Effect of Moxibustion on the Motility, Absorption and Activities of ATPase in Small Intestine of Spleen-deficiency Rats

Peng Yan, Yi Shou-xiang, Peng Fen, Long Yi-wen, Lin Ya-ping, Chang Xiao-rong, Xu Bin

1 Major Laboratory of Meridians and Viscera, Tertiary Laboratory of State Administration of Traditional Chinese Medicine, Institute of Acupuncture, Moxibustion and Massage, Hunan University of Traditional Chinese Medicine, Changsha 410007, China

2 Traditional Chinese Medicine Department of Nanjing University of Chinese Medicine, Nanjing 210046, China

BASIC STUDY

Effect of Moxibustion on the Motility, Absorption and Activities of ATPase in Small Intestine of Spleen-deficiency Rats

Peng Yan1, Yi Shou-xiang1, Peng Fen1, Long Yi-wen1, Lin Ya-ping1, Chang Xiao-rong1, Xu Bin2

1 Major Laboratory of Meridians and Viscera, Tertiary Laboratory of State Administration of Traditional Chinese Medicine, Institute of Acupuncture, Moxibustion and Massage, Hunan University of Traditional Chinese Medicine, Changsha 410007, China

2 Traditional Chinese Medicine Department of Nanjing University of Chinese Medicine, Nanjing 210046, China

Author: Peng Yan, M.D., associate professor

Objective: To observe the effect of moxibustion on intestinal propulsion rate, content of serum D-xylose, content of adenosine triphosphate (ATP) and activities of adenosine triphosphatases (ATPase) in jejunum tissues of spleen-deficiency rats, and to explore the effect of moxibustion on the production of ATP and activities of membrane proteins, for analyzing the mechanism of moxibustion in reinforcing the spleen-stomach.

Methods: Forty healthy Sprague Dawley rats were randomly divided into four groups, namely group A (blank control group), group B (model control group), group C (moxibustion group) and group D (herbs group). The animal model of spleen-deficiency was established by intragastric administration withDa Huang(Radix et Rhizoma Rhei) infusion. The rats in group C received moxibustion at Zusanli (ST 36), Zhongwan (CV 12), Guanyuan (CV 4), Pishu (BL 20) and Weishu (BL 21). The rats in group D received intragastric administration withSi Jun Zidecoction. The intestinal propulsion was measured by toner method, and the content of D-xylose in serum was detected by phloroglucinol method. Colorimetry method was used to detect the content of ATP and activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in jejunum tissues.

Results: Compared with group A, the intestinal propulsion rate, content of D-xylose in serum, content of ATP, activities of Na+-K+-ATPase and Ca2+-Mg2+-ATP in jejunum tissues decreased significantly in group B (P＜0.05 orP＜0.01). Compared with group B, the intestinal propulsion rate, content of D-xylose in serum, content of ATP, activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in jejunum tissues increased significantly in group C and D (P＜0.05 orP＜0.01), while there were no significant differences between group C and group D.

Conclusion: Moxibustion can increase the intestinal propulsion rate, content of D-xylose in serum and ATP in jejunum tissues, as well as the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase of spleen-deficiency rats, which suggests that moxibustion can enhance the motility and absorption functions of small intestine by promoting the production of ATP in intestinal epithelial cells, increasing the content of membrane proteins, and improving transmembrane transport.

Moxibustion Therapy; Spleen Deficiency; Adenosine Triphosphate (ATP); Adenosine Triphosphatases; Rats

Spleen deficiency syndrome refers to a group of complicated symptoms caused by various physiological functional insufficiency of the spleen,mainly manifested by disorders of digestion, absorption and nutrition. Moxibustion is an effective treatment for spleen-stomach disorders because it has the warming-supplementing effect. Modern physicians have explored the warming-supplementing effect of moxibustion on spleen-stomach disorders from different aspects, and found that moxibustion can improve the movement and absorption of gastrointestinal tract and regulate the activities of digestive enzymes to improve spleen deficiency syndrome[1-3]. The basic function of the spleen, governing transportation and transformation, is important to the digestion and absorption of nutrients. From the view of modern medicine, a variety of nutrients in the food, including water, are absorbed in the small intestine. After food is digested to be simple molecules, these molecules are transported to various cells of the body through blood. The transmembrane transport, i.e. the transportation of these substances into the cell membrane or mitochondrial membrane, is mediated by a series of relevant membrane proteins. In spleen-deficient conditions, the production of adenosine triphosphate (ATP) decreases, and the contents or activities of related membrane proteins change, which are manifested by decreased digestion and absorption of intestine. Therefore, the effects of moxibustion on the intestinal propulsion rate and content of D-xylose, ATP production and content of ion pump of jejunum tissue in the spleen-deficiency rats were observed in this study, to explore the relationship among warming-supplementing effect of moxibustion, transmembrane transport, digestive and absorptive functions of jejunum.

