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Abstract
Acne is a chronic inflammatory skin disease of the sebaceous glands of the hair follicles, caused by a variety of factors and tends to recur, causing skin damage and psychological stress to patients. Blue light (415nm) is a popular physical therapy for acne, however, studies on the effects of blue light on skin surface lipids (SSL) have not been exhaustively reported. So, we want to investigate the difference in SSL before and after acne treatment with blue light and to reveal the potential mechanism of acne treatment with blue light from the lipid level. SSL samples were collected and physiological indicators (moisture content, transepidermal water loss (TEWL), sebum content and pH) were measured. By using ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) with multivariate data analysis methods to obtain specific information on the lipid composition. Analysis of the physiological index data showed a significant increase in moisture content (p = 0.042), pH (p = 0.000) and a significant decrease in sebum content(p = 0.008) in the after treatment area (AT group), while there was no significant change in TEWL values. A total of 2398 lipids were detected by lipidomics analysis and 25 differential lipids were screened. Triradylglycerols (TGs), isoprenoids and hopanoids being the potential differential lipids. Among the lipids associated with the skin barrier, only monounsaturated fatty acids (MUFA) (p = 0.045) were significantly increased. This study revealed significant changes in SSL after blue light treatment for acne, suggesting that blue light exposure may cause changes in the relative lipid content and redistribution of lipid components, and that whether it damages the skin barrier requires further study.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Acne is a common chronic inflammatory skin disease of the sebaceous glands and hair follicles [1,2]. The main clinical manifestations are comedones, papules, pustules, and so on. The pathogenesis involves four key factors: increased sebogenesis, abnormal keratinization of the follicular sebaceous ducts, proliferation of Propionibacterium acnes (P.acnes) and an inflammatory response [3,4]. Of these, increased sebogenesis is thought to be a major causative factor in the development of acne [5–9]. And sebum is an important substrate and provides favorable conditions for the growth of P.acnes [10], it closely related to the development of acne.
Blue light (BL) therapy at 407∼ 420nm is a new physiotherapy method that has been gradually promoted in clinical in recent years. Mu YL [10], Deng JH [11], Huang L [12], Danile RO [13], Goldberg DJ [14], Kwon HH [15] et al. observed the efficacy of BL in the treatment of acne, they all concluded that visible light therapy was more effective in the inflammatory lesions of common acne, and no significant side effects were found. BL (415 nm) acts on endogenous porphyrins (mainly coproporphyrin III, with absorption wavelengths peaking at 320 nm UV and 415 nm BL) produced by P. acne metabolism to stimulate photodynamic effects and transform them into unstable porphyrins. Then they combine with trimorphic oxygen to form unstable monomorphic oxygen, which binds to compounds on the cell membrane and damages the cell membrane, leading to bacterial death and inhibiting the proliferation of P. acne [12]. However, the amount of P. acne was not directly related to the severity of acne, whereas the amount of sebum production was positively correlated with it [10]. It has also been found [16,17] that acne is associated with alterations in the composition of SSL and that quantitative and qualitative modifications of sebum may trigger inflammation and lead to the development of acne lesions. Above findings suggest that in addition to observing the inhibitory effect of BL on P. acne, we should also focus on its effect on abnormal sebum secretion, which is the main causative factor in the development of acne. However, studies and reports on this subject are still incomplete.
SSL is located on the outer layer of epidermal cells and is a mixture of sebaceous gland lipids (75%∼90%) and intercellular lipids (10%∼25%), and changes in its content and composition can cause skin problems and even skin diseases [18]. The concept of lipidomic was introduced firstly by Han and Gross in 2003 [19], which is the study of lipids in organisms at a systems level to reveal their interactions and interactions with other biomolecules. With the development of lipidomic analysis techniques and intensive research on lipid metabolism, it is becoming clear that many physiological disorders are closely related to abnormal lipid metabolism. Earlier studies in our laboratory using UPLC-QTOF-MS have revealed abnormality in SSL in young male acne patients [20] and differential lipids associated with the development of acne during adolescence [21]. Therefore, this project used UPLC-QTOF-MS technique to analysis the differential lipids on the SSL of acne patients before and after BL treatment to investigate the effect of BL irradiation on the SSL composition of acne, to reveal the potential mechanism of BL treatment for acne at the lipid level and provide new theoretical insights for the clinical treatment of acne.
2. Materials and methods
2.1 Chemicals and reagents
Methanol (MeOH), ammonium formate (HCO2NH4), isopropyl alcohol (iPrOH), acetonitrile (AN), and formic acid (HCOOH) were of LC-MS grade and were purchased from Thermo Fisher Scientific (Massachusetts, USA). Sebutape were purchased from Cuderm Corporation (Dallas, USA).
