Wang, Kun MD; Wu, Huaxing MD; Wang, Guonian MD; Li, Mingming MD; Zhang, Zhaodi MD; Gu, Guangying MD
Surgery and trauma induce changes in the immune response, showing a predominant pattern of activation through the Type 2 T helper cell (Th2) pathway to the detriment of Type 1 T helper cell (Th1) pathway activation. It is now thought that surgery (including surgical trauma, anesthesia, and stress) induces a Th1/Th2 balance shift toward a Th2 immune response by activating afferent signals from the operative site and releasing cytokines from damaged tissue.1 The Th1 response is suppressed as shown by diminished interleukin (IL)-2 and interferon (IFN)-γ after major injury, and the enhancement of the Th2 response is marked by increased IL-10 and IL-4.2 Trauma, surgical stress, and anesthesia are associated with postoperative immune suppression and increased susceptibility to infection.3 It thus appears important to prevent the suppression of T cell function and a shift to the Th2 cytokine response to maintain immune function and decrease postoperative infectious complications.
Electroacupuncture (EA), an integral part of the ancient Chinese medicine system, has been used to attenuate pain in clinical practice.4 Cumulative evidence suggests that EA may enhance immune function after surgical trauma both in animals and humans.5,6 EA at Zusanli (ST36) acupoint may increase lymphocyte proliferation and IL-2 production of surgically traumatized rats7–9 and IFN-γ production of the spleen in mice.10 Another study has demonstrated that the production of Th2 cytokine IL-4 in splenocytes is suppressed in ST36 acupoint electroacupunctured mice.11 These studies provide a clue that the immunoregulatory effects of EA are closely associated with Th1/Th2 cytokine production. However, no studies of the effect of EA on cytokine mRNA expression in splenic T cells of traumatized rats have been performed nor has the mechanism mediating this effect been investigated.
Mitogen-activated protein kinases (MAPKs) are signaling molecules that play an important role in the regulation of T cell activation and cytokine production.12 MAPKs include four subfamilies; the best characterized are extracellular-regulated protein kinase (ERK)1/2, p38, and c-Jun NH2-terminal kinase (JNK). Upon activation, the MAPKs phosphorylate various cytoplasmic effector proteins and are translocated to the nucleus where they participate in the regulation of gene expression by acting on transcription factors nuclear factor (NF)-κB and activator protein (AP)-1.13,14 IL-2 and IFN-γ production by splenic T cells is decreased after trauma-hemorrhage, and this is accompanied with a decrease in T cell MAPK, NF-κB, and AP-1 activation.15 However, it is unknown whether these kinases and transcription factors are involved in mediating the EA effect on splenic T cell cytokines after surgical trauma.
We hypothesized that EA could ameliorate surgical trauma-induced immunosuppression by modulating the splenic Th1/Th2 cytokine balance, and that this effect is mediated via MAPK signaling pathways. The purpose of this study was to investigate the effect of EA on the expression of Th1/Th2 cytokine protein and mRNA, as well as the activation of MAPKs, NF-κB, and AP-1 in splenic T cells of rats undergoing surgical trauma.
Male Sprague-Dawley rats weighting 220 ± 20 g, supplied by the Animal Center of Vital River Laboratories (Beijing, China), were acclimated in controlled conditions (12:12 h light/dark cycle, 23°C ± 0.5°C, relative humidity 40%–60%) for 1 wk. All experiments were approved by the local animal ethics committee of the Harbin Medical University and performed in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and the International Association for the Study of Pain.
