|
Cell Physiol Biochem:拔罐療法的科學原理是什麽?這個研究給出了答案
2018-2-23 作者:雍黎 來源:科技日報
中醫拔罐治療有什麽科學依據和作用原理?2月22日,記者從陸軍軍醫大學第二附屬醫院(重慶新橋醫院)獲悉,該院全軍腫瘤研究所李詠生團隊率先使用小鼠拔罐模型,運用超高效液相-質譜聯用儀建立的脂質代謝組學平台,揭示了拔罐療法導致體內抗炎/促炎脂質代謝譜的變化規律,為拔罐療法的潛在機製提供了科學支撐。研究論文已於20日在《Cell Physiol Biochem》雜誌發表。
據了解,在以往的報道中,拔罐療法研究者的關注點多在於拔罐處的皮膚溫改善、血壓、熱效應以及血氧含量或者受試者的客觀感受評分。該團隊使用小鼠拔罐模型,運用超高效液相-質譜聯用儀建立的脂質代謝組學平台研究發現,拔罐後健康小鼠體內抗炎脂質(如PGE1, 5,6-EET, 14,15-EET, 11 10S,17S-DiHDoHE, 17R-RvD1, RvD5和14S-HDoHE),顯著升高,而促炎脂質(如12-HETE和TXB2)明顯下調。
通過體外實驗,課題組進一步發現拔罐療法能減少脂多糖誘導的腹膜炎老鼠模型腹腔液中的促炎介質TNF-α及IL-6的產生。該研究說明拔罐可引起體內抗炎、促消退脂質成分的升高,促炎脂質的減少,為其促進機體 免疫自穩提供了科學依據。
原始出處:
|
Anti- Versus Pro-Inflammatory Metabololipidome Upon Cupping Treatment
Zhang Q.a · Wang X.a · Yan G.a · Lei J.a · Zhou Y.a · Wu L.a · Wang T.a ·Zhang X.a · Ye D.b · Li Y.a |
|
Cell Physiol Biochem 2018;45:1377–1389
Previous researches mainly focused on its role in improving skin temperature [5], plasma pressure [5], heat effect [12], and plasma oxygen in local sites [7], and subjective human feeling indices (e.g., pain scores [4], visual analogue scale [8], numerical rating scale [6]). Although these indices can quantify the therapeutic effect, the fundamental mechanism remains unclear.
Homeostasis is delicately regulated by pro- and anti-inflammatory lipids [13]. The temporal and differential levels of lipid mediators also represent the stage of inflammation [14]. The metabolites derived from ω-3, 6, and 9 polyunsaturated fatty acids (PUFAs; e.g. linoleic acid (LA), arachidonic acid (AA), eicosapntemacnioc acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA)) are essential lipid mediators involved in inflammation initiation and resolution. For example, ω-3 PUFAs potentially exert anti-inflammatory activities and have promising benefits in various inflammatory human diseases such as diabetes, atherosclerosis, asthma, and arthritis [15]. Deficiencies of ω-3 PUFAs contribute to several chronic inflammatory diseases, including obesity and diabetes [16]. Leukotriene B4 (LTB4), 5-HETE and prostaglandin E2 (PGE2) are pro-inflammatory, while lipoxin A4 (LXA4) and PGI2 are anti-inflammatory metabolites derived from AA, an ω-6 PUFA [13, 17, 18]. These relationships between tissue homeostasis and lipid metabolism inspire us to address whether cupping treatment modulates the metabolic balance between pro- and anti-inflammatory PUFAs.
Here, we employed reversed-phase ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), a sensitive and powerful technique, which provides a platform to identify the PUFA metabolome in nude mice. Our results demonstrated that several anti-inflammatory lipids (e.g. PGE1, 5, 6-EET, 14, 15-EET, 10S,17S-DiHDoHE, 17R-resolvin D1 (RvD1), RvD5 and 14S-HDoHE) were increased and many pro-inflammatory lipids (e.g. 12-HETE and Thromboxane B2 (TXB2)) were deceased in the skin and plasma post cupping treatment. Moreover, PGE1, 5, 6-EET, 5, 6-DHET and 12-HETE differentially regulated interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) production from RAW264.7 macrophages. These findings identified the anti- versus pro-inflammatory metabololipidome upon cupping treatment, and suggested potential PUFA-derived lipid mediators that function as diganostic biomakers and therapeutics compounds in cupping treatment.
