Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders.

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

Fast freezing inhibits melanin synthesis of melanocytes by modulating the Wnt/ß-catenin signalling pathway.

Skin hyperpigmentation is mainly caused by excessive synthesis of melanin; however, there is still no safe and effective therapy for its removal. Here, we found that the dermal freezer was able to improve UVB-induced hyperpigmentation of guinea pigs without causing obvious epidermal damage. We also mimic freezing stimulation at the cellular level by rapid freezing and observed that freezing treatments <2.5 min could not decrease cell viability or induce cell apoptosis in B16F10 and Melan-A cells. Critically, melanin content and tyrosinase activity in two cells were greatly reduced after freezing treatments. The dramatic decrease in tyrosinase activity was associated with the downregulation of MITF, TYR, TRP-1 and TRP-2 protein expression in response to freezing treatments for two cells. Furthermore, our results first demonstrated that freezing treatments significantly reduced the levels of p-GSK3ß and ß-catenin and the nuclear accumulation of ß-catenin in B16F10 and Melan-A cells. Together, these data suggest that fast freezing treatments can inhibit melanogenesis-related gene expression in melanocytes by regulating the Wnt/ß-catenin signalling pathway. The inhibition of melanin production eventually contributed to the improvement in skin hyperpigmentation induced by UVB. Therefore, fast freezing treatments may be a new alternative of skin whitening in the clinic in the future.

Development of an α-Klotho Recognizing High-Affinity Peptide Probe from In-Solution Enrichment.

The kidney, parathyroid gland, and choroid plexus express the aging-related transmembrane protein α-Klotho, a coreceptor of the fibroblast growth factor 23 (FGF23) receptor complex. Reduced α-Klotho levels are correlated with chronic kidney disease and other age-related diseases, wherein they are released from membranes into circulation. Klotho's potential physiological action as a hormone is of current scientific interest. Part of the challenges associated with advancing these studies, however, has been the long-standing difficulty in detecting soluble α-Klotho in biofluids. Here, we describe the discovery of peptides that recognize α-Klotho with high affinity and selectivity by applying in-solution size-exclusion-based affinity selection-mass spectrometry (AS-MS). After two rounds of AS-MS and subsequent N-terminal modifications, the peptides improved their binding affinity to α-Klotho by approximately 2300-fold compared to the reported starting peptide Pep-10, previously designed based on the C-terminal region of FGF23. The lead peptide binders were shown to enrich α-Klotho from cell lysates and to label α-Klotho in kidney cells. Our results further support the utility of in-solution, label-free AS-MS protocols to discover peptide-based binders to target proteins of interest with high affinity and selectivity, resulting in functional probes for biological studies.

Discovery of reactive peptide inhibitors of human papillomavirus oncoprotein E6.

Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, which is the fourth most common cancer in women worldwide. High-risk variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. This degradation is mediated by the HPV early protein 6 (E6), which recruits the E3 ubiquitin ligase E6AP and redirects its activity towards ubiquitinating p53. Targeting the protein interaction interface between HPV E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53. In this study, we designed a covalent peptide inhibitor, termed reactide, that mimics the E6AP LXXLL binding motif by selectively targeting cysteine 58 in HPV16 E6 with quantitative conversion. This reactide provides a starting point in the development of covalent peptidomimetic inhibitors for intervention against HPV-driven cancers.

Branched Multimeric Peptides as Affinity Reagents for the Detection of α-Klotho Protein.

α-Klotho, an aging-related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α-Klotho are a hallmark of age-associated diseases. Detecting or labeling α-Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by single-shot parallel automated fast-flow synthesis that recognize α-Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α-Klotho in physiological settings.

Hordenine Activated Dermal Papilla Cells and Promoted Hair Regrowth by Activating Wnt Signaling Pathway.

Hordenine is effective in treating hyperpigmentation, fighting diabetes and resisting fibrosis and acute inflammation. However, the role of Hordenine on hair growth has not been elucidated. Here, we found that Hordenine treatments significantly enhance proliferation of primary mouse dermal-papilla cells (DPCs) and increase the activity of DPCs in a dose-dependent manner. Additionally, Hordenine markedly promoted the elongation of the hair shaft in the model of in vitro-cultured mouse vibrissa follicle and accelerated hair regrowth in a mouse model of depilation-induced hair regeneration. Real-time PCR, Western Blot and immunofluorescent assays showed that nuclear ß-catenin and its downstream gene expression such as Lef1, Axin2, Cyclin D1 and ALP were greatly upregulated in DPCs and mouse hair follicles after Hordenine treatments. Moreover, the increased DPCs' proliferation and hair shaft elongation of cultured mouse vibrissa follicles induced by Hordenine treatments were rescued by a Wnt/ß-catenin signaling inhibitor, FH535. These data indicate that Hordenine can effectively enhance DPCs' activity and accelerate hair regrowth through activating the Wnt/ß-catenin signaling pathway. Therefore, these findings suggest Hordenine/its derivatives may be potentially used for preventing and treating alopecia in the future.

