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J. Acupunct. Tuina. Sci. 2011, 9 (6): 336-339 DOI: 10.

1007/s11726-011-0546-9

Special Topic Study

Experimental Research on the Anti-tumor Effect of Moxibustion Serum

Chen Yunfei ()1, Zhao Cuiying ()1, Lv Qiyong ()2 1 Shanghai Research Institute of Acupuncture and Meridian, Shanghai 200030, P. R. China 2 Clinic of Guizhou Fire Corps, Guiyang 550001, P. R. China

C57BL/6 R2-03 A Abstract Objective: To observe the inhibiting tumor effect of moxibustion serum on the tumor-bearing mice. Methods: The mice were transplanted with C57BL/6 mouse thymus cells to form solid tumor and were intraperitoneally injected with moxibustion serum. The tumor growth and survival time of tumor-bearing mice were recorded. Results: The moxibustion serum could significantly postpone the formation of tumor nodules. Compared with the tumor-bearing group, tumor nodules formation time of all treatment groups was delayed and the survival time of the tumor-bearing mice was prolonged. Besides, the inhibiting tumor effect of moxibustion serum in the pre-treatment group was better than that in the moxibustion serum treatment group. Moxibustion acts specifically on acupoints. Conclusion: The moxibustion serum had obvious anti-tumor effects. Key WordsMoxibustion Therapy; Direct Moxibustion; Immune Sera; Neoplasms; Mice Acupuncture-moxibustion has some therapeutic effect on malignant tumor. It can relieve clinical symptoms, reduce the side effects of radiotherapy and chemotherapy, and inhibit tumor growth in a certain extent[1-3]. Moxibustion serum is a kind of research ideas and methods proposed in acupuncture research[4]. Some studies showed that moxibustion serum contained various immune activity factors of non-specific immune function[5, 6]. We have observed the anti-tumor effect of moxibustion serum on inhibiting tumor, which supplied the solid clues for further research on the molecular biological and immunological mechanism of moxibustion serum in inhibiting tumor. Now, it was reported as follows.
Fund Item: National Natural Science Foundation of China (39570882) Author: Chen Yunfei, researcher, doctoral supervisor, icyf1968@yahoo.com.cn

1 Material and Methods


1.1 Serum preparation 1.1.1 Moxibustion serum Thirty normal healthy C57BL/6 mice (weighing 20-25 g) were applied moxibustion to Dazhui (GV 14) with small moxa cone made by refined moxa (1.5 mg/cone), 2 cones for each treatment, once every 2 d, for continuous 6 treatments. The blood was collected from the eyeballs on the next day after the final treatment. The serum was obtained after clotting and centrifugation, and then mixed with the other from the same group, filtrated, sterilized, packaged and frozen at -20. 1.1.2 Moxibustion serum with non-meridian point The serum was collected as the operations of the moxibustion serum except for the acupuncture point, not Dazhui (GV 14) but non-meridian point on the tails of mice.

