Wound healing is a strictly regulated process. Once the process is not well controlled, patients suffered from the pain and it may cause serious outcome, such as amputation. Finding drugs to accelerate wound healing process is necessary and urgent. Andrographolide (AND) is a major bioactive phytoconstituent in various parts of A. paniculata, particularly in the leaves. AND contains anti-inflammatory, antibacterial, and antifungal activities. Whether AND can accelerate wound healing is still not understood, this study explore the effects of AND on skin wound healing process and its underlying mechanisms.
C57BL/6 mice were used in the in vivo wound healing assay. Mice were anesthetized and two wounds were made on the back. Hydrogen gel containing DMSO (CTL) or AND was administered on the wounds every day. Pictures were taken every two days, and the surface wound areas were quantified by Image Pro. Wounds were then cut on day 3、5 and 7 to analyze the histological changes of skin with H&;E and trichrome stain. The wound size was smaller and the collagen synthesis was more in AND treated wound. With these analysis, AND can significantly accelerate wound healing.
Next, we further investigated the mechanisms underlying AND-accelerated wound healing. With in vitro cell model, macrophages and fibroblasts are key modulators of wound healing process in the inflammation and proliferation phases. Raw 264.7 and NIH3T3 were used to observe the AND-induced anti-inflammatory, cell proliferative, migratory, and collagen productive effects. AND can not only significantly attenuate LPS-induced COX-2 expression and NO production in macrophages, but also expedite the proliferation and migration in fibroblasts through ERK and PI3K/AKT signaling pathways.
With the results in the in vivo and in vitro models, AND has the potential to be a new drug for wound healing. Further development of AND may lead to a beneficial therapeutic agent for the treatment of wound healing disease and shorten the healing time of wound.

目 錄
誌謝………………………………………………………………………i
摘要……………………………………………………………………...ii
Abstract………………………………………………………………….iii
Abbreviations.………………………………………………………..….iv
壹、 緒論…..……………………………………………………………1
一、 引言…………………………………………………………1
二、 傷口癒合的過程與相關的細胞…………………………....1
三、 纖維母細胞和巨噬細胞與傷口癒合的關係………………3
四、 傷口癒合相關的訊息傳遞路徑……………………………4
五、 Andrographolide的藥理特性與應用………………………5
六、 研究目的……………………………………………………6
貳、 材料與方法………………………………………………………..7
參、 實驗結果…………………………………………………………16
肆、 討論………………………………………………………………22
伍、 附圖………………………………………………………………26
陸、 參考文獻…………………………………………………………43

1.Kanter, G., N.R. Connelly, and J. Fitzgerald, A system and process redesign to improve perioperative antibiotic administration. Anesth Analg, 2006. 103(6): p. 1517-21.
2.葉碧芳, 實用傷口護理 = Practical wound care. 2003, 臺北市: 華杏.
3.Beanes, S.R., et al., Skin repair and scar formation: the central role of TGF-beta. Expert Rev Mol Med, 2003. 5(8): p. 1-22.
4.Broughton, G., 2nd, J.E. Janis, and C.E. Attinger, The basic science of wound healing. Plast Reconstr Surg, 2006. 117(7): p. 12S-34S.
5.Chen, R.N., et al., Development of N,O-(carboxymethyl)chitosan/collagen matrixes as a wound dressing. Biomacromolecules, 2006. 7(4): p. 1058-64.
6.Singer, A.J. and R.A. Clark, Cutaneous wound healing. N Engl J Med, 1999. 341(10): p. 738-46.
7.Ruszczak, Z. and R.A. Schwartz, Modern aspects of wound healing: An update. Dermatol Surg, 2000. 26(3): p. 219-29.
8.Kim, J.B., et al., Inhibition of LPS-induced iNOS, COX-2 and cytokines expression by poncirin through the NF-kappaB inactivation in RAW 264.7 macrophage cells. Biol Pharm Bull, 2007. 30(12): p. 2345-51.
9.Bainbridge, P., Wound healing and the role of fibroblasts. J Wound Care, 2013. 22(8): p. 407-8, 410-12.
10.McDougall, S., et al., Fibroblast migration and collagen deposition during dermal wound healing: mathematical modelling and clinical implications. Philos Trans A Math Phys Eng Sci, 2006. 364(1843): p. 1385-405.
11.Darby, I.A. and T.D. Hewitson, Fibroblast differentiation in wound healing and fibrosis. Int Rev Cytol, 2007. 257(10): p. 143-79.
