Wei Xing (魏星), Lin Ya-ping (林亚平), Cao Jian-zhong (曹建中), Yang Jian-wen (杨建文), Chen Hai-jiao (陈海交), Zhang Cheng-cheng (张程程), Peng Yan (彭艳)

1 The Domestic First-class Discipline Construction Project of Chinese Medicine of Hunan University of Chinese Medicine, College of Acupuncture & Moxibustion and Tui-na, Hunan University of Chinese Medicine, Changsha 410000, China

2 College of Traditional Chinese Medicine, Hunan University of Chinese Medicine, Changsha 410000, China

Abstract

Keywords: Acupuncture Therapy; Electroacupuncture; Calcium-activated Chloride Channel; Interstitial Cells of Cajal; Diabetes Complications; Gastroparesis; Rats

Gastroparesis is a syndrome characterized by delayed gastric emptying in absence of mechanical obstruction of stomach. Gastroparesis occurs more frequently in patients with diabetes than in general population[1]. According to reports, 76% of outpatients with diabetes have one or more gastrointestinal (GI)-related symptoms such as nausea, vomiting, early satiety, abdominal pain, constipation, diarrhea and fecal incontinence[2]. Diabetic gastroparesis (DGP) influences the postprandial blood glucose level possibly via affecting the gastric emptying speed[3]. Therefore, it is of great significance to find effective methods to prevent and treat DGP. Recent evidence suggests that interstitial cells of Cajal (ICCs) may play an important role in controlling GI motility. The data also suggest that long-standing diabetes is associated with decreased ICCs volume, as well as the decreased inhibitory innervations and increased excitatory innervations. The changes in ICCs volume and enteric nerves may underlie the pathophysiology of GI complications associated with diabetes and indicate the future treatment targets[4]. Acupuncture is an effective therapy for bowel motility dysfunction[5]. Our previous study revealed that electroacupuncture (EA) at Zusanli (ST 36) significantly improved the symptoms in DGP, down-regulated the blood glucose level, and promoted gastric emptying in rat models[6]. The mechanism should be linked to ICCs regulators, such as stem cell factor (SCF), insulin released test (INS), insulin-like growth factor-1 (IGF-1), ghrelin, and the improvement of ICCs ultrastructure by EA[7-9]. ICCs generate pacemaker activity (slow waves) in GI smooth muscles, but the role of pacemaker activity is controversial. Several conductances, such as calcium- activated chloride channel (CaCC) and non-selective cation channels (NSCC) have been suggested to be involved in slow wave depolarization[10]. Anoctamin 1 (ANO1), encoded by transmembrane protein 16A (TMEM16A), was found to be a subunit of CaCC and specifically expressed in ICCs in GI[11-13]. Nevertheless, few reports focused on EA and its regulatory mechanisms related to ICCs. Therefore, the purpose of this research was to investigate the effects of acupuncture at Zusanli (ST 36), Liangmen (ST 21) and Sanyinjiao (SP 6) on the current intensity of calcium-activated chloride channel (ICaCC) and the expression of TMEM16A in ICCs in DGP rats. Furthermore, we were trying to explore the relationship between the effect of acupuncture on DGP and ICCs pacing mechanism to provide scientific basis for the prevention and treatment of DGP.

1 Materials and Methods

1.1 Animals and grouping

Forty Sprague-Dawley rats, weighing 220-240 g, half male and half female, were obtained from Hunan SJA Laboratory Animal Co., Ltd. [Certificate No.: SCXK (Xiang) 2016-0002]. All the rats were housed at the Experimental Animal Center of Hunan SJA Laboratory Animal Co., Ltd. The rats were kept at 20-22 ℃ with relative humidity 65%-70%, with free access to food and sterile water. After the rats received one week of adaptive feeding, the experiments began officially.

The rats were randomly assigned into four groups, namely a normal control group (group A), a model group (group B), an EA group (group C) and a metoclopramide group (group D), with 10 rats in each group.