Experimental Design and Trauma Procedure
Ninety-six rats were randomized into four groups by using a computer-generated random number table: 1: nontraumatized group (control, n = 24); 2: surgical trauma group (trauma, n = 24); 3: sham point EA applied on trauma stressed group (trauma [T] + sham EA, n = 24); 4: EA applied on trauma stressed group (T + EA, n = 24). Eight rats in each group (including control group) were killed simultaneously on postoperative day 1, 3, and 5, respectively, and processed to perform immunological analysis. Rats were fasted overnight but allowed water ad libitum before the experiment. Directly before surgery, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg/kg). As described by Wang et al.,5 rats were aseptically incised longitudinally to a length of 6 cm along the dorsal median line and 5 cm along the abdominal median line, and the abdominal viscera were exposed for 3 min. After ensuring there was no bleeding in the abdominal cavity, the wound was sutured and covered with adhesive dressing. Rats were placed into acrylic holders and allowed to awaken. Surgical trauma was performed starting at 8:00 am, and the duration of the surgery was 15–20 min. Rats were awake 5–10 min after surgery and allowed free access to water and food. Rats were kept warm under standard housing conditions, and no postoperative infection occurred. No analgesic was given throughout the experiment. Control rats were only anesthetized without surgical trauma, and the dorsal and abdominal regions were covered with adhesive dressing to maintain blinding conditions. Research assistants who collected the measurements and performed the assays were blinded to group assignments.
Two pairs of stainless steel needles (diameter, 0.3 mm; length, 50 mm) were inserted perpendicularly with a depth of 5 mm into the bilateral acupoints of ST36 (located 5 mm below and lateral to the anterior tubercle of the tibia) and Lanwei (Extra37) (located about 3.3 mm below ST36). We chose the acupoints of ST36 and Extra37 for this experiment because ST36 is one of the most frequently used points for acupuncture analgesia and immunomodulation,4,6,10 and Extra37 is an adjunct acupoint. EA stimulation applied to ST36 and Extra37 acupoints improves the immunosuppression induced by surgical trauma.5,16,17 The negative output lead from the EA apparatus (nerve and muscle stimulator SDZ-II, Suzhou Medical Appliance Factory, China) was connected to the handle of the needle inserted at ST36 acupoint, and the positive terminal was connected to the handle of the needle inserted at the ipsilateral Extra37 acupoint. EA variables were set as alternating strings of dense-disperse frequencies (60 Hz for 1.05 s and 2 Hz for 2.85 s alternately), and the intensity was adjusted to induce moderate muscle contract of the hindlimb (≤1 mA). Sham point is located 0.5 cm obliquely superior and lateral to ST36 or Extra37 acupoint and is not in the meridian. EA stimulation was applied 20 min after surgery and lasted for 30 min, and then performed once a day (30 min) on postoperative days 1–5 (9:00 am). Rats remained awake in acrylic holders during EA treatment. The rats of no-EA groups were restricted for 30 min in the holder to stimulate holder stress. EA was performed by an experienced acupuncturist who was not involved in data acquisition and analysis.
An observer blinded to group allocation performed pain threshold testing in each group (n = 8) before surgery (baseline), and on days 0 (20 min) and 1–5 after surgery. Surgical trauma induces inflammatory pain and hyperalgesia, which reflects a sensitization of the peripheral terminal and central facilitation evoked by persistent small afferent input.18 Postoperative acute pain may contribute to an increase in the response to noxious stimuli and a decrease in the pain threshold. Acupuncture may relieve pain by releasing endogenous opioids from central nervous system stores and by modulating the hypothalamic-limbic system.4 Electrical stimulation of the arcuate nucleus in the hypothalamus produces an analgesic effect as tested by paw withdrawal threshold (PWT).19 Therefore, PWTs were chosen in surgical trauma as a measure of pain relief for an axial incision.
Mechanical pain threshold was determined by measuring the PWT in response to von Frey filaments. Briefly, each von Frey filament (Stoelting, Wood Dale, IL) in ascending order of stiffness from 1.0 g was applied five times at 5-s intervals on the right hindpaw. Once this paw was withdrawn from the particular hair in three of five consecutive applications, the PWT was defined as the lowest hair force in grams. Licking or brisk withdrawal of the paw was considered a positive response.
Thermal pain threshold was assessed by measuring the paw withdrawal latency (PWL) in response to a radiant heat source. Latency was defined as the time (in seconds) required for the paw to briskly withdraw as detected by photodiode motion sensors (Life Science Instruments, Woodland Hills, CA). The right hindpaw was tested for three trials at each time period with 10-min intervals between each trial. The average of the three trials was determined.
