| ObjectiveTo explore the mechanisms of aging and the mechanism of chronic stress to promote aging from the perspective of autophagy,to provide a new idea and theoretical basis for anti-aging;from the mTOR signal pathway to illustrate the effect of acupoint catgut embedding on the autophagy function of hippocampus to delay the clinical application of aging to provide experimental basis through observing general condition,body weight,open field experiment and morris maze navigation experiment and space exploration,hippocampal neurons hematoxylin eosin staining,hippocampal neurons Nissl' s staining,and tem ultrastructure of hippocampal neuroni,expression of ULK1?Beclinl?LC3-??P62mRNA of hippocampus,expression of ULK1?Beclinl?LC3-II?P62?p-mTOR protein of hippocampus in aging rat model induced by multi-chronic stress from behavior,morphology and molecular biology,so as to provide theoretical and experimental basis to clarify the Scientificity of acupoint catgut embedding Delaying agingMethods60 healthy SPF SD rats aged 3 months were randomly divided in 5 groups:blank control group,model group 1,model group 2,acupoint catgut embedding group and sham acupoint catgut embedding group.The model group lwas given intraperitoneal injection with D-galactos(120mg/Kg/d)whilemodel group 2,acupoint catgut embedding group and shamacupoint catgut embedding group were given intraperitoneal injection with D-galactos(120mg/Kg/d)and multi-chronic stress.Otherwise,the acupoint catgut embedding group was given also was given acupointcatgut embedding once a day for 6 weeks while sham group was givensham acupointcatgut embedding once a day for 6 weeks.At the end of the experiment,body weight,open field experiment and morris maze navigation experiment and space exploration was carried out.The hippocampus was cut into sections to be stained with H.E.and nissl technique.Also,the rats were executed for the examination of ULK1?Beclinl?LC3-??P62 mRNA expression of hippocampus and expression of ULK1?Beclinl?LC3-??P62?p-mTOR protein of hippocampus.Results1.general conditionThe rats in model group 1,model group 2,sham cupoint catgut embedding group were messy-dark-haired,unresponsive,lack of mental fatigue,I have a loss of appetite and the auricles were dull.Aging phenomenon in model group 2,sham cupoint catgut embedding group was more obvious.The aging phenomenon of acupoint catgut embedding group had been improved obviously2.body weightDuring the modeling period,the body weight of each group increased.The weight gain of the blank control group and the model group 1 was the fastest,and the body weight of the rats in the catgut embedding group was slower than that in the blank control group.The body weight of the rats in the model group 2 and the sham catgut embedding group was the slowest.3.open fieldCompared with the blank control group,the activity,the central area distance,the central time and the total distance of model group 1,model group 2,sham cupoint catgut embedding group(P<0.05).Compared with the model group 2,the activity,the central area distance,the central time and the total distance of the acupoint catgut embedding group increased(P<0.05)4.morris mazeAfter four days of navigation training,the escape latency of each group was significantly reduced,among which the trend of blank control group and acupoint catgut group were the most obvious,the model group 1 was the less obvious,the trend of model group 2 and sham cupoint catgut embedding group is the slowest.On the fourth day of the navigation test,the escape latency of each group showed significant difference.Compared with the blank control group,the escape latency of both the model group and 2 the sham catgut embedding group increased(P<0.05),and the escape latency of the model group 1 showed an increasing trend(P>0.05).Compared with the model group 1,the escape latency of the model group 2 and the sham-embedding group showed an increasing trend(P>0.05).Compared with the model group 2,the escape latency of catgut embedding group was decreased(P<0.05).Compared with the blank control group,the number of groups crossing the platform in the model group 1,the model group 2 and the sham catgut embedding group decreased(P<0.05).The number of crossing points the platform of the catgut embedding group was higher than that of the model group 2,model group 2 and the sham catgut embedding group(P>0.05).There was no significant difference among the model group 2,model group 2 and the sham catgut embedding group(P>0.05).5.hematoxylin eosin stainingCompared with the control group,Hippocampal structure of model group lis loose and disordered,cellular level decreased,part of the neuron atrophied and austenite reduced.Hippocampal structure of model group 2 was more loose and disordered.A large number of neuronal atrophied,the number of neurons decreased further,austenite reduced or even disappear.In the acupoint catgut embedding group,hippocampal neurons are dense,intercellular space increased slightly,austenite was clear.Hippocampal structure of sham acupoint catgut embedding group was like that of model group6.Nissl' s stainingThe normal structure of normal neurons in the hippocampus of the blank control group was clear and