Effect of circadian rhythm changes on the expression of RORs and the RORs agonist SR1078 on corneal epithelial wound repair

Objective

To investigate the effect of circadian rhythm changes on the expression of retinoic acid-related orphan receptors (RORs) and the RORs agonist SR1078 on corneal epithelial wound repair.

Methods

A total of 228 SPF C57BL/6 female mice aged 6-8 weeks old were selected, and 180 mice were divided into the normal circadian rhythm group, full-day group, full-night group, 12-hour reversed circadian rhythm group and 3-week reversed circadian rhythm group, with 36 mice in each group.The remaining 48 mice were randomly divided into phosphate buffered saline (PBS) control group and SR1078 group by random number table method, with 24 mice in each group.According to grouping, the mice were placed in a light box where the light (light intensity of 300 lx) and dark time could be controlled.The light time of the normal circadian rhythm group, the PBS control group and the SR1078 group in the light box was from 7: 00 to 19: 00, and the dark time was from 19: 00 to 7: 00 the next day.According to the Zeitgeber Time method, the starting time of light at 7: 00 was recorded as ZT0, and the time of closing light at 19: 00 was recorded as ZT12.Real-time fluorescence quantitative polymerase chain reaction was used to detect the relative expression levels of RORα and RORγ mRNA at ZT1, ZT5, ZT9, ZT13, ZT17, ZT21 in the five groups.In the PBS control group and SR1078 group, a golf-like knife was used to establish the mouse corneal epithelial injury model, and the model eyes were administered with drugs once every 6 hours according to the grouping.The corneal epithelial defect area was measured with Adobe Photoshop CC2019 software, and the corneal epithelial defect rate was calculated and compared between the two groups.The correlation between the relative expression levels of RORα and RORγ mRNA in mice corneal epithelium of the five groups and corneal epithelial defect rate in the PBS control group and SR1078 group was analyzed.The corneal epithelium repair was observed by whole cornea spreading and immunofluorescence staining, and the number of corneal epithelial dividing cells in the PBS control group and the SR1078 group was calculated and compared.The use and care of animals complied with the ARVO statement.This study protocol was approved by the Laboratory Animal Ethics Committee of Jinan University (No.JN-A-2002-01).

Results

Compared with the normal circadian rhythm group, the relative expression levels of RORα/RORγ mRNA in the full-day group, full-night group, 12-hour reversed cirdian rhythym group and 3-week reversed cirdian rhythym group showed an overall decreasing trend.There was a statistically significant difference in the corneal epithelial defect rate between the PBS control group and the SR1078 group at different time points after modeling (F_group=74.01, P<0.001; F_time=5 171.48, P<0.001). Twelve hours after modeling, the corneal epithelial defect rate in the SR1078 group was significantly lower than that in the PBS control group, and the difference was statistically significant (P<0.05). The relative expression levels of RORα and RORγ mRNA in corneal tissue was moderately positively correlated with the corneal epithelial defect rate in mice (r=0.614, 0.537; both at P<0.01); The regression equation of the straight line between the relative expression level of RORα mRNA and the change in corneal epithelial defect rate was Y=33.153X-43.052 (F=20.58, P<0.001), and the linear regression equation between the relative expression level of RORγ mRNA and the change of corneal epithelial defect rate was Y=2.764X-1.364 (F=13.11, P<0.001). There was a significant overall difference in the number of corneal epithelial dividing cells at various time points following modeling between the PBS control group and the SR1078 group (F_group=160.55, P<0.001; F_time=83.57, P<0.001). The number of dividing cells in the SR1078 group was significantly less than that in the PBS control group at 24, 30, and 36 hours following modeling, and the differences were statistically significant (all at P<0.05).

Conclusions

Circadian rhythm changes reduce the expression of RORα and RORγ mRNA in the mouse cornea.SR1078 can promote the expression of RORα and RORγ mRNA in corneal epithelium to decrease the number of mouse corneal epithelial dividing cells, and inhibit the repair after corneal trauma.

Cornea; Circadian rhythm; Wound healing; SR1078; Clock gene