Activation of orphan receptor GPR132 induces cell differentiation in acute myeloid leukemia.

Blocked cellular differentiation is a critical pathologic hallmark of acute myeloid leukemia (AML). Here, we showed that genetic activation of the orphan GPCR GPR132 significantly induced cell differentiation of AML both in vitro and in vivo, indicating that GPR132 is a potential trigger of myeloid differentiation. To explore the therapeutic potential of GPR132 signaling, we screened and validated a natural product 8-gingerol (8GL) as a GPR132 agonist. Notably, GPR132 activation by 8GL promoted differentiation and reduced colony formation in human AML cell lines with diverse genetic profiles. Mechanistic studies revealed that 8GL treatment inhibits the activation of the mammalian target of rapamycin (mTOR), a regulator of AML cell differentiation blockade, via activating GPR132-Gs-PKA pathway. We further showed that the combination of 8GL and an mTOR inhibitor synergistically elicited AML cell differentiation in vitro. Importantly, 8GL alone or in combination with an mTOR inhibitor remarkably impaired tumor growth and extended mouse survival in an AML xenograft model accompanied by enhanced cell differentiation. Notably, genetic or pharmacological activation of GPR132 triggered the differentiation of human primary AML cells. In summary, this study demonstrated that activation of orphan GPR132 represents a potential strategy for inducing myeloid differentiation in AML patients.

Yohimbine hydrochloride inhibits skin melanin synthesis by regulating wnt/ß-catenin and p38/MAPK signal pathways.

BACKGROUND: Yohimbine hydrochloride (YH) is a prescription drug to treat erectile dysfunction. It also had potential in fighting high blood pressure and diabetic neuropathy as well as promoting weight loss. OBJECTIVE: The aim of the study is to investigate the anti-melanogenic function of yohimbine hydrochloride and reveal its underlying molecular mechanism. METHODS: B16F10 mouse melanoma cells, Melan-A murine melanocyte, Zebrafish embryos and C57BL/6 mouse ear skins were treated with different concentrations of YH. The extracellular and cellular melanin content was detected by spectrometry. The expression of microphthalmia-associated transcription factor (MITF), tyrosinase and the activities of Wnt/ß-catenin and p38/MAPK signal pathways were determined by RT-qPCR, Western blot analysis and immunofluorescent staining. RESULTS: Melanin production could be effectively inhibited by YH at the safe concentration in vitro and in vivo. Q-PCR and WB results showed that the expression of MITF and tyrosinase were strongly downregulated after YH treatments along with the reduction of tyrosinase activity. YH markedly inhibited ß-catenin nuclear accumulation and p38 phosphorylation in B16F10 cells compared with the untreated controls. Importantly, the increase of MITF expression induced by ß-catenin activator BIO and p38 activator anisomycin could be fully reversed by YH treatments. CONCLUSIONS: These results indicate that YH can function as an anti-melanogenic agent, at least in part, by inhibiting Wnt/ß-catenin and p38/MAPK signal pathways. Therefore, YH may be potentially used as a skin-whitening compound for preventing hyperpigmentation disorders in the future.

P2RY6 Has a Critical Role in Mouse Skin Carcinogenesis by Regulating the YAP and ß-Catenin Signaling Pathways.

