Effects of different doses of ginger-partitioned moxibustion on trefoil factor 1, mucin 5AC and epidermal growth factor receptor in rats with spleen deficiency syndrome

Bi Ding-yan (賁定嚴(yán)), Ning Jiong-jie (寧炯杰), Xu Yin (徐寅), Luo Yan (羅燕), Li Mu-qing (李木清) ,Wang Yun-hui (王云輝), Yi Zhan (易展)

1 Hunan University of Chinese Medicine, Changsha 410208, China

2 The First Hospital of Hunan University of Chinese Medicine, Changsha 410007, China

3 Hunan Academy of Chinese Medicine, Changsha 410006, China

Spleen deficiency syndrome is one of the common syndromes of traditional Chinese medicine. It is often seen in gastrointestinal mucosal diseases such as peptic ulcer and chronic gastritis. Studies have found that rats with spleen deficiency syndrome show damaged or shedding gastric mucosa, mucosal focal erosion,different degrees of reduction of gastric main cells,parietal cells and gastric glands, as well as the compensatory state of gastric parietal cell ultrastructure[1]. Therefore, some people think that spleen deficiency syndrome is the integration of multi-system and multi-functional abnormality based on the characteristics of digestive system morphology and dysfunction; gastrointestinal mucosal lesions and their corresponding dysfunction may be one of the pathological basis of spleen deficiency syndrome.

Studies have shown that trefoil factor 1 (TFF1) has the role in protection of gastric mucosa and repair of the injury[2-4]. On one hand, it plays a physiological function through the combination with Mucin 5ac(MUC5AC) ligand; on the other hand, it’s involved in the reconstruction of gastric mucosal injury by inducing the epidermal growth factor receptor (EGFR) mediated signaling pathway. A large number of clinical studies have shown that acupuncture treatment has exact therapeutic efficacy on deficient cold of spleen and stomach, and the role of moxibustion therapy is more obvious[5]. Our previous study showed that gingerpartitioned moxibustion could not only improve the clinical symptoms of patients with spleen deficiency syndrome, but also had antioxidant effect[6]. Focused on TFF1, our current study explored the protective effect and the dose-effect characterization of gingerpartitioned moxibustion on the gastric mucosa in rats with spleen deficiency syndrome on the basis of our previous studies, therefore, to provide evidence for the clinical application of ginger-partitioned moxibustion in treatment of spleen deficiency syndrome.

1 Materials and Methods

1.1 Animals and groups

Seventy-five SPF grade Sprague-Dawley (SD) rats,weighing 200-250 g, were purchased from Hunan SJA Laboratory Animal Co., Ltd., China [license: SCXK(Hunan) 2011-0003]. Rats were randomly divided into a blank control group (group A), a model group (group B),a 3 moxa-cone ginger-partitioned moxibustion group(group C1), a 6 moxa-cone ginger-partitioned moxibustion group (group C2) and a 9 moxa-cone ginger-partitioned moxibustion group (group C3), 15 rats in each group by random number table method.Disposal of experimental rats followed the guidance of theGuiding Opinions on the Treatment of Experimental Animalsissued by the Ministry of Science and Technology.

1.2 Reagents and instruments

Moxa stick (Nanyang Wolong Chinese medicine moxa factory, China); ginger slices (2-3 mm in thickness, 1.8 cm in diameter); EGFR kit (Proteintech Group, Inc., USA);paraffin, neutral gum (Sigma, USA); hematoxylin, PBS buffer, citrate buffer, two-step kit (Wellbio Ltd., USA);DAB kit (Beijing Zhongshan Jinqiao Biotechnology Co.,Ltd., China); conventional chemical reagents (Shanghai Sinopharm Biomedical Co., Ltd., China); shaker(Kylin-bell Instrument Manufacturing Co., Ltd., China);incubator (Beijing 61 Instrument Factory, China);microwave oven (Midea Group, China); slicers (Lycra,Germany); slicing machine (Zhejiang Jinhua City Yi Di Medical Equipment Co., Ltd., China); embedding machine (Changzhou Zhongwei Electronic Instrument Co., Ltd., China); ordinary refrigerator (Hefei Rongshida Sanyo Electric Co., Ltd., China); precision PH meter(Leici-Shanghai Instrument Technology Instrument Co.,Ltd., China); microscope (Motic Mike Audi Industrial Group Co., Ltd., China); coverslips and slides (Haimen Yuantai Experimental Equipment Factory, China).

