RJX Improves Wound Healing in Diabetic Rats

Abstract

Background: We recently reported the clinical safety profile of RJX, a well-defined intravenous GMP-grade pharmaceutical formulation of anti-oxidant and anti-inflammatory vitamins as active ingredients, in a Phase 1 study in healthy volunteers (ClinicalTrials.gov Identifier: NCT03680105) (Uckun et al., Front. Pharmacol. 11, 594321. 10.3389/fphar.2020.594321). The primary objective of the present study was to examine the effects of GMP-grade RJX on wound and burn injury healing in diabetic rats.

Methods: In the present study, a rat model of T2DM was used that employs HFD in combination with a single injection of STZ intraperitoneally (i.p) at a moderate dose level (45 mg/kg). Anesthetized diabetic rats underwent full-thickness skin excision on the back or were subjected to burn injury via a heated brass probe and then started on treatments with normal saline (NS = vehicle) or RJX administered via intraperitoneal injections for three weeks.

Findings: Notably, diabetic rats treated with the 1.25 mL/kg or 2.5 mL/kg RJX (DM+RJX groups) rapidly healed their wounds as fast as non-diabetic control rats. Inflammatory cell infiltration in the dermis along with fibrin and cell debris on the epithelial layer persisted for up to 14 days in the DM+NS group but not in RJX-treated groups. The histopathological score of wound healing on days 7 and 14 was better in diabetic rats treated with RJX than diabetic rats treated with NS and comparable to the scores for non-diabetic healthy rats consistent with an accelerated healing process. The residual wound area of RJX-treated rats was significantly smaller than that of NS-treated diabetic rats at each evaluation time point (P<0.001). The accelerating effect of RJX on diabetic wound healing was dose-dependent. We obtained similar results in the burn injury model. Our results demonstrate that RJX - at a dose level >10-fold lower than its clinical maximum tolerated dose (MTD) - accelerates the healing of excision wounds as well burn injury in diabetic rats.

Keywords: COVID-19; RJX; inflammation; nutraceuticals; nutrients; pharmacodynamics; pharmacology; vitamins.

Conflict of interest statement

Author FMU is employed by Ares Pharmaceuticals, and he served as a consultant for Reven Pharmaceuticals. FMU participated in the analysis of data as well as manuscript submission decisions. MV serves as a consultant for Reven Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Uckun, Orhan, Tuzcu, Durmus, Ozercan, Volk and Sahin.

Figures

Figure 1

(A–D) RJX Accelerates Cutaneous Wound Healing in Diabetic, Obese Rats. (A) The Effects of Rejuveinix (RJX) on Wound Closure in Healing Diabetic Wounds. Wistar albino rats were treated with i.p injections of RJX (1.25 mL/kg and/or 2.5 mL/kg), or vehicle (NS). Except for untreated control rats (Control), each rat was fed a high-fat diet (HFD) for four weeks and injected a single dose of streptozotocin (STZ, 45 mg/kg i.p.) to induce diabetes (DM). At the end of 4 weeks, an experimental wound with a diameter of 5 mm (initial wound areas= 19.625 mm2) was created in all rats. The closure of the wound area was measured by digital camera until day 21. The depicted wound area data represent the median and min-max. The depicted wound area data represent the median and min-max. Statistical significance between groups is shown by *p<0.05 compared to the control group, and #p<0.05; ##p<0.01 compared to the DM+NS group. ANOVA and Tukey’s post-hoc or Welch-ANOVA and Tamhane T2’s post-hoc test were used for comparing the results among different treatment groups. (E) Residual Wound Area in Diabetic Rats Treated with RJX vs NS. A,B,C,DMeans in a row with different superscripts are statistically different (p<0.05). a,bMeans in a column with different superscripts are statistically different (p<0.05). ANOVA and Tukey’s post-hoc or Welch-ANOVA and Tamhane T2’s post-hoc test were used for comparing the results among different treatment groups or different times. ###p<0.001 compared to the DM+NS group.

Figure 2

Effects of RJX on Cutaneous Wound Healing in Diabetic, Obese Rats. Representative wound bed images from rats in Figure 1. Healing of the wounds was significantly delayed in diabetic rats treated with NS compared to untreated non-diabetic control rats. Except for untreated control rats (Control), each rat was fed a high-fat diet (HFD) for 4 weeks and injected a single dose of streptozotocin (STZ, 45 mg/kg i.p.) to induce diabetes (DM). At the end of 4 weeks, an experimental wound with a diameter of 5 mm (initial wound areas=19.625 mm2) was formed in all rats. The macroscopic changes of the wound area were documented using a digital camera.

