| Hypoxic-ischemic encephalopathy is one of the brain injury which caused by perinatal neonatal hypoxia,and it is one of the important causes of neonatal death and neurological sequelae.The survivors are more likely to develop mental retardation,paralysis,epilepsy and other abnormalities of the nervous system.Therefore,timely and effective treatment is of great importance for children with HIE and their family.In recent years,in the treatment of HIE sequelae,through acupuncture,moxibustion,acupoint injection,pediatric massage,Chinese herb,fumigation and other Chinese medicine effect methods,especially acupuncture therapy,the effect is extremely significant.Objective"Jin three needle point of head"(Nao three needle,Nie three needle,Zhi three needle)is the main group to treat children with encephalopathy.Through clinical treatment of pediatric encephalopathy more than 30 years,its effect is certain.The mechanism of acupuncture treatment of HIE is not clear,so its in-depth study is extremely important.In recent years,many studies suggest that in the event of hypoxic-ischemic injury,brain tissue in addition to cell apoptosis,but also the presence of mitochondrial autophagy.In this study,we established the model of HIBD rats.The behavior of experimental rats was tested by open field test and Morris water maze test.The autophagy-related protein BNIP3 and Beclin-1 were detected by Western blot and Flow Cytometry,and neuronal apoptosis was detected by Tunel.The expression of mitochondrial autophagy and its morphological structure were observed by transmission electron microscopy.By exploring the possible mechanism of acupuncture on the treatment of hypoxic-ischemic brain damage through affecting mitochondrial autophagy provide a theoretical basis for using acupuncture to treat HIE.MethodsLiterature research mainly summarized the modern medicine on the pathogenesis and treatment methods of HIE,as well as Chinese medicine on the pathogenesis and the main treatment of HIE.The experimental study was divided into two parts.In the first part of the experiment,according to the experimental design,we established HIBD rat model,and divided the offspring of the natural childbirth group into normal group,normal acupuncture group randomly.The HIBD group was randomly divided into model group and model acupuncture group.The rats began to acupuncture on its 14th day,and acupuncture intervention was conducted every morning for 14 days.The behavioral detection of each group was performed on the second day after acupuncture intervention,and the autophagy-related protein BNIP3 and Beclin-1 were detected by Western blot and Flow Cytometry.In the second part,the offspring of the intrauterine distress group were randomly divided into HIBD model group and acupuncture group.The offspring of the natural childbirth group were set as normal group.And then they were randomly divided into dl,d14,d21,d28 normal group,dl,d14,d21,d28 model group,d14,d21,d28 acupuncture group.According to the different material time points and acupuncture points,each group of 8 rats,a total of 11 groups.Acupuncture intervention was conducted every morning for 7 days or 14 days.The neuronal apoptosis was detected by Tunel,and the expression of mitochondrial autophagy and its morphological structure were observed by transmission electron microscopy.And the content of autophagy protein Beclin-1 was detected by Western blot at different time points.Results1.Behavior test result:(1)Open field test result:Compared with the normal group,the central area and the total distance of the model group were significantly decreased(P<0.01,P<0.05).There was no significant difference between normal acupuncture group and normal group(P>0.05).Compared with the model group,the central area and the total distance of the model acupuncture group increased(P<0.05,P<0.05).(2)Morris Water maze test result:Compared with the normal group,the time of passage of the first passage of the model group was increased(P<0.01).There was no significant difference in the passage time between the normal acupuncture group and the normal group(P>0.05).Compared with the model group,the time of the first passage of the model acupuncture group was decreased(P<0.01).2.BNIP3 protein test result:Compared with the normal group,the expression of BNIP3 protein in hippocampus in the model group was higher than that in normal group(P<0.01).There was no significant difference between the normal acupuncture group and the normal group(P>0.05).Compared with model group,the expression of BNIP3 protein in hippocampus was higher in model acupuncture group(P<0.01).3.Beclin-1 protein test result:Compared with the normal group,the expression of Beclin-1 protein in hippocampus in the model group was higher than that in normal group(P<0.01).There was no significant difference between the normal acupuncture group and the normal group(P>0.05).Compared with model group,the expression of Beclin-1 protein in hippocampus was higher in model acupuncture group(P<0.01).Beclin-1 expression rate:Compared with the normal group,the positive expression rate of Beclin-1 in the model group was higher than that in the normal group(P<0.05).There was no significant difference between the normal acupuncture group and the normal group(P>0.05).Compared with the model group,the positive expression rate of Beclin-1 in the model acupuncture group was higher than that in the model group(P<0.01).4.Tunel staining result:Compared with dl normal group,the number of Tunel staining cells in dl model group increased(P<0.01).Compared with d28 normal group,the number of Tunel staining cells in the hippocampus of d28 model group increased(P<0.01).There was no significant difference between d28 acupuncture group and d28 normal group(P>0.05).Compared with d28 model group,the number of Tunel staining cells in hippocampus of d28 acupuncture group decreased(P<0.01).There was no significant difference in the number of Tunel staining cells between d28 normal group and dl normal group(P>0.05).Compared with dl model group,the number of Tunel staining cells in d28 model group decreased,but the difference was not statistically significant(P>0.05).5.The autophagy was observed by transmission electron microscopy:The neuron of dl normal group had no autophagosomes.The neuronal nucleus edge of dl model group was not smooth,the pulp was not dense,and autophagosome expressed occasionally.The neuron of d14 normal group had no autophagosomes.The neuronal nucleus of the d14 model group was not smooth,the interlobular space widened,the rough endoplasmic reticulum was expanded,and the phenomenon of mitochondrial autophagy appeared.The neuron of d21 normal group had no autophagosomes.In the d21 model group,the perivascular space of the neurons was widened,the rough endoplasmic reticulum was expanded,and autophagosome expressed occasionally.The expression of autophagy was significant in d21 acupuncture group.The neuron of d28 normal group had no autophagosomes.In the d28 model group,autophagosome expressed occasionally The expression of autophagy was significant in d28 acupuncture group.6.The expression of Beclin-1 protein at different time points:Compared with dl normal group,the expression of Beclin-1 protein in dl model group was higher than that in dl normal group(P<0.01).Compared with d14 normal group,the expression of Beclin-1 protein in d14 model group increased(P<0.01).Compared with d21 normal group,the expression of Beclin-1 protein in d21 model group and d21 acupuncture group increased(P<0.01).Compared with d28 normal group,the expression of Beclin-1 protein in d28 model group and d28 acupuncture group increased(P<0.01).The expression of Beclin-1 protein in d21 acupuncture group was higher than that in d21 model group(P<0.01).The expression of Beclin-1 protein in d28 acupuncture group was higher than that in d28 model group(P<0.01).Compared with dl model group,the expression of Beclin-1 protein in d14,d21 and d28 model groups decreased(P<0.01).D14,d21 and d28 model groups were not statistically different(P>0.05).Compared with d14 acupuncture group,the expression of Beclin-1 protein in d21 and d28 acupuncture group increased(P<0.01).There was no significant difference between d21 acupuncture group and d28 acupuncture group(P>0.05).Conclusion1.Acupuncture can promote the recovery of HIBD rats’ exercise and learning and memory ability.2.Acupuncture can improve the expression of autophagy-related proteins BNIP3 and Beclin-1,suggesting that acupuncture promotes the repair of damaged neurons that may be associated with BNIP3-mediated mitochondrial autophagy.3.Acupuncture can be appropriate to increase the expression of mitochondrial autophagy in the damaged area,while decreasing the incidence of apoptosis in damaged areas,suggesting that acupuncture may promote the development of mitochondrial autophagy,thereby reducing the incidence of apoptosis. |
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