1011 D3000 new imprint Word template Vol 13, No 1 (2025) ISSN 2167-8677 (online) DOI 10.5195/d3000.2025.1011 http://dentistry3000.pitt.edu Expression of IGF-1R in Skin Wound Healing Treated by Topical Appli- cation of Cinnamomum Zeylanicum Noor Na&k Raheem, Lobna K. Al-Khafaji, Lehadh M. Al-Azzawi, Dhifaf Kamil Kadhim Al-Thahab College of Den*stry, Mustansiriyah University, Iraq Abstract Objec&ve: The aim of this study was to evaluate the immunohistochemical expression of IGF- 1R on cutaneous wound healing. Material and Methods: A total of 18 male albino rats (RaSus norvegicus albinus) weighing nearly 300-400 gm were used in the present study. Two punch biopsies were done on the dorsum of each animal, and daily local applicaUon of cinnamon zeylanicum extract was performed on leW side, while the right one was allowed to heal nat- urally (control measure). The process of wound skin healing was tracked for periods (1, 3, and 7 days), all specimens were processed for immunohistochemistry examinaUon. Results: Based on immunohistochemical results, experimental groups had the highest mean values of IGF-1R expression in the stroma and epidermis when compared to control groups. These values increased with Ume, which was more noUceable on days three and seven of the ex- periments. Conclusion: PosiUve localizaUon of IGFF-1R in rat's skin indicated the effecUveness of cinnamon zeylanicum extract in acceleraUng the healing pro- cess of cutaneous layers of skin. Open Access Cita%on: Raheem NN, et al. (2025) Expression of IGF-1R in Skin Wound Healing in Rats Treated by Topical Appli- ca%on of Cinnamomum Zeylanicum. Den%stry 3000. 1:a001 doi:10.5195/d3000.2025.1011 Received: August 4, 2025 Accepted: August 26, 2025 Published: September 10, 2025 Copyright: ©2025 Raheem NN, et al. This is an open ac- cess ar%cle licensed under a Crea%ve Commons AXribu- %on Work 4.0 United States License. Email: noorna%kraheem@uomustansiriyah.edu.iq Introduc)on Cutaneous wound healing is a complicated natural process. Attempts to repair the de- fect caused by signi:icant damage start very early during in:lammatory response [1]. Ul- timately, they result in repair. Herbal thera- pies have been utilized to treat wounds and injuries for centuries [2]. Cinnamomum zeylanicum, an old botanical synonym for the cinnamon tree, comes from Sri Lanka's former name. Cinnamon has diverse biologi- cal properties, including anti-in:lammatory [2], antioxidant, [3,4], and anti-microbial [4]. The wound repair process requires close regulation of degradative and regenerative processes, a wide variety of cell types, and complicated interactions between many bio- chemical cascades needed to repair process without complications [5,6]. Growth factors released in the injured area encouraging cell migration to the wound site by chemotaxis, stimulate the growth of :ibro- blasts and epithelial cells, initiate angiogene- sis, and extracellular matrix ECM deposition and remodeling of the wounded area [1]. Growth hormone action, often mediated by (IGF-I) is predominantly seen in the dermis of skin, impacting the synthesis of collagen :i- ber, IGF activity has a signi:icant impact on both epidermal and dermal compartments, that enhance the proliferation, migration and survival of stem cells [7]. The insulin-like growth factor 1 receptor (IGF-1R) is a small, multifunctional molecule like pro-insulin, it is acting in an autocrine or paracrine way to aid in different and multi- ple functions, including the early phases of wound healing [6,8]. Many physiological functions rely on the important balance be- tween proliferation of mitotically active epi- dermal keratinocytes of the skin and differ- entiation of post-mitotic cells. This balance has a crucial role in skin formation and de- velopment [9]. The study aimed to evaluate the effects of locally applied cinnamon essen- tial oil on expression of IGF-1R in cutaneous wound healing in rats. Material and Methods Experimental design 18 male albino rats of about (300–350gm) and aging from (4-8) weeks were used [10]. All rats were maintained under good condi- tions with a standard laboratory environ- ment. Animals were housed in single, and separate cages for 7 days in equivalent set- tings. The rats were arbitrarily divided into 3 main groups (six rats each), based on period of healing (1, 3, and 7 days). Two full-thickness punch biopsies of 2 mm depth [10] were made on the dorsum of each rat with a steri- lized punch of 5 mm diameter [11]. The Expression of IGF-1R in Skin Wound Healing Treated by Topical ApplicaUon of Cinnamomum Zeylanicum Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.1011 http://dentistry3000.pitt.edu 2 wound on the right side was left untreated. While the other side (experimental) treated with local cinnamon essential oil (100% pu- rity) every day till scarifying. Surgical procedures All surgical instruments were sterilized by using the oven at 150ºC for one hour. A com- bination of 50 mg (40 mg/kg-B.W.). Keta- min HCL and 2% xylazine (0.4 mg/kg B.W.) was injected intramuscularly according to the animal’s weight. Hair clippers were used for removal of dorsal hair :irst, followed by disinfection using ethyl alcohol (70%). Two punch biopsies of 5 mm in diameter were done with a rotary device. 