Growth factors‐based platelet lysate rejuvenates skin against ageing through NF‐κB signalling pathway: In vitro and in vivo mechanistic and clinical studies

Abstract Introduction Platelets benefit tissue regeneration by secreting growth factors, and platelet products, for example, platelet lysate (PL), have been clinically applied for tissue rejuvenation. To determine the anti‐ageing efficacy and mechanism of human PL (hPL) on skin, this study conducted clinical retrospective analysis, nude mice‐based in vivo study and human dermal fibroblasts (HDFs)‐based in vitro study. Methods Flow cytometry was employed for quality control of hPL, and ELISA was used for quantification of growth factors (EGF, IGF‐1, PDGF and TGF‐β) in hPL. After d‐galactose modelling, skin texture grading, histopathological observation, immunofluorescence analysis and oxidative stress assays were conducted on nude mice, while SA‐β‐gal staining, CCK‐8 and wound healing assays were conducted on HDFs. qPCR and western blot were conducted to clarify hPL's mechanism. Results The clinical retrospective data showed that hPL obviously rejuvenated human skin appearances without adverse events. The animal data showed that hPL exerted rejuvenative effects on skin, and the cellular data showed that hPL significantly promoted the proliferation and migration of HDFs and suppressed senescence‐associated secretory protein secretion and senescence state of senescent HDFs by suppressing NF‐κB pathway. The NF‐κB‐dependent mechanism was verified positively by using P65 siRNA and negatively by using prostratin. Furthermore, EGF, IGF‐1, PDGF and TGF‐β were found as the main ingredients in hPL, which contributed to the efficacy and mechanism of hPL. Conclusion This study provided novel knowledge of hPL, making it ideal for skin rejuvenation.

Conclusion: This study provided novel knowledge of hPL, making it ideal for skin rejuvenation.

