MOLECULAR MEDICINE REPORTS 17: 8385-8390, 2018

The therapeutic effect of artesunate on rosacea through the inhibition of the JAK/STAT signaling pathway

TING LI1, QINGWEN ZENG2, XINGMING CHEN1, GUOJIANG WANG3, HAIQING ZHANG1, AIHUA YU1, HAIRUI WANG1 and YANG HU1

1Department of Dermatology, Nanxiang Hospital of Jiading, Shanghai 201802; 2Department of Pediatric Endocrinology/Genetics, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai Institute for Pediatric Research, Shanghai 200092; 3Department of Dermatology, Zhoupu Hospital of Pudong, Shanghai 201318, P.R. China

Received September 21, 2016; Accepted February 9, 2018

DOI: 10.3892/mmr.2018.8887

Abstract. Acne rosacea is a type of chronic dermatosis with infection may lead to inflammation through the JAK/STAT the characteristics of erubescence, angiotelectasis and pustule signaling pathway. In conclusion, the potential mechanism by formation. However, current treatment methods are limited due which damage occurs in rosacea was revealed and a promising to the side effects. Artesunate demonstrated a promising thera- therapeutic method against rosacea was demonstrated. peutic efficacy with a high safety margin. HaCaT cells were treated with antibacterial peptide LL‑37 to simulate rosacea Introduction caused by Demodex folliculorum (D. folliculorum) infection. Cell Counting kit 8 and flow cytometry assays were performed Rosacea, also known as acne rosacea, is a type of chronic to measure cellular proliferation, apoptosis, the stage of the dermatosis with the characteristics of erubescence, cell cycle and reactive oxygen species generation in order to angiotelectasis and pustule formation. Rosacea can be determine the level of cell damage. Then the damaged cells divided into erythematotelangiectatic rosacea (ERT) and were treated with different concentrations of artesunate and papulopustular rosacea (PPR) in the clinic (1). The cause to determine the therapeutic effect of artesunate. of rosacea remains unclear; however, it may be associated Pro‑inflammatory cytokines tumor necrosis factor‑α (TNF‑α), with Demodex folliculorum (D. folliculorum) infections, interleukin (IL)‑6, IL‑8 and C‑C motif chemokine 2 (MCP‑1) alcoholic drinks, spicy food and exposure to sunlight (2,3). were measured using an ELISA, while western blotting was Parasitic D. folliculorum is commonly demonstrated in used to detect the expression of Janus kinase 2 (JAK2) and human hair follicles and sebaceous glands; an overgrowth signal transducer and transcription activator (STAT3). As a of D. folliculorum may cause disease (4). The infection rate result, LL‑37 treated HaCaT cells decreased in cell viability, and density of D. folliculorum measured in damaged rosacea had an increased apoptotic rate and cell cycle arrest, indicating skin was demonstrated to be significantly increased compared that cell damage caused by rosacea was simulated. In addi- with healthy skin through microscopic or histopathological tion, upregulated concentrations of the pro‑inflammatory examination (5). Increased macrophages and Langerhans cytokines TNF‑α, IL‑6, IL‑8 and MCP‑1 were attenuated in cells were observed in positive specimens of D. folliculorum, the artesunate group in a dose‑dependent fashion, indicating indicating a hypersensitivity reaction was induced by the therapeutic effect of artesunate. Furthermore, higher mite‑associated follicular antigens, leading to the formation of concentrations of artesunate exhibited an improved effect a papulopustule (6). Previous studies have demonstrated that compared with the doxycycline group. In addition, increased the expression level of matrix metalloproteinase‑9 (MMP‑9) expression levels of JAK2 and STAT3 following treatment has a linear correlation to the density of D. folliculorum (7,8). with LL‑37 suggested that rosacea caused by D. folliculorum Upregulated MMP results in the activation of epidermal serine protease, leading to the generation of the antibacterial peptide LL‑37. Therefore, D. folliculorum infection is an important exacerbating factor for rosacea. Treatment against rosacea includes systemic drugs such Correspondence to: Dr Yang Hu, Department of Dermatology, as and doxycycline, as well as locally acting Nanxiang Hospital of Jiading, 495 Zhongren Road, Jiading, Shanghai 201802, P.R. China drugs including metronidazole and azelaic acid according to E‑mail: [email protected] the food and drug administration (FDA) administration on rosacea. However, certain issues exist in current therapies Key words: rosacea, artesunate, Janus kinase 1/signal transducer against rosacea. External preparations may cause local inflam- and transcription activator mation due to the damaged skin barrier function of patients with rosacea (9). In addition, oral administration of antibiotics for long‑term treatment may lead to body dysbacteriosis (10). 8386 LI et al: ARTESUNATE IN ROSACEA