1.3 Modeling and model evaluation

Models were prepared according to the method developed by Peng Y and others[7]: except rats in group A, all rats in other four groups received intragastric administration of 4 ℃ and 200% concentratedDa Huang(Radix et Rhizoma Rhei) [10 mL/(kg·bw)], twice a day for 14 continuous days. The body weight, diet and water consumption, coat color, stools, mental status and behavior change were observed.

Evaluation criteria of spleen deficiency syndrome animal model were made according to the literature[7].Main symptoms: diarrhea or prolapse of anus; eat less or anorexia. Secondary symptoms: ematiation, weight loss; dispirited appearance, loose limbs, coat haggard;twisted and stacking together; easy fatigue. Spleen deficiency syndrome model was successful when two main symptoms and two secondary symptoms appeared simultaneously (Table 1).

1.4 Acupoints

Point positioning was conducted according to the method normally used for animal and personification control method based onExperimental Acupuncture Science[8]. The navel is the intersection of the lower 1/4 and upper 3/4 on the line between sternoclavicular symphysis and pubic symphysis.

Zusanli (ST 36): At back lateral of the knee joint, and about 5 mm below the capitulum fibulae.

Zhongwan (CV 12): At midpoint on the line between the umbilicus and xiphoid process, about 20 mm above the navel.

1.5 Treatment and sampling

Group A: Rats were not subject to modeling, with a 15 min bundling each day but without moxibustion for 8 continuous days.

Group C1: Rat’s hair was shaved (around 2.0 cm ×2.0 cm) at bilateral Zusanli (ST 36) and Zhongwan (CV 12)to expose local skin tissue, and the points were disinfected with ethanol treated cotton ball. Rats were fixed in a supine position with a self-made rat clip, and subjected to moxibustion treatment when they were still. Fixed the ginger slices (2-3 mm in thickness, 1.8 cm in diameter) with moxa cones on the acupoints, 3 cones daily (about 15 min) for constant 8 d.

Group C2: The treatment before moxibustion,materials and acupoints used for moxibustion were all same as in group C1. Ginger-partitioned moxibustion was performed for 6 cones (about 30 min) each day for 8 continuous days.

Group C3: The treatment before moxibustion,materials and acupoints used for moxibustion were all same as in group C1. Ginger-partitioned moxibustion was performed for 9 cones (about 45 min) each day for 8 continuous days.

Rats in each group were fasted for 24 h and deprived of water for 6 h at the end of treatment. Blood samples were collected after anesthesia using 10% urethane(10 mL/kg·bw). After the belly was opened, stomachus pyloricus and gastric cardia were ligated with hemostatic forceps, then the esophagus and duodenum were cut off to separate the whole stomach. The stomach was opened along the arcus major ventriculi,and the stomach residue was rinsed with iced saline. On the super-clean bench, 1.0 cm × 0.5 cm size of tissue was sampled from the gastric mucosa appearing obvious injury, rinsed with saline, and fixed with 4%paraformaldehyde at 4 ℃ for 24 h. The 5 μm thick slices were used for enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry assay.

1.6 Observation items

1.6.1 General symptom observation

The stools, mental state and behavior changes were observed; the body weight, food intake and pulling tail resistance duration were recorded; and the score was quantified according to the syndromes of animal models with spleen deficiency syndrome[7]and the total points were calculated before the experiment, on the 14th and the 22th experimental day respectively.

With the two symptoms of loose stools and less diet,and the total score ≥4 points indicated the successful spleen deficiency modeling. The score ≤5 points was mild degree, 6 to 10 points was moderate degree, ＞10 points was severe degree.

Table 1. Quantitative score table for symptoms of animal model with spleen deficiency syndrome

1.6.2 Serum TFF1 and MUC5AC

Serum TFF1 and MUC5AC were determined by ELISA.

