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Effects Of Bloodletting Therapy On HSP70 And
IL-1 In Adjuvant Arthritic Rat Local Diseased Area
Yuxing Liu, OMD (MD)
Zheng Zeng, OMD (MD)
Fanrong Liang, OMD (MD)
Yi Fu, MS
Jie Chen
Xuezhi Li, OMD (MD)
ABSTRACT
Background Bloodletting therapy has been used for its pain-relieving and heat-reducing effects when treating adjuvant arthritis; however, the mechanism of these effects has not been studied.
Objective To explore the peripheral mechanism of reducing heat by bloodletting.
Design, Setting, and Subjects Thirty-two arthritic model rats were divided into 4 groups: control, model, bloodletting, and electroacupuncture. Acupoint Kunlun (BL 60) was selected. Heat shock protein 70 (HSP70) and interleukin-1 (IL-1) were measured by radioimmunoassay.
Main Outcome Measure Change in HSP70 and IL-1 levels by group.
Results The HSP70 content in the model group was much higher than that in the control group. There was no statistically significant difference among the bloodletting group, electroacupuncture group, and control group (for all, P>.05). The IL-1 levels in the inflammation local area of the model group was much higher than that of the control group; IL-1 levels in the bloodletting and electroacupuncture groups were also higher than that of the control group, but lower than levels in the model group; IL-1 in the bloodletting group did not differ significantly from that of electroacupuncture group.
Conclusion Bloodletting can relieve inflammation damage and protect the cell. It may reduce heat and pain through its regulating action on the content of HSP70 and IL-1 in the local diseased area. Further studies are needed to verify our findings and pinpoint the mechanism of action.
KEY WORDS
Adjuvant Arthritic, Bloodletting Therapy, HSP70, Heat Shock Protein 70, IL-1, Interleukin-1
INTRODUCTION
Bloodletting therapy is an ancient yet unique acupuncture therapy of Traditional Chinese Medicine (TCM). It has been used to treat more than 100 different diseases in internal medicine, surgery, pediatrics, and gynecology. Bloodletting therapy has been used for its pain-relieving and heat-reducing effects when treating adjuvant arthritis; the mechanism of these effects has not been studied.
We focused on heat shock protein 70 (HSP70) and interleukin-1 (IL-1) and observed the effects and mechanism of bleeding therapy on rheumatoid arthritis.
STUDY DESIGN AND METHODS
Selection of Animals
For our study, we selected 32 adult Sprague-Dawley rats, weighing a mean (SD) of 220 (20) g, aged 3-4 months, with an equal number of male and female animals.
The rats were provided by the Experiment Animal Center of Cheng Du University of TCM. They were fed for a week after adoption, during which the environmental temperature was maintained around 20° C and humidity 70%.
Animals were treated humanely and ethically, conforming to current practices.
Major Experimental Drugs
We used an IL-1 enzyme-linked immunosorbent assay (ELISA) kit (provided by Jingmei Biotech Co); Anti-HSPs (k-20), sc-10-60 goat polyclonal IgG at 200 mg/mL (provided by Santa Cruz Biotech Inc). Corresponding 2 antibody (provided by Peking-Zhongshan Biotech Co) was also used.
Group Division
Animals were randomly divided into 4 groups (8 animals in each group): normal control group, model group, bloodletting group, and electroacupuncture group.
Normal control group. Rats did not undergo intervention but were fastened with clips for 3 times longer than that done to the bloodletting group. Each time lasted for 20 minutes.
Model group. One day prior to the experiment, rats were injected with 0.1 mL of Freund's adjuvant at the pad of the right posterior foot toward the ankle. The rest of the manipulations were the same as in the normal control group.
Bloodletting group. One day prior to the experiment, the rats underwent intevention as those in the model group, then these rats were fastened by rat clips and during the experiment, pricked Acupoint Kunlun (BL 60)1 with a 3-edged needle at the diseased area of the rats. Each time, 1 drop of blood was released, once per day for a total of 3 times.
Electroacupuncture group. Modeled one day prior to the experiment. After the rats were fastened, we selected needles of 0.5 cun, punctured 0.3 to 0.5 cun into BL 60 at the right posterior foot, and then added the needles with a WQ-10 multifunctional electroacupuncture machine and twined the tail root with a reference electrode that had been dipped into the physiological saline. The device functioned with loose-dense wave, frequency: 20-100 HZ; voltage: 2-40; the intensity was increased from a small value until the posterior feet twitched. This treatment was repeated 3 times for 20 minutes each time.
