Li Tian, Yu-Xin Huang, Wei Gao, Qing Chang, Department of Gastroenterology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China
Min Tian, Department of Ultrasound, Heping Hospital of Changzhi Medical College, Changzhi 046000, Shanxi Province, China
Supported by the National Nature Science Foundation of China, No.39970888
Correspondence to: Dr Yu-Xin Huang, Department of Gastroenterology, Tangdu Hospital, 4th Military Medical University, Xi'an 710038, Shaanxi Province, China.  tianli72@263.net
Telephone: +86-29-3577597
Received: 2002-11-12    Accepted: 2002-12-30

Abstract
AIM: To investigate the regulatory effect of electroacupuncture (EA) at Zusanli (ST36) on tumor necrosis factor-alpha (TNF-a) in rats with ulcerative colitis (UC), and further elucidate the therapeutic mechanism of EA on UC.

METHODS: Thirty-two male Sprague-Dawley (SD) rats were randomly divided into four groups (n=8): normal control group, UC control group, UC+ST36 group and UC+non-acupoint group. A solution containing ethanol and 2,4,6-trinitrobenzenesulfonic acid (TNBS) was instilled into the distal colon in the rat (at a dose of 100 mg/kg) to set up UC rat model. Rats in wakefulness state of UC+ST36 group were stimulated at ST36 by EA once a day, while those of UC+non-acupoint group were done at 0.5 cm beside ST36. After 10 d treatment, all rats were sacrificed simultaneously. Colon musocal inflammation and damage were assessed by measuring colon mass, morphologic damage score, colonic myeloperoxidase enzyme (MPO) activity, serum TNF-a and colonic TNF-a mRNA level. Morphologic damage score was examined under stereomicroscope. Colonic MPO activity was measured by spectrophotometer method. Serum TNF-a concentration was determined by radioimmunoassay (RIA). Colonic TNF-a mRNA expression level was analyzed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR).

RESULTS: Ratio of colonic mass/body mass (mC/mB) and activity of colonic MPO (mkat/g tissue) markedly increased (8.5±2.6 vs 2.5±0.4; 145±25 vs 24±8, P<0.01 vs normal control group). Compared with normal control rats, serum TNF-a and colonic TNF-amRNA level in UC control group were increased 2.5 fold (2 278±170 vs 894±248,P<0.01) and 4.3 fold (0.98±0.11 vs 0.23±0.11, P<0.01) respectively. After EA at ST36, m_C/m_B and MPO activity were reduced significantly (5.3±2.0 vs 8.5±2.6; 104±36 vs 145±25, P<0.01, 0.05) compared with those of UC control group. Serum TNF-a and colonic TNF-a mRNA level were inhibited by EA stimulation at ST36 (P<0.01). The inhibitory rate was 16 % and 44 % respectively. Morphologic damage score was also increased markedly in rat with UC (P<0.01), whereas it was decreased by EA at ST36 (P<0.05). There was no significant difference between UC control group and UC+EA at non-acupoint (P>0.05). Furthermore, these parameters were highly correlated with each other (P<0.01).

CONCLUSION: Serum TNF-a concentration and colonic TNF-a mRNA expression level are increased significantly in UC rats in correlation with the severity of disease. It indicates that TNF-a is closely involved in the immune abnormalities and inflammatory responses in UC. EA at ST36 has therapeutic effect on UC by downregulating serum TNF-a and colonic TNF-a mRNA expression. High levels of TNF-a and its corresponding mRNA expression seem to be implicated in the pathogenesis of UC.

Tian L, Huang YX, Tian M, Gao W, Chang Q. Downregulation of electroacupuncture at ST36 on TNF-a in rats with ulcerative colitis. World J Gastroenterol  2003; 9(5): 1028-1033
http://www.wjgnet.com/1007-9327/9/1028.asp
 