Although doxycycline is used as a non‑antibiotic at a dosage of the treatment with LL‑37 for 24 h, then centrifugation was 40 mg/day, gastrointestinal adverse reactions are common in performed at 150 x g at 4˚C for 10 min. Cells were collected the clinic. In addition, therapy against papulopustular rosacea and resuspended with dyeing buffer containing 50 µM (PPR) recommended by the FDA demonstrated a limited dihydroethidium (Wieglas Biotech (Beijing) Co., Ltd., Beijing, curative effect on the persistent and diffuse face erythema (9). China). Then ROS were measured using a flow cytom­eter Artesunate is an innovative antimalarial drug with a low (Accuri C6; BD Biosciences, Franklin Lakes, NJ, USA) level of side effects that was developed by Chinese researchers. and analyzed with Accuri C6 software version 1.0.264 (BD It was recognized by the World Health Organization and Biosciences). enrolled in the 11th edition of essential drug list in 2002. To measure apoptosis, cultured HaCaT cells were collected Artesunate was demonstrated to exhibit anti‑parasitic and using 0.25% trypsin and washed with PBS, followed by the anti‑angiogenesis activity (11‑13). It was reported to inhibit centrifugation at 150 x g at 4˚C for 5 min. The supernatant was delayed type hypersensitivity reactions and MMP activity (14). then discarded and the cells were incubated with Annexin V Vascular and inflammatory reactions are the two components fluorescein isothiocyanate apoptosis detection kit (BD necessary for rosacea. Therefore, artesunate was chosen from Biosciences) for 10 min at room temperature without light. the anti‑parasitic drugs as an alternative medicine for the treat- The cellular apoptotic rate was measured and the data was ment of rosacea. Artesunate was expected to have a similar obtained using a flow cytom­eter (Accuri C6) and Accuri C6 therapeutic effect and lower side effects when compared with software version 1.0.264 (BD Biosciences). doxycycline. Artesunate as an inexpensive drug with a high In addition, HaCaT cells were digested with 0.25% trypsin safety profile which may benefit patients with rosacea. and washed with PBS, followed by the addition of 70% pre‑cooled ethanol at 4˚C for 12 h and the RNA was Materials and methods removed using 1 µg/ml RNaseA. Then cells were stained with propidium iodide (PI) for 10 min at room temperature Cell culture and damage simulation. Cultured HaCaT and the DNA content was measured using a flow cytom­eter cells (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) (Accuri C6) and Accuri C6 software Version 1.0.264 (BD were treated with LL‑37 (Shanghai Aladdin Biochemical Biosciences) to determine the proportion of cells in each stage Technology Co., Ltd., Shanghai, China) at various concen- of cell cycle. trations (0, 10, 20, 40, 60, 80 and 100 µg/ml) to establish a rosacea skin lesion model. LL‑37 treated HaCaT cells were ELISA. The ELISA method was adapted to measure the content measured for cell viability, reactive oxygen species (ROS) of interleukin (IL)‑6 (JYM0140Hu), IL‑8 (JYM0139Hu), C‑C content, cell cycle stage and apoptotic rate to evaluate the motif chemokine 2 (MCP‑1, JYM0136Hu) and tumor necrosis rosacea‑damage model. factor‑α (TNF‑α, JYM0110Hu) in HaCaT cells by determining Furthermore, artesunate was employed to treat the rosacea the absorbency at 450 nm. The ELISA kits were provided by skin lesion model to evaluate the therapeutic effect on rosacea. Wuhan Colorful Gene Biological Technology (Wuhan, China). Cultured HaCaT cells were digested with 0.25% trypsin A total of ~100 µl culture medium was processed according to (Solarbio, Beijing, China) and transferred into 6‑well plates at the manufacturer's protocol. The concentrations of IL‑6, IL‑8, 3x105 cells/well. Following incubation at 37˚C for 24 h, the MCP‑1 and TNF‑α were calculated according to the standard Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE curve, which was generated from a series of different known Healthcare Life Sciences, Logan, UT, USA) was discarded concentrations of standard bovine serum albumin. and LL‑37 with a final concentration of 60 µg/ml was added into each well except for the control group, which received an Western blot analysis. Cells were seeded at 3x105 cells/well in equal volume of medium instead. Different concentrations 6‑well plates and duplicated wells were treated with or without (1, 10 and 50 µM) artesunate (Shanghai Aladdin Biochemical (control group) 60 µg/ml LL‑37 at 37˚C for 48 h. Following Technology Co., Ltd.) and 20 µg/ml doxycycline (Shanghai this, cell lysates were prepared in a RIPA lysis buffer Aladdin Biochemical Technology Co., Ltd.) were added to (BYL40825; JRDUN Biotech, Shanghai, China). Total protein the experimental groups. Following incubation at 37˚C for concentration in each sample was assayed by the bicinchoninic 48 h, cells were digested with 0.25% trypsin, and collected for method (Thermo Fisher Scientific, Inc., Waltham, MA, USA). ELISA and western blotting assays. Aliquots (20 mg total protein) of each sample were then loaded on 12.5% SDS‑polyacrylamide gels. Following electrophoresis, Cell Counting kit‑8 (CCK‑8) assay. Cultured HaCaT cells the proteins were transferred onto a nitrocellulose blotting treated with different concentrations of LL‑37 (0, 10, 20, 40, membrane (EMD Millipore, Billerica, MA, USA), blocked 60, 80 and 100 µg/ml) were mixed with 100 µl serum‑free with 5% nonfat milk and immunoblotted with specific anti- DMEM (Hyclone; GE Healthcare Life Sciences) containing bodies overnight at 4˚C. The sources of the primary antibodies 10% CCK‑8 (Dojindo Molecular Technologies, Inc., were as follows: Anti‑Janus kinase 2 (JAK2; cat. no. 3230;

