Platelet‐rich plasma protects human keratinocytes from UVB‐induced apoptosis by attenuating inflammatory responses and endoplasmic reticulum stress

Although the role of platelet‐rich plasma (PRP) in ultraviolet light B (UVB)‐induced photoaging has been confirmed in many studies, the specific mechanism is still not clear. Therefore, we attempted to investigate the effect and mechanism of PRP on UVB‐induced human keratinocyte (HaCaT cells) apoptosis.

epidemiological data and related literature data, the occurrence and progression of common clinical skin diseases such as polymorphous light eruption, chronic actinic dermatitis, and basal cell carcinoma are associated with skin photoaging. 5 UV light contains UVB (290-320 nm) and UVA (320-400 nm). Generally, UVB has stronger energy and penetration than UVA, so it is more likely to induce homeostasis imbalance of the skin environment and is the leading cause of skin photoaging. 6 Photoaging is linked with the dose and duration of UV irradiation, and UV-induced apoptosis is the main pathophysiological basis of photoaging. 7 Apoptosis, also known as programmed cell death process, is a critical regulator of cell growth and homeostasis. Skin apoptosis plays an important role in maintaining normal cell proliferation, skin thickness, and keratogenesis. 8 The mechanism of apoptosis is very complex and usually relies on the mitochondrial apoptotic pathway and the death receptor (Fas-FasL)-mediated apoptotic pathway. 9 In addition, inflammation and endoplasmic reticulum (ER) stress are also involved in the pathway of apoptosis. On the one hand, excessive inflammatory response induces cell damage and affect the process of apoptosis. 10 On the other hand, there is a close link between the ER and mitochondria, so ER stress mediates apoptosis by activating the mitochondrial apoptotic pathway. 11 Platelet-rich plasma (PRP) is a platelet concentrate obtained from venous blood by two gradient centrifugations, in which the concentration of platelets is about five times that of blood. 12 In addition to generating gels through activators such as CaCl2 and thrombin, PRP can also release more than 20 high-concentration growth factors like platelet-derived growth factor (PDGF), transforming growth factorbeta (TGFβ), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) to promote tissue regeneration and repair.
Moreover, a large number of studies have confirmed that PRP can stimulate tissue regeneration and promote early healing through the above growth factors but not induce malformations and tumors. 13 The effectiveness of PRP in repairing photoaged skin and the wide application in cosmetic dermatology have been confirmed by several clinical trials. However, the specific mechanism of action for PRP is not clear. An increasing number of studies still focus on elucidating the molecular mechanism of PRP repairing photoaged skin. [14][15][16] Therefore, a UVB-induced photoaging model was established to observe and preliminarily explore the effect and mechanism of PRP on UVB-induced apoptosis of human keratinocytes (HaCaT cells) in this paper. Briefly speaking, the objective of this study was to provide a theoretical basis for PRP repair of skin photoaging.

| Cell culture
HaCaT cells were purchased from China Center for Type Culture Collection (Wuhan University). They were cultured in a DMEM (Dulbecco's modified Eagle's medium, Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), streptomycin (100 U/ml), and penicillin (100 U/ml) (Gibco, USA). The medium was placed in a humidified incubator with 5% CO 2 and 95% relative humidity at 37°C. All experiments were performed using cells between passages 2 and 7.

| Experimental grouping
HaCaT cells were divided into following four groups: (1) Sham group: HaCaT cells were cultured normally; (2) 5% PRP group: Cells were treated with 5% PRP for 24 h; (3) UVB group: Cells were treated with 100 mJ/cm 2 of UVB radiation at a distance of 10 cm; (4) UVB + 5% PRP group: Cells were treated with 5% PRP for 24 h after 100 mJ/ cm 2 of UVB radiation at a distance of 10 cm.
Ten young healthy male volunteers aged from 20 to 30 years donated 10 ml of whole blood, respectively. The donated blood was collected into anticoagulant tubes and centrifuged to collect plasma.
After re-centrifugation of the plasma, PRP was obtained, and the concentration of platelets was measured. Next, platelet poor plasma (PPP) was added until the concentration of platelets in PRP was adjusted to 5%. 17 Finally, PRP was activated by thrombin and calcium, The study was in line with the Declaration of Helsinki, and the informed consent was obtained from all volunteers before blood collection.

| MTT assay for cell proliferation
Cells in the logarithmic phase were seeded in a 96-well plate at 1 × 10 4 cells/well. Next, MTT kits (Beyotime, Shanghai) were applied to measure cell proliferation. Specifically, 10 μl MTT solution was added to the cells in each well, subsequent to 4 h of culture. After absorbing the medium in the wells, 100 μl Formanzan dissolving solution was added to the incubator for 4 h of incubation with cells.
Finally, absorbance values of each well were measured at 490 nm using a Multiskan FC microplate reader (thermo fisher, USA).

| Apoptosis detection by flow cytometry
Cells in logarithmic phase were seeded into 96-well plates at a density of 1 × 10 4 cells/well. After the above treatment, cells were washed with PBS buffer pre-warmed at 37°C and centrifuged twice.
Briefly, 1 × Annexin V Binding Solution was added and blown evenly; then, the suspension (1 × 10 6 cells/ml) was prepared with 1 × Binding buffer. Later, 100 μl cell suspension was added into the corresponding Falcon test tubes, and each tube was supplemented with 5 μl

| ELISA for detecting cellular inflammatory factor levels
Cells in the logarithmic phase were seeded in 6-well plates at 1 × 10 6 cells/well, and after the above treatments, they were re-cultured with fresh DMEM medium for 12 h. Then, the supernatant of each well was collected, and the levels of TNFα, IL-18, IL-6, and IL-1β were tested via an ELISA kit (Nanjing Jiancheng) in strict accordance with the manuals.

