| Objective:From the aspects of general state,behavior,morphology and molecular biology,whole body condition plus hippocampal neurons were observed by open field,rod rotation,water maze,Nissl staining,beta-galactosidase staining and Tunel staining.Using ultrastructure of mitochondria in CA1 region,the expression of ATP and ROS in hippocampus,mitochondrial membrane potential,to find out the effect of compound aging model on mitochondrial structure and function and the intervention effect of acupoint catgut embedding on mitochondrial function;Detecting mitophagy related proteins:LC3,P62,Pink1,Parkin,Mfn2,Drpl,Miro,and mitochondrial generation protein PGC1-Alpha,to observe the effects of compound aging model on the Pink1/Parkin pathway of mitophagy,balance between mitochondrial fusion and division,mitochondrial clearance and production,mitochondrial transport,to explore the changes of mitochondrial quality control during aging,and the intervention of acupoint implantation on mitochondrial related changes.From the perspective of mitochondrial quality control,especially Pink1/Parkin pathway of mitophagy,to expore the possible mechanism of acupuncture and moxibustion catgut embedding in delaying aging,which may provide the corresponding basis for clinical application.Method:The animals were randomly divided into control group,model group,acupoint catgut embedding group,sham catgut embedding group,rapamycin group and 3-MA group by SPSS17.0 statistical software.Rats in thecontrol group were given intraperitoneal injection of saline for 10 weeks;rats in the model group were given intraperitoneal injection of D-galactose(150mg/Kg/d)for 10 weeks and chronic unpredictable stress stimulation for 10 weeks from the first week;Catgut Embedding at acupoints was performed once a week,10 times on the basis of D-galactose injection plus stress;rats in the sham embedding group were given sham catgut embedding once a week from the first week on the basis D-galactose injection plus stress;baside the same operationwith model gruop,rats in rapamycin group were injected with rapamycin(1.5 nmol/5ul,5.49 ug/kg)into lateral ventricle at the beginning of the 9th week,while those in 3-MA group were injected with 3-MA(400 nmol/5ul)into lateral ventricle at the beginning of the 9th week.After the completion of the model,3-5 rats in each group were injected with LC3 lentivirus into bilateral hippocampus,3 UL on each side.Catgut embedding prescription:Baihui,Zusanli(Shuang),Hegu(Shuang),Taichong(Shuang)were selected as acupoint catgut embedding prescription.Among them,"Baihui","Zusanli(Shuang)" group,"Hegu(Shuang)" and "Taichong(Shuang)" group,two groups buried wires alternately.During the experiment,the general conditions of diet,hair,activity and mental state of rats were observed,and rats i were weighed once a week.At the end of 10 weeks,open field test,rotating rod test and Morris water maze were conducted.Once finishing the behavioral experiment,samples were taken and the aging of hippocampal neurons was observed by beta-galactosidase staining;the morphology and apoptosis of neurons were observed by Nissl and TUNEL staining;the ultrastructure of hippocampal CA1 region,such as mitochondria,autophagy formation were observed by transmission electron microscopy.Mitochondrial function changes such as ATP,ROS and mitochondrial membrane were observed by mito_detection;autophagy formation in hippocampus was detected by immunofluorescence;LC3?,P62,Pink1,Parkin,Mfn2,Drip1 and PGC1-alpha expression of classical autophagy protein and Pink1/Parkin pathway were detected by Western Blot.Result:1.General state changes of rats in each groupThe rats in model group and sham catgut embedding group lost weight,disordered hair,dim eyes,reduced diet,lazy and happy sleeping,often accompanied by phlegm sounds,such as rough breathing sound,early seizure irritation and fecal incontinence,late slow reaction;the aging phenomenon of rats in catgut embedding group and rapamycin group improved,but the state of rats in 3-MA group did not improve significantly.2.Weight Result of Rats in Each GroupDuring the process of establishing model,the weight of rats in each group increased,and the weight of rats in control group increased the most;compared with the control group,the growth degree was slightly lower in model group,acupoint catgut embedding group,sham catgut embedding group,rapamycin group and 3-MA group;compared with the model group,the weight of rats in acupoint catgut embedding group increased.3.Open-field experimental results of rats in each groupCompared with the control group,the activity,central area time and central area distance of model group and sham catgut embedding group decreased;compared with model group,the activity,central area time,central area distance and total distance of acupoint catgut embedding group increased,but false catgut