INTRODUCTION
The incidence of ulcerative colitis (UC) has become higher in China^[1]. It is characterized by intestinal inflammation and ulceration. Its pathogenesis has not been elucidated yet. A number of clinical and experimental studies, however, have suggested that imbalance between proinflammatory cytokines and anti-inflammatory cytokines is involved in the pathogenesis of UC^[5,6]. High levels of proinflammatory cytokines (interleukin-1b, interleukin-6, interleukin-8 and TNF-a) in the intestinal mucosa are thought to be the pivotal factors in the pathogenesis of intestinal inflammation and ulceration in UC. These proinflammatory cytokines concentrations and their corresponding mRNA expression levels elevated significantly in colonic mucosa, perfusion fluids, spleen and serum of UC patients. It has been suggested that TNF-a may be an important mediator involved in the initiation and perpetuation of intestinal inflammation in UC. TNF-a can cause inflammation directly, and indirectly by inducing the production of other proinflammatory cytokines.
     Nearly all drug treatments for UC have many side effects, which limit patients acceptance. Acupuncture is characteristic technique of traditional oriental medicine. It has been accepted by patients for its better clinical therapeutic effects and fewer side effects on many diseases including UC. Recent studies have demonstrated that acupuncture has immunoregulatory role. It can modulate the production and expression of many cytokines. But the therapeutic mechanism of acupuncture on UC is still uncertain. As an important acupoint, ST36 is not only for disorders of the lower limbs, but also for the whole digestive system, even with certain effect on immunity. The effect of EA at ST36 on UC has rarely been reported yet.
     In this study, we induced UC in rats and stimulated them with EA at ST36 (Zusanli). The aim of this study was to assess the effect of EA stimulation at ST36 on UC, further investigate its role of regulating TNF-a, explain the therapeutic mechanism of EA on UC and provide new thought to acupuncture and moxibustion.

MATERIALS AND METHODS
Materials
Male SD rats weighed 220±20 g were purchased from Experimental Animal Research Center, Fourth Military Medical University. The rats were housed in an air-conditioned animal room at 252 ℃ and 60 % humidity with food and water available ad libitum, and drinking water was changed every day. Hadecyltrimethylammonium bromide (HATB) was purchased from Xizhong Chemical Co (Beijing, China). TNBS was purchased from Sigma Chemical Co (St.Louis, MO). TNF-a RIA kit was purchased from Dongya Biotechnology (Beijing, China). Access RT-PCR System Kit was obtained from Promega (Madison, WI). TRIzol Reagents were purchased from Gibco BRL (Gercy-Pontoise, France).

Induction of ulcerative colitis
The rats were randomized into 4 groups (8 rats for each group): normal control group, UC control group, UC+ EA at ST36 group and UC+ non-acupoint group. Rats were fast for 24 hours. Being gently anesthetized by ether, a rubber catheter(OD, 2 mm) was inserted through anus into its colon whose length in the lumen was 8 cm proximally. TNBS dissolved in 300 ml/L ethanol was instilled into the lumen of the colon through the rubber catheter at the dose of 100 mg/kg^[28,29]. Rats of normal control group were treated by 90 mL/L NaCl solution at the same dose.

EA stimulation
Rats in wakefulness state of UC+ST36 group were stimulated at ST36 (bilateral) which lies just 0.5 cm below fibular head of hinder leg in rat, while those of UC+non-acupoint group were done at 0.5 cm beside ST36^[33,35,40]. These rats were immobilized in special cages, and then were stimulated by the intermittent pulse with 2Hz frequency, 4mA intensity for 30 minutes once a day and 10 times in all. Rats in other two groups were immobilized in the same way with sham acupuncture stimulation.

Preparation of the samples
All rats were weighed after 10 d treatment. Serum was separated from blood drawn from carotid and then stored at -20 ℃ until analysis. All rats were sacrificed simultaneously. The colon was taken from the region, which was 8 cm proximal to the anus. Along its mesenteric border, the colon was opened and gently rinsed out of its contents with an iced NaCl solution 90 mL/L. The colon was then placed flat, with mucosal surface upwards, on a plate chilled at 4 ℃. The colon was immediately examined under a stereomicroscope and any visible damage was scored on a 0-5 scale by two independent observers blinded to the treatment^[28]. There was a highly significant linear correlation between the scores assigned by the two observers(r=0.98, P<0.001). The colon tissue was weighed after being dried on a filter paper. Three tissue samples (1 mm³) were excised from affected region of each colon and then were fixed in 40 g/L glutaraldehyde for electro microscopic examination. The remaining colon was stored at -70 ℃.

Colonic MPO activity assay
The distal 6 cm segment of the colon was homogenized in 5 mL/L HTAB in 20 mmol/L phosphate buffer (pH=6.0, 50 mg of tissue/ml). Homogenates were sonicated and centrifuged for 15 min (15 000 r/min). The supernatant was assayed for MPO activity by using a spectrophotometer, and 0.1 ml of supernatant was mixed with 2.9 ml of 20 mmol/L phosphate buffer (pH=6.0) containing 20 mmol/L guaiacol and 5 mL/L hydrogen peroxide. The changes in absorbance at 460 nm were measured with an Uvikon 860 spectrophotometer (Kontron Instrument, St. Quentin, France). MPO activity was expressed as mkat/g. One mkat/g was defined as that degrading 1 mmol of peroxides per second at 25 ℃ for 1 g of tissue.

