The Effect of Topical Administration of an Ointment Prepared From Trifolium Repens Hydroethanolic Extract on the Acceleration of Excisional Cutaneous Wound Healing

Seied Kiavash Habibi Zadeh, DVM; Mohammad-Reza Farahpour, DVM, DVSc; Hamed Hamishe Kar, PhD


Wounds. 2020;32(9):253-261. 

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Phytochemical chemicals, such as flavonoids and triterpenoids, have been reported to promote the wound healing process.[25,26] The present findings showed that rutin and quercetin were major compounds in TRHE, which have been reported as major compounds in T repens extract by Xiong et al.[27] Tundis et al[19] showed TRHE had a DPPH radical scavenging ability with an IC50 value of 10.30 μg/mL. It can be stated that TRHE has a stronger antioxidant activity compared with ascorbic acid. Inflammation-exerted oxidative stress changes the physiologic activities and metabolism during the healing process.[28] In this regard, neovascularization is a defensive and physiologic structural procedure that reduces the free radicals and metabolite delivery of cells involved in the healing process.[29] The present biochemical analyses on the herbal composition of TRHE revealed that this plant contained high contents of phenols and flavonoids. The shortened wound healing process may be attributed to the phenol- and flavonoid-induced antioxidant properties.

Based on the present results, administration of T repens upregulated the expression of Bcl-2 and downregulated the expression of p53 and BAX; nevertheless, the animals in the control group showed a lower Bcl-2 expression and higher p53 and BAX expressions on day 7. The administration of T repens initiated cellular proliferation. BAX and p53 support the genome and prevent damages in cells through distinguishing and stopping the cell cycle.[24] After wounding, p53 and BAX expressions increased and promoted the immune cells apoptosis, thereby initiating the elimination of the immune cells. Following this phase, Bcl-2 prevented apoptosis and triggered cellular proliferation.[24,30] As previously mentioned, the Bcl-2 family inhibits apoptosis.[31,32] The results of reverse transcription PCR showed the administration of T repens increased Bcl-2 expression and reduced the expressions of p53 and BAX. It can be concluded that T repens increased the cellular proliferation via upregulating the Bcl-2 expression and reducing p53 and BAX mRNAs. Another important factor for cellular proliferation is the elimination of the immune cells from granulation tissues. More than 70 years ago, Mancini[33] reported that faulted fibroblast numbers increased pathological injuries for granulation tissue, formation of hypertrophied scar tissues, and keloids.

Mast cells play major roles in secreting fibroblast growth factor-2 and participating in collagen synthesis through increasing the physiologic interaction of fibroblasts in the wound healing process.[34,35] Mast cells secrete vascular growth factor, which stimulates the proliferation of endothelial cells and completes the upregulation of neovascularization in wound healing.[35,36] Even in the preliminary stages, downregulated inflammation associated with provoked angiogenesis promotes proliferative machinery, such as fibroblasts, fibrocytes physiologic, and/or compensatory functions.[37] The present findings showed the distribution of mast cells increased in T repens-treated animals. It can be stated that the topical administration of T repens promoted the wound healing process and the proliferation and maturation stages through provoking mast-cell proliferation and distribution and upregulating the neovascularization (Figure 7).

Figure 7.

Accelerative effect associated with the topical administration of Trifolium repens hydroethanolic extract on excisional wound healing. (A) T repens enhanced the angiogenesis and fibroblasts distribution (B) by upregulating the B-cell lymphoma 2 (Bcl-2) expression and reducing the p53 and B-cell lymphoma 2-like protein 4 (BAX) expression. T repens accelerated fibroblast proliferation, ultimately increasing the collagen biosynthesis as well as dermal maturation. The elevated Bcl-2 expression enhanced the epithelial cell proliferation and finally shortened the reepithelialization.

In the animals treated with 6% T repens, angiogenesis was upregulated and vascularization increased on day 3 following wound induction. Vascular distribution (per 1 mm2 of the tissue) in 6% T repens-treated groups was 3 to 4 times higher than those of the control group on day 7. Flavonoid contents of T repens upregulated the cellular profiles. Higher intracytoplasmic carbohydrate ratio increased the fibroblasts and fibrocytes; mast-cell distribution per 1 mm2 of the tissue increased in the animals treated with high doses (ie, 6%) of T repens.

The intracytoplasmic carbohydrate ratio differs among various cell types under different injury conditions.[2,33,38] Carbohydrates are used for producing hyaluronic acid, proteoglycan, and epithelium of glycosaminoglycan and injured skin (particularly in stratum spinosum).[30,32,38] The results of staining carbohydrates (PAS staining) showed that the administration of 3% and 6% T repens increased the expression of PAS-positive compared with the control animals. Furthermore, the stratum spinosum of the epidemis increased the intracytoplasmic carbohydrate storage in the animals treated with 6% T repens. From the results of this study, it is plausible that treatment with T repens increased carbohydrate ratio in dermal and epidermal cells, supplied the energy required for cells, and acted as a substrate necessary for the synthesis of structural materials.