Coated antibody: the specific antibody globulin was diluted with a coating buffer to the optimal concentration, and added into wells by 0.3 mL/well;kept at 4 ℃ overnight or 37 ℃ water bath for 3 h,then stored in refrigerator. Wash: removed the coating buffer and washed the wells with washing buffer for 5 min × 3 times. 0.2 mL of the TFF1 or MUC5AC containing buffer was respectively added to each well and incubated at 37 °C for 1-2 h. Wash: removed the buffer and washed the wells with washing buffer for 5 min × 3 times. Added 0.2 mL of the enzyme labeling specific antibody solution diluted with dilution buffer and incubated at 37 °C for 1-2 h. Wash: removed the buffer and washed the wells with washing buffer for 5 min × 3 times. Added 0.2 mL of substrate solution to each well, incubated at room temperature for 30 min(for the blank control, 0.4 mL substrate and 0.1 mL terminator). Adding terminator: added 0.05 mL 2 mol/L H2SO4or 2 mol/L citric acid per well. Observation of the results: visual observation or measured the integral optical density (IOD) value with a standard colorimeter at 492 nm [substrate: o-phenylenediamine (OPD)].

1.6.3 EGFR protein

The EGFR protein was determined by immunehistochemistry.

The slices were heated at 60 ℃ for 45 min; dewaxed to water: in xylene for 10 min × 2 times, then in 100%,95%, 85% and 75% ethanol for 5 min, and washed with distilled water for 5 min; repair of the antigen by heating: immersed the slices in 0.01 mol/L citrate buffer(pH 6.0) and heated to boiling by electric furnace or microwave oven and switched off the power, repeated twice with an interval of 5-10 min; washed with 0.01 mol/L PBS (pH 7.2-7.6) for 3 min × 3 times after cooling. Added 3% H2O2, incubated at room temperature for 10 min to inactivate endogenous enzyme, and washed 3 min × 3 times with PBS.Incubation with primary antibody: added appropriate dose of diluted EGFR primary antibody, incubated at 4 ℃ overnight, rinsed 5 min × 3 times with PBS;incubation with secondary antibody: added 50-100 μL anti-rabbit IgG antibody-HRP polymer, incubated at 37 ℃ for 30 min, washed with PBS for 5 min × 3 times.

DAB color: added 50-100 μL pre-prepared DAB working solution, incubated at room temperature for 1-5 min, and controlled the reaction time under microscope, and washed with distilled water.Counterstained with hematoxylin for 5-10 min × 2 times and rinsed with distilled water, blued with PBS;dehydrated with 60%, 70%, 80%, 90% and 100% alcohol,5 min for each concentration. Exposed to xylene for 10 min × 2 times, and sealed with neutral gum.Observed under microscope, and analyzed using Image-Pro-Plus image analysis software.

1.7 Statistical analysis

The SPSS 21.0 statistical software was used for data processing and analysis. Normally distributed measurement data were expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparison among groups. The least significant difference (LSD) and Student-Newman-Keuls (SNK)Q-test were used for data with homogeneous variance;Tamhane's T2 or Dunnett's T3 was used for data with heterogeneous variance; pairedt-test was used for self-comparison before and after intervention. Ranksum test was used if the data did not fit the normal distribution.P＜0.05 indicated that the difference was statistically significant.

2 Results

2.1 Comparison of syndrome scores in rats with spleen deficiency syndrome among groups

After modeling, in addition to rats in group A, all the other rats showed a slow increase of body weight,decreased appetite and activity, curled up, with loose stool and dull hair; the syndrome scores of spleen deficiency in group B, C1, C2 and C3 were higher than those before modeling, as well as in group A (P＜0.01),suggesting that the model was successful. After treatment with ginger-partitioned moxibustion, the symptoms of spleen deficiency were not obvious in group C1, C2 and C3, and the syndrome scores of spleen deficiency were lower than those of group B (allP＜0.01).

Compared with group C1, the syndrome scores of spleen deficiency were lower in rats of group C2 and C3(P＜0.01), but there was no significant difference in the syndrome score of spleen deficiency between group C2 and C3 (P＞0.05),(Table 2).

Table 2. Comparison of syndrome scores of spleen deficiency among groups (x±s, point)

2.2 Comparison of serum TFF1 and MUC5AC levels

After the intervention, the levels of serum TFF1 and MUC5AC in the other groups were significantly higher than those in group A (P＜0.01). Compared with the model group, the levels of serum TFF1 and MUC5AC in group C1, C2 and C3 were increased after treatment with ginger-partitioned moxibustion (P＜0.01).Compared with group C1, the levels of serum TFF1 and MUC5AC in the group C2 and C3 were increased(P＜0.01), while the difference was not statistically significant between group C2 and C3 (P＞0.05). This indicated that there was no significant difference in the regulation of serum TFF1 and MUC5AC levels between the 6 and 9 moxa-cone ginger-partitioned moxibustion groups (Table 3).