3 cm distant from each other. The control wound was irrigated with distilled water while left experimental (left side) was topically treated with one drop of cinnamomum zylanicum essential oil (10 µl, U.S.A.), applied by using a micropi- pette. Each rat received general anesthesia and was scari:ied at the end of (1, 3 and 7 days). The operation site was demarcated with nearly 5 mm of normal tissue surround- ing the wounded area, and then a complete excisional biopsy was obtained. For :ixation purposes. the specimens were placed in a plastic biopsy container :illed with ten per- cent formaldehyde and cut using a micro- tome to gain 5 µm thickness for immuno- histochemical study; polyclonal IGF-1R were used according to manufacturer instruc- tions. SPSS software version 24.0 was used for statistical analysis. We used ANOVA test after using a paired sample t-test, which established the data's normal distribution. Alpha was de:ined as 0.05. Results One-day duration Positive localization of IGF-1R expressed by :ibroblasts and collagen :ibers in control group is showed in Figure 1. Figure 2 shows the expression of experimental skin section group that positive reaction in hair follicles and :ibrous connective tissue. Figure 1. View of positive expression of IGF- 1R by :ibroblast (red arrow) and collagen :i- ber (arrow CF) in control group. DAB stain with counter stain hematoxylin, X40. Figure 2. View of positive expression of IGF- 1R by :ibroblast (red arrow) and collagen :i- bers (CF). DAB stain with counter stain he- matoxylin, X40. Three days duration Positive localization of IGF-R1 in the skin section of the control group is detected by epithelium, :ibroblasts, and vascular endo- thelium as shown in Figure 3. Experimental skin section group shows positive localiza- tion of IGF-1R by new epithelium and :ibro- blasts (Figure 4). Figure 3. Magni:ied view of positive expres- sion of IGF-1R by vascular endothelium (black arrow) and :ibroblasts (FB), epithe- lium (blue arrow). DAB stain with counter stain hematoxylin, X40. Figure 4. Positive localization of IGF-1R at new epithelium (blue arrow) and :ibroblast (red arrow). DAB stain with counter stain he- matoxylin, X40. Seven days duration Positive localization of IGF-1R in the control group is detected by new epithelium; posi- tive reaction is shown by dermal :ibroblasts and vascular endothelial cells (Figure 5). While the microphotograph view after cin- namomum zylanicum application shows positive localization of IGF-1R by epithelial cells, hair follicles, collagen :ibers, and :ibro- blasts (Figure 6). Figure 5. Photomicrograph shows positively stained endothelial cells (EN) and :ibroblast (red arrow). DAB stain with counter stain he- matoxylin X40. Figure 6. Positive expression of IGF-1R by epithelium (E), :ibroblasts (red arrow). DAB stain with counter stain hematoxylin, X40. Stromal expression Statistical analysis of positively stained cells for immunohistochemical localization of IGF-1R as measured in different healing pe- riods (1, 3, and 7 days) for studied groups is shown in Table 1. There was a decrease in mean values with time for the 1- and 3-day CF CF FB Epithelium EN Epithelium CF Expression of IGF-1R in Skin Wound Healing Treated by Topical ApplicaUon of Cinnamomum Zeylanicum Vol 13, No 1 (2025) DOI 10.5195/d3000.2025.1011 http://dentistry3000.pitt.edu 3 durations whereas values increased at day 7; the highest values were recorded at day 1 for the experimental group, the lowest value was detected at day 3 for the controls. A significant difference (P <0.05) was ob- served between control and experimental groups on days 1 and 7. There was a non- significant difference (P>0.05) at day three. Those findings were illustrated in Figure 7. Figure 7. Experimental groups versus and controls regarding IGF-1R expression (stro- mal). Epidermal expression Statistical analysis of epidermal immuno- histochemical localization of IGF-1R is demonstrated in Table 2. The mean value of positively stained cells increased with time at day 3 and 7 healing durations for control and experimental groups; the highest aver- age value of positively stained cells was rec- orded at day (7) for the experimental group, whereas the lowest values were noticed at day 3 for the control