1.2 Main reagents and apparatuses

Streptozocin (STZ, S0130-1G, Sigma, USA); bovine serum album (BSA, A2153, Sigma, USA); metoclopramide (MP, Shanxi Yunpeng Pharmaceutical Co., Ltd., China); EliVision plus kit (kit-9902, Maixin Biotechnology Co., Ltd., China); DAB Kit (DAB-1031, Maixin Biotechnology Co., Ltd., China); DMEM/F12 (Solarbio, China); FBS, PBS and D-Hanks (Hyclone, USA); BSA, Trypan blue (Sigma, USA); SCF (Peprotech, USA); Fluo-8 AM (ab142773, Abcam, USA); PE-labeled rabbit anti-rat CD117 (c-kit) fluorescent antibody (1 mL: 0.2 mg, eBioscience, USA); rat tail collagen type Ⅰ (2 mL: 10 mg, Solarbio, China); collagenase type , Ⅱtrypsin inhibitor (Solarbio, China); triphosadenine (ATP, Hubei Tianyao Co., Ltd., China); penicillin (10 000 U/mL, Hyclone, USA); streptomycin (10 000 μg/mL, Hyclone, USA).

Blood glucose meter and paper (Johnson & Johnson, USA); SDZ-Ⅱ EA apparatus (Suzhou Medical Appliance Factory Co., Ltd., China); UV1800 spectrophotometer (Chengdu Shimadzu Equipment Co., Ltd., China); RF-5000 fluorospectrophotometer (SHIMADZU, Japan); 700B patch-clamp equipment (Axon, USA).

1.3 Modeling

After overnight fasting but free access to water, rats were intraperitoneally injected with a single dose of 2% STZ [55 mg/(kg·bw)], freshly dissolved in 0.1 mmol/L citric acid-sodium citrate buffer solution (4 ℃, pH 4.5). At 72 h after injection, the development of diabetes was verified by measure of blood glucose level in tail venous blood. The rats with immediate blood glucose level ≥16.7 mmol/L were considered as successful diabetes models and were used in the experiment. These experimental rats were irregularly (morning on odd days and afternoon on even days) fed with high- glucose high-fat diet (ordinary diet: lard: sucrose: milk powder: egg = 58:15:20:5:2) for 8 weeks and the blood glucose level was monitored. The rats with blood glucose level ＜16.7 mmol/L were eliminated from the experiment.

Despite of blood glucose level, there were significant differences in GI propulsive parameters, such as gastric emptying rate and intestinal propulsion rate, between the models and the control group rats. In terms of comparing the rat injected with STZ to normal rats,P＜0.05 indicated that the DGP model was successfully established.

1.4 Interventions

Group A: The rats in group A were fixed for 20 min each time, once a day, for a total of 15 d.

Group B: After modeling, the rats in group B were fixed for 20 min each time, once a day, for a total of 15 d.

Group C: After modeling, the rats in group C were fixed and treated with EA at Zusanli (ST 36), Sanyinjiao (SP 6) and Liangmen (ST 21) for 15 d, with sparse-dense wave (sparse wave 10 Hz, dense wave 50 Hz), 2 mA, 20 min each time, once a day.

Group D: After modeling, the rats in group D were fixed for 20 min and intragastric administration of 1.7% MP, 10 mL/(kg·bw), once a day, for a total of 15 d.

During intervention, all the rats were fed with normal diet and allowed free access to water. After successive intervention for 15 d, they were fasted for 24 h, and then water-deprived for 2 h. On the 16th day, the rats received 2 mL phenol red solution (500 mg/L) by gavage, and 20 min later, specimens were taken under anesthesia. Rats in each group were tested for gastric emptying and intestinal propulsion rate. Then the rats were sacrificed, and the stomach was rinsed with cold 0.9% sodium chloride solution. A part of the antrum (3 mm × 3 mm) was quickly taken (about 0.5 cm from the pylorus). One part of the tissues was fixed in 2.5% glutaraldehyde and 4% paraformaldehyde fix solution (4 ℃) in order to measure the expression of TMEM16A protein in the gastric sinus of rats. The other part was used to isolate and culture ICCs. The change in Ca2+concentration in ICCs was detected by immunofluorescence assay. Patch-clamp method was used to detect the current of CaCC in ICCs. Limited to the funding of the experiment, 6 mice in each group were selected for TMEM16A detection, primary cell extraction and subsequent cell detection.