| INTRODUCTION
Skin ageing is the most intuitive consequence of ageing, affecting epidermis, dermis and subcutaneous layer. 1 Dermis layer, containing collagen fibres, elastic fibres and other extracellular matrix (ECM), suffers the most changes of ageing in skin. 2 Human dermal fibroblasts (HDFs) are the primary cell types in dermis layer and are responsible for synthesis of collagen and elastic fibres. 3 With ageing, the quantity and proliferation rates of HDFs are reduced. Once aged, HDFs lose their capacities to produce collagen and release senescence-associated secretory proteins (SASP) and matrix-degrading metalloproteinases (MMPs), resulting in degradation of ECM. 4,5 Phenotypically, skin wrinkles are increased, and meanwhile, skin elasticity and mechanical resistance are decreased. 6 Skin ageing contains extrinsic ageing and intrinsic ageing, both of which involve inflammation, DNA damage and oxidative stress. [7][8][9] Extrinsic ageing is mainly caused by environmental factors, such as UV radiation, while intrinsic ageing is closely associated with cellular senescence. 10 Inflammation and DNA damage are the main causes of cellular senescence in the intrinsic ageing, which produce SASP (e.g., IL-6 and IL-8) with activation of NF-κB signalling pathway. 11 Moreover, reactive oxygen species in oxidative stress causes inflammatory response and MMPs overexpression, resulting in acceleration of ageing process via NF-κB signalling pathway. 12,13 Therefore, NF-κB signalling pathway plays predominant role in the pathogenesis of cellular senescence and SASP release of intrinsic skin ageing, acting as an important target for anti-ageing treatment.
Many therapeutics and techniques have been explored to prevent or reverse skin ageing for several decades. Antioxidants, such as resveratrol and epigallocatechin gallate (EGCG), have been applied to scavenge ROS and suppress inflammation of aged skin. 14,15 Nonetheless, resveratrol is easily metabolized and needs to be packaged by vehicle for use, making it difficult for delivery in clinical applications. 10 The use of EGCG lacks standard of dose range, resulting in uncertain effectiveness in clinic. 12,15 Retinoids, such as retinol and tretinoin, have been applied to treat skin ageing by regulating MMPs activity and promoting collagen production to improve skin textures. 16,17 However, retinoids have risks of inducing dermatitis and erythema in clinic, since their use with inappropriate doses may induce skin irritation. 18 Injective techniques by using botulinum toxin are extensively employed for treating dynamic wrinkles of aged skin. 19,20 Nevertheless, this chemical may cause immediate vision loss and non-hypersensitivity reactions. 21,22 Botulinum toxin is gradually metabolized over time and finally disappears, the effect of which cannot last for a long time. 23 Recently, cell therapy-based techniques have attracted increasing attention in the field of anti-ageing. Injection of platelet-rich plasma (PRP) is the most popular one that can improve facial skin appearance (texture and lines), increase skin thickness and enhance collagen content. 24,25 However, this technique lacks standard of preparation, contains cellular residues and cannot be stored, resulting in less than 50% global improvements rate and unsatisfactory efficacy in clinic. 25,26 Therefore, it is necessarily needed to develop new techniques with certain effectiveness for skin ageing treatment.
Platelet lysate (PL) is a growth factor-released product of platelets and also the next generation of PRP, containing insulin-like growth factor (IGF-1), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor-β (TGF-β), and so forth, by freeze-thawing preparation from platelet concentrates. 27 PL is cell free with advantages over PRP, including: (1) the avoidance of heterologous material uses for PL preparation; (2) the availability of long-term storage of PL in low temperature for consecutive applications and (3) stable release of high concentrations of growth factors that exerts better efficacy and can easily be quality controlled for standardization. 28,29 Previous reports showed that PL exerted regenerative effects on various tissues and cells, such as skin, cartilage, tendon cells and nerve cells. [30][31][32] Recently, our studies demonstrated that PL exerted protective effects on chondrocytes and cartilage against osteoarthritis through regulation of NF-κB signalling pathway, and that PL benefited umbilical cord-derived MSCs (huc-MSCs) by promoting cell proliferation, cell cycle progression and cell migration through activation of AMPK/mTOR signalling pathway-mediated autophagy. 28 Moreover, we found that the growth factors (PDGF, IGF-1, TGF-β and EGF) contributed to the regenerative effects of PL in varying degrees. 28 Of these, PDGF and EGF are capable of promoting collagen deposition and tissue formation in the defect region of skin, resulting in re-epithelialization and skin regeneration. [32][33][34][35] Therefore, PL possesses pro-regenerative effect on skin, which has great potential for skin ageing treatment. To date, there is little study regarding the anti-ageing mechanism of PL.
To determine the anti-ageing efficacy and explore the underlying mechanism of human PL (hPL) on skin, we retrospectively analysed subjects treated with hPL and then established an aged skin model of nude mice and HDFs by using D-galactose (D-gal) for efficacy and mechanism study. D-gal-induced ageing model has been widely used to mimic intrinsic or natural ageing of human beings for anti-ageing studies, which causes oxidative stress and cell senescence by triggering inflammatory processes on skin. [36][37][38][39] In this study, the efficacy and mechanism of hPL on skin ageing were determined for the first time, providing a promising therapeutic strategy for anti-ageing treatment.

