Effect of hydrocinnamoyl-L-valyl pyrrolidine on healing quality of deep partial-thickness scald wound in mice
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摘要: 目的 观察Toll白细胞介素1受体同源区/BB环状拟似物——氢化肉桂基-L-缬氨酰吡咯烷(AS-1)对小鼠深Ⅱ度烫伤创面愈合质量的影响。 方法 将42只成年C57BL/6小鼠按随机数字表法分为假伤组、烫伤组、AS-1早期治疗组、二甲基亚砜(DMSO)早期对照组、AS-1晚期治疗组、DMSO晚期对照组,每组7只。假伤组小鼠模拟致假伤后不行其他处理;其余小鼠在背部造成约10%TBSA深Ⅱ度烫伤,伤后每日以生理盐水清洁创面并更换凡士林纱布。AS-1早期治疗组、AS-1晚期治疗组小鼠分别自伤后8 h、15 d起,每日腹腔注射20 mg/mL AS-1 50 mg/kg;DMSO早期对照组、DMSO晚期对照组小鼠分别自伤后8 h、15 d起,每日腹腔注射20 mg/mL DMSO 50 mg/kg。伤后21 d,观察烫伤小鼠创面愈合大体情况,计算创面愈合率;取烫伤小鼠创面愈合组织,分别行HE、Masson染色,观察组织形态学变化、胶原纤维化情况,并计算胶原纤维化百分比;取烫伤小鼠创面愈合组织,采用实时荧光定量RT-PCR法检测IL-1β、TNF-α、TGF-β1、基质金属蛋白酶1(MMP-1)、基质金属蛋白酶组织抑制剂1(TIMP-1)、结缔组织生长因子(CTGF)、Ⅰ型胶原、Ⅲ型胶原的mRNA表达,采用蛋白质印迹法检测Ⅰ、Ⅲ型胶原的蛋白表达。假伤组以与烫伤小鼠观察和取材相同区域的皮肤组织为对象,同期同前进行相关观测。对数据行单因素方差分析、Tukey检验。 结果 伤后21 d,假伤组小鼠皮肤组织未见异常;AS-1早期治疗组小鼠创面完全愈合,无瘢痕形成;其余4组小鼠创面均未完全愈合,均伴有不同程度的瘢痕形成。伤后21 d,AS-1早期治疗组小鼠创面愈合率为(97±4)%,与假伤组小鼠的100%相近(
q =1.753,P >0.05),均明显高于烫伤组、DMSO早期对照组、AS-1晚期治疗组、DMSO晚期对照组的(83±8)%、(87±6)%、(85±9)%、(85±7)%(q 值为4.819~6.803,P <0.05或P <0.01)。伤后21 d,假伤组小鼠皮肤组织形态未见明显异常;AS-1早期治疗组小鼠创面愈合组织形态与假伤组接近,表皮玻璃样变性少,新生胶原纤维较少,排列相对有序;其余4组小鼠创面愈合组织中表皮呈现带状或片状均匀一致的玻璃样变性,胶原纤维增生较为明显,排列紊乱,其中烫伤组有较多炎性细胞浸润。AS-1早期治疗组小鼠创面愈合组织胶原纤维化百分比为(30±3)%,与假伤组的(30±4)%相近(q =0.159,P >0.05),均明显低于烫伤组、DMSO早期对照组、AS-1晚期治疗组、DMSO晚期对照组的(86±9)%、(74±5)%、(82±4)%、(82±7)%(q 值为12.080~15.530,P 值均小于0.01)。伤后21 d,与假伤组比较,烫伤组小鼠创面愈合组织中IL-1β、TNF-α、TGF-β1、MMP-1、CTGF mRNA,DMSO早期对照组、DMSO晚期对照组小鼠创面愈合组织中TGF-β1 mRNA,AS-1晚期治疗组小鼠创面愈合组织中MMP-1 mRNA表达量明显增多(q 值为4.039~5.232,P 值均小于0.05);烫伤组小鼠创面愈合组织中TIMP-1 mRNA表达量明显减少(q =4.921,P <0.05)。与烫伤组比较,AS-1早期治疗组小鼠创面愈合组织中IL-1β、TNF-α、TGF-β1、MMP-1、CTGF mRNA及AS-1晚期治疗组小鼠创面愈合组织中IL-1β、CTGF mRNA表达量明显减少(q 值为4.418~6.402,P <0.05或P <0.01),AS-1早期治疗组和AS-1晚期治疗组小鼠创面愈合组织中TIMP-1 mRNA表达量明显增多(q 值分别为3.929、8.299,P <0.05或P <0.01)。与假伤组比较,其余各组小鼠创面愈合组织中Ⅲ型胶原mRNA及蛋白,烫伤组、DMSO早期对照组、AS-1晚期治疗组、DMSO晚期对照组小鼠创面愈合组织中Ⅰ型胶原mRNA及蛋白表达量均明显增多(q 值为7.054~11.650,P 值均小于0.01)。与AS-1早期治疗组比较,烫伤组、DMSO早期对照组、AS-1晚期治疗组、DMSO晚期对照组小鼠创面愈合组织中Ⅰ型胶原mRNA及蛋白表达量明显增多(q 值为5.156~7.451,P <0.05或P <0.01)。 结论 在小鼠深Ⅱ度烫伤后早期炎症反应阶段进行AS-1干预,可有效促进创面愈合并减轻纤维化程度,可能与其降低炎症反应相关因子IL-1β、TNF-α和纤维化相关因子TGF-β1、MMP-1、CTGF以及Ⅰ型胶原的表达有关。Abstract: 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)%, withq values from 4.819 to 6.803,P <0.05 orP <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)%, withq 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 (withq 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 (withq values from 4.418 to 6.402,P <0.05 orP <0.01), while the mRNA expressions of TIMP-1 in healed wound tissue of mice in groups EAT and LAT were significantly increased (withq values respectively 3.929 and 8.299,P <0.05 orP <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 (withq 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 (withq values from 5.156 to 7.451,P <0.05 orP <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.-
Key words:
- Burns /
- Wound healing /
- Fibrosis /
- Cicatrix /
- Hydrocinnamoyl-L-valyl pyrrolidine /
- Inflammatory response
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