1.5 Culture and identification of ICCs

Rats were fasted without water for 24 h, after ether inhalation anesthesia, and then sacrificed by cervical dislocation. The rats were immersed in 75% ethanol solution for 1 min, the abdominal cavity was opened, the stomach was cut and removed from the cardia and pylorus, and the stomach tissue was put into a sterile plate containing 0.01 mol/L PBS (containing 2% penicillin-streptomycin). The vascular and gastric serosal layers were removed by ophthalmological scissors, and the stomach was opened along the greater curvature. Afterwards, the gastric antrum was cut off and washed 3 times with D-Hanks (4 ℃). Then, gastric antrum mucosa, submucosa and serous layer were stripped off by sharp dissection under dissecting microscope. Followed by rinse twice with D-Hanks (4 ℃), the tissues were cut into pieces at about 1-2 mm3, treated with type Ⅱ collagenase solution, 3-4 times the volume of the tissues, for 60 min each at 37 ℃. The digestion was terminated by adding equal volume of DMEM/F12 containing 10% FBS. The solution was centrifuged at 1 000 r/min for 5 min, the supernatant was discarded, and the pellets were re-suspended and passed through cell strainer. The cell status was identified and counted by 0.4% trypan blue. The cell concentration was adjusted to 1×105/cm2. The cells were inoculated into a six-pore plate with DMEM/F12, and then put into the incubator for culture under the condition of 37 ℃ and 5% CO2. After 72 h, the medium was replaced by DMEM/F12 without penicillin and streptomycin. After that, the fluid was changed every 3 d, and the growth of cells was observed under an inverted microscope. In order to determine whether the cultured cells were ICCs or not, the internationally accepted c-kit immunofluorescence antibody was used for culture. At the second week, ICCs (P2) were selected to detect the calcium concentration, as well as in the patch-clamp experiments.

1.6 Detections

1.6.1 Blood glucose

Blood glucose was measured with OneTouch blood glucose meter and blood glucose test paper, and recorded every week.

1.6.2 Gastric emptying rate and intestinal propulsion rate

After overnight deprivation of food and 2 h water- deprivation, the rats received 2 mL phenol red solution (500 mg/L). Twenty minutes later after anesthesia, rats were sacrificed, followed by dissection and measurement of the total length of small intestine (pylorus-ileocecal valve). The furthest distance where NaOH (0.5 mol/L) made the small intestine turn purple served as the propelling distance of phenol red in the intestine. Intestinal propulsion rate = Propelling length of phenol red ÷ Total length of small intestine × 100%. The whole stomach was removed carefully, then cut along the greater curvature, and washed with 20 mL 0.9% normal saline. Next, 20 mL NaOH (0.5 mol/L) was poured into the washed contents. One hour later, 5 mL supernatant was taken into centrifuge tube, together with 0.5 mL 20% trichloroacetic acid. Optical density (OD) of the supernatant was read at 560 nm with spectrophotometer after centrifugation at 3 500 r/min for 10 min. Standard phenol red solution was prepared by mixing 2 mL phenol red (500 mg/L) with 18 mL distilled water, 20 mL NaOH (0.5 mol/L) and 4 mL 20% trichloroacetic acid, and then measured for its OD value. The gastric emptying rate was calculated according to the following formula: Gastric emptying rate (%) = (1 - X/Y) × 100, in which X represented the OD value of phenol red collected from the stomach of rats sacrificed 20 min after the test meal and Y represented the OD value of standard phenol red solution. 1.6.3 Expression of TMEM16A

