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Apigenin induces dermal Introduction Correspondence: Xiaoling Zhang and Qingfeng collagen synthesis via smad2/3 Li, Department of Plastic and Reconstructive signaling pathway Dermis consists of several structural compo- Surgery, Shanghai 9th People’s Hospital, nents, and collagen takes the major part. In Shanghai Jiao Tong University School of Y. Zhang,1 J. Wang,1 X. Cheng,2 B. Yi,3 addition to glycosaminoglycans and elastin Medicine, 639 Zhizaoju Road, Shanghai 200011, X. Zhang,4 Q. Li1 fibers, dermal matrix in adult skin are com- China. Tel. +86.21.23271699-5615 - Fax: +86.21.63089567. posed of type I (80-85%) and type III collagen 1Department of Plastic and E-mails: (X. Zhang) [email protected]; (Q. Li) (10-15%).1 It is of great importance that colla- Reconstructive Surgery, Shanghai 9th [email protected] gen plays a main role in the texture and People’s Hospital, School of Medicine, appearance of skin. Skin aging is inevitably Shanghai Jiao Tong University Key words: , , collagen I/III, associated with a disturbance in collagen 2 fibroblasts, smad2/3. Department of Urology, Renji Hospital, metabolism2 (due to the decreased activity of School of Medicine, Shanghai Jiao Tong fibroblasts and their collagen synthesis), as Contributions: YZ and JW contributed equally to University well as elastin. Increasing the collagen content this work; YZ, JW, experimental work, data collec- 3Clinical College of General Hospital of of dermis has been regarded as a well-effective tion and interpretation; XC, participation in Beijing Military Region, Anhui Medical way for anti-aging in skin. experimental work design and coordination, data University, Hefei Collagen is a kind of biomacromolecule and acquisition; BY, participation in study design, 4The Key Laboratory of Stem Cell it cannot be absorbed through the stratum data collection, analysis of data and manuscript preparation; QL, XZ, study design, data analysis Biology, Institute of Health Sciences, corneum. Recently, some researches demon- and interpretation, manuscript drafting. Shanghai Institutes for Biological strated that collagen hydrolysate ingestion Sciences, Chinese Academy of Sciences might be beneficial to slow chronological skin Conflict of interest: the authors declare no con- 3 4 and Shanghai Jiao Tong University School aging and photoaging in rats, and the density flict of interest. of Medicine, Shanghai, China of collagen fibrils increased compared with lac- talbumin and water controls.5 But the clinical Funding: this study was supported by grants from effect of oral collagen hydrolysate still lacks the key project of the National Natural Science convincing evidences. Up to now, injectable Foundation (No. 81230042), the National Key collagen1 or analogous composition6 filling Project of Scientific and Technical Supporting Abstract implants are recognized as the well-accepted Programs Funded by Ministry of Science & treatment modality for cosmetic purposes. Technology of China (No. 2012BAI11B03) (Q. Li) Decrease in fibroblast-produced collagen However, maintaining skin appearance relies and the Chinese Academy of Sciences (No. has been proven to be the pivotal cause of skin on expansive and complex treatment repeated- XDA01030102), Shanghai Municipal Commission of Health and Family Planning (No. aging, but there is no satisfactory drug which ly because of the short-term duration of exo- 2013ZYJB0501) (X. Zhang). directly increases dermal thickness and col- genic injected collagen. lage density. Here we found that a flavonoid Apigenin (4,5,7-trihydroxyflavone), a natural product, apigenin, could significantly Received for publication: 28 November 2014. flavone subclass of flavonoid widely distributed Accepted for publication: 9 March 2015. increase collagen synthesis. NIH/3T3 and pri- in