| Human PL preparation and quality control
hPL was obtained using a two-step procedure: (1) extraction and purification of platelet concentrates and (2) freeze-thawing lysis to produce hPL, as described by our previous studies. 28,29 The methodology was innovative and licensed by China National Invention Patent (ZL 2014 1 0508458.8). Briefly, after obtaining informed consent from healthy adult donors, whole blood was collected in tubes containing sodium citrate anticoagulant (3.2%, w/v; blood:citrate = 9:1). To obtain purified platelet concentrates (purity >99%), each 50 ml of whole blood was centrifuged at 210Âg for 10 min, and yellow plasma with buffy coat was collected in a new tube and centrifuged at 210Âg for 5 min. Residual erythrocytes were discarded, the supernatant plasma and platelet pellet were collected as platelet concentrates. All blood specimens used in this study were approved by Ethical Committee of the Zhejiang Chinese Medical University.
To determine the purity of platelet in our product before lysis, the human platelet surface marker (CD41a) was analysed as previously described. 28,29 Briefly, the sample was incubated with the antibody against CD41a, followed by incubation for 1 h. Then the sample was washed with PBS and loaded on flow cytometer (BD FACS Calibur, BD Biosciences, CA, USA) in triplicates. Fluorescent signal intensity was recorded and analysed by CellQuest software (Version 3.3, BD Biosciences, CA, USA). The platelet number was measured by Mindray BC-3000Plus Blood Cell Analyzer (Shenzhen, China) and standardized to 1 Â 10 8 platelets/ml, and then lysed by repeating freeze-thaw (À80 to 37 C) for three times. The residual platelet fragments were removed by centrifugation and the obtained supernatant was collected as hPL. The concentrations of IGF-1, TGF-β, PDGF and EGF in hPL were measured in triplicates with commercially available ELISA kits, according to each manufacturer's instructions. The absorbance was measured using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).   (Table 1).

| SOD and MDA analysis
The skin tissue sample is weighed, added physiological saline to produce a 10% homogenate and sonicated twice every 30 s. The homogenization and sonication were performed at 4 C. After sonication, the homogenate was centrifuged at 3000 rpm for 10 min and at 12,000 rpm for 15 min. An aliquot of the supernatant was used for further experiments. BCA protein determination kit was used to determine the protein content of aliquots. SOD and MDA kit were used to detect the content of SOD and MDA in the skin of nude mice, according to each manufacturer's instructions. The SOD in the cell supernatants were estimated following the kit instructions.

| Histopathological observation and immunofluorescence analysis
Each skin sample was fixed with formalin (10%) for 24 h at room temperature. Then each sample was embedded in paraffin and sectioned

| Senescence-associated-beta-galactosidase staining
HDFs were plated in six-well plates (1 Â 10 5 /well), modelled with D-gal at 20 g/L for 24 h and followed by hPL at the dilution rate of 1 The secondary lines are flattened, and the number is reduced. A few wrinkles are shallow coarse. 2 The primary line becomes uneven, the secondary line becomes obviously flat or deformed, and the number of intersections is reduced. Some wrinkles are coarse. 3 The texture is deteriorated, the primary lines are thick and deep, a large flat skin appears between the primary lines, and secondary lines are deformed and disappeared. Wrinkles are deeply coarse and wide.

| Real time PCR
The mRNA expression of targeted genes in HDFs cells was measured using a qPCR assay on an ABI QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems; Thermo Scientific, USA).
Total RNA was extracted with TRIzol reagent and quality controlled by NanoDrop2000 spectrophotometer (Thermo Scientific, USA).
cDNA reverse transcription was performed by using All-in-One

| Western blot analysis
Total cellular proteins of HDFs were extracted with RIPA buffer containing proteinase inhibitor cocktail for 30 min on ice. The targeted protein was separated by denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis (8%-12%) and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk for 2 h, which was followed by overnight incubation at 4 C with the following primary antibodies against β-actin, ATM, P65, p-P65, P62, interleukin-6 (IL-6), P16, P21, LaminB1, MMP3 and MMP9. Following incubation with peroxidase-conjugated goat anti-rabbit/mouse IgG at 4 C for 2 h, each protein was visualized using Western Lightning ® Plus ECL, detected using X-ray film and scanned. Each experiment was conducted in triplicate.

