Hypothesis / aims of study
Nocturia refers to waking up for urination at least once during the night, according to International Continence Society (ICS). Currently, 57.5% of Chinese people over 18 years old have at least one nighttime urination, and 24.7% have at least two [1]. A growing number of studies have found that nocturia is associated with clock genes. Meanwhile, it has been found that in mice with nocturia caused by restraint stress, the expression of clock genes such as Bmal1 increased prematurely in bladder tissue. MicroRNAs are regulator of clock genes, which could be loaded and delivered by extracellular vesicles, and could be regulated by melatonin. In this study, we explored the regulatory effect of melatonin preconditioned urinary stem cell extracellular vesicles (USC-EVs) on clock genes, evaluated its effect on calibrating bladder peripheral clock to improve nocturia.
Study design, materials and methods
USC-EVs were isolated and cultured from the urine of healthy adults and pretreated with melatonin. The expression difference of miRNA was detected through sequencing and rt-qPCR. The distribution of EVs was observed by in vivo imaging and immunofluorescence staining. A mouse model of nocturia was established by restraint stress[2]. Female twelve-week-old C57 mice were divided into three groups: normal group, control group (restraint stress + normal saline), and MT-USC-EVs group (restraint stress + MT-USC-EVs), with three mice in each group. Mice in control and MT-USC-EVs group were administered with saline or MT-USC-EVs single bladder perfusion during light/dark environment alternation on the third day after modeling. The changes of nocturia were evaluated by automatic voided stain on paper technique, and the expression of clock genes in mouse bladder tissues were evaluated by rt-qPCR.
Results
(1) Results of USC-EVs sequencing suggest high expression of miRNAs such as let-7a-3 and miR-4485, and KEGG enrichment analysis suggests a high correlation between these miRNAs and clock regulatory genes.(2) Let-7a and other miRNAs which could target clock genes are up-regulated in USC-EVs pretreated with melatonin.(3) MT-USC-EVs could be taken up by mouse bladder cells by bladder irrigation. 12 hours after the irrigation, in vivo imaging showed that MT-USC-EVs were effectively taken up; 24 hours after co-culturing with DIO-labeled MT-USC-EVs, DIO fluorescence was observed by fluorescence microscopy in nucleus of human bladder epithelial cells, suggesting that MT-USC-EVs could enter the nucleus to exert regulatory effects. (4) The nocturia of mice with MT-USC-EVs bladder irrigation was improved, and the expression of bladder Bmal1 decreased during the inactive(light) phase. Restraint stress caused circadian rhythm disorder and significant increase of urination frequency during the inactive period in mice. After MT-USC-EVs bladder irrigation, the frequency of urination during the inactive period in mice was decreased. Compared with normal mice, Bmal1 expression in bladder epithelial tissue of restraint stress mice were increased during the inactive period, which is similar to existing research. After a single bladder irrigation of MT-USC-EVs, Bmal1 expression during the inactive period tended to be consistent with normal rhythms but increased again during the inactive period 24 hours later.
Interpretation of results
In this study, we found that USC-EVs pretreated with melatonin could regulate the expression of clock gene Bmal1 to reconstruct peripheral clock rhythms in the mouse bladder and improve the symptom of nocturia.