P2RY6 is highly expressed in skin keratinocytes, but its function in skin diseases is unclear. We use a two-step chemical induction method to induce mouse skin tumor formation. Multiple in vitro and in vivo assays were used to explore the role of P2RY6 in skin tumors. We report that P2ry6-deficient mice exhibit marked resistance to 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin papilloma formation compared with wild-type mice. Consistent with these findings, epidermal hyperplasia in response to TPA was suppressed in the P2ry6-knockout or MRS2578 (P2RY6 antagonist)-treated mice. The dramatic decrease in hyperplasia and tumorigenesis due to P2ry6 disruption was associated with the suppression of TPA-induced keratinocyte proliferation and inflammatory reactions. Notably, P2ry6 deletion prevented the TPA-induced increase in YAP nuclear accumulation and its downstream gene expression in an MST/LATS1-dependent manner. On TPA stimulation, enhanced activation of MAPK/extracellular signal‒regulated kinase kinase 1 and ß-catenin were also impaired in P2ry6-knockout primary keratinocytes, tumor tissues, or MRS2578-treated HaCaT cells. Moreover, mutual promotion of the YAP and ß-catenin signaling pathways was observed in normal skin cells treated with TPA, whereas P2ry6 deletion could inhibit their crosstalk by regulating MAPK/extracellular signal‒regulated kinase kinase 1. Thus, P2RY6 is a critical positive regulator of skin tumorigenesis through the modulation of the Hippo/YAP and Wnt/ß-catenin signaling pathways.

Binary combinatorial scanning reveals potent poly-alanine-substituted inhibitors of protein-protein interactions.

Establishing structure-activity relationships is crucial to understand and optimize the activity of peptide-based inhibitors of protein-protein interactions. Single alanine substitutions provide limited information on the residues that tolerate simultaneous modifications with retention of biological activity. To guide optimization of peptide binders, we use combinatorial peptide libraries of over 4,000 variants-in which each position is varied with either the wild-type residue or alanine-with a label-free affinity selection platform to study protein-ligand interactions. Applying this platform to a peptide binder to the oncogenic protein MDM2, several multi-alanine-substituted analogs with picomolar binding affinity were discovered. We reveal a non-additive substitution pattern in the selected sequences. The alanine substitution tolerances for peptide ligands of the 12ca5 antibody and 14-3-3 regulatory protein are also characterized, demonstrating the general applicability of this new platform. We envision that binary combinatorial alanine scanning will be a powerful tool for investigating structure-activity relationships.

De Novo Discovery of High-Affinity Peptide Binders for the SARS-CoV-2 Spike Protein.

The ß-coronavirus SARS-CoV-2 has caused a global pandemic. Affinity reagents targeting the SARS-CoV-2 spike protein are of interest for the development of therapeutics and diagnostics. We used affinity selection-mass spectrometry for the rapid discovery of synthetic high-affinity peptide binders for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. From library screening with 800 million synthetic peptides, we identified three sequences with nanomolar affinities (dissociation constants K d = 80-970 nM) for RBD and selectivity over human serum proteins. Nanomolar RBD concentrations in a biological matrix could be detected using the biotinylated lead peptide in ELISA format. These peptides do not compete for ACE2 binding, and their site of interaction on the SARS-CoV-2-spike-RBD might be unrelated to the ACE2 binding site, making them potential orthogonal reagents for sandwich immunoassays. These findings serve as a starting point for the development of SARS-CoV-2 diagnostics or conjugates for virus-directed delivery of therapeutics.

Establishing the Structure-Activity Relationship of Daptomycin.

Daptomycin is effective in treating infections caused by antibiotic-resistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and vancomycin-resistant S. aureus (VRSA). Due to its distinct mechanism of action toward multidrug-resistant bacteria, daptomycin provides an attractive structural motif to generate new daptomycin-based antibiotics to combat the problem of bacterial resistance. In this study, we used the total synthesis method to produce daptomycin analogues with a variety in terms of types and sites of modifications. Five classes of daptomycin analogues were synthesized, and the antimicrobial activities of the analogues were analyzed by several biological assays. From this study, we established a comprehensive structure-activity relationship of daptomycin which will lay the foundation for the further development of daptomycin-based antibiotics.

Ultra-large chemical libraries for the discovery of high-affinity peptide binders.

High-diversity genetically-encoded combinatorial libraries (108-1013 members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only ~ 106 compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 108 members-a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 106-108. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3-19 nM-affinity, α/ß-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of ß-amino acids in facilitating a key binding contact.

Natural product corynoline suppresses melanoma cell growth through inducing oxidative stress.

Natural product corynoline is a unique isoquinoline alkaloid extracted from traditional Chinese medicine Corydalis bungeana Turcz, whereas its anticancer properties have not been investigated. In this study, we found that corynoline potently impairs the growth of melanoma cells, B16F10, and A375 in a concentration-dependent manner. Treatment of melanoma cells with corynoline results in G2 cell arrest accompanied by reduced cdc2 activation. Furthermore, corynoline triggers apoptosis of melanoma cells, which is associated with increased expression of Bax and cleaved caspase-3. Mechanistic study indicates that corynoline strongly induces reactive oxygen species (ROS) generation and subsequent DNA damage as evidenced by γ-H2AX accumulation. Notably, the effect of corynoline on melanoma cell cycle and apoptosis is abolished by a ROS scavenger N-acetyl cysteine (NAC), indicating a ROS-dependent mechanism. Finally, corynoline significantly inhibits in vivo B16F10 melanoma tumor growth accompanied by reduced expression of Ki-67 in tumor tissue. Taken together, our data suggest that corynoline suppresses melanoma cell growth in vitro and in vivo by inducing oxidative stress and represents a potential therapeutic agent for melanoma patients.

