Now, chronic psychological stress (CPS) related diseases are increasing. Many CPS patients have gastrointestinal complaints, immune suppression, and immune imbalance. Increasing evidence is indicating that acupuncture (AP) at the Zusanli point (ST36) can alleviate functional gastrointestinal disorders (FGID), immune suppression, and immune imbalance. However, few studies have investigated the potential mechanisms. In this study, CPS rat models were established, and electroacupuncture (EA) at ST36 was done for CPS rats. Daily food intake, weight, intestinal sensitivity, the morphology of interstitial cell of Cajal (ICC) in the small intestine, and serum indexes were measured. The study found that, in CPS rats, EA at ST36 could improve food intake, weight, visceral hypersensitivity, and immunity; in CPS rats, in small intestine, the morphology of ICCs was abnormal and the number was decreased, which may be part causes of gastrointestinal motility dysfunction. EA at ST36 showed useful therapeutic effects. The mechanisms may be partially related to its repairing effects on ICCs damages; in CPS rats, there were immune suppression and immune imbalance, which may be part causes of visceral hypersensitivity. EA at ST36 showed useful therapeutic effects. The mechanisms may be partially related to its regulation on immunity.
With the changes of the social environment and ways of life, job stress is increasing and the pace of life is accelerating. Meanwhile, chronic psychological stress (CPS) related diseases are growing rapidly.
The digestive system is very vulnerable to the influence of CPS. Functional gastrointestinal disorders (FGID) are very common in daily clinical practice, and they are characterized by disturbances in the motility patterns and/or visceral hypersensitivity [
Acupuncture (AP), a traditional Chinese medicine, has garnered significant attention due to its role in regulation of gastrointestinal function. In the clinic, growing evidences have shown that it could alleviate FGID [
The interstitial cell of Cajal (ICC) exhibits a highly branched morphology and forms unique network in the gastrointestinal tract (GI). ICCs serve as electrical pacemakers, active propagation pathways for slow waves, and mediators of enteric motor neurotransmission. They play an important role in generating and regulating gastrointestinal motility [
Visceral hypersensitivity and gastrointestinal inflammation are important pathophysiological factors for gastrointestinal motility dysfunction diseases, and they closely correlate with immunity [
Repeated exposure to water avoidance stress has been shown to successfully establish a CPS rat model for sustained visceral hyperalgesia [
Thirty seven-week-old male Wistar rats were provided by the experimental animal center of Dalian Medical University. The rats were maintained in a normal light-dark cycle, housed 3-4 rats per cage, and provided with free access to food and water. All protocols were approved by the Ethics Committee of The First Affiliated Hospital of Dalian Medical University.
The CPS rat models were established by the method of Bradesi et al. [
The experimental flow diagram.
At day 11, rats in the control group were housed separately for one day, and daily food intake (24 h) was measured. At day 12, the rats were fasted starting at 3 p.m. but provided free access to water. At day 13, the rats were weighed and intestinal sensitivity was evaluated. Then, the rats were sacrificed under anesthesia. Blood was collected from the inferior vena cava, and the proximal jejunum was cut and collected. The blood was centrifuged at 3000 r/min for 15 min, and the serum was segregated for subsequent analyses. The proximal jejunum was washed quickly with normal saline and then dissected into 1-2 cm fragments, fixed in 4% paraformaldehyde, and placed in the refrigerator. At day 11, 6 rats in the model group were selected according to the random number table method. The rats were processed the same way as the control group.
At day 11, rats in the model group (the remaining 6 rats), AP group, and sham-AP group were fed for another 15 days. In the AP group, every day, rats underwent binding and EA at ST36 for 20 minutes between 7 a.m. and 9 a.m. The acupoint was located at the posterior and lateral side of the knee joint, 5 mm below capitulum fibulae [
At day 24, in each group, rats were housed separately for one day, and daily food intake (24 h) was measured. At day 25, the rats were fasted starting at 3 p.m. but provided free access to water. At day 26, each rat was weighed and intestinal sensitivity was evaluated. Then, the blood and jejunum were processed as described above.
Abdominal withdrawal reflex (AWR) in response to acute visceral pain was used to assess the intestinal sensitivity following the method of Al-Chaer et al. [
A part of proximal jejunum was cut into 1-2 cm fragments, fixed in 4% paraformaldehyde, and placed in the refrigerator at 4°C overnight. The tissues were dissected with an anatomic microscope to strip the mucosa and submucosa as one layer and retain the whole intestinal muscularis.
