Objective To observe the effects of Huanglian ointment on wound healing of mice with full-thickness skin defect, and to explore the related mechanism.
Methods Thirty male C57BL/6J mice were divided into Huanglian ointment group and vehicle group according to the random number table after round wounds of full-thickness skin defect with diameter of 7.5 mm were inflicted on the back of each mouse, with 15 mice in each group. Wounds of mice in Huanglian ointment group and vehicle group were treated with Huanglian ointment and vehicle respectively from post injury day (PID) 1 on, 2 times each day. Five mice from each group were selected to observe wound changes on PID 0, 3, 7, 10, and 14, and wound healing rates were calculated. Five mice out of the 10 mice that hadn′t been used for general observation in each group were sacrificed on PID 3 and 7 respectively, and 5 mice after being used for general observation in each group were sacrificed on PID 14. Wound and skin tissue within 2 mm from the edge of wound was collected. Histologic scoring was conducted based on the histomorphological observation with HE staining. The expression of double positive cells of alpha smooth muscle actin (α-SMA) and Ki-67 (myofibroblast) in tissue of wounds of mice was observed by immunofluorescence staining. Protein expressions of transforming growth factor beta (TGF-β) and collagen in tissue of wounds of mice were determined by enzyme-linked immunosorbent assay. Data were processed with analysis of variance for repeated measurement, analysis of variance of factorial design,
t test of two independent samples, one-way analysis of variance, and Bonferronni test or correction.
Results (1) Wounds of mice in two groups were red and swollen on PID 0, while they were neither red nor swollen with scabs on PID 3 and 7. On PID 10, woundsof mice in Huanglian ointment group contracted obviously, while the contracted wounds of mice in vehicle group were smaller than those in Huanglian ointment group. On PID 14, wounds of most mice in Huanglian ointment group were healed, while wounds of some mice in vehicle group failed to heal. Wound healing rates of mice in two groups were close on PID 3 and 7 (with
t values respectively 0.64 and 1.90,
P values above 0.05). Wound healing rates of mice in Huanglian ointment group on PID 10 and 14 were (76±7)% and (93±5)% respectively, significantly higher than those of vehicle group [(48±9)% and (68±11)%, with
t values respectively 7.44 and 3.89,
P values below 0.01]. Wound healing rates of mice in two groups on PID 7, 10, and 14 were significantly higher than those on the previous time points of the same group (with
P values below 0.01). (2) Histologic scores of wounds of mice in two groups were close on PID 3 (
t=-0.76,
P>0.05). Histologic scores of wounds of mice in Huanglian ointment group on PID 7 and 14 were (7.0±1.6) and (11.6±2.1) points respectively, significantly higher than those of vehicle group [(4.2±1.3) and (7.2±1.3) points, with
t values respectively 1.96 and 2.50,
P<0.05 or
P<0.01]. Histologic scores of wounds of mice in two groups on PID 7 and 14 were significantly higher than those on the previous time points of the same group (with
P values below 0.01). (3) Percentages of double positive cells of α-SMA and Ki-67 in tissue of wounds of mice in Huanglian ointment group on PID 3 and 7 were (35±12)% and (62±10)% respectively, significantly higher than those of vehicle group [(17±12)% and (34±6)%, with
t values respectively -2.48 and -5.25,
P<0.05 or
P<0.01]. The percentage of double positive cells of α-SMA and Ki-67 in tissue of wounds of mice in Huanglian ointment group on PID 14 was (25±5)%, significantly lower than that of vehicle group [(44±17)%,
t=2.50,
P<0.05]. The percentage of double positive cells of α-SMA and Ki-67 in tissue of wounds of mice on PID 7 was significantly higher than that on PID 3 or 14 in Huanglian ointment group (with
P values below 0.01). Percentages of double positive cells of α-SMA and Ki-67 in tissue of wounds of mice on PID 7 and 14 were significantly higher than those on the previous time points in vehicle group (with
P values below 0.05). (4) Protein expressions of TGF-β in tissue of wounds of mice in Huanglian ointment group on PID 3 and 7 were (396±45) and (722±96) pg/mL respectively, significantly higher than those of vehicle group [(290±42) and (382±62) pg/mL, with
t values respectively -8.17 and -6.65,
P values below 0.01]. Protein expressions of TGF-β in tissue of wounds of mice in two groups were close on PID 14 (
t=1.60,
P>0.05). The protein expression of TGF-β in tissue of wounds of mice in Huanglian ointment group on PID 7 was significantly higher than that on PID 3 or 14 (with
P values below 0.01). Protein expressions of TGF-β in tissue of wounds of mice in vehicle group on PID 7 and 14 were significantly higher than those on the previous time points (with
P values below 0.05). Protein expressions of collagen in tissue of wounds of mice in two groups were close on PID 3 (
t=1.99,
P>0.05). Protein expressions of collagen in tissue of wounds of mice in Huanglian ointment on PID 7 and 14 were (47±10) and (70±14) ng/mL respectively, significantly higher than those of vehicle group [(34±10) and (42±12) ng/mL, with
t values respectively 3.15 and 3.52,
P<0.05 or
P<0.01]. Protein expressions of collagen in tissue of wounds of mice in two groups on PID 7 and 14 were significantly higher than those on the previous time points of the same group (
P<0.05 or
P<0.01).
Conclusions Huanglian ointment can promote wound healing of full-thickness skin defect of mice through increasing production of myofibroblasts and protein expressions of TGF-β and collagen.
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