Effects of electroacupuncture at Chize (LU 5) versus Shangjuxu (ST 37) in rats with ulcerative colitis

Luo Qin (罗钦), Li Zhi-tong (李志同),2, Yang Wei-ning (杨伟宁), Li Sheng-jie (李胜杰), Xie Jie-ping (解秸萍)

1 Department of Acupuncture and Massage, Beijing University of Chinese Medicine, Beijing 100029, China

2 Department of Acupuncture and Massage, Changping Hospital of Traditional Chinese Medicine, Beijing 100029, China

Basic Study

Effects of electroacupuncture at Chize (LU 5) versus Shangjuxu (ST 37) in rats with ulcerative colitis

Luo Qin (罗钦)1, Li Zhi-tong (李志同)1,2, Yang Wei-ning (杨伟宁)1, Li Sheng-jie (李胜杰)1, Xie Jie-ping (解秸萍)1

1 Department of Acupuncture and Massage, Beijing University of Chinese Medicine, Beijing 100029, China

2 Department of Acupuncture and Massage, Changping Hospital of Traditional Chinese Medicine, Beijing 100029, China

Objective:To compare the effects between electroacupuncture (EA) at Chize (LU 5, the He-Sea point of the Lung Meridian) and Shangjuxu (ST 37, the lower He-Sea point of the large intestine) in rats with ulcerative colitis (UC) on the variations of mesenteric microcirculation and vasoactive intestinal peptide (VIP) in the colon, lung, and hypothalamus. The relative specificity of acupoints was also explored.

Acupuncture Therapy; Electroacupuncture; Point, Chize (LU 5); Point, Shangjuxu (ST 37); Colitis, Ulcerative; Microcirculation; Vasoactive Intestinal Peptide

The He-Sea point is one of the five Shu-Transport points. Based on traditional Chinese medicine (TCM) theories, the He-Sea point is for diarrhea caused by adverse flow of qi. Among the He-Sea points, Chize (LU 5), Ququan (LR 8) and Zusanli (ST 36) are more frequently used in treating vomiting and diarrhea[1]. Chize (LU 5) is the He-Sea point of the Lung Meridian. Boodletting at Chize (LU 5) can cure acute gastro-enteritis[2]. The lung and large intestine are internalexternally connected through meridians, and there is an interaction between the lung diseases and large intestine diseases[3-4]. Applying TCM theory on the lung always produces good effects in treating intestinal diseases[5]. The lower He-Sea points refer to the six specific acupoints of the Three Yang Meridians of Foot where qi of the six Fu organs descend and gather. As the lower He-Sea points can treat corresponding diseases of Fu organs, Shangjuxu (ST 37), the lower He-Sea point of the large intestine, is specially used for large intestine diseases[6]. As stated above, both Chize (LU 5) and Shangjuxu (ST 37) can treat intestinal diseases. Our previous study[7]indicated that electroacupuncture (EA) at Chize (LU 5) and Shangjuxu (ST 37) could produce beneficial effects in rats with chronic obstructive pulmonary diseases (COPD), a model of lung disease affecting the intestine. Chize (LU 5) showed better effects and the change of hypothalamus vasoactive intestinal peptide (VIP) possibly promoted its action mechanism. However, what is the difference between the two points in treating primary intestinal microcirculation abnormalities? Therefore, in this study, the ulcerative colitis (UC) rat model was established to investigate the different effects of Chize (LU 5) and Shangjuxu (ST 37) on mesenteric microcirculation and VIP in the colon, lung and hypothalamus.

1 Materials and Methods

1.1 Materials and apparatuses

Acetic acid and urethane (Beijing Chemical Reagent Company, China); acupuncture needles (Beijing Zhongyantaihe Pharmaceutical Co., Ltd., China); Han’s nerve-acupoint stimulator (Nanjing Jisheng Medical Technology Co., Ltd., China); microcirculation microscopic tester XW-B-3 (Pathology Laboratory, Basic Medical College of Beijing University of Chinese Medicine, China); centrifuge 5810R (Eppendorf Company, Germany); VIP RIA Kits (Beijing Huanyataike Biomedical Technology Co., Ltd., China).

1.2 Animals and groups

Wistar rats [male, SPF-class, (200±20) g] were supplied by Xinglong Experimental Animal Breeding Factory of Haidian District, Beijing [SCXK-(JUN) 2007-004]. All rats were housed at (22±2) ℃ and humidity (60±5)% with free access to food and water. The study was performed in accordance with the guidelines approved by the Animal Ethics Committee of Beijing University of Chinese Medicine. After adaptive feeding for 3 d, the rats were randomly divided into four groups: a normal group, a model group, a Chize (LU 5) group and a Shangjuxu (ST 37) group, 7 in each group.

