The benefit of LZ-8 as immunomodulatory protein has been widely studied. LZ-8 reported might accelerate rat liver wound injury. However, the effect of LZ-8 on skin wound healing is less studied.This study aimed to evaluate the effectsof LZ-8 in mouse skin wound healing and determine the molecular mechanism of LZ-8-induced acceleration of wound healing.Different group of wound were generated at C57Bl/6 mice dorsal skin followed by wound histology observation. To evaluate the effect of LZ-8 for fibroblast and keratinocyte migration we performed In Vitro wound healing assay.Our studied revealed that an increase in PMN infiltrations and collagen depositions in the wound of animals treated with LZ-8 on day 2 and day 3 respectively. In addition,we found that both fibroblast and keratinocyte migration were significantly increase under treatment with LZ-8In Vitro study of wound healing. These results suggested that LZ-8 not only modulate PMN cell infiltration, but also play an active role in enhancing fibroblast and keratinocyte migration and increase collagen deposition.

Table of Content
Table of content i
List of figures iii
Abstract v
Introduction 1
1.1 Skin Anatomy and Physiology 2
1.1.1 Epidermis Surface of the Skin 2
1.1.1.1 Stratum Basale 3
1.1.1.2 Stratum Spinosum 3
1.1.1.3 Stratum Granulosum 3
1.1.1.1 Stratum Corneum 4
1.1.2 Dermis Surface of the Skin 4
1.2 Skin Wound and Types of Wound 5
1.3 Skin Wound Repair and Regeneration 6
1.3.1 Hemostasis and Coagulation Phase/ Lag Phase 6
1.3.2 Inflammation Phase 7
1.3.2.1 Neutrophil Role in Wound Healing 7
1.3.2.1 Macrophage Role in Wound Healing 8
1.3.1 Hemostasis and Coagulation Phase/ Lag Phase 8
1.3.3 Proliferation and Repair Phase 9
1.3.3.1 Fibroblast 10
1.3.3.2 Keratinocyte 11
1.3.4 Skin Remodeling 14
1.4 Ling Zhi-8 14
1.5 Aim of the Study 16
Experimental Section 17
Results 26
Discussion 30
Conclusion 34
Future Work 35
Reference 58

