关键词: miR-340-5p, Smad同源物2, 黄芪甲苷, 盆底功能障碍, 大鼠

Abstract:

AIM: To investigate the protective effect of astragaloside IV (ASIV) in pelvic floor dysfunction (PFD) rats and its effect on the expression of microRNA (miR)-340-5p and Smad homolog 2 (SMAD2). METHODS: PFD rat model was constructed. The successfully modeled rats were randomly divided into the PFD group, the ASIV-L group, the ASIV-M group, the ASIV-H group, the ASIV-H+miR-NC group, and the ASIV-H+miR-340-5p mimics group, with 12 rats in each group. Another 12 normal healthy rats were taken as the Control group. The relevant indicators such as pelvic floor function, urodynamics (bladder volume, intravesical pressure, leak point pressure), and mean myoelectric potential of muscle fibers of rats in each group were detected. HE staining and Masson staining were used to observe the pathological changes of the levator ANI muscle tissue. Immunohistochemical detection of the relative expressions of type Ⅰ Collagen (Collagen Ⅰ) and Elastin. qRT-PCR was used to detect the level of miR-340-5p. Western blot was used to detect the relative expression of SMAD2. The dual-luciferase reporter gene system was used to verify the targeting relationship between miR-340-5p and SMAD2. RESULTS: Compared with the Control group, the levator ani muscle in the PFD group exhibited hypertrophy, with atrophied cells and the presence of mast cells within the muscle fiber tissue. A large number of muscle fibers showed necrosis and disorganized arrangement. A small amount of blue collagen fibers was observed, and the overall muscle fiber content was significantly reduced (P<0.05). Bladder volume, intravesical pressure, leak point pressure, vaginal resting pressure, vaginal squeeze pressure, and the mean myoelectric potential of type Ⅰ and type Ⅱ muscle fibers were all decreased (P<0.05). The relative expressions of Collagen Ⅰ, Elastin, and protein SMAD2 were also decreased, while the bladder neck range of motion, urethral rotation angle, and relative level of miR-340-5p were increased (P<0.05). Compared with the PFD group, the ASIV-L, ASIV-M, ASIV-H, and ASIV-H+miR-340-5p mimics groups showed markedly improvements in muscle fiber tissues, with reduced necrosis, no apparent degeneration, and more orderly arranged fibers. Collagen fiber content increased to varying degrees, and the overall muscle fiber content was elevated (P<0.05). Bladder volume, intravesical pressure, leak point pressure, vaginal resting pressure, vaginal squeeze pressure, and the mean myoelectric potential of type Ⅰ and type Ⅱ muscle fibers were increased (P<0.05). The relative expressions of Collagen Ⅰ, Elastin and SMAD2 were also upregulated, whereas the bladder neck range of motion, urethral rotation angle, and relative level of miR-340-5p were decreased (P<0.05). In the ASIV-H+miR-340-5p mimics group, muscle fiber histopathological damage was more severe than in the ASIV-H, and ASIV-H+miR-NC groups, with a significant reduction in collagen fibers. Bladder volume, intravesical pressure, leak point pressure, vaginal resting pressure, vaginal squeeze pressure, and the mean myoelectric potential of type Ⅰ and type Ⅱ muscle fibers were decreased (P<0.05). The relative expressions of Collagen Ⅰ, Elastin and SMAD2 were decreased, while the bladder neck range of motion, urethral rotation angle and relative level of miR-340-5p were increased (P<0.05). Analysis using the Starbase database and dual-luciferase gene report assays indicated that miR-340-5p targeted and inhibited SMAD2 expression. CONCLUSION: ASIV improves PFD in rats through downregulation of miR-340-5p and upregulation of SMAD2.

Key words: miR-340-5p, SMAD2, astragaloside IV, pelvic floor dysfunction, rat