有氧运动调控TGF-β1/Smad/miR-21分子通路改善衰老小鼠肝纤维化的机制研究

关键词：有氧运动  肝纤维化  转化生长因子-β  miRNA-21  Smad family member 7

Keywords:aerobic exercise  liver fibrosis  transforming growth factor-β  miRNA-21  SMAD family member 7

摘要：

      摘要 目的：探讨有氧运动对自然衰老诱导的肝纤维化的改善作用及机制。 方法：SPF级的C57BL/6J雄性小鼠32只，其中，2月龄小鼠16只（年轻组），19月龄小鼠16只（衰老组）。衰老组与年轻组均适应性饲养1周。将动物随机分为四组：年轻对照组（Y组）、年轻运动组（YE组）、衰老对照组（A组）、衰老运动组（AE组），每组8只。实验以跑台方式进行运动干预，各组运动小鼠均进行1周的递增负荷适应性运动，随后根据方案进行12周的正式有氧运动干预。HE、Masson和天狼猩红染色观察肝组织病理形态；免疫组化染色观察肝脏内Collagen-Ⅰ、α-SMA水平；q-PCR法检测肝组织miR-21、Ⅰ型胶原蛋白（Collagen-Ⅰ）、α-平滑肌肌动蛋白（α-SMA）、转化生长因子-β1（TGF-β1）、CTGF、Smad7mRNA表达；Western Blot法检测肝组织Collagen-Ⅰ、α-SMA、CTGF、TGF-β1、TGF-β受体Ⅱ（TβR-Ⅱ）、Smad7、P-Smad2/3蛋白表达。 结果：与Y组比较，A组小鼠可见明显肝细胞变性和纤维沉积；其肝组织内Collagen-Ⅰ、α-SMA、CTGF、TGF-β1、TβR-Ⅱ、P-Smad2/3、miR-21的mRNA及蛋白水平显著升高（P＜0.05），Smad7水平显著降低（P＜0.05）。运动干预后，AE组小鼠肝组织变性及纤维沉积现象减轻，肝组织Collagen-Ⅰ、α-SMA、CTGF、TGF-β1、TβR-Ⅱ、P-Smad2/3、miR-21的表达显著降低（P<0.05），Smad7水平显著升高（P＜0.05）。 结论：有氧运动可有效改善衰老诱导的肝纤维化，其机制可能与其抑制TGF-β1过度表达，降低TGF-β1/Smad信号通路相关分子表达，抑制miR-21同时提高Smad7的活性，减少肝细胞纤维沉积有关。

Abstract:

      Abstract Objective： To investigate the effect and underlaying mechanism of aerobic exercise on aging-induced hepatic fibrosis. Method： Thirty-two SPF grade C57BL/6J male mice were used， including 16 mice aged 2 months （young group） and 16 mice aged 19 months （senescent group）. Both the aging group and the young group underwent one week of acclimatization. The animals were randomly divided into four groups： young control group （Y group）， young exercise group （YE group）， aging control group （A group） and aging exercise group （AE group）， with 8 animals in each group. The experiment carried out exercise intervention in the way of running on the treadmill. All the exercise mice in each group received adaptive exercise with increasing load for one week， and then received formal aerobic exercise intervention for 12 weeks according to the protocol. HE， Masson and Sirius scarlet staining were used to observe the pathological morphology of liver tissue. The levels of Collagen I and α-SMA in liver were observed by immunohistochemical staining. The mRNA expressions of miR-21， collagen I， α-smooth muscle actin （α-SMA）， transforming growth factor-β1 （TGF-β1） and SMAD7 in liver tissues were detected by q-PCR. Western Blot assay was used to detect the protein expressions of Collagen Ⅰ， α-SMA， CTGF， TGF-β1， TGF-β receptor Ⅱ （TβR-Ⅱ）， Smad7 and P-Smad2/3 in liver tissues. Result： Compared with Y group， hepatocyte degeneration and fiber deposition were observed in group A. The mRNA and protein levels of Collagen Ⅰ， α-SMA， CTGF， TGF-β1， TβR-Ⅱ， P-SMAD2/3 and miR-21 in liver tissues were significantly increased （P<0.05）， while the level of Smad7 was significantly decreased （P<0.05）. After exercise intervention， liver tissue degeneration and fiber deposition were alleviated in the AE group， and the expressions of Collagen Ⅰ， α-SMA， CTGF， TGF-β1， TβR-Ⅱ， P-Smad2/3 and miR-21 in liver tissue were significantly decreased （P<0.05）， the level of Smad7 was significantly increased （P<0.05）. Conclusion： Aerobic exercise can effectively improve age-induced hepatic fibrosis. The underlying mechanism may be related to inhibiting the overexpression of TGF-β1， reducing the expression of TGF-β1/Smad signaling pathway related molecules， inhibiting miR-21， improving the activity of Smad7， and reducing the deposition of liver cell fibers.