1.3 Modeling

The UC rat model was established by improved acetic acid method[8]. All rats fasted for 24 h but had free access to water before operation. After anesthetized with urethane, the rats were kept upside down. Rats in the model group, Chize (LU 5) group and Shangjuxu (ST 37) group were slowly injected with 10% acetic acid at 10 mL/(kg·bw) into the anus with a PVC tube of 2 mm in diameter, while rats in the normal group were injected with normal saline at 10 mL/(kg·bw). The solution was retained in the gut cavity at a depth of 8 cm, and then the rats were inverted by lifting their tails for 20 s.

1.4 Acupoints and interventions

The points Chize (LU 5) and Shangjuxu (ST 37) were located according to theExperimental Acupuncture Science[9].

The interventions began on the third day after successful modeling. The normal group and model group only received fixed for 15 min once daily for 7 d. The Chize (LU 5) group and Shangjuxu (ST 37) group were given EA at Chize (LU 5) (depth: 3 mm) and Shangjuxu (ST 37) (depth: 5 mm) respectively with Han’s nerve-acupoint stimulator at 2 Hz/100 Hz and 0.2-0.4 mA for 15 min, once daily for 7 d.

1.5 Microcirculation observation

After all interventions, each rat was narcotized with 20% urethane at 7 mL/(kg·bw) injected into its lateral thigh muscle. Then they were fixed on the operation table in a supine position and made a 1-2 cm incision along the ventral midline. Get the mesentery near to the ileocecum, spread it in a physiological saline mesentery observation box at 37 ℃. Switch on the microcirculation microscopic tester and select a mesenteric micro-vessel at a length of 20-100 μm with a concomitant artery-vein and without tanglesome vessels around to make observation. Record changes of the mesenteric microvascular calibers and collect the static image of microvascular blood flow status with ×10, ×20, and ×40 microscope and dynamic image with ×20 and ×40 microscope every 10 min. Make observation and records for 4 times in a row and get the average data. The blood flow status was graded on the basis of semi-quantitative determination of velocity classification method[10].

The grades criteria and their features are as follows. Grade 3: fastest blood flow, presenting as a smooth line strips and without particles. Grade 2: relatively faster blood flow, a few or even obvious particles. Grade 1: slow blood flow, presenting as sediment form. Grade 0: stasis of blood flow or even invisible.

1.6 Sample preparation and VIP measurement

The colon of over 3 cm from the anus, right lung and hypothalamus of each rat were separated and washed by iced saline. Cut each part at approximately 200 mg, and then put them into three slurry pipes with iced saline at 1:4 respectively. Grind the tissues on the ice tillcompletely mixed with the iced saline. Get them centrifuged at 4℃, 3 500 r/min for 15-20 min, then collect the supernate and store them at －20℃. VIP in the colon, lung and hypothalamus were measured by radioimmunoassay according to the VIP kit instructions.

1.7 Statistical analysis

2 Results

2.1 General condition

The normal group rats were in a good condition. The UC rats had loose stools with waste residue around the anus, accompanied by lassitude and a poor appetite since the third day after modeling. After the 7 d interventions, stools of the model group rats were still pasty, while this symptom was improved in Chize (LU 5) group and Shangjuxu (ST 37) group. As to Chize (LU 5) group rats, loose stools almost disappeared. Shangjuxu (ST 37) group rats had no loose stools and presented a good condition with good appetite and spirit.

2.2 Mesenteric microcirculations analysis

2.2.1 Mesenteric microvascular calibers

Compared with the normal group, the calibers were significantly expanded in the model group and Chize (LU 5) group (P＜0.05); there was no significant difference between the model group and Chize (LU 5) group (P＞0.05); compared with the model group and Chize (LU 5) group, the calibers were obviously shrunk in Shangjuxu (ST 37) group (P＜0.05), (Table 1 and Figure 1).

2.2.2 Blood flow status

The four groups showed no significant inter-group differences (P＞0.05), (Table 1).

2.3 Distribution of VIP in the colon, lung and hypothalamus

The colonic VIP levels in the model group and Chize (LU 5) group were significantly higher than that in the normal group (P＜0.01,P＜0.05); the level in Shangjuxu (ST 37) group was markedly lower than that in the model group (P＜0.01). VIP levels in lung and hypothalamus seemed no significant inter-group differences (P＞0.05), (Table 2).