12.Steinbrech, D.S., et al., Fibroblast response to hypoxia: the relationship between angiogenesis and matrix regulation. J Surg Res, 1999. 84(2): p. 127-33.
13.Enoch, S. and D.J. Leaper, Basic science of wound healing. Surgery (Oxford), 2008. 26(2): p. 31-37.
14.Svensjo, T., et al., Cultured autologous fibroblasts augment epidermal repair. Transplantation, 2002. 73(7): p. 1033-41.
15.Lamme, E.N., et al., Allogeneic fibroblasts in dermal substitutes induce inflammation and scar formation. Wound Repair Regen, 2002. 10(3): p. 152-60.
16.Breitbart, A.S., et al., Accelerated diabetic wound healing using cultured dermal fibroblasts retrovirally transduced with the platelet-derived growth factor B gene. Ann Plast Surg, 2003. 51(4): p. 409-14.
17.Huang, C., K. Jacobson, and M.D. Schaller, MAP kinases and cell migration. J Cell Sci, 2004. 117(Pt 20): p. 4619-28.
18.Kyriakis, J.M. and J. Avruch, Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10-year update. Physiol Rev, 2012. 92(2): p. 689-737.
19.Pearson, G., et al., Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev, 2001. 22(2): p. 153-83.
20.Schaeffer, H.J. and M.J. Weber, Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol, 1999. 19(4): p. 2435-44.
21.Ip, Y.T. and R.J. Davis, Signal transduction by the c-Jun N-terminal kinase (JNK)--from inflammation to development. Curr Opin Cell Biol, 1998. 10(2): p. 205-19.
22.Leppa, S. and D. Bohmann, Diverse functions of JNK signaling and c-Jun in stress response and apoptosis. Oncogene, 1999. 18(45): p. 6158-62.
23.Morgensztern, D. and H.L. McLeod, PI3K/Akt/mTOR pathway as a target for cancer therapy. Anticancer Drugs, 2005. 16(8): p. 797-803.
24.Ward, S.G. and P. Finan, Isoform-specific phosphoinositide 3-kinase inhibitors as therapeutic agents. Curr Opin Pharmacol, 2003. 3(4): p. 426-34.
25.Medforth, C.J., R.S. Chang, and G.-Q. Chen, A conformational study of diterpenoid lactones isolated from the chinese medicinal herb andrographis paniculata. Perkin Trans, 1990. 2(1): p. 1011-1016.
26.Verma, N. and M. Vinayak, Antioxidant action of Andrographis paniculata on lymphoma. Mol Biol Rep, 2008. 35(4): p. 535-40.
27.Puri, A., et al., Immunostimulant agents from Andrographis paniculata. J Nat Prod, 1993. 56(7): p. 995-9.
28.Kumar, R.A., et al., Anticancer and immunostimulatory compounds from Andrographis paniculata. J Ethnopharmacol, 2004. 92(2-3): p. 291-5.
29.Nugroho, A.E., et al., Anti-diabetic effect of a combination of andrographolide-enriched extract of Andrographis paniculata (Burm f.) Nees and asiaticoside-enriched extract of Centella asiatica L. in high fructose-fat fed rats. Indian J Exp Biol, 2013. 51(12): p. 1101-8.
30.Zhang, Y.Z., J.Z. Tang, and Y.J. Zhang, [Study of Andrographis paniculata extracts on antiplatelet aggregation and release reaction and its mechanism]. Zhongguo Zhong Xi Yi Jie He Za Zhi, 1994. 14(1): p. 28-30, 34, 5.
31.Jiang, C.G., et al., Andrographolide inhibits the adhesion of gastric cancer cells to endothelial cells by blocking E-selectin expression. Anticancer Res, 2007. 27(4b): p. 2439-47.
32.Lim, J.C., et al., Andrographolide and its analogues: versatile bioactive molecules for combating inflammation and cancer. Clin Exp Pharmacol Physiol, 2012. 39(3): p. 300-10.
33.Najib Nik, A.R.N., et al., Antimalarial activity of extracts of Malaysian medicinal plants. J Ethnopharmacol, 1999. 64(3): p. 249-54.
34.Yun, K.J., et al., Anti-inflammatory effects of sinapic acid through the suppression of inducible nitric oxide synthase, cyclooxygase-2, and proinflammatory cytokines expressions via nuclear factor-kappaB inactivation. J Agric Food Chem, 2008. 56(21): p. 10265-72.
35.Zaidan, M.R., et al., In vitro screening of five local medicinal plants for antibacterial activity using disc diffusion method. Trop Biomed, 2005. 22(2): p. 165-70.