Measurement of serum TNF-a concentration by TNF-a RIA kit
Measurement of TNF-amRNA expression in colonic tissue 
Total RNA of 100 mg colonic tissue was purified by TRIzol Reagents. Reverse transcription (RT) was performed in a final volume of 50 ml containing nuclease-free water, AMV/tfl 5×reaction buffer, dNTP mix (10 mM each dNTP), 50 pmol specific primers, 25 mmol/L MgSO4, 5Mu/L AMV Reverse Transcriptase, 5Mu/L tfl DNA Polymerase and 1 mg RNA. cDNA was synthesized by incubating the solution for 50 minutes at 48 ℃ and heating them for 2 minutes at 94 ℃. Then 45 cycles of PCR were performed in a thermal cycler, using the following conditions: denaturation, 30 seconds at 94 ℃; annealing, 1 minute at 55 ℃ for TNF-a and 57 ℃ for b-actin; and extension, 2 minutes at 68 ℃. At the end of the 45 cycles, further extension was continued for 7 minutes at 68 ℃. The primers were TNF-a sense, 5'-AGAACTCCAGGCGGTGTCT-3'; TNF-a antisense, 5'-TCCCTCAGGGGTGTCCTTAG-3'(484bp); b-actin sense, 5'-AACCCTAAGGCCAACCGTGAAAAG-3'; b-actin antisense, 5'-GCTCGAAGTCTAGGGCAACATA-3'(343bp). Amplification of b-actin was used for the determination of TNF-a mRNA expression level. 10 ml aliquots of the synthesized PCR products were separated by electrophoresis on a 22 g/L agarose gel and analyzed by Gel-Pro version 3.1 software (Media Cybernetics). The ratio of arbitrary unit (AU, D_area.D_density) of TNF-a over b-actin was used for expressing the relative level of mRNA expression.

 Statistical analysis
The results are expressed as mean  SEM. All data were analyzed by using ANOVA. P values <0.05 were considered significant. All statistical calculations were performed using the SPSS for windows version 10.0 software package.

RESULTS
Effect of EA on colonic morphology of UC rats
The levels of colonic tissue damage score and mC/mB of rats in UC control group were increased significantly compared with those of normal control group (P<0.01). In comparison with those of UC control group, EA at ST36 made them decreased markedly (P<0.01). EA stimulation at non-acupoint had few effects on these two parameters (P>0.05,Table 1, Figure 1).

Table 1  Effect of EA on colonic tissue damage score  

1: Normal control; 2: UC Control; 3: UC+EA at ST36; 4: UC+EA at non-acupoint. These classifications are qualified for all figures.

     Histological ultrastructure of colonic tissue was assessed by electron microscopy. Colonic ultrastructure manifestations of rats in UC control group and those in UC+non-acupoint group were similar: exiguous goblet cells, scanty microvilli, dilatations of endoplasmic reticula, mitochondria swelling and rounding, with loss of cristae and many inflammatory cells infiltration. While in UC+EA at ST36 group, the results of colonic electron microscopy were more microvilli with well organized appearance, more goblet cells filled with numerous mucous drop in colonic mucosa and only slight mitochondria swelling compared with those of UC control group (Figure 2-4).

Figure 1(PDF) Effect of EA on m_C/m_B. ^aP<0.05, ^bP<0.01 vs normal control; ^dP<0.01, ^fP>0.05 vs UC control; ^eP<0.05 vs UC+EA at non-acupoint.
Figure 2  Colonic ultrastructure of UC control group: A, goblet cells, microvilli, endoplasmic reticula and mitochondria(TEM10 000); B, inflammatory cells (TEM2 500).
Figure 3  Colonic ultrastructure of UC+EA at ST36 group: A, goblet cells (TEM4 000); B microvilli and mitochondria (TEM8 000).
Figure 4  Colonic ultrastructure of EA+non-acupoint group: A, goblet cells, microvilli and mitochondria (TEM8 000); B, endoplasmic reticula and mitochondria (TEM10 000).
Figure 5(PDF) Effect of EA on colonic MPO activity. ^bP<0.01vs normal control; ^cP<0.05, ^fP>0.05vs UC control; ^eP<0.05 vs UC+EA at non-acupoint.