Table 1. Comparison of mesenteric microvascular calibers and blood flow status

Figure 1. Variation of mesenteric microvascular caliber (×20)

Table 2. Distribution of VIP in different tissues

Table 2. Distribution of VIP in different tissues

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

Group n Colon Lung Hypothalamus Normal 7 2.34±0.35 3.08±0.58 4.45±0.37 Model 7 4.08±0.352) 2.32±0.75 4.38±0.26 Chize (LU 5) 7 3.36±0.191) 2.57±0.68 5.17±0.52 Shangjuxu (ST 37) 7 2.64±0.343) 2.76±0.70 5.03±0.45

3 Discussion

UC is a form of inflammatory bowel diseases (IBD), and its main clinical symptoms include frequent small-volume diarrhea with urgency and blood[11]. Among various UC modeling methods, acetic acid modeling is frequently used for its convenience, short modeling period, high success rate and presents with similar acute symptoms and colonic pathology to UC in human. In this study, after anal injection of aceticacid, UC rats had loose stool. In our another related study[12], the colonic pathology showed that the gland structures of colonic epithelium were damaged. The mesenchyme appeared obvious edema and congestion with heavy neutrophil infiltration invasion. This infers that anal injection of acetic acid has successfully and rapidly induced UC models.

The microcirculation mainly refers to the blood/fluid flow status of arterioles capillaries, lymphatic and tissue spaces. The microcirculation has a close correlation with the transmission of intercellular materials, information and energy[13]. Acupuncture can significantly affect the microcirculations of brain, nail fold, gastro-intestine, uterus[14]. The variation of mesenteric microcirculation can vividly show the differences of intestinal pathology among conditions of normal, UC and post-EA. This study found that the mesenteric microvascular calibers were significantly expanded after UC modeling, corresponding with the vessel variation in acute inflammation. Compared with the model group, the calibers of Shangjuxu (ST 37) group were significantly shrunk, while Chize (LU 5) group seemed no obvious change. We can infer that Shangjuxu (ST 37) helped relieve the change of mesenteric microvascular calibers along with the inflammation pathology, while Chize (LU 5) cannot. There was no significant difference in the blood flow status among the four groups. While in our previous study[7], both Chize (LU 5) and Shangjuxu (ST 37) could markedly reduce the fast blood flow of mesenteric microcirculation in COPD rats. This difference between our COPD and UC studies may be related to different disease models and disease inflammation processes. So, it indicates that Shangjuxu (ST 37) has a better regulation effect on mesenteric microcirculation than Chize (LU 5) in primary bowel diseases.

VIP is one of the most important neuropeptides in intestine[15]. Besides intestine, VIP exists in many organs and VIP receptors are specially expressed in the lung and brain[16-17]. VIP has a strong vasodilator function and can inhibit the peristaltic reflex in the circular muscle layer and control intestinal blood flow[18-19]. It possesses powerful anti-inflammatory effects and participates in intestinal mucosal immunity[15,20]. In this study, the colonic VIP increased in the model group, serving as a self-protective reaction in acute inflammation, and it corresponds with the trend of mesenteric microvascular caliber change. VIP in Shangjuxu (ST 37) group was significantly lower than that in the model group, and was also associated with the microvascular caliber change. VIP in Chize (LU 5) group showed a decline trend, but without a significant difference. It also prompts that Shangjuxu (ST 37) has a better anti-inflammatory effect on UC. It was found in our another related study[12]that both Chize (LU 5) and Shangjuxu (ST 37) significantly down-regulated colonic tumor necrosis factor (TNF)-α level in UC rats. So, the effect of Chize (LU 5) could not be denied. Chize (LU 5) might show relative lag effects on anti-inflammatory. These different effects may be associated with the different nerve segments[21]. The colonic nerve segment locates at the level of T6-L3, Shangjuxu (ST 37) at the level of L4-S2and Chize (LU 5) at C5-C6. The nerve segments of colon and Shangjuxu (ST 37) are relatively near, so Shangjuxu (ST 37) has a quicker nerveendocrine-immune system response than Chize (LU 5)[22].

‘The correlation between acupoints and Zang-fu organs’ is one of the basic contents of acupuncture theories. It’s been proven that an acupoint can treat different diseases of Zang-fu organs, meanwhile different acupoints can used for the same disease of Zang-fu organs[23-24]. This shows that acupoints should have relative specificity in treatment. The results of this study indicate that Shangjuxu (ST 37) should have better benefits in treating UC than Chize (LU 5). It can be concluded that when diarrhea results from intestine itself, the efficacy of Shangjuxu (ST 37, the lower He-Sea point of large intestine) seems better than that of Chize (LU 5, the He-Sea point of the Lung Meridian). The lower He-Sea point can treat its corresponding diseases of Fu organ and the He-Sea point can treat diarrhea caused by adverse flowing qi. Chize (LU 5) belongs to Water in the Five Elements, so it has the kidney characteristic for the five Zang organs and the Five Elements are correlated with each other. The kidney dominates the transportation and distribution of water from the stomach. If the kidney fails to transform qi, the water will go down and stay in the intestine, and diarrhea will appear. Therefore, both Chize (LU 5) and Shangjuxu (ST 37) can treat diarrhea, while Shangjuxu (ST 37) tends to work better for diarrhea caused by intestine itself such as UC, and Chize (LU 5) tends to diarrhea caused by disturbance of kidney qi.

