WORLD JOURNAL OF ACUPUNCTURE-MOXIBUSTION

Vol.10 No.2,  June, 2000

Effect of Electro-Acupuncture on Mouse Lymphocyte c-fosmRNA, ppENKmRNA, MEK-IR and Dyn-IR

by Tsogoev AS Wang Hongmei(王红梅) Wu Jinglan(吴景兰) Liu Qing(刘 清)

Molecular Cell Biology Research Center, Henan 

Medical University, Zhengzhou 4500 52, China

Abstract  To investigate the relation between acupuncture stimulation and cellular immunity and the relation between the expression of c-fosmRNA and ppENKmRNA or MEK-IR and Dyn-IR, the effect of acupuncture on the circulating lymphocyte c-fosmRNA, ppENKmRNA, MEK-IR and Dyn-IR was studied. 20 BALB/C mice were randomly divided into 2 groups: a) acupuncture group, treated with 5 Hz electroacupuncture(EA); b) control group, treated with restraint but no EA. The mouse pain threshold was detected by K+ ionophoresis before and after EA or restraining. The mouse lymphocyte c-fosmRNA and ppENKmRNA were demonstrated by in situ hybridization and RNA dot blot. The mouse lymphocyte MEK-IR and Dyn-IR were detected by immunohistochemistry and protein dot blot. The dot blot signals were scanned for statistical analysis. The results showed that all the signals of c-fosmRNA, ppENKmRNA, MEK-IR and Dyn-IR were localized in the lymphocyte cytoplasma and stronger in the acupuncture group than that in the control group( P<0.05). All of the signals were positively correlated with the analgesic effect in the EA group (P<0.05-0.01). Besides, there was a positive correlation on alteration between c-fosmRNA and ppENKmRNA or between c-fosmRNA and Dyn-IR(P<0.05), suggesting that c-fos may be involved in ppE or ppD gene transcription in the mouse lymphocyte.

Key Words c-fos ppENK MEK Dyn Lymphocyte Acupuncture

In the previous studies it was found that the E-rosette, especially active E-rosette, lymphocyte transformation, T4 subpopulation and the circulating lymphocyte with receptors for methionin enkephalin (MEK) were enhanced in the acupuncture group; and the potent MEK-immunoreactivity (IR) lymphocyte also showed α-naphthyl esterase (ANAE)-focal pattern, representing TH^[1-4]. Guo reported that c-fos could induce preprodynorphin (ppD) rather thanpreproenkephalin (ppE) gene expression^[5]. Cui reported that c-fos could involve ppE acting as a transactivating factor^[6]. Han reported that 2 Hz electroacupuncture stimulation could result in MEK release, while 100 Hz electrostimulation was associated with dynorphin (Dyn) release in the patients' cerebrospinal fluid^[7]. The expression of c-fosmRNA, ppENKmRNA, MEK-IR and dynorphin (Dyn)-IR in the mouse lymphocyte has not yet been reported. The acupuncture effect on gene expression of the mouse lymphocyte c-fos and ppENK was studied by using in situ hybridization and RNA dot blot, the immunohistochemistry and protein dot blot in the present study.

Materials and Methods

20 BALB/C mice with about 20-22 g of body weight were randomly divided into 2 groups: A. electroacupuncture(EA) group(EG) treated with 1.5 V, 5 Hz EA at bilateral “Zusanli” for 15 min; B. control grouup (CG) treated with restraint for 15 min but no EA. Before and after EA or restraining the pain threshold was detected by K+ ionophoresis method. The circulating lymphocyte was isolated from the peripheral blood by gradient density. The lymphocyte suspension in concentration of 1×105 cells/ml was divided into 2 aliquots: one aliquot was blotted onto the slide to detect the c-fosmRNA, ppENKmRNA by in situ hybridization an d to detect MEK-IR and Dyn-IR by immunohistochemistry. Another aliquot was blotted onto the nitrocellulose membrane (NCM), one dot for each animal, for detect ion of c-fosmRNA and ppENKmRNA by RNA dot blot and detection of MEK-IR and Dyn-IR by protein dot blot^[8]. The hybridization reagents were supplied by Promega, the antisera against MEK and Dyn were purchased from INC. The BCIP/N BT was used as the substrate to develop violet color for demonstration of hybridization signal, the DAB as the substrate to develop brownish color for immunoreactivity. The substitution of NS for the first antibody or predigesting with RN ase was performed as the negative control. The dot blot signals were scanned wit h 528-532 nm using a Shimadu TLC Scanner and analyzed statistically.

Results 

After acupuncture the mouse pain threshold was elevated by 0.38±0.04 mA, in the acupuncture group, when compared to that before acupuncture, (P<0.01) ; whereas the alteration of the pain threshold was not significant after restraining in the control group (0.02±0.03, P>0.05).

All the signals of c-fosmRNA, ppENKmRNA, MEK-IR and Dyn-IR were distributed in the lymphocyte cytoplasma and stronger in the acupuncture group than that in t he control group.

