Electroacupuncture attenuates depressive disorder and gastrointestinal dysfunction in myocardial hypertrophy via upregulation of 5-HT on Rats

Background: Acupuncture therapy has been described as a preventive and therapeutic technique with low or no risk for a number of conditions related to cardiovascular disease. However, its role in protecting against myocardial hypertrophy (MH) has not been elucidated. This study aimed to investigate whether electroacupuncture (EA) can attenuate MH by upregulating tryptophan 5-hydroxytryptamine (5-HT) metabolism, depressive disorder and gastrointestinal dysfunction in MH rats model. Methods: We assessed the anti-myocardial ischemia effects of EA by using the isoproterenol hydrochloride (ISO)-induced myocardial hypertrophy (MH) rats model. Forty 3-month-old Sprague-Dawley (SD) rats were randomly divided into normal, model, EA and sham EA groups, with ten rats in each group. Regression of MH was evaluated by ratio of heart weight/body weight (HW/BW) and ratio of left ventricular weight/body weight (LVW/BW). Ultrastructural observation and hematoxylin and eosin (H&E) staining were evaluated to cardiac pathology. The sucrose preference test (SPT), open eld test (OFT), and forced swim test (FSW) were conducted to examine the depression-like behavior. Gastric retention and intestinal transit were evaluated using the carbon powder labeling method, while immunohistochemical staining was used to detect 5-HT expression in the cerebral cortex and ileum.


Background
Myocardial hypertrophy (MH), a heart muscle thickening that causes the enlargement of ventricles, is the primary reaction of myocardium in response to mechanical overload or stress [1]. Depression is a frequent psychiatric symptom among patients with MH [2] and it effects the prognosis of MH as an independent risk factor. In addition to this, previous studies showed that MH has an in uence on intestinal barrier integrity and somatic psychiatric symptoms (e.g., gastrointestinal symptoms, fatigue, poor appetite, and nausea) that also re ects prognosis of the disease. It was demonstrated in the clinical studies that myocardial injury was reduced effectively by acupuncture pretreatment in patients with myocardial ischemia [3]. Meanwhile, according to a lot of experimental studies, results indicated that acupuncture can meaningfully diminish the incidence rates of sudden death [4], arrhythmias, and angina pectoris [5]. However, there is no research to explore that acupuncture can regulate the association between the mechanisms of somatic and psychiatric symptoms after MH at present, especially to focus on 5-Hydroxytryptamine (5-HT), which is a pivotal neurotransmitter in the central nervous system.
Meanwhile, most of 5-HT in the body is contained within gastrointestinal tract, and it is important for depression and gastrointestinal function.
In the early animal research [6], we observed that EA can improve cardiac function in MH rats by modulating upstream neuroendocrine cytokines that regulate the extracellular signal-regulated kinase signaling pathways. For further research, we hypothesize that electroacupuncture (EA) regulates depression and gastrointestinal disorders after MH by ameliorating central and enteric 5-HT dysfunction.

Chemicals and reagents
Rabbits anti-mouse 5-HT antibody and goat anti-rabbit secondary antibody were purchased from Invitrogen and Abcam (MA1-81821 and ab150077; California USA and Cambridge UK respectively).

Animals groups and Models
All experimental procedures comply with the guidelines of National Institutes of Health (NIH) "Guide for the Care and Use of Laboratory Animals and the legislation of the People's Republic of China for the use and care of laboratory animals. The experimental protocols were approved by the Animal Experimentation Ethics Committee of the Hubei University of Traditional Chinese Medicine, China. A total of forty 3-month-old female Sprague-Dawley (SD) rats, weighing 200 g to 220 g, were provided by Tongji Medical College of HUST (SCXK (E) No. 2016-0057). In accordance with the random number table, rats were divided into four groups which are normal, model, EA and sham EA groups, with ten rats in each group. Rats in the normal group received 3 mg/kg·d of physiologic saline injection into the back skin, whereas the other rats were injected with isoproterenol hydrochloride (ISO), as previously described [6].

EA stimulation
The acupuncture stimulation involved the insertion of stainless steel needles (0.2 mm in diameter and 13.0 mm in length) into the acupoint PC6 at a vertical depth of 2-3 mm in groups EA and sham EA.
Needles were manually twisted for one minute at a frequency of about 2 Hz before EA treatment. Rats in EA group received EA stimulation on bilateral acupoint PC6 with the following stimulus parameters: continuous-wave, frequency of 2 Hz, and current intensity of 1 mA, while rats in sham EA group received no electric current. They were treated for 20 min every day for 14 days.