enriched.In the model group 1,the model group 2 and sham acupoint catgut embedding group,the hippocampal neurons were disordered,the cell gap increased and the number of Nissl bodies decreased,and the degree of lesion in the model group 2 and sham acupoint catgut embedding group was worse than that in the model group 1,and the Nissl body decreased or even disappeared.In acupoint catgut embedding group hippocampus neurons arranged fairly neat,but slightly loosed,cell gap increased slightly,Nissl body was clear.7.ultrastructure of hippocampal neuroniIn the blank control group,Neuronal nuclei,mitochondria,endoplasmic reticulum,Golgi apparatus and other organelles in CA1 region were normal,there were autophagosomes;no lipofuscin was observed;there were large number of synaptosomes and they were all large,and the microtubules were rich in number.In the model group 1,neuronal cell nucleus isoided,mitochondria reduced,swelled and degenerated;rough endoplasmic reticulum disordered,or even degranulated;The number of autophagosomes was significantly higher than that of the blank control group.;The number of synaptosomes decreased,the volume decreased,dendritic microtubules and microfilament structure dissolved.There increased and deposited lipofuscin.In the model group 2 and sham acupoint catgut embedding group,neuronal ultras tructural damage was more severe than the model group,and autophagy increased.In the acupoint catgut embedding group,the morphology of the neurons was normal,the nucleus was round,the nucleolus wass intact,the nucleolus wass obvious,the chromatin quantity wass even and the distribution wass uniform;the number of mitochondria was still acceptable,the occasional mitochondrial crest irregularly disappeared with no vacuolar degeneration;The number of synaptosomes was normal,and the structure of the microfilaments in the dendrites was normal.8.expression of ULK1?Beclinl?LC3-II?P62 mRNA of hippocampusCompared with the control group,the expression of ULK1?Beclin1 mRNA inmodel group lpresented a rising trend,the expression ofLC3-??P62 mRNA increased.Compared with the model group 1,the expression ofLC3-??P62 mRNA inmodel group 2 increased futher,the expression of ULK1?Beclinl mRNApresented a rising trend.Compared with the model group 2,the expression of ULK1?Beclinl?LC3-??P62 mRNA in the acupoint catgut embedding group decreased while there was no diffrence between those mRNA in the sham group and model group9.expression of ULK1?Beclinl?LC3-??P62?p-mTOR protein of hippocampusCompared with the blank control group,the expression of P62 and Beclin1 protein in the hippocampal neurons of the model group 1 showed an increasing trend(P>0.05),and the expression of LC3-II and ULK1 mRNA was increased(P<0.05),the expression of LC3-II,P62,ULK1 and Beclin1 of the model group 2 increased(P<0.05).Compared with the model group 1,the expression of ULK1,P62 and Beclinl protein in hippocampal neurons of model group2 increased(P<0.05).Compared with model group 2,the expression of ULK1,Beclinl,LC3-II and P62 protein in hippocampal neurons of acupoint catgut embedding group decreased(P<0.05),and the hippocampal neurons ULK1,Beclinl,LC3-?,P62 mRNA expression showed no significant difference of sham acupoint catgut embedding group.Conclusion1.D-galactose model rats and D-galactose with chronic stress aging model rats induces aging changes in general state,autonomy ability,learning cognitive ability,hippocampus general histopathological structure and ultrastructure,hippocampus autophagy and so on.Chronic stress can cause D-galactose-aging-model rats more serious aging performance about the behavioral,morphological,molecular biologyabove,which suggestsing that chronic stress complex model of aging rats model was successful.2.Acupoint Catgut embedding has an improved effect on the behavior of rats with chronic stress complex aging model,including the general state,the open field experiment reflecting the ability of autonomous activity,and the water maze test,which reflects the cognitive memory ability.3.Acupoint catgut embedding can improve the general histopathological structure and ultrastructure of hippocampal neurons in rats with chronic stress and aging through soothing liver and strengthening spleen,tonifying kidney and brain.4.Acupoint catgut embedding inhibits of autophagy initiation and autophagy formation in hippocampal neurons,recovers autophagy-lysosomal scavenging function,repairs autophagy flux to be intact,removes false-folded proteins in hippocampal neurons and impaired or aged organelles,to make the hippocampal neurons return to normal physiological activities and delay the aging process of hippocampus.In conclusion,acupoint catgut embedding induces the general pathological morphology of hippocampus in rats and the ultrastructural structure be improved and the function be restored through inhibitting autopsy and autophagosome formation in the hippocampal neurons,restoring autocrine lysosomal scavenging and degrading function,repairing complete autophagy,so that the general state of the rats,self-activity ability,learning cognitive memory ability return to be normal.Finally aging process is delaying. |
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