groups Figure 8. Besides there was signi:icant difference (p<0.05) be- tween studied groups at 3,7 days as shown in Table 2. Figure 8. Comparison between experimental and control group regarding IGF-1R expres- sion (epidermal). Discussion One of the major concerns for medical staff in skin wounds that have a significant impact on people's health and lives is increasing morbidity and death rates [12]. The healing process is a complicated dynamic process in- cluding four overlapped phases: it starts with hemostasis, inflammation, and proliferation, and ends with tissue remodel- ing (maturation). The most important goal of the healing process is accelerating recovery of normal condition of tissues without scar- ring [13,14]. Growth factors are important regulators tak- ing part in orchestrating and promote cellu- lar responses, including: growth, migration, differentiation, and deposition of extracellu- lar matrix needed for successful wound heal- ing [5]. Insulin-like growth factor 1 acts as a regulator of vascular endothelial function adjusted vasoconstriction and /or vasodila- tion, and modulates inflammatory actins to conserve skin homeostasis [16]. The exact mechanism of the healing process of a wound is not clearly understood. There are several parameters that are involved in the healing of wounds, including histological results (re-epithelialization, granulation tis- sue, collagen accumulation, inflammatory re- action, angiogenesis, ulcer formation). The results showed a significantly increased level of IGF-1R expression during advances of the skin healing process as it stimulates the keratinocytes and fibroblast migration, especially during wound epithelialization [17]. While these cells express only small amounts of this protein. Thus, optimum heal- ing is related to IGF-1R levels. A few cells in the dermis and epidermis in normal skin express IGF-1R receptors (basal epidermal keratinocytes and undifferenti- ated epithelial cells related to the outer root sheath of hair follicle, and sebaceous glands. All epidermal cells, macrophages, and other cells of the skin showed positive expression of IGF-1R at day one to three after the injury in coincidence with [18]. In the present study, the highest mean values of positive expression for stromal (IGF-1R) were detected on day 1, when the process of re-epithelization is usually taking place. This is in agreement with Póvoa and Diniz (2011) [19], who studied growth hormone system: skin interactions and stated that the maxi- mum levels of IGF-1 in body :luids and local tissues occur early in the :irst hours or days following the injury since it acts as a chemo- attractant for endothelial cells correlated with the proliferation and migration of keratinocytes and :ibroblasts which play a major role in the healing of wounds [20,21], by stimulating collagen synthesis and secre- tion, thus accelerating wound healing and in- creasing the strength of the wound. It may originate from migrating keratinocytes from epithelial cells of adjacent hair follicles, :i- broblasts from granulation tissue, in:lamma- tory cells, or from plasma in accordance with Seeger and Paller (2015) [22]. Conclusion The present study concluded that using local cinnamomum zylanicum essential oil accel- erates wound healing as demonstrated in IGF-1R expression, which is involved in the healing process in different durations. Acknowledgements The authors extend their thanks to Mus- tansiriya University, Baghdad, Iraq, for providing support. Funding This research did not receive any speci:ic grant from funding agencies in the public, commercial, or not-for-pro:it sectors. Conflict of Interest The authors have no con:licts of interest to declare. Regulatory Statement This study was approved by the Research Ethical Committee of the College of Dentis- try, Mustansiriyah University (study num- ber: MUOPA9). References 1. Ganapathy N, Venkataraman SS, Daniel R, Ara- vind RJ, Kumarakrishnan VB. Molecular biology of wound healing. Journal of Pharmacy and Bioallied Science. 2012 Aug 4;(Suppl 2):S334-7. doi: 10.4103/0975-7406.100294. 2. 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MS SD P-value No. % No. % 1 Day 1 0 0 2.833 0.408 0 0 3.33 0.516 0.023 2 1 16.66 0 0 3 5 83.33 4 66.66 4 0 0 2 33.33 3 Days 1 3 50 1.5 0.547 3 50 1.83 0.983 0.097 2 3 50 1 16.66 3 0 0 2 33.33 4 0 0 0 0 7 Days 1 3 50 1.66 0.816 0 0 2.5 0.547 0.039 2 2 33.33 3 50 3 1 1.66 3 50 4 0 0 0 0 Table 2. Control groups in comparison to experimental groups regarding IGF-1R expression (epidermal). Period Score Control MS SD Exp. MS SD P-value No. % No. % 3 Days 1 0 0 2.5 0.547 0 0 3.16 0.752 0.036 2 3 50 1 16.66 3 3 50 3 50 4 0 0 2 33.33 7 Days 1 0 0 2.66 0.516 0 0 3.66 0.516 0.041 2 2 33.33 0 0 3 4 66.66 2 33.33 4 0 0 4 66.66