吴茱萸碱对大鼠摄食及下丘脑摄食肽表达的影响

Intragastric Administration of Evodiamine Reduces NPY and AGRP Gene Expression in The Hypothalamus andDecreases Food Intake in Rats1Shi Jinghong，Yan Jianqun *，Lei Qi，Zhao Junjie，Chen Ke，Yang Dejun，Zhao XiaolinDepartment of Physiology and Pathophysiology，Key Laboratory of Environment and Genes Related to Diseases，Ministry of Education，Xi’an Jiaotong University School of Medicine，Xi’an（710061）E-mail：jqyan@AbstractEvodiamine (Evo), an alkaloidal component extracted from the fruit of Evodiae fructus (Evodia rutaecarpa Benth., Rutaceae), decreases the body weight of rats through a poorly defined mechanism. The hypothalamus is responsible for controlling food intake and energy expenditure. We postulate that Evo mediates this activity by modulating feeding-related peptides in the hypothalamus. We investigated the effects of Evo on food intake, body weight and the mRNA expression of hypothalamic NPY, AGRP, Pmch, POMC, CART, and MC4R, in male rats. Evo (40 mg/kg or 4 mg/kg) was intragastrically administrated for 25 days, and the food intake and body weight of rats were recorded daily. Blood samples were collected for leptin radioimmunoassay (RIA). Real-Time PCR was used to analyze the mRNA expression. Our results show that intragastric administration of Evo (40 mg/kg) inhibits food intake, decreases body weight and reduces the mRNA expression of NPY and AGRP in the hypothalamus, while it increases the circulating level of leptin. Our results indicate that Evo decreases food intake, and therefore body weight, partly by down-regulating NPY and AGRP mRNA expression in the hypothalamus.Keywords：evodiamine，leptin，NPY，AGRP，food intake1. IntroductionEvodiamine (Evo) is a major alkaloidal component of the dried, unripe fruit of Evodia rutaecarpa Bentham (Rutaceae), and is an agonist of vanilloid receptor1 (VR1) and has no perceivable taste (including a peppery hot taste). Previous investigations have indicated that Evo has a number of pharmacological actions such as decreasing body weight, regulating body temperature, anti-tumor activity, pain relief, heart protection, decreasing blood pressure and inducing endocrine effects [1-6]. Previous study demonstrated that Evo exhibits Capsaicin-like anti-obese actions which may stimulate VR1 in the primary sensory neurons. Evo significantly decreased the body weight, perirenal fat weight and epididymal fat weight of rats on a high-fat diet. Also, the level of serum free fatty acid, total lipids, triglyceride, and cholesterol in the liver were significantly reduced as compared with the control diet group. Furthermore, both lipolytic activity in the perirenal fat tissue and specific GDP binding in brown adipose tissue mitochondria, as the biological index of enhanced heat production, were significantly increased in the Evo fed rats. The researchers concluded that Evo exerts anti-obesity effects by increasing energy expenditure and preventing the accumulation of perivisceral fat. They also thought that the anti-obesity effects of Evo may depend on the enhancement of UCP1 thermogenesis [1].Wang et al reported recently that dietary supplementation with Evo could ameliorate diet-induced obesity in mice and inhibit adipogenesis. They indicated that Evo inhibits adipogenesis by stimulating ERK/MAPK signaling which down-regulates the expression of adipocyte transcription factors and insulin-induced Akt signaling. Because Evo clearly showed anti-obesity effects in UCP1-deficient mice, this suggests that Evo triggered an UCP1-independent mechanism to prevent diet-induced obesity. Evo1This project was supported by the grants from National Natural Science Foundation of China (No.30670691) and from Ministry of Education of China (No.20060698046)。