many herbs, fruits, and vegetables, is a sub- mary human dermal fibroblasts (HDFs) were stantial component of the human diet and has This work is licensed under a Creative Commons incubated with various concentrations of api- been shown to possess a variety of biological Attribution NonCommercial 3.0 License (CC BY- genin, with dimethyl sulfoxide (DMSO) serv- characteristics, including anti-oxidative,7 anti- NC 3.0). ing as the negative control. Real-time reverse- inflammatory effect,8 tumor growth inhibition9 transcription polymerase chain reaction and promoting neurogenesis.10 It has been ©Copyright Y. Zhang et al., 2015 (PCR), Western Blot, and Toluidine blue stain- Licensee PAGEPress, Italy shown that apigenin could enhance wound European Journal of Histochemistry 2015; 59:2467 ing demonstrated that apigenin stimulated 11 healing and tissue repair in diabetic rat skin. doi:10.4081/ejh.2015.2467 type-I and type-III collagen synthesis of fibrob- In the process of wound healing, fibroblasts lasts on the mRNA and protein levels. secreted collagen and the formation of colla- Meanwhile, apigenin did not induce expres- gen-rich granulation tissue are vital patho- sion of alpha smooth muscle actin (α-SMA) in physiological mechanisms for wound closure.12 Materials and Methods vitro and in vivo, a fibrotic marker in living tis- Given this, we wonder what effect would Cell culture sues. Then the production of collagen was con- apigenin have on fibroblasts and whether api- firmed by Masson’s trichrome stain, genin could induce collagen synthesis in nor- Primary human dermal fibroblasts were Picrosirius red stain and immunohistochem- mal human dermal fibroblasts. Consequently, obtained from adolescent foreskin tissue of ten istry in mouse models. We also clarified that we examined its effects on collagen synthesis people (aged 8-12 years), and none of them this compound induced collagen synthesis by in normal human dermal fibroblasts in vitro had a history with skin diseases. Skin tissue activating smad2/3 signaling pathway. Taken and tested its function in the skin aging mouse was obtained after obtaining informed consent together, without obvious influence on fibrob- model induced by D-Galactose. Furthermore, from the patients, with the approval of the lasts’ apoptosis and viability, apigenin could we investigated the potential mechanism ethics committee of Shanghai 9th People’s promote the type-I and type-III collagen synthe- involved in the positive effects of apigenin on Hospital and in conformity with the Helsinki sis of dermal fibroblasts in vitro and in vivo, collagen expression in fibroblasts. guidelines. NIH/3T3 and human dermal fibrob- thus suggesting that apigenin may serve as a lasts (HDFs) were maintained in DMEM potential agent for esthetic and reconstructive (Hyclone, Thermo Fisher Scientific, Waltham, skin rejuvenation. MA, USA) supplemented with 10% fetal bovine

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serum (Hyclone, Thermo Fisher Scientific), Flow cytometric analysis Western blot 100 U/mL penicillin, and 100 mg/L strepto- Cell apoptosis was assessed by flow cytome- Cultured cells were lysed using radioim- mycin. Both HDFs and NIH/3T3 were incubated try using the Alexa Fluor® 488 Annexin V/Dead munoprecipitation assay (RIPA) lysis buffer. at 37°C in a humidified atmosphere with 5% Cell Apoptosis Kit (Invitrogen, Carlsbad, CA, Protein concentrations were determined using CO . Primary fibroblasts of passages 6-8 were 2 USA). Following apigenin (5 mol/L or 1 a micro bicinchoninic acid (BCA) assay used. Toluidine blue (Sigma-Aldrich, St. Louis, μ mol/L) or dimethyl sulfoxide (DMSO) treat- (Thermo Fisher Scientific). Twenty micro- MO, USA) staining was used to assess extra- μ ment, harvested cells were suspended in 100 grams total protein extract was separated by cellular