Isolation of Splenic T Cells
Rats were killed by overanesthetizing with pentobarbital sodium (60 mg/kg, IP). Spleens were removed aseptically and teased into single-cell suspensions. The erythrocytes were lysed with buffer elution (Qiagen, Valencia, CA). The remaining cells were washed and loaded into a nylon wool column. After 1 h of incubation (37°C, 5% CO2), T cells were eluted from the column and suspended (1 × 106 cells/mL) in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum and antibiotics. T cells obtained were found to be >95% positive for anti-CD3.15
T Lymphocyte Proliferation
Based on previous studies showing that the spleen lymphocyte proliferation and IL-2 production of rats were inhibited on days 1, 3, and 5 after surgical trauma, and EA stimulation increased lymphocyte proliferation at these time points,7–9,20 we measured lymphocyte proliferation on postoperative days 1, 3, and 5.
Cell viability was estimated using methyl thiazolyl tetrazolium assay. Splenic T cells (1 × 106/well) were incubated in a 96-well plate (Sigma, St. Louis, MO) in triplicate cultures in medium alone (control media) or in concanavalin A (Sigma) 0.5 μg/mL. Four hours before the end of treatment, 50 μg methyl thiazolyl tetrazolium was added. Finally, cells were lysed in 0.1 M HCl in isopropanol, and optical density (OD) was measured in an enzyme-linked immunosorbent assay (ELISA) autoreader. Lymphocyte proliferation was expressed as: stimulation index (%) = test group (OD values)/control media (OD values).
Enzyme-Linked Immunosorbent Assays
The isolated T cells were cultured in 24-well plates with anti-CD3 (2 μg/mL) at 37°C in a 5% CO2 incubator for 24 h. The supernatants were collected and analyzed for the concentrations of IL-2, IFN-γ, IL-4, and IL-10 using specific ELISA (Shanghai Senxiong Science and Technology Company, Shanghai, China).
Quantitative Real-Time Polymerase Chain Reaction
Isolated T cells were harvested, and then total RNA was extracted using Trizol reagent (Invitrogen). Total RNA (1 μL) was reverse-transcribed to cDNA, and 20 μL reverse transcription reaction system (Invitrogen) placed in 42°C water for 60 min and heated in 70°C water for 15 min to deactivate reverse transcriptase. The primers were designed and synthesized in the Shanghai Institute of Biochemistry (China) (Table 1). Polymerase chain reaction amplification was performed on a DNA Engine thermal cycler (Bio-Rad, Hercules, CA) using SYBR Green I (Invitrogen). All samples were run in triplicate for each experiment. mRNA expression of the target gene was normalized against glyceraldehyde-3-phosphate dehydrogenase, and cytokine relative mRNA levels were calculated as described by Pfaffl.21
Western Blot Analysis
Briefly, splenic T cells were isolated and protein was separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane. The membranes were saturated with blocking buffer for 1 h at room temperature and incubated with the antibody to p38 protein, phospho-p38, ERK1/2 protein, phospho-ERK1/2, JNK protein, and phospho-JNK or β-actin (Santa Cruz, CA) at 4°C overnight. After the membranes were washed three times in 0.3% Tween 20/phosphate buffered saline for 10 min, they were incubated with an IRDye800 conjugated secondary antibody (Biotrend Chemikalien GmbH, Köln, Germany) in blocking buffer. Protein-antibody complexes conjugated with IRDye800 were visualized on the Odyssey Infrared Imaging System (LI-COR Biosciences, Bad Homburg, Germany).
