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Is cupping blister harmful?—A proteomical analysis of blister fluid induced by cupping therapy and scald
Complementary Therapies in Medicine 36 (2018) 25–29
Contents lists available at ScienceDirect
Complementary Therapies in Medicine journal homepage: www.elsevier.com/locate/ctim
Is cupping blister harmful?—A proteomical analysis of blister fluid induced by cupping therapy and scald
T
Zhidan Liua,1, Chunlan Chena,1, Xiaoyan Lia, Chuang Zhaoa, Zunyuan Lia, Wei Lianga, ⁎ Yufang Linb, a Department of Acupuncture, Shanghai Baoshan Hospital of Integrated Traditional Chinese Medicine and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 201999, China b Department of Acupuncture, Zhejiang Hospital, Hangzhou, 310013, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Proteomics Cupping therapy Blister fluid
Objective: Cupping therapy has a long history in traditional medicine especially in Asian countries. It was controversial whether cupping induced blisters are beneficial to healing effects, and the formation and content in the blisters remain unexplored. We aimed to identify and compare the molecular components of the blister fluid from the cupping therapy and the scalds to explore the necessary of inducing cupping induced blisters. Methods: Fluid sample of blisters from fifteen patients receiving cupping therapy (Cupping group) and scald burns (Scald group) were collected in this study. Proteins from the blisters were separated by two-dimensional electrophoresis (2D-gel) and further analyzed by mass spectrometry. In addition, the changes in particular proteins were confirmed by Western blotting. Results: The protein components are significantly different between blister from cupping therapy and scalds. The immune responses, oxidative stress and metabolic related proteins (Ig lambda-2 chain C regions, Ig gamma-1 chain C region, hemopexin, prdx2, calmodulin, succinyl-CoA ligase and tetranectin) were increased, whereas the hemoglobin subunit beta was decreased in the Cupping group compared with the Scald group. Conclusions: Cupping induced blisters contain several proteins which relate to the activation of certain immune pathways including anti-oxidation, anti-apoptosis, tissue repairing and metabolic regulation. This proteomic analysis may indicate a significant clue to the mechanism study of cupping.
1. Introduction Cupping therapy which utilize a glass or bamboo cup to create suction on the skin has a long history in the traditional medicine in Asia, especially in China.1–3 The efficacy has been shown in treating dysmenorrheal, pain, osteoarthritis, et al. 4–6. The skin where cupping therapy applied would usually be marked with a spot in pink, dark purple or red, as we can see on the skin of Olympic athletes, and sometimes be accompanied by the appearance of small blisters on the skin due to the generation of negative pressure by combustion (Fig. 1). Some opinion holds that cupping-induced blisters are embodiment of “damp evil” suction to excrete the “toxin” in vivo,7 so it’s beneficial to recovery, and symptoms including edema, crepitation and stiffness were significantly relieved after blister formation. But some others argue that cupping-induced blisters might be similar to blisters caused
by scalds which manifest the production of severe skin damage, and the blisters could leave skin scars and will affect the patient’s appearance, or induce erythematous based vesiculobullous plaque.8 So practitioners should try to avoid blisters when applying cupping therapy. Thus it’s unclear whether the composition of the cupping-induced blisters will promote therapy efficiency; and if it is, what is the molecular mechanisms underline. Therefore, it should be answered that is there something different be produced in cupping-induced blisters? So it is necessary to firstly compare the protein components between blister fluid samples caused by cupping therapy and scalds. We characterized the molecular elements of the blister fluid by proteomic analysis.
Abbreveations: MS, mass spectrometry; TCM, Traditional Chinese Medicine; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; GTP, guanosine triohosphte; ATP, adenosine triphosphate; CaMKII, Calcium/calmodulin-dependent protein kinase II; GABAA R, γ-aminobutyric acid type A receptor ⁎ Corresponding authors. E-mail address: [email protected] (Y. Lin). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ctim.2017.11.002 Received 2 August 2017; Received in revised form 2 October 2017; Accepted 1 November 2017 Available online 14 November 2017 0965-2299/ © 2017 Elsevier Ltd. All rights reserved.
Complementary Therapies in Medicine 36 (2018) 25–29
Z. Liu et al.
Fig. 1. Cupping spot, cupping-induced and scald-induced blisters. Cupping spot usually shows in pink, dark purple or red color related to the maintaining time and intensity of negative pressure. Cupping-induced blisters appears when cups retains a long time more than fifteen minutes or in other conditions of some sick individuals, and its’ exterior are similar to scald-induced blisters.
2. Materials and methods
2.3. Two-dimensional (2-D) PAGE
2.1. Subjects
300 μg samples were loaded into IPG Runner cassettes and a dry strip (GE Healthcare，24cm, pH = 3–10 isoelectric focusing gel) was placed into the well. Rehydration was carried out for 8 h, after which the strips were transferred. Isoelectric focusing was carried out at 20 °C as follows: 100 V, 1 h; 200 V, 1 h; 500 V, 1 h; 1000 V, 1 h; 1000–10000 V-h, 108000 V, 12 h; 500 V, 12 h. After focusing, the strips were placed into gels and sealed in place with agarose with bromophenol blue to be fractionated. Signals were visualized by silver staining. The difference between group B and group S was calculated as fold ratio. A threshold of p ≤ 0.05 and fold change ≥2 or ≤0.5 was set to select differentially protein spots.
This study was conducted from January 2013 to December 2015. Fluid sample of cupping group were collected from fifteen patients suffered from shoulder and back pain who were given cupping therapy in the Department of Acupuncture, Shanghai Baoshan Integrated Hospital of Traditional Chinese Medicine and Western Medicine, Shanghai University of Traditional Chinese Medicine (TCM). And fluid sample of scald group were collected from twenty burned patients in the Department of Burn and Plastic Surgery, Ninth People's Hospital of Shanghai Jiaotong University. All patients were between 20 to 60 years old with males and females. This study was reviewed and approved by the ethical committee in Shanghai Baoshan Integrated Hospital of Traditional Chinese and Western Medicine, Shanghai University of TCM. Participants with the following conditions were excluded: (1) serious comorbid conditions (e.g., life-threatening condition or severe neurological defects); (2) patients who cannot communicate reliably with the investigator or who are not likely to obey the instructions of the trial; (3) severe infection; (4) pregnancy. Patients in Cupping group received dry cupping therapy with glass pot (5 cm caliber) on related acupuncture points or regions like DU14(Da-zhui), LI15(Jian-yu), SJ14(Jian-liao), BL11(Da-zhu), BL12(Feng-men), BL13(Fei-shu) and Ashi points. The patient was in prone position. Routine disinfection of back skin was applied. The sucked glass pots were kept for ten minutes. Once blisters appeared, the glass pots were removed and blister liquid was collected immediately. For patients in Scald group, routine clinical care was applied at the Department of Burn and Plastic Surgery. Skin blister liquid was collected prior to appropriate treatment.