| Histopathological and immunofluorescence analyses of aged skin with hPL treatment
Histopathological observation was performed on dermal thickness and collagen fibres of nude mice by HE and Masson's trichrome staining. As shown in Figure 3A,B, the dermal thickness levels of F I G U R E 1 Clinical retrospective study on human platelet lysate (hPL). Wrinkle, texture and pore appearance of subjects with VISIA ® imaging system before and after hPL treatment (A). Objective assessments and score of skin characteristics before and after hPL treatment (B). Data expressed as mean ± SD. *p < 0.05 vs. NC group by paired-sample t-test model group were significantly decreased as compared with that of NC group (p < 0.01 vs. NC), while that of hPL groups were significantly restored to normal levels (p < 0.01 vs. model). Masson's trichrome staining was used to visualize the thickness of collagen fibres. As shown in Figure 3A,B, the collagen fibres were obviously disrupted with significant decrease of thickness levels, when compared with that of NC group (p < 0.01 vs. NC). In contrast, the abnormalities were remarkably restored with significant increase of collagen thickness levels in hPL-treated groups (p < 0.01 vs. model). Immunofluorescence result of VEGF was shown in Figure 3B,C. The skin

| In vitro effects of hPL on senescent HDFs
D-gal was applied to establish senescence model of HDFs, and CCK-8 assay, wound healing assay and SA-β-gal staining were respectively performed to evaluate the pro-migrative, proliferative, and anti-senescent effects of hPL on HDFs. As shown in Figure 4B, hPL at a dilution rate ranging from 1/200 to 1/10 exerted significantly proliferative effect on HDFs in a dose-dependent manner after 24 and 48 h treatment. Accordingly, hPL at a dilution rate of 1/40 was chosen as an effective dose for further use. As shown in Figure 4A, the blank ratios of wound area at 24 and 48 h to the area without treatment (0 h) of model group were significantly higher than that of NC group (both p < 0.01), and that of hPL group were significantly lower than that of model group (both p < 0.01). SA-β-gal staining is a way to exhibit senescent cells. As shown in Figure 4C, the number of SAβ-gal stained cells (in blue colour) of model group was significantly higher than that of NC group (p < 0.01), and that of hPL group was significantly lower than that of model group (both p < 0.01). The above data revealed that hPL effected on HDFs by inducing proliferation and migration, as well as suppressing senescence.

| Molecular actions of hPL on senescent HDFs
qPCR and WB analyses were conducted to elucidate molecular actions of hPL on senescent HDFs. As shown in Figure 5A, the mRNA F I G U R E 5 The relative mRNA expressions of genes of human dermal fibroblasts (HDFs) with human platelet lysate (hPL) treatment detected by qPCR (A). Protein bands and protein expression in HDFs with PL treatment (B). Data expressed as mean ± SD. ## p < 0.05 or ## p < 0.01 vs. NC group; *p < 0.05 or **p < 0.01 vs. model group by one-way ANOVA followed by least significant difference multiple comparison. We repeated the experiments three times to ensure the accuracy of the experiments 3.6 | Verification of NF-κB pathway-dependent mechanism of hPL Positive and negative verifications were conducted to determine the NF-κB pathway-dependent mechanism of hPL. First, P65-siRNA was constructed to knockdown the NF-κB pathway of senescent HDFs, mimicking the molecular action of hPL. As shown in Figure 6A, with D-gal plus siNC (non-targeting control siRNA) treatment, the expression changes of senescence-related proteins (IL-6, P16, P21, P62 and MMP3) were similar to that of model group (all p < 0.05 or <0.01 vs. NC). With D-gal plus siP65 (P65-siRNA) treatment, the expression and phosphorylation of P65 were successfully blocked and the abnormal expressions of senescence-related proteins were significantly restored towards normal levels (all p < 0.01 vs. siNC). Moreover, as shown in Figure 6B, the cell viability of HDFs was significantly inhibited by D-gal plus siNC treatment (p < 0.01 vs. NC) and was significantly improved by D-gal plus siP65 treatment (p < 0.01 vs. siNC). The above data indicated that siP65 exerted similar effects as hPL on molecular targets of senescence as well as cell viability of HDFs, positively verifying the NF-κB pathway-dependent mechanism of hPL.
As an agonist of NF-κB, prostratin (PKC) was applied to counteract the NF-κB pathway-dependent action of hPL for negative verification. Four groups were designated as follows: model group with D-gal