Table 3. Comparison of serum TFF1 and MUC5AC levels among groups (x±s, ng/mL)

2.3 Comparison of EGFR protein expression in gastric mucosa

At the end of the intervention, the EGFR protein expression in gastric mucosa of the other groups was significantly higher than that of group A (allP＜0.01);compared with group B, the EGFR protein expression in rat gastric mucosa of group C1, C2 and C3 was increased(P＜0.01).

EGFR protein expression in gastric mucosa of group C2 and C3 was higher than that in group C1 (P＜0.01),but there was no significant difference between group C2 and C3 (P＞0.05), indicating no significant difference in regulation of EGFR protein expression in gastric mucosa between 6 and 9 moxa-cone ginger-partitioned moxibustion (Table 4).

Table 4. Comparison of EGFR protein expression in gastric mucosa of rats among groups (x±s)

3 Discussion

Gastric mucosal injury is the main pathological factor leading to acute and chronic gastritis, gastric ulcer and other diseases, mainly reflected by the increased gastric mucosal attack factors and decreased gastric mucosal self-protection/defense, while the gastric mucosal injury is closely related to both the pathological process and syndrom of spleen deficiency[9]. With the establishment of the diagnostic standard of spleen deficiency syndrome, various animal models of spleen deficiency were successfully replicated. At home and abroad, many institutes have confirmed that feeding rhubarb decoction to animals is the most stable method to establish of the spleen deficiency model[10], after study and exploring of the functional changes,physiological and biochemical changes, as well as the immunocompromice.

Chinese medicine indicates that ‘a(chǎn) strong spleen helps to defend against invasion of pathogen’,suggesting that the spleen and stomach are closely related to the defensive function, meanwhile, spleen and stomach are intrinsically linked with the digestive system, especially the local defensive function of the gastric mucosa.

Therefore, it should focus on the spleen and stomach to correct the pathological basis of spleen and stomach deficiency, and improve the gastric mucosal selfprotection/defensive function during the treatment.

Moxibustion is one of the external therapies in traditional Chinese medicine. The warm stimulation by moxibustion can produce a good warm and steady effect, thus playing a role in disease prevention and treatment[11]. Ginger-partitioned moxibustion is the comprehensive treatment combining pungent-warm and moving nature of ginger with dispelling cold to circulate blood and warm to dredge the meridian of moxibustion. Ginger and moxibustion produce synergies to complement each other. Experiments have confirmed that ginger partitioned moxibustion could significantly improve the general symptoms of experimental spleen deficiency rats, and correct the indiscriminate plasma β-endorphin (β-EP), motilin (MTL)and somatostatin (SS) levels in rats with spleen deficiency, which was better than conventional mild moxibustion[10]. Moxibustion must reach a certain dose to produce the best therapeutic effect. And the moxibustion time is an important factor affecting the dose of moxibustion, therefore, to explore the effect of different dose of moxibustion in ginger-partitioned moxibustion is essential to improve the clinical efficacy.

TFF1 is a class of small molecules secreted by the gastrointestinal tract, and its specific site can be bound by the carbohydrate chain of the mucin to form a stable gel complex and stabilize the gastrointestinal slime layer,thus to play an important role in protection and repair of the gastrointestinal tract[12]. Ren JL,et al[13]pointed out that TFF1 expression was higher in human peptic ulcer and drug-induced rabbit gastric ulcer, suggesting that it plays an important role in gastric mucosal protection and epithelial reconstruction. Li TL,et al[14]found that moxibustion treatment could heal the gastric ulcer caused by spleen deficiency by increase of the TFF1 expression level and repair of the damaged gastric mucosa. MUC5AC is a family of mucin, and secreted by epithelial cells. The main role of MUC5AC is to maintain lubrication of the cavity organs (such as the gastrointestinal tract, respiratory lumen). MUC5AC plays an important role during the repair of the damaged gastrointestinal mucosal barrier, and is also an important biological marker for predicting the development, progression and prognosis of gastric cancer[15]. EGFR has a small amount of expression in normal gastric mucosa epithelial cells, intrinsic membrane cells and mucosal muscle cells. Of which,the distribution is the most in the gastric mucosal epithelial cells, playing a very important role during the repair of gastric mucosal injury. EGFR expression is increased during the gastric mucosal injury[16]. Liu XL,et al[17-18]showed that MUC5AC and EGFR protein expressions were significantly decreased, when the self-defense ability of the gastric mucosal was decreased in rats with spleen deficiency.Bu Zhong Yi Qidecoction can improve the rat spleen syndrome and stimulate the mechanism of gastric mucosal reconstruction, thus to achieve gastric mucosal repair,by increasing the expression of MUC5AC and EGFR proteins.