The samples were baked in incubator for 60 min at 60 ℃, then dewaxed in absolute-95%-85%-70% ethanol gradient, and washed 3 times with PBS. The slices were placed in an antigen repair buffer and heated to boil for 20 min. Then the samples were cooled in running water. After washed 3 times, the slices were treated with 3% H2O2-methanol solution for 15 min, and then washed 3 times again. The sections were incubated with 100 μL primary antibody for 2 h at 37 ℃. After 3 times of wash, the slices were treated with 50 μL reinforcing agent for 20 min at 37 ℃. The slices were covered with 50 μL of HRP-conjugated secondary antibody for 20 min at 37 ℃, added with 2 drops of freshly-prepared DAB solution, and the dyeing intensity was observed under microscope. The sections were then washed gently with distilled water for 15 min to stop the color reaction. Afterwards, put the sections into hematoxylin for 10 min, and washed it with distilled water. The sections were then soaked in 70%-85%-95%-absolute ethanol gradient for 5 min in turn, in xylene for 10 min, and in xylene for another 10 min after replacement. After drying, the slices were added with neutral gum and the slides were covered. The sections were observed under optical microscope.

1.6.4 Concentration of Ca2+

Determination of intracellular Ca2+concentration in ICCs by Fluo 8-Am. The concentration of ICCs was adjusted to 1×105/cm2and the cells were moved into a 96-well plate with DMEM/F12, incubated overnight under the condition of 37 ℃ and 5% CO2. After the medium was removed, 4 μm Fluo-8 AM dissolved in 100 μL Hank's buffer with Hepes was added into the plate and the plate was incubated at 37 ℃ under 5% CO2for 1 h. The final concentration was adjusted to 4 μm and the tubes were shaken slightly. The tubes were wrapped by black paper, incubated at 37 ℃ for 40 min while the tubes were gently shaken to accelerate the entry of Fluo-8 AM into the cells. After incubation, the tube was centrifuged at 1 500 r/min for 6 min and the supernatant was discarded. Finally, the cells were observed and taken photos under fluorescence microscope.

1.6.5 Patch-clamp experiments

Patch-clamp experiment was carried out after all cells were attached to the dish. To perform the experiment, the culture medium was removed and the cells were washed gently 2-3 times with Tyrode’s solution (140 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L CaCl2, 1.2 mmol/L MgCl2, 10 mmol/L glucose, 10 mmol/L hepes, pH 7.4 with 2 mmol/L NaOH). Laser confocal culture plate was placed in the chamber dedicated to diaphragm clamp. Glass electrode was horizontally pulled into glass microelectrode and electrode resistance should be 3-5 mΩ. Pipette solution contained 140 mmol/L N-Methyl-D-glucamine (NMDG), 2 mmol/L MgCl2, 5 mmol/L CaCl2, and 10 mmol/L Hepes (pH 7.4 with 2 mmol/L NMDG). The Petri dish was irrigated with 2 mL Tyrode’s solution under microscope. The visual field was adjusted to the center under the microscope to find suitable ICCs. With positive voltage, the electrodes were slowly moved into the bottom layer of the solution, and then quickly approached to the cells. When resistance increased suddenly, negative voltage was added onto the cells and finally formed 1-3 GΩ high resistance sealing. After the diaphragm was broken, the whole cell recording was formed by compensating capacitor current and electrode series resistance. Experimental data were collected and analyzed using Axon 1440A. Macroscopic currents were filtered at 10 kHz and digitized at 3 kHz with patch-clamp amplifier. Series resistance was maintained near 5 MΩ and compensated by 65%-70%.

