ADH5 K113 Lactylation is Associated with Enhanced Glycolysis and Bladder Epithelial Cell Proliferation in Cystitis...

Cystitis glandularis (CG) is a common inflammatory proliferative disease of the bladder urothelial epithelium. Histologically, it is marked by Brunn 's nest formation and adenoid metaplasia. At present, the molecular mechanism of CG development is not yet clear, and the key signaling pathways regulating the abnormal proliferation of bladder epithelial cells remain to be explored. Lactate, a byproduct of anaerobic glycolysis, serves as a substrate for lactylation, a recently identified post-translational modification, which has been proved to play a regulatory role in inflammatory response and cell proliferation. However, whether this modification occurs in CG bladder epithelial cells and its potential role are still unknown.

Rat CG model induced by lipopolysaccharide (LPS) and SV-HUC-1 human bladder epithelial cell line treated with LPS were used. Lactic acid detection kits were used to evaluate the lactate content in cells, animal tissues and human serum, and the glycolytic ATP yield, oxygen consumption rate and extracellular acidification rate were detected by Seahorse analyzer. Quantitative lactylation omics was used to screen differentially lactylated proteins and sites in bladder epithelial cells of CG rats. Pan-lysine lactylation (Pan Kla) and ADH5-K113 lactate levels were detected by Western blot. The mRNA and protein expressions of PKM2 and LDHA were detected by qPCR and Western blot. The expression of PKM2, LDHA in rat bladder was detected by immunofluorescence. Cell proliferation activity was measured by CCK-8 kit, and cell migration function was evaluated by scratch test. HE staining was used to observe the formation of Brunn 's nest in rat bladder.

Lactate levels in CG rats and LPS-treated SV-HUC-1 cells were significantly increased, glycolytic ATP production rate and extracellular acidification rate were increased, and glycolysis was enhanced. In vitro and in vivo experiments confirmed that the mRNA and protein levels of PKM2 and LDHA were up-regulated. Omics analysis further revealed that there were extensive differential lactylation in the glycolytic pathway, among which the up-regulation of alcohol dehydrogenase 5 lysine 113 lactylation (ADH5-K113la) was the most significant (log2FC = 2.463, FC = 5.52, P = 0.000101). The levels of pan-lysine lactylation and ADH5-K113 lactylation in CG were significantly increased. CCK-8 and scratch experiments confirmed that LPS treatment promoted the proliferation and migration of SV-HUC-1 cells. HE staining showed that the number of Brunn nests in the bladder of CG rats increased.

Lactate accumulation and glycolysis are enhanced in CG, and the level of ADH5-K113 lactylation is significantly up-regulated, which is related to the increase of PKM2, LDHA expression and cell proliferation, suggesting that ADH5-K113 lactylation is associated with enhanced glycolysis and proliferation in CG, suggesting its potential involvement in CG pathogenesis.