The results of this study showed that the serum TFF1 and MUC5AC levels, as well as the expression of the EGFR protein in the gastric mucosa of the model group were increased in the successful rat model of spleen deficiency, which indicated that the gastric mucosa had some self-repair function when it was damaged by initiation of cell proliferation. It’s similar to the findings of Li TL,et al[14], while it’s different from that of Liu XL,et al[17-18]. The reason may be related to the duration of intragastric administration of concentratedDa Huang(Radix et Rhizoma Rhei), which was 4 d less in the study conducted by Liu XL,et al[17]than our current study, and may not reach enough stimuli to induce gastric mucosal self-repair function. In addition, this may also be related to the quality of herbs, constitution of the experimental animals, the physical existence of a certain relationship,which are to be further validated in the future experiments. The levels of serum TFF1 and MUC5AC, as well as the EGFR protein in the gastric mucosa were further increased in rats of each group after ginger-partitioned moxibustion at Zusanli (ST 36) and Zhongwan (CV 12), therefore, we inferred that gingerpartitioned moxibustion promoted the proliferation and repair of gastric mucosal injury in rats with spleen deficiency syndrome, by up-regulation of TFF1 expression and binding with the specific mucin MUC5AC, as well as co-activation of EGFR.

At the same time, the results showed that the levels of TFF1 and MUC5AC, as well as the expression of EGFR protein in gastric tissue were higher in the 6 and 9 moxa-cone ginger-partitioned moxibustion groups versus the 3 moxa-cone ginger-partitioned moxibustion group, suggesting that in a certain range, the proliferation and repair of gastric mucosa will be enhanced with the dose increase of moxibustion.However, the effect was similar in 6 and 9 moxa-cone ginger-partitioned moxibustion groups, indicating that it is not that the greater the amount of moxibustion, the stronger the effect.

In summary, ginger-partitioned moxibustion can promote the proliferation and repair of gastric mucosa in rats with spleen deficiency syndrome. The mechanism may be related to the increase of serum TFF1 and MUC5AC levels, as well as activation of EGFR protein expression. Within a certain range, the proliferation and repair of gastric mucosa will be enhanced with the dose increase of moxibustion.

Conflict of Interest

The authors declared that there was no potential conflict of interest in this article.

This work was supported by Fund Project of Hunan Province Education Office (湖南省教育廳科研計(jì)劃項(xiàng)目,No.13C685); Graduate Student Research Innovation Project of Hunan Province (湖南省研究生科研創(chuàng)新項(xiàng)目,No. CX2016B351); Undergraduate Student Research Innovation Project of Hunan Province (湖南省大學(xué)生科研創(chuàng)新項(xiàng)目, No. 201409060207).

Statement of Human and Animal Rights

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

[1] Duan YQ, Cheng WD, Cheng YX, Yang XY, Li LZ, An YR,Tian R, Wang Y. Investigation of syndrome mode connotation for the ‘spleen deficiency syndrome body generalization effect’ based on the principle of spleen deficiency syndrome. Zhongguo Zhongyiyao Xinxi Zazhi,2013, 20(7): 3-5.

[2] Cheng YM, Lu MT, Yeh CM. Functional expression of recombinant human trefoil factor 1 byEscherichia coliand Brevibacillus choshinensis. BMC Biotechnol, 2015, 15(1):1-13.

[3] Rao J, Yang XJ, Fan DM, Chen WQ, Wang JH. Effect the treatment ofJianpi Qingre Huayudecoction with gastric precancerous lesions and its influence on mucin 5AC protein expression. Zhongguo Shiyan Fangjixue Zazhi,2014, 20(4): 183-187.