Materials and Methods
Chemicals and Reagents
Formic acid (>99%), methyl formate, hexane, 2-propanol, acetonitrile, chloroform and methanol (all HPLC-MS grade) were purchased from Honeywell (New Jersey, USA). SepPak C18 SPE Cartridges (500 mg, 6mL) were purchased from Waters (Hertsfordshire, UK). Lipid mediators including 12-HETE (12-hydroxy-5Z,8Z,10E,14Z -eicosatetraenoic acid), 14, 15-EET (14, 15-epoxy-5Z,8Z,11Z-eicosatrienoic acid), 5, 6-EET (5, 6-epoxy-8Z,11Z,14Z-eicosatrienoic acid), 5, 6-DiHET (5, 6-dihydroxy -8Z,11Z,14Z-eicosatrienoic acid), 10S,17S-DiHDoHE (10(S),17(S)-dihydroxy -4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid), 14(S)-HDoHE (14S-hydroxy -4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid), 17(R)-RvD1 (7S,8R,17R -trihydroxy-4Z,9E,11E,13Z,15E19Z-docosahexaenoic acid), RvD5 (7S,17S -dihydroxy-4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid), PGE1 (9-oxo-11α,15S -dihydroxy-prost-13E-en-1-oic acid), TXB2 (9α,11, 15S-trihydroxythromba -5Z,13E-dien-1-oic acid), 20-HDoHE (20-hydroxy-4Z,7Z,10Z,13Z,16Z,18E -docosahexaenoic acid), and lipid standards were obtained from Cayman Chemicals (Ann Arbor, MI, USA). All the lipid standards were dissolved in methyl formate or methanol as a premixed solution and stored at -80°C in glass tubes.
Results
Lipidomics analysis in mice before and after cupping treatment
To investigate the effect of cupping treatment on lipid metabolism, we used UPLC-MS/MS to analyze the PUFA metabolome in mice before and after cupping treatment. The skin and plasma from untreated nude mice were collected as control (Ctrl). The dark red spots (treated skin, TS) and adjacent skin (AS), as well as the plasma of the cupping treated nude mice were harvested as treatment group. 64 representative metabolites of AA, EPA, DHA and other PUFAs in mice were evaluated, with a total of 30 kinds of lipids including LTB4, 11-HETE, 20-HDoHE, TXB2, 5, 6-EET, 14, 15-DHET 5, 6-DHET, 12-HETE, 5-HETE, PGF1α, PGD1, PGD2, 11, 12-DHET, LXB4, 14, 15-EET, 12-HEPE, 8S-HEPE, 15-HEPE, PGE1, 4-HDoHE, 17R-RvD1, RvD5, 14S-HDoHE, 16-HDoHE, 17-HDoHE, 7-HDoHE, 10S,17S-DiHDoHE, PGE2, 5S,15S-DiHETE, 15S-HETrE in skin and 24 kinds of lipids including LXB4, 12-HETE, PGE2, TXB2, 5, 6-EET, 5, 6-DHET, 14, 15-EET, 14, 15-DHET, 11, 12-DHET, 11-HETE, 5-HETE, 8, 9-DHET, 5-HEPE, 18-HEPE, 11-HEPE, 12-HEPE, 4-HDoHE, 7-HDoHE, 17-HDoHE, 20-HDoHE, 16-HDoHE, 13-HDoHE, 14S-HDoHE, and 9-HODE in plasma were unambiguously identified and quantified in this study (Table 1 and 2, Fig. 2 and (for all online suppl. material, see www.karger.com/doi/10.1159/000487563) Fig. S1). The levels of PUFA metabolites were quantified and subjected to heatmaps by using MetaboAnalyst 3.0. The heatmaps showed that the levels of these PUFA metabolites were differentially changed in the skin and plasma of the nude mice after cupping treatment (Fig. 2B).
Table 1.
The levels of PUFA metabolites (ng/ml) in mice plasma before and after cupping treatment
Table 2.
The levels of PUFA metabolites (ng/mg protein) in control (Ctrl), cupping treated-(TS) and adjacent-skin (AS)
Fig. 2.