Figure 3

The Effects of Rejuveinix (RJX) on Histological Changes in Diabetic Wound Healing. Groups of 20 Wistar albino rats were treated with i.p injections of RJX (1.25 mL/kg and/or 2.5 mL/kg), or vehicle (NS). Except for untreated control rats (Control), each rat was fed a high-fat diet (HFD) for 4 weeks and injected a single dose of streptozotocin (STZ, 45 mg/kg i.p.) to induced diabetes (DM). At the end of 4 weeks, an experimental wound with a diameter of 5 mm was formed in all rats. On days 3, 7, 14, and 21, five rats in each group were randomly selected. Photomicrographs Demonstrating Rapid Re-epithelialization of Cutaneous Wounds In RJX-Treated Diabetic Rats. Columns 1-4: Blue arrow (E): Re-epithelialization; Red arrow (I): Inflammation; Black arrow (F): Fibrin, Fibrinogen; White arrow (G): Granulation; Green arrow (C): Collagen; Yellow arrow (Fb): Fibrosis. H&E X40. Column 5: Amplified Collagen Deposition in Skin Wounds of RJX-treated Diabetic Rats on Day 21. Masson’s trichrome staining. X40.

Figure 4

Histopathological Scores of Wound Healing by Presence of Diabetes and Treatment Group. (A–D) Histopathological scores for wound healing for the healthy control vs. diabetic rats. The depicted scores represent the median and min-max. Statistical significance between groups is shown by *p<0.05 as compared to the healthy control group, and #p<0.05; ##p<0.01 as compared to the DM+NS group. Kruskal Wallis and Mann Whitney U tests were used for comparing the results among the different treatment groups. (E) Histopathological scoring system for wound healing. (E) Histopathological Scores of Wound Healing. On days 3, 7, 14, and 21, five rats in each group were randomly selected. The depicted histopathological score data represent the mean and standard deviation. A,B,C,DMeans in a row with different superscripts are statistically different (p<0.05). a,b Means in a column with different superscripts are statistically different (p<0.05). Kruskal Wallis and Mann Whitney U tests were used for comparing the results among different treatment groups or different times.

Figure 5

The Effects of Rejuveinix (RJX) on Malondialdehyde (MDA, A), Superoxide Dismutase (SOD, B), Skin Total Protein (C), and Hydroxyproline (D) in Diabetic Wound Healing. Groups of 20 Wistar albino rats were treated with i.p injections of RJX (1.25 mL/kg and/or 2.5 mL/kg), or vehicle (NS). Except for untreated control rats (Control), each rat was fed a high-fat diet (HFD) for 4 weeks and injected a single dose of streptozotocin (STZ, 45 mg/kg i.p.) to induce diabetes (DM). At the end of 4 weeks, an experimental wound with a diameter of 5 mm was formed in all rats. On day 21, five rats (n=5) in each group were selected. The depicted Whisker plots represent the median and min-max values. ANOVA and Tukey’s post-hoc test were used for comparing the results among different treatment groups. Statistical significance between groups is shown by *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 as compared to control group, and ###p<0.001; ####p<0.0001 as compared to DM+NS group, and +++p<0.001; ++++p<0.0001 pairwise comparisons between the groups.

Figure 6

The Effects of Rejuveinix (RJX) on Interleukin-6 (IL-6, A) and Tumor Necrosis Factor-α (TNF-α, B) in Diabetic Wound Healing (A, B) or Burn Wounds (C, D) Induced Diabetic Rats. Groups of 20 Wistar albino rats were treated with i.p injections of RJX (1.25 mL/kg and/or 2.5 mL/kg), or vehicle (NS). Except for untreated control rats (Control), each rat was fed a high-fat diet (HFD) for 4 weeks and injected a single dose of streptozotocin (STZ, 45 mg/kg i.p.) to induce diabetes (DM). At the end of 4 weeks, an experimental wound with a diameter of 5 mm was formed in all rats. On day 21, five rats in each group were selected and skin tissues were removed and processed for Western blot analyses. The depicted bars represent the relative levels for IL-6 and TNF-a in skin wound or burn tissues of NS or RJX-treated diabetic rats in comparison to the levels for IL-6 and TNF-a in tissue specimens from healthy control rats (control). Results are expressed as percent of control with the expression level in the skin tissue samples from untreated healthy control rats taken as 100% for comparisons. Each bar represents the mean and standard deviation. Immunoblotting with an anti-actin antibody was used to ensure equal protein loading. ANOVA and Tukey’s post-hoc test were used for comparing the results among different treatment groups. Statistical significance between groups is shown by ****p<0.0001 as compared to control group, and #p<0.05; ####p<0.0001 as compared to DM+NS group, and ++++p<0.0001 pairwise comparisons between the groups.