Sampling and Management
We sampled IL-1 at the local area from each animal. Afterward, we humanely destroyed each rat, then severed the foot at the level of cut ankle, 0.1 g of subcutaneous tissue and muscle from the feet, added some physiological saline, and made it even. Finally, we sampled and stored the upper supernatant liquid for further investigation.
We sampled HSP70 at the local area from each animal.We cut a 1 x 1 cm square of skin and subcutaneous tissue at the diseased ankle. This material was dipped into 80 mL of paraformaldehyde and soaked for 12 hours, after which paraffin sections were made.
Immunohistochemical staining was performed as recommended by the manufacturer (Santa Cruz Biotech, Inc). Integral calculus of optical density was determined as the main index (the bigger of this score, the more positive substance examined). Local IL-1 consistency was determined per the directions of the ELISA kit at the Central Laboratory of Sichuan People's Hospital (China).
RESULTS
Effects of Bloodletting Therapy on HSP70
The effects of bloodletting therapy on HSP70 content in arthritic rats and the comparisons with model and electroacupuncture groups are shown in Table 1.The HSP70 content in the model group was much higher than that in the control group. There was no statistically significant difference among the bloodletting group, electroacupuncture group, and control group (for all, P>.05).
Effects of Bloodletting Therapy on IL-1
The effects of bloodletting therapy on IL-1 levels in arthritic rats and comparisons with model and electroacupuncture groups are shown in Table 2.
The IL-1 levels in the inflammation local area of the model group was much higher than that of the control group, and IL-1 levels in the bloodletting and electroacupuncture groups were also higher than that of the control group but lower than levels in the model group; IL-1 in the bloodletting group did not differ significantly from that of electroacupuncture group. Our results suggest that bloodletting therapy can reduce local IL-1 content.
DISCUSSION
In our research, arthritis model rats manifested inflammatory symptoms that deteriorated while being heated. Therefore, these desirable arthritis febrile models can be differentiated as Heat Bi Syndrome in TCM. During the experiment, bloodletting and electroacupuncture treatment was carried out at the moment when inflammatory symptoms peaked (18 hours). According to some scientific research, a certain level of heat stress as well as much other stimulation such as physiological, chemical, and biological stress, can lead to various cellular productions.
There are many new proteins, among which HSP70 is measured ahead of the others. The expression of the human HSP70 gene is in-duced by a wide range of physiological stresses including exposure to heat shock and heavy metals, or under nonstress conditions such as in response to serum stimulation.2 Similarly, bloodletting therapy, a physiological stress, can also activate the production of HSP70 which then expresses HSP in the body. Immunization with HSPs has protective effects in models of induced arthritis. Analyses have shown a reduced synovial inflammation in such protected animals.3 The increase in HSP70 has been proven to promote the survival rate of cells as well as to ease the cellular lesion; this may be how HSP70 protects cells.4
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Table 1. Heat Shock Protein 70 (HSP70) by Study Group
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Group
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HSP70
Photoconsistency
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Integral
Mean (SD)
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P Value
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Control (n=8)
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5976669
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-1448310.93
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<.05 vs Model
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Model (n=8)
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14619345
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-3203940.68
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<.05 vs Control
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Electroacupuncture (n=8)
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20842297
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-6755590.66
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>.05 vs Model
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Bloodletting (n=8)
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17522378
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-1144305.2
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>.05 vs Model
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HSP70 is involved in the regulation of lipopolysaccharide-induced cytokine production, and this family of proteins plays a role in mitigating adverse effects of endotoxin during infection or other pathological stresses.5
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Table 2. Levels of Interleukin-1 (IL-1) by Study Group
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Group
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IL-1 IntegralPhotoconsistency
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Mean (SD)
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P Value
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Control (n=8)
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124.5833
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-38.88
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Model (n=8)
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557.5
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-260.4169
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<.01 vs Control
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Electroacupuncture (n=8)
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247.95
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-46.3229
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<.01 vs Control and vs Model
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Bloodletting (n=8)
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254.4
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-33.0511
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<.01 vs Control and vs Model
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It is known that cellular factors are a class of small molecular polypeptides existing outside the cells. They are the important medium of inflammation, infection, and immune diseases. These factors include the inflammatory monocyte factor IL-1, produced mainly by monocyte and tumor necrosis factor, etc. Among them, IL-1 activates vascular endotheliocytes in active rheumatoid arthritis and enhances the expression of adhesive molecules from endotheliocytes, interacting with leukocytes in the blood that is collected to the articular cavity in arthritis models. At the same time, IL-1 stimulates synovial cells and chondrocytes, which leads to the increase of degradation, reduction of synthesis, and the release of osteocalcium; hence, inducing damage to the bones and cartilage.6 In addition, the general symptoms of fever and anemia in rheumatoid arthritis also relate to IL-1. Interleukin-1 is a kind of pyrogen, a cellular factor that acts as a leading inflammation stimulant in the pathology of rheumatoid arthritis. Interleukin-1 and tumor necrosis factor-a are potent, multifunctional cytokine mediators of inflammation and immune responses produced primarily by activated monocytes and macrophages.7
CONCLUSION
We concluded that bloodletting therapy leads to a remarkable increase of local HSP70 expression and reduction of IL-1, and this therapy reaches much the same effects with electroacupuncture. Thus, bloodletting therapy as a kind of physiological therapy is effective in adjusting disorders of immune functions. The specific mechanism may be a process to activate the production of HSP70, to inhibit the secretion of IL-1, and thus reduce IL-1 content in the diseased local area and the circulation, so as to restrain the pathological process of rheumatoid arthritis and avoid damage as well as distortion of the joints.