NF-κB and AP-1 Activity Assay
NF-κB and AP-1 DNA binding activity was measured using Trans-AM ELISA-based kits (Active Motif, Carlsbad, CA). Nuclear proteins were extracted using a Nuclear Extract kit. Biotinylated double-stranded oligonucleotides containing the NF-κB or AP-1 consensus site were precoated onto 96-well plates. Activated transcription factors bound to the respective immobilized oligonucleotide were detected using the antibodies to NF-κB p65, c-Fos, or c-Jun (AP-1) followed by a secondary antibody conjugated to horseradish peroxidase in an ELISA-like assay.
Data Analysis and Statistics
Power analysis was based on our results of preliminary experiments comparing pain thresholds and immune variables on postoperative day 3 among groups and yielded a sample size of n = 8 (α = 0.05; 1 − β = 0.9) for each group at each time point. Data were analyzed with SPSS 13.0 software (serial 5031432, Stats Data Mining Co., China) and are presented as means ± sd. Data from the PWT and PWL were analyzed using analysis of general linear model with repeated measures followed by Student-Newman-Keuls test as a post hoc test for multiple comparisons. Baseline values were compared using unpaired Student’s t-tests. Other data were analyzed using one-way analysis of variance with the Student-Newman-Keuls test. P <0.05 was considered significant.
Baseline values of PWT and PWL did not differ in the four groups. Compared with the control group, PWT and PWL in the trauma group were significantly lower on postoperative day 0 (1.9 ± 0.2 vs 9.3 ± 0.6 g; 3.5 ± 0.5 vs 12.3 ± 0.4 s), day 1 (3.0 ± 0.7 vs 9.3 ± 0.7 g; 4.5 ± 0.5 vs 12.8 ± 0.4 s), day 2 (3.8 ± 0.6 vs 9.5 ± 0.6 g; 5.0 ± 0.5 vs 12.5 ± 0.4 s), day 3 (4.8 ± 0.7 vs 9.3 ± 0.7 g; 6.0 ± 0.5 vs 12.5 ± 0.4 s), day 4 (5.2 ± 0.6 vs 9.5 ± 0.6 g; 6.5 ± 0.5 vs 12.6 ± 0.4 s), and day 5 (5.5 ± 0.6 vs 9.3 ± 0.6 g; 7.0 ± 0.8 vs 12.8 ± 0.4 s) (P < 0.01–0.001). On postoperative days 0–2, PWT (1.9 ± 0.3, 5.8 ± 0.7, and 6.8 ± 0.7 g, respectively) and PWL (3.5 ± 0.5, 7.0 ± 0.8, and 8.5 ± 0.8 s) in the T + EA group were significantly decreased compared with control (P < 0.01). On postoperative days 1–5, PWT (5.8 ± 0.7, 6.8 ± 0.7, 8.3 ± 0.6, 8.8 ± 0.6, and 9.0 ± 0.6 g, respectively) and PWL (7.0 ± 0.8, 8.5 ± 0.8, 10.5 ± 0.5, 11.3 ± 0.5, and 12.0 ± 1.3 s) in the EA group were significantly increased compared with the trauma group (P < 0.001), whereas PWT (3.8 ± 0.7, 4.4 ± 0.7, 5.3 ± 0.6, 5.8 ± 0.7, and 6.3 ± 0.7 g) and PWL (5.0 ± 0.5, 6.3 ± 0.5, 7.3 ± 0.5, 7.5 ± 0.5, and 7.6 ± 0.5 s) in the T + sham EA group were not increased.
T Lymphocyte Proliferation
T lymphocyte proliferation in the trauma group was decreased on postoperative days 1, 3, and 5 compared with the control group (P < 0.001), and the most obvious decrease appeared on postoperative day 3. T lymphocyte proliferation in the EA group on postoperative days 1, 3, and 5 was significantly increased compared with the trauma group (P < 0.001; Fig. 1), whereas lymphocyte proliferation did not increase in the T + sham EA group.
Production of IL-2 and IFN-γ in splenic T cells in the trauma group was significantly decreased on postoperative day 3 compared with control (37.4 ± 2.0 vs 76.1 ± 3.7 pg/mL; 30.6 ± 1.3 vs 49.9 ± 2.6 pg/mL, P < 0.001). Compared with the trauma group, the production of IL-2 (58.3 ± 1.1 vs 37.4 ± 2.0 pg/mL, P < 0.01) and IFN-γ (39.2 ± 1.5 vs 30.6 ± 1.3 pg/mL, P < 0.05; Fig. 2A) in the EA group was significantly increased.
