The Effects of Aloe vera Cream on the Expression of [CD4.sup.+] and [CD8.sup.+] Lymphocytes in Skin Wound Healing.
Aloe vera is the one of potential herbal medicines and could be found in the tropical area , such as Indonesia. Aloe vera leaf contains gel . Its gel contains flavonoid, terpenoid, lectin, anthraquinone, tannin, and saponin. And these contents have pharmacological properties to promote wound  and skin burns healing  and have antibacterial , anti-inflammatory , and cosmetic characteristics; this gel could also be used as a moisturizer and in dermatological products .
Damage to the normal anatomical structure and function of tissues is defined as wound . Normally, each wound will be healed through a process involving hemostasis, inflammation, proliferation, maturation, and remodeling [8, 9]. T lymphocytes, especially [CD4.sup.+], play important role in wound healing by secreting lymphokines to activate fibroblast, as wound anti-inflammatory agent, and activate factor in major histocompatibility complex (MHC) class II. On the other hand, [CD8.sup.+] lymphocytes play a role as proinflammatory and activate factor in MHC class I . Effect of Aloe vera on wound healing, especially role of [CD4.sup.+] and [CD8.sup.+] lymphocytes, is not fully known. In this study, the effects of Aloe vera on the percentage of the wound area, epidermal thickness, neutrophil infiltration, fibroblast, angiogenesis, and [CD4.sup.+] and [CD8.sup.+] lymphocytes were observed.
2. Material and Methods
This study was taken in Laboratory of Pathology, Faculty of Veterinary Medicine, Gadjah Mada University. All animal procedures in this study were approved by ethical clearance committee of Gadjah Mada University, with license numbers 00052/04/LPPT/VII/2016. Aloe vera from Kalimantan, Indonesia, was used in this study. Aloe vera leafs were peeled, cut, put in a blender mix, and extracted by alcohol 70%. Aloe vera extract was made into 1% and 2% cream concentration. The cream was made from the mixture of Aloe vera extract, stearic acid, potassium hydroxide (KOH), glycerin, methyl paraben, propylparaben, and water .
Thirty-six male Sprague-Dawley rats weighing 150-200 grams were divided into four groups. All groups were shaved and exposed to round 4 mm full-thickness punch biopsy on the back under ketamine 50 mg/kg BW and xylazine 4 mg/kg BW anesthesia. Next, group I was the control group; group II was treated with topical application of 1% Aloe vera cream; group III was treated with 2% Aloe vera cream; and group IV was treated with madecassol. The treatments were given once a day, for fifteen days. Three rats from each group were euthanized with cervical dislocation on days 5, 10, and 15 to collect skin specimen. The collected specimen was stored in neutral buffer formalin 10%, dehydrated, blocked in paraffin, and cut using microtome for H&E and IHC stain. IHC stain used monoclonal antibodies for [CD4.sup.+] (antirat [CD4.sup.+], Novocastra, RTU-CD4-1F6, Cat. number PA0427) and [CD8.sup.+] (anti-rat [CD8.sup.+], Novocastra, RTU-CD8-295, Cat. number PA0183).
The data was divided into macroscopical and microscopical data. Macroscopical data was the percentage of the wound area. Microscopical data was epidermal thickness, neutrophils infiltration, fibroblast, angiogenesis, and [CD4.sup.+] and [CD8.sup.+] lymphocytes. Microscopical examination was performed by ImageJ software. All data were analyzed by SPSS 16. Epidermal thickness, neutrophils infiltration, fibroblast, angiogenesis, and [CD8.sup.+] lymphocytes data were analyzed with Kruskal-Wallis and Mann-Whitney U Test. [CD4.sup.+] lymphocytes were analyzed with two-way ANOVA and Post hoc test.
3. Result and Discussion
There is significant difference between group II, group III, and group IV compared with group I (p [less than or equal to] 0,05). It shows that topical application of 1% and 2% Aloe vera cream and madecassol has potential effect on wound healing to the percentage of wounds area (Figures 1(a) and 1(b)). All groups showed that epidermal thickness increased; however, neutrophils infiltration and angiogenesis decreased during the wound healing process. Group II and group III showed significant epidermal thickness increasing (p [less than or equal to] 0,05) (Figure 2(a)) and neutrophils infiltration decreasing (p [less than or equal to] 0,05) (Figure 2(b)), as well as angiogenesis compared with the other group (p [less than or equal to] 0,05) (Figure 3(a)). Proliferating of fibroblast did not show the significant difference in all groups (p [greater than or equal to] 0,05). Fibroblast increased significantly during the wound healing process (p [less than or equal to] 0,05) (Figure 3(b)). This result shows that 1% and 2% Aloe vera cream has better effect than madecassol against the epidermal thickness, neutrophil infiltration, and angiogenesis; however, it was not different regarding fibroblast.
