Study On The Mechanism Of Mongolian Moxibustion Therapy On Senile Dementia Model Rats

Objective1. This paper proposed a hypothesis that Mongolian Moxibustion might have a positive effect on the cranial nerves damages and cranial nerves’functional rehabilitation of Alzheimer Disease (AD) rat model based on literature review and previous research. By using methods of treating AD rat with Mongolian Mixibustion, the paper demonstrated the therapeutical effect and mechanism of treating AD with Mongolian Moxibustion.2. The research established AD model rat by injecting D-Galactose subcutaneously and Aβ25-35into bilateral hippocampal zones. By using Mongolian medicine’s DINGHUIXUE, XINXUE, and WEIXUE as a break-through point, divided the rats into normal group, sham-operated group, model group, moxibustion group and western medicine group to observe Mongolian moxibustion’s effect on AD rats’faculty of learning and memory, relative choline, oxidation, content of free radical enzyme, brain tissue nerve cells’pathological change, the effect on brain tissue’s relevant genetic protein’expression, and clarified the therapeutical effect and mechanism of treating AD with Mongolian Moxibustion.Method1. Animal select and screen method:The body weight of220±20(g), healthy male SD rats of clean grade,then the rats are taken part in Morris water maze test separately to eliminate natural dementia rats,at last70qualified rats will be picked out.2.Rats grouping and model manufacture:Dividing70rats into5groups randomly according to body constitution, normal group, sham operation group, model group, a moxibustion group and a western medicine group,14rats in each group. In the first6weeks, keeping neck-back subcutaneous injection to the rats from model group, moxibustion group and western medicine group with D-galactose (150mg/kg. D) and neck subcutaneous injection to the rats from sham-operated group with Physiological saline (150mg/kg. D) once a day,the normal group is not be processed. From the seventh week, using the Stereotaxic hippocampal technique to the rats except the normal group and undergoing craniocerebral surgery, the rats from model group, moxibustion group and western medicine group are injected into each hippocampus with prepared state of aggregation Aβ25-35slowly. each side of the luL within the5min injection. the rats from sham-operated group are injected into each hippocampus with isodose Sterile Saline Solution,the normal group is not be processed.3.Model detecting method:Having water maze test after finishing models in two days, then concluding the rats’ average escaping latency and average number of passing through the platform area after manufacturing model.Only the rats whose average escaping latency and average number of passing through the platform area are drop by one-third before the manufacturing model are called successful models.Only the winner models get into the next tests. The death and unqualified rats are eliminate, and follow the random principle to supply the animals.4.Therapies:Two days after craniocerebral operation,starting the therapies. Taking the "cave","heart" and "stomach" acupoints of rats from moxibustion group,every day an acupoint,15min per treatment.using the moxa cone moxibustion of self sticking method, alternating three acupoints,10days for a period of treatment, one day rest after a period of treatment, and then begining treatment again, the total treatment is two procedures. Western medicine treatment group is given gavage with clinical medecine-Pyrazole raschig temple (brain convalescent pills)0.92mg/kg which cures the senile dementia,the treatment lasts20days. The model group has the same grabbing and fixing method with moxibustion group,and is given gavage with isodose Saline as western medicine group. The sham-operated group has the same grabbing and fixing method with moxibustion group,and is given gavage with isodose Saline as western medicine group.The normal group is not be processed.5. Materials drawing and index detecting:After the therapies,all rats are taken part in the maze test、Materials drawing and index detecting.(1)14rats in each group are taken part in the Morris water maze experiment to test behavior, then calculating every rat’s average escaping latency、the number of passing through the platform and the ratio of passing through effective area;(2) After taking the cardiac perfusion fixation for4rats in each group,having Nissl and TUNEL staining to detect changes of hippocampal neurons,then observing changes of hippocampal neurons、Injury and apoptosis situation under the Microscope.