Effect of electro-acupuncture on basic fibroblast growth factor protein and mRNA expression in hippocampal dentate gyrus of spleen 
deficiency rats*☆
Zhuoxin Yang1, Yuanyuan Zhuo2, Haibo Yu1, Min Pi1, Mumin Shao3

1Department of Acupuncture and Moxibustion, Shenzhen Hospital of Traditional Chinese Medicine, Shenzhen  518033, Guangdong Province, China
2Shenzhen Hospital of Traditional Chinese Medicine, Guangzhou University of Chinese Medicine, Shenzhen  518033, Guangdong Province, China
3Department of Pathology, Shenzhen Hospital of Traditional Chinese Medicine, Shenzhen  518033, Guangdong Province, China

Zhuoxin Yang☆, Doctor, Professor, Chief physician, Doctoral supervisor, Department of Acupuncture and Moxibustion, Shenzhen Hospital of Traditional Chinese Medicine, Shenzhen  518033, Guangdong Province, China

Corresponding author: Zhuoxin Yang, Department of Acupuncture and Moxibustion, Shenzhen Hospital of Traditional Chinese Medicine, Shenzhen  518033, Guangdong Province, China
qinyuan64@yahoo.com.cn

Yuanyuan Zhuo and Zhuoxin Yang contributed equally to this study.

Supported by: Science and Technology Plan Program of Social Development of Guangdong Science and Technology Department in 2007, No. 73127*

Abstract
BACKGROUND: Spleen deficiency in traditional Chinese medicine refers to the functional disorder of spleen, pancreas, intestines, and nervous system in modern medicine.
OBJECTIVE: To test whether electro-acupuncture could alter basic fibroblast growth factor (bFGF) protein and mRNA expression in the hippocampal dentate gyrus of spleen deficiency rats.
DESIGN, TIME AND SETTING: The randomized, controlled, in vivo animal experiment was performed at the National Level-B Laboratory of Clinical Cell Molecule and Biology in Shenzhen Hospital of Traditional Chinese Medicine, between March and November in 2008.
MATERIALS: Reserpine injection was produced by Guangdong Bangmin Pharmaceutical Co. Rhubarb extract granule preparation was produced by Guangdong Yifang Pharmaceutical. Huanqiu Brand sterile acupuncture pin was provided by Suzhou Acupuncture Supplies, China. Huatuo Brand electroacupuncture instrument (type SDZ-II) was purchased from Suzhou Medical Appliance Factory, China.
METHODS: A total of 96 male Sprague Dawley rats were randomly assigned to control (n = 32) and induction (n = 64) groups. Spleen deficiency was induced via intraperitoneal injection of reserpine and intragastric administration of rhubarb. The successful models were randomized into two groups: model and electro-acupuncture, with 32 rats in each group. Elec-tro-acupuncture was administered at Zusanli (ST 36) and Sanyinjiao (SP 6) acupoints using a condensation wave and rarefaction (condensation wave 15 Hz) at a strength of 6–15 V for 20 minutes, once per day. The appearance of a slight shiver in the corresponding locus was taken as the standard. According to electro- acupuncture time points, each group was assigned to four subgroups at 7, 14, 28, and 49 days, respectively, with eight rats in each subgroup. Im-munohistochemical staining, image analysis, and reverse-transcription polymerase chain reac-tion were performed at different time points.
MAIN OUTCOME MEASURES: bFGF protein and mRNA expression in the hippocampal den-tate gyrus of spleen deficiency rats.
RESULTS: After 7 days of electro-acupuncture therapy, bFGF protein and mRNA expression significantly increased compared with the model and control groups (P < 0.05). After 14 days, bFGF protein and mRNA expression decreased until 28 days, where levels were then equal to the model group and greater than the control group (P < 0.05). After 49 days, the above indices remained increased in the electro-acupuncture group compared to the model and control groups (P < 0.05).
CONCLUSION: Continuous electro-acupuncture maintained a high level of bFGF protein and mRNA expression in the hippocampal dentate gyrus of spleen deficiency rats.
Key Words: spleen deficiency; electro-acupuncture; basic fibroblast growth factor; mRNA; protein; nerve factor; neural regeneration