1.7 Statistical analyses

All data were analyzed by SPSS 20.0 statistical software. Normal distribution test and homogeneity test for variance of the measurement data were performed first. Data with homogeneity of variance were expressed as mean ± standard deviation (±s); one-way analysis of variance was used for between- group comparisons; the least significant difference method was used for data with homogeneity of variance while Dunnett T3 method was used for data with heterogeneity of variance. Rank-sum test was used when the normality was not satisfied.P＜0.05 indicated a statistically significant difference.

2 Results

2.1 Blood glucose

Before modeling, there was no significant difference in the blood glucose level among the four groups. After modeling, the blood glucose levels increased significantly in groups B, C and D compared with group A (allP＜0.01). It’s suggested that the DGP model was successfully established. Compared with group B, the blood glucose levels decreased markedly in group C and group D after intervention (P＜0.01,P＜0.05). When compared with the blood glucose level after modeling, only group C showed marked intra-group difference after treatment (P＜0.01). The results suggested that the control of blood glucose was more effective in group C than in group B and group D (Table 1).

2.2 Gastric emptying rate and intestinal propulsion rate

Compared with group A, the gastric emptying rate in group B showed a significant reduction (P＜0.01), indicating that the DGP model was successful. Compared with group A, the gastric emptying rate in group D decreased markedly (P＜0.01). Compared with group B, the gastric emptying rate in group C and group D increased significantly (bothP＜0.01).

Compared with group A, the intestinal propulsion rate in the other 3 groups declined significantly (allP＜0.01). Compared with group B, there was a significant increase in the intestinal propulsion rate in both group C and group D (bothP＜0.01), (Table 2).

Table 1. Comparison of the blood glucose level ( ±s, mmol/L)

Table 1. Comparison of the blood glucose level ( ±s, mmol/L)

Note: Compared with the normal control group, 1) P＜0.01; compared with the model group, 2) P＜0.01, 3) P＜0.05; comparison between before and after modeling in the same group, 4) P＜0.01; comparison between after modeling and after intervention in the same group, 5) P＜0.01

Group n Before modeling After modeling After intervention Normal control (A) 10 6.09±1.00 5.29±0.50 5.61±1.24 Model (B) 10 5.91±1.54 25.33±5.231)4) 26.50±3.831) EA (C) 10 5.98±1.28 25.51±6.121)4) 21.32±3.641)2)5) Metoclopramide (D) 10 5.98±1.21 25.42±4.031)4) 23.21±3.281)3)

Table 2. Comparison of gastric emptying rate and intestinal propulsive ratio (±s, %)

Table 2. Comparison of gastric emptying rate and intestinal propulsive ratio (±s, %)

Note: Compared with the normal control group, 1) P＜0.01; compared with the model group, 2) P＜0.01

Group n Gastric emptying rate Normal control (A) 10 77.95±4.92 69.11±5.93 Model (B) 10 46.10±7.081) 39.64±5.721) EA (C) 10 71.94±10.242) 54.65±7.191)2) Metoclopramide (D) 10 63.00±12.151)2) 51.13±7.591)2) Intestinal propulsion rate

2.3 Morphology of ICCs

After 72 h of culture, the ICCs were stably attached to the wall of culture dish with clear cell boundaries. Under microscope, the cells showed fusiform, triangular or stellate, large nucleus and small cytoplasm. Some cells had two or three short protrusions, but there was no obvious connection between the protrusions. Besides, the unique network structure of ICCs was not typical (Figure 1-A). One week later, the morphology of ICCs did not change, which may be related to the high differentiation in adult rats (Figure 1-B). Two weeks later, the cell structure became clearer, and the protuberances became longer and thinner and were connected with each other to form an obvious network (Figure 1-C). The expression of C-kit is the typical feature of ICCs. To test if the isolated cells were ICCs, C-kit expression was examined by immunofluorescence with P2 cells. As shown in Figure 1-D, most of the cells had strong C-kit staining, indicating that the cells were ICCs.

