Epidermal Growth Factor and Tocotrienol-Rich Fraction Cream Formulation Accelerates Burn Healing Process Based on Its Gene Expression Pattern in Deep Partial-Thickness Burn Wound Model.

Our previous study has demonstrated that epidermal growth factor (EGF) with tocotrienol-rich fraction (TRF) cream formulation accelerating postburn wound healing with deep partial-thickness burn in rats. Current study was conducted to determine the gene expression levels related to burn wound healing process. A total of 180 Sprague-Dawley rats were randomly divided into 6 groups: untreated control, treated with Silverdin cream, base cream, base cream with 0.00075% EGF, base cream with 3% TRF or base cream with 0.00075% EGF, and 3% TRF, respectively. Burn wounds were created and the above-mentioned creams were applied once daily. Six animals from each group were sacrificed on days 3, 7, 11, 14, and 21 postburn. RNA was extracted from wound tissues and quantitative real-time polymerase chain reaction was performed to analyze the 9 wound healing-related genes against time postburn. Results demonstrated that topically applied EGF + TRF formulation downregulated the expression levels of IL-6 (interluekin-6), TNF-α (tumor necrosis factor-α) and iNOS (inducible nitric oxide synthase) throughout the whole healing process. TGF-ß1 (transforming growth factor-ß) and VEGF-A (vascular endothelial growth factor-A) were reduced on day 14 postburn. On the contrary, increased expression of Collagen-1 in the early stage of wound healing was observed with no effects on epidemal growth factor receptor (EGFR). The results showed beneficial application of EGF + TRF cream in the treatment of burn wound since it accelerated wound healing by relieving oxidative stress, decreasing inflammation, and promoting proper tissue modelling in the burn wound.

A new histological score grade for deep partial-thickness burn wound healing process.

Burns are injuries on the skin or other tissues. Burns are divided into superficial, partial, and full-thickness, characterized by the depth of the affected tissues. Histological analysis is critical to assess the burn wound healing process. Thus, a systematic evaluation system is imperative for burn research. In the present study, a total of thirty Sprague-Dawley rats were randomly divided into five groups. Deep partial-thickness burn wound was induced on the dorsal part of the rats. Six animals from each group were sacrificed on the 3rd, 7th, 11th, 14th and 21st day post-burn, respectively. Half of the wound tissue was immediately fixed in buffered neutral formalin for hematoxylin & eosin staining. The healing of the epidermis was evaluated with scores ranging from 0 to 7 based on the state of crust on wound surface, the degree of epithelialization as well as the formation of rete ridges. Meanwhile, healing of the dermis was also evaluated with scores ranging from 0 to 7 according to the proportion of adipose cells, inflammatory cells and fibroblasts, the state of collagen deposition as well as the formation of hair follicles. Furthermore, temporal changes of histological score of epidermis and dermis in the skin tissue with deep partial-thickness burn was evaluated. In conclusion, a new comprehensive system for assessing microscopic changes in the healing process of deep partial-thickness burn wound in hematoxylin & eosin staining slides was established, which simplified the scoring process and helped to obtain reproducible and accurate results in the burn study.

Analysis of expression of vitamin E-binding proteins in H2O2 induced SK-N-SH neuronal cells supplemented with α-tocopherol and tocotrienol-rich fraction.

Natural α-tocopherol (α-TCP), but not tocotrienol, is preferentially retained in the human body. α-Tocopherol transfer protein (α-TTP) is responsible for binding α-TCP for cellular uptake and has high affinity and specificity for α-TCP but not α-tocotrienol. The purpose of this study was to examine the modification of α-TTP together with other related vitamin E-binding genes (i.e., TTPA, SEC14L2, and PI-TPNA) in regulating vitamin E uptake in neuronal cells at rest and under oxidative stress. Oxidative stress was induced with H2O2 for an hour which was followed by supplementation with different ratios of α-TCP and tocotrienol-rich fraction (TRF) for four hours. The cellular levels of vitamin E were quantified to determine bioavailability at cellular levels. The expression levels of TTPA, SEC14L2, and PI-TPNA genes in 0% α-TCP were found to be positively correlated with the levels of vitamin E in resting neuronal cells. In addition, the regulation of all the above-mentioned genes affect the distribution of vitamin E in the neuronal cells. It was observed that, increased levels of α-TCP secretion occur under oxidative stress. Thus, our results showed that in conclusion vitamin E-binding proteins may be modified in the absence of α-TCP to produce tocotrienols (TCT), as a source of vitamin E. The current study suggests that the expression levels of vitamin E transport proteins may influence the cellular concentrations of vitamin E levels in the neuronal cells.