36.Wiart, C., et al., Antiviral properties of ent-labdene diterpenes of Andrographis paniculata nees, inhibitors of herpes simplex virus type 1. Phytother Res, 2005. 19(12): p. 1069-70.
37.Thamlikitkul, V., et al., Efficacy of Andrographis paniculata, Nees for pharyngotonsillitis in adults. J Med Assoc Thai, 1991. 74(10): p. 437-42.
38.Iruretagoyena, M.I., et al., Andrographolide interferes with T cell activation and reduces experimental autoimmune encephalomyelitis in the mouse. J Pharmacol Exp Ther, 2005. 312(1): p. 366-72.
39.Panossian, A., et al., Effect of Andrographis paniculata extract on progesterone in blood plasma of pregnant rats. Phytomedicine, 1999. 6(3): p. 157-61.
40.Handa, S.S. and A. Sharma, Hepatoprotective activity of andrographolide from Andrographis paniculata against carbontetrachloride. Indian J Med Res, 1990. 92(8): p. 276-83.
41.Woo, A.Y., et al., Andrographolide up-regulates cellular-reduced glutathione level and protects cardiomyocytes against hypoxia/reoxygenation injury. J Pharmacol Exp Ther, 2008. 325(1): p. 226-35.
42.Zhu, X.K., et al., Anti-AIDS agents. Part 61: Anti-HIV activity of new podophyllotoxin derivatives. Bioorg Med Chem, 2004. 12(15): p. 4267-73.
43.Uttekar, M.M., et al., Anti-HIV activity of semisynthetic derivatives of andrographolide and computational study of HIV-1 gp120 protein binding. Eur J Med Chem, 2012. 56(3): p. 368-74.
44.Akbarsha, M.A. and P. Murugaian, Aspects of the male reproductive toxicity/male antifertility property of andrographolide in albino rats: effect on the testis and the cauda epididymidal spermatozoa. Phytother Res, 2000. 14(6): p. 432-5.
45.Jayakumar, T., et al., Experimental and Clinical Pharmacology of Andrographis paniculata and Its Major Bioactive Phytoconstituent Andrographolide. Evid Based Complement Alternat Med, 2013. 2013(2013) p. 846740.
46.Al-Bayaty, F.H., et al., Effect of Andrographis paniculata leaf extract on wound healing in rats. Nat Prod Res, 2012. 26(5): p. 423-9.
47.Castilho, R.M., C.H. Squarize, and J.S. Gutkind, Exploiting PI3K/mTOR signaling to accelerate epithelial wound healing. Oral Dis, 2013. 19(6): p. 551-8.
48.Jiang, Q., et al., EGF-induced cell migration is mediated by ERK and PI3K/AKT pathways in cultured human lens epithelial cells. J Ocul Pharmacol Ther, 2006. 22(2): p. 93-102.
49.Hough, C., M.R. , and J.J.E.D., TGF-Beta Induced Erk Phosphorylation of Smad Linker Region Regulates Smad Signaling. PLoS ONE, 2012. 7(8). p. 1-10
50.Chen, J.C., et al., NGF Accelerates Cutaneous Wound Healing by Promoting the Migration of Dermal Fibroblasts via the PI3K/Akt-Rac1-JNK and ERK Pathways. Biomed Res Int, 2014. 32(2) p. 547187.
51.Regan, M.C., et al., The wound environment as a regulator of fibroblast phenotype. J Surg Res, 1991. 50(5): p. 442-8.
52.Ehrlich, H.P. and T.M. Krummel, Regulation of wound healing from a connective tissue perspective. Wound Repair Regen, 1996. 4(2): p. 203-10.
53.Gabbiani, G., The myofibroblast in wound healing and fibrocontractive diseases. The Journal of pathology, 2003. 200(4): p. 500-503.
54.Pomerantz, R.J., et al., Lipopolysaccharide is a potent monocyte/macrophage-specific stimulator of human immunodeficiency virus type 1 expression. J Exp Med, 1990. 172(1): p. 253-61.
55.Li, W., et al., Activation of Nrf2-antioxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis. Biochem Pharmacol, 2008. 76(11): p. 1485-9.
56.Zhang, B., et al., CHP1002, a novel andrographolide derivative, inhibits pro-inflammatory inducible nitric oxide synthase and cyclooxygenase-2 expressions in RAW264.7 macrophages via up-regulation of heme oxygenase-1 expression. Int Immunopharmacol, 2013. 15(2): p. 289-95.