Is cupping blister harmful?

ABSTRACT

Objective: Cupping therapy has a long history in traditional medicine especially in Asian countries. It was controversial whether cupping induced blisters are bene?cial to healing e?ects,and the formation and contentin the blisters remain unexplored. We aimed to identify and compare the molecular components of the blister ?uid from the cupping therapy and the scalds to explore the necessary of inducing cupping induced blisters. Methods: Fluid sample of blisters from ?fteen 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 con?rmed by Western blotting. Results: Theproteincomponents aresigni?cantlydi?erentbetweenblisterfromcupping therapyand 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 signi?cant 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 e?cacy 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 bene?cial to recovery, and symptoms including edema, crepitation and sti?ness were signi?cantly 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 a?ect 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 e?ciency; and if it is, what is the molecular mechanisms underline. Therefore, it should be answered that is there something di?erent be produced in cupping-induced blisters? So it is necessary to ?rstly compare the protein components between blister ?uid samples caused by cupping therapy and scalds. We characterized the molecular elements of the blister ?uid by proteomic analysis.

Fig. 1. Cupping spot, cupping-induced and scald-induced blisters. Cupping spotusually showsin pink,darkpurple orredcolor related tothe maintainingtimeandintensity ofnegativepressure. Cupping-inducedblisters appearswhencupsretains along time more than ?fteen minutes or in other conditions of some sick individuals, and its』 exterior are similar to scald-induced blisters.

2. Materials and methods

2.1. Subjects

This study was conducted from January 2013 to December 2015. Fluid sample of cupping group were collected from ?fteen patients su?ered 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 ?uid 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.

2.2. Sample preparation and protein extraction

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 for20 minat roomtemperature, andsupernatant wascollectedandstoredat?70 °Cforanalysis.Supernatant(toremove 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 bu?er 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. 10μg total protein was loaded on the gel and subject to western blot analysis.

2.3. Two-dimensional (2-D) PAGE

300μg 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 di?erence 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 di?erentially protein spots.

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 re?ectorusingaCalMix5standardtocalibrate theinstrument (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 identi?ed based on 95% or higher con?dence 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 ?xed modi?cation, variable modi?cations 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

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 with0.5% skimmilkinTTBS(0.1% Tween20inTris-bu?eredsaline)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,andthemembraneswereincubatedfor1 hatroomtemperature. After washing with TTBS, the hybridized bands were detected using an ECL detection kit and Hyper?lm-ECL reagents (Amersham Pharmacia).

Fig. 2. General protein di?erence 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 di?erent expressions between two groups, eleven of them were identi?ed 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, proteinname): IGLC2, Ig lambda-2 chainCregions; IGHG1,Ig gamma-1 chainCregion;CALM1, Calmodulin 1;CLEC3B, Tetranectin; PRDX2, Peroxiredoxin-2; HPX, Hemopexin; SUCLG1, Succinyl-CoA ligase; HBB, Hemoglobin subunit beta.

2.6. Data analysis and statisticsThe 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 signi?cance 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 signi?cant. Data are shown as mean values±SEM.

3. Results

3.1. Distinct expression patterns of the ?uid components in 2-D gel electrophoresis

We ?rst 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 di?erent expression patterns between 2 groups were identi?ed (Fig. 2A).

3.2. Identi?cation of di?erent proteins using mass spectrometry (MS)

Subsequently, in order to identify what proteins are di?erently expressedbetweentwogroups,MS/MSwasconducted.Proteinspotswere excised from 2-D gels and analyzed by peptide mass ?ngerprinting by MALDI-TOF MS. Among the above twenty three protein spots, eleven of them were identi?ed by MALDI-TOF MS matching to eight corresponding proteins (Fig. 2B). Compared with the scald group (control group),atotalofsevenproteinsinthecuppinggroupwereincreased(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 ?ve 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.

3.3. Validation of di?erential 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 ?uid from individual patient and the equal amount of protein was loaded on the gel. Consistent with MS results, we found a signi?cant 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

Skin injuries induce a complex network of signaling systems, rendering them applicable to analysis using advanced analytical approaches, such as proteomics and metabolomics, to pro?le the biological processes in response to burn injuries. Blister ?uid is an ideal matrix for proteomics and metabolomics investigations of burn injury.10 Proteins in the blister ?uid 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 ?uid proteome comparing with a serum proteome and constructed a prejudiced list of 34 proteins relatively highly and uniquely expressed in suction blister ?uid. The arti?cial 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, di?erentially expressed proteins from blister ?uid induced by cupping therapy and burn injury were investigated, and some particular proteins with clinical signi?cance were identi?ed which may refer to the value of the clinical application of blistering cupping therapy.

4.1. Positive immune responses might be stimulated by blistering cupping therapy

Immunoglobulin (Ig) is a protein secreted by B lymphocytes, which couldformat antigen-antibody complexandtriggersimmune responses. We found that Ig gamma C region was increased in cupping-induced blisters, indicating that the expression of IgG was up-regulated after cuppingtherapy. Iglambda-2 chain Cregion hasstable 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 protectiverole againstcells apoptosisduring 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 ?nding was consistent with Chinese Traditional Medicine theory of 「dispelling dampness」.

4.3. Tissue repair process might start after blistering cupping therapy

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 in?ammatory 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) Quanti?cation 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.

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 ?uid is already excreted from the skin, which means the ?uid is not reintroduced into the body to lead to any therapeutic or harmful e?ect. Thus changes in super?cial blister ?uid are indirect evidences for its" therapeutic e?ect. Protein transformations in peripheral blood which directly presents in vivo reactions still need further study.

5. Conclusion and prospect

This work identi?ed several proteins in super?cial blisters induced by cupping therapy which relate to the activation of certain immune pathways including anti-oxidation, anti-apoptosis, tissue repairing and metabolicregulation. Thisproteomicanalysismayindicateasigni?cant 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 bene?t of blistering cupping therapy.

Con?ict of interest statement

The authors have declared no con?ict 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 ?nal 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 the Department of Burn and Plastic Surgery, Ninth People"s Hospital of Shanghai Jiaotong University for sample collecting from scald patients.

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 e?cacy 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 e?ectiveness 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. MacLeanB,TomazelaDM,ShulmanN,etal.Skyline:anopensourcedocumenteditor 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 ?uid 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 ?uid isolated from human skin. Clin Exp Dermatol. 2006;31(3):445–448. 12. Kool J, Reubsaet L, Wesseldijk F, et al. Suction blister ?uid as potential body ?uid 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 e?ect 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 e?ects 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 neuroin?ammation. 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 di?erentiation in vitro. Dev Biol. 1998;200(2):247–259. 19. Caterer NR, Graversen JH, Jacobsen C, et al. Speci?city 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 e?ects of intraganglionic injection of calcium/calmodulin-dependent protein kinase II inhibitors on pain-related behavior in diabetic neuropathy. Neuroscience. 2014;256:302–308. 22. CrownED,GwakYS,YeZ,etal.Calcium/calmodulin dependent kinaseIIcontributes 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 spinaldorsalhorncontributestothepainhypersensitivityinducedbyγ-aminobutyric acid type a receptor inhibition. J Neurosci Res. 2013;91(11):1473–1482.

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