The Effect Of Constant Light Exposure On POMC-induced Skeletal-muscle Wasting In Septic Rats

Objective:Constant light exposure is common in intensive care units(ICUs)and is always ignored by clinical staff.In normal organisms,excessive light exposure could lead to metabolism problems such as insulin resistance and impaired glucose tolerance.Sepsis is a common complication in the ICU,and it typically exhibits hypercatabolism characterized by skeletal muscle wasting.Severe skeletal muscle atrophy and malnutrition are leading causes of high mortality and poor prognosis in patients with sepsis.So,would constant light exposure further aggravate the metabolic disorders in sepsis patients?Light is an important timing factor for circadian rhythm and could regulate the expression of clock genes in the suprachiasmatic nucleus(SCN),which is the center of circadian rhythm system.And the activation of proopiomelanocortin(POMC)neurons in the hypothalamic arcuate nucleus(ARC)is considered to be a key to regulating the metabolism of skeletal muscle in sepsis.The deacetylase SIRT1 can participate in the regulation of the circadian rhythm in the SCN,and it also plays a role in the POMC neurons of hypothalamic ARC based on its nutrient sensitivity.Therefore,this study fouced on the LPS-induced septic rats and study on the effects of constant light exposure on hypothalamic clock gene CLOCK,POMC neuropeptide and skeletal muscle wasting in septic rats,explore the mechanism of constant light exposure through CLOCKSIRT1 on POMC-induced skeletal muscle wasting in sepsis by interventing the expression of SIRT1 and POMC and determine if the exogenous melatonin supplement could improve muscle wasting via the pathway.This study aims to explore the effect of constant light exposure on skeletal muscle wasting in sepsis,which should be paid dequate attention to.What’s more,it may provide new ideas and experimental basis for melatonin on regulating circadian rhythm and alleviating skeletal muscle wasting in sepsis with constant light exposure in the clinical.Methods:1.Correlation among constant light exposure,hypothalamic POMC neuropeptide expression and skeletal muscle wasting in rats with sepsisFifty-four adult male Sprague-Dawley rats(220±20g)adapted to the environment for a week were randomLy divided into three groups:CON-LD group,LPS-LD group and LPS-LL group.All animals were housed with a single cage and exposed to 200 lux light through incandescent lamps for 7 days.Rats in the CON-LD group and LPSLD group maintained a 12h/12h light-dark cycle,i.e.turning on the light at 8:00 am(defined as ZT 0)and turning off the light at 8:00 pm(defined as ZT 12).LPS-LL group maintains constant light illumination for 7 days.Rats in the LPS-LD group and the LPS-LL group were given intraperitoneal injection of LPS(5 mg/kg on the first day and 2 mg/kg next six days)as a chronic sepsis model and rats in the CON-LD group were given equivalent normal saline.Body weight changes and food intake were measured every 24 hours.On the 8th day,at the 6 time points of ZT 0,ZT 4,ZT 8,ZT 12,ZT 16 and ZT 20,3 rats were randomLy selected from every group and sacrificed.The bilateral extensor digitorum longus were weighed and the level of skeletal muscle wasting was measured,as the level of 3-methylhistidine(3-MH),tyrosine and the expression of two muscle atrophy genes MuRF1 and MAFbx.Hypothalamus was collected for the measurement of the expression of the clock genes,SIRT1,POMC and the expression of the hypothalamic inflammatory factors IL-1β and TNF-α.2.Constant light exposure affects skeletal muscle wasting in septic rats through CLOCK-SIRT1-POMC pathwayNinty adult male Sprague-Dawley rats(220±20g)adapted to the environment for a week,and the third ventricle catheterization was performed.After 3 days were given intraperitoneal injection of LPS(method ibid)and randomLy divided into five groups:LPS-LL+EX527 group,LPS-LL+RES group,LPS-LL+siRNA group,LPSLL group and LPS-LD group.SIRT1 specific inhibitor EX-527,SIRT1 specific activator resveratrol(RES)and lentiviral vector containing siRNA against POMC were respectively injected into the first three groups through the third ventricle cannulation at 8:00 a.m.everyday while the later two groups with equal artificial cerebrospinal fluid.Rats in the four