The production of IL-4 and IL-10 was higher on postoperative day 3 in the trauma group than in the control group (70.5 ± 3.1 vs 26.8 ± 3.0 pg/mL; 97.3 ± 3.3 vs 42.7 ± 2.0 pg/mL, P < 0.001). Compared with the trauma group, the production of IL-4 (50.9 ± 3.4 vs 70.5 ± 3.1 pg/mL, P < 0.001) and IL-10 (65.9 ± 1.9 vs 97.3 ± 3.3 pg/mL, P < 0.001; Fig. 2B) in the EA group was significantly decreased.
Cytokine mRNA Expression
mRNA expression of IL-2 and IFN-γ in splenic T cells was significantly decreased on postoperative day 3 in the trauma group compared with control (P < 0.001). Compared with the trauma group, mRNA expressions of IL-2 (P = 0.01; Fig. 3A) and IFN-γ (P = 0.008; Fig. 3A) in the EA group were significantly increased.
mRNA expression of IL-4 and IL-10 was significantly increased on postoperative day 3 in the trauma group compared with control (P < 0.001). Compared with the trauma group, mRNA expressions of IL-4 (P = 0.001; Fig. 3B) and IL-10 (P = 0.002; Fig. 3B) in the EA group were significantly decreased.
Activation of ERK1/2 and p38 was significantly lower on postoperative day 3 in splenic T cells in the trauma group than in the control group (P < 0.001). Compared with the trauma group, ERK1/2 and p38 activation (P < 0.001; Figs. 4A and B) in the EA group was significantly increased.
NF-κB and AP-1 Activity
DNA binding activity of NF-κB and AP-1 in splenic T cells in the trauma group was decreased on postoperative day 3 compared with control (P < 0.001). Compared with the trauma group, the activity of NF-κB and AP-1 in the EA group was significantly increased (P < 0.001; Fig. 5).
Our findings indicate that production of Th1 cytokines (IL-2 and IFN-γ) and mRNA expression in splenic T cells of traumatized rats were decreased after surgical trauma, whereas Th2 cytokine (IL-4 and IL-10) production and mRNA expression were increased. This was accompanied by suppressed activity of ERK1/2, p38, NF-κB, and AP-1. EA administration increased expressions of Th1 cytokine proteins (IL-2, 56%; IFN-γ, 28%) and mRNA (IL-2, 83%; IFN-γ, 85%), suppressed expressions of Th2 cytokine proteins (IL-4, 28%; IL-10, 32%) and mRNA (IL-4, 77%; IL-10, 68%), and increased the activities of ERK1/2 (85%), p38 (46%), NF-κB (80%), and AP-1 (c-Fos, 34%; c-Jun, 85%) in the EA group compared with trauma group. Overall, EA administration exhibits similar potency in the enhancement of IL-2 and IFN-γ mRNA expression, ERK1/2 and p38 activation, as well as NF-κB and AP-1 DNA binding activity, suggesting that these effects are mechanistically related.
Our results showed that surgical trauma induced postoperative pain and suppressed T lymphocyte proliferation. EA administration increased PWT and PWL and restored suppressed lymphocyte proliferation. These results suggest again that acupuncture may not only alleviate pain but also attenuate immunosuppression after surgical trauma. In the current study, the most obvious decrease in T lymphocyte proliferation appeared on day 3 after operation. This result is consistent with the finding that the minimum level of the spleen lymphocyte proliferation is on day 3 after operative trauma.7,8 Therefore, we selected a single time point (i.e., postoperative day 3) to measure the expression of cytokine and MAPKs.