matrix synthesis.13 μL Annexin-binding buffer. Then, 5 μL Alexa 8% or 10% sodium dodecyl sulfate-polyacry- Real-time PCR analysis Fluor® 488 Annexin V and 1 μL PI working lamide gel electrophoresis (SDS-PAGE) under solution were added and incubated with the reducing conditions and electroblotted onto The total RNA of cells was isolated using cells for 15 min in the dark. After the incuba- polyvinylidene difluoride membranes TRIzol reagent (Invitrogen, Carlsbad, CA, USA) (Millipore, Bedford, MA, USA). The membrane tion period, 400 μL 1X Annexin-binding buffer and subjected to reverse transcription with was added and mixed gently. The stained cells was then blocked and then incubated with pri- Oligo (dT) and M-MLV Reverse Transcriptase were analysed directly by flow cytometry using mary antibodies overnight at 4°C. (Thermo Fisher Scientific). Synthesized com- the Cell Quest program (Becton Dickinson, CA, The primary antibodies used included the plementary DNA (cDNA) was analysed with USA). Data were analysed using FlowJo soft- followings: anti-Col1a2, anti-Col3a1, anti-α- quantitative real-time PCR using SYBR® ware. SMA (Abcam; 1:1000), anti-cyclin-dependent Premix (Takara, Dalian, China) and Roche480 kinase (CDK) family, anti-cyclin D1, anti- system. Glyceraldehyde 3-phospate dehydroge- Colony formation assay cyclin E1, anti-smads, anti-MAPK family (Cell nase (GAPDH) was used as a reference gene. Anchorage-dependent growth of HDFs were Signaling Technology, Beverly, MA, 1:1000), Primers sequences were as follows: collagen, investigated by monolayer colony formation anti-GAPDH (Sigma-Aldrich; 1:10,000). type I, alpha 2 (Col1a2; mouse), 5-GGTGAGC- assay.14 Cells were cultured in a 6-well plate Immunoreactive bands were quantitatively CTGGTCAAACGG-3 (forward) and 5- ACTGT- (500 per well) and treated with 5 mol/L or 1 analyzed with ImageJ software. GTCCTTTCACGCCTTT-3 (reverse); Col1a2 μ μmol/L apigenin or DMSO. After cultured for D-galactose-induced skin aging (human), 5-GGCCCTCAAGGTTTCCAAGG-3 14 days, surviving colonies were stained 5 min (forward) and 5-CACCCTGTGGTCCAA- with Gentian Violet (Sigma-Aldrich) after 4% mouse model and animal CAACTC-3 (reverse); collagen, type III, alpha 1 paraformaldehyde fixation. experiments (Col3a1; mouse), 5- CTGTAACATG- Six-week-old female C57BL/6 mice were GAAACTGGGGAAA-3 (forward) and 5- Immunofluorescence cell staining purchased from the Shanghai Slac Laboratory CCATAGCTGAACTGAAAACCACC-3 (reverse); Human dermal fibroblasts at a density of Animal (Slac, Shanghai, China). All animal Col3a1 (human), 5-TTGAAGGAGGATGTTCC- 3 2×10 cells per well were seeded on cover studies have been approved by the Animal CATCT-3 (forward) and 5- ACAGACA- slides in 24-well plates and incubated Care and Use Committee of Shanghai Jiao CATATTTGGCATGGTT-3 (reverse); matrix overnight. Cells were fixed with 4% Tong University. All efforts were made to mini- metalloproteinases 1 (MMP1; human), 5- paraformaldehyde and blocked with 5% goat mize animal suffering. AAAATTACACGCCAGATTTGCC-3 (forward) serum in PBST (0.1% TritonX-100 in phos- A total of 18 mice were randomly assigned to and 5-GGTGTGACATTACTCCAGAGTTG-3 phate buffered saline) for 1 h. For F-actin three groups (n=6). Two groups of animals (reverse); matrix metalloproteinases 2 staining, cells were incubated with Alexa Fluor received daily subcutaneous injection of D- (MMP2; human), 5-TACAGGATCATTGGCTA- 488 Phalloidin (Cytoskeleton Inc., Denver, CO, galactose (D-gal; 1000 mg/kg) for 8 weeks.15 CACACC-3 (forward) and 5-GGTCA- USA; 1:200) for 1 h at room temperature. For The third group received phosphate buffered CATCGCTCCAGACT-3 (reverse); matrix metal- α-SMA staining, cells were incubated with pri- saline (PBS) as a negative control. Two weeks loproteinases 9 (MMP9; human), 5-TGTAC- mary antibodies against -SMA (Abcam, α later, DMSO