Ceftriaxone and Melittin Synergistically Promote Wound Healing in Diabetic Rats

Abstract

High glucose levels in diabetic patients are implicated in delay wound healing that could lead to more serious clinical complications. The aim of the present work was to examine the formulation of ceftriaxone (CTX) and melittin (MEL) as nanoconjugate (nanocomplex)-loaded hydroxypropyl methylcellulose (HPMC) (1.5% w/v)-based hydrogel for healing of acute wounds in diabetic rats. The CTX-MEL nanoconjugate, formulated by ion-pairing at different molar ratio, was characterized for size and zeta potential and investigated by transmission electron microscopy. CTX-MEL nanoconjugate was prepared, and its preclinical efficacy evaluated in an in vivo model of acute wound. In particular, the potential ability of the innovative CTX-MEL formulation to modulate wound closure, oxidative status, inflammatory markers, and hydroxyproline was evaluated by ELISA, while the histopathological examination was obtained by using hematoxylin and eosin or Masson's trichrome staining techniques. Quantitative real-time PCR (qRT-PCR) of the excised tissue to measure collagen, type I, alpha 1 (Col1A1) expression and immunohistochemical assessment of vascular endothelial growth factor A (VEGF-A) and transforming growth factor beta 1 (TGF-β1) were also carried out to shed some light on the mechanism of wound healing. Our results show that the CTX-MEL nanocomplex has enhanced ability to regenerate epithelium, also giving better keratinization, epidermal proliferation, and granulation tissue formation, compared to MEL, CTX, or positive control. The nanocomplex also significantly ameliorated the antioxidant status by decreasing malondialdehyde (MDA) and increasing superoxide dismutase (SOD) levels. The treatment of wounded skin with the CTX-MEL nanocomplex also showed a significant reduction in interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) pro-inflammatory cytokines combined with a substantial increase in hydroxyproline, VEFG-A, and TGF-β1 protein expression compared to individual components or negative control group. Additionally, the CTX-MEL nanocomplex showed a significant increase in mRNA expression levels of Col1A1 as compared to individual compounds. In conclusion, the ion-pairing nanocomplex of CTX-MEL represents a promising carrier that can be topically applied to improve wound healing.

Keywords: ceftriaxone; inflammation; ion pairing; mellitin; nanocomplex; oxidative status; wound healing.

Figure 1

Particle size (A), zeta potential (B), and TEM image (C) of the CTX–MEL complex.

Figure 2

(A) Wound closure in diabetic rats belonging to the 5 experimental groups at day 0, 3, 7, 10, and 14. (B) Wound closure % at day 14. Negative control refers to rats that received topical plain vehicle consisting of HPMC-based hydrogel (1.5% w/v) on the wound area, while positive control refers to animals that received 0.5 g of Mebo^TM ointment on the wound area. Data are expressed as mean (n = 6) ± SD. *** Significantly different vs. negative control, p < 0.001; ^### significantly different vs. positive control, p < 0.001; ^ϕϕϕ significantly different vs. CTX, p < 0.001; ^θθθ significantly different vs. CTX, p < 0.001.

Figure 3

Histopathological effects of CTX, MEL, or CTX–MEL nanocomplex on wound healing on day 14. Negative control refers to rats that received topical plain vehicle consisting of HPMC-based hydrogel (1.5% w/v) on the wound area, while positive control refers to animals that received 0.5 g of Mebo^TM ointment on the wound area. H&E = hematoxylin and eosin (scale bar = 50 µm); MT = Masson’s trichrome (scale bar = 50 µm).

Figure 4

Effect of CTX, MEL, or CTX–MEL nanocomplex on (A) MDA and (B) SOD levels in wounded skin of diabetic rats. Negative control refers to rats that received topical plain vehicle consisting of HPMC-based hydrogel (1.5% w/v) on the wound area, while positive control refers to animals that received 0.5 g of Mebo^TM ointment on the wound area. Data are presented as mean (n = 6) ± SD. ** Significantly different vs. negative control, p < 0.01; *** significantly different vs. negative control, p < 0.001; ^ϕ significantly different vs. CTX, p < 0.05; ^θ significantly different vs. CTX, p < 0.05.

Figure 5

Effect of CTX, MEL, or CTX–MEL nanocomplex on (A) IL-6 and (B) TNF-α content in wounded skin of diabetic rats. Negative control refers to rats that received topical plain vehicle consisting of HPMC-based hydrogel (1.5% w/v) on the wound area, while positive control refers to animals that received 0.5 g of Mebo^TM ointment on the wound area. Data are expressed as mean ± SD, (n = 6). ** Significantly different vs. negative control, p < 0.01; *** significantly different vs. negative control, p < 0.001; ^# significantly different vs. positive control, p < 0.05; ^## significantly different vs. positive control, p < 0.01; ^### significantly different vs. positive control, p < 0.001; ^ϕϕ significantly different vs. CTX, p < 0.01; ^ϕϕϕ significantly different vs. CTX, p < 0.001; ^θθ significantly different vs. CTX, p < 0.01; ^θθθ significantly different vs. CTX, p < 0.001.

Figure 6

Effect of CTX, MEL, or CTX–MEL nanocomplex on mRNA expression of (A) Col1A1 and (B) hydroxyproline content at day 14. The abundance of Col1A1 mRNA is expressed relative to the abundance of GAPDH mRNA. Negative control refers to rats that received topical plain vehicle consisting of HPMC-based hydrogel (1.5% w/v) on the wound area, while positive control refers to animals that received 0.5 g of Mebo^TM ointment on the wound area. Data are expressed as mean (n = 6) ± SD. *** Significantly different vs. negative control, p < 0.001; ^### significantly different vs. positive control, p < 0.001; ^ϕϕ significantly different vs. CTX, p < 0.01; ^ϕϕϕ significantly different vs. CTX, p < 0.001; ^θθθ significantly different vs. CTX, p < 0.001.

Figure 7

Immunohistochemical assessment of VEGF-A expression in skin tissues obtained from diabetic rats belonging to the different experimental groups. (A) Negative control, (B) positive control, (C) CTX, (D) melittin, (E) CTX–MEL, (F) quantitative representation of VEGF-A expression. Negative control refers to rats that received topical plain vehicle consisting of HPMC-based hydrogel (1.5% w/v) on the wound area, while positive control refers to animals that received 0.5 g of Mebo^TM ointment on the wound area. Data are expressed as mean ± SD, (n = 6). ** Significantly different vs. negative control, p < 0.01; *** significantly different vs. negative control, p < 0.001; ^## significantly different vs. positive control, p < 0.01; ^### significantly different vs. positive control, p < 0.001; ^ϕ significantly different vs. CTX, p < 0.05; ^ϕϕϕ significantly different vs. CTX, p < 0.001; ^θθθ significantly different vs. CTX, p < 0.001.

Figure 8

Immunohistochemical assessment of TGF-β1 expression in skin tissues obtained from diabetic rats belonging to the different experimental groups. (A) Negative control, (B) positive control, (C) CTX, (D) melittin, (E) CTX–MEL, (F) quantitative representation of TGF-β1 expression. Negative control refers to rats that received topical plain vehicle consisting of HPMC-based hydrogel (1.5% w/v) on the wound area, while positive control refers to animals that received 0.5 g of Mebo^TM ointment on the wound area. Data are expressed as mean ± SD, (n = 6). *** Significantly different vs. negative control, p < 0.001; ^### significantly different vs. positive control, p < 0.001; ^ϕϕϕ significantly different vs. CTX, p < 0.001; ^θθθ significantly different vs. CTX, p < 0.001.