INTRODUCTION
  
In traditional Chinese medicine, the spleen is associated with functions described as being of the of spleen, pan-creas, intestines, and nervous system in modern medi-cine[1]. Neuroendocrinological studies have demon-strated that more than 20 types of gut peptides can also be detected in brain tissues. Results have shown a close correlation between the nervous system and the gastro-intestinal tract in terms of source and function[2]. Accord-ing to the Chinese medicine theory, spleen deficiency may alter cerebral neurotransmitters, neurotrophic fac-tors, signal transductions, and gene expression[3-10]. Spleen deficiency can influence brain functions by in-ducing changes in brain tissues, such as the cortex and hippocampus, as a result of cellular differentiation and proliferation and tissue reconstruction capacity[11]. It is important to understand whether recovery of cerebral function can be promoted by rectifying spleen deficiency symptoms and improving spleen deficiency.
Some traditional Chinese medicine studies have dem-onstrated that the spleen is closely associated with neu-rophysiology and neuropathology[12], thereby suggesting an integral theory for traditional Chinese medicine. However, few reports have focused on the relationship between the spleen and brain development, in particular whether splenic functions can be used as a reliable index for brain developmental states.
Acupuncture therapy has been shown to induce basic fibroblast growth factor (bFGF) and mRNA expression, as well as promote the production of new neurons[13-17]. Accordingly, this study aimed to observe changes in bFGF protein and mRNA expression in the hippocampal dentate gyrus of spleen deficiency rats following elec-tro-acupuncture to Zusanli (ST 36) and Sanyinjiao (SP 6) acupoints.

MATERIALS AND METHODS

Design
Randomized, controlled, in vivo experiment.
Time and setting     
The experiment was performed at the National Level-B Laboratory of Clinical Cell Molecule and Biology in Shenzhen Hospital of Traditional Chinese Medicine, between March and November in 2008.
Materials
A total of 96 healthy, male, Sprague Dawley rats, weigh-ing (50 ± 5) g and aged 4 weeks, were offered by Guangdong Provincial Experimental Animal Center (Li-cense No. 2008A003). All rats were housed at 26 °C and 60% humidity, with free access to food and water. All experimental protocols were in accordance with Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[18].
Main reagents and instruments are as follows:

Methods
Grouping and model establishment
A total of 96 male Sprague Dawley rats were randomly assigned to control (n = 32) and induction (n = 64) groups. Spleen deficiency was induced using the multiple factor complex method[19] via intraperitoneal injection of reserpine and intragastric administration of rhubarb.
The successfully established models were randomized into two groups: model and electro-acupuncture, with 32 rats in each group.
According to electro-acupuncture time points, the groups were assigned to four subsets at 7, 14, 28, and 49 days, respectively, with eight rats in each subset.
Intragastric administration of rhubarb was modified by staff from the Spleen Institute of Guangzhou University of Traditional Chinese Medicine. Rhubarb extract granule preparations were soaked and completely dissolved in equal volumes of hot water. Each rat was administered 10 mL/kg rhubarb extract, once per day, for 2 weeks. Intraperitoneal injection of reserpine was initially admin-istered as follows: rats were treated with      0.15 mL/kg reserpine once daily after 1–9 days of model estab-lishment, followed by 0.25 mL/kg at 10–12 days once per day, and 0.5 mL/kg at 13–14 days, twice per day.  
The control group was treated with physiological saline using the same methods and equal volumes.
Model success was dependent upon symptoms of rat spleen deficiency[20]: (1) severe diarrhea or even prolapse of anus; (2) anorexia; (3) poor appetite, weight loss; (4) somnolent, poor hair quality; (5) crouched; (6) lethargy. Of these, severe diarrhea or even prolapse of anus and anorexia are primary symptoms, while the others are accompanying symptoms. The appearance of two primary and accompanying symptoms indicated successful model establishment (Figure 1).

