Effects of Topical Paiteling in Nude Mice Implanted with Human Condyloma
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bioRxiv preprint doi: https://doi.org/10.1101/563536; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Effects of topical PaiTeLing in nude mice implanted with human condyloma 2 acuminatum tissue infected with HPV 6, 31, and 81: comparison with imiquimod and 3 interferon-α-2b 4 Rong Xu1, Li Wang2, Jianmei Hou3, Jun Li3, Zhiyan Fan4, Liangcai Wu5, Congzhong 5 Zhu6, Miaomiao Ma1, Huiping Wang1, Shuping Hou1* 6 7 1. Department of Dermatovenereology, Tianjin Medical University General Hospital, 8 154 Anshan Road, Heping District, Tianjin 300052, China 9 2. Department of Hematology, The Affiliated Yantai Yuhuangding Hospital of 10 Qingdao University, Shandong Province, P.R. China 11 3. Department of Pharmacy, Chinese Traditional Medicine Hospital of Lanling County, 12 Shandong Province, P.R. China 13 4. Department of Pharmacy, The Third People’s Hospital of Linyi, Shandong Province, 14 P.R. China 15 5. Department of Dermatology, The Sixth Affiliated Hospital of Sun Yat-sen 16 University, Guangzhou, P.R. China bioRxiv preprint doi: https://doi.org/10.1101/563536; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 17 6. Department of Obstetrics and Gynecology, Tianjin Medical University General 18 Hospital, Tianjin, P.R. China 19 Rong Xu and Li Wang contribute equally to this manuscript. 20 *Corresponding author: Email: [email protected]; Tel: +86-13821150956 21 The total number of words, figures, tables and references: This paper includes 22 1947 words, 3 Figures and 42 references. 23 Sources of Financial Support: This work was supported by the National Natural 24 Science Foundation (#31770194; to Shuping Hou). Science and technology projects 25 of Guangdong province (No. 2014A020212434 to Wu. L) 26 Author email addresses: Rong Xu, [email protected]; Li Wang, 27 [email protected]; Jianmei Hou, [email protected]; Jun Li, 28 [email protected]; Zhiyan Fan, [email protected]; Liangcai Wu, 29 [email protected]; Congzhong Zhu, [email protected]; Miaomiao Ma, 30 [email protected]; Huiping Wang, [email protected]; Shuping Hou, 31 [email protected] bioRxiv preprint doi: https://doi.org/10.1101/563536; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 32 33 Abstract 34 BACKGROUND: The standard treatment for condyloma acuminatum is topical 35 imiquimod. In the current study, we used a mouse model to compare the effects of an 36 herbal medication PaiTeLing. 37 METHODS: Lesion tissue was obtained from a woman with genital condyloma 38 acuminatum. DNA genotyping revealed HPV6, 31, and 81. Tissue prism (0.5 cm3) 39 was implanted to BALB/C nude mice, 22 days after the implantation, mice began to 40 receive topical treatment with imiquimod, interferon-α-2b gel, or PaiTeLing over the 41 site of implantation for 2 consecutive weeks. Mice receiving tissue implantation but 42 no other intervention was included as a control. Skin tissue was collected for H&E 43 staining and anti-CD207 immunohistochemistry. Blood was collected to determine a 44 panel of cytokines. 45 RESULTS: H&E staining showed lower number of koilocytes and higher number of 46 Langerhans cells in the treatment groups, particularly in mice receiving imiquimod or bioRxiv preprint doi: https://doi.org/10.1101/563536; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 47 PaiTeLing. Blood levels of TNFα, IL-2, INF-γ and IL-12p70 were increased in the 48 treatment groups, particularly in mice receiving imiquimod or PaiTeLing. 49 CONCLUSION: Immune response in nude mice infected with HPV6, 31, and 81 is 50 enhanced by treatment with imiquimod, interferon-α-2b and PaiTeLing. Effects of 51 imiquimod and PaiTeLing seems to be stronger than interferon-α-2b. 52 INTRODUCTION 53 Condyloma acuminatum (CA) is a wide-spread sexually transmitted disease with 54 increasing prevalence during the past decades [1]. CA is caused by human 55 papillomavirus (HPV) infection. Infection with high-risk HPV strains is closely 56 associated cervical cancer as well as other type of cancer in the anogenital region [2, 57 3]. HPV16 is a major risk for the development of oropharyngeal 58 squamous-cell-carcinoma [4, 5]. HPV infection causes mental distresses and produces 59 negative impact on sexual life and social well-being of infected individuals. Clearance 60 of HPV virus is the goal for the treatment of genital warts and could prevent cancers. 