Evaluation of regression of cardiac ischemia and cardiac hypertrophy
Reversion of cardiac hypertrophy was assessed by ratio of heart weight/body weight (HW/BW) and ratio of left ventricular weight/body weight (LVW/BW). Regression of cardiac ischemia was recorded by electrocardiogram (ECG) recording electrode resettlement as performed previously [6]. For recording the standard -lead ECG, we used BL-420 physiological function experiment system. Its negative and positive stainless steel electrodes were placed beneath the skin close to bilateral left and right forelimb horizontal grain respectively, and the reference electrode was placed beneath the skin of the right hind limb. In process of recording, we selected a resolution ratio of 500nv/mv, chart speed of 50ms/div, and taken ten cardiac cycle into calculation.

Ultrastructure observation
Rats were quickly decapitated after anesthesia with intraperitoneal injection of 10% chloral hydrate (100 g/0.3 ml), and cardiac tissues were separated. The central ventricle muscle of left ventricular free wall was removed and chopped into 1mm×1mm×1mm size, xed in 2.5% glutaraldehyde for 48 hours, then in 1% osmium tetrachloride for 1.5 hour, dehydration, embedding, sectioning, and examined on Hitachi H-600 transmission electron microscopy (Hitachi Company, Tokyo, Japan).

Infarct size measurement
Rats were euthanized and hearts were removed after 2 weeks modeling. In order to histological assessment, a small fragment from each heart was xed in 10% buffered formalin solution and dehydrated with alcohol and xylene. After dehydration, each sample was embedded in para n wax and divided into slides of 5 μm thickness with a Leica rotation microtome. Groups of slides were stained with hematoxylin and eosin (H&E) based on standard methods. Areas of myocardial infarction were observed using ImageJ software (NIH, Bethesda, MD, USA).

Sucrose preference test
Rats were single placed in a quiet room with two bottles of sucrose solution (1%, w/v) for the rst 24 h period. Then one bottle was replaced with tap water for the second 24 h period. After this initial adaption phase, rats were deprived of food and water for 24 h and then permitted access to two bottles for 3 h, one containing 100 ml of 1% sucrose solution and the other 100 ml of tap water. Sucrose preference was de ned as the ratio of consumption of sucrose solution and the consumption of both pure water and sucrose solution during the test phase, and calculated as follows: percentage (%) = total sucrose consumption / (total sucrose consumption + total water consumption) × 100%.

Forced swim test
Brie y, each rat was subjected to pre-swimming habituation for 15 minutes on day 15 and tested 24 hours later. Rats were individually placed into plexiglas cylinders (internal diameter 21 cm; height 50 cm) lled with water (depth 30 cm; 25 °C) for 5 min of forced swimming in the training session. Time spent on swimming, striving, and immobile and the latency to immobility were recorded, using a swimming analysis system (EthoVision XT, Noldus, the Netherlands) to analyze.

Open-eld test
Brie y, rats were placed individually at center of the eld and were allowed to explore any areas freely in open eld test. Then, behavior was recorded using a detection system (JLBehv-LAR-1; Shanghai Jiliang Software Technology Co., Ltd., Shanghai, China). The total and central distances explored during the 5minute test were used to analyzed the behaviors.

Determination of gastric residual and gastrointestinal transit rates
Before commencing the experiment, animals were deprived of food for 24h but allowed free access to water. Each rat was received 1 mL/100 g nutritional black semi-solid paste and was sacri ced 20min later. Afterward, opening the abdominal cavity, stomach and small intestine were excised after ligation of the pylorus and the cardia, then the stomach was obtained, dried, and weighed. It was cut along the greater curvature, the contents were rinsed, and the stomach was weighed again. The difference in gastric weight showed the weight of the gastric residues. The gastric residual rate was calculated as follows: gastric residual rate (%) = (total gastric weight − gastric net weight) / intragastric volume × 100.
Immediately after excision of the stomach, the small intestine (from the pylorus to the ileocecum) was completely freed from its mesenteric attachments. Its total length and the carbon propulsion distance were measured by a meter scale. The gastrointestinal transit rate was calculated as follows: intestine propulsion rate (%) = carbon propulsion distance /total length of small intestine × 100%.

