Electroacupuncture and Moxibustion Promote Neutrophil Apoptosis and Improve Ulcerative Colitis in Rats
The purpose of this study was to investigate the effect of electroacupuncture (EA) and moxibustion on promoting neutrophil apoptosis. A rat model of ulcerative colitis was established by immunological methods using human colonic mucosa as antigen. All rats were randomly assigned to the model control (MC) group, EA group, or herbs-partition moxibustion (HPM) group. Normal rats were used as the normal control (NC) group. Peripheral blood mononuclear cells (PBMCs) from all rats and circular neutrophils from NC rats were isolated and cultured. Circular neutrophils were incubated with cultured supernatants of PBMCs from the MC, NC, EA, and HPM groups, respectively. Neutrophil apoptosis and concentration of IL-1β, IL-6, and TNF-α from induced cultured supernatants were detected by cell cytometry and ELISA, respectively. Compared with MC, HPM, and EA rats, mucosal inflammatory lesions abated remarkably. No hyperemia or edema was seen in the lamina propia, inflammatory cell infiltration decreased, neutrophil infiltration disappeared, and epithelial and crypt cells proliferated and repaired the ulceration of the mucosa. Neutrophil apoptosis was promoted. Concentrations of IL-1β, IL-6, and TNF-α were decreased, respectively. We conclude that EA and HPM therapy can improve ulcerative colitis rats histologically, which may be due to promoting neutrophil apoptosis and down-regulating monocyte cytokines. EA and moxibustion are effective for treating ulcerative colitis.
KeywordsElectroacupuncture Moxibustion Ulcerative colitis Neutrophil apoptosis Rats
The infiltration of neutrophils is one of the characteristic features in the mucosal lesions of ulcerative colitis (UC) . Although their presence represents a first line of hosdt defense against microbial agents, it is also possible that they are involved in the pathogenesis of UC because of their potential to cause tissue injury and inflammatory responses . During the inflammatory response, circulating neutrophils enter the inflamed area to exert their biological functions. Neutrophils are assumed to be linked with mucosal injury as a consequence of epithelial damage through the production of oxidants  and transepithelial migration  or as a result of microvascular damage caused by emigration through the endothelium . The biological effectiveness of neutrophils is regulated through their secretory capacity, their life span, and the recruitment of juvenile cells from the bone marrow. In this light, apoptosis of neutrophils represents a physiologic clearance mechanism in the circulation to create and maintain homeostasis of neutrophil numbers. In healthy humans, mature neutrophils have a short half-life of 6 to 7 hr and undergo rapid spontaneous apoptosis within 24 hr . It is believed that this process minimizes the risk of colon injury release of neutrophil-derived toxic mediators such as proteinase and oxygen free radicals. However, inhibition or delay of neutrophil apoptosis during systemic inflammation may lead to an increased life span of neutrophils, with excessive release of toxic products causing exacerbation of colon injury and, finally, organ dysfunction. Thus, the mechanisms of promoting clearance of neutrophils may be important in the control of inflammatory tissue injury in UC and the subsequent resolution process in UC [7, 8].
Recent studies have demonstrated the ability of peripheral blood monocytes (PBMCs) from patients with UC to secrete enhanced amounts of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, which may be particularly important for inducing and sustaining intestinal inflammation in UC [9, 10, 11, 12]. The inhibition/delay of neutrophil apoptosis by these agents not only increases the life span of cultured neutrophils, but also prolongs their functional longevity as assessed by a number of parameters including secretion of toxic products. Because pro-inflammatory cytokines have been detected at increased concentrations in the circulation of UC patients, these mediators may be responsible for the imbalance of neutrophil apoptosis. Therefore, a rat model of UC was established by immunological methods using human colonic mucosa as antigen. After treatment with electroacupuncture (EA) and herbs-partition moxibustion, the number of circular neutrophil apoptosis induced by PBMC supernatants and the IL-1β, IL-6, and TNF-α levels were determined by TUNEL and ELISA, respectively, to elucidate the mechanism of eletroacupuncture and moxibustion underlying neutrophil apoptosis in rat UC.
Materials and methods
Experimental animals and materials
Two hundred male SD rats (weighting 150±20 g) were provided by the Experimental Animal Center of Shanghai University of TCM. Fetal calf serum (FCS) and RPMI 1640 medium were purchased from GIBCO (USA). Cytokine ELISA kits were purchased from R&D (Germany). Twenty-four-well round-bottom plates were purchased from Corning (USA).