Kumamoto, Japan) and incubated in 5% CO2 at 37˚C for 1 h. 1:1,000; CST Biological Reagents Co., Ltd., Shanghai, The optical density (OD)450 nm of the cell suspension was China), phosphorylated (p)‑JAK2 (cat. no. 8082; 1:800; CST measured using a spectrophotometer in order to evaluate the Biological Reagents Co., Ltd.), signal transducer and transcrip- cell viability of HaCaT cells. tion activator (STAT3; cat. no. Ab50761; 1:100), p‑STAT3 (cat. no. Ab76315; 1:2,000; both Cambridge, UK Abcam) Flow cytometry assay. Cultured HaCaT cells were washed and GAPDH (cat. no. 5174; 1:1,500; CST Biological Reagents using PBS and digested at 37˚C with 0.25% trypsin following Co., Ltd.). The membranes were then washed with TBST MOLECULAR MEDICINE REPORTS 17: 8385-8390, 2018 8387

Artesunate attenuates cell damage caused by LL‑37. The concentrations of IL‑6, IL‑8, MCP‑1 and TNF‑α were measured using an ELISA. As the results exhibit in Fig. 3, the concentrations of all the indices increased significantly in the LL‑37 treated groups compared with the control, while they were attenuated by treatment with various concentrations of artesunate (1, 10 and 50 µM) in a dose‑dependent manner. HaCaT cells treated with 50 µg/ml artesunate demonstrated a significantly improved therapeutic effect on LL‑37 treated cells compared with the doxycycline group (P<0.001; Fig. 3).

Figure 1. HaCaT cells were cultured and cell viability was measured. A Cell Artesunate inhibits cell damage through the JAK‑STAT3 Counting kit‑8 assay was performed to evaluate the viability of HaCaT cells treated with different concentrations (0, 10, 20, 40, 60, 80 and 100 µg/ml) of signaling pathway. Western blotting was employed to measure LL‑37 for 0, 24, 48 and 72 h. The cell viability of HaCaT cells declined in a the expression level of proteins associated with the JAK‑STAT dose‑dependent manner following treatment with LL‑37. Data are expressed *** signaling pathway. The upregulated expression levels of as the mean ± standard deviation (n=3). P<0.001 vs. the 0 µg/ml group. p‑JAK2/JAK2 and p‑STAT3/STAT3 were attenuated by artesunate (1, 10 and 50 µM) dose‑dependently (Fig. 4).

