[Effects of inhibition of Rho/ROCK pathway on proliferation and migration of airway smooth muscle cells and related mechanisms]

Abstract

Objectives: To investigate the effects and molecular mechanisms of inhibition of the Ras homolog gene (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) pathway on the proliferation and migration of airway smooth muscle cells involving myocardin (MYOCD).

Methods: Human airway smooth muscle cells were infected with the adenoviral vector Ad-ZsGreen-shRNA-hROCK1 in vitro. The cells were randomly divided into four groups: ROCK1 gene silencing control (shNC) group, shNC + arachidonic acid (AA, Rho/ROCK pathway activator) group, ROCK1 gene silencing (shROCK1) group, and shROCK1 + AA group (n=3 each). Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression levels of ROCK1 and MYOCD mRNA and protein. ELISA was employed to measure the levels of globular actin and filamentous actin, while immunofluorescent staining and scratch assays were utilized to assess cell proliferation and migration.

Results: Compared to the shNC + AA group, the shROCK1 + AA group exhibited decreased levels of ROCK1 and MYOCD mRNA and protein expression, reduced expression levels of globular actin and filamentous actin, and diminished cell proliferation and migration capabilities (P<0.05).

Conclusions: Inhibition of the Rho/ROCK pathway suppresses the proliferation and migration of airway smooth muscle cells, which may be associated with the downregulation of MYOCD.

Keywords: Airway smooth muscle cell; Asthma; Myocardin; Proliferation; Rho/ROCK pathway.

图1. Western blot法检测各组细胞中ROCK1、MYOCD蛋白表达电泳图 1：shNC组；2：shROCK1组；3：shROCK1+AA组；4：shNC+AA组。

图2. 免疫荧光染色检测沉默ROCK1对HASMC增殖的影响（共聚焦显微镜，×200） 蓝色荧光标记细胞核，红色荧光标记细胞质中α-SMA，粉色荧光标记增殖细胞的细胞核中Ki67。

图3. 各组细胞在划痕后的0 h、6 h、24 h的细胞迁移区域（倒置显微镜，×100） 虚线表示0 h时细胞划痕区域边界。6 h时，shROCK1+AA组细胞划痕覆盖区域较shNC+AA组明显减少；24 h时，与shNC+AA组比较，shROCK1+AA 组划痕区域仍未完全愈合。shROCK1+AA 组细胞迁移能力明显低于 shNC+AA 组。

图4. Rho/ROCK/MYOCD通路对气道平滑肌细胞增殖与迁移的相关机制　　［GPCR］G蛋白偶联受体；［ROCK］Rho相关卷曲螺旋形成蛋白激酶；［RhoA］Ras同源基因家族成员A；［GDP］二磷酸鸟苷；［GTP］三磷酸鸟苷；［G-actin］球状肌动蛋白；［F-actin］丝状肌动蛋白；［MYOCD］心肌素；［SRF］血清反应因子；［MLCK］肌球蛋白轻链激酶；［MLCP］肌球蛋白轻链磷酸酶；［MYPT1］肌球蛋白磷酸酶靶亚基1；［MLC］肌球蛋白轻链；［P］磷酸化。