Reference


1.Kanitakis J. Anatomy, histology and immunohistochemistry of normal human skin. European Journal of Dermatology. 2002;12(4):390–401.
2.Reinke JM, Sorg H. Wound repair and regeneration. European surgical research Europaische chirurgische Forschung Recherches chirurgicales europeennes. 2012;49(1):35-43
3.Sinno H, Prakash S. Complements and the wound healing cascade: an updated review. Plastic surgery international. 2013;2013:146764
4.Summers C, Rankin SM, Condliffe AM, Singh N, Peters AM, Chilvers ER. Neutrophil kinetics in health and disease. Trends in immunology. 2010;31(8):318-24
5.Dovi JV, Szpaderska AM, DiPietro LA. Neutrophil function in the healing wound: adding insult to injury? Thrombosis and haemostasis. 2004;92(2):275-80
6.Dovi JV, He LK, DiPietro LA. Accelerated wound closure in neutrophil-depleted mice. Journal of leukocyte biology. 2003;73(4):448-55
7.Koh TJ, Novak ML, Mirza RE. Assessing macrophage phenotype during tissue repair. Methods Mol Biol. 2013;1037:507-18.
8.Rodero MP, Khosrotehrani K. Skin wound healing modulation by macrophages. International journal of clinical and experimental pathology. 2010;3(7):643-53.
9.Scull CM, Fischer TH. Macrophage pro-inflammatory cytokine secretion is enhanced following interaction with autologous platelets. Journal of Inflammation. 2010;7(53):1-9.
10.Mirza R, DiPietro LA, Koh TJ. Selective and specific macrophage ablation is detrimental to wound healing in mice. The American journal of pathology. 2009;175(6):2454-62.
11.Sorrel JC, AI. Fibroblast heterogeneity: more than skin deep. Journal of Cell Science. 2004;117:667-75.
12.Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 2008;16(5):585-601
13.Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiological reviews. 2003;83(3):835-70
14.Isaac C, Paggiaro AO, Conduta JL, Aldunate B, Herson MR, Altran SC, Mathor MB, et al. Role of keratinocytes in wound contraction: an impact assessment using a model of collagen matrix populated with fibroblasts. Rev Bras Cir Plast. 2011;26(3):402-6.
15.Haase I ER, Pofahl R, Watt F.M. Regulation of keratinocyte shape, migration andwound epithelialization by IGF-1- and EGF-dependent signalling pathways. Journal of Cell Science. 2003;116:3227-38.
16.Pivarcsi A BL, Reathi B, Szaboa AK,, Koreck A MSM, Beer Z, Csoergo ZB,, Magoacsi M RE, Dobozy A, Kemeany L. Expression and function of Toll-like receptors 2 and 4 in human keratinocytes. International Immunology. 2003;15(6):721-30.
17.Kohsuke Kino AY, Kyoko Yamaoko, Junko watanabe, Shizuko Tanaka, Kerry Ko, Kimiko Shimizu, and Hajime Tsunoo. Isolation and Characterization of a New Immunomodulatory Protein, Ling Zhi-8 (LZ-8), from Ganoderma lucidium. J Biol Chem. 1989;264(1):472-8.
18.Liang Huang FS, Chongyang Liang,Yong-Xing He,Rui Bao,Lixia Liu,Cong-Zhao Zhou. Crystal structure of LZ-8 from the medicinal fungus Ganoderma lucidium. Proteins. 2008;75(2):524-7.
19.Tanaka S KK, Kino K. Complete amino Acid Sequence of an Immunomodulatory Protein, Ling Zhi-8 (LZ-8). J Biol Chem. 1989;264(28):16372-7.
20.Huang W.-N YC-Y, Chen D.-C, Chuang L.-T Correlation of structure and bioactivity of recombinant fungal immunomodulatory protein, ling-zhi-8 (LZ-8) following exposure to denaturing condition. Journal of food biochemistry. 2013.
21.Wu CT, Lin TY, Hsu HY, Sheu F, Ho CM, Chen EI. Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway. Carcinogenesis. 2011;32(12):1890-6
22.Sun J, He H, Xie BJ. Novel antioxidant peptides from fermented mushroom Ganoderma lucidum. Journal of agricultural and food chemistry. 2004;52(21):6646-52
23.Hsu HY, Kuan YC, Lin TY, Tsao SM, Hsu J, Ma LJ, et al. Reishi Protein LZ-8 Induces FOXP3(+) Treg Expansion via a CD45-Dependent Signaling Pathway and Alleviates Acute Intestinal Inflammation in Mice. Evidence-based complementary and alternative medicine : eCAM. 2013;2013:513542
24.Lin Y-L, Liang Y-C, Tseng Y-S, Huang H-Y, Chou S-Y, Hseu R-S, et al. An immunomodulatory protein, Ling Zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and MAPK pathways. Journal of leukocyte biology. 2009;86(4):877-89
25.Richards H, Williams A, Jones E, Hindley J, Godkin A, Simon AK, et al. Novel role of regulatory T cells in limiting early neutrophil responses in skin. Immunology. 2010;131(4):583-92
26.Lin H.-J CY-S, Lin L.-H, Haung C.-F, Chia-Yu Wu aK-LO. An Immunomodulatory Protein (Ling Zhi-8) froma Ganoderma lucidum Induced Acceleration of Wound Healing in Rat Liver Tissues after Monopolar Electrosurgery. Evidence-Based Complementary and Alternative Medicine. 2014;2014:1-12.
27.Kim MH, Liu W, Borjesson DL, Curry FR, Miller LS, Cheung AL, et al. Dynamics of neutrophil infiltration during cutaneous wound healing and infection using fluorescence imaging. The Journal of investigative dermatology. 2008;128(7):1812-20
28.Nishio N, Okawa Y, Sakurai H, Isobe K. Neutrophil depletion delays wound repair in aged mice. Age (Dordr). 2008;30(1):11-9.
29.Darby IA, Hewitson TD. Fibroblast differentiation in wound healing and fibrosis. International review of cytology. 2007;257:143-79
30.McDougall S, Dallon J, Sherratt J, Maini P. Fibroblast migration and collagen deposition during dermal wound healing: mathematical modelling and clinical implications. Philosophical transactions Series A, Mathematical, physical, and engineering sciences. 2006;364(1843):1385-405
31.Baker BS, Ovigne JM, Powles AV, Corcoran S, Fry L. Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis. The British journal of dermatology. 2003;148(4):670-9
32.Pivarcsi A, Bodai L, Rethi B, Kenderessy-Szabo A, Koreck A, Szell M, et al. Expression and function of Toll-like receptors 2 and 4 in human keratinocytes. Int Immunol. 2003;15(6):721-30