Effect of hydrocinnamoyl-L-valyl pyrrolidine on healing quality of deep partial-thickness scald wound in mice

Objective To observe the effect of Toll interleukin-1 recptor homology/BB-loop mimetic hydrocinnamoyl-L-valyl pyrrolidine (AS-1) on the healing quality of deep partial-thickness scald wound in mice. Methods Forty-two adult C57BL/6 mice were divided into sham injury group (SI), scald group (S), early AS-1 treatment group (EAT), early dimethyl sulfoxide (DMSO) control group (EDC), late AS-1 treatment group (LAT), late DMSO control group (LDC) according to the random number table, with 7 mice in each group. Mice in group SI were sham injured without other treatment. Deep partial-thickness scald model with 10% total body surface area was reproduced on the back of the other mice, and the wound was treated by daily wound cleaning with saline and dressing changing with vaseline gauze after injury. Mice in group EAT and those in group LAT were intraperitoneally injected with 20 mg/mL AS-1 50 mg/kg each day respectively from post scald hour (PSH) 8 and post scald day (PSD) 15 on. Mice in group EDC and those in group LDC were intraperitoneally injected with 20 mg/mL DMSO 50 mg/kg each day respectively from PSH 8 and PSD 15 on. On PSD 21, the gross condition of wound healing of mice with scald was observed, and the wound healing rate was calculated. Tissue samples of healed wound were collected and stained with HE and Masson respectively to observe the histomorphological change and fibrosis of collagen, and the percentage of fibrosis of collagen was calculated. The mRNA expressions of interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), transforming growth factor β₁ (TGF-β₁), matrix metalloproteinase-1 (MMP-1), tissue inhibitors of metalloproteinase 1 (TIMP-1), connective tissue growth factor (CTGF), type Ⅰ collagen and type Ⅲ collagen in healed wound tissue were detected by real time fluorescent quantitive reverse transcription polymerase chain reaction. The protein expressions of type Ⅰ collagen and type Ⅲ collagen in healed wound tissue were detected by Western blotting. Skin tissue of mice in group SI at the same area as that observed and collected in mice with scald was performed with the same observation and detection as mentioned above at the same time. Data were processed with one-way analysis of variance and Tukey test. Results On PSD 21, no abnormal appearance was found in skin tissue of mice in group SI. Wounds of mice in group EAT were healed completely without scar formation, while those in the other four groups were not completely healed with scars formed in different degree. The wound healing rate of mice in group EAT was (97±4)%, close to that of group SI (100%, q=1.753, P>0.05), and both of them were obviously higher than those of groups S, EDC, LAT, and LDC [respectively (83±8)%, (87±6)%, (85±9)%, and (85±7)%, with q values from 4.819 to 6.803, P<0.05 or P<0.01]. On PSD 21, no abnormal appearance was found in morphology of skin tissue of mice in group SI. The morphology of healed wound tissue of mice in group EAT was close to that in group SI, with little epidermis hyalinosis and few newly formed collagen fibers arranged orderly. Epidermis hyalinosis in band- or flake-shape and obvious proliferation of collagen fibers arranged disorderly were observed in healed wound tissue of mice in the other four groups. Much infiltration of inflammatory cells was found in group S. The percentage of fibrosis of collagen in healed wound tissue of mice in group EAT was (30±3)%, close to that of group SI [(30±4)%, q=0.159, P>0.05], and both of them were obviously lower than those of groups S, EDC, LAT, and LDC [respectively (86±9)%, (74±5)%, (82±4)%, and (82±7)%, with q values from 12.080 to 15.530, P values below 0.01]. On PSD 21, compared with those of group SI, the mRNA expressions of IL-1β, TNF-α, TGF-β₁, MMP-1, and CTGF in healed wound tissue of mice in group S, the mRNA expressions of TGF-β₁ in healed wound tissue of mice in groups EDC and LDC, and the mRNA expression of MMP-1 in healed wound tissue of mice in group LAT were significantly increased (with q values from 4.039 to 5.232, P values below 0.05), while the mRNA expression of TIMP-1 in healed wound tissue of mice in group S was significantly decreased (q=4.921, P<0.05). Compared with those of group S, the mRNA expressions of IL-1β, TNF-α, TGF-β₁, MMP-1, and CTGF in healed wound tissue of mice in group EAT and the mRNA expressions of IL-1β and CTGF in healed wound tissue of mice in group LAT were significantly decreased (with q values from 4.418 to 6.402, P<0.05 or P<0.01), while the mRNA expressions of TIMP-1 in healed wound tissue of mice in groups EAT and LAT were significantly increased (with q values respectively 3.929 and 8.299, P<0.05 or P<0.01). Compared with those of group SI, the mRNA and protein expressions of type Ⅲ collagen in healed wound tissue of mice in the other groups and the mRNA and protein expressions of type Ⅰ collagen in healed wound tissue of mice in groups S, EDC, LAT, and LDC were significantly increased (with q values from 7.054 to 11.650, P values below 0.01). Compared with those of group EAT, the mRNA and protein expressions of type Ⅰ collagen in healed wound tissue of mice in groups S, EDC, LAT, and LDC were significantly increased (with q values from 5.156 to 7.451, P<0.05 or P<0.01). Conclusions AS-1 can effectively promote wound healing and reduce fibrosis degree in the early stage of inflammation response after deep partial-thickness scald in mice, which may be related to its effect in decreasing the expression of inflammation related factors IL-1β and TNF-α and fibrosis related factors TGF-β₁, MMP-1, CTGF, and type Ⅰ collagen.