also improve leptin resistance and insulin sensitivity in the mice [2]. Evo increased the plasma concentration of CCK in a dose-dependent manner and inhibited both gastric emptying and gastrointestinal transit [7]. CCK is obviously implicated as a factor regulating feeding and obesity. The mechanism involved in the process is not clear yet and needs further investigation.VR1, or the transient receptor potential vanilloid subtype 1 channel (TRPV1), was the first member characterized of a family of six vanilloid receptors named TRPV1–6, which are themselves part of a larger superfamily of calcium permeable cation channels. VR1 is not only expressed in primary sensory neurons, but has rather widespread distribution in mouse, rat and human brain tissue. Mezey et al observed VR1-expressing neurons throughout the neuroaxis in the rat, including several areas of the hypothalamus. There were immunopositive cells in the suprachiasmatic nucleus, numerous weakly immunostained cells in anterior hypothalamic nuclei, the paraventricular nuclei among the magnocellular hypothalamic neurons, the dorsomedial hypothalamic nucleus, and also the arcuate nuclei [8]. Jennifer et al reported that VR1 expression was densely localized in the arcuate, the dorsal medial hypothalamic nucleus, periventricular and ventromedial hypothalamic nuclei in the mouse. In contrast, there was very little binding in the paraventricular hypothalamic nucleus [9]. Some studies indicated that capsaicin has a number of pharmacological actions in the brain. In the hypothalamus, the capsaicin-sensitive neurons are thought to carry 5-hydroxytryptamine receptors [10]. Capsaicin was shown to evoke glutamate release from slices of hypothalamus [11] and deplete the β-endorphin content of the hypothalamus [12], an effect which might be mediated through the VR1 receptors in the arcuate nucleus. Evo has some pharmacological effects same as those of capsaicin. However, the effects of Evo in the brain, especially in the hypothalamus, have not yet been reported. The expression area for VR1 and NPY, AGRP peptides are similarly widespread and very similar in their distribution in the hypothalamus. We postulated that Evo not only acts on peripheral sensory neurons, but also on the neural centers.The hypothalamus and its neural circuits play an important role in regulating feeding behavior and responding to signaling in the homeostatic system [13,14]. The hypothalamus is the center responsible for regulating food intake and energy expenditure. Hypothalamic nuclei and other areas, such as the arcuate, ventromedial, dorsomedial, paraventricular nuclei and lateral hypothalamic areas, are associated with regulation of energy balance. The hypothalamus regulates many aspects of energy homeostasis, adjusting both the drive to eat and expenditure of energy in response to a wide range of nutritional and other signals. It is becoming clear that various neural circuits operate to different degrees and probably serve specific functions under particular conditions of altered feeding behavior [14]. Furthermore, long-term energy-related signals (insulin and leptin) and short-term signals (intestinal nutrients) are transmitted to balance and control energy metabolism, food intake and endocrine response.The hypothalamus has numerous neuropeptides regulating appetite [14,15,16] these include; orexinergic factors; NPY, AGRP, orexin, MCH, and ghrelin, and anti-orexinergic factors; leptin, α-MSH, urocortin, and CART. Arcuate nuclei are the most important hypothalamic nuclei regulating food intake and body weight, and involve several neural functional areas that stimulate or inhibit feeding. Williams et al [14] described both neuropeptide Y (NPY) and agouti gene-related protein (AGRP), as potent stimulators of food intake, and are co-localized in a population of neurons in the ARC [17,18]. Meanwhile pro-opiomelanocortin (POMC; the precursor of a-melanocyte-stimulating hormone [a-MSH]) and cocaine- and amphetamine-regulated transcript (CART), which induce an anorexic response, are co-localized in an adjacent subset of ARC neurons [19,20]. Leptin inhibits food intake and increases energy expenditure through interactions with its specific receptors in the hypothalamus. Hypothalamic α-MSH is implicated strongly in the control of food intake and body weight through binding with high affinity to melanocortin receptor-3 (MC3) and MC4 [21,22]. AGRP is a MC4 antagonist and binds with MC4 receptors to compete with α-MSH to enhance the effect ofNPY [23]. The PVN is an integrating center involving many neural pathways that influence energy homeostasis. It is richly supplied by axons projecting from the ARC-NPY/AGRP and POMC/CART neurons and from the orexin neurons of the lateral hypothalamus [24,25]. PVN is very sensitive to feeding-related peptides such as NPY and α-MSH. MCH is mainly expressed in the lateral hypothalamus, and the central administration of MCH may stimulate feeding [26]. MCH over expression in the hypothalamus results in obesity [27]. MCH is an