摘要: Objectives It has been widely reported that maternal diabetes impairs oocyte quality. However, the responsible mechanisms remain to be explored. In the present study, we focused on whether SIRT3-GSK3 beta pathway mediates the meiotic defects in oocytes from diabetic mice. Materials and methods GSK3 beta functions in mouse oocyte meiosis were first detected by targeted siRNA knockdown. Spindle assembly and chromosome alignment were visualized by immunostaining and analysed under the confocal microscope. PCR-based site mutation of specific GSK3 beta lysine residues was used to confirm which lysine residues function in oocyte meiosis. siRNA knockdown coupled with cRNA overexpression was performed to detect SIRT3-GSK3 beta pathway functions in oocyte meiosis. Immunofluorescence was performed to detect ROS levels. T1DM mouse models were induced by a single intraperitoneal injection of streptozotocin. Results In the present study, we found that specific depletion of GSK3 beta disrupts maturational progression and meiotic apparatus in mouse oocytes. By constructing site-specific mutants, we further revealed that acetylation state of lysine (K) 15 on GSK3 beta is essential for spindle assembly and chromosome alignment during oocyte meiosis. Moreover, non-acetylation-mimetic mutant GSK3 beta-K15R is capable of partly preventing the spindle/chromosome anomalies in oocytes with SIRT3 knockdown. A significant reduction in SIRT3 protein was detected in oocytes from diabetic mice. Of note, forced expression of GSK3 beta-K15R ameliorates maternal diabetes-associated meiotic defects in mouse oocytes, with no evident effects on oxidative stress. Conclusion Our data identify GSK3 beta as a cytoskeletal regulator that is required for the assembly of meiotic apparatus, and discover a beneficial effect of SIRT3-dependent GSK3 beta deacetylation on oocyte quality from diabetic mice.

摘要: Objectives Runx3, a member of the Runx family of transcription factors, has been studied as a tumour suppressor and key player of organ development. In a previous study, we reported differentiation failure and excessive angiogenesis in the liver of Runx3 knock-out (KO) mice. Here, we examined a function of the Runx3 in liver, especially in iron metabolism. Methods We performed histological and immunohistological analyses of the Runx3 KO mouse liver. RNA-sequencing analyses were performed on primary hepatocytes isolated from Runx3 conditional KO (cKO) mice. The effect of Runx3 knock-down (KD) was also investigated using siRNA-mediated KD in functional human hepatocytes and human hepatocellular carcinoma cells. Result We observed an iron-overloaded liver with decreased expression of hepcidin in Runx3 KO mice. Expression of BMP6, a regulator of hepcidin transcription, and activity of the BMP pathway were decreased in the liver tissue of Runx3 KO mice. Transcriptome analysis on primary hepatocytes isolated from Runx3 cKO mice also revealed that iron-induced increase in BMP6 was mediated by Runx3. Similar results were observed in Runx3 knock-down experiments using HepaRG cells and HepG2 cells. Finally, we showed that Runx3 enhanced the activity of the BMP6 promoter by responding to iron stimuli in the hepatocytes. Conclusion In conclusion, we suggest that Runx3 plays important roles in iron metabolism of the liver through regulation of BMP signalling.

摘要: Objectives Idiopathic pulmonary fibrosis (IPF) is marked by the excessive accumulation of extracellular matrix, which participates in a variety of chronic diseases or injuries and seriously threatens human health. Due to the side effects of clinical drugs, there is still a need to develop novel and less toxic drugs to treat pulmonary fibrosis. Materials and Methods SKLB-YTH-60 was developed through computer-aided drug design, de novo synthesis and high-throughput screening. We employed the bleomycin (BLM)-induced lung fibrosis animal models and used TGF-beta(1) to induce the epithelial-mesenchymal transition (EMT) of A549 cells in vitro. Meanwhile, the protein expression of collagen I and the alpha-smooth muscle actin (alpha-SMA), E-cadherin, p-FGFR1, p-PLC gamma, p-Smad2/3 and p-Erk1/2 was detected by western blot. Results YTH-60 has obvious anti-proliferative activity on fibroblasts and A549 cells. Moreover, YTH-60 could impair the EMT of A549 cells and suppressed fibrosis by inhibiting FGFR and TGF-beta/Smad-dependent pathways. Intraperitoneal administration of preventive YTH-60 could significantly reduce the degree of fibrosis in mice and regulate the imbalance of the immune microenvironment. In addition, we observed that therapeutic YTH-60 treatment attenuated fibrotic changes in mice during the period of fibrosis. Importantly, YTH-60 has shown an acceptable oral bioavailability (F = 17.86%) and appropriate eliminated half-life time (T-1/2 = 8.03 hours). Conclusions Taken together, these preclinical evaluations suggested that YTH-60 could be a promising drug candidate for treating IPF.

摘要: Objectives Various factors could interfere the biological performance of DPSCs during post-thawed process. Yet, little has been known about optimization of the recovery medium for DPSCs. Thus, our study aimed to explore the effects of adding recombinant bFGF on DPSCs after 3-month cryopreservation as well as the underlying mechanisms. Materials and methods DPSCs were extracted from impacted third molars and purified by MACS. The properties of CD146(+) DPSCs (P3) were identified by CCK-8 and flow cytometry. After cryopreservation for 3 months, recovered DPSCs (P4) were immediately supplied with a series of bFGF and analysed cellular proliferation by CCK-8. Then, the optimal dosage of bFGF was determined to further identify apoptosis and TRPC1 channel through Western blot. The succeeding passage (P5) from bFGF pre-treated DPSCs was cultivated in bFGF-free culture medium, cellular proliferation and stemness were verified, and pluripotency was analysed by neurogenic, osteogenic and adipogenic differentiation. Results It is found that adding 20 ng/mL bFGF in culture medium could significantly promote the proliferation of freshly thawed DPSCs (P4) through suppressing apoptosis, activating ERK pathway and up-regulating TRPC1. Such proliferative superiority could be inherited to the succeeding passage (P5) from bFGF pre-stimulated DPSCs, meanwhile, stemness and pluripotency have not been compromised. Conclusions This study illustrated a safe and feasible cell culture technique to rapidly amplify post-thawed DPSCs with robust regenerative potency, which brightening the future of stem cells banking and tissue engineering.

摘要: Circular RNAs (circRNAs), a type of non-coding RNA, are single-stranded circularized molecules characterized by high abundance, evolutionary conservation and cell development- and tissue-specific expression. A large body of studies has found that circRNAs exert a wide variety of functions in diverse biological processes, including cell cycle. The cell cycle is controlled by the coordinated activation and deactivation of cell cycle regulators. CircRNAs exert mutifunctional roles by regulating gene expression via various mechanisms. However, the functional relevance of circRNAs and cell cycle regulation largely remains to be elucidated. Herein, we briefly describe the biogenesis and mechanistic models of circRNAs and summarize their functions and mechanisms in the regulation of critical cell cycle modulators, including cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors. Moreover, we highlight the participation of circRNAs in cell cycle-related signalling pathways and the clinical value of circRNAs as promising biomarkers or therapeutic targets in diseases related to cell cycle disorder.