治伤巴布剂对大鼠急性软组织损伤模型P38MAPK、AKT信号通路的影响

Effects of Injury-curing Cataplasm on P38MAPK and AKT Signaling Pathways in Rat Acute Soft Tissue Injury Model

目的观察治伤巴布剂对急性软组织损伤(acute soft tissue injury,ASTI)模型p38丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)、丝/苏氨酸蛋白激酶(AKT)信号通路的影响,探讨治伤巴布剂干预ASTI的可能作用机制。方法将40只雄性SD大鼠按照随机数字表法分为正常对照组、模型对照组、治伤巴布剂组、p38MAPK信号通路抑制剂组、AKT信号通路抑制剂组,每组8只。除正常对照组外,其余四组均予以左侧后肢小腿ASTI造模。造模成功后,治伤巴布剂组于标记部位立即予治伤巴布剂(修剪成1.5x3cm大小)外敷,并用胶布固定;其余四组均予等剂量赋形剂(修剪成1.5×3 cm大小)外敷处理,胶布固定;持续外敷,共持续24 h。p38MAPK信号通路抑制剂组在造模前30 min予腹腔注射p38MAPK信号通路抑制剂SB203580(400μg/kg/天)1次;AKT信号通路抑制剂组在造模前30 min予腹腔注射AKT信号通路抑制剂perifosine(20 mg/kg/天)1次。分别于0(造模前)、2h、4h、8h、12h、24h测量受伤小腿肌肉处的周长,并计算肌肉肿胀率(muscle swelling rate,MSR)。24 h药物干预结束后,采用颈椎脱臼法处死大鼠。后将左侧后肢小腿损伤中心部位进行取材,分成三份。一部分用于观察组织病理学形态变化;一部分用于逆转录聚合酶链反应(RT-PCR)检测核因子-κB(nuclear factor kappa-B,NF-κB)p65 mRNA、肿瘤坏死因子-α(TNF-α)mRNA、白细胞介素-1β(IL-1β)mRNA表达水平;剩下部分用酶联免疫吸附试验(ELISA)法检测骨骼肌组织TNF-α、IL-1β含量水平及蛋白质免疫印迹(Western-blot)法测定p38MAPK、AKT、NF-κB p65、核因子抑制蛋白α(inhibitor kappa B alpha,IκBα)表达水平。结果与正常对照组相比,模型对照组MSR显著增加(P<0.01);病理形态学上,骨骼肌组织可见大面积肌细胞排列紊乱,肌细胞变性坏死,间质内可见红细胞聚集及大量炎症细胞浸润;骨骼肌组织TNF-α、IL-1β含量水平显著升高(P<0.01);磷酸化p38MAPK(p-p38)/总p38MAPK(t-p38),磷酸化-AKT(p-AKT)/总-AKT(t-AKT)明显升高(P<0.01),NF-κB p65及NF-κB p65mRNA表达水平明显升高(P<0.01)。与模型对照组相比,治伤巴布剂组MSR在治疗第8 h、12 h、24 h显著下降(P<0.01),且在治疗第24 h,其MSR较p38MAPK、AKT信号通路抑制剂组下降更明显(P<0.05);病理学评分显著下降(P<0.01),且较p38MAPK、AKT信号通路抑制剂组下降更显著(P<0.05);骨骼肌组织TNF-α、IL-1β含量水平明显下降(P<0.01),且较p38MAPK、AKT信号通路抑制剂组更显著(P<0.05);p-p38/t-p38及p-AKT/t-AKT明显下降(P<0.01),NF-κB p65及NF-κB p65 mRNA表达水平显著下降(P<0.01),且较p38MAPK、AKT信号通路抑制剂组在降低NF-κB p65及NF-κB p65 mRNA相对表达值方面更显著(P<0.01)。结论治伤巴布剂可能同时对p38MAPK、AKT信号通路产生了一定的抑制作用,引起NF-κB活性下调,NF-κB p65蛋白的表达下调,进而引起骨骼肌组织TNF-α、IL-1β炎性细胞因子含量水平下调,减轻ASTI炎症反应,从而改善ASTI。