Animal model and therapeutic methods
The animal model was established according to the Experimental Methodology of Pharmacology , and the UC rat model was established by immunological methods using human colonic mucosa as antigen. In brief, fresh human colonic mucosa was prepared from surgical colonic specimens, homogenized by adding an appropriate amount of normal saline, and centrifuged for 30 min at 3000 rpm. The supernatant was removed for measurement of the protein concentration and then mixed with Freund's adjuvant, as antigen. The antigen fluid was first injected into the anteroplanta pedis of the model group rats, then into the posteroplanta pedis, dorsum, inguen, and abdominal cavity (without Freund's adjuvant in the last injection) on days 10, 17, 24, and 31, respectively. When a certain titer of serum anti-colonic antibody was reached, 3 ml of 3% formalin and 2 ml of antigen fluid (without Freund's adjuvant) were administered by enema successively. Normal control (NC) rats were administered normal saline by the same procedure as for model control (MC) rats.
After the UC rat model was built, the animals were randomly divided into the MC group (n=8), EA group (n=8), herbs-partition moxibustion (HPM) group (n=8), and NC group (n=8).
In the HPM group, moxa cones (0.5 cm in diameter and 0.6 cm high) made of refined mugwort floss were placed on the medicinal formula (medicinal formula dispensing [radix] Aconiti praeparata, [cortex] Cinnamomi, etc.) for Qihai (RN6) and Tianshu (ST 25; bilateral) and ignited. RN6 is a point of the conception vessel and it can strengthen the original qi and improve immune function. ST25 is the mu point of the large intestine meridian of hand Yangming, which regulates the function of the large intestine, spleen, and stomach. RN6 and ST25 are the two efficacious points chosen by the authors based on clinical treatment of patients with UC since the 1980s. Two moxa cones were used for each treatment once a day and 14 times as a course.
In the EA group, Tianshu (bilateral) and Qihai were acupunctured and then stimulated with an intermittent pulse of 2-Hz frequency and 4-mA intensity for 20 min once a day and 14 times as a course.
After treatment, the four groups of rats were sacrificed simultaneously. The distal 6-cm-long colons were dissected and reserved in formaldehyde solution.
Cell isolation and culture
PBMC isolation and supernatant fluid collection
PBMCs from NC, MC, HPA, and EA rats were isolated by centrifugation on Ficoll-Hypaque gradients as previously described : 2×106 PBMCs were cultured in 24-well round-bottom plates and incubated for 24 hr at 37°C in a humidified atmosphere containing 5% CO2 in 1.5 ml of RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) containing 20 U/ml penicillin and 20 μg/ml streptomycin. Then supernatants were collected and stored at −20°C until required.
Neutrophils from NC rats were isolated from peripheral blood using a standard technique as previously described . Briefly, peripheral blood was collected from NC rats in 8-ml sterile pyrogen-free glass tubes containing heparin sodium as an anticoagulant and then centrifuged (300 g, 10 min). Isolation of neutrophils was performed with a buffy coat technique. Briefly dextran sedimentation was performed to remove erythrocytes, followed by centrifugation through a Ficoll-Hypaque (Pharmacia, Kalamazoo, MI) density gradient. The resulting neutrophil suspension was washed twice and resuspended at a concentration of 2×106/ml, which were cultured in 48-well round-bottom plates and incubated at 37°C in a humidified atmosphere containing 5% CO2 in 1.5 ml of RPMI 1640 medium supplemented with 10% FCS containing 20 U/ml penicillin and 20 μg/ml streptomycin. Cell viability was determined by trypan blue exclusion, whereas purity was determined by cytocentrifugation and differential staining. These were routinely >98% and >95%, respectively.
Induction of neutrophil apoptosis by PBMC supernatants in different groups
Measurement of neutrophil apoptosis
Detached cells were rinsed with phosphate-buffered saline (PBS) and centrifuged at 1500 rpm for 5 min. After centrifugation, the cells were incubated in permeabilization solution, 0.1% Triton X-100 in 0.1% sodium citrate, for 10 min at room temperature, centrifuged at 1500 rpm for 5 min at room temperature, and rinsed with PBS again. Cells were treated with DNase-free RNase (50 μg/ml) at 37°C and propidium-iodide (50 μg/ml) for 15 min. Samples were kept at 4°C in the dark until analysis. FACS analysis was performed using the FACSCalibur system (Becton Dickinson) and the CellQuest software (BD Biosciences, San Jose, CA). The data were analyzed by q test, using the SPSS statistical package.
