Effect And Mechanism Of PDE5 Inhibitor TF Regulating Circadian Rhythm Disorder

Background:The circadian rhythm refers to a circadian rhythm in which life activities exhibit a cycle of approximately 24 hours.The normal circadian rhythm can maintain a variety of physiological functions of the body and adjust the body to respond to changes in the external environment.The circadian rhythm is maintained and regulated by the body's internal factors(such as hormone secretion,disease influence,etc.)and external factors(such as light conditions,temperature changes,etc.).The circadian clock is mainly produced by the negative feedback regulation loop of transcription and translation composed of circadian clock genes.When the body's internal source factors or external environmental factors suddenly change,such as changes in external light conditions caused by cross-time zone flying and shift work,the balance of the biological clock will be broken at this time,and the circadian rhythm will be disturbed.Biological clock phase imbalance caused by shift work and cross-time zone flight is the most important cause of biological clock-related diseases.Therefore,the phase shift adjustment of the biological clock is an important means to treat diseases related to phase imbalance,so it is very important to research and explore new drugs that specifically adjust the phase of the biological clock.In addition,hypoxia is an important factor leading to circadian rhythm disorders.The key genes of the biological clock system and oxygen sensing pathways in mammals are members of the PAS family,so hypoxia is closely related to the biological clock.Therefore,studying the characteristics of the biological clock changes under high altitude hypoxic environment and developing effective intervention therapy drugs are of great significance for improving the military operations capabilities of officers and soldiers in hypoxic environments.Phosphodiesterase 5(PDE5)inhibitors regulate cGMP levels by inhibiting the activity of PDE in the body.It has been reported in the literature that the PDE5 inhibitor sildenafil promotes the phase shift of the circadian rhythm in mice by regulating the suprachiasmatic nucleus(SCN)circadian clock gene^[1].TF is a new type of PDE5inhibitor independently researched and developed in my country,belonging to a category 1.1 new drug.Compared with Sildenafil,it has the advantages of higher selectivity and longer half-life.In terms of circadian rhythm adjustment,whether TF is superior to sildenafil is currently unclear.As one of the most important metabolic organs of the body,the liver has its own oscillating regularity.It can respond to the regulation of SCN and maintain a stable circadian rhythm together with other peripheral organs.Therefore,whether TF can function by regulating the liver biological clock genes has not been reported.In addition,because PDE5 inhibitors have significant anti-hypoxia effects,whether they can alleviate circadian rhythm disorders under hypoxic conditions has not been reported in the relevant literature.Objective:This topic studies the effects of drugs on the peripheral circadian clock circadian clock organ liver in both in vivo and in vitro experiments under normoxia and hypoxia conditions,and explores related mechanisms.It is hoped to provide new ideas for the treatment of circadian rhythm disorders caused by shift work,flying across time zones,and circadian rhythm disorders in special environments such as high altitude low pressure and hypoxia.Methods and Results:1.Under normoxic conditions,firstly,at the cellular level,Lumicycle equipment is used to dynamically monitor the effects of PDE5 inhibitors on the main characteristics of the human-derived osteosarcoma cell line(U2OS)biological clock cycle,amplitude,and phase shift.The results show that PDE5 inhibitors has no effect on the cycle,phase,and amplitude of the biological rhythm of C26(Bmal1::luc U2OS)cells,but has a phase shift of about 3.6 h on D15(Per2::luc U2OS)cells.2.Through the mouse running wheel behavior experiment,the day and night light time was controlled to maintain the 12h/12h light and dark circadian rhythm in the C57BL/6J mice.After the mice formed a stable rhythm,the light was turned on 8h in advance,and the activity of the mice was recorded.To reflect the daily phase shift of each group of mice and the time required for each group to synchronize to the new rhythm cycle,the experimental results show that compared with the control group,PDE5 inhibitors can promote the circadian phase of mice move forward,and TF has a better promoting effect than sildenafil.3.At the molecular level,consistent with behavioral conditions,after the mice have formed a stable rhythm,the time to turn on the lights is advanced 8 hours,and the PDE5 inhibitor is administered at the same time.Use qPCR and WB to reflect the result of the effects of PDE5 inhibitors on mouse liver circadian clock genes,compared with the control group,PDE5 inhibitors can significantly up-regulate the m RNA and protein expression levels of the circadian clock positive regulators Bmal1,Naps2,Clock,and Ror?in the mouse liver.Regulatory factors Per2 and Rev-erb?m RNA and protein expression levels were significantly down-regulated.4.Under hypoxic conditions,firstly give proline hydroxylase inhibitor DMOG at the cellular level to simulate the hypoxic environment,and study the effects of PDE5inhibitor on the biological clock cycle,amplitude and phase shift of U2OS cells under hypoxic environment.The influence of characteristics.The results showed that hypoxia can significantly reduce the amplitude of cells(by about 87.5%)and cause cell death.PDE5 inhibitors can restore the amplitude of the cell's circadian clock and prolong the time of death,but cannot prevent cell death.5.In terms of molecular mechanism,qPCR was used to detect the effect of PDE5inhibitors on cellular clock genes under hypoxic conditions.The results showed that hypoxia can lead to high expression of negative regulators of the biological clock PER1,PER2,CRY1,CRY2,and PDE5 inhibitors can be significant reduce the expression of these genes.6.The mice were exposed to hypoxia(ZT0-ZT8)for 8 hours by simulating the high altitude hypoxia environment(altitude 6000m).The qPCR results showed that the expression of the biological clock genes Per1 and Per2 in the liver of mice significantly increased after 8 hours of hypoxia,the mouse lung tissue circadian clock gene cry1 is highly expressed,Per1,Per2,Cry2 and Rev-erb?are low expressed.7.Perform an 8-hour hypoxic administration experiment on mice.According to the different administration time of PDE5 inhibitor,there are two models:hypoxia 8 hours after drug administration during the day(ZT6-ZT14)and hypoxia 8 hours after drug administration at night(ZT18-ZT4).The results of hypoxia for 8 hours after daytime administration showed that hypoxia can increase the expression of Cry2 m RNA in the liver,and PDE5 inhibitors can significantly reduce the expression of Cry2.The qPCR results of the 8-hour hypoxic administration experiment at night showed that hypoxia can increase the expression of Per1 and Cry2 genes in the liver,and decrease the expression of Cry1.PDE5 inhibitors can significantly reduce the expression of Per1(compared with the hypoxia group).And there is a decreasing trend in Cry2 without any difference.Conclusions:The above conclusions suggest that PDE5 inhibitors can promote the phase shift of circadian rhythm under normoxia conditions,and can regulate circadian rhythm disorders caused by hypoxia under hypoxia conditions.For the special needs of the current market and the military to regulate circadian rhythm disorders,TF,as a low-toxicity 1.1 new drug independently developed by my country,has a good development prospect in the prevention and treatment of circadian rhythm disorders.