and apigenin (5 μmol/L) was CGCTATGGTTACACTCG-3 (forward) and 5- Cambridge, UK, 1:200) for 2 h at room temper- delivered by microneedles16 [MTS-Roller GGCAGGGACAGTTGCTTCT-3 (reverse); tissue ature, followed by an Alexa Fluor 555-conjugat- Model: CR2 (0.2 mm)] to the dermis of D- inhibitor of metalloproteinases1 (TIMP1; ed secondary antibody. For smad3 staining, galactose treated mice once a day for 4 weeks, human), 5-CTTCTGCAATTCCGACCTCGT-3 cells were incubated with primary antibodies respectively. Mice were sacrificed at the end of (forward) and 5-ACGCTGGTATAAGGTG- against smad3 (Cell Signaling Technology, treatment, and skin tissue was harvested for GTCTG-3 (reverse); α-SMA (human), 5- Beverly, MA, USA; 1:200) for 2 h at room tem- further analyses. AAAAGACAGCTACGTGGGTGA-3 (forward) and perature, followed by an Alexa Fluor 488-conju- 5-GCCATGTTCTATCGGGTACTTC-3 (reverse). gated secondary antibody. Immunofluore - Histology and immunohistochemistry Cell viability assay scence signals were captured using confocal Paraformaldehyde-fixed paraffin-embedded microscopy (LSM 510, META Laser Scanning tissue sections (5 μm) were stained with For the cell viability assay, HDFs were seed- Microscope; Zeiss, Jena, Germany). hematoxylin and eosin (H&E), Masson’s ed on 96-well plates (100 mL per well), fol- trichrome (Trichrome stain LG solution, lowed by apigenin (cat. no. 42251; Sigma- Smad2/3 knockdown by siRNA HT10316; Sigma-Aldrich) and Picrosirius red Aldrich) or DMSO (Sigma-Aldrich) treatment. RNA interference was performed using (Fluka, Buchs, Switzerland). For immunohis- After 3 or 5 days, cell culture medium was smad2/3 siRNA (human) (sc-37238; Santa tochemical staining, the sections were detect- replaced by Thiazolyl Blue Tetrazolium Cruz Biotechnology, Dallas, TX, USA), target- ed with primary antibodies against collagen (MTT) working solution, followed by ing human smad2/3 and control siRNA (sc- I/III (Millipore; 1:1,000) and α-SMA (Abcam; a 4-hour incubation at 37°C in a 5% CO2 incu- 37007) as negative control. Transfection for 1:200) overnight at 4°C. After incubation with bator. After MTT working solution was HDFs was conducted using Lipofectamine the appropriate secondary antibodies, the sec- removed and DMSO added, the absorbances at RNAiMAX reagent (Invitrogen, Carlsbad, CA, tions were developed with diaminobenzidine 490 nm were detected. USA) according to the manufacturer’s protocol. and counterstained with hematoxylin.

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Statistical analysis mal cells in the dermis, and their function is Results strongly implicated in dermatology. To study Statistical differences were calculated using apigenin’s effect on fibroblasts (Figure 1A), Friedman’s analysis of variance (ANOVA), with Apigenin stimulated collagen NIH/3T3 and HDFs were administered with post-hoc least significant difference (LSD) test synthesis but had no effect on apigenin at the concentrations of 5 mol/L. As as appropriate. A significant difference among μ shown in Figure 1B, apigenin could potently groups was set at P<0.05. matrix metalloproteinases in vitro increase Toluidine blue staining after api- Fibroblasts are the predominant mesenchy- genin treatment for 5 days, which mean the

Figure 1. Apigenin stimulated collagen synthesis of fibroblasts. A) The molecular structure of apigenin. B) Toluidine blue staining in NIH/3T3 and HDFs for 5 days. C) Dose-dependent effects of apigenin on mRNA expression of Col1a2 and Col3a1 in NIH/3T3 and HDFs for 3 days. D) The protein level of Col1a2 and Col3a1 was measured by Western Blot at 5 days after apigenin was applied at concentrations of 0.1 μmol/L to 10 μmol/L. E) The expression of MMP1, MMP2, MMP9 and TIMP1 were also assessed by real-time PCR. Data are presented as mean ± SD, n≥3; NS, not significant; *P<0.05; **P< 0.01; ***P<0.001.