Healing Properties of Epidermal Growth Factor and Tocotrienol-Rich Fraction in Deep Partial-Thickness Experimental Burn Wounds.

Background: An experimental study was undertaken to determine the efficacy of the epidermal growth factor (EGF) with tocotrienol-rich fraction (TRF) cream in the wound-healing process on skin with deep partial-thickness burn in rats. Methods: A total of 180 Sprague-Dawley rats were randomly divided into six groups of six each and were: untreated control, treated with Silverdin® cream, base cream, base cream with c% EGF, base cream with 3% TRF or base cream with c% EGF and 3% TRF, respectively. Creams were applied once daily for 21 consecutive days. Six animals from each group were sacrificed using anaesthetic overdose on the third, seventh, 11th, 14th and 21st day post-burn. Skin tissues with the wound to be examined were excised for macroscopic and microscopic evaluation and biochemical analyses. Results: EGF + TRF formulation decreased the number of neutrophils, lymphocytes and myofibroblasts post-burn. However, no effects on the number of adipose cells in the healing process were recorded. In addition, lipid peroxidation and nitrite production were found to be reduced post-burn, reducing oxidative stress. Conclusions: Results of the present study indicate that the addition of EGF with TRF have ameliorating effects on deep-partial thickness burn healing parameters.

Ardisia crispa root hexane fraction suppressed angiogenesis in human umbilical vein endothelial cells (HUVECs) and in vivo zebrafish embryo model.

Ardisia crispa Thunb. A. DC. (Primulaceae) has been used extensively as folk-lore medicine in South East Asia including China and Japan to treat various inflammatory related diseases. Ardisia crispa root hexane fraction (ACRH) has been thoroughly studied by our group and it has been shown to exhibit anti-inflammatory, anti-hyperalgesic, anti-arthritic, anti-ulcer, chemoprevention and suppression against inflammation-induced angiogenesis in various animal model. Nevertheless, its effect against human endothelial cells in vitro has not been reported yet. Hence, the aim of the study is to investigate the potential antiangiogenic property of ACRH in human umbilical vein endothelial cells (HUVECs) and zebrafish embryo model. ACRH was separated from the crude ethanolic extract of the plant's root in prior to experimental studies. MTT assay revealed that ACRH exerted a concentration-dependent antiproliferative effect on HUVEC with the IC50 of 2.49 ±â€¯0.04 µg/mL. At higher concentration (10 µg/mL), apoptosis was induced without affecting the cell cycle distribution. Angiogenic properties including migration, invasion and differentiation of HUVECs, evaluated via wound healing, trans-well invasion and tube formation assay respectively, were significantly suppressed by ACRH in a concentration-dependent manner. Noteworthily, significant antiangiogenic effects were observed even at the lowest concentration used (0.1 µg/mL). Expression of proMMP-2, vascular endothelial growth factor (VEGF)-C, VEGF-D, Angiopoietin-2, fibroblast growth factor (FGF)-1, FGF-2, Follistatin, and hepatocyte growth factor (HGF) were significantly reduced in various degrees by ACRH. The ISV formation in zebrafish embryo was significantly suppressed by ACRH at the concentration of 5 µg/mL. These findings revealed the potential of ACRH as antiangiogenic agent by suppressing multiple proangiogenic proteins. Thus, it can be further verified via the transcription of these proteins from their respective DNA, in elucidating their exact pathways.