2.4. Mass spectrometry analysis Gels were stained, destained and subjected to tryptic digestion, followed by MALDI-TOF/TOF Plus mass spectrometer analysis (Applied Biosystems, Foster City, USA). Data were acquired in a positive MS reflector using a CalMix5 standard to calibrate the instrument (ABI5800 Calibration Mixture). Data were integrated and processed by using the GPS Explorer V3.6 software (Applied Biosystems, USA) with default parameters. Proteins were successfully identified based on 95% or higher confidence interval of their scores in the MASCOT V2.3 search engine (Matrix Science Ltd., London, U.K.). We used NCBInr-Homo Sapiens database, Carbamidomethyl (C) was set as fixed modification, variable modifications included protein N-terminal acetylation (Protein N-term), Deamidated (NQ), Dioxidation (W), Oxidation (M), 100 ppm for precursor ion tolerance and 0.3 Da for fragment ion tolerance.
2.5. Western blot 2.2. Sample preparation and protein extraction
Protein samples were resolved by 10% sodium dodecyl sulfate–polyacrylamide electrophoresis (SDS–PAGE), and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Uppsala, Sweden). For western hybridization, the membranes were preincubated with 0.5% skim milk in TTBS (0.1% Tween 20 in Tris-buffered saline) at room temperature for 2 h. The primary antibody to phospho-Src, or phospho-PKC (Cell Signaling Technology Inc., Danvers, MA) was diluted 1:1000 in TTBS containing 5% bovine serum albumin (BSA), and incubated overnight at 4 °C. The membranes were washed four times with TTBS, horseradish peroxidase-conjugated secondary antibody was added, and the membranes were incubated for 1 h at room temperature. After washing with TTBS, the hybridized bands were detected using an ECL detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia).
Blister liquid of all participants was extracted using 2 ml clean syringe and was stored at room temperature for 30 min. Samples were centrifuged at 2000xg for 20 min at room temperature, and supernatant was collected and stored at −70 °C for analysis. Supernatant (to remove serum albumin and IgG) was cleaned using ProteoExtract™ Albumin/ IgG Removal Kit (CALBIOCHEM, USA), and was centrifuged at 5000xg at 4 °C for 30 min. Pellets were resuspended in lysis buffer containing protease inhibitors at 30 °C for 1 h. Extracts were centrifuged at 15000×g for 15 min to remove debris. Protein concentrations were determined using the Bradford assay. 10 μg total protein was loaded on the gel and subject to western blot analysis.
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Complementary Therapies in Medicine 36 (2018) 25–29
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Fig. 2. General protein difference between cupping group and scald group. Representative images of 2D-gel electrophoresis of cupping group and scald group. (A) Among the twenty three protein spots with different expressions between two groups, eleven of them were identified by MALDI-TOF MS. (B) The eleven protein spots matched to eight corresponding proteins. Seven proteins in the cupping group were increased (Ig lambda-2 chain C regions, Ig gamma-1 chain C region, hemopexin, prdx2, calmodulin, succinyl-CoA ligase, and tetranectin), while one protein (hemoglobin subunit beta) was decreased (see Table 1). Abbreviations(Gene name, protein name): IGLC2, Ig lambda-2 chain C regions; IGHG1, Ig gamma-1 chain C region; CALM1, Calmodulin 1; CLEC3B, Tetranectin; PRDX2, Peroxiredoxin-2; HPX, Hemopexin; SUCLG1, Succinyl-CoA ligase; HBB, Hemoglobin subunit beta.
Table 1 Statistically significant protein alterations between cupping group and scald group. #
Gene name
Protein description
CG/SG Ratio
MS score
Mass (kDa)
Function
1
5103 IGLC2 8217 IGHG1 10 CALM1 4008 CLEC3B 4009 PRDX2 4604 HPX 2109
Ig lambda-2 chain C regions GN = IGLC2 PE = 1 SV = 1 Ig gamma-1 chain C region GN = IGHG1 PE = 1 SV = 1 Calmodulin 1 GN = CALM1 PE = 1 SV = 2 Tetranectin GN = CLEC3 B PE = 1 SV = 3 Peroxiredoxin-2 GN = PRDX2 PE = 1 SV = 5 Hemopexin GN = HPX PE = 1 SV = 2 Succinyl-CoA ligase [ADP/GDP-forming] subunit alpha, mitochondrial GN = SUCLG1 PE = 1 SV = 4 Hemoglobin subunit beta GN = HBB PE = 1 SV = 2
207.4375
106
11,294
Enhance immunity
29.02965
330
36,106
Enhance immunity
8.623531
149
16,838
Enhance pain sensitivity
11.52837
202
22,537
Promote tissue repairing
6.078076
198
21,892
Anti oxidative stress, enhance immunity, anti apoptotic
8.816424
462
51,676
Anti oxidative stress, enhance immunity, anti apoptotic
14.23714
346
36,250
Regulate metabolism
0.117406
147
15,998
Iron ions binding and oxygen binding, participate in oxygen transporting
2 3 4 5 6 7
8
SUCLG1 9003 HBB
MS score: Mass.
2.6. Data analysis and statistics
3. Results
The SRM data were analyzed using the Skyline software.9 All data where the AUC value was less than 10 were excluded from further analyses. Statistical significance of western blot results was determined by the Kruskal-Wallis test followed by the Dunn’s comparison test. A pvalue ≤0.05 was considered statistically significant. Data are shown as mean values ± SEM.