In this study, VIP in lung and hypothalamus showed insignificant inter-group differences, which is different from our COPD study[7]. This might be associated with the modeling method. Even though acetic acid modeling could present with similar human acute UC symptoms and pathological changes[25], the complex pathogenesis of UC and the chronic recurrent characteristics could not be reflected. The complex pathogenesis might involve the interactions among genes, environment, microorganism and the immune system[26]. Therefore, the lung and hypothalamus were not influenced yet in a short time. In the further study, the modeling methods need more careful consideration.

In conclusion, our results suggest that the effects of Chize (LU 5) and Shangjuxu (ST 37) are different in treating UC. Shangjuxu (ST 37) showed better efficacies in reducing VIP in the colon and regulating mesentericmicrocirculation, while the effects of Chize (LU 5) were not significant. The results of our COPD[7]study and this study jointly support that acupoints have relative specificity in treatment.

Conflict of Interest

There was no conflict of interest in this article.

Acknowledgments

This work was supported by the Independent Subject Project of Beijing University of Chinese Medicine for Postgraduate (北京中医药大学在读研究生自主选题项目, No.2011-JYBZZ-XS099).

Statement of Informed Consent

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

Received: 25 December 2014/Accepted: 28 January 2015

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电针尺泽及电针上巨虚对溃疡性结肠炎大鼠的影响比较

目的：比较电针尺泽(肺经合穴)与上巨虚(大肠下合穴)对溃疡性结肠炎(ulcerative colitis, UC)大鼠肠系膜微循环及结肠、肺、下丘脑中血管活性肠肽(vasoactive intestinal peptide, VIP)的不同影响, 并探讨腧穴相对特异性规律。方法：将28只Wistar雄性大鼠随机分为正常组、模型组、尺泽组及上巨虚组, 每组7只。采用乙酸灌肠法制备UC模型。灌肠后第3天起, 尺泽组与上巨虚组每日分别电针尺泽穴、上巨虚穴15 min, 连续治疗7天后取材。微循环仪观察肠系膜微血管管径及血液流态变化; 放射免疫法测定结肠、肺及下丘脑VIP含量。结果：模型组肠系膜微循环管径较正常组显著扩大(P＜0.05); 模型组与尺泽组无显著差异(P＞0.05); 上巨虚组较模型组、尺泽组显著缩小(P＜0.05)。各组血液流态无统计学差异(P＞0.05)。模型组、尺泽组结肠VIP含量较正常组显著升高(P＜0.01,P＜0.05)；上巨虚组较模型组显著下降(P＜0.05)。各组肺、下丘脑VIP含量无显著差异(P＞0.05)。结论：尺泽穴和上巨虚穴对UC效应不同, 上巨虚穴能降低UC大鼠结肠VIP, 改善肠道微循环, 尺泽穴效应不明显。

针刺疗法; 电针; 穴, 尺泽; 穴, 下巨虚; 结肠炎, 溃疡性; 微循环; 血管活性肠肽

R2-03 【

】A

Author: Luo Qin, 2012 master degree candidate

Xie Jie-ping, M.D., professor.

E-mail: xiejieping@sina.com.

Methods: A total of 28 male Wistar rats were randomized into a normal group, a model group, a Chize (LU 5) group and a Shangjuxu (ST 37) group, 7 rats in each group. The UC model was established by enema with acetic acid. Since the third day after modeling, rats in the Chize (LU 5) group and Shangjuxu (ST 37) group respectively received EA at Chize (LU 5) and Shangjuxu (ST 37), 15 min each time for successive 7 d. The variations of mesenteric microvascular calibers and blood flow status were observed by a microcirculation microscopic tester; VIP in the colon, lung and hypothalamus was measured by radioimmunoassay.

Results:Compared with the normal group, the mesenteric microvascular calibers were significantly expanded in the model group (P＜0.05); there was no significant difference between the model group and Chize (LU 5) group (P＞0.05); compared with the model group and Chize (LU 5) group, the calibers were obviously shrunk in Shangjuxu (ST 37) group (P＜0.05). The four groups showed no significant inter-group differences in comparing blood flow status (P＞0.05). The colonic VIP levels in the model group and Chize (LU 5) group were significantly higher than that in the normal group (P＜0.01,P＜0.05); the VIP level in Shangjuxu (ST 37) group was markedly lower than that in the model group (P＜0.01). There were no significant differences among the four groups in comparing VIP level in lung and hypothalamus (P＞0.05).

Conclusion:The effects of Chize (LU 5) and Shangjuxu (ST 37) were different in treating UC. Shangjuxu (ST 37) showed a more significant efficacy in down-regulating VIP in the colon and regulating mesenteric microcirculation, while the effects of Chize (LU 5) were not obvious.