The Healing Capability of Clove Flower Extract (CFE) in Streptozotocin-Induced (STZ-Induced) Diabetic Rat Wounds Infected with Multidrug Resistant Bacteria

Abstract

Treatment of diabetic foot ulcer (DFU) is of great challenge as it is shown to be infected by multidrug resistant bacteria (MDR bacteria). Sixty four bacterial isolates were isolated from DFU cases; antibiotic susceptibility tests were carried out for all of them. One bacterial isolate (number 11) was shown to resist the action of 8 out of 12 antibiotics used and was identified by both a Vitek-2 system and 16S rRNA fingerprints as belonging to Proteus mirabilis, and was designated Proteus mirabilis LC587231 (P. mirabilis). Clove flower extract (CFE) inhibited distinctively the P. mirabilis bacterium obtained. GC-MS spectroscopy showed that this CFE contained nine bioactive compounds. The effect of CFE on wound healing of Type 1 diabetic albino rats (Rattus norvegicus) was studied. The results indicated that topical application of CFE hydrogel improved wound size, wound index, mRNA expression of the wound healing markers (Coli1, MMP9, Fibronectin, PCNA, and TGFβ), growth factor signaling pathways (PPAR-α, PGC1-α, GLP-1, GLPr-1, EGF-β, EGF-βr, VEGF-β, and FGF-β), inflammatory cytokine expression (IL8, TNFα, NFKβ, IL1β, and MCP1), as well as anti-inflammatory cytokines (IL4 & IL10), pro-apoptotic markers (FAS, FAS-L, BAX, BAX/BCL-2, Caspase-3, P53, P38), as well as an antiapoptotic one (BCL2). Furthermore, it improved the wound oxidative state and reduced the wound microbial load, as the cefepime therapy improved the wound healing parameters. Based on the previous notions, it could be concluded that CFE represents a valid antibiotics alternative for DFU therapy since it improves diabetic wound healing and exerts antibacterial activity either in vitro or in vivo.

Keywords: MDR-Proteus mirabilis; Syzygium aromaticum; diabetic foot ulcer; growth factor; inflammatory markers.

Figure 1

Chemical structure of the GC-MASS-screened Syzygium aromaticum ethanolic extract bioactive compounds (A–J).

Figure 2

Glycemic (A,B) and wound parameters of Type 1 diabetic rats throughout 21 days. Fasting blood glucose level (mg/dL) (A), serum insulin level (mIU/I) (B), wound diameter (cm) (C) and wound index (D).

Figure 3

Wound imaging follow-up on the 1st, 3rd, 6th, 9th, 12th, 15th, and 18th days. (three days post diabetic wound curation and infection with P. mirabilis).

Figure 4

Effect of the hydrogel topical application of cefepime and CFE once daily for 14 successive days on the mean fold change of the, mRNA relative expression of regenerative markers (PCNA, Fibronectin, COL1, and MMP9) to internal control gene Gapdh (A–D) and collagen deposition (E,F) of a diabetic wound infected with clinical isolates of P. Mirabilis infected with LC587231 in Type 1 diabetic rats. (A) mRNA relative expression of PCNA to internal control gene Gapdh, (B) mRNA relative expression of Fibronectin to internal control gene Gapdh, (C) mRNA relative expression of COL1 to internal control gene Gapdh, (D) mRNA relative expression of MMP9 to internal control gene Gapdh, (E) a photomicrograph of Masson blue staining of: control group (left panel), cefepime-treated group (middle panel), and CFE-treated group (right panel), and (F) for the level of the collagen deposition.

Figure 5

Effect of the hydrogel topical application of cefepime and CFE once daily for 14 successive days on the mean fold change of the mRNA relative expression of growth factors signaling pathway (PPAR-α, PGC1-α, GLP-1, GLPr-1, EGF, EGFr, VEGF, and FGF) to internal control gene Gapdh (A–D) and collagen deposition (E,F) of a diabetic wound infected with P. Mirabilis in Type 1 diabetic rats. (A) mRNA relative expression of PPAR-α to internal control gene Gapdh, (B) mRNA relative expression of PGC1-α to internal control gene Gapdh, (C) mRNA relative expression of GLP-1 to internal control gene Gapdh, (D) mRNA relative expression of GLPr-1 to internal control gene Gapdh, (E) mRNA relative expression of EGF to internal control gene Gapdh, (F) mRNA relative expression of EFGr to internal control gene Gapdh, (G) mRNA relative expression of VEFG to internal control gene Gapdh, and (H) mRNA relative expression of FGF to internal control gene Gapdh. **** very distinctive difference; *** Distinctive difference; ** Moderate difference; * Scant difference.