Immuno uorescence staining of 5-HT
The rat brain and terminal ileum was removed and sections (5 μm thick) were obtained consecutively. The sections were washed with phosphate-buffered saline (PBS), and processed with green-uorescent Alexa Fluor 488 (1 : 500) to incubate. The nuclear marker DAPI was used. The uorescence microscope (Leica, Germany) was used to observed and imaged these sections. SABC method was used to perform immunohistochemical staining of 5-HT protein. Positive staining of protein was shown in the cytoplasm. NeuN-positive cells were counted in three random images from each section by a technician blinded to this study.

Statistical analysis
Data were presented as mean ± standard error of mean (S.E.M) in this study. Unpaired student's t-test was used to analyze two sets of data. Differences among groups were tested using one way or repeated measures analysis of variance (ANOVA). The Tukey-Kramer test was used to differentiate means. A Pvalue of less than 0.05 was considered statistically signi cant.

EA prevented cardiac hypertrophy induced by pressure overload
We rst determined the degree of intervention for cardiac hypertrophy induced by ISO after treatment with EA for two weeks. Results in g.1 show that the ratios of HW/BW and LVW/BW were evidently increased in groups model and sham EA compared to the group Control, which were meaningfully suppressed by EA treatment (P<0.05, Fig. 1A and 1B). ECG analysis detected that ST segment and R-R interval was decreased in cardiac hypertrophic rats treated with EA compared with those rats in model and sham EA groups (P<0.05, Fig. 1C, 1D and 1E).
Ultrastructural of cardiac cell was observed by transmission electron microscopy. In model group, the cardiac cell exhibited mitochondria swelling, excessive cell apoptosis, endothelial cell interstitial hyperplasia, muscle plasma nets expansion, out [1]sync contraction of contraction band, and intercalated disc deformation. Compared EA group with model and sham EA groups, myocardial ischemia and hypertrophy was improved. Ultrastructure observation suggested that treatment with EA signi cantly reduced pressure overload-induced increase cardiomyocyte cross-sectional areas ( Fig. 1F).
Histological observation of control group (after H&E staining) presented that cardiac muscle cells were arranged in order, the nucleus was clearly visible and myocardial bers were compact. However, rats in model group showed a great number of structurally ber tissues were disordered, with widened gaps and local brosis. Cells were ruptured and exhibited dissolution and a distribution disorder in more than 50% of the area of ischemic necrosis, which indicated that myocardial cells were minimally damaged. Sham EA group did not obviously differ from the model group on histological evaluation. In contrast, compared to model group, the cardiac muscle of EA group presented obvious improvement, with lighter myocardial brosis, smaller nuclei and signi cantly reduced areas of ischemia ( Fig. 1G).

Sucrose preference test results
A one-way analysis of variance exhibited signi cant differences in sucrose intake among 4 groups at 2 weeks modeling (F[3, 36] = 23.84; P < 0.01). In addition, Tukey's multiple comparisons suggested that sucrose intake in model and sham EA groups was decreased relative to normal group (both P < 0.01).
However, EA increased the sucrose intake while compared with groups model and sham EA (both P < 0.01). There were no signi cant differences between model and sham EA groups (P 0.05, Fig 2B).

Open-eld test results
A one-way analysis of variance showed signi cant differences in the total and central distance among 4 groups (F [3, 36] = 63.34, P < 0.01; F [3, 36] = 303.2, P < 0.01, respectively). Further Tukey's multiple comparisons showed that the total and central distances were signi cantly reduced in model and sham EA groups relative to normal group (both P < 0.01). However, EA increased the total and central distances while compared with groups model and sham EA (both P < 0.01). There were no signi cant differences between model and sham EA groups (P 0.05, Fig 2C and 2D).

Forced swim test results
A one-way analysis of variance revealed signi cant differences in immobility time, swimming time,  Fig 3A and 3B). Compared to normal and sham EA groups, EA group exhibited increased 5-HT expression in the cerebral cortex region and the distal ileum (all P < 0.05). There were no signi cant differences between model and sham EA groups (both p 0.05; Fig 4C and 4D).

