Based On The Circadian Clock Protein CRY Associated Inflammatory Signaling To Explore The Common Molecular Mechanism Of Jiao-tai-wan In Improving Sleep And Insulin Resistance

SECTION I The effects of Jiao-tai-wan on sleep structure of obesity-resistant rats with chronic partial sleep deprivationObjectiveThe present study aimed to selectively breed and screen obesity-resistant（OR）rats as the basic rat model for chronic partial sleep deprivation,and to figure out the effect of Jiao-tai-wan on sleep structure of obesity-resistant rats with chronic partial sleep deprivation.MethodsJiao-tai-wan formula was prepared and the fingerprint of the granule was analyzed using（High Performance Liquid Chromatography,HPLC）method.After a total of 200male Sprague-Dawley rats were fed with high-fat and high-energy diet for 2 weeks,80 of them with the lowest body weight increase rate（the lowest 40%of total rats）were selected as obesity resistant（OR）rats.Among them 30 OR rats received brain electrodes implantation one week before chronic sleep deprivation.The other 50 OR rats were left for further use in section II and section III.The 30 OR rats were randomly divided into normal group,model group,low dose of Jiao-tai-wan group,high dose of Jiao-tai-wan group and Estazolam group.Except for those of normal group,the rats were subjected to noise interference for chronic partial sleep deprivation.Meanwhile,during the whole PSD period,drugs（JTW or Estazolam）were orally given to the rats separately in the treating groups.After 4 weeks,the EEGs of OR rats from each group were collected for sleep structure analysis.ResultsThe main constituents of JTW were berberine hydrochloride,cinnamon acid,cinnamaldehyde,jatrorrhizine hydrochloride,coptisine hydrochloride and palmatine chloride.Compared with normal group,total sleep time（TST）,total slow wave time(SWS_total),Slow-wave sleep stage I time（SWS₁）and Slow-wave sleep stage II time（SWS₂）were decreased in rats of model group（P<0.01）.Compared with model group,JTW（both JTW1 and JTW2）and Estazolam treatment dramatically increased TST and SWS_totalotal in OR rats with PSD（P<0.01）,especially SWS₁（P<0.01,P<0.01 and P<0.05）.JTW slightly increased the SWS₂ time compared with the model group but there was no statistical difference.ConclusionsJiao-tai-wan has a obvious effect of improving the sleep of obesity-resistant rats with chronic partial sleep deprivation.SECTION II The effects of Jiao-tai-wan on intestinal mucosal damage in obesity-resistant rats with chronic partial sleep deprivationObjectiveInadequate sleep can lead to intestinal damage.The present study aimed to explore the effect and possible mechanism of JTW on intestinal damage in rats with chronic partial sleep deprivation.MethodsOR rats（from section I）were randomly divided into normal group,model group,low dose of JTW group,high dose of JTW group,and Estazolam group.PSD rat models were established by environmental noise interference from10:00 to 14:00,every day for 4 weeks.At the same time of PSD,JTW of different doses and Estazolam were orally given to the rats separately in the treating groups.After 4 weeks,plasma and small intestine tissues were collected.Concentrations of LPS in plasma were examined.Morphological changes of intestinal epithelium were observed by HE-staining.The protein expression of Occludin（Ocln）in the intestine was measured by immunofluorescence technique and Western blot methods.The expressions of circadian proteins CRY1 and CRY2 in the intestine were also determined by Western blot method.ResultsCompared with normal group,LPS level was significantly increased in rats of model group（P<0.05）.Meanwhile,rats of model group had intestinal damage,with the menifastations of shorter,sparse and incomplete villus,wide gap between the villus,mucosal swelling and congesting,with decreased expressions of Occludin（P<0.01）,CRY1 and CRY2 proteins in intestine（P<0.01）.Compared with model group,LPS level was significantly decreased in high dose of JTW group rats（P<0.05）.Intestinal damage in rats of two JTW groups was attenuated compared with model group,and the expressions of Ocln protein,circadian proteins CRY1 and CRY2 in the