3.1. Distinct expression patterns of the fluid components in 2-D gel electrophoresis We first chose 2-D gel electrophoresis to evaluate the protein mixtures from two groups. Proteins were resolved on pH3-10 linear immobiline stripes and then separated by SDS-PAGE. Protein expression patterns were distinct from cupping therapy and scalds, both were heterogeneous groups. These protein spots average matching rate was 90%-93%. Twenty three protein spots with different expression patterns between 2 groups were identified (Fig. 2A). 27
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particular proteins with clinical significance were identified which may refer to the value of the clinical application of blistering cupping therapy.
3.2. Identification of different proteins using mass spectrometry (MS) Subsequently, in order to identify what proteins are differently expressed between two groups, MS/MS was conducted. Protein spots were excised from 2-D gels and analyzed by peptide mass fingerprinting by MALDI-TOF MS. Among the above twenty three protein spots, eleven of them were identified by MALDI-TOF MS matching to eight corresponding proteins (Fig. 2B). Compared with the scald group (control group), a total of seven proteins in the cupping group were increased (Ig lambda-2 chain C regions, Ig gamma-1 chain C region, hemopexin, prdx2, calmodulin, succinyl-CoA ligase, and tetranectin), one protein (hemoglobin subunit beta) was decreased (Table 1). These proteins can be divided into five categories: (1) the immune related proteins: Ig lambda-2 chain C regions, Ig gamma-1 chain C region; (2) against oxidative stress associated protein: hemopexin, prdx2; (3) pain related proteins: calmodulin; (4) metabolism associated proteins: hemoglobin subunit beta, succinyl-CoA ligase; (5) tissue repairing related protein: tetranectin.
4.1. Positive immune responses might be stimulated by blistering cupping therapy Immunoglobulin (Ig) is a protein secreted by B lymphocytes, which could format antigen-antibody complex and triggers immune responses. We found that Ig gamma C region was increased in cupping-induced blisters, indicating that the expression of IgG was up-regulated after cupping therapy. Ig lambda-2 chain C region has stable antigenicity and is positively correlated with the immune function of the body. Its high expression in patients accepting cupping therapy suggested that humoral immunity mediated by B lymphocyte was enhanced. 4.2. Anti oxidative stress and anti apoptotic functions might be activated by blistering cupping therapy Peroxiredoxin-2/Prdx2 is involved in redox regulation of the cell metabolism. Prdx2 increases the intracellular antioxidant capacities of monocyte-derived dendritic cells, making them better equipped to resist oxidative stress and initiate the T-cell responses.13 Prdx2 is protective role against cells apoptosis during oxidative stress.14 Hemopexin transports the toxic free heme to liver for decomposition and recovery of iron. Hemopexin is cell protective from oxidative stress.15 The enhanced expression of prdx2 and hemopexin in cupping-induced blisters indicated that they had enlarged antioxidant capacity and stable internal environment. This finding was consistent with Chinese Traditional Medicine theory of “dispelling dampness”.
3.3. Validation of differential expressed proteins by western blot To verify the changes of these selected proteins using MS, we performed western blot to examine protein expression between cupping therapy and scald groups. Total protein was extract from blister fluid from individual patient and the equal amount of protein was loaded on the gel. Consistent with MS results, we found a significant increase of calmodulin, succinyl-CoA ligase, tetranectin, Ig lambda-2 chain C regions in cupping group (Fig. 3). Our results suggested that cupping therapy promotes selected protein expression at translational level. 4. Discussion
4.3. Tissue repair process might start after blistering cupping therapy Skin injuries induce a complex network of signaling systems, rendering them applicable to analysis using advanced analytical approaches, such as proteomics and metabolomics, to profile the biological processes in response to burn injuries. Blister fluid is an ideal matrix for proteomics and metabolomics investigations of burn injury.10 Proteins in the blister fluid may present as potential biomarkers involved in biological pathways of particular diseases,11 the measurement of them could be advantageous in facilitating the diagnostic process or therapy. Kool et al.12 analyzed the suction blister fluid proteome comparing with a serum proteome and constructed a prejudiced list of 34 proteins relatively highly and uniquely expressed in suction blister fluid. The artificial negative pressure suction device used to produce suction blister in this study was quite familiar to cupping spots used in cupping therapy. In this work, differentially expressed proteins from blister fluid induced by cupping therapy and burn injury were investigated, and some
Tetranectin is a plasmiraogen binding protein, causing local thrombus dissolution and matrix repair.16,17 Increased tetranectin was also correlated to the osteoarthritis progression and inflammatory responses.18,19 Our study found that the tetranectin was highly expressed in cupping patients, suggesting that cupping therapy may promote tissue repairing and enhance blood circulation. 4.4. Local substances in pain sensation might be eliminated by blistering cupping therapy Calmodulin is involved in drug-induced anti-nociception, which could mediate emotional responses to pain and tolerance to morphine analgesia.20 Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a primary role in nociception in diabetic neuropathy.21 and spinal cord injury 22 CaMKII signaling contributes to pain hypersensitivity by reducingγ-aminobutyric acid type A receptor (GABAA R) inhibition.23
Fig. 3. Selective proteins were up-regulated in cupping group. (A) Representative of western blot analysis of scald group and cupping group (3 samples per group). (B) Quantification of protein expression after normalization to GAPDH. * P < 0.05; ** P < 0.01. S:scald group; C: cupping group. Abbreviations(Gene name, protein name): CALM1, Calmodulin 1; SUCLG1, Succinyl-CoA ligase; IGLC2, Ig lambda-2 chain C regions; CLEC3B, Tetranectin.
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the Department of Burn and Plastic Surgery, Ninth People's Hospital of Shanghai Jiaotong University for sample collecting from scald patients.
We discovered that calmodulin was up-regulated in blisters from cupping patients for neck pain, suggesting that calcium/CaMK II may mediate the pain sensation and cupping changes the pain signaling transduction. The decreased hemoglobin subunit beta suggested that the oxygen transporting is altered after blistering cupping therapy. But nonetheless, limitations are obvious since the fluid is already excreted from the skin, which means the fluid is not reintroduced into the body to lead to any therapeutic or harmful effect. Thus changes in superficial blister fluid are indirect evidences for its' therapeutic effect. Protein transformations in peripheral blood which directly presents in vivo reactions still need further study.