| Molecular actions of hPL-contained growth factors on senescent HDFs
As shown in Figure 7A, flow cytometrical analysis showed 90.95% positive expression of CD41a in hPL production before freeze-thaw lysis. As shown in Figure 7B, after lysis, hPL was obtained containing The SASP cytokines IL-6 and IL-8 are hallmarks of inflammation that reinforce the senescent growth arrest and induce inflame-ageing phenotype of nearby cells. 51 in senescent cells. 63,64 It has been reported that activation of NF-κB in young HDFs mimicked a replicative senescence of HDFs and resulted in degradation of collagen type I, suggesting an important role of NF-κB pathway in skin ageing. 63 In this study, the protein expressions of ATM, P65 and phosphorylated P65 were up-regulated in senescent HDFs and reversed by hPL treatment, indicating NF-κB pathway as the mediator of hPL's anti-senescent mechanism on skin ( Figure 5). ATM is a DDR kinase acting as an initial driver of NF-κB pathway. It reduces P62 expression and then activates the transcription factor NF-κB to facilitate senescence and initiate SASP. 64 Figure S1).
Interestingly, this study discovered that hPL-contained IGF-1, TGF-β, PDGF and EGF synergistically contributed to the anti-ageing efficacy and mechanism of hPL. Each of these growth factors at the corresponding concentration in hPL exerted hPL-like anti-ageing efficacy and molecular actions via NF-κB signalling pathway, and none of them could achieve or surpass the efficacy of hPL (Figure 7). It is reasonable because these growth factors all have potential of inducing skin rejuvenation, and their combinations of them may synergistically result in the best outcome. For instance, IGF-I is capable of maintaining skin surface lipids and thickness, 79 and EGF, PDGF and TGF-β are capable of proliferating HDFs, producing collagens and inhibiting MMPs of ECM, benefiting wrinkle removal and skin smooth. [80][81][82][83] Moreover, PDGF is a major component of adipose derived stem cells (ADSCs) secretome, which endows ADSCs antiageing function on skin. 84 To date, the concrete roles and contributions of these grown factors in hPL or PRP remain to be elucidated.
Whether some other growth factors or ingredients in hPL also contribute to hPL, warrants further investigations. As the next generation of PRP, hPL possesses better bioactivity and bioavailability than PRP, since hPL contains higher concentrations of growth factors. 28 Nevertheless, the comparison between hPL and PRP in anti-ageing or regenerative applications has never been reported. Hypodermic injection of PRP has been reported to promote the production of elastin and fibrillin of skin. 85,86 However, PRP may also cause skin erythema, inflammation and fibrosis, which indirectly accelerates ageing process in some cases and sometimes exerts ineffective effect on skin ageing. [86][87][88] Such defects may be due to the remain of heterologous activators and cell residuals (WBC and RBC) in PRP. In this study, the lysis and purification processes in hPL preparation not only released high concentrations of growth factors with no need of heterologous activators, but also removed cell residuals completely, thereby overcoming the defects of PRP. The clinical data also verified the safety of hPL in use on subjects. Moreover, hPL exerted anti-ageing effects on skin from various aspects including dermal ECM regeneration and anti-oxidization, wrinkle removal and texture smooth, and HDFs protection from inflammation, and senescence, whereas PRP can only ameliorate wrinkles and texture of skin in clinic with little report on anti-senescent and anti-oxidative effects on skin cells. 26,86 Therefore, hPL is an ideal therapeutic for anti-ageing application on skin.

| CONCLUSION
This study demonstrated hPL's efficacy on skin ageing of nude mice and clarified its mechanism relying on NF-κB signalling pathway of improved the experimental design and methodology; Huiling Wu provided ideas and designed this study; Letian Shan designed, drafted, and funded this study; Thomas Efferth improved the design and writing of this study.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.