γ干扰素对小鼠骨骼肌钝挫伤后转化生长因子-β-Smad信号通路表达的影响

目的 观察γ干扰素(IFN-γ)局部注射对小鼠骨骼肌钝挫伤后纤维化以及转化生长因子-β(TGF-β)-Smad信号通路表达的影响.方法 将30只成年雌性C57bl小鼠随机分为正常组(A组)、磷酸盐缓冲液(PBS)注射组(B组)和hIFN-γ注射组(C组),每组10只.B、C组采用50g钢球于30cm高度下砸的方法 造成双侧腓肠肌钝挫伤,分别于伤后1、2、3周局部皮下注射0.1ml PBS或hIFN-γ,A组不行任何处理;4周后取双侧腓肠肌检测.以Masson染色后计算胶原纤维面积评估骨骼肌纤维化程度,Western blot检测TGF-β1、Smad2、Smad3、Smad4、Smad7蛋白表达水平.结果 Masson染色显示B组胶原面积最大为(14074.0±15.2)μm2(P<0.05),C组为(4019.0±13.4)μm2高于A组(1741.0±10.1)μm2(P<0.05);TGF-β1在B、C组表达增高(P<0.05),但两组之间表达差异无统计学意义(P>0.05);3组间Smad4、7的表达差异无统计学意义(P>0.05);Smad2、3及其磷酸化蛋白在B、C组表达升高(P<0.05),其中磷酸化的Smad2、3 在C组较B组表达明显降低(P<0.05).结论 局部注射hIFN-γ,未发现影响TGF-β1的表达,但能够有效降低Smad2、3磷酸化蛋白的表达,可能是其抑制损伤骨骼肌纤维化的作用机制.

Abstract：

Objective To observe the influence of local injection of human interferon γ (hIFN-γ) on the fibrosis and transforming growth factor-β1 (TGF-β1)-Smad signal pathway in the skeletal muscle following acute contusion. Methods Thirty female C57bl mice were randomly divided into 3 groups as A, B and C. The mice in groups B and C were injured by heavy hit of a 50 g ball from 30 cm height to establish gastrocnemius contusion models, and then subcutaneously injected with 0.1 ml PBS and hIFN-γ respectively. No interference was given to group A. All mice were sacrificed 4 weeks later to harvest the gastrocnemius. Fibrosis was estimated by calculating collagen areas on masson staining section. Expression of the proteins as TGF-β1, Smad2, Smad3, Smad4 and Smad7 was detected by Western blotting. Results The collagen area on masson staining section of group B was the greatest as (14 074.0±15.2) μm2, and that in group C was (4019.0±13.4) μm2, greater than that in group A (1741.0±10.1) μm2 (P<0.05). The level of TGF-β1 was increased in both groups B and C, but without statistically significant difference between them (P>0.05). No significant difference was found in Smad4 and Smad7 protein expression among three groups (P>0.05). Total Smad2 and Smad3 were increased in groups B and C but without significant difference (P>0.05). Phosphorylated Smad2/3 level was also increased in both groups B and C, and that was lower in group C (P<0.05). Conclusion Without influence on the expression of TGF-β1, local injection of hIFN-γ could reduce the phosphorylation of Smad2/3 significantly, which probably contributed to the suppression of fibrosis within injured skeletal muscle.