336 Shanghai Research Institute of Acupuncture and Meridian and Springer-Verlag Berlin Heidelberg 2011

J. Acupunct. Tuina. Sci. 2011, 9 (6): 336-339

1.2 Experimental animal Female C57BL/6 mice were 6-8 weeks old. The recovery EL-4 tumor lines (activity > 95%) were washed with sterile Hanks solution for 3 times, transferred into 5105/mL cell suspension and inoculated to the axillary subcutaneous of mice at the dose of 0.1 mL per mouse. 1.3 Groups The successful tumor-bearing mice were randomly divided into 5 groups with 9 mice in each group. Tumor-bearing group (group A): The mice were only intraperitoneally injected with 0.1 mL physiological saline at the 2nd and 7th day after tumor cell inoculation, without any other treatment. Moxibustion serum pre-treatment group (group B): The mice were intraperitoneally injected with 0.1 mL non-diluted moxibustion serum per mouse before tumor cell inoculation. Moxibustion serum treatment group (group C): The mice were intraperitoneally injected with 0.1 mL non-diluted moxibustion serum per mouse on the 2nd and 7th day after tumor cell inoculation. Cyclophosphamide group (group D): The mice were intraperitoneally injected with Cyclophosphamide at 30 mg/(kgbw) on the 2nd and 7th day after tumor cell inoculation. Non-meridian point moxibustion serum group (group E): The mice were intraperitoneally injected with 0.1 mL non-diluted non-meridian moxibustion serum per mouse on second and 7th day after tumor cell inoculation. 1.4 Measurement of outcome 1.4.1 Time of tumor nodules formation According to the pre-test, the tumor nodules formed at the part of mice where the tumor cell injected were closely observed after the treatment to calculate average tumor formation time of mice in each group. 1.4.2 Dynamic changes of tumor size The maximum diameter (a) and minimum diameter (b) were measured for once every other day since the 14th day of post-inoculation. Then the diameter (D) of tumor nodules were calculated as the formula D=ab. 1.4.3 Survival time The death time of each mouse in each group was recorded everyday, and calculated for mean survival time, the prolonging rate of life span and 50% survival time.

The prolonging rate of life span (%) = (Mean survival time of treatment group - Mean survival time of group A) / Mean survival time of group A 100%.

2 Results
2.1 comparison of tumor nodules formation The moxibustion serum could postpone the tumor nodules formation significantly. Compared with that of group A, the tumor nodules formation time of all the treatment groups were delayed except group E, especially group B (P<0.01). The tumor nodules formation time of group B was much later than group D (P<0.05). However, the difference of tumor nodules formation time between group C and group D was not significant (P>0.05). Compared with group E, group B and group C showed statistical difference (P<0.05) (table 1).
Table 1. Tumor nodules formation time (Day) Groups Group A Group B Group C Group D Group E n 9 8 8 9 9 Tumor nodules formation time 14.331.01 23.751.281)2) 3) 19.501.193) 20.221.23 14.891.05

Note: Compared with group A, 1) P<0.01; compared with group D, 2) P<0.05; compared with group E, 3) P<0.05

From the 15th to 30th day after the operation, the differences among group A, group E and group D were not obvious, resulting in the nearly overlapping curves. However, the tumor nodules formation time of group B, followed by group E, was notably later than those of the other groups, indicating the slow tumor growth. From the 30th to 35th day after the operation, the difference between group B and group A was significant (P<0.05) (Fig.1).

Days
Group A Group D Group B Group E Group C

Fig.1. Growth curves of tumor nodules formation

Shanghai Research Institute of Acupuncture and Meridian and Springer-Verlag Berlin Heidelberg 2011 337

J. Acupunct. Tuina. Sci. 2011, 9 (6): 336-339

2.2 Comparison of survival time Parts of the mice in each group died on the 29th day post-operation. The death time of mice in each group was recorded everyday till the 58th day, which was calculated for the mean survival time, the prolonging rate of life span and 50% survival time of each group. Compared with group A, the differences of all groups (except group E) were significant, especially group B. The mean survival time of group B was 47.01 d, markedly higher than that of group A, group E (P<0.01), group C and group D (P<0.05). The survival time of group C was lower than that of group D without significant difference (P>0.05). Regarding 50% survival time, group B ranked the top (43.5 d), followed by group D. As far as the prolonging rate of life span, group B was still the best one (44.67%) and the next one was group D, suggesting that the significant inhibiting tumor effect in group B which prolong the survival time of tumor-bearing mice and group B was prior to group C referring to anti-tumor effect (table 2, Fig.2).
Table 2. Survival time of tumor-bearing mice Groups Group A Group B Group C Group D Group E n 9 8 8 9 9 ST (day) 32.662.64 47.016.041)2) 38.633.99 42.114.86 32.442.29 50% ST (day) Prolonging RLS (%) 31.5 43.5 34.1 39.0 31.2 44.67 18.28 28.93 -

Note: ST= Survival time; RLS=Rate of life span Compared with group A and E, P<0.01; compared with group C and D, P<0.05
Group A Group B Group C Group D Group E

