Diabetic ulcers (DUs) are a major global health concern, significantly impacting patient quality of life. Conventional treatments, focusing on debridement, offloading, and antibiotics, often prove insufficient, leading to complications like amputations. The rise of antibiotic resistance further exacerbates this problem. The wound-healing process is complex, involving hemostasis, inflammation, proliferation, and re-epithelialization, with fibroblasts playing a crucial role in collagen synthesis and tissue repair. Vascular endothelial growth factor (VEGF) also significantly contributes to angiogenesis and epithelialization. Given the limitations of current DU treatments, alternative therapies are urgently needed. Lactic acid bacteria (LAB), particularly lactobacilli, have shown promise as probiotics due to their immunomodulatory, gut microbiota-modulating, and antipathogenic properties. Lactobacilli can inhibit pathogens like *S. aureus* and *P. aeruginosa*, potentially through the production of organic acids like lactic acid. Additionally, they can stimulate VEGF production and fibroblast migration, influencing collagen synthesis and wound healing. This study aimed to evaluate the therapeutic potential of an oleogel-based formulation containing four Lactobacillus species (*L. rhamnosus*, *L. casei*, *L. fermentum*, and *L. acidophilus*) in a diabetic rat model.

Numerous studies have explored the beneficial effects of probiotics in various medical applications, including wound healing. The mechanisms of action are multifaceted, often involving modulation of the immune system and intestinal barrier function, along with antipathogenic effects. Specific lactobacilli strains have demonstrated the ability to inhibit infections caused by Gram-positive and Gram-negative bacteria. This activity is often linked to the production of organic acids such as lactic acid, which can alter bacterial membrane structures. Furthermore, probiotics can stimulate VEGF expression and fibroblast migration, key processes in wound healing. Studies have reported that probiotics can enhance re-epithelialization, collagen synthesis, and overall wound healing rates in animal models and even in human clinical studies focused on the treatment of ulcers. However, the specific mechanisms underlying these beneficial effects often require further investigation. The application of probiotics in wound care offers a potential alternative to traditional treatments, particularly in the context of increasing antibiotic resistance.

An oleogel-based formulation was developed, incorporating each of four Lactobacillus strains (*L. rhamnosus*, *L. casei*, *L. fermentum*, and *L. acidophilus*) individually at 1 × 10⁸ CFU/ml. The formulation's components included glycerol, polyethylene glycol (PEG) 400 and 4000, and paraffin. Diabetes was induced in male Sprague-Dawley rats using streptozotocin (STZ). Forty-eight rats were randomly divided into eight groups (n=6 per group): a control group (non-diabetic, untreated); a diabetic control group (DM, untreated); a diabetic group treated with the base gel (DM + B.g); a diabetic group treated with 3% tetracycline ointment (DM + TC); and four groups treated with the oleogel formulation containing each of the four Lactobacillus strains (DM + LBF, DM + LBR, DM + LBC, DM + LBA). A 2 cm diameter wound was created on the dorsal skin of each rat. The antibiofilm activity of *L. rhamnosus* against *E. coli*, *L. monocytogenes*, and *S. typhimurium* was assessed using a 12-well microtiter plate assay. Wound closure was morphologically assessed by photographing wound sites at days 0, 3, 7, and 14. Tissue samples were collected at days 7 and 14 for biochemical (hydroxyproline content) and histopathological analyses. Histopathological analyses included assessment of re-epithelialization, hair follicle formation, fibroblast population, collagen deposition, and neovascularization using stereological methods (Delesse and optical dissector). Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test.

*L. rhamnosus* exhibited strong antibiofilm activity against all three tested pathogens (*E. coli*, *L. monocytogenes*, and *S. typhimurium*), with the highest inhibition observed at 24 hours. All Lactobacillus groups (except *L. casei*) showed increased wound closure compared to the diabetic control group. *L. acidophilus* and *L. rhamnosus* demonstrated the highest wound closure rates, exceeding that of the tetracycline-treated group at day 14. Biochemical analysis revealed significantly higher hydroxyproline content in the *L. acidophilus* and *L. rhamnosus* groups compared to the diabetic control and tetracycline groups at both day 7 and day 14. Histopathological analysis showed significantly higher re-epithelialization, hair follicle formation, fibroblast population, collagen deposition, and neovascularization in the *L. acidophilus* and *L. rhamnosus* groups compared to the other groups. Microscopic images supported these findings, showing improved epidermis thickness, collagen fiber organization, and overall tissue regeneration in the *L. acidophilus* and *L. rhamnosus* treated groups. *L. casei* showed the least effect on wound healing parameters compared to other lactobacillus groups.

The results demonstrate the significant potential of the developed probiotic formulation, particularly those containing *L. acidophilus* and *L. rhamnosus*, for treating DUs. The improved wound healing observed is likely multifactorial, involving antibiofilm activity, stimulation of VEGF production and fibroblast migration, increased collagen synthesis, and enhanced angiogenesis. The significant improvement observed with *L. acidophilus* and *L. rhamnosus* compared to the tetracycline-treated group suggests the probiotic approach may offer advantages over conventional antibiotic treatment. The lower efficacy of *L. casei* might be due to the release of certain bioactive compounds that could interfere with the coagulation cascade. Further research is needed to clarify the specific mechanisms of action of each probiotic strain and to investigate the optimal formulation for clinical application.

This study provides strong evidence for the potential of a novel oleogel-based probiotic formulation, particularly those containing *L. acidophilus* and *L. rhamnosus*, for treating diabetic ulcers. The combination of antibiofilm activity and stimulation of key wound-healing processes makes this a promising alternative therapy. Future studies should focus on large-scale clinical trials to confirm these findings and to optimize the formulation for clinical use. Further investigation into the mechanisms of action of different probiotic strains and the identification of biomarkers for treatment response would also be valuable.

This study used a rat model, which may not fully reflect the complexities of human DU healing. The study focused on a limited number of Lactobacillus strains and did not explore the potential synergistic effects of combining different strains. Furthermore, the long-term effects of the probiotic formulation on wound healing were not investigated. While the study included the assessment of microbiological load, a more detailed analysis of the wound microbiome during treatment would provide further insights.