However, the particular process of HSP70 that restrains the secretion of IL-1 remains unknown, and whether HSP70 can also inhibit the content level of other inflammatory cellular factors should bestudied. To deal with these problems, further efforts are needed.
REFERENCES
- Chinese Acupuncture and Moxibustion Association of Animal Husbandry and Veterinarian. Chinese Acupuncture of Veterinary Medicine. Peking, People's Republic of China: Agriculture Press House; 1984.
- Wu BJ, Kingston RE, Morimoto RI. Human HSP70 promoter contains at least two distinct regulatory domains. Proc Natl Acad Sci USA. 1986;83(3): 629-633.
- VE W, Wendling U, Paul L, et al. Arthritis protective regulatory potential of self-heat shock protein cross-reactive T cells. Cell Stress Chaperones. 2000;5 (5):452-457.
- Sun J. Distribution, adjusting, and functions of HSP70. Chinese Foreign Medical Science. 1997;1:8.
- Ding XZ, Fernandez-Prada CM, Bhattacharjee AK, et al. Over-expression of hsp-70 inhibits bacterial lipopolysaccharide-induced production of cytokines in human monocyte-derived macrophages. Cytokine. 2001;16(6): 210-219.
- Qing C. IL-1, TNF and rheumatoid arthritis. Shanghai J Immunol. 1998;1: 62-63.
- Hall TJ. Role of hsp70 in cytokine production. Experientia. 1994;50(11-12):1048-1048-1053.
AUTHORS' INFORMATION
Dr Yuxing Liu is a Medical Doctor (China), and is Dean of Research and faculty member at Academy of Oriental Medicine (AOMA) in Austin, Texas. Dr Liu previously served as Director and Associate Researcher of the Central Lab of Acupuncture and Tuina College of Chengdu University of TCM in P.R. China.
Yuxing Liu, OMD (MD), PhD, LAc*
Academy of Oriental Medicine at Austin
2700 W. Anderson Lane, Suite 204
Austin, TX 78757
Phone: 512-454-1188 • Fax: 512-454-7001
E-mail: dryuxingliu@yahoo.com
Dr Zheng Zeng is a Medical Doctor (China), and is a faculty member of Academy of Oriental Medicine in Austin, Texas.
Zheng Zeng, OMD (MD), MS, LAc
Academy of Oriental Medicine at Austin
2700 W. Anderson Lane, Suite 204
Austin, TX 78757
Phone: 512-454-1188 • Fax: 512-454-7001
E-mail: zeng_zh_99@yahoo.com
Dr Fanrong Liang is Professor, PhD advisor, and Vice President at Chengdu University of TCM in P.R. China.
Fanrong Liang, OMD (MD)
Chengdu University of Traditional Chinese Medicine
37# Shi-er-qiao Road, Chengdu, China
610075 P.R.China
E-mail: lfr@cdutcm.edu.cn
Yi Fu, MS, is a PhD student in Acupuncture at Tuina College of Chengdu University of TCM in P.R. China.
Jie Chen is a postgraduate student in Acupuncture at Tuina College of Chengdu University of TCM in P.R. China.
Xuezhi Li, OMD (MD), MS, is a lecturer of TCM at Chongqing Medical University in P.R. China.
*Correspondence and reprint requests
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