1 Materials and Methods

1.1 Materials and apparatuses

Moxa stick (Wolong Hanyi Moxa Factory of Nanyang); toner (Chemical Plants of Taishan City, Guangdong); gum arabic (Sigma company), D-xylose (Guangfu Fine Chemical Research Institute of Tianjin), phloroglucinol (Fuchen Chemical Reagent Factory of Tianjin); advanced micro amount of ATP enzyme test kits (Nanjing Jiancheng Bioengineering Institute); ATP content assay kit (Nanjing Jiancheng Bioengineering Institute).

Si Jun Zidecoction:Ren Shen(Radix Ginseng) 9 g,Bai Zhu(Rhizoma Atractylodis Macrocephalae) 9 g,Fu Ling(Poria) 9 g, andGan Cao(Radix Glycyrrhizae) 6 g, boiled in water, and concentrated to decoction containing 1 mL of crude drugs[4].

1.2 Grouping

Forty healthy Sprague Dawley rats were randomly assigned into four groups, namely group A (blank control group), group B (model control group), group C (moxibustion group), and group D (herbs group), 10 in each group.

1.3 Modeling

The spleen-deficiency rat model was established by intragastric administration with 200%Da Huang(Radix et Rhizoma Rhei) infusion at 10 mL/(kg·BW) each time, twice a day, for 14 d. The rats in group A were given distilled water via intragastric administration.

1.4 Interventions

Group A: The rats in group A were only restricted without other treatments.

Group B: After modeling, the rats in group B were restricted without any other treatments.

Group C: After modeling, the rats in group C were treated by moxibustion at Zhongwan (CV 12), Guanyuan (CV 4), unilateral Zusanli (ST 36), Pishu (BL 20), and Weishu (BL 21). The acupoints were located according to theExperimental Acupuncture Science[5].

Group D: After modeling, the rats in group D were givenSi Jun Zidecoction via intragastric administration at 6 mL/(kg·BW) for 14 d.

At the end of the experiments, all the rats were fasted for 24 h. Serum was collected for D-xylose detection and jejunum tissues for ATP and ATPase detection on the 15th day.

1.5 Determination of intestinal propulsion rate

After fasting for 24 h, the rats in each group were orally administered with 10% toner suspension (10% activated carbon and 10% gum arabic) at l mL/100 g. The rats were sacrificed 30 min later and the intestine was collected. The length from the front end of toner to pyloric sphincter (L1) and the length from pyloric sphincter to the end of small intestine (L2) were measured. The ratio between L1 and L2 indicated gastrointestinal propulsion.

Intestinal propulsion rate ＝ L1 ÷ L2 × 100%.

1.6 Determination of D-xylose content

The blood was collected from abdominal aorta 1 h after the rats were administered with 3 g/l00 mL D-xylose at 0.2 mL/(kg·BW) via gavage. The clear supernatant liquid was separated by centrifugation at 3 000 rpm at 4 ℃ for 15 min after laying aside for 2 h to 3 h at room temperature. The content of D-xylose in serum was detected by phloroglucinol method.

1.7 Determination of ATP, Na+-K+-ATPase andCa2+-Mg2+-ATPase

The jejunum tissues of 400 mg were collected and rinsed with ice saline. Samples of jejunum tissues were homogenized and treated with the test kits according to the manufacturers’ instructions.

1.8 Statistical analysis

The SPSS 16.0 version software was used for data analyses. All data were expressed asMultiple-group comparisons were performed by using One-way ANOVA. The Least Significant Difference (LSD) and Student-Newman-Keuls (SNK) tests were used when the variances were equal, and Tamhane’s T2 or Dunnett’s T3 tests were used when the variances were not equal.P＜0.05 indicated a significant difference.

2 Results

2.1 Intestinal propulsion rate and D-xylose content in serum

The intestinal propulsion rate and content of D-xylose in group B were significantly lower than those in group A (P＜0.05,P＜0.01), suggesting decreased intestinal motility and absorption in the spleen-deficiency rats. The intestinal propulsion rate and content of D-xylose in group C and D were significantly higher than those in group B (P＜0.05 orP＜0.01). There were no significant differences between group C and D. It suggests that moxibustion can promote intestinal propulsion and increase the content of D-xylose in serum to improve the digestive function of spleen-deficiency rats, and the effect is equal to that ofSi Jun Zidecoction (Table 1).

2.2 ATP in jejunum tissues

The content of ATP, activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in group B were significantly lower than those in group A (P＜0.05), suggesting that the production of ATP is decreased in jejunum tissues of spleen-deficiency rats. The content of ATP, activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in group C and D were significantly higher than those in group B (P＜0.05 orP＜0.01). There were no significant differences between group C and D. It suggests that moxibustion andSi Jun Zidecoction can promote the production of ATP, improve the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in jejunum tissues (Table 2).

