Effects Of Different Moxibustion Factors On Mitochondrial Function - Energy Metabolism In APP / PS1 Double Transgenic AD Mice

Purpose:To determine the effects and further investigate the mechanism of two different parts of moxibustion, namely moxa smoke and heat, on learning and memory behavior, mitochondrial activities (ATP, mitochondrial respiratory chain (RC) complexes Ⅰ, Ⅱ, Ⅲ), oxidative state, cell apoptosis via mitochondria pathway in brain and urinal metabolism in APP/PS1 male mice.Methods:Ten wild-type C57BL/6 normal mice served as normal control while 40 APP/PS1 mice were randomly divided into 4 groups (n=10/group),1) traditional moxibustion, 2) heat only produced by a smokeless moxa,3) moxa smoke and 4) no-treatment control. Treatments were given 20-min,6 days a week for 8 weeks. In groups 1 and 2, the moxa stick was aimed above acupoint Guanyuan (CV4). In group 3, mice were exposed to moxa smoke (10-15mg/m3), while in groups 4 and normal control, no treatment was given. The step-through test, Morris water maze (MWM) test and open-field lest were conducted in the 9th week. Urine samples of each mice were collected and stored for UPLC-MS analysis. Mice were sacrificed at the end of week 11. ELISA techniques was used to detect the level of ROS, 8-OhdG,4-HNE,3-NT, ATP and activity of mitochondrial RC complexes I, II, III both in prefrontal cortex and hippocampus.Bcl-2、Bax、Caspase-3、Caspase-9 levels were evaluated using immunohistochemical staining. β-amyloid wasevaluated using Congo red staining. Urine metabolites fingerprint analysis aimed to distinguish the moxibustion and no-treatment control from other groups, then screened the potential biomarkers.Results:1. Behavioral testComparing to normal control group, mice of group 4 demonstrated lower auditory system excitation (P=0.004) and worse ability of learning and memory (P=0.026). Mice of group eat more food than that of group 4 at the beginning of experiment (P<0.01). In the open field test, mice in group 2 moved longer distance than that in group 3 and group 4 (P=0.004, P=0.019), stood up more often than that in group 3 (P=0.007). In the step-through test,on the aspect of learning record the groupl (P=0.012) demonstrated shorter reaction time than group 3. On the aspect of memory record, group2,3,4 showed longer memory latency time (P=0.000、P=0.000、 P=0.007) and both group 2 and 3 committed less (P=0.001,P=0.004) than group 4. All mice in 5 groups tended to perform better in MWM test with the training went on, but there was no significant difference between them. There was no significance of memory latency time between them. On the memory aspect of MWM, only normal control got better memory than group 4.2. Oxidative status of brainComparing to normal control group, mice in group 4 had significant higher levels of ROS 8-OhdG,4-HNE,3-NT in prefrontal cortex and hippocampus (P<0.05).Comparing to group 4, mice in group 1 got lower level in ROS (P=0.002), mice in group 1,2,3 showed lower levels in 8-OhdG (P=0.002,P=0.003,P=0.001),4-HNE (P=0.001,P=0.000,P=0.002),3-NT (P=0.009,P=0.002,P=0.004) of prefrontal lobe. Mice of group 1 showed lower level of ROS than that of group 4 (P=0.001) and 3 (P=0.017). Comparing to group 4, mice in group 1,2,3 had lower levels of 8-OhdG (P=0.001,P=0.000,P=0.000) and 3-NT (P=0.007,P=0.0001,P=0.0001) in hippocampus.3. Function of mitochondria in brainComparing to normal control group, mice in group 4 had significant lower levels of ATP, RC Ⅰ, Ⅱ,Ⅲ (P<0.05). Comparing to group 4, mice in group 1,2,3 got significant higher level of ATP (P=0.008), RC I (P=0.000,P=0.000,P=0.023) and RC Ⅲ (P=0.000,P=0.000,P=0.000) in prefrontal cortex. The activity of RC Ⅱ in prefrontal cortex was remarkably