| The research objectiveThis study took A?1-42 induced AD model rats as the object,and used moxibustion method to observe the effects of moxibustion heat sensitivity on cognitive behavior,hippocampal neuron morphology and A?metabolic transporter enzyme in AD model rats,and to explore the intervention effect and action mechanism of heat sensitivity moxibustion on AD model rats.The research methodsIn this study,male SD rats were used as the object.After adaptation for 3 days,they were randomly divided into blank control group(group A),sham operation group(group B),and the rest were induced by A?1-42for modeling.AD model establishment method:use 3%sodium pentobarbital anesthesia in rats,shaving after anesthesia in parietal bits of hair in order to further surgery,again USES the fixed rats brain stereotaxic instrument department,the wounds of the rats with iodine volts disinfection,after the subcutaneous tissue separated from fur,with clean dry cotton swab to wipe the residual blood,before the skull midline and anterior fontanelle exposure.Reference Bao Xin Min stereotaxic atlas rats,lateral ventricle injection site for anterior fontanelle basis points before,back 0.8 mm,near the center line 1.2 mm,with dental drill drilling skull,with 10?L trace syringe needle straight down into 4 mm since the brain surface,back to pin 1 mm,set aside A certain injection space,left and right sides of the slow injection respectively 5?L A?1-42(including 5?g A?1-4 2),5 min after injection,injection of retaining needle after 5 min after fully to ensure that the solution dispersion slowly withdraw the needle.At the end of the operation,the bone window was sealed with bone wax and then sprayed with penicillin to disinfect it.Finally,the separated skin was sutured.It is worth noting that the body temperature of the rats undergoing surgery should be kept within a constant range of 36.5 to 37.5?during the entire operation.Through the water maze experiment,AD model rats were further screened out and divided into AD model group(group C),positive control group(group G),and other rats were treated with moxibustion.The moxibustion group was moxibustion once a day for 40 minutes each time;The positive control group was given drugs once a day;The treatment lasted for 28 days.According to the degree of moxibustion thermal sensitivity,it is naturally divided into moxibustion thermal sensitivity group(group D),moxibustion non-thermal sensitivity group(group E),moxibustion late-onset thermal sensitivity group(group F).Moxibustion thermosensitive group(group D):The average tail temperature increased by more than 1?during moxibustion treatment;Before the treatment of AD model rats by moxibustion,the hair at“Dazhui”acupoint of rats was shaved off in advance according to the positioning of the acupoint map of the experimental rats.During the whole treatment process,the moxa stick(made by Affiliated Hospital of Jiangxi University of Chinese medicine,size:1cm in diameter,12cm in length)was vertically suspended at the height of about 3cm at“Dazhui”point for mild moxibustion.At the same time,the room temperature was controlled at a constant temperature of 25±2?.Before moxibustion,the rats were placed in a special rat cage for 30 minutes.The tail temperature in the middle section of rats was measured every 5 minutes before the preparation of moxibustion and after the start of moxibustion,respectively,with the medical infrared frontal temperature meter(adjusted to the object temperature mode),until the 40th minute,the end of moxibustion.The tail temperature of rats was the average temperature of 3 measurements at the same time point.Moxibustion inthermosensitive group(group E):the average tail temperature rise during moxibustion treatment was less than 1?;The operation procedure is the same as that of moxibustion heat sensitive group.Late moxibustion heat sensitivity group(group F):the average tail temperature rise in the first 7 days during moxibustion treatment was less than 1?,and then with the extension of treatment time,the average tail temperature rise is greater than or equal to 1?.The operation procedure is the same as that of moxibustion heat sensitive group.Positive control group(group G):model rats were given intraperitoneal injection of donepezil 0.5mg/kg/d.After 28 days of treatment,the behavior of each group was observed by water maze method.HE tissue section staining was used to observe the morphology and arrangement of neurons in the hippocampus of each group of rats.The contents of?-secretase,?-secretase and?-secretase in brain tissues of rats in each group were determined by ELISA.Meanwhile,the protein expressions of IDE,NEP,ECE-1,ACE2,LPL,Apo E,LRP1,RAGE and RAP in the hippocampal tissues of rats in each group were detected by Western Blot.The results of the study1.Observation results of water maze method:Compared with the control group,there were no significant differences in the water maze escape latency,The Times of crossing on the sixth day and the proportion of platform quadrant time in the sham operation group.Compared with the model group,the escape latency of moxibustion thermosensitive group from the 1st to the 5th day was significantly decreased,and the time of crossing the sixth day maze and the ratio of platform quadrant time were significantly increased,which were the same as the positive control group.There was no significant change in the water maze escape latency of moxibustion inthermosensitive group and moxibustion late thermosensitive group.The ratio of The Times of crossing the water maze on the sixth day to the platform quadrant time of moxibustion late thermosensitive group was significantly increased,and the ratio of platform quadrant time of moxibustion