The regulatory mechanism on granulosa cells (GCs) oxidative injury is becoming increasingly important in polycystic ovary syndrome (PCOS) studies. Serine/threonine kinase mitogen-activated protein 4 kinase 4 (Map4k4) is linked with oxidative injury and possibly associated with premature ovarian failure and ovarian dysgenesis. Herein, we investigated the function and mechanism of Map4k4 in a PCOS rat model. A microarray from GEO database identified Map4k4 was up-regulated in the ovarian of PCOS rats, and functional enrichments suggested that oxidative stress-associated changes are involved. We verified the raised Map4k4 expression in an established PCOS rat model and also in the isolated PCOS-GCs, which were consistent with the microarray data. Map4k4 knockdown in vivo contributed to regular estrous cycle, restrained steroid concentrations and ovarian injury in PCOS rats. Both Map4k4 silencing in vivo and in vitro attenuated the PCOS-related GC oxidative stress and apoptosis. Mechanically, Map4k4 activated the JNK/c-JUN signaling pathway. Importantly, a JNK agonist restored the suppressive effects of Map4k4 silencing on PCOS-induced granulosa cell injury and oxidative stress. Besides, Map4k4 may be a target gene of miR-185-5p. In conclusion, Map4k4, a potential target of miR-185-5p, is up-regulated and induces ovarian GC oxidative injury by activating JNK/c-JUN pathway in PCOS. The Map4k4/JNK/c-JUN mechanism may provide a new idea on the treatment of PCOS.
Purpose: The deposition of hydroxyapatite (HAp) crystals plays an important role in the development of vascular calcification (VC). This study aimed to demonstrate the effects of nanosized HAp (nHAp) on vascular smooth muscle cells (VSMCs) and VC progression.Methods: Transmission electron microscopy (TEM) was used to examine cellular uptake of nHAp. Cell viability was determined using CCK-8 assay kit. Mitochondrial impairment and reactive oxygen species were detected by TEM and fluorescence dye staining, respectively. Cell apoptosis was detected by Western blot analysis and Annexin V staining. Mouse model of VC was built via applying nHAp on the surface of abdominal aorta. Calcification was visualized by Alizarin red and von Kossa staining.Results: We found that nHAp could promote osteogenic transformation of VSMCs by elevating expression of runt-related factor 2 (Runx2), osteopontin (OPN) and alkaline phosphatase (ALP), impairing function and morphology of mitochondria and inducing apoptosis of VSMCs. More phosphorylation of c-Jun N-terminal protein kinase/c-JUN (JNK/c-JUN) in VSMCs was detected after mixing nHAp with VSMCs. HAp-induced osteogenic transformation of VSMCs was blocked by JNK inhibitor SP600125, resulted in decreased ALP activity, less Runx2 and OPN expressions. SP600125 also inhibited apoptosis of VSMCs. Application of nHAp to outside of aorta induced osteogenic transformation and apoptosis of VSMCs, and significant deposition of calcium on the vessel walls of mice, which can be effectively attenuated by SP600125.Conclusion: JNK/c-JUN signaling pathway is critical for nHAp-induced calcification, which could be a potential therapeutic target for controlling the progression of VC.
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