基于ILK信号通路探究马桑水提取物影响大鼠烧伤创面愈合和瘢痕增生的机制

Effects of Coriaria sinica Maxim’s extract on burn wound healing and scar hyperplasia in rats based on ILK signaling pathway

目的:基于整合素连接激酶(ILK)调控的转化生长因子β1(TGF-β1)和PI3K/AKT调控的ILK等互通信号通路,探究马桑水提取物(CSME)促进大鼠烧伤创面早期愈合和抑制后期瘢痕过度增生的机制。方法:随机将180只SD雌性大鼠(180~200 g)分成正常对照(NC)组、凡士林(VL)组、磺胺嘧啶银(SS)组及CSME低、中和高剂量(CSME-L、CSME-M和CSME-H)组,每组各30只。大鼠以水合氯醛麻醉,腰背部II°烫伤造模后,每天分别在创面涂擦VL、SS和3种剂量的CSME烧伤软膏,计算创面愈合率(HR),于第7、14和21天随机选取各组动物10只,水合氯醛麻醉,切取创面皮肤,病理学观查,Western blot检测相关蛋白表达,RT-qPCR检测相关mRNA表达,培养成纤维细胞检测其胶原收缩率(SK)。结果:在第7天,CSME各组创面中ILK、纤维连接蛋白(FN)、TGF-β1、α-平滑肌肌动蛋白(α-SMA)和整合素β1(integrin-β1,ITG-β1)的蛋白与mRNA表达呈现剂量依赖性强于VL和SS组,第21天,弱于VL和SS组(P<0.05);第14天,CSME各组组织中I型胶原蛋白(Col I)的蛋白与mRNA表达呈剂量依赖性强于VL和SS组,第21天则弱于VL和SS组(P<0.05),而III型胶原蛋白(Col III)的蛋白与mRNA表达呈剂量依赖性弱于VL和SS组,第21天则强于VL和SS组(P<0.05);随时间推移,VL和SS组成纤维细胞的SK持续升高,96 h达高峰,而CSME各组在24 h和48 h呈剂量依赖性高于VL和SS组,在96 h呈剂量依赖性低于VL和SS组(P<0.05)。结论:CSME早期促进烧伤创面愈合,后期抑制创面瘢痕过度增生,其机制与其多成分多靶点影响ILK调控的TGF-β1和PI3K/AKT调控的ILK信号通路及ColⅠ与Col III表达比的调控相关。

AIM:To explore the mechanism of Coriaria sinica Maxim’s extract(CSME)promoting burn wound healing in the early stage and inhibiting excessive scar hyperplasia in the later stage,based on the signaling pathways,such as transforming growth factor-β1(TGF-β1)regulated by integrin-linked kinase(ILK)and ILK regulated by PI3K/AKT.METHODS:Female SD rats(n=180;180~200 g)were randomly divided into 6 groups:normal control(NC)group,vaseline(VL)group,silver sulfadiazine(SS)group and low-,medium-and high-dose of CSME(CSME-L,CSME-M and CSME-H)groups,with 30 rats in each group.Except for the rats in NC group,VL,SS,and 3 doses of CSME were applied to the wound surface of the rats in the corresponding groups every day after II°burn model was made on their waist-back in the condition of chloral hydrate anesthesia.To calculate the healing rate(HR),10 rats in each experimental group were randomly selected to remove their wound skin for observing the pathologic change,detecting the expression of related proteins by Western blot and RT-qPCR,and checking the collagen shrinkage(SK)by fibroblast culture at the 7th,14th,and 21st days.RESULTS:The expression of ILK,fibronectin(FN),TGF-β1,α-smooth muscle actin(α-SMA)and integrin-β1(ITG-β1)at protein and mRNA levels in wound skin of CSME groups was stronger than that in VL group and SS group at the 7th day in a dose-dependent manner,but weaker than that in VL group and SS group at the 21st day(P<0.05).Meanwhile,the protein and mRNA expression of collagen type I(Col I)in CSME groups was stronger than that in VL group and SS group from the 7th day to the 14th day,but weaker than that in VL group and SS group at the 21st day in a dose-dependent manner(P<0.05).However,the protein and mRNA expression of collagen type III(Col III)in CSME groups was weaker than that in VL group and SS group from the 7th day to the 14th day,but stronger than that in VL group and SS group at the 21st day in a dose-dependent manner(P<0.05).The SK of fibroblasts in VL group and SS group was increased continuously over time and reached its peak at 96 h.SK in CSME groups was only higher than that in VL group and SS group at 24 h and 48 h in a dose-dependent manner,but lower than that in VL group and SS group at 96 h(P<0.05).CONCLUSION:CSME promotes burn wound healing in the early stages and inhibits the scar hyperplasia in the later stages.The mechanisms may be related to its multicomponents or multiple-targets to intervene in the signaling pathways such as TGF-β1 regulated by ILK and ILK regulated by PI3K/AKT.It may also be related to the ratio of Col I and Col III expression.