Table 1. Comparison of intestinal propulsion rate and serum D-xylose

Table 1. Comparison of intestinal propulsion rate and serum D-xylose

Note: Compared with group A, 1) P＜0.05, 2) P＜0.01; compared with group B, 3) P＜0.05, 4) P＜0.01

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Table 2. Comparison of ATP, Na+-K+-ATPase and Ca2+-Mg2+- ATPase in jejunum tissues

Table 2. Comparison of ATP, Na+-K+-ATPase and Ca2+-Mg2+- ATPase in jejunum tissues

Note: Compared with group A, 1) P＜0.05; compared with group B, 2) P＜0.05, 3) P＜0.01

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3 Discussion

Moxibustion is an external therapy using moxa as the primary material to heat, burn, and/or fumigate acupoints or other specific areas on the surface of the body. Through the functions of the meridians and acupoints, moxa warms and dredges the meridians and vessels, assists healthy qi, expels evil qi, and regulates the body's physiological functions, thus achieving the goals of treating disease and cultivating health. Moxibustion is an effective treatment for spleen-stomach disorders because it has the warming and supplementing effect. Clinically, spleen-deficiency syndrome is often treated by moxibustion[1-3,6-8]. Spleen-deficiency syndrome is mainly manifested by dysfunction of digestion, peristalsis and absorption.SiJun Zidecoction is often used for treatment of spleen-deficiency syndrome and has definite curative effect, so it was applied to the rats in group D as a control treatment in this study. Moxibustion can warm meridians to dissipate cold, activate yang qi to resolve dampness, dispel stasis to dissipate nodules, and promote the flow of qi to relieve pain. Moxibustion with proper acupoints can regulate qi movement of the spleen and stomach. Clinically, the acupoints used for moxibustion to warm and supplement the spleen and stomach are as follows. The first group should be Zhongwan (CV 12), Qihai (CV 6) and Zusanli (ST 36)[1,6], which are the basic points for supplementing the spleen suggested by Luo Tian-yi, a medical expert from Yuan Dynasty. The second group should be the Front-Mu and Back-Shupoints of spleen and stomach, such as Pishu (BL 20), Zhangmen (LR 13), Weishu (BL 21), and Zhongwan (CV 12)[7-8]. In this study, Zusanli (ST 36), Zhongwan (CV 12), Guanyuan (CV 4), Pishu (BL 20) and Weishu (BL 21) were chosen. Moxibustion at the five acupoints can tonify and replenish the spleen and stomach.

The most important and basic function of spleen is transportation and transformation, closely related to the gastrointestinal movement and intestinal absorption. There were various reports on gastrointestinal motility of spleen-deficiency animals. Hu LL, et al found the enhanced gastric emptying and intestinal propulsion in spleen-deficiency rats induced respectively by overstrain, intragastric administration withDa Huang(Radix et Rhizoma Rhei) infusion and exhausting swimming, and considered it caused by gastrointestinal hyperactivity in deficient conditions[9]. While Tao L, et al found that the gastric emptying and intestinal propulsion rate of spleen-deficiency rats decreased[10]. Gastrointestinal motility is very important to digestion and absorption of intestine, hyper- or hypo- gastrointestinal motilities are not conducive to digestion and absorption. In our study, the intestinal propulsion rate decreased in spleendeficiency rats induced by intragastric administration withDa Huang(Radix et Rhizoma Rhei) infusion, indicating the decreased gastrointestinal motility.

D-xylose is a kind of pentose which is diffused and absorbed in the duodenum and jejunum after oral administration. D-xylose is a water-soluble monosaccharide which often excretes as prototype from urine, and there is no detectable xylose in blood in general cases[11]. Therefore, determination of content of D-xylose in urine or blood can reflect the superficial area and absorptive function of the small intestine[12-13]. The previous researches showed that the urine-D-xylose excretion rate in patients and animals of spleen-deficiency syndrome all decreased to varying degrees[14-15], and the content of D-xylose in blood serum in the animals of spleen-deficiency syndrome decreased, too[16]. The results of this study showed that the content of D-xylose in blood serum decreased in the model control group, which is consistent with the previous researches. The serum D-xylose concentration in the moxibustion group was significantly higher than that in the model control group, which suggests that moxibustion can improve the absorptive function of small intestine in spleen-deficiency rats, and there was no significant difference between the effects of moxibustion andSi Jun Zidecoction.