groups would suffer from constant light exposure of 200 lux for 7 days without a break.LPS-LD group was also under light of 200 lux for 7 days,which maintains a 12h/12h light-dark cycle.On the 8th day,samples were obtained as described above.3.Effects of melatonin treatment on circadian rhythm and skeletal muscle wasting in septic rats with constant light exposureSeventy-two adult male Sprague-Dawley rats(220±20g)adapted to the environment for a week were randomLy divided into four groups:CON-LD group,LPS-LD group,LPS-LL group and LPS-LL+MT group.Every rat was housed in a single cage and exposed to 200 lux light from incandescent lamps for 7 days.CONLD group and LPS-LD group maintained a 12h/12h light-dark cycle,while rats in LPS-LL group and LPS-LL+MT group maintained constant light exposure for 7 days without a break.Rats in LPS-LD group,LPS-LL group and LPS-LL+MT group were given intraperitoneal injection of LPS(method ibid)and CON-LD group were given equivalent normal saline.Rats in the LPS-LL+MT group accepted hypodermic injection with melatonin(0.2 mg/kg)at 7:00 p.m.everyday.On the 8th day,samples were obtained as described above.Results:1.Correlation among constant light exposure,hypothalamic POMC neuropeptide expression and skeletal muscle wasting in rats with sepsisCompared with the CON-LD group,intraperitoneal injection of LPS caused significant skeletal muscle wasting while a decrease in SIRT1 levels and a significant increase in POMC neuropeptide expression were observed in the hypothalamus.Compared with the LPS-LD group,constant light exposure could cause abnormal rhythm of CLOCK and BMAL1 expression in the hypothalamus,as well as decreased SIRT1 expression and elevated levels of POMC neuropeptides in the hypothalamus and aggravated the hypercatabolism of skeletal muscle.2.Constant light exposure affects skeletal muscle wasting in septic rats through CLOCK-SIRT1-POMC pathwayCompared with the LPS-LD group,constant light exposure caused an arrhythmia of CLOCK expression,decreased the level of SIRT1 level and increased POMC expression in the hypothalamus.Compared with LPS-LL group,the expression of SIRT1 in the hypothalamus was little and the level of POMC neuropeptide was significantly increased in LPS-LL+EX527 group,and the skeletal muscle wasting was aggravated.The results were just the reverse in the LPS-LL+RES group.In LPSLL+siRNA group,the expression of POMC was significantly inhibited,the skeletal muscle wasting was alleviated,but the level of SIRT1 has no significant change.What’s more,the expression of CLOCK in the hypothalamus had no circadian rhythm in the four groups with constant light exposure.3.Effects of melatonin treatment on circadian rhythm and skeletal muscle wasting in septic rats with constant light exposureCompared with the LPS-LD group,constant light exposure could significantly decrease the serum melatonin level,while an arrhythmia of hypothalamic CLOCK expression,reduced SIRT1,increased POMC and aggravated muscle wasting were also observed in the LPS-LL group.Compared with the LPS-LL group,melatonin supplement could increase the level of serum melatonin and help to recover the circadian rhythm of CLOCK.Meanwhile,the expression of SIRT1 was increased,the activation of POMC was reduce and skeletal muscle wasting was alleviated in the LPS-LL+MT group.Conclusion:1.Constant light exposure could aggravate skeletal muscle wasting in septic rats,which is associated with the circadian rhythm of hypothalamic clock gene expression,SIRT1 and POMC neuropeptide.2.In septic rats,constant light exposure may affect skeletal muscle wasting through the hypothalamic CLOCK-SIRT1-POMC pathway.Constant light exposure could cause an arrhythmia of CLOCK clock gene expression in the hypothalamic SCN region followed with decreased expression of SIRT1 in the hypothalamus and the activation of POMC neurons,and ultimately aggravates skeletal muscle wasting.3.Constant light exposure could reduce serum melatonin levels in sepsis rats.Exogenous melatonin supplementation can effectively alleviate skeletal muscle wasting in sepsis rats with constant light exposure.And this process is associated with the expression of hypothalamic clock genes CLOCK,SIRT1 and POMC,but the exact mechanism is not very clear and needs more investigations.