Figure 1. Morphology of ICCs (×400)

2.4 Expression of TMEM16A in gastric antrum

TMEM16A is a subunit of CaCC, and CaCC is the most important part in ICCs generating pacemaker potential. Moreover, in GI system, TMEM16A is an ICC-specific biomarker. As shown in Figure 2, the expression of TMEM16A in gastric antrum in group A showed brown- yellow staining between muscle layers. Compared with group A, the expression of TMEM16A in group B reduced significantly (P＜0.01), suggesting that there was a reduction of TMEM16A expression in DGP rats. Compared with group A, the expression of TMEM16A in group C decreased markedly (P＜0.05). Compared with group B, there was an obvious increase in the expression of TMEM16A in group C (P＜0.05). The detail is shown in Table 3.

Figure 2. Expression of TMEM16A in gastric antrum (Envision, ×800)

Table 3. Comparison of expression of TMEM16A protein among groups ( ±s)

Table 3. Comparison of expression of TMEM16A protein among groups ( ±s)

Note: E5=105; E4=104; compared with the normal control group, 1) P＜0.01, 2) P＜0.05; compared with the model group, 3) P＜0.01, 4) P＜0.05.

Group n TMEM16A Normal control (A) 6 3.92E5±6.62E4 Model (B) 6 1.86E5±4.99E41) EA (C) 6 2.86E5±8.89E42)3) Metoclopramide (D) 6 3.10E5±7.57E44)

2.5 Intracellular Ca2+ concentration in ICCs

Compared with group A, the fluorescence intensity of Ca2+in group B reduced significantly (P＜0.01). Compared with group B, the fluorescence intensity of Ca2+in group C and group D increased significantly (P＜0.01,P＜0.05). Therefore, we suppose that a certain amount of intracellular Ca2+concentration was necessary for ICCs to maintain the function of pacemaker and the generated current of pacemaker relied on intracellular Ca2+oscillation[14]. In addition, it was also suggested that the way EA and metoclopramide promoted the gastrointestinal motility in DGP should be related to the increase of Ca2+concentration in ICCs (Table 4 and Figure 3).

Table 4. Fluorescence intensity of Ca2+ ( ±s)

Table 4. Fluorescence intensity of Ca2+ ( ±s)

Note: Compared with the normal control group, 1) P＜0.01; compared with the model group, 2) P＜0.01, 3) P＜0.05

Group n Fluorescence intensity of Ca2+ Normal control (A) 6 30.2±6.0 Model (B) 6 19.2±5.11) EA (C) 6 29.4±5.82) Metoclopramide (D) 6 26.2±6.73)

Figure 3. Fluorescence intensity of Ca2+ under microscope (Fluo 8-AM, ×200)

2.6 ICaCC in ICCs

Whole-cell patch-clamp technique was used to record the current characteristics of CaCC in primary ICCs. Figure 4 A-D showed that the current increased with time. It was clear to see that the current was positively correlated with time except that the current in group B was significantly reduced, while ICaCC in group C and group D increased. Figure 5 showed the relationship between voltage and current. When the voltage was 0 mV, the current was 0 nA. With the increase of voltage, the current also increased, which indicated that the current was positively correlated with the voltage, and the current was greater in group C and group D than in group B. As shown in Figure 4 (A’-D’), the current was inhibited by chloride channel blocker NFA, indicating that the current in ICCs was chloride current. The current was consistent with classic current of TMEM16A channel. In a word, it can be confirmed that the slow wave current generated by ICCs is the current of CaCC. When ICaCC in DGP was significantly reduced, the GI motility was weakened, while EA could enhance the current and improve the GI motility.

Figure 4. Current changes in each group (ICaCC in ICCs)

Figure 5. Relationship between voltage and current in each group

3 Discussion

DGP is a common complication of poorly controlled diabetes, affecting 20%-50% of the diabetic population, especially those with type 1 diabetes mellitus or those with long-standing (≥10 years) type 2 diabetes mellitus[15]. Gastroparesis is more common in type 2 diabetes, and 30% of the patients with type 2 diabetes suffer from delayed gastric emptying[16]. Acupuncture therapy is an effective treatment for GI disorders, such as functional dyspepsia[5]and postoperative GI dysfunction[17-18]. Nevertheless, few reports focused on EA and its mechanisms in regulating ICCs.