Point location and acupuncture
Electro-acupuncture was administered at the Zusanli (ST 36) and Sanyinjiao (SP 6) acupoints, according to loca-tions of Experimental Acupuncture Science[21]. Zusanli (ST 36): 5 mm below the capitulum fibulae of the hind limb knee joint; Sanyinjiao (SP 6): 10 mm above inner malleolus tip of hind limb. Bilateral acupuncture was performed at a wave of condensation and rarefaction (condensation wave 15 Hz) and strength of 6–15 V for 20 minutes, once per day.
Specimens
The rats were anesthetized with 3.5% chloral hydrate via peritoneal injection, and brain coronal slices containing hippocampus were cut into two pieces for immunohisto-chemistry and reverse-transcription RT-PCR determina-tion. Immunohistochemical stained specimens were fixed in 4% paraformaldehyde for      6 hours at 4 °C, and RT-PCR reaction specimens were stored in TrizoL Re-agent at –80 °C until further use.
Immunohistochemistry
The embedded paraffin tissues were cut into 1-μm con-tinuous slices and were baked at 80 °C for 24 hours. Following a series of procedures, including xylene de-waxing, gradient ethanol hydration and tap water wash-ing, the slices were boiled in citrate antigen retrieval so-lution (pH 6.0). The glass slides were allowed to cool to room temperature and were rinsed three times with phosphate-buffered saline (PBS). The sections were incubated in rabbit anti-bFGF polyclonal antibody over-night at 4 °C. The following day, the slices were washed three times with PBS for 5 minutes each time. Each slice was incubated with 50 μL MaxVision? reagent at room temperature for 20 minutes, followed by three PBS rinses for 5 minutes each. Each slice was supplemented with 100 μL fresh DAB solution, then observed under light microscopy for 1–2 minutes. The sections were then rinsed with tap, counterstained with hematoxylin, and rinsed with tap water. The sections were then processed by gradient ethanol hydration, xylene transparency, and mild gum mounting.
Image analysis
Image analysis was performed using Leica DC software digital image analysis system software package. One brain slice was selected from each rat, and 5 adjacent visual fields were randomly selected from the same loci of hippocampal dentate gyrus under 200 × light micros-copy. bFGF-positive cells were then quantified. 
RT-PCR
Homogenized tissue was mixed with 1 mL Trizol reagent and 200 μL chloroform. The solution was shaken vigor-ously by hand for 15 seconds and centrifuged    (13 000 r/min) for 15 minutes at 4 oC, followed by precipita-tion of RNA with 0.5 mL isopropyl alcohol per   1 mL Trizol Reagent via vortex and centrifugation for   10 minutes. The white precipitate was the RNA.
RNA was used as a template to synthesize the first chain using the RT-PCR kit and MyCycler PCR instrument. Equal volumes of brain tissue total RNA (2 μg) were combined with 1 μL oligo(dT) primer and water to a final volume of 12 μL. The solution was heated at 70 °C for 5 minutes, then 4 μL RT buffer, 2 μL 10 mmol/L dNTP, 1 μL RNA enzyme inhibitor, and water were added to a final volume of 19 μL, followed by incubation at 70 °C for 5 minutes. A total of 1 μL AMV RT was added and the sample was incubated at 42 °C for 60 minutes, followed by 10 minutes at 70 °C for elongation. The samples were then stored on ice. 
Primer source: rat bFGF gene mRNA sequence was obtained from NCBI, designed by Primer 5, and synthe-sized by Shanghai Invitrogen Biotechnology. The primer sequences are as follows: 

  
  
bFGF: Basic fibroblast growth factor.

PCR reaction was performed using rat cDNA as a tem-plate, and cycle parameters are as follows: 94 °C pre-degeneration for 2 minutes, 94 °C degeneration for 30 seconds, 58 °C annealing for 30 seconds, 72 °C elongation for 45 seconds, for a total of 32 cycles. The samples were then incubated at 72 °C for an additional 10 minutes, followed by storage at 4 °C. PCR primers and internal reference GAPDH primers were designed and synthesized by Shanghai Invitrogen Biotechnology, China.
Semiquantitative analysis of RT-PCR products
A total of 6 μL amplification products from each specimen were electrophoretically separated on a 1% agarose gel containing GV nucleic acid dyes. The DNA Marker served as a reference of standard relative molecular mass for the gel image system and absorbance scanning, as well as GAPDH internal standard correction and rela-tive amount analysis. The ratio of two absorbances was used to determine the final result.
Main outcome measures
The bFGF protein and mRNA expression in the hippo-campal dentate gyrus of spleen deficiency rats at differ-ent time points.
Statistical analysis
Measurement data were analyzed using STATA 10.0 statistical software (Computer Resource Center, USA) and were expressed as Mean ± SD. Measurement data were processed into completely randomized one-way analysis of variance, and homogeneity of variance was tested by Levene according to the standard of α = 0.10. For homogeneity variance, the least significant difference t-test was used, and a level of P < 0.05 was considered statistically significant. For non-homogeneity variance, the Cochran & Cox similar t-test was used, and a level of P < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of experimental analysis
A total of 96 rats were included in the final analysis without loss.
Influence of electro-acupuncture on bFGF expres-sion in the hippocampal dentate gyrus of spleen de-ficiency rats
bFGF-positive cells were observed with a yellow or brown cytoplasm.
Electro-acupuncture group: There were more bFGF-positive cells than the control group at 7, 14, 28, and 49 days (P < 0.05). However, the elec-tro-acupuncture group exhibited increased numbers of bFGF-positive cells compared with the model group only at 7 and 49 days (P < 0.05) (Figures 2, 3).
Control group: There were no significant differences in the number of positive cells between the various time points (Figures 2, 3).
Electro-acupuncture group: numbers of bFGF-positive cells were highest at 7 days, but were dramatically at-tenuated by 14 days (P < 0.05). There was no significant difference in cell numbers between 28, 49, or 14 days (Figures 2, 3).  
Following model establishment, numbers of bFGF-positive cells were lowest at 7 days and signifi-cantly increased by 14 days (P < 0.05). There was no significant difference between 14 and 28 days, but cell numbers significantly decreased between 28 days and 49 days (P < 0.05, Figures 2, 3).