References 1. Mehta P, Dhapte V. Cupping therapy: a prudent remedy for a plethora of medical ailments. J Tradit Complement Med. 2015;5(3):127–134. 2. Abbas Zaidi SM, Jameel SS, Jafri K, Khan SA, Ahmad E. Ilaj bil hijamah (cupping therapy) in the Unani system of medicine: anecdotal practice to evidence based therapy. Acta Med Hist Adriat. 2016;14(1):81–94. 3. Yoo SS, Tausk Cupping F. East meets west. Int J Dermatol. 2004;43(9):664–665. 4. Inanmdar W, Sultana A, Mubeen U, Rahman K. Clinical efficacy of Trigonella foenum graecum (Fenugreek) and dry cupping therapy on intensity of pain in patients with primary dysmenorrhea. Chin J Integr Med. 2016(May)http://dx.doi.org/10.1007/ s11655-016-2259-x [Epub ahead of print]. 5. Chi LM, Lin LM, Chen CL, Wang SF, Lai HL, Peng TC. The effectiveness of cupping therapy on relieving chronic neck and shoulder pain: a randomized controlled trial. Evid Based Complement Alternat Med. 2016;2016:7358918. 6. Khan AA, Jahangir U, Urooj S. Management of knee osteoarthritis with cupping therapy. J Adv Pharm Technol Res. 2013;4(4):217–223. 7. Qureshi NA, Ali GI, Abushanab TS, et al. History of cupping (Hijama): a narrative review of literature. J Integr Med. 2017;15(3):172–181. 8. Benli AR, Aktas H. A complication of wet cupping therapy: vesiculobullous plaque on an erythematous base. J Integr Med. 2017;15(3):252–254. 9. MacLean B, Tomazela DM, Shulman N, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26(7):966–968. 10. Zang T, Broszczak DA, Broadbent JA, Cuttle L, Lu H, Parker TJ. The biochemistry of blister fluid from pediatric burn injuries: proteomics and metabolomics aspects. Expert Rev Proteomics. 2016;13(1):35–53. 11. Macdonald N, Cumberbatch M, Singh M, et al. Proteomic analysis of suction blister fluid isolated from human skin. Clin Exp Dermatol. 2006;31(3):445–448. 12. Kool J, Reubsaet L, Wesseldijk F, et al. Suction blister fluid as potential body fluid for biomarker proteins. Proteomics. 2007;7(20):3638–3650. 13. Van Brussel, Schrijvers DM, Martinet W, et al. Transcript and protein analysis reveals better survival skills of monocyte-derived dendritic cells compared to monocytes during oxidative stress. PLoS One. 2012;7(8):e43357. 14. Zhao F, Wang Q. The protective effect of peroxiredoxin II on oxidative stress induced apoptosis in pancreatic β-cells. Cell Biosci. 2012;2(1):22. 15. Grinberg LN, O'Brien PJ, Hrkal Z. The effects of heme-binding proteins on the peroxidative and catalatic activities of hemin. Free Radic Biol Med. 1999;27(1–2):214–219. 16. Chora AA, Fontoura P, Cunha A, et al. Heme oxygenase-1 and carbon monoxide suppress autoimmune neuroinflammation. J Clin Invest. 2007;117(2):438–447. 17. Iba K, Hatakeyama N, Kojima T, et al. Impaired cutaneous wound healing in mice lacking tetranectin. Wound Repair Regen. 2009;17(1):108–112. 18. Wewer UM, Iba K, Durkin ME, et al. Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro. Dev Biol. 1998;200(2):247–259. 19. Caterer NR, Graversen JH, Jacobsen C, et al. Specificity determinants in the interaction of apolipoprotein (a) kringles with tetranectin and LDL. Biol Chem. 2002;383(11):1743–1750. 20. Jackson KJ, Damaj MI. Calcium/calmodulin-dependent protein kinase IV mediates acute nicotine-induced antinociception in acute thermal pain tests. Behav Pharmacol. 2013;24(8):689–692. 21. Jelicic Kadic A, Boric M, Kostic S, Sapunar D, Puljak L. The effects of intraganglionic injection of calcium/calmodulin-dependent protein kinase II inhibitors on pain-related behavior in diabetic neuropathy. Neuroscience. 2014;256:302–308. 22. Crown ED, Gwak YS, Ye Z, et al. Calcium/calmodulin dependent kinase II contributes to persistent central neuropathic pain following spinal cord injury. Pain. 2012;153(3):710–721. 23. Suo ZW, Fan QQ, Yang X, Hu XD. Ca2+/calmodulin-dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ-aminobutyric acid type a receptor inhibition. J Neurosci Res. 2013;91(11):1473–1482.