Next, (1) the specimens were incubated in 0.5% Triton-X in 0.05 mol/L Tris-HCl buffer (pH 7.6) solution at 37°C for 4 h; (2) after rinsing twice in PBS, specimens were incubated for 1 h at room temperature in 1% BSA to prevent nonspecific binding; (3) after rinsing twice in PBS, specimens were incubated for 48 h at 4°C in rabbit anti-rat c-kit polyclonal antibody (c-19, Santa Cruz Biotech, USA); (4) specimens were rinsed twice and incubated 2 h at 4°C in the dark in Cy3-mouse anti-rabbit IgG antibody (Santa Cruz Biotech, USA); (5) specimens were embedded in fluorescence mounting medium and examined under a confocal laser scanning microscope [
In step (3), the control group was incubated in only antibody diluent instead of antibody.
Specimens were observed using a TCS-SP2 laser scanning confocal microscope (Leica, Germany) with immersion objectives (
The serum immunoglobulin G (IgG) and immunoglobulin M (IgM) levels were measured using commercially available sandwich ELISA kits (Xiang Sheng Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Absorbance was read at 492 nm. The serum interleukin-2 (IL-2) and interleukin-6 (IL-6) levels were measured using commercially available sandwich ELISA kits (Boster Biotechnology, Wuhan, China) in accordance with the manufacturer’s instructions. Absorbance was read at 450 nm.
The measurements of weight and food intake, the assessment of intestinal sensitivity, and the measurement of ICCs and serum immune indexes were performed by blinded researchers.
The food intake, weight, scores of AWR, IOD of ICCs, and immune indexes data were analyzed by a commercial software package (SPSS13.0). Data was expressed as the mean
In the model group, food intake and weight were significantly lower than those in the control group (Figure
Food intake and weight. In the model group, daily food intake is significantly lower than that in the control group (
In the model group, when the pressure of CRD was 20, 40, 60, or 80 mmHg, score of AWR was significantly higher than that in the control group (Figure
Scores of AWR. In the model group, when the pressure of CRD was 20, 40, 60, or 80 mmHg, score of AWR is significantly higher than that in the control group (
The control group (Figure
ICCs in intestinal tissue. The control group (a) shows that ICCs are spindle-shaped cells with 2-3 synapses; they are connected to each other by synapses and form network structure. The model group (b) shows that the number of ICCs and the synapses of ICCs are decreased, and the network integrity is damaged.
The model group (Figure
IOD of ICCs. In the model group, the IOD of ICCs is significantly lower than that in the control group (
In the model group, the serum IgG, IgM, and IL-2 levels were significantly lower than those in the control group (Figures
Serum IgG, IgM, IL-2, and IL-6 levels. In the model group, the serum IgG, IgM, and IL-2 levels are significantly lower than those in the control group ((a)
In the AP group, food intake (Figure
Food intake and weight. There is significant difference in daily food intake among the three groups (
When the pressure of CRD was 20 mmHg, scores of AWR were not significantly different among the model group, AP group, and sham-AP group; when the pressure of CRD was 40, 60, or 80 mmHg, in the AP group, score of AWR was significantly lower than that in the model group, and there was no significant difference between the model group and the sham-AP group (Figure
Scores of AWR. When pressure of CRD was 20 mmHg, there is no significant difference in scores of AWR among the three groups (
The model group (Figure
ICCs in intestinal tissue. The model group (a) shows that the number of ICCs and the synapses of ICCs are low, and the network integrity is damaged. The AP group (b) shows that the number of ICCs and the synapses of ICCs are significantly increased. ICCs are connected to each other by synapses and form network structure. The sham-AP group (c) shows that the number of ICCs and the synapses of ICCs are low, and the network integrity is damaged.
The AP group (Figure
IOD of ICCs. There is significant difference in the IOD of ICCs among three groups (
The sham-AP group (Figure
In the AP group, the serum IgG, IgM, and IL-2 levels were significantly higher than those in the model group (Figures
Serum IgG, IgM, IL-2, and IL-6 levels. Whatever the serum IgG, IgM, IL-2, or IL-6 level, there is significant difference among the three groups ((a)
Nowadays, with the accelerated pace of life, we tend to bear more pressure in our profession and family life. Meanwhile, chronic psychological stress (CPS) related diseases are growing.
