Moxibustion, the burning of moxa cones or sticks (made of mugwort, usually
The SAMP8 is the commonly used animal model for aging study and senescence-accelerated mouse/resistance (SAMR1) mice were usually used as a normally aging control for SAMP strains. In SAMP8 mice, senescence naturally occurs four to six months after birth; typical symptoms resemble Alzheimer’s disease [
After acclimation in the animal room for one week, 10 SAMR1 mice were used as normal control, and the 70 SAMP8 mice were randomly assigned (
2 × 3 factorial design.
Factor and level | Concentration | ||
---|---|---|---|
Low concentration (5~15 mg/m3) | Middle concentration (25~35 mg/m3) | High concentration (85~95 mg/m3) | |
Time | |||
15 min | L1 | M1 | H1 |
30 min | L2 | M2 | H2 |
Custom-designed mouse cages that can accommodate a single mouse and in which the mouse can turn around in the cage and make itself comfortable were used during the moxa smoke interventions. Moxa smoke was generated by burning moxa sticks (three-year-old pure moxa, 0.5 cm × 12 cm, Nanyang Hanyi Moxa Co., Ltd., China). The moxa smoke was contained within a custom-designed glass box (80 cm × 80 cm × 60 cm). Its upper cover, with a circular hole 0.6 cm in diameter, can be shifted. A light-scattering digital dust tester (DT, Beijing BINTA Green Technology Co., Ltd) was used to monitor smoke concentration by detecting levels of PM10 (particulate matter < 10
Groups receiving the same concentration of moxa smoke were exposed together. The main procedure was as follows. The DT was placed in the middle of the glass box to monitor concentration. Mice were individually put into the custom-designed cages, and groups with the same concentration, such as group L1 and group L2, were put into custom-designed cages, and subsequently, groups with the same concentration were placed on opposite sides of the DT. The burning end of a moxa stick was inserted into the glass box from the upper hole while the other end was held in the investigator’s hand. When the box filled with the predetermined amount of smoke, which took about 15, 32, and 82 seconds for low, middle, and high concentration, respectively, the stick was withdrawn and the hole was quickly closed. After 15 minutes, the mice that belonging to the 15-minute exposure group were removed, while the 30-minute exposure group continued for another 15 minutes.
To ensure concentrations of moxa smoke in the specified ranges, moxa smoke concentration was monitored dynamically every three minutes by DT. When the concentration exceeded the upper range, the upper cover of the glass box was moved to release some of the smoke and when it fell below the lower range, the burning moxa stick was reinserted to the box for a few seconds.
The model and normal groups were not exposed to moxa smoke. Mice in those two groups were caged and put on opposite sides of the glass box for 30 minutes with no exposure to moxa smoke.
Twenty-four hours after the last intervention, the mice were sacrificed, and cerebrum samples were quickly dissected on an ice board. Using enzyme-linked immunosorbent assay kits of 5-HT, DA, and NE (produced by Nanjing Jiancheng Bioengineering Institute, Nanjing, China), values of absorbance were strictly measured by microplate reader (Multiskan MK3, Finland) at 450 nm wavelength. The concentrations of 5-HT, DA, and NE were determined by comparing the O.D. of the samples to the standard curve.
Data were analyzed by analysis of variance (ANOVA), and post hoc analyses were conducted using the Student-Newman-Keuls test. For that of the six smoke groups, ANOVA for factorial data was used. All values were reported as means ± standard error. Analyses were performed with SPSS software version 13.0;
Compared to the normal group, the model group showed a remarkable decrease in cerebral 5-HT, DA, and NE levels (Figure
Differences in 5-HT (a), DA (b), and NE (c) levels, normal group (SAMARI mice) versus model group (SAMP8 mice). Note:
Moxa smoke groups showed a higher level of monoamine neurotransmitters than model group. Compared to the model group, 5-HT and NE were significantly increased in L2, M1, and M2, while DA was significantly increased in L2 and M1 (Figure
Cerebral levels of 5-HT (A), DA (B), and NE (C) of SAMP8 mice exposed to different concentrations of moxa smoke for various lengths of time. Note: any two groups without a common alphabet (a or b) are significantly different
Using ANOVA for factorial data, there was a significant interaction between length of exposure and concentration effects on cerebral monoamine neurotransmitter levels (Figure
Cerebral levels of 5-HT (a), DA (b), and NE (c) of SAMP8 mice exposed to moxa smoke at different concentrations and for different lengths of time.
Multiple comparisons showed that moxa smoke intervention for the M1 group, middle concentration for 15 minutes, manifested the highest effect in increasing cerebral 5-HT, DA, and NE levels among the different combinations between concentration and exposure length (Figure
In this study, we explored the anti-aging effects of moxa smoke and found that moxa smoke may increase monoamine neurotransmitter levels in SAMP8 mice and the effects were related to exposure length and concentration of moxa smoke. This indicated that moxa smoke may be one of the effective components of moxibustion. 5-HT, DA, and NE were important neurotransmitters in the central nervous system and were closely related to neural functions and aging [
Moxa floss is made from mugwort leaf (
In this study, we also find that the anti-aging effect is linked to smoke concentration, and there is an interaction between concentration and length of exposure. According to our results, monoamine neurotransmitter levels were highest in the M1 group (i.e., middling concentration for 15 minutes), which suggested that these may be the optimum specifications for raising monoamine neurotransmitter levels. There is probably a nonlinear relationship between dose (concentration and exposure length) and effect of moxa smoke based on the present results, however; further study is needed to determine the dose-effect curve.
There exist some limitations in this study. Oxygen concentration was not monitored during the intervention procedure. However, the combustion of moxa stick in the glass box was only for a very short duration from 15 to 82 seconds and the glass box was not completely sealed. Methods to monitor the oxygen supply should be applied in subsequent studies. Secondly, behavioral tests of learning and memory should be recommended for future studies.
In conclusion, our preliminary observation showed that moxa smoke may increase monoamine neurotransmitter in central nerve system and the middle concentration of moxa smoke for 15 minutes seemed most beneficial. However, further investigation to confirm our findings and to explore possible mechanisms of action is warranted.
The authors declare that they have no Conflict of Interests.
The authors thank Dr. Lyn Lowry at the Center for Integrative Medicine, University of Maryland School of Medicine, for her English edit. This study was supported by the National Natural Science Foundation of China (no. 81072862) and the National Basic Research Program of China (no. 2009CB522906).