Food is digested in the small intestine to simple molecules and transported to cells through blood. The cell membrane is a selective permeability barrier between the cells and the extracellular environment. The transmembrane transport of the substance is essential to the survival and growth of cells, and closely related to energy transformation and the generation of cells. This process requires energy supplied by ATP and is mediated by membrane proteins[17]. ATP is the most primary and direct energy supply of living activities, and also as a bridge linking the energy production, conversion, storage and utilization of cells. Two-thirds of the total cellular energy consumption is used in the material transport. The process that the substances get through the cell membrane or mitochondrial membrane (transmembrane transport) also needs a series of associated membrane proteins to mediate. ATPase, also known as the ion pump, is an enzyme that hydrolyzes ATP to ADP and mediates the transmembrane transport of ions by using the energy stored in high-energy phosphate bond. The ubiquitous ion pump in mammalian cell membrane is the sodium-potassium pump (Na+-K+-ATPase). Material transmembrane transport is directly driven by the ion electrochemical concentration gradient on both sides of the membrane. The active transport of sugars and amino acids in many tissues also depends on the reasonable distribution of Na+and K+. In addition, Na+-K+-ATPase can regulate calcium concentration, thereby controlling the excitability of glands and cells to regulate the secretion by sodium-calcium exchange[18]. Calcium pump, also known as Ca2+-ATPase, is also an active transport supplied by the energy from ATP. The Ca-dependent ATPase operates as electrogenic Ca2+/H+and Mg2+exchanger[19]. Ca2+-Mg2+-ATPase activity reduction can cause ion transmembrane transport obstacles and intracytoplasmic Ca2+accumulation, which leads to abnormal cell morphology, structure and functions[20]. The study on substance transmembrane transport in patients and animals with spleen-deficiency syndrome focused on the activities of sodium-potassium pump and calcium pump. The study found that the intestinal absorption of nutrients, such as carbohydrates and proteins, decreased in the spleen-qi-deficiency state. The carbohydrates, protein and other nutrients are mainly absorbed by the small intestinal epithelium, and Na+-K+-ATPase provides the conditions for this transepithelial absorption. The study found less activity, decreased body temperature, weakened intestinal absorptive function and decreased Na+-K+-ATPase activity of liver in model rats with cold dampness and spleen-deficiency syndrome; while increased activity and body temperature, enhanced intestinal absorptive function and increased Na+-K+-ATPase activity of liver were found in model rats with intestinal heat and bowel excessive syndrome. It suggests that Na+-K+-ATPase activity is closely related to the intestinal absorptive function[21].Peng C, et al found that the Na+-K+-ATPase activity of gastrointestinal cell membrane decreased in spleen-deficiency model rats induced by vinegar. Water molecules entered the cell membrane due to osmotic pressure caused by intracellular shift of Na+, which caused gastrointestinal cell swelling and damage of the cell structure. It might be the molecular foundation of the spleen-deficiency syndrome[22]. Wang CX, et al found that the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase of erythrocyte membrane in spleen-qi-deficiency, spleen-yin-deficiency and spleen-yang-deficiency young rats were lower than those in normal rats. The activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase of erythrocyte membrane decreased more obviously in deficient aged rats compared with young rats. It suggests that oxidative and anti-oxidative imbalances, including accumulation of a large number of free radicals, increased peroxidation, destroyed biological membrane structure and decreased activities of membrane proteins (enzymes, receptors, etc.) result in the abnormal material transport and energy conversion in spleen-deficiency animals[23]. Reduced energy supplied by ATP also affects the material transport. Guo WF, et al found that the intestinal digestive and absorptive functions changed in spleen-deficiency rats, accompanied by the reduction of the ATP concentration in intestinal mucosa[24], suggesting that the material transmembrane active transport is affected. The results of our study also showed that the decreased activities of membrane proteins and energy supplied by ATP can cause transmembrane transport obstacles.

In summary, the spleen-deficiency syndrome is mainly manifested by decreased gastrointestinal digestive and absorptive functions. In the process of absorption, transmembrane transport is essential to the survival and growth of cells, and is closely related to energy conversion and the generation of cells. The process requires ATP to supply energy and membrane proteins to mediate. Our study showed that the content of D-xylose in serum, the content of ATP, the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in jejunum tissues decreased in the model control group, while moxibustion increased the content of D-xylose in serum, the content of ATP, the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in jejunum tissues. The results suggest that moxibustion can warm and supplement the spleen and stomach to relieve spleen-deficiency syndrome by increasing ATP generation of jejunum tissues to provide sufficient energy for transmembrane transport, and by regulating the content of membrane proteins to promote intestinal absorptive function.

Conflict of Interest

The authors declare that there is no conflict of interest.

Acknowledgments

The study was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Open Project Program of Traditional Chinese Medicine Department of Nanjing University of Chinese Medicine (No. YS2012ZYX414); Subject of Hunan Education Department (No.11C0965); Hunan University Innovation Fund Project Open Platform (No.12K088).

All the procedures in this study were approved by the Laboratory Animals Management Committee of Hunan University of Chinese Medicine.

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Xu Bin, M.D., professor.

E-mail: xuuuux@sina.com

R2-03

: A

Date:December 20, 2013