(3) After10rats in each group having the anesthesia,putting them on the ice box to separate cortex and left side hippocampus、weighing and cryopreserved, then using ultraviolet spectrophotometer to determinate MDA content of cortex and hippocampus organization、active content of SOD, ChAT, AchE.Using real-time quantitative RT-PCR method to detect the expression of hippocampus organization Bcl-2、Bax mRNA;Using Western Blot method to determinate expression of hippocampus organization Bel-2and Bax protein.6. statistical treatment:using the SPSS19.0software for statistical treatment, and the data is showed by mean±standard deviation (x±s), Comparison between groups using F test, LSD test comparison between the two groups, P<0.05means significant difference.Results1.Mongolian moxibustion’s effect on AD rats’ability of learning and memory ethology(1) navigation test on each group of rats after treatment "1-5day average latency period"and comparison "effective area ratio",The model group has a significant extension "1-5day average latency period".(P<0.05); the model group,and comparison shem-operation group’s "effective area ratio" has a apparent shortening (p<0.05); has a significant extension,"1-5day average latency period"(P<0.01) while compared "ration of passing valid area" has a significant reduction(p<0.01) while compared to the Moxibustion group and model group’s ration of passing valid area has a comparatively significant increase (p<0.01); the western medicine group’s comparison with the model group,"ration of passing valid area "has no significant difference (p>0.05), Mongolian moxibustion’s and Western medicine have great effect on AD rats’ability of learning and memory ethology.(2) spatial probe test on each groups of rat after treatment " the frequency of passing platform area"the model group has a significant decrease compared to normal group(P<0.05), the comparison of the frequency of passing platform area between shem-operation group and model group has a significant shortening (p<0.01); it showed that the AD rats model succeed.the moxibustion group’s comparison with the model group on "the ration of passing valid area" has a significant increasing (p<0.01); the western medicine group’s comparison with model group,"the frequency of passing platform area" both have a signifigant extension,(p<0.01);"on the ration of passing valid are"a has no significant differences (p>0.05), the western medicine group’s comparison with moxibustion group’s "on the ration of passing valid area" has a significant increasing (p<0.05); Mongolian moxibustion’s effect on AD rats’ability of learning and memory ethology is superior to Western medicine(piracetam).2.Mongolian moxibustion’s effect on AD rat’s hippocampus nerve cells(1)Nissl coloring:Model group comparing to blank and shen-operation group, hippocampus Nissl are shortening,(p<0.01). blue granular Nissl cytoryctes, the stronger the activity of the neurons the more the number of Nissl cytoryctes, and the deeper the color; on the contrary, the lesser the number of Nissl cytoryctes, the lighter the color, and it demonstrate the severer damaged the neurons. The result shows that, compared to the blank group and shen-operation group, the model group’s brain hippocampus area’s number of Nissl cytoryctes is relatively less (p<0.05), the coloring is lighter and disposed irregularly. After brain rehabilitation treatment, the Nissl cytoryctes’morphological observation standard has a significant improvement (p<0.01) compared to model group; after Mongolian moxibustion treatment, the rat’s brain hippocampus area’s Nissl coloring is dipper, the number of Nissl cytoryctes increased (p<0.05), and disposed regularly.(2) TUNEL coloring:the more TUNEL positive cells indicate the more of the apoptosis cells. The TUNEL coloring test of this research shows, the blank group and shem-operation group’s hippocampus appear a TUNEL positive cell on occasion. But the model group’s hippocampus appears relatively more TUNEL positive cell (P<0.01); after brain rehabilitation treatment, the rat’s hippocampus positive cell has a certain decrease (p <0.01) compared to the model group. In Mongolian moxibustion treatment occurs positive cells in occasion, compared to the model group has a significant decrease (p<0.01), while comparing Mongolian moxibustion group with western medicine group, the TUNEL positive cells has a significant decrease in Mongolian moxibustion (p<0.05). Mongolian moxibustion’s effect on AD rat’s brain tissue is superior to Western medicine.3. The Mongolian medicine moxibustion of AD rat brain tissue MDA, SOD and ChAT, the influence of the AchE(1) each rat cerebral cortex and hippocampus in the determination of SOD and MDA①each group rats cortex and hippocampus tissue SOD activity, compared with normal group and control group, model group of cortex and hippocampus SOD activity decreased significantly (P<0.01), the moxibustion group, western medicine group compared with model group, SOD activity of cortex and hippocampus were significantly increased (P<0.01), the comparison between the western medicine group and moxibustion group had no significant difference (P>0.05); Compared with normal group, control group of cortex and hippocampus SOD activity is significantly increased (P<0.01, P<0.05).②groups of the MDA content of cortex and hippocampus of rats, compared with normal group and control group, model group and the hippocampus cortex MDA content were significantly increased (P<0.01), the moxibustion group, western medicine group compared with model group, cortex and hippocampal MDA were significantly reduced (P <0.01), the comparison between the western medicine group and moxibustion group had no significant difference (P>0.05); Compared with normal group, control group of cortex MDA content significantly decreased (P<0.01), MDA content in hippocampus no significant difference (P>0.05).