1 in the hippocampal CA3 region were further up-regulated in the conventional model group and the antagonist(cirs-7-KD)group,while P62 was down-regulated.Shenfu injection can inhibit the expression of LC3 and Beclin-1in the hippocampus of model group mice,and promote the expression of P62.(2)At 24 h after resuscitation,Nissl staining showed that neurons in the conventional model group were missing and loosely arranged.The neuronal arrangement in the cirs-7-KD group was looser than that in the conventional model group.Compared with the conventional model group,the neurons in the control group were arranged neatly and the deletion was reduced.Neurons in cirs-7s-Kd +SF group were loosely arranged and tended to be missing compared with those in the control group.9.Effects of Shenfu Injection on Autophagy and Apoptosis of Oxygen and Sugar Deprivation(OGD/R)PC12 Cells(1)The apoptosis of PC12 cells was increased in OGD/R group;Apoptosis in Shenfu injection group was lower than that in model group.Cirs-7-KD and mTOR activator(MHY1485)promoted the apoptosis of PC12 cells in Shenfu injection group.(2)Compared with the normal group,LC3 protein expression and autophagy lysosome expression were increased in PC12 cells of OGD/R group;LC3expression in Shenfu injection group was lower than that in model group.Cirs-7-KD and mTOR activator(MHY1485)promoted LC3 expression in PC12 cells of Shenfu injection group.Conclusion1.Shenfu injection can improve the survival rate of cardiac arrest/cardiopulmonary resuscitation(CA/CPR)mice and reduce the neurological severity score,and has a neuroprotective effect on brain injury.2.By replicating a mouse model of brain injury after cardiac arrest induced by cardiac arrest/cardiopulmonary resuscitation(CA/CPR),cerebral ischemia and reperfusion may down-regulate the expression of cirs-7,thereby reducing the inhibition of its downstream miR-7,while up-regulating the protein expression of miR-7/mTOR/p70S6 K signaling pathway,which leads to autophagy and apoptosis of nerve cells.3.Shenfu injection may up-regulate the expression of cirs-7,enhancing the inhibitory effect on miR-7,down-regulate the expression of downstream miR-7/mTOR/p70S6 K protein,thereby reducing the occurrence of autophagy and apoptosis of nerve cells in mice after cardiopulmonary resuscitation,and plays a protective role of nerve cells.This study provides a new therapeutic target of Shenfu injection on neuroprotection of cerebral resuscitation at molecular level. |
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