Fig.2. Comparison of survival rate

3 Discussion
In the mid-1980s, Japanese scholars suggested the concept of serum pharmacology, which argued that the filtrated serum of the animals that treated with some drugs for the pharmacological experiment could remove all obstacles in some way that greatly

enhanced the credibility of the results. The concept and method were introduced by the researchers in traditional Chinese medicine and improved to a new method "Chinese herbal medicine serum", revealing the grand improvement in Chinese herbal medicine in vitro methodology[7]. Basing on the method and thinking of "Chinese herbal medicine serum", the researchers in acupuncture-moxibustion science provided the concept and method of "acupuncture serum", in order to overcome the limitation of in vivo experiment for more intuitive and more accurate research information and data. "Moxibustion serum" is advised as a research idea and method in acupuncture research. In the experiment of "moxibustion inhibiting tumor", Pei J cultured the splenic lymphocyte with "moxibustion serum" and the results showed the content of a biologically active substance in the moxibustion serum, improving the activity of natural killer cell (NK) and lymphokine activated killer cells (LAK) and promoting the proliferation of splenic lymphocyte induced by concanavalin A (ConA), was quite different from the content in tumor-bearing mice[6]. The experiment primarily confirming the "moxibustion serum" would help broaden the field of acupuncture research, make up for the lack of in vivo experiments, promote the study of mechanism of acupuncture and deepen the understanding of the laws and characteristics of acupuncture. In our study, the moxibustion serum prepared from Dazhui (GV 14) of normal mice could obviously postpone the formation of tumor nodules and prolong survival time of the tumor-bearing mice. The maximum prolonging rate of life span of group B was 44.67%, which was much better than that in group C and the inhibiting tumor effect of non-meridian point moxibustion serum was not obvious on the tumor-bearing mice, indicating the acupoint speciality of moxibustion. The former experiments of moxibustion inhibiting tumor, moxibustion was used directly to treat tumor-bearing mice but the survival time and tumor growth of tumor-bearing mice treated with moxibustion did not changed significantly as reported[8]. However, some studies reported that moxibustion could markedly influence the tumor growth and survival time of tumor-bearing mice[9]. This difference, in addition to the strains and quality

338 Shanghai Research Institute of Acupuncture and Meridian and Springer-Verlag Berlin Heidelberg 2011

J. Acupunct. Tuina. Sci. 2011, 9 (6): 336-339

of modeling animals, the different tumor types, the different selected points, stimulation parameters and others factors, may also be related to the treatment approach and timing. Generally, the tumor-bearing mice are often in immunosuppression with an unbalanced body environment. Moxibustion, an external artificial stimulus, is bound to cause stress reaction in mice and the stress response is closely related to the ability of the stress. When the tumor is in the bud, the stress ability in mice is still strong, thus the stress response helps to improve the immune function of tumor-bearing mice and to inhibit tumor growth. However, stress response failed to improve immune function with the continually deteriorating tumor, declining stress ability and seriously unbalanced internal environment. Additionally, the adrenal glucocorticoid hormones produced by stress response increased the immune suppression[10]. This may be the reason for the inconsistent results of inhibiting tumor experiment for tumor-bearing mice treated with moxibustion. Therefore, it is necessary to adjust the administration route and timing of moxibustion treatment for tumor. We proposed to prevent and treat transplanted tumors of mice with moxibustion serum made from normal mice taking moxibustion. The method that moxibustion directly operated on tumor-bearing mice was changed into moxibustion serum injected into the tumor-bearing mice. The change from direct moxibustion to indirect moxibustion could significantly release the stress response in mice. Meanwhile, the treatment time was also adjusted that moxibustion serum was injected on the 4th day before tumor cell inoculation. The results confirmed that the inhibiting tumor effect of moxibustion serum pre-treatment group was much

better than that of the other groups, providing new clues and inspiration for improving the treatment and method of moxibustion for inhibiting tumor.

References
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