Slow waves are in charge of the rhythmic contraction of GI muscles and mediate GI motility. Slow waves are caused by the release of Ca2+, which is stored inside cells, through the IP3 receptor, leading to the spontaneous transient inward current (STIC)[19-20]. The accumulation of STIC leads to spontaneous transient depolarization (STD) which causes slow waves. In low Cl-solution or 4, 4’-diisothiocya nostilbene-2, 2’ disulfonic acid (DIDS) solution containing CaCC inhibitor, the plateau phase was weakened[1,21]. As shown by previous studies, TMEM16A has been identified as a CaCC subunit[22-23]. Meanwhile, in GI tract, TMEM16A is specifically expressed in ICCs, but not in GI smooth muscle cells[22]. Moreover, contraction of smooth muscle cells (SMCs) in TMEM16A knockout mice was defective[22]. Based on the above facts, we speculated that the treatment effect of EA for DGP may be related to CaCC.

STD in ICC is closely related to Ca2+[23]. We measured the concentration of intracellular Ca2+in ICCs, and found that the concentration of Ca2+was much higher in groups C and D than in group B. And the intracellular concentration of Ca2+in group C was comparable to that in group A. Therefore, the improvement of GI motility in groups C and D should be related to the enhancement of Ca2+.

This study showed that EA therapy could effectively regulate blood glucose level and GI motility. We studied the TMEM16A expression to determine whether EA improved GI motility by regulating CaCC. In this study, the intracellular Ca2+concentration in group C was obviously higher than that in group B. In other words, it was highly possible that EA and MP increased the Ca2+concentration in ICCs to improve gastrointestinal movement, and a certain amount of Ca2+concentration should be necessary for GI smooth muscle contraction. The generation of pacemaker current depends on intracellular Ca2+oscillation. The expression of average gray value of TMEM16A decreased in group B, indicating the reduction of CaCC in DGP, which contributed to the abnormal pacemaker of ICCs and affected gastric motility. TMEM16A in group C was greatly increased compared with group B. The current in group C was obviously increased than that in group B. The current and voltage were positively correlated in all groups, and the current intensity was higher in groups C and D than in group B. Besides, the results showed that the current could be blocked by NFA, a chloride channel blocker. Therefore, it could be further confirmed that the detected current in ICCs was CaCC current. Moreover, EA improved gastric motility by increasing ICaCC, and it depends on, to some extent, the concentration of intracellular Ca2+. MP, as a commonly used medication for gastroparesis, can effectively relieve the symptoms in gastroparesis. In this study, it was further proved that it can also improve gastric motility by regulating ICaCC. It can be inferred that CaCC is in the downstream of regulating gastric movement, while MP and EA interfere different upstream regulatory pathways, and finally Ca2+is used as a mediator to directly affect the contraction of smooth muscle. However, its specific mechanism needs further study.

In conclusion, our data supported that EA can effectively improve GI motility, and EA can improve GI motility by regulating ICaCC, which was positively correlated with the concentration of Ca2+in ICCs. In-depth experiments are needed to explore the molecular pathways involved in EA treatment of DGP.

Conflict of Interest

There is no potential conflict of interest in this article.

Acknowledgments

This work was supported by National Natural Science Foundation of China (国家自然科学基金项目, No. 81774431); Open Fund of the Domestic First-class Discipline Construction Project of Chinese Medicine of Hunan University of Chinese Medicine (湖南中医药大学中医学一流学科开放基金, No. 2018ZYX35).

Statement of Human and Animal Rights

The treatment of animals conformed to the ethical criteria in this experiment.

Received: 25 December 2019/Accepted: 16 April 2020