intestine were increased at the same time（P<0.01）.ConclusionsJTW has the beneficial effect on improving intestinal damage in rats with chronic partial sleep deprivation.The mechanism appears to be related to the up-regulation of the expressions of circadian proteins CRY1 and CRY2,as well as Occludin protein in the intestine,thereby attenuating the inflammation caused by intestinal damage.SECTION III Based on the circadian clock protein CRY associated inflammatory signaling to explore the common molecular mechanism of Jiao-tai-wan in improving sleep and insulin resistance in rats with chronic partial sleep deprivationObjectiveThe present study aimed to explore the common molecular mechanism of Jiao-tai-wan in improving sleep and insulin resistance in rats with chronic partial sleep deprivation based on the circadian clock protein CRY associated inflammatory signaling.MethodsOR rats were randomly divided into normal group,model group,low dose JTW group,high dose of JTW group,and Estazolam group.PSD rat models were established by environmental noise interference from10:00 to 14:00,every day for 4 weeks.At the same time of PSD,JTW of different doses and Estazolam were orally given to the rats separately in the treating groups.After 4 weeks,plasma and tissues including hypothalamus,liver and adipose were collected.The amount of food intake and metabolic parameters such as body weight increase rate,fasting plasma glucose（FPG）,fasting insulin（FINS）and homeostasis model assessment-insulin resistance（HOMA-IR）were measured.Inflammatory markers including interleukin-6（IL-6）,interleukin-1β（IL-1β）,high-sensitivity C reactive protein（hs-CRP）and tumor necrosis factor-α（TNF-α）levels were examined in the rat plasma using ELISA method.The m RNA expressions of IL-1β,IL-6 and TNF-α in hypothalamus,liver and adipose tissues were measured using Real-time PCR method.We used Western Blot method to determine the activity of nuclear factor kappa B（NF-k B p65）protein,the expressions of insulin PI3K/AKT signaling pathway proteins such as insulin receptor（Ins R）,phosphoinositide 3-kinase（PI3K）,the serine/threonine kinase AKT,phosphorylated AKT（p-AKT）,and glucose transporters,and the expressions of circadian proteins CRY1 and CRY2 in the hypothalamus,adipose and liver tissues.Additionally,cyclic adenosine 3’,5’-monophosphate（c AMP）level and activity of vasodilator-stimulated phosphoprotein（VASP）in hypothalamus tissue were also determined using Western Blot method.ResultsCompared with normal group,rats in model group had higher amount of food intake and body weight increased rate（P<0.01,P<0.05）;FPG,FINS and HOMA-IR levels were also increased（P<0.01）;the levels of plasma inflammatory markers were significantly increased（P<0.01）.Compared with model group,the amount of food intake and body weight increased rate were significantly decreased（JTW1 P<0.01,JTW2 P<0.05）;FPG,FINS and HOMA-IR levels were significantly decreased in JTW2 group（P<0.01）and the levels of plasma inflammatory markers were also decreased（P<0.01）.Meanwhile,the activation of NF-k B and m RNA expressions of inflammatory markers in hypothalamus,liver and adipose tissues was significantly decreased（P<0.01）.In high dose of JTW group,PI3 K expression,AKT activation and GLUT3 expression in hypothalamus tissue were significantly increased（P<0.05,P<0.01）;Ins R expression,PI3 K expression,AKT activation and GLUT4 expression in liver tissue were significantly increased（P<0.05,P<0.01）;Ins R expression,PI3 K expression,AKT activation and GLUT2 expression in adipose tissue were increased as well（P<0.05,P<0.01）.The expressions of proteins CRY1 and CRY2 were increased in high dose of JTW group compared with model group（P<0.05,P<0.01）.Meanwhile,the c AMP/PKA signaling in hypothalamus tissue was also inhibited in high dose of JTW group rats（P<0.01）.ConclusionsJTW improves sleep and ameliorates insulin resistance in OR rats with PSD.The mechanism may be related to the up-regulation of the expressions of circadian proteins CRY1 and CRY2,which inhibits the downstream inflammatory pathways of c AMP/PKA and NF-k B,and then improves the defect of PI3K/AKT signaling pathway of insulin signaling transduction.