Objective To observe the effects of injury-curing cataplasm on p38MAPK and AKT signaling pathways in acute soft tissue injury model, and to explore the possible mechanism of injury treatment babu agent on ASTI.Methods Forty male SD rats were divided into normal control group, model control group, injury-curing cataplasm group,p38MAPK signaling pathway inhibitor group and AKT signaling pathway inhibitor group according to random number table method, with 8 rats in each group. Except for the normal control group, the other four groups underwent ASTI modeling. After successful modeling, curing-injury cataplasm was externally applied with curing-injury cataplasm(pruned to 3 x 1.5 cm size) and fixed with adhesive tape;the other four groups were externally applied with the same dose of excipient(pruned to 3 x 3 cm size) and fixed with adhesive tape. This process lasted for 24 hours. The p38MAPK signaling pathway inhibitor group was intraperitoneally injected with p38MAPK signaling pathway inhibitor SB203580(400 μg/kg/day) once 30 minutes before modeling;AKT signaling pathway inhibitor group was intraperitoneally injected with AKT signaling pathway inhibitor perinosine(20 mg/kg/day) once 30 minutes before modeling. The circumference of the injured calf muscle was measured at 0(before modeling), 2 h, 4 h, 8 h, 12 h and 24 h, and the muscle swelling rate(MSR) was calculated. After 24 hours of drug intervention, the rats were sacrificed by cervical dislocation. Then the center of the left hind leg injury was taken and divided into three parts. One part was used to observe histopathological morphological changes;the other part was used for detecting the mRNA expression levels of nuclear factor kappa-B(NF-κB) p65, tumor necrosis factor-α(TNF-α) and interleukin-1 β(IL-1β) by reverse transcription polymerase chain reaction(RT-PCR);the remaining part was used to detect the contents of TNF-α and IL-1β in skeletal muscle tissue and determine the expression level of p38MAPK, AKT, NF-κB p65 and Inhibitor Kappa B-α(IκBα) by Western-blot.Results Compared with normal control group, The MSR of model control group increased significantly(P<0.01);In terms of histomorphology, a large area of muscle cells were disordered in skeletal muscle tissue, with degeneration and necrosis of muscle cells, and red blood cell aggregation and inflammatory cell infiltration were observed in the interstitium;The levels of TNF-α and IL-1β in skeletal muscle were significantly increased(P<0.01);The phosphorylation of p38MAPK(p-p38)/total p38MAPK(t-p38) and phosphorylation of p-AKT(p-AKT)/total Akt(tAKT) in model control group were significantly increased(P<0.01). The mRNA expression of NF-κB p65 and NF-κB p65 were significantly increased(P<0.01). Compared with the model control group, the MSR in the injury-curing cataplasm group was significantly decreased at 8 h, 12 h and 24 h(P<0.01), and the MSR in the treatment group was more significantly decreased than that in the p38MAPK and AKT signaling pathway inhibitor groups at 24 h(P<0.05);The pathological score decreased significantly(P<0.01), and decreased more significantly than p38MAPK and AKT signaling pathway inhibitor groups(P<0.05);The levels of TNF-α and IL-1β in skeletal muscle were significantly decreased(P<0.01), and were more significant than those in p38MAPK and AKT signaling pathway inhibitor groups(P<0.05);The p-p38/t-p38 and p-AKT/t-AKT in the injury-curing cataplasm group were significantly decreased(P<0.01),and the mRNA expression levels of NF-κB p65 and NF-κB p65 were significantly decreased(P<0.01). Compared with p38MAPK and AKT signaling pathway inhibitor group, the relative expression of NF-κB p65 and NF-κB p65mRNA were significantly decreased(P<0.01) in the injury-curing cataplasm group.Conclusion The injury-curing cataplasm might inhibit the p38MAPK and AKT signaling pathways to a certain extent, leading to the down-regulation of NF-κB activity and the expression of NF-κB P65 protein, and then lead to the down-regulation of TNF-α and IL-1βinflammatory cytokines in skeletal muscle tissue, and reduce the inflammatory response of ASTI, thereby improving ASTI.