Thiamet-G

As one of the most important posttranslational modifications, glycosylation participates in various cellular activities in organisms and is closely associated with many pathogeneses. It has been reported that glycosylation affects the liver, spinal cord, and heart tissue regeneration. The zebrafish fin has become a valuable model due to its high regenerative capacity. The molecular mechanism of regeneration has been a hot research topic in the field for a long time. However, studies on the influence of glycosylation during limb regeneration in zebrafish are relatively scarce. We discovered that N-acetylglucosamine (O-GlcNAc) expression, identified by WGA, was elevated during the regeneration of the injured fin in zebrafish using lectin microarray. This phenomenon is due to the upregulation of the expression of OGT enzymes and elevated O-GlcNAcylation levels. To investigate the effects on the fin regeneration when O-GlcNAcylation changes, we used OSMI-1 or alloxan unilateral microinjection to decrease O-GlcNAcylation and observed that it prevented the fin regeneration. Conversely, the O-GlcNAcylation was impressed by a unilateral microinjection of thiamet-G or glucose into the fin, leading to a stimulation of the fin regeneration. To further understand the role of O-GlcNAcylation in fin regeneration, liquid chromatography-tandem mass spectrometry technology was performed to identify O-GlcNAc-glycoproteins. The results demonstrated that the O-GlcNAc glycoproteins, such as thrombospondin 4 and heparan sulfate proteoglycans, were involved in the regulation of zebrafish fin regeneration process and were closely associated with certain biological processes, such as stem cell differentiation, extracellular matrix-receptor interaction pathway, tissue remodeling, and so on. We demonstrated that O-GlcNAc glycoproteins are crucial for zebrafish fin regeneration, during which OGT promotes the process by upregulating the O-GlcNAcylation levels in the zebrafish fin.

This study aimed to investigate the effects of O-linked N-acetylglucosamine modification (O-GlcNAcylation) on astroglial-mesenchymal transition through connexin43 (Cx43) pathway under high-glucose conditions. The primary rat astrocytes were cultured under normal and high-glucose conditions, and level of GFAP, α-SMA and Cx43 was investigated. To explore the influence of O-GlcNAcylation on astroglial-mesenchymal transition, Thiamet G treatment was employed to enhance O-GlcNAcylation, while Alloxan was used to decrease it. Cx43 knockdown was acquired through lentivirus constructs to explore its role in astrocyte transition. The levels of GFAP and α-SMA expressions were examined, while astrocyte proliferation was evaluated using the CCK-8 assay, and migration was assessed through wound healing assays. The results showed that primary rat astrocytes were identified by GFAP antibody staining. Under high-glucose conditions, the levels of GFAP, α-SMA, and Cx43 increased, as confirmed by Western blot and immunofluorescence. O-GlcNAcylation augmentation induced by Thiamet G treatment significantly increased the expression of GFAP, α-SMA, and Cx43 compared to both normal and high-glucose conditions. Conversely, the inhibition of O-GlcNAcylation reversed the high-glucose-induced increase in GFAP and α-SMA. Cx43 knockout led to the downregulation of GFAP and α-SMA compared to high-glucose and O-GlcNAcylation-augmented conditions. Additionally, levels of O-GlcNAcylation and VEGF were reduced in Cx43 knockout group. Consistently, CCK8 and wound healing assays demonstrated that Cx43 knockout could inhibit astrocyte proliferation and migration compared to the high-glucose and O-GlcNAcylation augmented groups. These findings demonstrate that astroglial-mesenchymal transition occurs under high-glucose conditions, and can be promoted by O-GlcNAcylation augmentation, but suppressed by Cx43 knockout. The study underscores the significant role of Cx43 in this transition and its potential involvement in diabetic complications.