Lgr4 Deletion Delays the Hair Cycle and Inhibits the Activation of Hair Follicle Stem Cells.

It is known that LGR4 plays an important role in hair follicle (HF) development, but the impact of LGR4 on the hair cycle is still unclear. In this study, we have found that K14-Cre-mediated skin epithelia-specific deletion of Lgr4 results in delayed anagen entry during the physiological hair cycle and compromised HF regeneration upon transplantation. We show that, although Lgr4 deletion does not appear to affect the number of quiescent HF stem cells, it leads to reduced numbers of LGR5+ and actively proliferating stem cells in the HFs. Moreover, LGR4-deficient HFs show molecular changes consistent with decreased mTOR and Wnt signaling but upregulated BMP signaling. Importantly, the reactivation of the protein kinase B pathway by injecting the protein kinase B activator SC79 in Lgr4-/- mice can effectively reverse the hair cycle delay. Together, these data suggest that LGR4 promotes the normal hair cycle by activating HF stem cells and by influencing the activities of multiple signaling pathways that are known to regulate HF stem cells. Our study also implicates LGR4 as a potential target for treating hair disorder in the future.

Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis.

BACKGROUND: Increasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis. PURPOSE: The purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis. METHODS: A gaussia princeps luciferase-based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and ß-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining. RESULTS: Celastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions. CONCLUSION: Taken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.

MicroRNA-34 Family Enhances Wound Inflammation by Targeting LGR4.

Venous ulcers are the most common type of human chronic nonhealing wounds and are stalled in a constant and excessive inflammatory state. The molecular mechanisms underlying the chronic wound inflammation remain elusive. Moreover, little is known about the role of regulatory RNAs, such as microRNAs, in the pathogenesis of venous ulcers. We found that both microRNA (miR)-34a and miR-34c were upregulated in the wound-edge epidermal keratinocytes of venous ulcers compared with normal wounds or the skin. In keratinocytes, miR-34a and miR-34c promoted inflammatory chemokine and cytokine production. In wounds of wild-type mice, miR-34a-mimic treatment enhanced inflammation and delayed healing. To further explore how miR-34 functions, LGR4 was identified as a direct target mediating the proinflammatory function of miR-34a and miR-34c. Interestingly, impaired wound closure with enhanced inflammation was also observed in Lgr4 knockout mice. Mechanistically, the miR-34-LGR4 axis regulated GSK-3ß-induced p65 serine 468 phosphorylation, changing the activity of the NF-κB signaling pathway. Collectively, the miR-34-LGR4 axis was shown to regulate keratinocyte inflammatory response, the deregulation of which may play a pathological role in venous ulcers.

Fargesin inhibits melanin synthesis in murine malignant and immortalized melanocytes by regulating PKA/CREB and P38/MAPK signaling pathways.

BACKGROUND: Fargesin is commonly used in the treatment of allergic rhinitis, inflammation, sinusitis and headache. OBJECTIVE: The aim of the study is to investigate a new function of fargesin against melanin production and its underlying molecular mechanism. METHODS: B16F10 mouse melanoma cells, Melan-a and human epidermal melanocytes were treated with different concentrations of fargesin for the indicated time. The extracellular and cellular melanin content was detected by spectrometry at 490 nm and 405 nm, respectively. RT-qPCR and Western blot analysis were used to exam the expression of melanogenic enzymes and the activities of PKA/CREB and p38 MAPK pathway components. Zebrafish was used as an in vivo model for studying the function of fargesin in regulating melanogenesis. RESULTS: Fargesin effectively inhibited melanin production at moderate dose in mouse B16F10 melanoma cells, normal melanocyte cell lines and zebrafish. The expression of microphthalmia-associated transcription factor (MITF), its downstream melanogenic enzymes and tyrosinase activity were also strongly reduced by fargesin. Moreover, the increase of melanin production induced by UVB and forskolin could be fully reversed by fargesin treatment. Fargesin also effectively inhibited the activation of PKA/CREB and p38 MAPK as well as their interactions, which in turn is responsible for the expression of MITF and melanogenic enzymes. CONCLUSIONS: These results show that fargesin can function as an anti-melanogenic agent, at least in part, by inhibiting PKA/CREB and p38/MAPK signaling pathways. Therefore, fargesin and its derivatives may potentially be used for preventing hyperpigmentation disorders in the future.