The scanning OD mean values of c-fosmRNA, ppENKmRNA, MEK-IR and Dyn-IR signals were as following (Tab.1).

Tab.1 Comparison of the Scanning OD Mean Values

 of c-fosmRNA ppENKmRNA, MEK-IR and Dyn-IR 

between EG and CG in the Mouse Lymphocyte

The dot blot signals of c-fosmRNA, ppENKmRNA, MEK-IR, Dyn-IR increased in the acupuncture group, and were positively correlated with the analgesic effect shown as the following (Tab.2).

Tab.2 The Correlation between the Signal OD Mean 

Value and Analgesic Effect in the Lymphocyte of EG

Besides, there was a positive correlation in alteration between c-fosmRNA and ppENKmRNA (r=0.60, P<0.05) or between c-fosmRNA and Dyn-IR (r=0.67, P<0.025); while there was no correlation in alteration between ppENKmRNA and MEK (r=0.44, P<0.05) in the lymphocyte.

Discussion

In the spinal cord the c-fos gene expression peaked at 2 h and ppENK gene expression initiated at 4h and peaked at 48h following acupuncture^[5,6]. The present results showed that under 5Hz EA stimulation the gene expression of the lymphocyte c-fosmRNA and ppENKmRNA in the acupuncture group was up regulated compared with the control group, and positively correlated with the analgesic effec t. Moreover, there was a positive correlation in alteration between c-fosmRNA signals and ppENKmRNA signals with a similar intensity in the lymphocyte, suggesting that c-fos protooncogen and ppENK gene may express about at the same time, 4h after EA. The expression signals of both c-fos and ppENK genes without similar intensity in the spinal cord were found 4h after EA in our another study. It suggests that the existing phosphorylated CREB(c-AMP response element binding protein) instead of novo synthesized c-fos (such as in the spinal cord) may act as transactivating factor for both c-fos and ppENK genes in the lymphocyte, a different pattern of gene expression from that in the spinal cord.

The lymphocyte MEK-IR could demonstrate both ir-MEK in the lymphocyte cytoplasm and the bound MEK receptors at the lymphocyte cytomembrane. The lymphocyte irMEK may be released out of cells to affect the nervous system and pituitary axis as a “mobile brain”, especially after acupuncture. Thus, in the present series of experiments the MEK-IR intensity did not correspond to the ppENKmRNA sign al intensity (with out correlation) in the mouse lymphocyte, in spite of that ppENK is the precursor of MEK.

The precursor of dynorphin (Dyn) is preprodynophin (ppD). Guo reported that Fos and Jun proteins were involved in ppD transcription, rather than ppE gene expression^[5]. The present result showed that the circulating lymphocyte Dyn-IR was stronger in the acupuncture group than that in the control group, positively correlated with c-fosmRNA signals, suggesting that the c-Fos protein may be involve not only in ppE, but also in ppD transcriptions. The alteration of Dyn-IR was not so significant as that of MEK-IR, suggesting that the irDyn may be released less than irMEK in the lymphocyte under 5 Hz EA stimulation, a lower frequency of EA, which primarily corresponded to what Han reported in the cerebrospinal fluid^[7]. As to the ppDmRNA in the mouse circulating lymphocyte after acupuncture, it remains to be further studied.

Martin reported that the ppENKmRNA existed in T cells, not in B-cell lymphoma^[9]. Eurawski^[10] reported that activation of mouse T-helper cells induced abundant ppENKmRNA synthesis. In our previous work^[3]  acupuncture could induce abundant MEK-IR in the lymphocyte corresponding to ANA E-focal pattern(TH). The present results showed that the lymphocyte ppENKmRNA and MEK-IR were more enhanced in the acupuncture group than that in the control group. It implicates that acupuncture may promote cell-mediated immunity, especially T-helper cells to be involved in neuro-immunological modulation through the neuroendocrinoimmune network.

Reference

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2 Wu J, et al. Acupuncture effects upon α-naphthyl acetate esterase staining patterns of circulating lymphocytes and E-rosette forming cells. Chine se Med J, 1985, 98(10):753-8.

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4 Wu J, et al. Effects of the analgesic opiate drug and electrical stimulation at the acupoint on the organism. J Henan Med Univ, 1996,31(1):18-22.

5 Guo HF, et al. C-Fos proteins are not involved in activation of preproenkephaline gene expression in rat brain by peripheral stimulation (EA). Neurosci Lett 1996,207(3): 163-6.

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7 Han JS, et al. Effect of low and high frequency TENS on metenkephalin-Arg-Phe and dynophine, An immunoreactivity in human lumbar cerebrospinal flu id. Pain, 1991,47(3):295-8.

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9 Martin J, et al. Preproenkephaline mRNA in T cells, macrophages, and mast cells. J Neurosci Res, 1978,18(1):82-7.

10 Zurawski G, et al. Activation of mouse T-helper cells induces abundant preproenkephaline mRNA synthesis. Science, 1986, 232(4751):772-5.