Effect of EA on colonic MPO activity
Colonic MPO activity of rats in UC control group was significant higher than that in normal control group (145±25 vs 24±8, P<0.01). After EA stimulation at ST36, colonic MPO activity became lower than that in UC control group (104±36 vs 145±25, P<0.05). There was no significant difference between UC control group and UC+non-acupoint group (142±45 vs 145±25, P>0.05, Figure 5).

Effect of EA on the production and expression of TNF-a
Compared with normal control rats, serum TNF-a concentration and colonic TNF-a mRNA expression level increased 2.5 fold (2 278±170 vs 894±248, P<0.01) and 4.3 fold (0.98±0.11vs 0.23±0.11, P<0.01). Serum TNF-a and colonic TNF-a mRNA expression were inhibited by EA stimulation at ST36. The inhibitory rate was 16 % (1 913±232 vs 2 278±170, P<0.01) and 44 % (0.55±0.13 vs 0.98±0.11, P<0.01), respectively. While the production of serum TNF-a and the level of TNF-a mRNA expression were not affected by EA stimulation at non-acupoint (2 183±209 vs 2 278±170,0.92±0.17 vs 0.98±0.11, P>0.05, Figure 6, 7).

Figure 6(PDF) Effect of EA on TNF-amRNA expression. A: Representative pictures of RT-PCR. Lane 1, 4, 7, 9:b-actin. Lane 2, 3, 6, 8: TNF-a. Lane 5, 10: DNA Marker (DL2000). B: Relative level of TNF-a mRNA expression. ^bP<0.01 vs normal control; ^dP<0.01, ^fP>0.05 vs UC control; ^gP<0.01 vs UC+EA at non-acupoint.
Figure 7(PDF) Effect of EA on serum TNF-a concentration. ^bP<0.01 vs normal control; ^dP<0.01, ^fP>0.05 vs UC control; ^eP<0.05 vs UC+EA at non-acupoint.

Correlation among parameters quantified above
There were significant correlations between serum TNF-a concentration and colonic MPO activity (r=0.815, P<0.01), serum TNF-a and damage scores (r=0.877, P<0.01), serum TNF-a and m_C/m_B (r=0.691, P<0.01). colonic TNF-a mRNA expression level was highly correlated with colonic MPO activity (r=0.791, P<0.01), damage scores (r=0.827, P<0.01), and mC/mB (r=0.686, P<0.01). The correlation matrix is shown in Table 2.

Table 2  Correlations among the parameters (r)  