2.2 Experimental design
This study was approved by the Air Force Medical Center Ethic Committee (2021-162-PJ01) and was conducted in agreement with the principles expressed in the Declaration of Helsinki. All volunteers were fully informed about the purpose of the experiment and signed informed consent forms. A total of eleven volunteers with mild to moderate acne participated in the study (aged 25.27 ± 6.21 [range, 19∼43], four males and seven females).
The experiment was divided into three groups according to the samples collected at different times: Before Treatment area (BT group), Untreated area (UT group) and After Treatment area (AT group). BT group lipid samples were collected and basic physiological indicators such as moisture content, TEWL, sebum content and pH were measured. All subjects received a total of eight therapy treatments at Air Force Medical Center. BL treatment was two times per week for the four treatment weeks: BL (415 ± 5 nm) (Omnilux blue, Photo Therapeutics Led., Fazeley, UK) treatment, therapy session was 20 minutes duration, and irradiance was 40 mW/cm2. So, all subjects received dose of 48J/cm2 per treatment. The experiment was a random half-face control and the volunteers were numbered, with a single number treating the left face and a double number treating the right face. UT and AT group lipid samples were collected and the above physiological indicators were measured at 24 hours after the 8th treatment. The specific sampling sites and times are shown in Fig. 1 and Table S1.
2.3 Sample collection and preparation
Before sample collection, participants were asked to cleanse their faces with water, and sit at a constant temperature of (21 ± 2)°C and relative humidity of (50 ± 10)% for 30 min. Then the experimenter placed a Sebutape test strip on volunteer’s cheek, and removed strip after 3 minutes. Samples were stored at -80°C. The SLL on Sebutape were collected by a modified Blight and Dyer [22] mothed as previous study [20,23].
2.4 Physiological indicators measurement
All physiological indicators measurements were performed at subject’s cheek. Using Corneometer (CM825; CK, Germany) measured skin moisture content. After the detection probe was placed on the target area, five consecutive readings were collected from the same site and averaged for each participant. Using Tewameter (TM300; CK, Germany) measured transepidermal water loss (TEWL). After the detection probe was placed on the target area, three consecutive readings were collected from the same site and averaged for each participant. Using Sebumeter (SM815; CK, Germany) measured sebum content. And Skin-pH-Meter (pH905; CK, Germany) measured pH.
2.5 Instrument parameters
SSL were analyzed using Waters ACQUITY UPLC I-Class. Injected 2.0µL sample in Waters UPLC CSH C18 (2.1mm×100mm, 1.7µm) and kept column at 60°C. The flow rate was maintained at 0.4mL/min. The gradient elution program is shown in Table 1
Table 1. UPLC elution program
The mass spectrometer was performed using Waters Xevo G2-XS QTOF-MS (Waters Corporation, Milford, Massachusetts, USA) for high resolution mass detection with an electrospray ionization (ESI) in positive ion mode. Leucine encephalin (m/z = 554.2771) was used as an external standard for accurate mass calibration. Quality control (QC) were prepared by each of SSL with equal volumes for evaluation of system stability. The parameters of QTOF-MS were shown in Table 2.
Table 2. Selected QTOF-MS parameters
2.6 Data extraction and analysis
The raw data collection was used MassLynx 4.1 (Waters Corporation) software and extraction and analysis were performed by Waters Progenesis QI V2.0 and Ezinfo 3.0 (Waters Corporation, Milford, Massachusetts, USA). A comparison with the LIPID MAPS (http://www.lipidmaps.org/) Structure Database (LMSD) was performed to identify the compound ID of lipid composition of each sample. Variable influence on projection (VIP) > 1, p < 0.05 and fold change > 2 were used for selection criteria to find the potential differential metabolites. Statistical significance was computed by T-test of SPSS v21.0 and p-values were obtained, *p < 0.05, **p < 0.01, ***p < 0.001, error bars represent standard errors of the means.
3. Results
3.1 Fine stability of UPLC-QTOF-MS
Results showed that QCs almost coincided with each other in Score plots analysed by Principal Component Analysis (PCA) (Fig.S1), which indicated the fine stability of instrumentation. 2398 lipids were identified and they were classified into 8 categories by LIPID MAPS: fatty acyls [FA], glycerolipids [GL], glycerophospholipids [GP], sphingolipids [SP], sterd lipids [ST], prenol lipids [PR], saccharolipids [SL], polyketides [PK].
3.2 Reliability of research design
A total of eleven volunteers were participated for this study (four male and seven female), with a total sample of 33. The PCA plot (Fig. S1) indicates that there were not separated between BT and UT groups and clearly separated between BT and AT groups. And the relative total lipid content was significantly higher in the AT group compared to the BT (p = 0.017) and UT (p = 0.022) groups, and there was no difference between UT and BT group (Fig. S2). So, following the BT group was used as a control for its own before and after analysis with the AT group.
