Fig 1.
Photographs show the appearance of ML and rat skin over the Achilles tendon before and after surgery.
A: The rat skin over the Achilles tendon (red arrow labeled area) was prepared for surgery after removing the hair by a shaver. B: A wound (2 cm) was created in the skin over the Achilles tendon (black arrow labeled area). C: The skin wound was sutured (green arrow labeled area). D: The ML was applied on the sutured skin wound area (yellow arrow labeled material). E: The appearance of the 0% ML shows a white cream-like product. F: The appearance of the 6% ML shows a white cream-like product. G: Gross inspection shows some scar-like tissue appeared in the inside of the skin over the Achilles tendon in 0%ML. H: Gross inspection shows some scar-like tissue appeared in the inside of the skin over the Achilles tendon in 6%ML.
Fig 2.
Metformin lotion is anti-inflammatory in serum following skin wounds.
The levels of HMGB1 (A) and IL-1β (B) in the serum of the rats with skin wounds treated with 6% ML for 10 days are significantly lower compared to 0% ML treated group. ML: Metformin Lotion. *p < 0.05, n = 3–5. Analysis was conducted by ELISA.
Fig 3.
Metformin lotion inhibits the release of HMGB1 from cell nuclei to the skin tissue matrix in rats.
High levels of HMGB1 are present in the tissue matrix of rats treated with 0% ML (white arrows in D). However, in the 6% ML treated group (E-H), HMGB1 is predominantly localized within the cell nuclei (yellow arrows in H). Semi-quantification results agree with this finding (I). C: Merged images of A and B. G: Merged images of E and F. D: Enlarged yellow box area in image C. H: Enlarged yellow box area in image G. ML: Metformin Lotion. *p < 0.001, compared to 0% ML treated wounds. Scale bars: 200 μm (white), 50 μm (yellow). Analysis was conducted by immunostaining.
Fig 4.
Metformin lotion inhibits scar tissue formation in rat skin after 10 days of wound healing.
The results show a dense and thick epidermis formed at the wound area treated with 0% ML (yellow dashed line and arrows in A-C). In contrast, normal skin-like tissues formed at the wound area treated with 6% ML (green dashed line and arrows in D-F). Semi-quantification indicates that the epidermis thickness at the wound area treated with 0% ML is six times greater than at the wound area treated with 6% ML (G). There is no significant difference in epidermis thickness between normal skin and 6% ML-treated wounded skin. ML: Metformin Lotion. *p < 0.05 compared to 0% ML treated wounds. Scale bars: 1 mm (black), 500 μm (red), and 200 μm (yellow). Analysis was conducted using H&E staining.
Fig 5.
Metformin lotion inhibits scar tissue formation in rat skin after 10 days of wound healing.
The results show a large gap area (black arrows in A-C) found at the wound area treated with 0% ML (yellow dash line area in A-C). However, no gap is found at the wound area treated with 6% ML (green dash line area in D-F). Some normal skin-like tissues with hair follicles (yellow arrows in F) and blood vessels (black arrows in F) form at the wound area treated with 6% ML (D-F). Few hair follicles are found in the wound area treated with 0% ML (A-C). Many fibrosis-like tissues are found in the 0% ML treated wound area (yellow dash line area in A-C). However, there are much less fibrosis-like tissues found in the 6% ML treated wound area (green dash line area in D-F) compared to 0% ML treated wound. ML: Metformin Lotion. Scale bars: 1 mm (black), 500 μm (green), and 200 μm (red). Analysis was conducted by MT staining.
Fig 6.
Metformin lotion decreases collagen III levels in the wounded skin of rats after 10 days of wound healing.
Under a bright-field microscope, a large gap (black arrow in A) is found in the 0% ML treated wound areas (black dashed line in A), while no gap is found in the wound area treated with 6% ML (blue dashed line in C). Under polarized light microscopy, high levels of collagen III are found in the 0% ML treated wound area (green fluorescence in B), whereas the wound area treated with 6% ML is positively stained for collagen I (red fluorescence in D). Semi-quantification confirms these results (E). *p < 0.001, compared to the 0% ML treated skin wound. ML: Metformin Lotion. Scale bars: 500 μm (yellow). Analysis was conducted using Picro-Sirius Red staining.
Fig 7.
Metformin lotion decreases collagen III levels in the wounded skin of rats after 10 days of wound healing.
The results show that 6% ML decreased collagen III expression in wounded skin areas (E-H). In contrast, wounds treated with 0% ML exhibited higher levels of collagen III expression compared to the 6% ML-treated wounds (A-D). Semi-quantification results are in agreement with these findings (I). *p < 0.001 compared to the 0% ML treated wound. ML: Metformin Lotion. Scale bars: 500 μm (white), 200 μm (yellow). Analysis was conducted using immunostaining.
Fig 8.
Metformin lotion reduces α-SMA levels in the wounded skin of rats after 10 days of wound healing.
The results show that 6% ML decreases α-SMA expression in wounded skin areas (E-H). In contrast, 77% of the cells in the wounded skin areas treated with 0% ML express α-SMA (A-D, I), whereas about 16.7% of the cells in the wounded skin areas treated with 6% ML express α-SMA (E-H, I). *p < 0.05, compared to the 0% ML treated wounds. ML: Metformin Lotion. Scale bars: 500 μm (white), 200 μm (yellow). C: Merged images of A and B; G: Merged images of E and F; D: Enlarged yellow box area in C; H: Enlarged yellow box area in G. Analysis was conducted using immunostaining.
Fig 9.
Metformin lotion decreases TGF-β1 levels in the wounded skin of rats after 10 days of wound healing.
The results show that 6% ML inhibits TGF-β1 expression in wounded skin areas (E-H). In contrast, the wounds treated with 0% ML express higher levels of TGF-β1 (A-D) compared to 6% ML-treated wounds. Semi-quantification results agree with these findings (I). *p < 0.001, compared to the 0% ML treated wound. ML: Metformin Lotion. Scale bars: 500 μm (white), 200 μm (yellow). C: Merged images of A and B; G: Merged images of E and F; D: Enlarged yellow box area in C; H: Enlarged yellow box area in G. Analysis was conducted using immunostaining.
Fig 10.
Metformin lotion elevates p-AMPK in the wounded skin of rats after 10 days of wound healing.
The results show that 6% ML elevates p-AMPK expression in wounded skin areas (E-H). In contrast, the wounds treated with 0% ML express much lower levels of p-AMPK compared to the 6% ML-treated wounds (A-D). Semi-quantification results confirm these findings (I). *p < 0.001, compared to the 0% ML treated wound. ML: Metformin Lotion. Scale bars: 500 μm (white), 200 μm (yellow). C: Merged images of A and B; G: Merged images of E and F; D: Enlarged yellow box area in C; H: Enlarged yellow box area in G. Analysis was conducted using immunostaining.
Fig 11.
Metformin lotion increases collagen I expression in the wounded skin of rats after 10 days of wound healing.
The results show that 6% ML increases collagen I expression in wounded skin areas (E-H). In contrast, wounds treated with 0% ML express much lower levels of collagen I compared to 6% ML-treated wounds (A-D). Semi-quantification results confirm these findings (I). C: Merged images of A and B; G: Merged images of E and F; D: Enlarged yellow box area in C; H: Enlarged yellow box area in G. *p < 0.001, compared to the 0% ML-treated wound. ML: Metformin Lotion. Scale bars: 500 μm (white), 200 μm (yellow). Analysis was conducted using immunostaining.