IL-2 is a representative Th1 cytokine produced by activated T cells, which leads to T cell proliferation.22 Decreased IL-2 production is an important cause of postoperative depression in immune responses.23 IFN-γ, another Th1 cytokine, is a key cytokine for cell-mediated immune responses. IL-2 and IFN-γ production by splenic T cells is decreased after trauma-hemorrhage.15,24 IL-2 mRNA expression in splenocytes is also decreased after trauma.25 Our results showed that surgical trauma induced a decrease in production of IL-2 and IFN-γ as well as their mRNA expression in splenic T cells on postoperative day 3, which were significantly increased by EA stimulation. This implied that EA administration may increase Th1 cytokine (IL-2 and IFN-γ) production and their mRNA expression in splenic T cells of rats after surgical trauma and enhance cell-mediated immunity.
Th2 cells are associated with humoral immunity and production of cytokines such as IL-4 and IL-10. IL-4 production in the spleen was higher in trauma-hemorrhage mice,26 and its mRNA expression in the spleen was increased at 3 h after operative trauma in a simple laparotomy model.27 IL-10 has been described as a cytokine synthesis inhibitory factor, inhibiting the production of Th1 cytokines. A significant increase in IL-10 production and mRNA expression in the spleen has been observed at 3–6 h after operative trauma.27 Our results showed that the production of IL-4 and IL-10 and their mRNA expression in splenic T cells of rats was markedly increased after surgical trauma. These results, together with changes in IL-2 and IFN-γ, implied that surgical trauma may result in dysregulation of the Th1/Th2 cytokine profile, break the Th1/Th2 cytokine balance, and then contribute to immune suppression. EA administration indeed effectively inhibited the increase of IL-4 and IL-10 expression. This indicated that EA administration may modulate Th1 and Th2 cytokine production, moving toward an immunologically balanced state.
To further delineate the mechanism of EA on cytokine gene expression, we attempted to determine whether MAPKs (i.e., ERK1/2, p38, and JNK) play any role in these effects. Activation of ERK is essential for nuclear translocation of c-Rel (a member of the NF-κB), and an ERK-dependent signaling process regulates IL-2 expression in a late phase of T cell activation.28 The MAPK p38 kinase pathway regulates the expression of IFN-γ in Th1 cells.29 Studies of Jurkat cells also indicate that JNK may be involved in regulating IL-2 gene transcription and in IL-2 mRNA stability.30 A major consequence of MAPK phosphorylation is the activation of nuclear transcription factors that serve as downstream substrates of MAPKs. NF-κB and AP-1 are known as pleiotropic regulators of various genes that are involved in immune and inflammatory responses.31,32 It has been shown that the activation of MAPKs in splenic T cells is decreased after trauma-hemorrhage, and NF-κB and AP-1 activities are also suppressed.15 The activation of ERK1/2 and p38 in our results was suppressed in splenic T cells of rats after surgical trauma, NF-κB and AP-1 DNA binding activities were also inhibited, and EA administration increased activities of these signaling pathways. These novel findings indicate that the MAPK signal pathway could be one of the signaling mechanisms accounting for immunomodulatory effects of EA.
There are several limitations in this study. First, this study did not use specific MAPK inhibitors to further elucidate the role of the MAPK pathway; therefore, no direct proof of this idea can be drawn from our data. Second, this study used measurement at a single time point, i.e., on postoperative day 3, and thus it remains unclear whether the effects of EA on cytokine protein and mRNA expressions as well as MAPK activation are evident on other posttrauma days. It is also unknown how long after trauma EA must be administered to produce beneficial effects. To be clinically useful, therefore, additional studies are required to further elucidate the immunomodulatory effects of EA.
In summary, this study shows that EA, in addition to its analgesic effects, could prevent deleterious immunological changes in the postoperative state by regulating the balance of Th1/Th2 cytokine. The protective effects of EA are likely connected with the activation of ERK1/2, p38, NF-κB, and AP-1. Our findings have important implications in the understanding of EA in immunomodulatory mechanisms and clinical practice.
The authors thank Dr. Jialan Shi (Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School) for help in preparing the manuscript.
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