Research Of Changes Of Autophagy Related Factors Of The Senescence Accelerated Mouse (SAMP8) And The Regulatory Effect Of Acupuncture On It

Objective:This study observed the changes of autophagy related factors(autophagosome formation,autophagy lysosome formation and autophagosome degradation)in senescence accelerated mouse/prone8(SAMP8)and explored the effects of "Sanjiao" acupuncture on the above factors.From the perspective of autophagy,we discussed the possible mechanism of dementia and the therapeutic mechanism of "Sanjiao" acupuncture in the treatment of AD.Methods:There were 27 SAMP8 mice aged 8 months used as the AD model animal,and divided randomly into three groups: P8 group(the model group),acupuncture group(P8 +acupuncture),non acupoints group(P8 + acupuncture non acupoints).And 9 senescence accelerated mouse/resistance 1(SAMR1)of the same old were selected as the blank control group of this experiment.The mice of the blank group and model group were grabbed every day and without other additional treatments,the acupuncture group was treated with acupuncture at "Danzhong","Zhongwan","Qihai",bilateral "Blood Sea" and bilateral "Zusanli",and the non acupoint group mice was treated with acupuncture at bilateral subcostal fixed non meridian non acupoints.It is worth noting that six consecutive days as a course of treatment and all interventions of three groups were performed once a day for 2consecutive courses.Morriswatermaze was used to observe the learning ability and memory ability of mice in each group to explore the effect of "Sanjiao" acupuncture on learning and memory ability of AD model mice.After the end of the last water maze,brain tissues were taken.The m RNA expressions of LC3 B,p62,LAMP1,TFEB and CTSD in mouse hippocampus were detected by q-PCR,and the expressions of LC3 B,p62 and CTSD proteins in mouse hippocampus were observed by WB.Results:1.Changes of SAMP8 neurological behavior and the intervention effect of acupuncture:The results of Morriswatermaze showed that in the blank group totle required time of mice to the hidden platform(escape latency)shortened with the adding of artificial training days,and the academic performance was significantly improved every day,mice showed excellent learning and memory ability.While the model group did not show this trend,its escape latency is kept at a relatively high level: The escape latency of AD model mice was significantly prolonged compared with that of black normal mice(P<0.01)."Sanjiao" acupuncture can effectively amplify the learning and memory abilities of AD model mice:there was an apparent amelioration in escape latency of acupuncture group mice after after a few days of training,like the performance of mice in the black group,besides,the escape latency was significant shorter than that of AD model mice(P<0.05).Almost no changes in escape latency of mice can be seen in non acupoints group mice(P>0.05).2.Changes of autophagy related factor m RNA expression in SAMP8 hippocampus and the intervention effect of Acupuncture:(1)LC3B: The expression of LC3 B m RNA in mice hippocampus showed no significant difference among blank group,AD model group,acupuncture group and non acupoints group(P>0.05).(2)p62: The expression of p62 m RNA in mice hippocampus showed no significant difference among blank group,AD model group,acupuncture group and non acupoints group(P>0.05).(3)LAMP1: The expression of LAMP1 m RNA in hippocampus of AD model mice was significantly lower than that of normal blank mice(P>0.05)."Sanjiao" acupuncture can effectively improved the decreased LAMP1 m RNA expression: compared with AD model mice,the LAMP1 m RNA expression in hippocampus of acupuncture group mice was significantly increased(P<0.05).Non acupoints acupuncture could not show the similar effect of "Sanjiao" acupuncture: the expression of LAMP1 m RNA in hippocampus between AD model mice and non acupoints group mice was no significant difference(P>0.05).(4)TFEB: The expression of TFEB m RNA in mice hippocampus showed no significant difference among blank group,AD model group,acupuncture group and non acupoints group(P>0.05).(5)CTSD: the m RNA expression of CTSD in hippocampus of AD model mice was significantly declined compared with that of normal blank mice(P>0.05)."Sanjiao" acupuncture can effectively improved the decreased CTSD m RNA expression: compared with AD model mice,a marker rise of CTSD m RNA expression in hippocampus could be seen in the acupuncture group mice(P<0.05).Non acupoints acupuncture could not show the similar effect of "Sanjiao" acupuncture: a significant difference of CTSD m RNA in hippocampus coundn’t be found between AD model mice and non acupoints group mice(P>0.05).3.Changes of autophagy related factor protein expression in SAMP8 hippocampus and the intervention effect of acupuncture:(1)LC3B: The expression of LC3 B protein in mice hippocampus showed no significant difference among blank group,AD model group,acupuncture group and non acupoints group(P>0.05).(2)p62: The expression of p62 protein in mice hippocampus showed no significant difference among blank group,AD model group,acupuncture group and non acupoints group(P>0.05).(3)CTSD: Consistent with the results of CTSD m RNA,expression,the protein expression of CTSD in hippocampus of AD model mice was significantly declined compared with that of normal blank mice(P<0.01).After acupuncture treatments with "Sanjiao" acupuncture,the protein expression of CTSD was significantly up-regulated(P<0.01,compared with AD model mice);Non acupoints acupuncture could not significantly increase the expression of CTSD protein in AD model mice(P>0.05,compared with AD model mice).Conclusion:(1)SAMP8 mice showed similar characteristics to AD: significant decline in learning and memory abilities."Sanjiao" acupuncture can ameliorate both spatial learning ability and memory acquisition ability of SAMP8 mice,and play a benign regulatory role in the damage of learning and memory ability,but an non-significant change of the learning and memory ability can be seen in the non acupoints acupuncture group.(2)At the transcriptional level and protein expression: the expression of LAMP1 and CTSD in the hippocampus of model group mice decreased significantly,indicating that lysosome formation and function were damaged in the early stage of AD,which affected lysosome formation and autophagy degradation in the later stage of autophagy."Sanjiao" acupuncture can significantly improve the expression of LAMP1 and CTSD of AD model mice,indicating that "Sanjiao" acupuncture can play a role in the treatment of AD by affecting the formation of lysosomes and autophagy degradation in the later stage of autophagy,so as to improve the acquisition ability of ad spatial learning.