| Protein expression levels detected by Western blot
Cells in the logarithmic phase were seeded in a 6-well plate at a density of 1 × 10 6 cells/well, and after different treatments, they were washed twice with PBS then lysed with RIPA lysis buffer and PMSF (BioSharp, China). Later, cell lysate was centrifuged at 12 000 g for

| Statistical analysis
Statistical analysis was performed using SPSS 20.0 software. Oneway analysis of variance was used for comparisons among multiple groups and Tukey post hoc test for those between two groups.
The results were expressed as mean ± standard deviation (SD), and p < 0.05 was recorded as statistically significant.

| Platelet-rich plasma (PRP) promotes proliferation levels of ultraviolet light B (UVB)irradiated HaCaT cells
Based on MTT assay results, the proliferation level of cells was much lower in the UVB group than in the sham group (p < 0.05) while significantly higher in the UVB + 5% PRP group than in the UVB group (p < 0.05). Additionally, though 5% PRP group showed slightly higher proliferation level of cells than the sham group, the difference was not statistically significant (p > 0.05) (Figure 1).

| Platelet-rich plasma decreases ultraviolet light B-induced inflammatory responses in HaCaT cells
As the results of ELISA test shown in Figure 3A

| Platelet-rich plasma attenuates ultraviolet light B-induced endoplasmic reticulum stress in HaCaT cells
According to the outcomes of Western blot ( Figure 4A-D

| DISCUSS ION
Long-term exposure to UVB can lead to severe oxidative stress and inflammatory reactions in the skin, thereby inducing skin aging, impaired barrier function, and acute damage such as erythema, edema, blisters, and pain. By virtue of the functions in decomposing collagen and elastin, UVB also causes some chronic changes such as dry and rough skin, deepened wrinkles, and pigmentation; as for people with serious chronic symptoms, long-time UVB irradiation even may induce cancer. 19 Therefore, the study of photodamaged skin regeneration has always been much focused in the cosmetic medicine industry. PRP, containing bioactive components like PDGF, TGFβ, VEGF, EGF, and basic fibroblast growth factor (bFGF), is involved in regulating processes such as cell migration, proliferation, and differentiation. 20  as the mitochondrial pathway) is believed as one of the mechanisms of UVB radiation-induced apoptosis; notably, the release of Cyt.c is considered as one of the important links in this mechanism. 22 Many scholars claimed that mitochondrial Cyt.c is released through permeability transition pore (PTP), and Bcl-2 family proteins directly regulate PTP. In fact, Bax is a pro-apoptotic protein which can eliminate the membrane potential of mitochondria and promote the release of Cyt.c by affecting PTP. As for Bcl-2, it is an anti-apoptotic protein that can preserve the membrane potential and block the release of Cyt.c. 23,24 In this study, PRP significantly decreased the protein levels of Bax and Cyt.c while notably and activation of neutrophil and other phagocyte; besides, they also result in increased inflammatory cells in the dermis, trigger inflammatory damage to the skin, induce HaCaT cell damages, and accelerate skin aging. 26,27 Furthermore, UVB irradiation can increase the secretion of TNFα protein and significantly expand total mRNA expression. Once the increased TNFα binds to the corresponding specific receptor, the process of apoptosis in HaCaT cells will be accelerated. 28 In this study, PRP significantly decreased the levels of TNFα, IL-18, IL-6, and IL-1β induced by UVB in HaCaT cells, suggesting that PRP could improve the apoptosis by alleviating the inflammatory response triggered by UVB-induced HaCaT cells.
ER stress is also one of the mechanisms of UVB-induced apoptosis in photoaging model cells. Prolonged UVB irradiation causes ER stress, and excessive or prolonged ER stress leads to apoptosis by activating the mitochondrial apoptotic pathway. Generally speaking, ER stress activates different apoptotic signaling cascades mainly through mediating the activation and expression of the C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153 pathway, c-Jun N-terminal kinase pathway, and ER-associated caspase pathway. 29,30 GRP78, CHOP, and ATF4 are key factors involved in ER stress. GRP78 is an ER chaperone, and increased GRP78 expression due to ER stress can further activate ATF4 and ATF46 to bind to related response elements of ERS as transcription factors, thereby modifying or regulating the folding of proteins. 31 CHOP is a specific mediator for the ER stress-induced apoptosis pathway. 32 In this study, PRP could improve UVB-induced apoptosis by significantly reducing the GRP78, ATF4, and CHOP protein expression levels and the ER stress in UVB-induced HaCaT cells.

| CON CLUS ION
In summary, PRP can promote the proliferation level of UVBirradiated HaCaT cells, protect HaCaT cells from UVB-induced apoptosis, thereby repairing cellular photoaging. The mechanism of PRP affecting cellular photoaging may be achieved through inhibiting UVB-induced inflammatory response and ER stress in HaCaT cells.

AUTH O R CO NTR I B UTI O N S
Guang-Hua Duan and Ai-Cui Du conceived and designed study.

Bin Du and Wen Shao collected and analyzed the data. Zhao-Qi
Ren and Hai-Jiao Dong drafted the article. All authors reviewed the manuscript.

ACK N OWLED G M ENT
Not applicable.

FU N D I N G I N FO R M ATI O N
None.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C A L A PPROVA L
Not applicable.