(0.1% Tween) buffer and incubated with horseradish peroxidase Discussion conjugated secondary antibodies (A0208 and A0216; 1:1,000; Beyotime Institute of Biotechnology, Shanghai, China) for Vascular and inflammatory reactions are the two required 1 h at room temperature. The membranes were then washed components for rosacea (15). Expansion of blood vessels with TBST (0.1% Tween) buffer and developed according to and lymphatics were demonstrated in patients with rosacea the enhanced chemiluminescence protocols (EMD Millipore). according to data obtained from deformation analysis, The films of western blotting were then scanned using a immunohistochemistry analysis, gene microarray and Bio‑Rad imaging densitometer (Model GS‑700) and the densi- reverse transcription‑polymerase chain reaction; an increase ties of the bands were semi‑quantified using Quantity One in vascular endothelial growth factor, vessel marker cluster software version 4.2.1 (Bio‑Rad Laboratories, Inc., Hercules, of differentiation‑31 and lymphatic marker D2‑40 were also CA, USA). observed (16‑19). Patients with PPR or ETR are sensitive to various stimuli including vasodilators and spicy food. The Statistical analysis. Data were expressed as the neuropeptides released from nerve endings may cause partial mean ± standard deviation of at least three independent repli- congestion, edema and erythema, along with the recruitment of cates and analyzed using analysis of variance and Tukey's neutrophil granulocytes and the release of mastocytes, leading post‑hoc test. GraphPad Prism 5.0 (GraphPad Software, Inc., to an inflammatory response. These results associated with the San Diego, CA, USA) software was used to perform the vascular reaction concur with the persistent face erythema and analysis of data. P<0.05 was considered to indicate a statisti- angiotelectasis exhibited in the clinic (7,20). cally significant difference. Innate immune response enhancement is involved in the early stages of rosacea. Previous studies demonstrated that Results toll‑like receptor 2, antibacterial peptide LL‑37, serine protease and MMP were upregulated in areas of skin damage in patients LL‑37 causes cell damage in a rosacea skin model. HaCaT with rosacea (7,21). Furthermore, except for the increase in cells treated with LL‑37 were used in the CCK‑8 assay to antibacterial peptides and kallikrein‑related peptidase 5, the measure cell viability (Fig. 1). As illustrated in Fig. 1, the mRNA expression level of neutrophil chemotactic agent IL‑8 proliferation of HaCaT cells treated with 10 µg/ml LL‑37 rose significantly (7,21). A synergistic effect exists between was significant at 24, 48 and 72 h (P<0.001, respectively) the innate immune response and novel blood vessels/neural compared with the control (0 µg/ml), indicating that immune disorders in the angiectasis and the inflammatory the viability of HaCaT cells was inhibited by LL‑37 in cascade (22). Angiectasis contributes to the progression of the a time‑dependent manner. In addition, the HaCaT cells inflammatory response, while LL‑37 exhibits high angiogenic treated with various concentrations of LL‑37 demonstrated activity and variety of signaling pathways which stimulate a decreased proliferation rate in a dose‑dependent fashion. alterations in the endothelial cells (7). Then flow cytometry was used to measure the levels of The JAK‑STAT signaling pathway is a signal transduction ROS, cell apoptosis and cell cycle progression (Fig. 2). ROS pathway stimulated by cytokines (23). It is involved in prolif- content was measured to evaluate the level of cell damage eration, differentiation, apoptosis and immunoregulation. in the model. As exhibited in Fig. 2A, the fluorescence JAK is a type of non‑transmembrane form of tyrosine kinase, intensity of LL‑37 treated cells was increased compared with while STAT is called a signal transducer and transcription the control cells, indicating a high level of ROS content in activator. STAT serves a critical role in signal transduction rosacea damaged cells. In addition, the induced apoptotic and gene transcription. The JAK‑STAT signaling pathway rate and cell cycle arrest demonstrated cell damage caused has a wide range of functions, which are focused on the by LL‑37, and indicated that the rosacea skin damage model immune response and immunoregulation. Chemokines are was appropriate (Fig. 2B and C). secreted by leukocytes and stromal cells in the hematopoietic 8388 LI et al: ARTESUNATE IN ROSACEA