The PUFA metabolome in mice skin and plasma before and after cupping treatment. A. Representative MRM chromatograms show the retention times for each identified bioactive LMs: Q1, M-H (parent ion); and Q3, diagnostic ion in the tandem mass spectrometry (MS/MS) (daughter ion). Representative metabolites of AA, EPA, DHA and other PUFAs. B. The heatmap of PUFA metabolome in mice skin (left panel) and plasma (right panel) with or without cupping treatment. Results were expressed as mean of n=5 mice each group.
Anti- and pro-inflammatory lipids in skin after cupping treatment
To analyze the PUFA metabolites in mice skin tissue with or without cupping treatment, we further compared the above identified lipids in TS, AS and Ctrl. PCA was used to distinguish the differences of PUFA metabolites in all analyzed mice skin samples in 2- and 3-dimensional scatter plots. The principal component 1 (PC1) was the axis, which contained the largest possible amount of information and PC2 was perpendicular to PC1. The principal components were orthogonal and linear combinations of the original variables. PCA score plots were used to reflect the relationship of PUFA metabolites in mice skins with different treatments. The score plots of PCA provided a clear discrimination of these three groups. PC1 and PC2 were able to describe respectively 65.8% and 19.3% of total variance. They accounted for 85.1% of total variance (Fig. 3A). Samples with cupping treatment, including treated skin and adjacent skin were grouped in small regions in the score plot. And the responses of controls are clustered away from those corresponding to the cupping treated groups.
Fig. 3.
The significantly altered PUFA metabolites in mice skin tissue with or without cupping treatment. A. Score plot of principal component analysis based on PUFA metabolites profiling analysis of all mice skin tissue samples (n=5). B. Projection of variables in a two-dimensional loading plot for all measured samples, showing the major variables representing PUFA metabolites concentrations. C. The levels of 12-HETE, 5,6-DHET, PGE1, 5(6)-EET, 14(15)-EET, 17-RvD1, RvD5, 14S-HDoHE and 10S, 17S-DiHDoHE in control (Ctrl), cupping treated-(TS) and adjacent-skin (AS). Results were expressed as mean ± SEM of n=5 mice each group. *P<0.05, **P<0.01, and ****P<0.0001, Ctrl vs TS; #P<0.05, ##P<0.01, ###P<0.001, Ctrl vs AS; *P<0.05, **P<0.01, ***P<0.001, TS vs AS.
We also analyzed the variables (PUFA metabolites contents) in our PCA. The PUFA metabolites with significantly different levels in each treatment were analyzed and scattered at the edges of the loading plot, whereas PUFA metabolites with similar levels in each treatment were gathered in the middle right part of the loading plot. Of note, the PUFA metabolite contents, such as the amounts of 12-HETE, 5, 6-DHET, 17R-RvD1, RvD5, 14, 15-EET, 5, 6-EET, 14S-HDoHE, PGE1 and 10S,17S-DiHDoHE, were the most statistically significant variables (Fig. 3B).
The levels of PUFA metabolites in Ctrl, TS and AS were also accessed with GraphPad Prism 7 software using one-way ANOVA. In accordance with the results of PCA (Fig. 3A and 3B), various lipids increased in both TS and AS, including 5, 6-EET, 14, 15-EET, 10S,17S-DiHDoHE and 14S-HDoHE; while 5, 6-DHET, PGE1, 17R-RvD1 and RvD5 were only up-regulated in AS, but not in TS. In contrast, 12-HETE was down-regulated in AS (Fig. 3C). It has been acknowledgeable that 14, 15-EET [23], 5, 6-EET [24], 5, 6-DHET [25], 10S,17S-DiHDoHE[26], 17R-RvD1 [27], RvD5 [27] and PGE1 [28] are anti-inflammatory while 12-HETE is pro-inflammatory [29]. Together these results showed that cupping treatment increased anti-inflammatory lipids and decreased pro-inflammatory lipids.
Discussion
Cupping treatment is an ancient and traditional physical approach to improve health and maintain homeostasis [7]. To explore its underlying mechanism, we monitored the PUFA metabololipidome with a nude mice model. We quantified the levels of fatty acids in skin or plasma of nude mice before and after cupping treatment and found that numerous fatty acids were differentially regulated. Among these lipids, 14, 15-EET, 5, 6-EET, 5, 6-DHET, 14S-HDoHE 10S,17S-DiHDoHE, 17R-RvD1, RvD5 and PGE1 were increased while 12-HETE was decreased in the skin. 14, 15-EET increased while TXB2 and 20-HDoHE decreased in the plasma. Indeed, cupping treatment reduced the IL-6 and TNF-α production induced by LPS in vivo. We also identified 14, 15-EET, 14S-HDoHE, 17-RvD1, RvD5, PGE1, TXB2 and 12-HETE as potential biomarkers and therapeutic compounds in cupping treatment.