Supernatant concentrations of TNF-α, IL-1β, and IL-6 were assessed using a commercial ELISA according to the manufacturer's instructions. All PBMC supernatant samples were stored at –80°C and were assayed at the same time by the same ELISA to avoid variation of assay conditions. Briefly, bring all reagents and samples to room temperature (18°–25°C) before use. (All standards and samples must be run at least in duplicate.) Add 100 μl of each standard and sample into appropriate wells. Cover well with plate holder and incubate for 2.5 hr at room temperature. Discard the solution and wash four times with 1× wash solution (200 μl each). Add 100 μl of 1× prepared biotinylated anti-IL-6/IL-1β/TNF-α to each well. Incubate for 1 hr at room temperature. Discard the solution and wash four times with 1× wash solution (200 μl each). Add 100 μl of prepared streptavidin solution to each well. Incubate for 45 min at room temperature. Discard the solution and wash five times with 1× wash solution (200 μl each). Add 100 μl of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 min at room temperature in the dark. Add 50 μl of stop solution (Item I) to each well. Read at 450 nm immediately.
Data were analyzed using the q test. Data are given as mean ± standard deviation (SD). The SPSS statistical package was used.
Improvement of colonic ulceration in rats treated by EA and HPM
For these results, see Fig. 1.
Decreased cytokine levels in monocytes from EA- and HPM-treated rats
For these results, see Table 1.
Increased neutrophill apoptosis in vitro induced by PBMC supernatant from EA- and HPM-treated rats
For these results, see Table 2.
IL-1β, IL-6, and TNF-α concentrations in PBMC supernatant in the four groups (mean ± SD)
TNF-α c (pg/ml)
Neutrophil apoptosis induced by PBMC supernatant in the four groups
No. of apoptotic cells
Recent studies have demonstrated the ability of peripheral blood monocytes from patients with UC to secrete enhanced amounts of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α and so on, which may be particularly important for increasing neutrophils survival by inhibiting apoptosis of UC and promoting intestinal inflammation in UC [9, 10, 11, 12]. In the present study, the authors chose IL-1β, IL-6, and TNF-α as candidates for such procytokines found in increased quantities in UC; the results show that the concentrations of IL-1β, IL-6, and TNF-α were significantly increased compared with those of NC rats. After treatment with EA and HPM, the concentrations of IL-1β, IL-6, and TNF-α were markedly decreased compared with those of MC rats, and the concentration of IL-6 was equal to that of NC rats. This suggested that EA and moxibustion can regulate the expression of IL-1β, IL-6, and TNF-α and promote the apoptosis of neutrophils in UC in rats.
In our previous research in rat models of UC, we found that acupuncture and moxibustion reduced the hypercontent of C3 , circulating immune complex, and IgM in blood serum to normal levels. Acupuncture and moxibustion also enhanced the transformation of lymphocytes and the IL-2 level in blood serum significantly . We also found that acupuncture combined with medicine (salicylazosulfapyridine) therapy suppressed experimental UC-induced abnormal expression of interferon-γ and IL-12 effectively, which may contribute to its effect in relieving UC . Additionally, we found that acupuncture and HPM decreased the abnormal epithelial cell apoptosis significantly .
In clinical research, authors found that HPM down-regulated the expression of TNF-α and COX-2 in colonal mucous membranes , eliminated the expression of HLA-DR antigen of colonal epithelial cells, enhanced the number of T8+ cells, and adjusted the ratio of T lymphocyte subgroups . Wang et al.  reported that drug-separated moxibustion decreased the IgG content significantly and increased peripheral blood T-cell subgroups and natural killer cells to a certain extent, which was correlated positively with the therapeutic effect.
In summary, therapy with EA and moxibustion is efficacious in treating UC, which may be due to regulation of immunological function, reduction of expression of inflammatory cytokines, and promotion of neutrophil apoptosis.
This work was supported by the Shanghai Leading Academic Discipline Project (Project No. T0302), the National Basic Research Program of China (973 program; Grant No. 2005 CB523306), and the National Natural Science Foundation of China (Grant No. 30371806). Many thanks go Dr. Zean Zhang of Shanghai University of TCM for his invaluable suggestions and advice during the final stages of the preparation of this paper.
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