Figure 6

Effect of the hydrogel topical application of cefepime and CFE once daily for 14 successive days on the mean value of the oxidative status of the diabetic wound infected with P. Mirabilis in Type 1 diabetic rats (A–D). (A) lipid peroxidation marker (MDA nmol/mg), (B) GPx ng/mg, (C) CAT ng/mg, (D) SOD U/mg. *** Distinctive difference; ** Moderate difference; * Scant difference.

Figure 7

Effect of the hydrogel topical application of cefepime and CFE once daily for 14 successive days on the mean fold change of the mRNA relative expression of inflammatory and anti-inflammatory markers to internal control gene Gapdh of the diabetic wound infected with P. Mirabilis in type 1 diabetic rats (A–H). (A) mRNA relative expression of MCP1 to internal control gene Gapdh, (B) mRNA relative expression of TNF-α to internal control gene Gapdh, (C) mRNA relative expression of TGF-β to internal control gene Gapdh, (D) mRNA relative expression of NF-κβ to internal control gene Gapdh, (E) mRNA relative expression of IL-1β to internal control gene Gapdh, (F) mRNA relative expression of IL-8 to internal control gene Gapdh, (G) mRNA relative expression of IL-4 to internal control gene Gapdh, and (H) mRNA relative expression of IL-10 to internal control gene Gapdh. *** Distinctive difference; ** Moderate difference.

Figure 8

Effect of the hydrogel topical application of cefepime and CEF once daily for 14 successive days on the mean fold change of the mRNA relative expression of the apoptosis signaling pathway (FAS, FAS-L, BAX, BCL-2, BAX/BCL-2, Caspase-3, P53, and P38) to internal control gene Gapdh (A–D) and collagen deposition (E,F) of diabetic wound infected with clinical isolates of P. Mirabilis infected LC587231 in type 1 diabetic rats. (A) mRNA relative expression of FAS to internal control gene Gapdh, (B) mRNA relative expression of FAS-L to internal control gene Gapdh, (C) mRNA relative expression of BAX to internal control gene Gapdh, (D) mRNA relative expression of BCL-2 to internal control gene Gapdh, (E) mRNA relative expression of BAX/BCL-2 to internal control gene Gapdh, (F) mRNA relative expression of Caspase-3 to internal control gene Gapdh, (G) mRNA relative expression of P53 to internal control gene Gapdh, and (H) mRNA relative expression of P38 to internal control gene Gapdh. *** Distinctive difference; ** Moderate difference; * Scant difference.

Figure 9

Effect of the hydrogel topical application of cefepime and CFE for successful 14 days on the histopathological changes of the clinical isolates of P. mirabilis-infected diabetic wound in rats (A–I). (A) a photomicrograph of the diabetic infected untreated wound showing sloughed epidermal layer (black arrow), degenerated sebaceous gland (black circle), and smooth muscle (yellow arrow) H&E ×100, (B) a photomicrograph of cefepime-treated diabetic infected wound showing indefinite thin epidermal layer (black arrow), few degenerated sebaceous glands (black circle) and hair follicles (blue circle), and smooth muscle (yellow arrow) H&E, ×100, (C) a photomicrograph of clove ethanolic-extract treated diabetic infected wound showing thick well defined multicellular epidermal layer (black arrow), numerous regenerated hair follicles (blue arrow) and sebaceous glands (black circle), and well defined smooth muscle fiber (yellow arrow), H&E, ×100, (D) a photomicrograph of diabetic infected untreated wound showing narrow dermal layer with few depositions of collagen and granulation tissue (black line) and a severely degenerated muscular layer (black circle), H&E, ×100, (E) a photomicrograph of diabetic cefepime-treated diabetic infected wound showing moderate; dermal thickness (black line) and muscular degeneration (black circle), H&E, ×100, (F) a photomicrograph of clove ethanolic extract-treated diabetic wound showing heavy deposition of both collagen and granulation tissue with increasing dermal thickness (black line) and unaffected muscular tissue (black circle), H&E, ×100, (G) a photomicrograph of untreated diabetic infected wound showing severely congested blood vessels (black arrow), degenerated sebaceous glands (black circle), and abundant leukocytic infiltration with few fibroblastic ones (red circle), H&E, ×100, (H) a photomicrograph of cefepime-treated diabetic wound showing moderate blood vessel congestion (black arrow), leukocytic infiltration (red circle), and few sebaceous glands, H&E, ×100, (I) a photomicrograph of clove ethanolic extract-treated diabetic infected wound showing mild leukocytic infiltration (red circle), blood vessel congestion (black arrow), and numerous sebaceous glands with obvious filtration of fibroblast cells (yellow arrow), H&E, ×100.