5. Conclusion and prospect This work identified several proteins in superficial blisters induced by cupping therapy which relate to the activation of certain immune pathways including anti-oxidation, anti-apoptosis, tissue repairing and metabolic regulation. This proteomic analysis may indicate a significant clue to the mechanism study of cupping. In future, proteomic study on serum proteins should be carried out to provide direct evidence proving the therapeutic benefit of blistering cupping therapy. Conflict of interest statement The authors have declared no conflict of interest in this work. Fundings This work was supported by grants from the Excellent TCM Doctor Training Project (ZY3-RCPY-2-2041) in the three-year traditional Chinese medicine action plan from the Shanghai Health and Family Planning Commission. It was also supported by the Youth Medical Talents Training Project (BSWSYQ-2014-A05) from Baoshan District Health and Family Planning Commission of Shanghai. The funding bodies had no role in the study design or the decision to submit the manuscript for publication. Author’s contributions YFLwas responsible for the study concept and design. ZDL conducted the experiment and wrote the manuscript. CLC, XYL, CZ, ZYL conducted the experiments and collected the data. WL helped to analyze the data. All authors approved the final version of the manuscript. Ethics approval and consent to participate The experimental procedures were received approval from the Medical Ethics Committee Board of Baoshan Integrated Traditional Chinese Medicine and Western Medicine Hospital of Shanghai (permission No. 201512-08). And written consent from all participators were received for publication of their involved pictures. Acknowledgement We would like to express our thanks to Doctor Peng-Gao Yang form
29
Contents lists available at ScienceDirect
Complementary Therapies in Medicine journal homepage: www.elsevier.com/locate/ctim
Is cupping blister harmful?—A proteomical analysis of blister fluid induced by cupping therapy and scald
T
Zhidan Liua,1, Chunlan Chena,1, Xiaoyan Lia, Chuang Zhaoa, Zunyuan Lia, Wei Lianga, ⁎ Yufang Linb, a Department of Acupuncture, Shanghai Baoshan Hospital of Integrated Traditional Chinese Medicine and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 201999, China b Department of Acupuncture, Zhejiang Hospital, Hangzhou, 310013, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Proteomics Cupping therapy Blister fluid
Objective: Cupping therapy has a long history in traditional medicine especially in Asian countries. It was controversial whether cupping induced blisters are beneficial to healing effects, and the formation and content in the blisters remain unexplored. We aimed to identify and compare the molecular components of the blister fluid from the cupping therapy and the scalds to explore the necessary of inducing cupping induced blisters. Methods: Fluid sample of blisters from fifteen patients receiving cupping therapy (Cupping group) and scald burns (Scald group) were collected in this study. Proteins from the blisters were separated by two-dimensional electrophoresis (2D-gel) and further analyzed by mass spectrometry. In addition, the changes in particular proteins were confirmed by Western blotting. Results: The protein components are significantly different between blister from cupping therapy and scalds. The immune responses, oxidative stress and metabolic related proteins (Ig lambda-2 chain C regions, Ig gamma-1 chain C region, hemopexin, prdx2, calmodulin, succinyl-CoA ligase and tetranectin) were increased, whereas the hemoglobin subunit beta was decreased in the Cupping group compared with the Scald group. Conclusions: Cupping induced blisters contain several proteins which relate to the activation of certain immune pathways including anti-oxidation, anti-apoptosis, tissue repairing and metabolic regulation. This proteomic analysis may indicate a significant clue to the mechanism study of cupping.
1. Introduction Cupping therapy which utilize a glass or bamboo cup to create suction on the skin has a long history in the traditional medicine in Asia, especially in China.1–3 The efficacy has been shown in treating dysmenorrheal, pain, osteoarthritis, et al. 4–6. The skin where cupping therapy applied would usually be marked with a spot in pink, dark purple or red, as we can see on the skin of Olympic athletes, and sometimes be accompanied by the appearance of small blisters on the skin due to the generation of negative pressure by combustion (Fig. 1). Some opinion holds that cupping-induced blisters are embodiment of “damp evil” suction to excrete the “toxin” in vivo,7 so it’s beneficial to recovery, and symptoms including edema, crepitation and stiffness were significantly relieved after blister formation. But some others argue that cupping-induced blisters might be similar to blisters caused
by scalds which manifest the production of severe skin damage, and the blisters could leave skin scars and will affect the patient’s appearance, or induce erythematous based vesiculobullous plaque.8 So practitioners should try to avoid blisters when applying cupping therapy. Thus it’s unclear whether the composition of the cupping-induced blisters will promote therapy efficiency; and if it is, what is the molecular mechanisms underline. Therefore, it should be answered that is there something different be produced in cupping-induced blisters? So it is necessary to firstly compare the protein components between blister fluid samples caused by cupping therapy and scalds. We characterized the molecular elements of the blister fluid by proteomic analysis.
Abbreveations: MS, mass spectrometry; TCM, Traditional Chinese Medicine; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; GTP, guanosine triohosphte; ATP, adenosine triphosphate; CaMKII, Calcium/calmodulin-dependent protein kinase II; GABAA R, γ-aminobutyric acid type A receptor ⁎ Corresponding authors. E-mail address: [email protected] (Y. Lin). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ctim.2017.11.002 Received 2 August 2017; Received in revised form 2 October 2017; Accepted 1 November 2017 Available online 14 November 2017 0965-2299/ © 2017 Elsevier Ltd. All rights reserved.
Complementary Therapies in Medicine 36 (2018) 25–29
Z. Liu et al.
Fig. 1. Cupping spot, cupping-induced and scald-induced blisters. Cupping spot usually shows in pink, dark purple or red color related to the maintaining time and intensity of negative pressure. Cupping-induced blisters appears when cups retains a long time more than fifteen minutes or in other conditions of some sick individuals, and its’ exterior are similar to scald-induced blisters.
2. Materials and methods
2.3. Two-dimensional (2-D) PAGE
2.1. Subjects
300 μg samples were loaded into IPG Runner cassettes and a dry strip (GE Healthcare，24cm, pH = 3–10 isoelectric focusing gel) was placed into the well. Rehydration was carried out for 8 h, after which the strips were transferred. Isoelectric focusing was carried out at 20 °C as follows: 100 V, 1 h; 200 V, 1 h; 500 V, 1 h; 1000 V, 1 h; 1000–10000 V-h, 108000 V, 12 h; 500 V, 12 h. After focusing, the strips were placed into gels and sealed in place with agarose with bromophenol blue to be fractionated. Signals were visualized by silver staining. The difference between group B and group S was calculated as fold ratio. A threshold of p ≤ 0.05 and fold change ≥2 or ≤0.5 was set to select differentially protein spots.
This study was conducted from January 2013 to December 2015. Fluid sample of cupping group were collected from fifteen patients suffered from shoulder and back pain who were given cupping therapy in the Department of Acupuncture, Shanghai Baoshan Integrated Hospital of Traditional Chinese Medicine and Western Medicine, Shanghai University of Traditional Chinese Medicine (TCM). And fluid sample of scald group were collected from twenty burned patients in the Department of Burn and Plastic Surgery, Ninth People's Hospital of Shanghai Jiaotong University. All patients were between 20 to 60 years old with males and females. This study was reviewed and approved by the ethical committee in Shanghai Baoshan Integrated Hospital of Traditional Chinese and Western Medicine, Shanghai University of TCM. Participants with the following conditions were excluded: (1) serious comorbid conditions (e.g., life-threatening condition or severe neurological defects); (2) patients who cannot communicate reliably with the investigator or who are not likely to obey the instructions of the trial; (3) severe infection; (4) pregnancy. Patients in Cupping group received dry cupping therapy with glass pot (5 cm caliber) on related acupuncture points or regions like DU14(Da-zhui), LI15(Jian-yu), SJ14(Jian-liao), BL11(Da-zhu), BL12(Feng-men), BL13(Fei-shu) and Ashi points. The patient was in prone position. Routine disinfection of back skin was applied. The sucked glass pots were kept for ten minutes. Once blisters appeared, the glass pots were removed and blister liquid was collected immediately. For patients in Scald group, routine clinical care was applied at the Department of Burn and Plastic Surgery. Skin blister liquid was collected prior to appropriate treatment.