3.3 Analysis of physiological indicators
Observation changes of skin moisture content, TEWL, sebum content and skin surface pH before and after BL treatment for acne (Fig. 2), which showed a significant increase in skin moisture content (p = 0.042) and pH (p = 0.000) in AT group, and a significant decrease in sebum content (p = 0.008), but no significant change in TWEL.
Fig. 2. Physiological parameters. (A) Moisture content of the skin between BT and AT group. (B) TEWL of BT and AT group. (C) Sebum content of BT and AT group. (D) Skin pH of BT and AT group. There was a significant increase of moisture content and pH, and a decrease of sebum in AT group. ***p < 0.001; ** p < 0.01; * p < 0.05.
3.4 Differential lipid analysis before and after (after 24 h) blue light (415 nm) treatment
The OPLS-DA score plot (Fig. 3) showed better separation between the BT and AT groups. The relative contents of the eight categories in the BT and AT groups are shown in Fig. 4, of these, five categories of lipids, [FA], [GP], [ST], [PR] and [PK], were significantly different compared to before treatment (p < 0.05).
Fig. 3. There was a significant difference in the relative content of total lipid between the BT and AT groups. The OPLS-DA scores of BT and AT groups. Training set model obtained from lipid species of SSL, which showed obvious separation between BT (blue squares) and AT (green squares).
Fig. 4. A total of 2398 kinds of lipids were identified by matching the data analysis results with LMSD, which were divided into eight categories (fatty acyls [FAs], glycerolipids [GLs], glycerophospholipids [GPs], sphingolipids [SPs], sterol lipids [STs], prenol lipids [PRs], saccharolipids [SLs] and polyketides [PKs]). The variations eight category of lipid were calculated for the two groups. ***p < 0.001; ** p < 0.01; * p < 0.05.
Subsequently, we analyzed the categories of the five lipids with significant changes. A total of sixteen main classes were identified with significant differences (p < 0.05), of which fourteen main classes (other fatty acyls [FA00], fatty acids and conjugates [FA01], fatty alcohols [FA05], fatty amides [FA08], other glyceropholipids [GP00], Glycerophosphoglycerols [GP04], Glycerophosphoinositols [GP06], sterols [ST01], bile acids and derivattives [ST04], Isoprenoids [PR01], Hopanoids [PR04], polyether antibiotics [PK09], flavonoids [PK12], phenolic lipids [PK15]) shared the same trend with their categories ([FA], [GP], [ST], [PR], [PK]), as shown in Fig. S3.
As shown in Table 3, 25 individual lipid species were identified with VIP >1, p < 0.05, Fold Change >2. They were divided into 5 categories ([FA], [GL], [GP], [SP], [PR]), as shown in Fig. 5(A). There was significant difference in two categories ([GP] and [PR]), among them, Glycerophosphoglycerols [GP04], Glycerophosphoinositols [GP06], Isoprenoids [PR01] and Hopanoids [PR04] were identified with significant differences (p < 0.05) (Fig. 5(B)) and shared the same trend with their categories.
Fig. 5. 25 individual lipid species were identified. (A) The important individual lipid species between BT and AT groups. (B) The main classes of two categories significantly. (C) Pie chart showing the proportion of each important lipid. Among them, the TGs is the most important lipid. (D)The relative abundance of TGs of BT and AT groups. ***p < 0.001; ** p < 0.01; * p < 0.05.
Table 3. 25 individual lipid species were identified
25 individual lipids were divided 11 main classes, and triradylglycerols (TGs) dominated these lipids (Fig. 5(C)), which is necessary to pay attention to. Through statistical comparisons, we observed that the relative content of TGs was higher in AT group than BT group (Fig. 5(D)).
3.5 Analysis of lipids related to skin barrier
An important feature of the skin of people with acne is the reduced skin barrier function. The carbon length and content of ceramides (Cers) and fatty acids (FFAs) in SSL, and saturation of fatty acids (SFAs) were considered to play an important role. Our laboratory observed that [20] Chinese men and women aged 18∼25 years with acne showed a significant decrease in the carbon chain length of Cers, a significant decrease in the relative content of SFAs and a significant increase in the relative content of unsaturated fatty acids (UFAs). After 24h of BL treatment for acne (AT group), there was no significant change in the relative content and mean carbon chain length of Cers (Fig. 6(A)/B), no significant change in the carbon chain length of FFAs (Fig. 6(D)/E/F), no significant change in the relative content of SFAs and polyunsaturated fatty acids (PUFAs) (Fig. 6(C)), but a significant increase in the relative content of monounsaturated fatty acids (MUFAs) (p = 0.045).