(2) each group’s the determination of AchE,ChAT in rat cerebral cortex and hippocampus①each group ChAT activity of rat cerebral cortex and hippocampus, compared with normal group and control group, cortex and hippocampus ChAT activity of model group were significantly lower (P<0.01), the moxibustion group, western medicine group compared with model group, cortex and hippocampus ChAT activity were significantly increased (P<0.01), the comparison between the western medicine group and moxibustion group had no significant difference (P>0.05); Control group no significant difference compared with normal group (P>0.05); The moxibustion group compared with normal group, cortex ChAT activity have significant difference (P<0.05). The western medicine group compared with normal group and control group, cortex and hippocampus ChAT activity were significantly reduced (P<0.01). ②groups of rat cerebral cortex and hippocampus of AchE activity, compared with normal group and control group, cortex and hippocampus AchE activity of model group were significantly lower (P<0.01), the moxibustion group, western medicine group compared with model group, cortex and hippocampus AchE activity were significantly increased (P<0.01), the comparison between the western medicine group and moxibustion group had no significant difference (P>0.05); Control group no significant difference compared with normal group (P>0.05).4. The Mongolian medicine moxibustion therapy of AD rat hippocampal tissue Bel-2, the influence of Bax mRNA and protein expression(1) each group rats hippocampal tissue Bel-2mRNA and protein expression, compared with normal group and control group, model group the Bel-2mRNA and protein expression level decreased significantly (P<0.01), the moxibustion group, western medicine group compared with model group, the Bel-2mRNA and protein expression level increased significantly (P<0.01, P<0.05), the comparison between the western medicine group and moxibustion group had no significant difference (P>0.05); Compared with normal group, control group no significant difference (P>0.05).(2) groups of rat hippocampus Bax mRNA and protein expression, compared with normal group and control group, model group of Bax mRNA and protein expression level increased significantly (P<0.01), the moxibustion group and western medicine group compared with model group, Bax mRNA and protein expression levels were significantly reduced (P<0.01), the comparison between the western medicine group and moxibustion group had no significant difference (P>0.05); Compared with normal group, control group no significant difference (P>0.05).Conclusion1.This research established AD model rat by injecting D-Galactose subcutaneously and Aβ25-35into bilateral hippocampal zones, which can better simulate ethological and pathological features such as AD learning and memorizing disability, neurons damage, nerve cell apoptosis. It can demonstrate closer AD pathological phenotype, and has a better polarization and practicality. 2.Mongolian moxibustion can decrease the AD rat’s hippocampus nerve cell damage, inhibit the apoptosis of nerve cell, has a prevention and curing effect on AD.3.Mongolian moxibustion could Improve AD rat’s ethological fuction, improve it’ s learning and memorizing ability, has a prevention and curing effect on AD.4.Mongolian moxibustion could improve the AD rat’s brain tissue’s SOD activity, reduce MDA content, relive the effect of lipid peroxidation, increase anti-oxidant enzyme’s activity, remove the free radicals, stay caducity, and to prevent the AD’s occurring and development.5.Mongolian moxibustion could increase AD rat’s brain tissue’s ChAT, TchE’s activity, to protect the brain tissue’s enzyme’s activity, and imprve brain tissue’s energy metaboltion disorder.6. Mongolian moxibustion has a up-regulating effect on AD rat’s hippocampal tissue’s Bel-2’s mRNA and protein level. And have a down-regulating effect on Bax’s mRNA protein level and to increase Bcl-2/bax, to inhibit the AD’s brain tissue apoptosis.7.For AD rat’s Mongolian Moxibustion’s DINGHUIXUE, XINXUE, and WEIXUE theapy treatment has a positive effect on it’s learning and memorizing ability, to reduce the hippocampal nerve’s damage, and inhibit the brain tissue apptosis. It’s functioning mechanism has a close relation with improving brain tissue’s SOD activity, decreasing MDA content, enhancing ChAT, TchE’s activity and up-regulating hippocampus tissue’s Bcl-2’s mRNA and protein expression, down regulating Bax’s mRNA and protain expression.8. In comparison with the effect of western medicine (NAO FU KANG), Mongolian moxibustion has a apparent function on improving learning and memorizing ability, hippocampal nerve cell’s anti-apoptosis. It can prove that Mongolian moxibustion has a better function than NAO FU KANG in curing AD.