Modulation of cancer signalling pathway(s) in two -stage mouse skin tumorigenesis by annonacin.

BACKGROUND: Annonacin, an annonaceous acetogenin isolated from Annona muricata has been reported to be strongly cytotoxic against various cell lines, in vitro. Nevertheless, its effect against in vivo tumor promoting activity has not been reported yet. Therefore, this study was aimed to investigate antitumor-promoting activity of annonacin via in vivo two-stage mouse skin tumorigenesis model and its molecular pathways involved. METHODS: Mice were initiated with single dose of 7,12-dimethylbenz[α]anthracene (DMBA) (390 nmol/100 µL) followed by, in subsequent week, repeated promotion (twice weekly; 22 weeks) with 12-O-tetradecanoylphorbol-13-acetate (TPA) (1.7 nmol/100 µL). Annonacin (85 nM) and curcumin (10 mg/kg; reference) were, respectively, applied topically to DMBA/TPA-induced mice 30 min before each TPA application for 22 weeks. Upon termination, histopathological examination of skin, liver and kidney as well as genes and proteins expression analysis were conducted to elucidate the potential mechanism of annonacin. RESULTS: With comparison to the carcinogen control, Annonacin significantly increased the tumor latency period and reduced the tumor incidence, tumor burden and tumor volume, respectively. In addition, it also suppressed tumorigenesis manifested by significant reduction of hyperkeratosis, dermal papillae and number of keratin pearls on skin tissues. Annonacin also appeared to be non-toxic to liver and kidney. Significant modulation of both AKT, ERK, mTOR, p38, PTEN and Src genes and proteins were also observed in annonacin-targeted signaling pathway(s) against tumorigenesis. CONCLUSIONS: Collectively, results of this study indicate that annonacin is a potential therapeutic compound targeting tumor promoting stage in skin tumorigenesis by modulating multiple gene and protein in cancer signaling pathways without apparent toxicity.

Evaluation of Chondroprotective Activity of Channa striatus in Rabbit Osteoarthritis Model.

OBJECTIVES: The objective of the study is to evaluate the chondroprotective activity of Channa striatus (Channa) and glucosamine sulphate (glucosamine) on histomorphometric examinations, serum biomarker, and inflammatory mediators in experimental osteoarthritis (OA) rabbit model. DESIGN: Anterior cruciate ligament transection (ACLT) was performed to induce OA in thirty-three male New Zealand white rabbits and were randomly divided into three groups: Channa, glucosamine, and control group. The control group received drinking water and the Channa and glucosamine groups were orally administered with 51.4 mg/kg of Channa extract and 77.5 mg/kg of glucosamine sulphate in drinking water, respectively, for eight weeks and then sacrificed. The articular cartilage was evaluated macroscopically and histologically using semiquantitative and quantitative methods. Serum cartilage oligomeric matric protein (COMP), cyclooxygenase 2 (COX-2) enzyme, and prostaglandin E2 (PGE2) were also determined. RESULTS: Macroscopic analysis revealed that Channa group have a significantly lower severity grade of total macroscopic score compared to the control (p < 0.001) and glucosamine (p < 0.05) groups. Semiquantitative histology scoring showed that both Channa and glucosamine groups had lower severity grading of total histology score compared to the control group (p < 0.001). In comparison with the control, Channa group had lower histopathological changes in three compartments of the joint compared to glucosamine group which had lower histological scoring in two compartments only. The cartilage thickness, area, and roughness of both Channa (p < 0.05) and glucosamine (p < 0.05) groups were superior compared to the control group. However, the Channa group demonstrated significantly less cartilage roughness compared to the glucosamine group (p < 0.05). Serum COMP levels were lower in both Channa (p < 0.05) and glucosamine (p < 0.05) groups compared to the control group. CONCLUSION: Both oral administration of Channa extract and glucosamine exhibited chondroprotective action on an ACLT OA-induced rabbit model. However, Channa was superior to glucosamine in maintaining the structure of the cartilage.