| Objective: To establish an animal model of HIBD and OGD / R model of primary cells in vitro to analyze the occurrence of autophagy in brain tissue and primary cells of neonatal rats at different time points,and to investigate the effects of different autophagy interventions on fading Role of melanin in early mitochondrial autophagy in neonatal rats with hypoxic-ischemic brain damage.Methods: Eighty 7-day-old Sprague-Dawley(SD)rats were randomly divided into sham operation group(Sham group)and model group(HIBD group).The brain tissues were harvested at 0,2,4,6,8,12,24 and 48 h after model preparation.Western blot was used to detect the expressions of Bnip3 and LC3-II.The primary cultured rat cortical cells were cultured for 17-19 days.The mitochondrial membrane potential was detected by JC-1 staining and the mitochondrial autophagy was observed by immunofluorescence.After the OGD / R model was prepared,autophagy inhibitor 3-MA and mitophagy inhibition Mdivi-1,Rapamycin and Melatonin were used to detect the expression of Bnip3 and LC3-II.CCK8 was used to detect the cell viability.TTC staining in different treatment factors in the size of cerebral infarction.Result:1.Compared with Sham group,the expression of mitochondrial autophagy-related protein Bnip3 was up-regulated in HIBD group at 0h(P <0.05),and the expression of autophagy-related protein LC3 High expression,high expression at 4h,8h(P <0.05),more than the Sham group(P <0.05).2.Compared with the normal group(Con),the same mitochondrial autophagy-related protein Bnip3 was expressed in OGD / R group at 0h,and continued to be highly expressed at later time points(P <0.05);autophagy-related protein LC3-II expression began to overexpress from 0h,down 2h,and continued to show high expression after 4h(P <0.05).3.Mitochondrial membrane potential test of JC-1: Compared with the normal group(Con),the mitochondrial membrane potential of OGD / R group decreased.The mitochondrial membrane potential of 3-MA and Mdivi-1 treatment group decreased significantly compared with OGD / R group P <0.05).Compared with OGD / R group,apoptosis in Rapa and MT groups decreased(P <0.05).4.Detection of mitochondrial autophagy by immunofluorescence: The mitochondrial autophagosomes labeled by both Bnip3 and LC3 were all red fluorescence.Compared with Con group,the number of red fluorescence of primary cells after OGD / R increased(P <0.05).After treatment with OGD / R,the expression of Bnip3 and LC3 in 3-MA and Mdivi-1 groups decreased(P <0.05).The levels of Rapa and MT Group Bnip3 and LC3 red fluorescence were significantly increased(P <0.05).5.The results of CCK8 cell activity test showed that the cell viability decreased in OGD / R group compared with that in Con group.The cell viability in 3-MA and Mdivi-1 groups decreased on the basis of OGD / R treatment(P < 0.05).The cell viability in Rapa group was not significantly increased(P <0.05),and the cell viability in MT group was increased(P <0.05).The cell viability was increased in all intervention groups except MT group.The differences were statistically significant(P <0.05)..6.TTC staining results: Compared with Sham group,obvious white infarction was observed in HIBD group;the infarction area percentage in 3-MA group was more obvious than that in HIBD group(P <0.05),suggesting that 3-MA increased brain injury;Mdivi(P <0.05).Compared with HIBD group,the percentage of infarct size in Rapa treated group decreased slightly(P <0.05),and the area of cerebral infarction affected contralateral hemisphere.(P <0.05).Compared with HIBD group,the percentage of infarct size in HIBD treated group decreased significantly(P <0.05).Conclusions:1.Compared with Sham group,Bnip3 of mitochondrial autophagy in HIBD group was expressed at 0h,and continued to be highly expressed at later time points(P <0.05).The expression of autophagy-related protein LC3-II The expression was highly expressed at 0h,4h,8h(P <0.05),and the expression at the remaining time point was higher than that in Sham group(P <0.05).The difference was statistically significant.2.Compared with the normal group(Con),the same mitochondrial autophagy-related protein Bnip3 in OGD / R group was expressed in primary cultured cortical cells at 0h,and continued to be highly expressed at each subsequent time point(P < 0.05).The expression of LC3-II,an autophagy-related protein,began to show high levels at 0h,down after 2h,and remained high at 4h(P <0.05).The differences were statistically significant.3.3-MA and Mdivi-1 can aggravate brain tissue and cell injury in the early stage of hypoxia-ischemia(glycogen),MT can reduce this injury and may have a synergistic protective effect with Rapa early in brain injury.4.MT can enhance the occurrence of mitochondrial autophagy and alleviate brain damage caused by Mdivi-1 inhibition of mitochondrial autophagy,which plays a protective role in early brain injury. |
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