embedding group had no such effect,rapamycin group only increased in central area time,and 3-MA group was similar to model group.4.Rotary rod test results of rats in each groupCompared with the control group,the model group,rapamycin group and 3-MA group had shorter stay time on the rotating rod and more dropping times;compared with the model group,the acupoint catgut embedding group had longer stay time and less dropping times;compared with the acupoint catgut embedding group,the sham catgut embedding group,rapamycin group and 3-MA had shorter'stay time on the rotating rod and more dropping times.5.Water maze test results of rats in each groupWith the prolongation of training time,the escape latency of rats in each group decreased gradually.Compared with the control group,the escape latency increased in model group,sham catgut embedding group and 3-MA group.Compared with model group,the latency shortened in acupoint catgut embedding group and rapamycin group;compared with acupoint catgut embedding group,the latency prolonged in sham catgut embedding group and 3-MA group.6.The results of beta-galactosidase staining in rats of each groupIn the control group,the cells arranged neatly without obvious aging cells;in the model group,there were widely dispersed blue-stained cells;in the acupoint catgut embedding group,the senescent cells of hippocampal neurons were reduced,and almost no senescent cells were found;in the sham catgut embedding group,there were more blue-stained cells,no difference compared with the model group;compared with the rapamycin group,the senescent cells in the rapamycin group were significantly reduced,and the senes There was no significant difference between model group and 3-MA group.7.Nissl staining results of rats in each groupIn the control group,the neurons were arranged neatly,the structure was normal and the distribution of axonal Cunninghamia was uniform;in the model group,there were atrophic and deformed cells in the dentate nucleus of the hippocampus(DG area);in the acupoint catgut embedding group,the neurons were arranged as a whole,and the morphology was comparable to that in the model group,with only a few scattered degenerated cells;in the sham catgut embedding group,there were more deep-stained cells in the hippocampus DG The neurons in the mycin group were orderly arranged and the number of hyperchromic deformed neurons decreased.In the 3-MA group,the number of hyperchromic neurons was similar to that in the model group,and there were more polymorphic hyperchromic neurons in the DG region.8.Tunel staining results of rats in each groupThere were more green fluorescent cells in the hippocampus of control group,especially in DG area of dentate nucleus;the hippocampus of model group and acupoint catgut embedding group showed scattered green fluorescence;there were green positive cells in CA1 area and DG area of hippocampus of sham catgut embedding group;the positive apoptotic cells of hippocampus of rapamycin group and 3-MA group were less than sham catgut embedding group.9.The results of transmission electron microscopy in hippocampal CA1region of rats in each groupIn the control group,the cell ultrastructure was normal,the nuclear membrane was smooth,the organelles were more,the morphology was normal,the synapses were abundant,and t he microtubule filaments were stable;in the model group,the nuclear membrane was solid and inflexible,the organelles were degenerated and arranged disorderly,the mitochondria were less,the swelling and deformation of the mitochondria,the cristae rupture disappeared,the endoplasmic reticulum was swelled and degranulated,the synapse of theskeleton structure was less,and more lipofuscin In the embedding group,the nucleus was rounder,the mitochondria was slightly swollen,and there were more normal mitochondria and lysosomes in each stage.There was no difference between the sham embedding group and the model group.In the rapamycin group,the cell structure was still acceptable,the nucleus was round,the nuclear membrane was smooth and intact,the endoplasmic reticulum and Golgi body were abundant,the number of mitochondria was acceptable,the mitochondrial ridge was slightly edema,and the mitochondrial ridge was sparse Lysosomes at all levels were occasionally deposited with lipofuscin,and the morphology of 3-MA group was similar to that of model group.10.ATP staining results of rats in each groupCompared with the model group,ATP production in acupoint catgut embedding group increased,and ATP production was mainly in neuron-intensive areas.There was no difference between the sham catgut embedding group and the model group.ATP production in rapamycin group increased slightly compared with the model group,and ATP production in 3-MA group was the least.11.ROS staining results of rats in each groupThe expression of ROS in the hippocampus of