Figure 2. Flow cytometry was used to measure the ROS content, cell apoptosis and cell cycle distribution in HaCaT cells following treatment with LL‑37 at a concentration of 60 µg/ml. (A) Compared with the control group, ROS content measured by dihydroethidium demonstrated a significant increase following treatment with LL‑37. (B) Early apoptotic cells were identified using Annexin V/PI double staining and are presented in the lower right‑hand quadrant. (C) The cell cycle of HaCaT cells was arrested by LL‑37. The cell cycle distribution of cells was blocked at the G2 phase. Data are presented as the mean ± standard deviation (n=3). ***P<0.001 vs. the control group. ROS, reactive oxygen species; PI, propidium iodide. microenvironment (24). Chemokines attached to endothelial granulocytes. The level of inflammatory factor IL‑8 increases cells have a chemotactic, activatory effect on neutrophil granu- significantly at inflammatory sites, and in serum and bodily locytes, mononuclear leucocytes, lymphocytes, eosinophilc fluids during infection or immunological disease. Therefore, granulocytes and basophilic granulocytes. IL‑8 representing the measurement of IL‑8 levels was used for the diagnosis and the a subfamily, exhibits a chemotaxis effect on neutrophilic prognosis of inflammatory diseases (26). granulocytes, while MCP‑1, which represents the b subfamily, In the present study, cell damage in rosacea caused induces mononuclear leucocytes (25). IL‑8, known as a neutro- by D. folliculorum infection was simulated using HaCaT phil chemotactic factor, is an important factor in inflammatory cells treated with LL‑37 and the therapeutic effect of arte- disease. It serves a vital role in anti‑infection, anti‑cancer and sunate against rosacea was demonstrated. In addition, the immune response regulation. IL‑8 is strongly chemotactic to results obtained from the present study revealed the injury specific and non‑specific immune cells, particularly neutro- mechanisms involved in rosacea. LL‑37 treated HaCaT cells philic granulocytes, as well as lymphocytes and basophilic demonstrated cell damage including lower cell proliferation, MOLECULAR MEDICINE REPORTS 17: 8385-8390, 2018 8389

