Yang M L, Li Y H, Tan Q, Li J T, Que L L
Gulou Clinical Medical College of Nanjing Medical University, Nanjing 210008, China.
Zhonghua Shao Shang Za Zhi. 2016 Nov 20;32(11):658-666. doi: 10.3760/cma.j.issn.1009-2587.2016.11.005.
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. 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 β (TGF-β), 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. 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%, =1.753, >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 values from 4.819 to 6.803, <0.05 or <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)%, =0.159, >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 values from 12.080 to 15.530, values below 0.01]. On PSD 21, compared with those of group SI, the mRNA expressions of IL-1β, TNF-α, TGF-β, MMP-1, and CTGF in healed wound tissue of mice in group S, the mRNA expressions of TGF-β 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 values from 4.039 to 5.232, values below 0.05), while the mRNA expression of TIMP-1 in healed wound tissue of mice in group S was significantly decreased (=4.921, <0.05). Compared with those of group S, the mRNA expressions of IL-1β, TNF-α, TGF-β, 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 values from 4.418 to 6.402, <0.05 or <0.01), while the mRNA expressions of TIMP-1 in healed wound tissue of mice in groups EAT and LAT were significantly increased (with values respectively 3.929 and 8.299, <0.05 or <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 values from 7.054 to 11.650, 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 values from 5.156 to 7.451, <0.05 or <0.01). 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-β, MMP-1, CTGF, and type Ⅰ collagen.
观察Toll样白细胞介素-1受体同源性/BB环模拟物氢化肉桂酰-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。将42只成年C57BL/6小鼠按随机数字表分为假伤组（SI）、烫伤组（S）、AS-1早期治疗组（EAT）、早期二甲基亚砜（DMSO）对照组（EDC）、AS-1晚期治疗组（LAT）、晚期DMSO对照组（LDC），每组7只。SI组小鼠仅进行假伤，不做其他处理。对其余小鼠背部制备10%体表面积的深Ⅱ度烫伤模型，伤后每日用生理盐水清洗创面并更换凡士林纱布。EAT组和LAT组小鼠分别从烫伤后8小时（PSH）和烫伤后15天（PSD）起，每天腹腔注射20mg/mL AS-1 50mg/kg。EDC组和LDC组小鼠分别从PSH 8和PSD 15起，每天腹腔注射20mg/mL DMSO 50mg/kg。在PSD 21时，观察烫伤小鼠创面愈合的大体情况，计算创面愈合率。收集愈合创面的组织样本，分别进行HE染色和Masson染色，观察组织形态学变化及胶原纤维化情况，并计算胶原纤维化百分比。采用实时荧光定量逆转录聚合酶链反应检测愈合创面组织中白细胞介素-1β（IL-1β）、肿瘤坏死因子α（TNF-α）、转化生长因子β（TGF-β）、基质金属蛋白酶-1（MMP-1）、金属蛋白酶组织抑制剂1（TIMP-1）、结缔组织生长因子（CTGF）、Ⅰ型胶原和Ⅲ型胶原的mRNA表达。采用蛋白质免疫印迹法检测愈合创面组织中Ⅰ型胶原和Ⅲ型胶原的蛋白表达。对SI组小鼠与烫伤小鼠观察及取材相同部位的皮肤组织，同时进行上述相同的观察及检测。数据采用单因素方差分析和Tukey检验进行处理。在PSD 21时，SI组小鼠皮肤组织未见异常外观。EAT组小鼠创面完全愈合，无瘢痕形成，而其他四组小鼠创面均未完全愈合，有不同程度瘢痕形成。EAT组小鼠创面愈合率为（97±4）%，接近SI组（100%，F =1.753，P>0.05），且两组均明显高于S组、EDC组、LAT组和LDC组[分别为（83±8）%、（87±6）%、（85±9）%和（85±7）%，F值为4.819至6.803，P<0.05或P<0.01]。在PSD 21时，SI组小鼠皮肤组织形态未见异常外观。EAT组小鼠愈合创面组织形态接近SI组，表皮透明变性轻微，新生胶原纤维排列有序。其他四组小鼠愈合创面组织可见带状或片状表皮透明变性，胶原纤维明显增生且排列紊乱。S组有大量炎性细胞浸润。EAT组小鼠愈合创面组织中胶原纤维化百分比为（30±3）%，接近SI组[（30±4）%，F =0.159，P>0.05]，且两组均明显低于S组、EDC组、LAT组和LDC组[分别为（86±9）%、（74±5）%、（82±4）%和（82±7）%，F值为12.080至15.530，P值均低于0.01]。在PSD 21时，与SI组相比，S组小鼠愈合创面组织中IL-1β、TNF-α、TGF-β、MMP-1和CTGF的mRNA表达，EDC组和LDC组小鼠愈合创面组织中TGF-β的mRNA表达，以及LAT组小鼠愈合创面组织中MMP-1的mRNA表达均显著升高（F值为4.039至5.232，P值低于0.05），而S组小鼠愈合创面组织中TIMP-1的mRNA表达显著降低（F =4.921，P<0.05）。与S组相比，EAT组小鼠愈合创面组织中IL-1β、TNF-α、TGF-β、MMP-1和CTGF的mRNA表达以及LAT组小鼠愈合创面组织中IL-1β和CTGF的mRNA表达均显著降低（F值为4.418至6.402，P<0.05或P<0.01），而EAT组和LAT组小鼠愈合创面组织中TIMP-1的mRNA表达均显著升高（F值分别为3.929和8.299，P<0.05或P<0.01）。与SI组相比，其他组小鼠愈合创面组织中Ⅲ型胶原的mRNA和蛋白表达以及S组、EDC组、LAT组和LDC组小鼠愈合创面组织中Ⅰ型胶原的mRNA和蛋白表达均显著升高（F值为7.054至11.650，P值低于0.01）。与EAT组相比，S组、EDC组、LAT组和LDC组小鼠愈合创面组织中Ⅰ型胶原的mRNA和蛋白表达均显著升高（F值为5.156至7.451，P<0.05或P<0.01）。AS-1可有效促进小鼠深Ⅱ度烫伤后炎症反应早期的创面愈合并降低纤维化程度，这可能与其降低炎症相关因子IL-1β和TNF-α以及纤维化相关因子TGF-β、MMP-1、CTGF和Ⅰ型胶原的表达有关。