2.4. Mass spectrometry analysis Gels were stained, destained and subjected to tryptic digestion, followed by MALDI-TOF/TOF Plus mass spectrometer analysis (Applied Biosystems, Foster City, USA). Data were acquired in a positive MS reflector using a CalMix5 standard to calibrate the instrument (ABI5800 Calibration Mixture). Data were integrated and processed by using the GPS Explorer V3.6 software (Applied Biosystems, USA) with default parameters. Proteins were successfully identified based on 95% or higher confidence interval of their scores in the MASCOT V2.3 search engine (Matrix Science Ltd., London, U.K.). We used NCBInr-Homo Sapiens database, Carbamidomethyl (C) was set as fixed modification, variable modifications included protein N-terminal acetylation (Protein N-term), Deamidated (NQ), Dioxidation (W), Oxidation (M), 100 ppm for precursor ion tolerance and 0.3 Da for fragment ion tolerance.
2.5. Western blot 2.2. Sample preparation and protein extraction
Protein samples were resolved by 10% sodium dodecyl sulfate–polyacrylamide electrophoresis (SDS–PAGE), and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Uppsala, Sweden). For western hybridization, the membranes were preincubated with 0.5% skim milk in TTBS (0.1% Tween 20 in Tris-buffered saline) at room temperature for 2 h. The primary antibody to phospho-Src, or phospho-PKC (Cell Signaling Technology Inc., Danvers, MA) was diluted 1:1000 in TTBS containing 5% bovine serum albumin (BSA), and incubated overnight at 4 °C. The membranes were washed four times with TTBS, horseradish peroxidase-conjugated secondary antibody was added, and the membranes were incubated for 1 h at room temperature. After washing with TTBS, the hybridized bands were detected using an ECL detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia).
Blister liquid of all participants was extracted using 2 ml clean syringe and was stored at room temperature for 30 min. Samples were centrifuged at 2000xg for 20 min at room temperature, and supernatant was collected and stored at −70 °C for analysis. Supernatant (to remove serum albumin and IgG) was cleaned using ProteoExtract™ Albumin/ IgG Removal Kit (CALBIOCHEM, USA), and was centrifuged at 5000xg at 4 °C for 30 min. Pellets were resuspended in lysis buffer containing protease inhibitors at 30 °C for 1 h. Extracts were centrifuged at 15000×g for 15 min to remove debris. Protein concentrations were determined using the Bradford assay. 10 μg total protein was loaded on the gel and subject to western blot analysis.
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Fig. 2. General protein difference between cupping group and scald group. Representative images of 2D-gel electrophoresis of cupping group and scald group. (A) Among the twenty three protein spots with different expressions between two groups, eleven of them were identified by MALDI-TOF MS. (B) The eleven protein spots matched to eight corresponding proteins. Seven proteins in the cupping group were increased (Ig lambda-2 chain C regions, Ig gamma-1 chain C region, hemopexin, prdx2, calmodulin, succinyl-CoA ligase, and tetranectin), while one protein (hemoglobin subunit beta) was decreased (see Table 1). Abbreviations(Gene name, protein name): IGLC2, Ig lambda-2 chain C regions; IGHG1, Ig gamma-1 chain C region; CALM1, Calmodulin 1; CLEC3B, Tetranectin; PRDX2, Peroxiredoxin-2; HPX, Hemopexin; SUCLG1, Succinyl-CoA ligase; HBB, Hemoglobin subunit beta.
Table 1 Statistically significant protein alterations between cupping group and scald group. #
Gene name
Protein description
CG/SG Ratio
MS score
Mass (kDa)
Function
1
5103 IGLC2 8217 IGHG1 10 CALM1 4008 CLEC3B 4009 PRDX2 4604 HPX 2109
Ig lambda-2 chain C regions GN = IGLC2 PE = 1 SV = 1 Ig gamma-1 chain C region GN = IGHG1 PE = 1 SV = 1 Calmodulin 1 GN = CALM1 PE = 1 SV = 2 Tetranectin GN = CLEC3 B PE = 1 SV = 3 Peroxiredoxin-2 GN = PRDX2 PE = 1 SV = 5 Hemopexin GN = HPX PE = 1 SV = 2 Succinyl-CoA ligase [ADP/GDP-forming] subunit alpha, mitochondrial GN = SUCLG1 PE = 1 SV = 4 Hemoglobin subunit beta GN = HBB PE = 1 SV = 2
207.4375
106
11,294
Enhance immunity
29.02965
330
36,106
Enhance immunity
8.623531
149
16,838
Enhance pain sensitivity
11.52837
202
22,537
Promote tissue repairing
6.078076
198
21,892
Anti oxidative stress, enhance immunity, anti apoptotic
8.816424
462
51,676
Anti oxidative stress, enhance immunity, anti apoptotic
14.23714
346
36,250
Regulate metabolism
0.117406
147
15,998
Iron ions binding and oxygen binding, participate in oxygen transporting
2 3 4 5 6 7
8
SUCLG1 9003 HBB
MS score: Mass.
2.6. Data analysis and statistics
3. Results
The SRM data were analyzed using the Skyline software.9 All data where the AUC value was less than 10 were excluded from further analyses. Statistical significance of western blot results was determined by the Kruskal-Wallis test followed by the Dunn’s comparison test. A pvalue ≤0.05 was considered statistically significant. Data are shown as mean values ± SEM.