Figure 3. An ELISA was performed to measure the concentrations of the pro‑inflammatory cytokines TNF‑α, IL‑6, IL‑8 and MCP‑1. (A) The concen- trations of IL‑6, IL‑8 and MCP‑1 increased significantly and were attenuated in the groups pre‑treated with artesunate in a dose‑dependent manner. (B) TNF‑α was upregulated following treatment with LL‑37 (60 µg/ml) and attenuated by artesunate in a dose‑dependent manner. Data are presented Figure 4. Western blotting was used to measure the expression levels of JAK2 as the mean ± standard deviation (n=3). ***P<0.001 vs. the control group; and STAT3. (A) The expression levels of JAK2, p‑JAK2, STAT3 and p‑STAT3 ##P<0.01, ###P<0.001 vs. the LL‑37 group; $P<0.05, $$P<0.01 vs. the doxycy- were determined by SDS‑PAGE. (B) The expression of JAK2 and STAT3 cline group. IL, interleukin; TNF‑α, tumor necrosis factor‑α; MCP‑1, C‑C was analyzed by western blotting. Data are presented as the mean ± standard motif chemokine 2. deviation (n=3). *P<0.05 and ***P<0.001 vs. the control group; ##P<0.01 and ###P<0.001, vs. the doxycycline group. JAK, Janus kinase; p‑, phosphorylated; STAT3, phosphorylated signal transducer and transcription activator. increased apoptotic rate and ROS levels, and induced cell cycle arrest, indicating damage caused by LL‑37 on HaCaT cells. Furthermore, the production of pro‑inflammatory cytokines, was demonstrated. The present study provided reliable data including TNF‑α, IL‑6, IL‑8 and MCP‑1, in LL‑37 treated for future animal experimentation and clinical research. HaCaT cells was increased when compared with the control Artesunate may be a promising novel therapeutic method for group, while the levels were attenuated in the artesunate groups the treatment of rosacea. in a dose‑dependent manner, corresponding to the expression level of JAK2 and STAT3 measured by the western blotting Acknowledgements assay. The activation of the JAK/STAT signaling pathway upregulated the expression of pro‑inflammatory cytokines Not applicable. including TNF‑α, IL‑6, IL‑8 and MCP‑1, which resulted in inflammation, and was attenuated by the treatment with arte- Funding sunate. The deactivation of the JAK/STAT signaling pathway detected in the artesunate groups suggested that the activation The present study was supported by grants from Shanghai of JAK/STAT signaling pathway was inhibited by artesunate, City Key Medical Specialties in B Construction Projects followed by the suppressed expression of pro‑inflammatory (grant no. ZK2012B18), the Special Construction Project of cytokines TNF‑α, IL‑6, IL‑8 and MCP‑1. Weak Field of Traditional Chinese Medicine in Shanghai In addition, it was demonstrated that the concentration of Municipal Health Planning Commission (grant no. TNF‑α, IL‑6, IL‑8 and MCP‑1 in HaCaT cells treated with BR2K2016014) and the Medical Key Subject Construction a high level of artesunate was lower than those in the doxy- Project of Jiading District of Shanghai Municipality (grant cycline treated group, which was indicative of the improved no. ZD06). therapeutic and antibacterial effect of artesunate. This result revealed a promising treatment for rosacea and provides Availability of data and materials experimental data for future clinical research. In conclusion, the therapeutic and antimicrobial effect of All data generated or analyzed during the present study are artesunate on rosacea caused by D. folliculorum infection included in this published article. 8390 LI et al: ARTESUNATE IN ROSACEA