rats in control group was still acceptable,the overall fluorescence was dim,and the fluorescence was the most intensive in the dentate nucleus;the fluorescence in the model group was significantly enhanced;compared with the model group,the expression of ROS in the hippocampus of rats in the acupoint catgut embedding group was lower and the fluorescence was weaker;the expression of ROS in the sham catgut embedding group was higher than that in the acupoint catgut embedding group;The red fluorescence is weak in rapamycin group and 3-MA group.12.Mitochondrial Membrane Potential Results of Rats in Each GroupIn control group,the ratio of red fluorescence to green fluorescence in hippocampus was the highest,the mitochondrial membrane potential was normal and the cell condition was better;in model group,the mitochondrial membrane potential decreased and the ratio of red to green fluorescence decreased significantly;in acupoint catgut embedding group and sham catgut embedding group,the membrane potential recovered and the ratio increased slightly compared with model group;in rapamycin group,the mitochondrial membrane potential in 3-MA group was the lowest.13.Immunofluorescence staining of LC3 in hippocampus of rats in each groupCompared with the model group,the green fluorescence of acupoint catgut embedding group decreased,the red fluorescence increased,and the red/green fluorescence ratio increased;compared with the catgut embedding group,the hippocampal fluorescence of rats in the sham embedding group increased;compared with the catgut embedding group,the green fluorescence of acupoint catgut embedding group decreased,and the red/green fluorescence ratio increased.Compared with the model group,the red fluorescence of rapamycin group was relatively enhanced,and the red/green fluorescence ratio was reversed.The total fluorescence of 3-MA group decreased,while the red/green fluorescence ratio increased slightly.14.Expression of rat protein LC3?,P62,Pink1,Parkin,Mfn2,Drip1,PGC1-alpha in each groupCompared with the control group,the levels of LC3,P62,Pink1,Mfn2 proteins in hippocampus of model group increased,while the levels of PGC1-alpha in mitochondrial production decreased.Compared with model group,the expression of PGC1-alpha in acupoint catgut embedding group was similar,while the expression of PGC1-alpha proteins increased.The expression of LC3,P62,Pink1,Mfn2 and PGC1-alpha in pseudo-embedding group decreased.The general trend of protein expression in rapamycin group was the same as that of Catgut Embedding at acupoints,and the trend of 3-MA protein expression was similar to that of model group.Conclusion:1.D-galactose combined with chronic stress compound aging model rats in general state,autonomous activity,motor coordination,muscle endurance,spatial memory,cognitive ability of aging degeneration,hippocampal histopathology,cellular ultrastructure of degeneration,aging,apoptosis and other manifestations,suggesting that the model of compound aging model rats was established successfully.2.Mitochondrial structure and function were impaired in D-galactose combined with chronic stress aging model rats,suggesting that the damage mechanism of compound aging model was related to mitochondria.3.Acupoint catgut embedding can improve the general condition,behavior,hippocampal histopathology,neuronal ultrastructure and mitochondrial function of compound aging model rats.This effect is similar to that of rapamycin,an autophagy promoter,and can be eliminated by 3-MA,which suggests that autophagy is at least partially involved in the anti-aging mechanism of acupoint catgut embedding.4.Using LC3 double-fluorescent lentivirus to label autophagy activity in hippocampus,the red/green fluorescence of aging rats was lower than that of contr ol group,suggesting that aging could affect the fusion process of autophagosome and lysosome and hinder the fluency process of autophagy.The acupoint catgut embedding group and rapamycin group can be improved,suggesting that acupoint catgut embedding can restore complete autophagic flow and maintain intracellular homeostasis by promoting autophagic viscle transport,fusion with lysosomes,degradation and recycling.5.The expression of mitophagy-related protein increased while the mitochondrial production protein decreased in the model group,combine with degradation of autophagosomes decreased in the aging process,suggesting that mitochondrial clearance was blocked and the production of mitochondria was insufficient in the aging process.In a word,acupoint catgut embedding can protect neurons,resist stress injury and delay the aging process by restoring the function of clearing and degrading autophagic lysosomes,repairing complete autophagic flow,clearing damaged mitochondria in cells and increasing mitochondrial production. |
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