3.1. Distinct expression patterns of the fluid components in 2-D gel electrophoresis We first chose 2-D gel electrophoresis to evaluate the protein mixtures from two groups. Proteins were resolved on pH3-10 linear immobiline stripes and then separated by SDS-PAGE. Protein expression patterns were distinct from cupping therapy and scalds, both were heterogeneous groups. These protein spots average matching rate was 90%-93%. Twenty three protein spots with different expression patterns between 2 groups were identified (Fig. 2A). 27
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particular proteins with clinical significance were identified which may refer to the value of the clinical application of blistering cupping therapy.
3.2. Identification of different proteins using mass spectrometry (MS) Subsequently, in order to identify what proteins are differently expressed between two groups, MS/MS was conducted. Protein spots were excised from 2-D gels and analyzed by peptide mass fingerprinting by MALDI-TOF MS. Among the above twenty three protein spots, eleven of them were identified by MALDI-TOF MS matching to eight corresponding proteins (Fig. 2B). Compared with the scald group (control group), a total of seven proteins in the cupping group were increased (Ig lambda-2 chain C regions, Ig gamma-1 chain C region, hemopexin, prdx2, calmodulin, succinyl-CoA ligase, and tetranectin), one protein (hemoglobin subunit beta) was decreased (Table 1). These proteins can be divided into five categories: (1) the immune related proteins: Ig lambda-2 chain C regions, Ig gamma-1 chain C region; (2) against oxidative stress associated protein: hemopexin, prdx2; (3) pain related proteins: calmodulin; (4) metabolism associated proteins: hemoglobin subunit beta, succinyl-CoA ligase; (5) tissue repairing related protein: tetranectin.
4.1. Positive immune responses might be stimulated by blistering cupping therapy Immunoglobulin (Ig) is a protein secreted by B lymphocytes, which could format antigen-antibody complex and triggers immune responses. We found that Ig gamma C region was increased in cupping-induced blisters, indicating that the expression of IgG was up-regulated after cupping therapy. Ig lambda-2 chain C region has stable antigenicity and is positively correlated with the immune function of the body. Its high expression in patients accepting cupping therapy suggested that humoral immunity mediated by B lymphocyte was enhanced. 4.2. Anti oxidative stress and anti apoptotic functions might be activated by blistering cupping therapy Peroxiredoxin-2/Prdx2 is involved in redox regulation of the cell metabolism. Prdx2 increases the intracellular antioxidant capacities of monocyte-derived dendritic cells, making them better equipped to resist oxidative stress and initiate the T-cell responses.13 Prdx2 is protective role against cells apoptosis during oxidative stress.14 Hemopexin transports the toxic free heme to liver for decomposition and recovery of iron. Hemopexin is cell protective from oxidative stress.15 The enhanced expression of prdx2 and hemopexin in cupping-induced blisters indicated that they had enlarged antioxidant capacity and stable internal environment. This finding was consistent with Chinese Traditional Medicine theory of “dispelling dampness”.
3.3. Validation of differential expressed proteins by western blot To verify the changes of these selected proteins using MS, we performed western blot to examine protein expression between cupping therapy and scald groups. Total protein was extract from blister fluid from individual patient and the equal amount of protein was loaded on the gel. Consistent with MS results, we found a significant increase of calmodulin, succinyl-CoA ligase, tetranectin, Ig lambda-2 chain C regions in cupping group (Fig. 3). Our results suggested that cupping therapy promotes selected protein expression at translational level. 4. Discussion
4.3. Tissue repair process might start after blistering cupping therapy Skin injuries induce a complex network of signaling systems, rendering them applicable to analysis using advanced analytical approaches, such as proteomics and metabolomics, to profile the biological processes in response to burn injuries. Blister fluid is an ideal matrix for proteomics and metabolomics investigations of burn injury.10 Proteins in the blister fluid may present as potential biomarkers involved in biological pathways of particular diseases,11 the measurement of them could be advantageous in facilitating the diagnostic process or therapy. Kool et al.12 analyzed the suction blister fluid proteome comparing with a serum proteome and constructed a prejudiced list of 34 proteins relatively highly and uniquely expressed in suction blister fluid. The artificial negative pressure suction device used to produce suction blister in this study was quite familiar to cupping spots used in cupping therapy. In this work, differentially expressed proteins from blister fluid induced by cupping therapy and burn injury were investigated, and some
Tetranectin is a plasmiraogen binding protein, causing local thrombus dissolution and matrix repair.16,17 Increased tetranectin was also correlated to the osteoarthritis progression and inflammatory responses.18,19 Our study found that the tetranectin was highly expressed in cupping patients, suggesting that cupping therapy may promote tissue repairing and enhance blood circulation. 4.4. Local substances in pain sensation might be eliminated by blistering cupping therapy Calmodulin is involved in drug-induced anti-nociception, which could mediate emotional responses to pain and tolerance to morphine analgesia.20 Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a primary role in nociception in diabetic neuropathy.21 and spinal cord injury 22 CaMKII signaling contributes to pain hypersensitivity by reducingγ-aminobutyric acid type A receptor (GABAA R) inhibition.23
Fig. 3. Selective proteins were up-regulated in cupping group. (A) Representative of western blot analysis of scald group and cupping group (3 samples per group). (B) Quantification of protein expression after normalization to GAPDH. * P < 0.05; ** P < 0.01. S:scald group; C: cupping group. Abbreviations(Gene name, protein name): CALM1, Calmodulin 1; SUCLG1, Succinyl-CoA ligase; IGLC2, Ig lambda-2 chain C regions; CLEC3B, Tetranectin.
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the Department of Burn and Plastic Surgery, Ninth People's Hospital of Shanghai Jiaotong University for sample collecting from scald patients.
We discovered that calmodulin was up-regulated in blisters from cupping patients for neck pain, suggesting that calcium/CaMK II may mediate the pain sensation and cupping changes the pain signaling transduction. The decreased hemoglobin subunit beta suggested that the oxygen transporting is altered after blistering cupping therapy. But nonetheless, limitations are obvious since the fluid is already excreted from the skin, which means the fluid is not reintroduced into the body to lead to any therapeutic or harmful effect. Thus changes in superficial blister fluid are indirect evidences for its' therapeutic effect. Protein transformations in peripheral blood which directly presents in vivo reactions still need further study.