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synthesis of extracellular matrix was were observed when NIH/3T3 and HDFs were analysis. When HDFs were treated with api- increased.13 Further real-time PCR analysis treated with apigenin at the concentration of 5 genin for 5 days, the protein level of Col1a2 showed that, in NIH/3T3 and HDFs, apigenin μmol/L, and the increase of Col3a1 was more and Col3a1 were higher than that of cells treat- (0.1 μmol/L - 10 μmol/L) dose-dependently obvious than Col1a2 (Figure 1C). These upreg- ed with DMSO (Figure 1D). In addition, we stimulated endogenous expression of Col1a2 ulation effect of apigenin on collagen expres- next examined the effect of apigenin on and Col3a1. The most significant changes sion were then confirmed by Western blot matrix metalloproteinases (MMPs) and

Figure 2. No obvious cytotoxicity exerted by apigenin on fibroblasts viability, apoptosis, proliferation, cell cycle and activation. A) Cell viability was examined by MTT assays at 3 or 5 days after apigenin was applied in HDFs. B) Apoptosis was evaluated after treating HDFs with 5 μmol/L or 1 μmol/L apigenin or DMSO; flow cytometry profile represents Alexa Fluor® 488 Annexin V staining in X axis and PI in Y axis. C) The effect of apigenin on fibroblasts growth was investigated by monolayer colony formation assay. D) The expression of cyclin E1, CDK4, cyclin D1, CDK2 and p-CDK2 proteins was analysed using Western blot in HDFs. E-F) The levels of α-SMA mRNA and protein expression were measured by real-time PCR and Western blot. G) Immunofluorescence cell staining for α- SMA and F-actin in cultured HDFs after incubation with apigenin or DMSO for 72 h; F-actin is shown by green fluorescence and α- SMA is shown by red fluorescence; nucleus (blue) was stained with DAPI; scale bar: 50 μm. Data are presented as mean ± SD, n≥3; NS, not significant; **P<0.01; ***P<0.001.

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TIMP1, well-known proteases that degrade col- smad3 in a dose-dependent manner, whereas er and compact dermis (Figure 4 E,F). The in lagen proteins. The expression of MMP1, total smad2, smad3 and smad4 did not obvi- vivo study also demonstrates that apigenin MMP2, MMP9, and their inhibitor TIMP1 were ously alter. It also showed that apigenin had does not show any effect on activation of unchanged (Figure 1E). sustained effect on promoting phosphorylation fibroblasts (Figure 4G). of smad2 and smad3 after a 3-day treatment Apigenin did not effect fibroblasts (Figure 3B). Yoon et al.19 revealed that MAPK viability and activity pathway was involved with peptide-induced Afterwards, we studied the effect of api- collagen synthesis of fibroblasts. However, Discussion genin on fibroblasts viability, apoptosis, prolif- when fibroblasts were treated with apigenin eration and activation. In vitro, MTT assays for 12 h, the expression of total and phospho- Dermal atrophy is the major causes of aging showed that the viability of HDFs was similar rylated JNK, ERK and p38 protein remained appearance.20 In vivo and in vitro studies show with those incubated with DMSO, when incu- unchanged, compared with DMSO (Figure that decline in the production of collagen in 3C). Immunofluorescence experiments de - bated with apigenin (1 to 10 μmol/L) for 3 or 5 aging fibroblasts is mainly responsible for days, respectively (Figure 2A). In addition, no monstrated that after treatment with apigenin decreasing in dermal thickness seen in extrin- significant differences in percentages of apop- for 12 h, smad3 protein (labeled by green) was sically aging skin, which reveals dermal atro- totic cells was observed after the exposure of significantly increased and mostly translocat- phy, fragmentation, and irregular collagen fibroblasts to apigenin (Figure 2B). To investi- ed into the nucleus (labeled by blue) (Figure bundles.21 Since the ‘70s, animal and human gate the effects of apigenin on proliferation 3D). By contrast, in the DMSO groups, smad3 derived collagens have been studied for soft and cell cycle, colony formation assay and were retained in the cytoplasm. Once targeted tissue augmentation.22 Injectable filling Western Blot analysis of cell cycle related pro- knockdown smad2/3 by specific siRNA, the up- implants are now widely used for cosmetic pur- teins were performed. Colony forming ability regulation effect of apigenin on the expres- poses. However, exogenic injectable collagen of HDFs was similar in apigenin-treated group sion of collagen type-I and type-III protein was often presented various complications such as to DMSO-treated group (Figure 2C). The obviously reduced (Figure 3E), which con- allergy, ecchymosis, local necrosis and infec- expression levels of cell cycle associated pro- firmed that smad2/3 is required for the trans- tions of bacteria or virus. Scientists have tried teins remained unchanged between apigenin duction of apigenin effect on collagen expres- for decades to find other alternative to stimu- and DMSO treated groups (Figure 2D). These sions. late endogenous collagen synthesis. There are results suggested that apigenin had no obvi- Apigenin stimulated collagen syn- several anti-oxidants, such as vitamins C and ous cytotoxicity on fibroblasts’ viability, apop- E, co-enzyme Q10 and retinoids used for treat- tosis and proliferation. thesis in the D-galactose-induced ing UV-induced skin aging and wrinkles.23, 24 Fibroblast overactivation leads to pathologi- skin aging mouse model However, only few compounds are able to cal collagen deposition or scar formation.17 The in vivo effects of apigenin on collagen induce type I collagen synthesis25-27 and none Myofibroblasts, known as activated fibroblasts, synthesis was investigated in the D-galactose- of them can stimulate both type I and type III are marked by α-SMA expression. To deter- induced skin aging mouse model. The collagen collagen synthesis according to the record in mine the effects of apigenin on fibroblasts’ expression was showed by H&E, Masson’s literatures. activation, we evaluated the levels of α-SMA trichrome stain, Picrosirius red stain and Apigenin, a plant flavone, has gained con- mRNA and protein expression (Figure 2 E,F) immunohistochemistry. Histology showed sig- siderable attention due to its health benefits, in cultured HDFs treated with apigenin. We nificant changes in dermal thickness and den- chemopreventive properties and wide distribu- 28 found that α-SMA mRNA expression had no sity in samples obtained from D-gal-treated tion in the plant kingdom. Many studies have obvious change in apigenin-treated cells com- mice compared with PBS control group demonstrated that apigenin possesses a wide pared with DMSO-treated cells. We also (Figure 4 A-D). After 1 month of apigenin range of biological activities to the skin. It has showed that apigenin did not affect α-SMA administration at the concentration of 5 been reported that apigenin can stimulate expression in vitro by immunofluorescence nucleotide excision repair genes to protect μmol/L, the mice exhibited obviously staining (Figure 2G). These findings suggest- increased dermal thickness and collagen den- skin keratinocytes29 against UVB-induced skin ed that apigenin did not cause fibroblasts to sity compared with DMSO-treated mice inflammation.30 Dietary apigenin attenuates overactivate into myofibroblasts while colla- (Figure 4 A-D). Magnified images showed that the development of atopic dermatitis-like skin gen synthesis was increasing. dermis in the apigenin-treated group exhibit- lesions in atopic dermatitis model.31 Apigenin Induction of collagen synthesis was ed compact and clearly evident staining, could also effectively reduce the incidence and whereas collagens were loosely distributed in size of skin tumors caused by ultraviolet B mediated by smad2/3 activation DMSO-treated dermis of the aging skin model (UVB) exposure through the enhancement of To further explore the underlying mecha- (Figure 4 A-D), in both Masson’s trichrome UVB-induced apoptosis.32 In this study, we nism of how apigenin activated type-I and stain, Picrosirius red stain and immunohisto- investigated the effect of apigenin on dermal type-III collagen gene expression, transform- chemistry examinations. Dermal collagen fibroblasts’ function. At first, we found apigenin could increase ing growth factor beta 1 (TGF-β1) and mito- could be subdivided into type I and type III col- gen-activated protein kinase (MAPK) signal- lagen after Picrosirius red staining under the mRNA expression of Col1a2 and Col3a1 in NIH/3T3 and HDFs. With extracellular matrix ing pathway were analysed. TGF-β1 is a proto- polarized light. As shown in Figure 4F, api- typic fibrogenic cytokine, enhancing extracel- genin could significantly increase both colla- staining and Western Blot analysis, the stimu- lular matrix gene expression. Previous studies gen type I and type III density in the dermis of lative effect of collagen on protein level was proved that Col1a2 and Col3a1 were direct the skin aging mouse model. Quantitative data more significant. Although as reported in the literature, basal levels of Col1a1 and -SMA TGF-β1/smad3 targets in human dermal of dermal thickness and collagen density α fibroblasts.18 As shown in Figure 3A, when showed that mice subcutaneously injected mRNAs were reduced in fibroblasts treated HDFs were treated for 12 h, apigenin (1 with D-gal showed thinner skin and less colla- with high concentration of apigenin (20 mol/L),33 our research confirmed that api- μmol/L or 5 μmol/L) markedly increased the gen compared to control mice and apigenin- μ expression of phosphorylated smad2 and treated mice demonstrated significantly thick- genin of 25 μmol/L showed obvious cytotoxici-

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Figure 3. Apigenin-mediated collagen synthesis increase via smad2/3 signaling pathway. A,B) Western blot analysis and quantification of phosphorylated and total smad2, smad3 and total smad4 in HDFs with or without apigenin stimulation for 12 h or 3 days. C) Western Blot analysis of JNK, ERK and p38 in HDFs with or without apigenin stimulation for 12 h. D) Immunofluorescence experi- ments: smad3 was labeled as green; nucleus (blue) was stained with DAPI; scale bar: 50 μm. E) Western blot showed the expression of Col1a2 and Col3a1 in HDFs when cells were treated with DMSO, apigenin or apigenin with specific siRNA of smad2/3. Data are pre- sented as mean ± SD, n≥3; NS, not significant; *P<0.05; **P<0.01; ***P<0.001.