Fig. 6. Lipid analysis in relation to the skin barrier. (A) Ceramides relative abundance of BT group and AT group. (B) Dot plots of the calculated average ceramides chain length for BT group and AT group. There was no significant change in average ceramides chain length in two groups. (C) SFA, MUFA and PUFA relative abundance of BT group and AT group. There was a significant decrease of relative abundance of MUFA in AT group with BT group. (D) (E) (F) Dot plots of the calculated average free fatty acid chain length for AT group with BT group. ***p < 0.001; ** p < 0.01; * p < 0.05.
4. Discussion
There are many factors that influence the occurrence of acne. Milk and dairy intake, body mass index (BMI), length of sleep, and stress are all potential factors. It is generally accepted that increased lipid secretion, abnormal keratinization of the follicular sebaceous ducts, proliferation of P.acnes, and an inflammatory response are the four key factors. Among them, increased sebum production considered to be one of the main factors contributing to acne [6–9]. BL (415nm) is considered safe and effective in the clinical treatment of acne [24]. Total sebum was significantly reduced after BL treatment (Fig. 2(C)), consistent with previous studies.
From the analysis of 2398 lipids and 25 potentially differential lipids, we identified TGs, Isoprenoids and Hopanoids as important lipids contributing to the grouping. It is generally believed that [25,26] P.acnes produces a variety of enzymes that break down TGs in sebum, producing FFAs that induce skin inflammation and abnormal follicular keratinization. This was also demonstrated in our previous study, where acne samples had lower relative levels of TGs than healthy samples. In contrast, the relative content of TGs increased after BL exposure, indirectly suggesting that BL may kill P.acnes and thus prevent it from using TGs. Therefore, inhibition of the conversion of TGs to FFAs by BL damage to P.acnes may be an effective way to improve acne.
Compared to the healthy population, acne patient samples had significantly lower [PR] [27], while BL treatment resulted in significantly higher [PR]. This is a category of lipids found to be restored to the level of skin lipids in healthy people, and it is speculated that BL treatment of acne may have stimulated the resynthesis of this category lipids.
Our previous study found that both young men and women with acne had significantly higher [GP] compared to the healthy population, and the results of the present study showed a trend towards a significant increase in [GP] following BL treatment of acne. The correlation between [GP] and the occurrence and development of acne needs to be further investigated.
Visible light therapy for acne is considered a safe and effective physical therapy because it specifically kills P.acnes in a short period of time while protecting other skin tissues from being affected [11]. Cers and FFAs are important lipids associated with the skin barrier. Long chains of Cers maintain the skin barrier and a weakened Cers chain length will result in increased TEWL and impaired skin barrier function [28,29]. In contrast, no reduction in Cers chain length (Fig. 6(B)) or increase in TEWL (Fig. 2(B)) was found before or after BL treatment for acne, suggesting that the process of BL treatment for acne does not affect changes in Cers or further damage to the skin barrier via Cers. SFAs are involved in the immune response and have antibacterial properties, while UFAs induce inflammation, increase TEWL and lead to reduced skin barrier function [26,30]. The results of the study showed (Fig. 6(C)) no significant change in the relative levels of SFAs and PUFAs, while MUFAs increased significantly, but this did not cause an increase in TEWL. Therefore, it remains to be investigated whether MUFAs are a contributing factor to skin barrier damage.
In addition, it was found that [31] P.acnes is found in the sebaceous gland area, with an optimum growth temperature of 37°C and a pH of 4.20 to 5.90. The pH of the skin surface of acne patients was 4.44∼5.56, which is the suitable environment for the growth of P.acnes. After a course of BL irradiation, the pH of the skin surface changed to 5.40∼6.20 and showed a significant change (Fig. 2(D)), thus indicating that BL changed the growth environment of P.acnes and controlled the occurrence of acne. In addition, pH can alter lipid synthesis in the epidermis [32], demonstrating that BL treatment of acne caused a change in lipid content and redistribution.
5. Conclusion
In summary, our study clearly reveals significant changes in SSL after one cycle of BL (415nm) treatment for acne patients. Analysis of 25 differential lipids indicated TGs, Isoprenoids and Hopanoids as potential differential lipids. BL treatment at 415 nm did not cause an increase in TEWL although it caused an increase in the relative content of MUFAs. Whether BL treatment for acne can damage the skin barrier needs further study.
Funding
Beijing Technology and Business University.
Acknowledgments
Author contributions. Wenyu Ding, Yiqiong Hu and Xiaoqian Yu performed the experiments. Wenyu Ding analysed the date and wrote the manuscript. Congfen He and Yan Tian designed and supervised the study, revised the manuscript.
Ethical approval. This study was approved by the Air Force Medical Center Ethic Committee (2021-162-PJ01) and was conducted in agreement with the principles expressed in the Declaration of Helsinki.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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