61 Most of the currently available treatments focus on clearing the externally visible bioRxiv preprint doi: https://doi.org/10.1101/563536; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 62 warts rather than targeting the viruses [6,7]. An animal model for CA that look into 63 both the local and systemic immune responses is essential for development of new 64 treatments [8,9]. 65 Host immune responses and chronic inflammation have been shown to be 66 important risk factors for HPV-related carcinogenesis [10]. Increased duration of HPV 67 infection could influence disease development, indicating the importance of the host 68 immune response to HPV clearance and HPV-related carcinogenesis. Immune 69 responses to invading HPV consist of innate and adaptive immune systems. 70 Langerhans cells are immature dendritic cell (mDC), and the only 71 professional antigen-presenting cells in the epidermis. [11,12] HPV infection results 72 in a net loss of Langerhans cells at the site of infection. [13] HPV also interferes with 73 antigen presentation and processing machinery in Langerhans cells, [14, 15] and alters 74 chemokine and cytokine expression by LC. [16, 17] Cytokines TNF-α, IL-2, IFN-γ 75 and IL-12p70 are associated with HPV clearance. [18-21] More than 90% of cervical bioRxiv preprint doi: https://doi.org/10.1101/563536; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 76 squamous cancers and 75% of cervical lesions with koilocytes harbor HPV DNA [21]. 77 As a result, koilocytes in CA may be associated with HPV infection. 78 Imiquimod is an immuno-modulating agent with actions at multiple levels of the 79 adaptive immune system. Imiquimod activates the cells of the immune system via toll 80 like receptor, and increase the secretion of cytokines, including TNF-α, IL-1β, and 81 IL-6. [22] PaiTeLing (Beijing Paitborn Company, the Chinese Academy of Medical 82 Sciences) is an herbal medication widely used to treat genital warts in China. The 83 main components are honeysuckle and fructus cnidii, Indigowoad Leaf, sophorae 84 flavescentis, Brucea javanica, and Hedyotis diffusa. [23] A previous study suggested 85 that PaiTeLing could promote HPV clearance and regression of cervical lesions. [24] 86 PaiTeLing selectively destroys cancer cell membranes, including cytoplasmic and 87 mitochondrial membranes, thereby suppressing cancer cell proliferation. In addition, 88 PaiTeLing could also destroy viruses that reside inside the cells [25]. Interferons are a 89 group of natural messenger proteins with antiviral actions. Systemic administration of 90 interferons after ablative treatment for anogenital warts has been advocated to bioRxiv preprint doi: https://doi.org/10.1101/563536; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 91 increase clearance and decrease recurrence rate. [26] Human interferons are a class of 92 small proteins and glycoprotein cytokines (15-28 kD) produced by T cells, fibroblasts, 93 and other cells in response to viral infection and other biologic stimuli. Interferons 94 bind to specific receptors on cell membranes to produce a variety of biological actions, 95 include inducing enzymes, suppressing cell proliferation, inhibiting viral proliferation, 96 enhancing the phagocytic activity of macrophages, and augmenting the cytotoxic 97 activity of T lymphocytes.[27] In the current study, we used a mouse model of human 98 CA to examine the potential effects of topical imiquimod, PaiTeLing and 99 interferon-α-2b. 100 101 MATERIALS AND METHODS 102 1.1 Experimental animals 103 Female BALB/C-nu nude mice (4-6 weeks of age; Experimental Animal Center, the 104 Chinese Academy of Medical Sciences Hematology Hospital and Institute of bioRxiv preprint doi: https://doi.org/10.1101/563536; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 105 Hematology) were used in this study. The mice were housed in a specific-pathogen 106 free facility. 107 1.2 Human condyloma acuminatum tissue and genotyping 108 Human condyloma acuminatum tissue was obtained from an elderly woman with 109 informed consent prior to any treatment. The diagnosis was based on acetic acid white 110 test and pathological examination. HPV genotyping was carried out using a 23-type 111 polymerase chain reaction-based kit from Yanneng Biotechnology (YZB/County 112 3881-2014). The results showed HPV6, HPV31 and HPV81. 113 1.3 Tissue implantation 114 The wart tissue was removed under aseptic condition, transferred into EP tubes 115 containing Ringer lactate solution, and soaked in a solution containing 1-ml D-hanks 116 solution and 2.5-ml streptomycin/penicillin for 45 minutes prior to slicing into 117 0.5-cm3 tissue blocks.