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Figure 4. Apigenin increased dermal thickness and collagen density in the D-galactose-induced skin aging mouse model. A-D) H&E, Masson’s trichrome, Picrosirius red and immunohistochemistry stained dermis of control mice and D-gal-treated mice respectively received apigenin and DMSO; scale bars: 100 μm; zoom scale bars: 20 μm. E) Quantification of dermal thickness. F) Picrosirius red staining under polarized light and quantification of type I and type III collagen density; collagen type I is shown as red fibers and col- lagen type III is shown as green fibers; scale bars: 100 μm. G) Immunohistochemistry staining of α-SMA of control mice and D-gal- treated mice respectively received apigenin and DMSO; scale bars: 100 μm; zoom scale bars: 20 μm. Data are presented as mean ± SD, n=6/6/6; *P<0.05; **P<0.01; ***P<0.001.

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ty in fibroblasts. We believed that the attenuat- 2. Fisher GJ, Wang ZQ, Datta SC, Varani J, Srivastava G, et al. Epigenetic silencing of ing effect of high concentration of apigenin on Kang S, Voorhees JJ. Pathophysiology of a Ca(2+)-regulated Ras GTPase-activating phenotypic transitions in the analyzed cell pop- premature skin aging induced by ultravio- protein RASAL defines a new mechanism ulations were not independent of its cytotoxic let light. N Engl J Med 1997;337:1419-28. of Ras activation in human cancers. Proc activity. We detected the markers related to 3. Liang JA, Pei XR, Zhang ZF, Wang N, Wang Natl Acad Sci U S A 2007;104:12353-8. extracellular matrix degradation and found JB, Li Y. The Protective Effects of Long- 15. Zhang S, Dong Z, Peng Z, Lu F. Anti-aging that apigenin had no effect on the balance of Term Oral Administration of Marine effect of adipose-derived stem cells in a MMPs/TIMPs. Collagen Hydrolysate from Chum Salmon mouse model of skin aging induced by D- We further observed that apigenin directly on Collagen Matrix Homeostasis in the galactose. PLoS One 2014;9:e97573. activated smad2/3-dependent signaling path- Chronological Aged Skin of Sprague- 16. Prausnitz MR. Microneedles for transder- way. This is not surprising since this flavonoid Dawley Male Rats. J Food Sci 2010;75: mal drug delivery. Adv Drug Deliv Rev displays considerable muti-effect. It targets a H230-8. 2004;56:581-7. number of secondary messengers, including 4. Hou H, Li BF, Zhang ZH, Xue CH, Yu GL, 17. Wang J, Dodd C, Shankowsky HA, Scott PG, those potentially involved in TGF-β1 signaling Wang JF, et al. Moisture absorption and Tredget EE, Wound Healing Research G. pathway, such as NF-kB,34 MAPK/ERK,35 retention properties, and activity in allevi- Deep dermal fibroblasts contribute to FAK,36,37 PKC38 and PI3K-Akt39 in a cell context- ating skin photodamage of collagen hypertrophic scarring. 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