DISCUSSION
UC is a non-specific inflammatory bowel disease. Many factors including infection, environment and immune abnormality may   involve in the pathogenesis of UC, in which abnormal immunoregulation plays an important role^[2-4]. There is strong evidence that inflammatory immunoregulation in the intestinal mucosa is characterized by increased concentrations of proinflammatory cytokines with apparent inability to adequately downregulate immune activation. UC showed significantly increased mRNA expression of interleukin-1beta (IL-1b), interleukin-6 (IL-6), interleukin-8 (IL-8) and TNF-a in large numbers of cells throughout the inflamed intestine but also in some macroscopically unaffected tissue specimens. Elevated concentrations of proinflammatory cytokines were also found in serum, colonic mucosa, spleen and colorectal perfusion fluids in UC^[7-12]. There is significant correlation between the production of these cytokines and the activity of UC. The increased production of proinflammatory cytokines is thought to be a pivotal factor in the pathogenesis of UC. It is accepted that TNF-a may be particularly important for inducing and sustaining intestinal inflammation in UC. It is known from many studies that TNF-a is expressed in human gastrointestinal mucosa, and that the expression is strongly enhanced in the inflammatory course of UC. TNF-a production in the gut has been attributed to monocytes, macrophages, natural killer cells, T lymphocytes and mast cells. TNF-a is known to induce the synthesis of IL-6 and IL-8. TNF-a and IL-1b induce each other. The effects of IL-1b and TNF-a appear synergistic. These cytokines regulate many nuclear factor kappaB inducible genes that control expression of other cytokines, cell adhesion molecules, immunoregulatory molecules, and proinflammatory mediators^[13]. Enhanced production of TNF-a and IL-1bmay induce some key enzymes of the inflammation cascade and neutrophils chemotaxis. TNF-a can also induce more production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), which further promotes inflammation than IL-1b^[14-20]. High levels of proinflammatory cytokines in the mucosa lead to the excessive production of matrix degrading enzymes by gut fibroblasts, loss of mucosa integrity and ulceration^[21].
      MPO is an enzyme found predominantly in the azurophilic granules of polymorphonuclear neutrophils (PMN) and has been used as a quantitative index of inflammation in several tissues, including intestine. PMN are the most abundant cell type in intestinal lesions in UC. PMN carry the capacity to secret increased amounts of TNF-a and IL-1b in active UC and infectious colitis. Neutrophils may be important contributors to the initiation and perpetuation of mucosa inflammation^[27].
      Quantitative indexes of inflammation (damage scores, colon mass and MPO activity) were elevated significantly in UC^[22-26]. This study showed that in UC control group, erosion and ulceration induced by TNBS were so particularly severe in rectum and even extended to the proximal colon that damage scores and colon mass were increased markedly, neutrophil infiltration was characteristically present in the lesions and surrounding mucosa, MPO activity at lesions sites was increased, serum TNF-a concentration and colonic TNF-a mRNA expression level elevated significantly compared with those of normal control rats. All these parameters correlated significantly with each other. So TNF-a is a key inflammatory mediator in the pathogenesis of UC.
      Based on recent studies, several new therapeutic strategies are currently tested in clinical practice including inhibitors of proinflammatory cytokines (TNF-a, IL-12) and their receptors (TNF-a, IL-6R), in which anti-TNF-a has been impressive. These new therapeutic strategies have demonstrated efficacy in refractory UC patients. But there are still many problems to solve including the best way of therapy and side effects before they are formally applied to UC patients. Other potent medications with side effects limit patients acceptance. While the advantages of acupuncture treatment for UC have been obvious^[39].
      Acupuncture is one of the most important part of traditional Chinese medicine (TCM) which possesses a unique theoretical systems, rich clinical experience and excellent clinical effects. TCM theory says: inharmony between Qi and blood, and imbalance between Yin and Yang can lead to disease. EA at acupoint is able to stimulate meridians to transport Qi and blood, regulate Yin and Yang keeping the functions and activities of all parts of the body in harmony and balance relatively.
     As an important acupoint in TCM, ST36 is the lower He-(Sea) point. This point has a tonifying function. It is an important point for health maintenance and disorder of stomach and abdomen. Recent studies have shown that EA stimulation at ST36 may regulate nerve-endocrine-immune network by influencing the production and expression of neurotransmitters, hormones and cytokines^[30-37]. EA stimulation at ST36 significantly restores brain-derived neurotrophic facto (BDNF) mRNA expression declined by emergency^[38]. EA stimulation at ST36 improves the immune function by inducing interlukin-2 (IL-2) and interferon-gamma (IFN-a) production of spleen lymphocytes in traumatized rats. ST36 stimulated by EA can also inhibit abnormal IL-1 increment induced by trauma and even disease. Li et al^[40] found that high levels of serum IL-1b, IL-6, TNF-a and NO induced by lipopolysaccharide in rats were decreased significantly by EA stimulation at ST36.
      Acupuncture at acupoints such as Tianshu(ST25), Guanyuan (Ren4) has a marked curative effects with few side ones on UC, and therefore was readily acceptable to the patients^[39]. But its therapeutic mechanism is still unclear. There are only a few studies on its corresponding theory. Some studies showed that EA stimulation at Qihai(RN6) and Tianshu(ST25) may downregulate the expression of proinflammatory cytokines mRNA (IL-1bmRNA, IL-6 mRNA) and iNOS mRNA of spleen and colon in UC rats, whereas it could upregulate IL-1b mRNA expression. It is reported that acupuncture stimulation at acupoint reduces the excessive production of TNF-a positive cells of colonic mucosa in rats with UC. The study of effect of EA stimulation at ST36 on UC has rarely been reported yet.
     This study showed that in UC+EA at ST36 group, lesion formation was inhibited grossly and microscopically, neutrophil infiltration and MPO activity in and around lesions were lessened, serum TNF-a concentration and colonic TNF-a mRNA expression level were decreased significantly. These results suggest that EA stimulation at ST36 can inhibit effectively inflammation cascade in UC. But these parameters were not restored to normal levels by EA stimulation at ST36 without being accompanied by other acupoint.
     In summary, this study showed EA stimulation at ST36 has therapeutic effect on UC by reducing serum TNF-a concentration and colonic TNF-a mRNA expression level, decreasing colonic MPO activity and alleviating colonic inflammation damage. Its therapeutic mechanism is attributed to its downregulation effect on TNF-a, which is a key proinflammatory cytokine. It is still unknown whether EA can keep the balance between proinflamamtory cytokines and anti-inflammatory cytokines by upregulating the key anti-inflammatory cytokines (IL-4, IL-10, IL-13) when it downregulates proinflammatory cytokines. Its mechanism of regulating cytokines needs further study.

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Edited by Ren SY