The homeostasis is governed by the balance between pro- and anti-inflammatory mediators [31]. In this study, we identified numerous significantly altered PUFA derived metabolites by cupping treatment. The increased lipids included 14, 15-EET, 10S,17S-DiHDoHE, 17R-RvD1, RvD5, 14S-HDoHE, 5, 6-EET, PGE1, while the decreased lipids were 12-HETE and TXB2. The biofunctions of most of these lipid mediators were investigated previously and introduced below.
14, 15-EET was reported to protect nucleus pulposus cells from death induced by TNF-α in vitro via the NF-κB pathway, and reduced a variety of pro-inflammatory cytokines (e.g., TNF-α, IL-1, IL-6, IL-8) [32]. Furthermore, 14, 15-EET could stimulate the production of 15-epi LXA4 [33], a dual anti-inflammatory and specialized pro-resolving mediator (SPM) that exert an essential role in inhibiting neutrophil activation and restoring homeostasis [13]. Although we did not observe the increase of lipoxins after cupping treatment for 24 hrs, 14, 15-EET significantly reduced IL-6 and TNF-α and in LPS-stimulated macrophages. The up-regulation of 14, 15-EET in both the skin and plasma might lead to their production at subsequent intervals.
10S,17S-DiHDoHE, 17R-RvD1 and RvD5 belong to SPM [26, 34] and 14S-HDoHE is a precursor to maresin 1, another SPM [35]. Although we did not observe significant change in IL-6 and TNF-α production from RAW264.7 cells after these SPM treatment for 12 hrs, 14S-HDoHE, 7R-RvD1 and RvD5 showed their anti-inflammatory function in LPS-stimulated BM macrophages. 14S-HDoHE could decrease the PMN infiltration into inflammatory sites [35]. 10S,17S-DiHDoHE was reported to reduce the severity of colitis via attenuating neutrophil infiltration and decreasing levels of pro-inflammatory cytokines (e.g. TNF-α, IL-1β, IL-6)[26]. 17R-RvD1 is an aspirin-triggered epimer of RvD1 that reduces human PMN migration [36]. It shares the same function of RvD1 which was reported to target pro-inflammatory cytokines (IL-1β, IL-8 IL-6 and TNF-α) and genes [i.e., Chemokine (C-X-C motif) ligand (CXCL9)] as well as persistent STAT3 activation in human inflamed adipose tissue [37]. RvD5 significantly reduced pro-inflammatory cytokines (keratinocyte chemoattractant and TNF-α) and enhanced the human macrophage phagocytosis of E. coli and bacterial killing in mice [34]. The increase of these above SPM delineated the beneficial actions of cupping treatment.
In addition, 5, 6-EET was reported to be anti-inflammatory, it suppressed various pro-inflammatory cytokines such as TNF-α [24]. PGE1 was used to treat some chronic inflammatory diseases [38]. It was reported to protect cells from renal ischemia/reperfusion injury-induced oxidative stress and inflammation [39]. Consistently, in our study, 5, 6-EET and PGE1 significantly suppressed TNF-α production in macrophages.
On the other hand, 12-HETE and TXB2 are well-known pro-inflammatory lipid mediators [29, 30]. Our study showed that they both significantly promoted TNF-α and IL-6 production in macrophages. The reduction of them in the mice plasma suggested the anti-inflammation effect of cupping treatment.
The function of 20-HDoHE and 5, 6-DHET were not clearly elucidated yet. 20-HDoHE is biosynthesized from DHA and was increased during early period of oxidative stress in vitro [40] indicating that it probably played a pro-inflammatory role. It was reported that the nonsteroidal anti-inflammatory drug (NSAID) diclofenac elevated the level of 5, 6-DHET in inflammatory status associated with obesity [25], suggesting the anti-inflammatory role of 5, 6-DHET. In our study, we found 5, 6-DHET decreased IL-6 in RAW264.7 macrophages in vitro, while 20-HDoHE did not significantly alter the levels of IL-6 and TNF-α.
|
|
|