Effect Of Acupuncture On The Expression Of Beclin-1 And P62 In Brain Tissue Of HIBD Model Rats

Neonatal hypoxic-ischemic encephalopathy is one of the common neonatal brain injury,due to perinatal asphyxia,hypoxia caused by hypoxic ischemic damage.The pathogenesis of HIBD is not yet clear,there is no complete clinical treatment.The Chinese medicine in the treatment of children with encephalopathy has made a breakthrough,especially acupuncture in the treatment of HIE sequelae,the effect is significant.Purpose The treatment of encephalopathy in children includes mental retardation,autism,attention deficit hyperactivity disorder(ADHD)and ADHD for more than30 years,and the curative effect is definite.However,the effect of acupuncture on HIE Mechanism of the study is not yet clear,so in-depth study of acupuncture treatment of HIE mechanism is very necessary.In recent years,autophagy is a new direction for the study of nervous system diseases.Many studies suggest that there is also mitochondrial autophagy in the body due to hypoxic-ischemic injury.Mitochondrial autophagy was found to improve HIE brain as a self-protection mechanism Histopathological changes.Previous studies showed that acupuncture can promote the expression of autophagy-related protein Beclin-1 and LC3 in HIBD model mice.At the same time,acupuncture can induce mitochondrial autophagy by increasing BNIP3 protein level.In our experiment,the model of neonatal rats with intrauterine distress after hypoxic ischemic brain injury was used to improve the survival rate of newborn rats and ensure the consistency of hypoxia-ischemia in HIBD rats.The effect of acupuncture on rapamycin and 3-MA autophagy was completely inhibited and overexpressed,indicating the importance of acupuncture promoting mitochondrial autophagy in acupuncture repair neurons.Methods The intrauterine distress HIBD rat model was constructed by modeling the delayed cesarean section by simulating human perinatal uterine distress.The rats in model group,acupuncture group,activator group,inhibitor group,acupuncture plus activator group and acupuncture plus inhibitor group were given intraperitoneal injection of rapamycin,3-MA and acupuncture intervention,acupuncture group,acupuncture and activator group,acupuncture plus inhibitor group acupuncture intervention every day,model group,activator group,activator group acupuncture group and other two groups were empty The control group,the activator group,the inhibitor group,the acupuncture plus activator group and the acupuncture plus inhibitor group were intraperitoneally injected with the corresponding solutions every day.The model group and the acupuncture group were injected with normal saline intraperitoneally.At the end of 14 days after intervention,rats were sacrificed at 28 days after birth,and the morphological changes of midbrain were observed by HE staining.The contents of autophagy-related protein Beclin-1 and P62 in each sample were detected by Western Blot,And the structure of autophagosomes was observed by transmission electron microscopy.Results1、HE staining The results of HE staining showed that the organization of rat brain tissue in the model group was slightly disordered and the cell gap widened.Acupuncture group: Tissue cells arranged more compact,glial cells increased,necrotic cells decreased,some neuron morphology close to normal.Activator group: slightly larger intercellular space,a slight disorder in the arrangement of nerve cells,but the degree was significantly reduced compared with the model group.Inhibitor group: the number of layers decreased,the nuclei of pyramidal neurons were deeply stained,the cells were arranged in a scattered manner,and no neurons of normal morphology were found in the whole brain layers.Acupuncture plus activator group: Tissue cells arranged in a relatively compact,we can see some structural disorder cells.Acupuncture plus inhibitor group: rat neurons irregular cell morphology,cell arrangement scattered.2、Western Blot test results The results of Beclin-1 expression showed that Beclin-1 protein content in acupuncture group was significantly higher than that in model group(P<0.05),indicating that acupuncture can promote the expression of autophagy-related protein Beclin-1;Compared with model group,the expression of Beclin-1 in the activator group was not different.The expression of Beclin-1 in the inhibitor group was significantly decreased(P <0.05),indicating that the inhibitor can effectively inhibit the expression of Beclin-1 and inhibit the autophagy.Compared with the activator,acupuncture plus activator group Beclin-1 expression was significantly increased,indicating that acupuncture can significantly promote Beclin-1 expression;The results of P62 expression: Compared with the model group,P62 expression decreased in acupuncture group,the difference was statistically significant(P <0.05)that acupuncture can promote autophagy,and P62 is an indicator of autophagy negative feedback,autophagy(P <0.05).Compared with the model group,the expression of P62 in the inhibitor group was higher than that in the model group The difference was statistically significant(P<0.05).3、 Transmission electron microscope observation autophagosome results of each group In the model group,the edges of the nucleus of the neurons were slightly regulated and the autophagic bodies were seen occasionally.Neuronal cells in the acupuncture group had a large nucleus,showing vacuolar autophagic lysosomes around the cells.The edge of the neuronal cells in the activator group was visible In the autophagosome,the number of autophagosomes increased compared with the model group.In the inhibitor group,the edges of the neurons were irregular,with unclear borders with the surrounding tissues,almost no autophagosomes.The number of neurons in the acupuncture plus activator group The edge of the rule,nuclear large positive circle,visible autophagosomes;acupuncture plus inhibitor group of neurons edge irregular,no autophagy bodiesConclusion(1)In this study,the model of neonatal rat with intrauterine distress caused by pre-experimental clamping of bilateral uterine artery was used to ensure the consistency of HIBD model preparation.(2)The intervention of acupuncture intervention in brain tissue of HIBD neonatal rats with autophagy activator(rapamycin)and inhibitor(3-MA)Autophagy-related proteins Beclin-1,P62 have different expression.(3)From the results of TEM observation of autophagosome,acupuncture can promote the expression of mitochondrial autophagosome in brain tissue of HIBD rats.