[CD4.sup.+] lymphocytes in this study showed that there is no difference between all groups (p [greater than or equal to] 0,05), although [CD4.sup.+] lymphocytes in group II and group III increased maximally on day 5 compared with other groups. [CD4.sup.+] lymphocytes in group I increased on day 10, while the other groups decreased (Figure 4(a)). Low [CD4.sup.+] lymphocytes infiltration in group I has an adverse effect on wound healing when compared with the others. In every examination period (days 5,10, and 15), group I and group IV showed higher [CD8.sup.+] lymphocytes infiltration compared with group II and group III (p [less than or equal to] 0,05). It proved that Aloe vera has a better effect on decreasing [CD8.sup.+] lymphocytes infiltration in the wound area. Nevertheless, there is a sequential decreasing [CD8.sup.+] lymphocytes infiltration in all groups (Figure 5(a)). Both [CD4.sup.+] and [CD8.sup.+] lymphocytes show a trend of decreasing when it approaches the end of the healing period (p [less than or equal to] 0,05) (Figures 4 and 5). This study showed that Aloe vera may shorten healing period by increasing [CD4.sup.+]/[CD8.sup.+] lymphocytes infiltration ratio earlier on wound tissue.
Aloe vera contains anthraquinone, sterol, and saponin. Those active ingredients have a role as antibacterial and wound healing promotor . Topical application of Aloe vera cream increases the number of [CD4.sup.+] lymphocytes and decreases [CD8.sup.+] lymphocytes. Increasing the number of [CD4.sup.+]/[CD8.sup.+] lymphocytes ratio in group II and group III induces the other healing factors to promote wound . Aloe vera application on wound has a potential effect as the antiinflammatory agent. Its ability decrease leucocytes adhesion on wound via cyclooxygenase and prostaglandin route . The inflammatory brief period during wound will make faster healing process to the next phase. On proliferation phase, [CD4.sup.+] lymphocytes induce keratinocytes to release IL-1 in the wound area. Keratinocytes have a potential role on epithelization, proliferation, and maturation of epidermis . IL-1 that has been released by keratinocytes induces endothelial cells to form angiogenesis and fibroblast to form extracellular matrix .
Angiogenesis was formed to supply nutrition and others healing factors to wound area . Failure of angiogenesis impaired wound healing . In this study, increasing number of [CD4.sup.+]/[CD8.sup.+] lymphocytes ratio in group II and group III activates angiogenesis maximally on day 5 and it makes healing faster. Angiogenesis mediates fibroblast migration on wound tissues. Fibroblast-like cells appeared in group II and group III on day 5. Fibroblast-like cells are one indicator that healing process occurred faster . It proves that Aloe vera has a potential effect on healing. Increasing of fibroblast as a result of lymphokine induced which secreted by [CD4.sup.+] lymphocytes . [CD4.sup.+] lymphocytes have a role as healing promotor to cellular immune response . [CD4.sup.+] lymphocytes depletion can decrease skin tension and extracellular matrix component . On the other hand, [CD8.sup.+] lymphocytes have a role as proinflammatory agent  and as pain receptor.
Increasing of [CD8.sup.+] lymphocytes impaired wound healing. In this study, decreasing of [CD8.sup.+] lymphocytes on wound area was followed by increasing of [CD4.sup.+] lymphocytes. Decreasing of [CD8.sup.+] lymphocytes on wound area can decrease IL-3, so that healing process will be faster . Healing process could be identified macroscopically by percentage of wound area (Figure 1(b)).
Aloe vera has a potential effect to increase the ratio of [CD4.sup.+]/[CD8.sup.+] lymphocytes, which is mechanism influence on wound healing by decreasing percentage of the wound area, neutrophils infiltration, and angiogenesis and, however, increasing epidermal thickness and fibroblast in wound tissue. 1% and 2% Aloe vera cream have similar potential effect and both of 1% and 2% Aloe vera cream can be used as alternative therapy on the wound.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
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Yos Adi Prakoso (iD) (1) and Kurniasih (2)
(1) Faculty of Health, Muhammadiyah University of Sidoarjo, Sidoarjo, East Java, Indonesia
(2) Department of Pathology, Faculty of Veterinary Medicine, University of Gadjah Mada, Yogyakarta, Indonesia
Correspondence should be addressed to Yos Adi Prakoso; firstname.lastname@example.org
Received 3 November 2017; Accepted 23 January 2018; Published 15 February 2018
Academic Editor: Marcel Tanner
Caption: Figure 1: Percentage of wounds area (a); macroscopical examination of wound area from each group (b).
Caption: Figure 2: The average thickness of epidermis (a); the average number of neutrophils infiltration (b) on skin wound.
Caption: Figure 3: The average score of angiogenesis (a); the average number of fibroblast (b) on skin wound.
Caption: Figure 4: The average number of [CD4.sup.+] lymphocytes (a); microscopical examination of [CD4.sup.+] lymphocytes on skin wound (IHC antibody anti [CD4.sup.+], DAB, scale bar 50 [micro]m).
Caption: Figure 5: The average number of [CD8.sup.+] lymphocytes (a); microscopical examination of [CD8.sup.+] lymphocytes on skin wound (IHC antibody anti [CD8.sup.+], DAB, scale bar 50 [micro]m).
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|Title Annotation:||Research Article|
|Author:||Prakoso, Yos Adi; Kurniasih|
|Publication:||Journal of Tropical Medicine|
|Date:||Jan 1, 2018|
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