Plant natural product plumbagin presents potent inhibitory effect on human cytochrome P450 2J2 enzyme.

BACKGROUND: Cytochrome P450 2J2 (CYP2J2) is not only highly expressed in many kinds of human tumors, but also promotes tumor cell growth via regulating the metabolism of arachidonic acids. CYP2J2 inhibitors can significantly reduce proliferation, migration and promote apoptosis of tumor cells by inhibiting epoxyeicosatrienoic acids (EETs) biosynthesis. Therefore screening CYP2J2 inhibitors is a significant way for the development of anti-cancer drug. PURPOSE: The aim of this study was to identify a new CYP2J2 inhibitor from fifty natural compounds obtained from plants. STUDY DESIGN: CYP2J2 inhibitor was screened from a natural compounds library and further the inhibitory manner and mechanism were evaluated. Its cytotoxicity against HepG2 and SMMC-7721 cell lines was also estimated. METHODS: The inhibitory effect was evaluated in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant CYP2J2 (rCYP2J2), using astemizole as a probe substrate and inhibitory mechanism was illustrated through molecular docking. The cytotoxicity was detected using SRB. RESULTS: In all candidates, plumbagin showed the strongest inhibitory effect on the CYP2J2-mediated astemizole O-demethylation activity. Further study revealed that plumbagin potently inhibited CYP2J2 activity with IC50 value at 3.82 µM, 3.37 µM and 1.17 µM in RLMs, HLMs and rCYP2J2, respectively. Enzyme kinetic studies showed that plumbagin was a mixed-type inhibitor of CYP2J2 in HLMs and rCYP2J2 with Ki value of 1.88 µM and 0.92 µM, respectively. Docking data presented that plumbagin interacted with CYP2J2 mainly through GLU 222 and ALA 223. Moreover, plumbagin showed strongly cytotoxic effects on hepatoma cell lines, such as HepG2 and SMMC-7721, with lower toxicity on rat primary hepatocytes. Plumbagin had no effect on the protein expression of CYP2J2 in HepG2 and SMMC-7721, while down-regulated the mRNA level of anti-apoptosis protein Bcl-2. CONCLUSION: This study found out a new CYP2J2 inhibitor plumbagin from fifty natural compounds. Plumbagin presented a potential of anti-cancer pharmacological activity.

Potential role of S100A8 in skin rejuvenation with the 1064-nm Q-switched Nd:YAG laser.

The 1064-nm Q-switched Nd:YAG laser is demonstrated to be effective for non-ablative skin rejuvenation, but the molecular mechanism by which dermis responses to laser-induced damage and initiates skin remodeling is still unclear. HaCaT cells and 3T3 skin fibroblasts were irradiated with the 1064-nm Q-switched Nd:YAG laser at the different doses. Then, cells were collected and lysed for PCR and Western blot analysis. Cell viability was detected by Cell Counting Kit-8 (CCK-8) before and after laser irradiation. The expressions of S100A8, advanced glycosylation end product-specific receptor (RAGE) and inflammatory cytokines in two cell lines were markedly upregulated after laser treatments. The PCR, Western blot, and ELISA analysis showed the significant increase of type I and III procollagen in the 3T3 cells treated with the 1064-nm laser. Interestingly, si S100A8 effectively inhibited the expression of cytokines and collagen, while S100A8 treatments significantly increased them. P-p38 and p-p65 levels were also elevated after the 1064-nm laser irradiation, which is positively related with S100A8. Cell viability and reactive oxygen species (ROS) levels were not changed, while the content of superoxidase dismutase (SOD) in two cells was increased after laser irradiation. Our results demonstrated that the overexpression of S100A8 induced by the 1064-nm laser irradiation triggered inflammatory reactions in skin cells. The inflammatory microenvironment and improvement of skin antioxidant capacity contribute to new collagen synthesis in the skin cells. Thus, S100A8 was required for laser-induced new collagen synthesis in skin cells. p38/MAPK and NF-κB signal pathways were involved in S100A8-mediated inflammatory reactions in response to laser irradiation.