| ObjectiveDiscussion on Jin's three needle head acupoint on intrauterine hypoxic ischemic brain damage(HIBD)on expression of BNIP3 and LC3/Atg8 in rats brain tissue,studing the mechanism of acupuncture in the early stage with Jin's three needle head points on the treatment of neonatal hypoxic ischemic brain injury whether related with BNIP3 mediated mitochondrial autophagy pathway,by adding autophagy pathway upstream protein activator and inhibitor to explore the onset pathway and acupuncture intensity by promoting mitochondrial autophagy in improving brain injury of neonatal hypoxic ischemic encephalopathy,provide objective scientific basis for the mechanism research and clinical treatment of neonatal hypoxic ischemic encephalopathy.MethodsThe pregnant rats were anesthetized with isoflurane for 21 days.The uterine arteries of the pregnant rats were clamped by forceps.After 10 minutes of complete occlusion of blood flow,the rats were delivered.then promote baby rats waking up.The rats were fed to the day 14 from the suckling rats.The rats were randomly divided into six groups: model group,acupuncture group and activator group,inhibitor group,the acupuncture plus activator group and the acupuncture plus inhibitor group.Acupuncture intervention was carried out in acupuncture group,acupuncture plus activator group,acupuncture plus inhibitor group,and the other three groups were treated with grasping at the same time.The activator group,inhibitor group,acupuncture plus activator group,acupuncture plus activator group and acupuncture plus inhibitor group were given corresponding drug intervention.The model group and acupuncture group were intraperitoneally injected with normal saline at the same time.The rats in the six groups started to treat 14 days after birth,once a day,for 14 days,and then killed after 28 days after birth.The left brain tissue of 3 rats in each group was examined by electron microscopy,the right brain tissue by HE staining,hippocampus of 8 rats by western blotting of autophagy related protein BNIP3 and LC3.Results1.Transmission electron microscope: compared with the model group,acupuncture group,activator group,acupuncture plus activator group autophagy increased,inhibitor group,acupuncture plus inhibitor group decreased.2.HE staining: Compared with the model group,the number of cells in the acupuncture group and the acupuncture plus activator group increased,and the shape was improved,the number of activator groups increased,the number of the inhibitor group and the acupuncture plus inhibitor group decreased and the morphology was not improved obviously.3.Western blot: Expression of BNIP3 protein: Compared with model group,the expression of BNIP3 protein in acupuncture group was higher than that in model group(P<0.05),In the activator group,there was no significant change(P>0.05),the expression of the inhibitor group was decreased,and the expression was statistically significant(P<0.05);Compared with activator group,the expression of Acupuncture plus Activator group was higher than that of Activator group(P < 0.05);Compared with the inhibitor group,the acupuncture plus inhibitor group had no significant change(P > 0.05);Expression of LC3 protein: compared with model group,the expression of LC3 protein in acupuncture group was higher than that in model group(P<0.05),In the activator group,there was no significant change(P > 0.05),the expression of the inhibitor group was decreased,and the expression was statistically significant(P < 0.05);Compared with activator group,the expression of Acupuncture plus Activator group was higher than that of Activator group(P<0.05);Compared with the inhibitor group,the acupuncture plus inhibitor group had no significant change(P>0.05).Conclusion1.Acupuncture can promote the increase of autophagosome in neural stem cells,improve the damage of mitochondria,and improve the morphological recovery of nucleus and various organelles.Autophagy activator can also increase the number of autophagosome.Both acupuncture and activator can increase the number of autophagosome in neural stem cells,promote the generation of autophagy and play a protective role in autophagy.The expression of autophagosome was reduced by inhibitor and acupuncture plus inhibition.2.The activation of mitochondrial autophagy induced by acupuncture was mediated by promoting the expression of BNIP3 and LC3,while inhibitor 3-MA inhibited the process of autophagy by inhibiting the expression of BNIP3 and then LC3 expression.Acupuncture could not reverse the inhibition of autophagy in upstream and downstream of autophagy pathway mechanism.The activator RAPA did not promote autophagy by promoting the expression of BNIP3.Acupuncture plus activator can effectively promote the expression of BNIP3 and LC3,suggesting that acupuncture can promote autophagy by promoting the expression of autophagy related protein BNIP3 and LC3,repair damaged neurons and promote the recovery of cerebral ischemic state. |
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