ABSTRACT
Objective:to explore the mechanism of modified Tianwang Buxindan in improving abnormal glucose and lipid metabolism in mice with chronic sleep deprivation from the signal pathway of orexin A/ orexin receptor 1（OX1R）. Method:The 50 6-week-old male C57BL/6 mice were randomly divided into blank group ， model group ， estazolam group and Tianwang Buxindan low and high dose groups ，for ten mice of each group. Except the blank group， rats were deprived of sleep for 8 weeks by the method of multi-platform water environment. In the last 4 weeks， Tianwang Buxindan （8.5，17 g·kg-1）and estazolam solution（9.1 mg·kg-1）were given to the stomach， and the blank group and the model group were fed with pure water of the same volume. The food intake and body weight of mice were measured twice a week， on the 49th day， blood samples were collected from the tail vein for glucose tolerance test （GTT），on the 52nd day for insulin tolerance test（ITT）， was used to detect the expression of total cholesterol （TCH）， triglyceride（TG）and free fatty acid（FFA）in serum， and enzyme-linked immunosorbent assay（ELISA）was used to detect the expression of orexin A in serum and hypothalamus. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）and Western blot were used to detect the mRNA and protein expression of OX1R in hypothalamus. Result:After administration， the food intake of mice in each group was different， compared with the blank group， the body weight of model group was significantly reduced（P<0.05）， the glucose tolerance was significantly abnormal， and the TCH， TG， FFA values were significantly increased（P<0.01）. The expression of orexin A in serum and hypothalamus increased significantly（P<0.01）， and the mRNA and protein expression levels of OX1R in hypothalamus increased significantly（P<0.01）. Compared with the model group， the body weight of each group of Tianwang Buxindan was significantly increased（P<0.05）， with better glucose tolerance and insulin sensitivity， TCH， TG， FFA values were significantly reduced（P<0.05，P<0.01）， accompanied by serum and the expression of orexin A in the hypothalamus was significantly decreased（P<0.05，P<0.01）， the mRNA and protein expression levels of OX1R were significantly decreased（P<0.05，P<0.01）. Conclusion:Tianwang Buxindan can protect mice from abnormal glucose and lipid metabolism induced by chronic sleep deprivation， and its mechanism may be related to the down-regulation of orexin A/OX1R signal expression.
ABSTRACT
Objective: To observe the mRNA levels of Orexin and its receptors in the hypothalamus of ovariectomized osteoporosis rats, in order to explore the pathogenesis of postmenopausal osteoporosis(PMOP) and the mechanism Zuoguiwan. Method: An osteoporosis model induced by ovariectomy was established in rats. Totally 32 female Sprague-Dawley (SD) rats were randomly divided into sham-operated group, ovariectomized model group, 17β-estradiol treated positive group, and Zuoguiwan group, with 8 rates in each group. After 12 weeks of intragastric administration, the bone mineral density (BMD) and trabecular microstructural changes of femur were detected by micro-CT (μ-CT), and the morphological changes of bone tissue were observed by hematoxylin-eosin staining (HE) staining. The markers of bone turnover in serum osteocalcin (OCN), N-terminal propeptide of type Ⅰ procollagen (PINP), tartrate-resistant acid phosphatase (TRAP) were measured using enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of orexin, orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) were measured by Real-time PCR. Result: Compared with sham-operated group, the μ-CT showed that BMD, bone volume fraction(BV/TV), trabecular thickness(Tb. Th)and trabecular number(Tb. N)in ovariectomized model group were significantly decreased (PPN-terminal propeptide of type Ⅰ procollagen (PINP) levels decreased, whereas tartrate-resistant acid phosphatase (TRAP) content increased (PPPPPPPPPPConclusion: Decreased mRNA levels of Orexin and its receptors in the hypothalamus may be one of the mechanisms of PMOP. Zuoguiwan may correct the imbalance of bone metabolism, improve the trabecular microstructure and improve bone by up-regulating the mRNA expressions of Orexin and its receptors in the hypothalamus, density, so as to show a therapeutic effect on PMOP.
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Objective To investigate the interference effects of orexin A on cell proliferation of the insulin?secreting beta?cell line(INS?1 cells) through the orexin receptor 1(OX1R)and the AKT/PKB signaling pathway. Methods INS?1 cells were exposed to different concentrations of orexin A in vitro,and treated with OX1R antagonist(SB334867),PI3K antagonist(wortmannin),or AKT antagonist(PF?04691502). The INS?1 cell proliferation and apoptosis,insulin secretion,OX1R protein activity and AKT phosphorylation level were determined. Results Orexin A(10-10 to 10-6 mol/L)stimulated the proliferation and activation of INS?1 cells,prevented apoptpsis,and increased insulin secretion. Additionally,AKT phosphorylation was stimulated by orexin A(10-10 to 10-6 mol/L). The OX1R antagonist SB334867(10-6 mol/L),the PI3K antagonist wortmannin (10-8 mol/L)and the AKT antagonist PF?04691502(10-6 mol/L)weakened the effects of orexin A. Conclusion Orexin A activated the AKT sig?naling pathway through the mediation of orexin A?OX1R,and promoted cell proliferation in INS?1 cells.
ABSTRACT
Objective To investigate changes in the expression of prepro-orexin and orexin receptor-1 ( OX1R) following permanent middle cerebral artery occlusion ( MCAO ) with or without preconditioning through electrical stimulation of the cerebellar fastigial nucleus (FNS). Methods Wistar rats were subjected to permanent MCAO and randomly divided into 5 groups: a sham-operated control group (PO), an FNS preconditioning + shamoperated control group (FNS-PO) , an ischemia group, an FNS preconditioning + ischemia group (FNS-PI) and a cerebellar fastigial nucleus injury + FNS preconditioning + ischemia group (FNL-FNS-PI). Each group was divided into 5 subgroups according to the time at which the animals were sacrificed after the MCAO ( 1, 3, 6, 12 and 24 h).RT-PCR was used to detect expression of OX1R mRNA, and ELISA to measure the levels of orexin-A in the hypothalamus and plasma. Results The immunoreactivity of prepro-orexin decreased significantly in the PI groups, with further decreases over time. At the 12th h after MCAO, the immunoreactivity of prepro-orexin reached a minimum.There were significant differences between the rats in the PO and FNS-PO groups. On the contrary, the immunoreactivity of OX1R increased significantly in the PI groups, with further increases continuing over time, peaking at 12 h after the MCAO. There were significant differences between the PO and FNS-PO groups. In the rats with FNS preconditioning (PI-FNS) , the decrease in prepro-orexin and the increase in OX1R were significantly inhibited compared to the PI subgroups at the 6th and 12th hour. There was no significant difference between the FNL-PIFNS group and the PI group. The expression of OX1R mRNA increased significantly in the PI group, with further increases continuing over time, peaking at 24 hours. The plasma levels of orexin-A were not significantly different among the groups, but the levels of orexin-A in the hypothalamus decreased significantly in the PI and FNL-PI-FNS groups, with further decreases continuing over time. At the 12th h after the MCAO the levels were significantly different compared with the PO and PO-FNS groups. While in the rats with FNS preconditioning (PI-FNS) , the decrease in orexin-A level was reversed and there was no significant difference compared with PO and PO-FNS groups. Conclusions The orexinergic system is altered following cerebral ischaemia. FNS preconditioning may be able to regulate these changes.