References 1. Mehta P, Dhapte V. Cupping therapy: a prudent remedy for a plethora of medical ailments. J Tradit Complement Med. 2015;5(3):127–134. 2. Abbas Zaidi SM, Jameel SS, Jafri K, Khan SA, Ahmad E. Ilaj bil hijamah (cupping therapy) in the Unani system of medicine: anecdotal practice to evidence based therapy. Acta Med Hist Adriat. 2016;14(1):81–94. 3. Yoo SS, Tausk Cupping F. East meets west. Int J Dermatol. 2004;43(9):664–665. 4. Inanmdar W, Sultana A, Mubeen U, Rahman K. Clinical efficacy of Trigonella foenum graecum (Fenugreek) and dry cupping therapy on intensity of pain in patients with primary dysmenorrhea. Chin J Integr Med. 2016(May)http://dx.doi.org/10.1007/ s11655-016-2259-x [Epub ahead of print]. 5. Chi LM, Lin LM, Chen CL, Wang SF, Lai HL, Peng TC. The effectiveness of cupping therapy on relieving chronic neck and shoulder pain: a randomized controlled trial. Evid Based Complement Alternat Med. 2016;2016:7358918. 6. Khan AA, Jahangir U, Urooj S. Management of knee osteoarthritis with cupping therapy. J Adv Pharm Technol Res. 2013;4(4):217–223. 7. Qureshi NA, Ali GI, Abushanab TS, et al. History of cupping (Hijama): a narrative review of literature. J Integr Med. 2017;15(3):172–181. 8. Benli AR, Aktas H. A complication of wet cupping therapy: vesiculobullous plaque on an erythematous base. J Integr Med. 2017;15(3):252–254. 9. MacLean B, Tomazela DM, Shulman N, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26(7):966–968. 10. Zang T, Broszczak DA, Broadbent JA, Cuttle L, Lu H, Parker TJ. The biochemistry of blister fluid from pediatric burn injuries: proteomics and metabolomics aspects. Expert Rev Proteomics. 2016;13(1):35–53. 11. Macdonald N, Cumberbatch M, Singh M, et al. Proteomic analysis of suction blister fluid isolated from human skin. Clin Exp Dermatol. 2006;31(3):445–448. 12. Kool J, Reubsaet L, Wesseldijk F, et al. Suction blister fluid as potential body fluid for biomarker proteins. Proteomics. 2007;7(20):3638–3650. 13. Van Brussel, Schrijvers DM, Martinet W, et al. Transcript and protein analysis reveals better survival skills of monocyte-derived dendritic cells compared to monocytes during oxidative stress. PLoS One. 2012;7(8):e43357. 14. Zhao F, Wang Q. The protective effect of peroxiredoxin II on oxidative stress induced apoptosis in pancreatic β-cells. Cell Biosci. 2012;2(1):22. 15. Grinberg LN, O'Brien PJ, Hrkal Z. The effects of heme-binding proteins on the peroxidative and catalatic activities of hemin. Free Radic Biol Med. 1999;27(1–2):214–219. 16. Chora AA, Fontoura P, Cunha A, et al. Heme oxygenase-1 and carbon monoxide suppress autoimmune neuroinflammation. J Clin Invest. 2007;117(2):438–447. 17. Iba K, Hatakeyama N, Kojima T, et al. Impaired cutaneous wound healing in mice lacking tetranectin. Wound Repair Regen. 2009;17(1):108–112. 18. Wewer UM, Iba K, Durkin ME, et al. Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro. Dev Biol. 1998;200(2):247–259. 19. Caterer NR, Graversen JH, Jacobsen C, et al. Specificity determinants in the interaction of apolipoprotein (a) kringles with tetranectin and LDL. Biol Chem. 2002;383(11):1743–1750. 20. Jackson KJ, Damaj MI. Calcium/calmodulin-dependent protein kinase IV mediates acute nicotine-induced antinociception in acute thermal pain tests. Behav Pharmacol. 2013;24(8):689–692. 21. Jelicic Kadic A, Boric M, Kostic S, Sapunar D, Puljak L. The effects of intraganglionic injection of calcium/calmodulin-dependent protein kinase II inhibitors on pain-related behavior in diabetic neuropathy. Neuroscience. 2014;256:302–308. 22. Crown ED, Gwak YS, Ye Z, et al. Calcium/calmodulin dependent kinase II contributes to persistent central neuropathic pain following spinal cord injury. Pain. 2012;153(3):710–721. 23. Suo ZW, Fan QQ, Yang X, Hu XD. Ca2+/calmodulin-dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ-aminobutyric acid type a receptor inhibition. J Neurosci Res. 2013;91(11):1473–1482.
5. Conclusion and prospect This work identified several proteins in superficial blisters induced by cupping therapy which relate to the activation of certain immune pathways including anti-oxidation, anti-apoptosis, tissue repairing and metabolic regulation. This proteomic analysis may indicate a significant clue to the mechanism study of cupping. In future, proteomic study on serum proteins should be carried out to provide direct evidence proving the therapeutic benefit of blistering cupping therapy. Conflict of interest statement The authors have declared no conflict of interest in this work. Fundings This work was supported by grants from the Excellent TCM Doctor Training Project (ZY3-RCPY-2-2041) in the three-year traditional Chinese medicine action plan from the Shanghai Health and Family Planning Commission. It was also supported by the Youth Medical Talents Training Project (BSWSYQ-2014-A05) from Baoshan District Health and Family Planning Commission of Shanghai. The funding bodies had no role in the study design or the decision to submit the manuscript for publication. Author’s contributions YFLwas responsible for the study concept and design. ZDL conducted the experiment and wrote the manuscript. CLC, XYL, CZ, ZYL conducted the experiments and collected the data. WL helped to analyze the data. All authors approved the final version of the manuscript. Ethics approval and consent to participate The experimental procedures were received approval from the Medical Ethics Committee Board of Baoshan Integrated Traditional Chinese Medicine and Western Medicine Hospital of Shanghai (permission No. 201512-08). And written consent from all participators were received for publication of their involved pictures. Acknowledgement We would like to express our thanks to Doctor Peng-Gao Yang form
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