Authors' contributions 11. Ho WE, Peh HY, Chan TK and Wong WF: : Pharmacological actions beyond anti‑malarial. Pharmacol Ther 142: 126‑139, 2014. TL and YH conceived and designed the study. TL, QZ, XC, 12. Van Agtmael MA, Eggelte TA and van Boxtel CJ: GW, HZ, AY and HW performed the experiments. TL and drugs in the treatment of : From medicinal herb to regis- tered medication. Trends Pharmacol Sci 20: 199‑205, 1999. YH wrote the manuscript. All authors read and approved the 13. der Meersch Van H: Review of the use of artemisinin and its manuscript. derivatives in the treatment of malaria. J Pharm Belg 60: 23‑29, 2005 (Article in French). 14. Rasheed SA, Efferth T, Asangani IA and Allgayer H: First Ethics approval and consent to participate evidence that the antimalarial drug artesunate inhibits invasion and in vivo metastasis in lung cancer by targeting essential extra- Not applicable. cellular proteases. Int J Cancer 127: 1475‑1485, 2010. 15. Cribier B: Pathophysiology of rosacea: Redness, telangiectasia, and rosacea. Ann Dermatol Venereol 138 (Suppl 3): S184‑S191, Consent for publication 2011. 16. Gomaa AH, Yaar M, Eyada MM and Bhawan J: Lymphangiogenesis and angiogenesis in non‑phymatous rosacea. Not applicable. J Cutan Pathol 34: 748‑753, 2007. 17. Yamasaki K and Gallo RL: The molecular pathology of rosacea. Competing interests J Dermatol Sci 55: 77‑81, 2009. 18. Steinhoff M, Buddenkotte J, Aubert J, Sulk M, Novak P, Schwab VD, Mess C, Cevikbas F, Rivier M, Carlavan I, et al: The authors declare they have no competing interests. Clinical, cellular, and molecular aspects in the pathophysiology of rosacea. J Investig Dermatol Symp Proc 15: 2‑11, 2011. 19. Rosina P, Zamperetti MR, Giovannini A, Chieregato C and References Girolomoni G: Videocapillaroscopic alterations in erythemato- telangiectatic rosacea. J Am Acad Dermatol 54: 100‑104, 2006. 1. Wilkin J, Dahl M, Detmar M, Drake L, Feinstein A, Odom R 20. Drummond PD and Su D: Blushing in rosacea sufferers. and Powell F: Standard classification of rosacea: Report of J Psychosom Res 72: 153‑158, 2012. the national rosacea society expert committee on the classifica- 21. Yamasaki K, Di Nardo A, Bardan A, Murakami M, tion and staging of rosacea. J Am Acad Dermatol 46: 584‑587, Ohtake T, Coda A, Dorschner RA, Bonnart C, Descargues P, 2002. Hovnanian A, et al: Increased serine protease activity and 2. Del Rosso JQ: Management of cutaneous rosacea: Emphasis cathelicidin promotes skin inflammation in rosacea. Nat Med 13: on new medical therapies. Expert Opin Pharmacother 15: 975‑980, 2007. 2029‑2038, 2014. 22. Del Rosso JQ: Advances in understanding and managing rosacea: 3. Tüzün Y, Wolf R, Kutlubay Z, Karakuş O and Engin B: Rosacea Part 2: The central role, evaluation, and medical management of and rhinophyma. Clin Dermatol 32: 35‑46, 2014. diffuse and persistent facial erythema of rosacea. J Clin Aesthet 4. Lazaridou E, Giannopoulou C, Fotiadou C, Vakirlis E, Trigoni A Dermatol 5: 26‑36, 2012. and Ioannides D: The potential role of microorganisms 23. Bernardino L, Xapelli S, Silva AP, Jakobsen B, Poulsen FR, in the development of rosacea. J Dtsch Dermatol Ges 9: 21‑25, Oliveira CR, Vezzani A, Malva JO, Zimmer J: Modulator 2011. effects of interleukin‑1beta and tumor necrosis factor‑alpha on 5. Roihu T and Kariniemi AL: Demodex mites in acne rosacea. AMPA‑induced excitotoxicity in mouse organotypic hippo- J Cutan Pathol 25: 550‑552, 1998. campal slice cultures. J Neurosci 25: 6734‑6744, 2005. 6. Georgala S, Katoulis AC, Kylafis GD, Koumantaki‑Mathioudaki E, 24. Oberbach A, Schlichting N, Blüher M, Kovacs P, Till H, Georgala C and Aroni K: Increased density of Demodex Stolzenburg JU and Neuhaus J: Palmitate induced IL‑6 and folliculorum and evidence of delayed hypersensitivity reaction MCP‑1 expression in human bladder smooth muscle cells in subjects with papulopustular rosacea. J Eur Acad Dermatol provides a link between diabetes and urinary tract infections. Venereol 15: 441‑444, 2001. PLoS One 5: e10882, 2010. 7. Del Rosso JQ: Advances in understanding and managing 25. Deshmane SL, Kremlev S, Amini S and Sawaya BE: Monocyte rosacea: Part 1: Connecting the dots between pathophysiological chemoattractant protein‑1 (MCP‑1): An overview. J Interferon mechanisms and common clinical features of rosacea with Cytokine Res 29: 313‑326, 2009. emphasis on vascular changes and facial erythema. J Clin 26. Bailey C, Negus R, Morris A, Ziprin P, Goldin R, Allavena P, Aesthet Dermatol 5: 16‑25, 2012. Peck D and Darzi A: Chemokine expression is associated with 8. Bonamigo RR, Bakos L, Edelweiss M and Cartell A: Could the accumulation of tumour associated macrophages (TAMs) and matrix metalloproteinase‑9 be a link between Demodex follicu‑ progression in human colorectal cancer. Clin Exp Metastasis 24: lorum and rosacea? J Eur Acad Dermatol Venereol 19: 646‑647, 121‑130, 2007. 2005. 9. Parodi A, Drago F, Paolino S, Cozzani E and Gallo R: Treatment of rosacea. Ann Dermatol Venereol 138 (Suppl 3): S211‑S214, 2011. 10. Sanchez J, Somolinos AL, Almodóvar PI, Webster G, Bradshaw M and Powala C: A randomized, double‑blind, placebo‑controlled trial of the combined effect of doxycycline hyclate 20‑mg tablets and metronidazole 0.75% topical lotion in the treatment of rosacea. J Am Acad Dermatol 53: 791‑797, 2005.