CPS can cause various symptoms related to the digestive system. FGID are digestive diseases with high morbidity rates [
AP, a physical intervention which involves placement of small needles in the skin at different acupoints, has been practiced in China for 2000 years. In the clinic, growing evidences have shown that it could alleviate FGID [
In this study, CPS rat models were established. Food intake, weight, intestinal sensitivity, ICC of small intestine, and serum immune indexes were measured, and therapeutic effects of EA at ST36 were investigated.
In clinic, most CPS patients eat less, and some patients show significant weight loss. In this study, food intake and weight were significantly decreased in CPS rats than in the control group, and this is consistent with the clinic. This partially indicates that the models were established successfully.
In clinic, many CPS patients make complaints about the gastrointestinal disorder. In this study, was there abnormality of gastrointestinal function in CPS rats? AWR in response to acute visceral pain was used to assess the intestinal sensitivity following the method of Al-Chaer et al. [
Studies have shown that CPS is associated with suppression of both cellular and humoral measures [
In this study, after EA at ST36, food intake and weight were significantly increased; the scores of AWR were significantly decreased, which indicates the visceral hypersensitivity was improved; the serum IgG, IgM, and IL-2 levels of CPS rats increased markedly, and the serum IL-6 level decreased markedly. It indicates that immune suppression and immune imbalance were improved.
In conclusion, EA at ST36 could improve food intake and weight, could improve the symptom of visceral hypersensitivity, and could regulate immune suppression and immune imbalance in CPS rats. It may be a useful therapeutic approach.
Studies have shown that CPS plays an important role in the development and exacerbation of symptoms in FGID [
EA at ST36 showed useful therapeutic effects on visceral hypersensitivity of CPS rats. After treatment, ICCs in intestinal tissues were investigated using confocal laser scanning microscopy, and the results showed that the number of ICCs was increased and cellular morphology resumed normal. This indicated that the mechanisms underlying these effects may be partially related to its repairing effects on ICCs damage.
Studies have shown that brain-gut interactions play an important role in CPS related FGID. The brain receives a constant stream of interoceptive input from the GI, integrates this information with the other interoceptive information from the body and with contextual information from the environment, and sends an integrated response back to various target cells within the GI [
Studies have shown that neuroendocrine signaling plays a key role in AP treatment. Specifically, AP sends signals to the CNS and the CNS then sends signals to various target cells [
In CPS rats, there were visceral hypersensitivity, immune suppression, and immune imbalance. After EA at ST36, the immune suppression and immune imbalance were improved, and the visceral hypersensitivity was improved either. Studies have shown that visceral hypersensitivity closely correlates with immunity [
In CPS rats, neuroendocrine signaling may also play an important role in changes in immunity. Repeated stress will send signals to the CNS. Correspondingly, the CNS will send signals to the thymus, spleen, and gastrointestinal tissue via the neuroendocrine system to modulate the immune system. This process is very complex. At present, the hypothalamic-pituitary-adrenal axis, sympathetic nerve-adrenal medulla axis, and some hormones, peptides, and cytokines are thought to be involved [
The study indicates that therapeutic mechanisms of EA at ST36 may be multifaceted and target multiple components. Now, the exact mechanisms by which CNS affects ICCs in GI are not fully understood, and the pathophysiologic immunity changes in CPS rats are complex. They remain to be further investigated.
In CPS rats, EA at ST36 could improve daily food intake and weight as well as the symptom of visceral hypersensitivity. Also, EA at ST36 can regulate immune suppression and immune imbalance. It may be a useful therapeutic approach.
In CPS rats, in small intestine, the morphology of ICCs was abnormal and the number was decreased, which may be part causes of gastrointestinal motility dysfunction. EA at ST36 showed useful therapeutic effects on gastrointestinal motility dysfunction. The mechanisms may be partially related to its repairing effects on ICCs damage.
In CPS rats, there were immune suppression and immune imbalance, which may be part of reasons of visceral hypersensitivity. EA at ST36 showed useful therapeutic effects on visceral hypersensitivity. The mechanisms may be partially related to its regulation on immune suppression and immune imbalance.
The authors declare that they have no competing interests.
This work was supported by the National Natural Science Foundation of China (no. 81273920).