[4] Dahlhoff M, Gerhard M, Rad R, Lindén S, Wolf E,Schneider MR. A new mouse model for studying EGFR-dependent gastric polyps. Biochim Biophys Acta,2012, 1822(8): 1293-1299.

[5] Yi Z, Chang XR, Yi SX, Yan J, Xie H, Lin YP, Song J,Peng F. Evidence-based medicine analysis of clinical studies survey on the acupuncture-moxibustion treatment of spleen-stomach vacuity cold. Zhonghua Zhongyiyao Xuekan, 2010, 28(9): 1866-1868.

[6] Yi Z, Liu M, Chang XR, Yan J, Xie H, Wang DJ, Ai K, Liu WA. Influence of serum SOD and MDA by moxibustion on ginger therapy for chronic superficial gastritis spleenstomach vacuity cold patients with different time.Zhongguo Shiyan Fangjixue Zazhi, 2012, 18(23): 301-304.

[7] Peng Y, Peng F, Yi SX, Lin YP, Chang XR, Long YW,Zhang HG. Effect of moxibustion on motility, absorption and content of ATP in small intestine of spleen-deficiency rats. Zhongguo Zhen Jiu, 2012, 32(3): 246-250.

[8] Li ZR. Experimental Acupuncture Science. 2nd Edition.Beijing: China Press of Traditional Chinese Medicine,2007: 253.

[9] Shi T, Zhu FS, Xu TT. The correlation analysis between spleen deficiency and gastric mucosal injury. Yixue Yu Zhexue (Linchuang Juece Luntan Ban), 2011, 32(12):59-60.

[10] Tan J, Chang XR, Yan J, Yi SX, Lin YP, Yue ZH, Liu M,Peng Y. Effect of moxibustion on plasma levels of β-EP,MTL and SS in rats with spleen deficiency. Shijie Huaren Xiaohua Zazhi, 2011, 19(34): 3498-3502.

[11] Chang XR, Liu M, Yan J, Yi SX, Yue ZH, Zhang GS, Liu ML, Sun GJ, Wang LL, Hu L, Wu HG. Research on mechanisms and principles of warm-unblock and warmtonic effects on moxibustion. Shijie Zhongyiyao, 2013,8(8): 875-879.

[12] Sun Y, Wang LX. The research progress of trefoil factor family. Changwai Yu Changnei Yingyang, 2015, 22(1): 49-52.

[13] Ren JL, Lu YP, Wang L, Chen JM, Shi HX, Ye ZS, Wu YH,Zhong Y, Lin XT, Lin H, Pan JS, Luo JY. Express change of TFF1 in normal and injured gastric mucosa. Shijie Huaren Xiaohua Zazhi, 2003, 11(11): 1809-1810.

[14] Li TL, Su YM, Qi F, Ni W, Zhang H, Zhang YC, Guo H.Effect of moxibustion on serum TFF and gastric mucosa ERK1/2 and PCNA in gastric ulcer model rats with spleen deficiency symptom. Hunan Zhongyiyao Daxue Xuebao,2015, 35(2): 49-51.

[15] Song J, Guo RF, Su RL, Qin EP, Wang JL. Expressions and significance of TFF2, CLDN18 and MUC5AC protein in gastric mucosal lesions. Wei Chang Bing Xue, 2015, 20(7):394-397.

[16] Zhang H, Guo H, Zhang YC, Liu M, Ai K, Su YM, Li MH,Li TL. Effect of moxibustion intervention on expression of gastric epidermal growth factor receptor and extracellular signal regulated kinase 1/2 expression in rats with gastric ulcer. Zhen Ci Yan Jiu, 2014, 39(5): 351-357.

[17] Liu XL, Wang RJ, Zhao QQ. Effect on expressions of MUC5AC mRNA and protein in gastric mucosa ofBu Zhong Yi Qidecoction for rats with spleen deficiency.Zhongyao Yaoli Yu Linchuang, 2012, 28(4): 7-9.

[18] Liu XL, Wang RJ, Zhang QQ. Effect on EGFR protein expression in gastric mucosa ofBu Zhong Yi Qidecoction for rats with spleen deficiency. Zhongyao Yaoli Yu Linchuang, 2012, 28(5): 12-14.