inthermosensitive group was significantly increased.Compared with moxibustion non-thermosensitive group,the escape latency of moxibustion heat-sensitive group was significantly shortened,and the number of crossing the sixth Tianshui maze and the proportion of platform quadrant time were significantly increased,while the escape latency of moxibustion late thermosensitive group had no significant change,and the number of crossing the sixth Tianshui maze and the proportion of platform quadrant time were significantly increased.2.The observation results of HE tissue section staining showed that there were no abnormal phenomena in the hippocampal neurons and glial cells of the rats in the control group and the sham operation group,and their arrangement was uniform and orderly,with abundant cytoplasm,no interstitial hemorrhage and no necrosis.And AD model group rats brain tissue appears the phenomenon of serious damage,embodied in the arrangement of the neurons in a certain extent,the disorder,and the cells form severe swelling and deformation,lost a lot of neurons at the same time,the massive death up neuron,and is accompanied by some,the phenomenon of the dissolution,pyknotic nucleus.Compared with AD model group,after treatment,the brain tissue damage of rats in each group was alleviated,and the moxibustion thermosensitive group and positive control group were significantly alleviated.The brain tissue morphology was normal,no inflammatory cells were infiltrated and arranged neatly.The degree of cell disturbance and inflammatory cell infiltration was also reduced in the delayed heat sensitivity group.There was no obvious improvement in moxibustion group without heat sensitivity.HE semi-quantitative score showed that compared with model group,HE semi-quantitative score of brain tissue of rats in moxibustion heat sensitivity group,moxibustion late-onset heat sensitivity group and positive control group decreased significantly,but there was no significant change in the semi-quantitative score of brain tissue of rats in moxibustion non-heat sensitivity group.Compared with the non-thermosensitivity group,the semi-quantitative score of HE in the brain tissue of rats in the moxibustion thermosensitivity group and the late moxibustion thermosensitivity group decreased significantly,and the moxibustion thermosensitivity group was better than the late moxibustion thermosensitivity group.3.ELISA results:Compared with the control group,the contents of?-secretase,?-secretase and?-secretase in brain hippocampal tissues of model group were significantly increased;Compared with model group,moxibustion heat sensitive group,the moxibustion late thermal group and positive control group rats in?-secretase and?-secretase content reduced to some extent,the content of alpha secrete enzymes have the varying degree to rise,and moxibustion not thermal group was increased,while?-secrete increased,?-secretase and?-secretase drop,but the change is not obvious.Compared with non-thermosensitive moxibustion group,the content of?-secretase in thermosensitive moxibustion group was significantly increased,while the content of?-secretase and?-secretase were significantly decreased.The content of?-secretase in delayed moxibustion group was significantly decreased,but the content of?-secretase was not significantly decreased.4.WB results show that compared with blank control group rats,control of the NEP in the rat hippocampus,IDE,ECE-1,ACE2,LPL,Apo E,LRP1,RAGE and RAP protein expression level had no significant change,while the IDE in the model group,the NEP,ECE-1,Apo E,ACE2 and LRP1 protein expression decreased significantly,RAGE and RAP protein expression level of all present a significant rise.At the same time,the expression level of LPL protein did not show an obvious trend.Than AD model group rats,moxibustion heat sensitive group,the moxibustion late thermal group and positive control group of the NEP,IDE,ECE-1,the expression of ACE2 and Apo E and LRP1 protein levels are showing a significant rise,and RAP in the hippocampus,and RAGE protein expression levels were significantly lower,moxibustion heat sensitive group big mouse brains hippocampus in NEP,ECE-1,ACE2,LPL,Apo E and LRP1 and RAP protein expression level had no significant change,the IDE a significant rise in expression,RAGE expression significantly reduced.Do not thermal group compared with moxibustion,moxibustion heat sensitive group and moxibustion late thermal group,ACE2,Apo E,NEP,ECE-1,LRP1 protein expression levels were significantly elevated,RAP protein expression levels were significantly decreased,but RAGE protein expression is not obvious,moxibustion IDE protein expression significantly higher thermal group,moxibustion group delay thermal rising IDE protein expression is not obvious.Conclusion1.Thermosensitive moxibustion intervention can improve the spatial learning and memory ability of AD model rats.2.Thermosensitive moxibustion intervention can improve the spatial learning and memory ability of AD model rats,which may be related to the improvement of the arrangement structure and morphology of neurons and glial cells in the hippocampus of AD model rats.3.Thermosensitive moxibustion intervention can improve the spatial learning and memory ability of AD model rats,which may be related to the decrease of A?deposition by increasing the content of?-secretase and decreasing the content of?-secretase and?-secretase in the hippocampal tissue of AD model rats.4.Thermosensitive moxibustion intervention can improve the spatial learning and memory ability of AD model rats,which may be related to the regulation of proteins related to A?transport and metabolism. |
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