基于IGF-1/PI3K/Akt信号通路探讨象皮生肌膏对压力性损伤大鼠模型的影响及机制研究

Effects and mechanisms of Xiangpi Shengji Ointment on pressure induced injury rat model based on IGF-1/PI3K/Akt signaling pathway

目的通过动物实验观察并基于类胰岛素一号生长因子(insulin-like growth factors-1,IGF-1)/磷脂酰肌醇3激酶(phosphoinositide3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)信号通路探讨象皮生肌膏对压力性损伤大鼠模型的影响。方法将40只SD大鼠(SPF级,雌雄各半)复制压力性损伤大鼠模型,造模成功后随机均分为模型组(生理盐水纱布外敷)、阳性对照组(三乙醇胺乳膏外用)、象皮生肌组(象皮生肌膏外用)、联合给药组(三乙醇胺乳膏与象皮生肌膏联合外用)。另取10只健康SD大鼠作为空白组,仅同部位置入磁性装置,不予施加压力。实验过程中,每天记录各组大鼠愈合情况。实验结束时,HE染色法观察创面病理性损伤情况,ELISA法检测血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)水平,Western blot法及RT-PCR法检测创面组织中PI3K、Akt、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、IGF-1蛋白及mRNA的表达情况。结果与空白组比较,模型组大鼠创面面积明显增大(P<0.05);与模型组比较,阳性对照组、象皮生肌组、联合给药组大鼠的创面面积均减小(P<0.05);与阳性对照组及象皮生肌组比较,联合给药组创面面积缩小(P<0.05)。HE染色结果显示,空白组大鼠创面病理结构较完整,组织细胞排列较规则且病理致密,未见明显组织坏死碎片、水肿及炎性浸润;模型组可见明显的肌肉纤维坏死碎片,空泡变性,纤维肿大、横断;阳性对照组、象皮生肌组、联合给药组可见不同程度的病理缓解。与空白组比较,模型组大鼠血清炎性因子TNF-α、IL-1β、IL-6水平明显升高(P<0.05),PI3K、Akt、mTOR、IGF-1蛋白及mRNA表达明显升高(P<0.05);与模型组比较,阳性对照组、象皮生肌组、联合给药组大鼠血清炎性因子TNF-α、IL-1β、IL-6水平显著降低(P<0.05),PI3K、Akt、mTOR、IGF-1蛋白及mRNA表达显著升高(P<0.05);与阳性对照组及象皮生肌组比较,联合给药组血清炎性因子TNF-α、IL-1β、IL-6水平以及PI3K、Akt、mTOR、IGF-1蛋白及mRNA表达水平明显降低(P<0.05)。结论象皮生肌膏可改善压力性损伤大鼠模型创面病理情况,可能与象皮生肌膏上调IGF-1/PI3K/Akt信号通路相关蛋白表达,抑制炎性因子表达相关。

Objective To investigate the effects of Xiangpi Shengji Ointment on pressure induced injury rat models through animal experiments and based on the insulin-like growth factor-1(IGF-1)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway.Methods A total of 40 SD rats(SPF grade,half male and half female)were firstly set as the pressure induced injury rat models,and then randomly divided into model group(physiological saline gauze,external application),positive control group(Triethanolamine Ointment,external application),Xiangpi Shengji Ointment group(Xiangpi Shengji Ointment,external application),and combined administration group(Triethanolamine Ointment combined with Xiangpi Shengji Ointment,external application).Another 10 healthy SD rats were selected as the blank group,and only the same position was inserted into the magnetic device without pressure.The healing status of each group was recorded daily.HE staining was used to observe the pathological damage of wound surface,and ELISA was taken to measure serum tumor necrosis factor-α(TNF-α),Interleukin-1β(IL-1β),and interleukin-6(IL-6)levels.Western blot and RT-PCR methods were taken to check the expressions of PI3K,Akt,mammalian target of rapamycin(mTOR),IGF-1 protein,and mRNA in wound tissue.Results Compared with blank group,the wound area of model group rats increased(P<0.05);compared with model group,the wound area of rats in the positive control group,Xiangpi Shengji Ointment group,and combined administration group decreased(P<0.05);compared with the positive control group and Xiangpi Shengji Ointment group,the combined administration group showed a decrease in wound area(P<0.05).The HE staining showed that the pathological structure of the wound in the blank group rats was relatively complete,with regular arrangement of tissue cells and dense pathology.No obvious tissue necrosis fragments,edema,and inflammatory infiltration were observed;in the model group,obvious muscle fiber necrosis fragments,vacuolar degeneration,fiber enlargement,and transection were observed;the positive control group,Xiangpi Shengji Ointment group,and combined administration group showed varying degrees of pathological relief.Compared with blank group,the levels of serum inflammatory factors of TNF-α,IL-1β,and IL-6 in the model group rats increased(P<0.05),and the expressions of PI3K,Akt,mTOR,IGF-1 protein,and mRNA were higher(P<0.05);compared with the model group,the levels of serum inflammatory factors of TNF-α,IL-1β,and IL-6 of rats in the positive control group,Xiangpi Shengji Ointment group,and combined administration group decreased(P<0.05),while the expressions of PI3K,Akt,mTOR,IGF-1 protein,and mRNA increased(P<0.05);compared with the positive control group and the Xiangpi Shengji Ointment group,the levels of serum inflammatory factors of TNF-α,IL-1β,and IL-6,and the levels of PI3K,Akt,mTOR,IGF-1 protein,and mRNA expression of combined administration group were lower(P<0.05).Conclusion Xiangpi Shengji Ointment can alleviate the wound pathology of pressure induced injury rat models.It may be related to upregulating the related protein expression of IGF-1/PI3K/Akt signaling pathway and inhibiting the inflammatory factor expression.