Discussion
Ischemic heart disease is known to be one of the major causes of death in the world and it can directly lead to myocardial infarction [7]. Modern study has demonstrated that EA possesses therapeutic effects on cardiovascular system [8]. Previous research has indicated that acupoint PC6 was preferred and considered effective in treating cardiovascular disorders, which can attenuate cardiac injury, through regulating heart rate variability [9], apoptosis [10], and myocardial ischemia [11]. Our previous researches had con rmed that EA could effectively promote myocardial brosis and protect myocardial tissue against ischemia injury [6]. In the rats with ISO induced MH, we found a depression of ST segment of the ECG, which is indicative of ischaemia, repeated EA at PC6 for 20 min a day for 14 days improved cardiac function in MH rats model. Moreover, according to results of SPT, OFT and FWT, we observed that EA could signi cantly ameliorate depression-like symptoms. In addition, the gastric retention rate and gastrointestinal transit rate were improved, and expression of 5-HT was increased in the cerebral cortex and ileum after EA treatment. Depressive disorder and gastrointestinal dysfunction after MH are almost accompanied by abnormal 5-HT metabolism. This provides a new mind for why the somatic, cognitive, and psychiatric symptoms of depression commonly appear after MH.
Cardiovascular disease, including ischemic heart disease and cerebrovascular disease, has been identi ed as a leading provider to both mortality and disease burden globally [12]. Furthermore, depression may bring about autonomic nervous system disorders such as decreased heart rate variability, increased heart rate, excessive heart rate response to physical stress, and increased ventricular depolarization variability, thereby increasing the risk of cardiovascular disease [13]. MH is a potential target for therapy to improve outcomes among depressed non-ST elevation acute coronary syndrome patients [14], with high rates of morbidity and mortality and is often comorbid with gastrointestinal dysfunction [15]. Remarkably 5-HT, it is considered as a good communicator between central nervous system (CNS) and peripheral system, because of its signal transporting role in CNS transduction when releasing by presynapic vesicles, and it plays an important role in regulating emotional station and depression [16]. More and more evidence reveals that the decline of prefrontal cortex 5-HT release is highly contribute to etiology of depression and anxiety as some suicide victims [17]. Depression, the most prevalent mood disorder, is characterized by low mood, lack of motivation, reduced energy, anhedonia and despair. In this study, we assessed the degree of depressive-like behavior after MH in rats. Sucrose preference test was an indicator of anhedonia-like behavioral change, the open eld test was a way to evaluate exploratory activity, and the forced swimming re ected the degree of despair. Through our experiment, we discovered that, after MH, the preference of sucrose was notably decreased and the total and central distance traveled in the open eld test were remarkably decreased as well. Furthermore, immobility time in forced swimming test was increased, and swimming time and latency to immobility were shortened. Changes in 5-HT were assayed immunchemistry with uorescence detection. 5-HT in cerebral cortex and terminal ileum tissues were signi cantly decreased in MH rats model, wile EA treatment on PC6 increased 5-HT expression both in cerebral cortex and terminal ileum. Meanwhile, these data from this study are correspond to the 5-HT hypothesis of depression. To the best of our knowledge, the current study is the rst to report that EA can improve MH by regulating 5-HT expression both in brain and gastrointestinal.
In our body, 95% of 5-HT is synthesized in the intestinal tract and 90% is distributed to intestinal chroma n cells, whereas the remaining 5% is found in the enteric nervous system [18]. The enteric cavity stimulates to secret 5-HT in a stressful environment, which acts on intrinsic primary afferent neurons in the mucosal membrane. These afferent neurons can activate interneurons in the dorsal horn and initiate intestinal tract movement through receptor pathways [19]. Increases in 5-HT have associated with gastrointestinal motility dysfunction in pathological state. Low metabolism of 5-HT brings about abnormalities in brain regions linked with learning, memory, and emotion. It functions through the binding of its receptors, including the G-protein coupled 5-HT1A receptor, which is a predominant contributor to the regulation of emotion [20].

Conclusion
In summary, taking the viewpoint that acupuncture therapy is of some notable value for MH, we believe that there is a close relationship between PC6 (acupoint) and heart (organ). In summary, 5-HT acts as a bridge between gut and brain and is an critical regulator involved in EA regulation. Abnormal 5-HT expression in the intestinal tract plays a crucial role in presentation of depression after MH, and is provides a theoretical basis for acupuncture treatment of MH.  bers. With reference to normal group, compared electroacupuncture (EA) group with model group, EA signi cantly reduced cardiac muscular tissue injury. Ultrastructure features in normal group (a×17000, b×15000), model group (c×8000, d×6000), EA group (e×8000, f×17000) and sham EA group (g×8000, h×12000). (G) Effect of electroacupuncture (EA) on area of myocardial infarction in rats with myocardial hypertrophy (MH). Histological observation after hematoxylin and eosin (H&E) staining in the control group (a, b), model group (c, d), EA group (e, f) and sham EA group (g, h). All images are ×200 magni cation.