| Purpose:To observe the effects of eye acupuncture therapy on expression of autophagy-related proteins in rats brain tissue of cerebral ischemia reperfusion injury,and to explore the relationship between eye acupuncture therapy and neuronal autophagy in cerebral ischemia reperfusion injury.Material and method:120 rats were randomly divided into three groups:the normal group(n=16),the sham operation group(n=16),and the model group(n=88).The model group included pre-injection of the inhibitor 3-MA 44 rats and 44 normal rats.Modified thread plug method was used to replicate the model of model rats.The rats in the normal group were not treated,and the rats in the sham operation group were modeled with the same model,except that the fishing line was not inserted.After model evaluation,all rats that meet the experimental requirements were divided into six groups:normal group,sham operation group,model group,3-MA group,eye acupuncture group and 3-MA+eye acupuncture group.The rats in the normal group and the sham operation group were bred routinely until the end of the experiment,and the rats in the model group were bred until the reperfusion was completed;the rats in the 3-MA group used stereotaxic technique in the lateral ventricle(1mm behind bregma,1.5mm laterally,3.5mm deep)were injected with autophagy inhibitor3-MA0.6?L(600nmol),and then reared until reperfusion.In the eye acupuncture group,the acupuncture points were selected from the liver and gallbladder area,the kidney bladder area,the upper focal area,and the lower focal area.The needle was inserted from the 1-2mm area of the orbital rim of the rat's orbit,and the needle insertion direction was along the acupoint area.From the initial positioning line to the end positioning line,puncture the subcutaneous tissue and insert the needle about3mm(clockwise for the left eye and counterclockwise for the right).The injection of inhibitors in the 3-MA+eye acupuncture group was the same as that of the 3-MA group.After the completion of reperfusion,the eye acupuncture intervention was the same as the eye acupuncture group.The intervention time was immediately,12h and half an hour before the samples were taken for the two groups of rats.There are 3treatments in total,each treatment lasts for 20 minutes.When it reaches ten minutes,the scraping handle is used to scrape the needle once,5 times each time,which can enhance the stimulation.After the last intervention,the rats in each group were scored and recorded for neurological deficits.The rats were killed by over-abdominal anesthesia(2%sodium pentobarbital solution was used for anesthesia,the dose was200 mg·kg-1),and the formation of CIRI autophagosomes before and after the eye acupuncture intervention was observed by transmission electron microscope;Western blot methods and immunohistochemical methods were used to observe the expression levels of LC3,ATG1 and ATG4 in nerve cells.Results:1.Changes in the scores of nerve defects in each groupThe normal group and the sham operation group did not have neurological deficit symptoms,and the score was 0.Rats in the model group showed different degrees of neurological dysfunction,such as turning or dumping to the contralateral side when walking;unable to fully extend the contralateral front paw,etc.,indicating that the CIRI rat model was successfully replicated.Compared with the model group,the neurological deficit scores of rats in the 3-MA group,the eye acupuncture group and the 3-MA+eye acupuncture group were significantly decreased,and the difference was statistically significant(P<0.05).2.Transmission electron microscope observation of changes in the number of autophagosomesCompared with the normal group and the sham operation group,more autophagosomes were seen in the model group;compared with the model group,the autophagosomes were reduced in the eye acupuncture group,suggesting that eye acupuncture may reduce ischemic brain injury by reducing the number of autophagy in brain tissue.3.Western blot method to detect the expression of LC3,ATG1 and ATG4 protein in the brain tissue of each group of ratsUsing?-actin as the internal control,compared with the normal group,the expression of LC3,ATG1 and ATG4 protein in the brain tissue of the sham operation group did not change significantly,and the difference was not statistically significant(P>0.05);compared with the sham operation group,the expression of LC3,ATG1 and ATG4 protein in the brain tissue of the model group was significantly increased,and the difference was statistically significant(P<0.05);compared with the model group,the brain tissue LC3,ATG1 and ATG4 protein expressions of rats in the 3-MA group,eye acupuncture group and 3-MA+eye acupuncture group decreased significantly,and the difference was statistically significant(P<0.05).4.Immunohistochemistrymethod to detect the expression of LC3,ATG1 and ATG4protein in the brain tissue of each group of ratsCompared with the normal group,the expression of LC3,ATG1 and ATG4 protein in the brain tissue of the sham operation group did not change significantly,and the difference was not statistically significant(P>0.05);compared with the sham operation group,the expression of LC3,ATG1 and ATG4 protein in the brain tissue of the model group was significantly increased,and the difference was statistically significant(P<0.05);compared with the model group,the brain tissue LC3,ATG1,and ATG4 protein expressions of rats in the 3-MA group,eye acupuncture group and3-MA+eye acupuncture group decreased significantly,and the difference was statistically significant(P<0.05).Conclusion:1.Eye acupuncture can improve the neurological deficit score of rats with cerebral ischemia reperfusion injury;2.Eye acupuncture therapy can reduce the expression of autophagosomes in brain tissue of rats with cerebral ischemia reperfusion injury;3.Eye acupuncture may reduce the expression of LC3,ATG1 and ATG4 protein in brain tissue of rats with cerebral ischemia reperfusion injury to regulate excessive autophagy of nerve cells,so as to improve the cerebral ischemia-reperfusion injury of rats. |
PDF Full Text Request