Expression of bFGF mRNA in the hippocampal den-tate gyrus of the different rat groups
Compared with the control group, bFGF mRNA expres-sion in the hippocampal dentate gyrus was greater in the electro-acupuncture group at 7, 14, 28, and 49 days (P < 0.05), and was greater than the model group at 7 and 49 days (P < 0.05) (Figure 4). bFGF mRNA was expressed in the hippocampal dentate gyrus of all groups, as shown in Figure 5.
Control group: There were no significant differences in bFGF mRNA expression between the various time points (Figures 4, 5).
Electro-acupuncture group: bFGF mRNA expression levels were highest at 7 days, but were attenuated by 14 days (P < 0.05). There was no significant difference in bFGF mRNA expression between 28 days, 49 days, or 14 days (Figures 4, 5).  
Model group: bFGF mRNA expression was lowest at 7 days and significantly increased by 14 days (P < 0.05). The expression decreased between 28 days and 49 days (Figures 4, 5).

DISCUSSION

The mechanism of reserpine-induced spleen deficiency depends on reserpine, a peripheral sympatholytic agent, to prevent amine pumps from taking up noradrenalin and blocking monoamine oxidase metabolism. As a result of dysfunctional noradrenalin re-uptake, transmitter syn-thesis is reduced, catecholamines are accordingly ex-hausted, gangliated nerve function decreases, and parasympathetic nerve function accentuates, ultimately resulting in diarrhea[22]. Therefore, intraperitoneal injec-tion of reserpine ensures reliable and reproducible spleen deficiency models.
Studies have shown that bFGF promotes tissue regen-eration, provides neuronal nutrition, and facilitates de-velopment and differentiation of embryonic tissue[23-24]. In addition, it has been used to maintain neuron behavior[25]. bFGF has neuroprotective properties, provides nutrition for glial cells, and promotes neovascularization[26-28].
Results from the present study demonstrated that elec-tro-acupuncture therapy increases bFGF protein and mRNA expression, thereby playing a positive role in protection and repair of neurons in spleen deficiency young rats.
To investigate the modulation effect of elec-tro-acupuncture treatment on bFGF in spleen deficiency young rats, the present study utilized the Sanyinjiao (SP 6) acupoint, which is the Spleen Channel of Foot-Taiyin and the convergent acupoint of the three Yin meridians of the foot. This acupoint is associated with spleen-stomach weakness, strengthening spleen and replenishing qi, warming the middle warmer and reinforcing the defi-ciency. The spleen and stomach, in combination with the Zusanli (ST 36) acupoint of the stomach meridian, can enhance the therapeutic effect on spleen deficiency. Studies have demonstrated that the clinical effect of the Sanyinjiao (SP 6) acupoint corresponds with modulation and controlling functions in brain regions where glucose metabolism is altered following acupuncture[29]. Elec-tro-acupuncture at the Zusanli (ST 36) acupoint activates the brain regions associated with cognition in the pre-frontal lobe or temporal lobe[30]. Results from the present study demonstrated that Sanyinjiao (SP 6) elec-tro-acupuncture exhibited an influence on brain func-tions[31].
The mechanisms of bFGF protection and repair of cerebral neurons are not entirely understood. The effect of electro-acupuncture on brain development in spleen deficient rats can be further investigated using double-labeling methods.

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 (Edited by Yang XF, Tang Y/Yang Y/Song LP)