enhanced in group 1 than that of group 4.Comparing to group 4, mice in group 1,2,3 got significant higher level of ATP (P=0.000, P=0.000,P=0.001), RC Ⅰ (P=0.000,P=0.000,P=0.023) and RC Ⅲ (P=0.000,P=0.000, P=0.001) in hippocampus. Comparing to group 3,mice of group 1 and group 2 both got higher level of ATP (P=0.004,P=0.018), RC I (P=0.011,P=0.001;P=0.026, P=0.020), RCⅢ (P=0.000,P=0.001;P=0.018,P=0.025) in prefrontal cortex and hippocampus.4. Cell apoptosis via mitochondria pathway in brainComparing to normal control group, mice in no treatment control group had higher expression of Bax, Caspase-3, Caspase-9, lower expression of Bcl-2 in both prefrontal cortex and hippocampus (P<0.05). Comparing to group 4, mice in group 1,2,3 got significant higher expression of Bcl-2 (P=0.000,P=0.001,P=0.002;P=0.000,P=0.000,P=0.012), lower expression of Bax (P=0.000,P=0.000,P=0.001; P=0.001,P=0.002,P=0.012) both in prefrontal cortex and hippocampus CA3 district. The Caspase-3 of prefrontal cortex and hippocampus showed higher activity in groupl (P=0.009,P=0.035),2 (P=0.012) than that of group 4.Mice in group 4 expressed a lotaggregation of β-amyloid accompanying with neuron loss and irregular arrangement in hippocampus. Mice of normal control had none aggregation of (3-amyloid. Mice in group 1,2,3 got less aggregation of β-amyloid, more clear viewed structure and rare loss of nuron.5. Urine metabolism analysisUrine metabolites of no treatment control, normal control, real moxibustion, moxa smoke, smokeless moxibustion groups clustered well. There were 16 types of biological markers contributed to the difference between 5 groups, most realeted to amino acid metabolism, the synthesis and metabolism of carbohydrate, nitrogen delivering pathways. Comparing normal control group with group 4,17 types of biological markers were identified as metabolites contributing to the separation,15 of them were increased:Indoxyl sulfate, Methylhippuric acid,2-Methylhippuric acid, Heptanoylglycine, Indolelactic acid, Heptylmalonic acid, Creatine, Hippuric acid,2-Methylbutyrylglycine, Isobutyrylglycine,N 1-Methyl-4-pyridone-3-carboxamide, Propenoylcarnitine, N-Heptanoylglycine, Ureidopropionic acid, Tiglylglycine, only Hypoxanthine was decreased. They were most related to amino acid metabolism, protium nitrogen delivering pathway and energy metabolism.Comparing group 1 with group 4,7 types of biological markers were identified as metabolites contributing to the separation, p-Cresol sulfate, Hexanoylglycine, Muricholic acid,2-Methylhippuric acid,3alpha-Hydroxy-5beta-chola-8,14-dien-24-oic acid, Creatine,5beta-Chola-3,8(14),11-trien-24-oic Acid. While 4 were decreased:Methylhippuric acid, Xanthurenic acid, Butenylcarnitine, N-Formyl-L-methionine. They were most related to the synthesis and decomposition of amino acid and carbohydrate.Conclusions:1.Moxibustion, moxa smoke and smokeless moxibustion play equal role of delaying the pathological process Alzheimer’s disease by protecting the function of mitochondria, reducing the oxidative status, inhibiting cell apoptosis via mitochondria pathway.Comparing to smokeless moxibustion, moxa smoke contributes more to protecting the function of mitochondria. Both of them have a great impact on the upsteam components of cell apoptosis viamitochondriapathway.2. Moxibustion, moxa smoke and smokeless moxibustion are capable of enhancing auditory system excitation and cognitive abilities of APP/PS1 mice, and moxibustion plays the leading role.3. Moxibustion, moxa smoke and smokeless moxibustion can regulate the related compounds in metabolic pathways including amino acid, starch and sucrose, nitrogen metabolism,pentose and glucuronate interconversions, Aminoacyl-tRNA biosynthesis.