antagonist of melanocortin. MCH and NPY interact with anorexic neural peptides in the hypothalamus [28]. Orexic peptides interact with anorexic peptides to balance energy metabolism.Here, we examined the mRNA expression levels of hypothalamic NPY, AGRP, Pmch, POMC, CART, and MC4R in rats after Evo administration. We wanted to investigate what role the hypothalamus plays in the previously observed anti-obesity effects of Evo.2. Methods and Materials2.1. AnimalsMale Sprague-Dawley rats (70 g) were housed in individual cages with access to water and chow pellets, and maintained at 20–22°C with a dark: light cycle of 12-hours light, 12-hours dark (lights on at 8:00 AM). Animals acclimatized to these conditions and were handled frequently one week before the beginning of the experiment. Animals (n = 9) were randomly divided into three treatment groups; (1) Evo-treated with 40 mg/kg; (2) Evo-treated with 4 mg/kg; (3) control treatment. Evodiamine (Shaanxi Huisheng Medical Technology Co. Ltd, China) was prepared as a suspension using double-distilled water. The food intake and body weight of the rats were recorded daily at 9:00 AM. Intragastric administration of Evo, or water for controls, was performed at 10:00 AM. The intragastric administration was done once a day for 25 days according to the weight of the rats at the solution volume of 1ml/100g. The animals were maintained in accordance with the principles of The Council of Xi’an Jiaotong University Communities.After 25 days of treatment, animals were sacrificed and anesthetized with urethane (1.2 g/kg, ip) between 10:00 AM and 12:00 noon. Next, 4 ml blood was collected from abdominal aorta and allowed to clot on ice. Serum was obtained and stored at -80°C until analysis. The hypothalamus was dissected en bloc under a binocular microscope and stored at -80°C until analysis.2.2. Serum leptin assaySerum leptin was measured by radioimmunoassay (RIA) (rat leptin RIA kit: No. R-28，Beijing Puerweiye BioTech Co. Ltd, China). The sensitivity of the assay was 0.4 ng/ml.2.3. RNA extractionTotal RNA was extracted from the hypothalamus using Trizol reagent (Invitrogen, USA), according to manufacturer’s recommendations. Pellets were suspended in 20–30 µl of DEPC-treated water. The quantity and integrity of isolated RNA was determined for each sample by using UV absorbance (260/280) as well as 1% agarose gel electrophoresis.2.4. Reverse transcription and Real-Time quantitative PCRTotal RNA from the hypothalamus was reverse transcribed using a commercial RT-PCR Kit (AmpliQ, Cat. No.: 260802, Germany). Total RNA from the hypothalamus (2 µg) was reverse transcribed in a final volume of 25 µl. In a sterile RNase-free microcentrifuge tube, we added 2 µg total RNA, 1 µg random hexamer (1 µg/µl) in a total volume of 15 µl of RNase free water. We heated the tube to 70°C for 10 minutes to melt secondary structures within the template. Then the following components were added: 20 units RT Enzyme (20 U/µl), 2.0 µl dNTP mixture (12.5 mM), 0.6 µlRNasin Inhibitor (40 units/µl) and 2.5µl 10× RT Buffer, to a total volume of 25 µl with RNase free water. After incubation (42°C, 45 minutes), the mixture was heated (95°C, 3 min). RT-PCR (AmpliQ, Cat. No.: 260802, Germany) was performed in 25 µl containing 1 µl of the RT reaction, 12.5 µl 2× master mix, 1 µl of each forward and reverse primer, 3.5 µl Green DNA dye I (1:2000) and 6 µl dH2O. The AmpliQ Real Time PCR master mix included TEMPase Hot Start DNA polymerase, a modified Taq DNA polymerase with hot start capabilities. Each forward and reverse primer for hypothalamic neuropeptides and internal are shown in Table 1. Thermal cycling parameters were as follows: 1 cycle of 95°C for 10 minutes followed by 40–45 cycles of 95°C for 30 seconds, 58–60°C for 1 minute, 72°C for 30 seconds, performed on ABI Prism 7000.2.5. Statistical analysisData are presented as the mean ± S.E.M. One-way ANOVA followed by Post Hoc test was used to detect significance among groups. P < 0.05 was considered to be statistically significant. The formula used to calculate the normalized expression of quantitive PCR target genes was as the follows: Normalized expression = (E target)∆Ct target (control – sample) /(E ref)∆Ct ref (control – sample)E target represents the expression of the target gene; E ref the expression of the reference gene ; ∆Ct the difference between the experimental group and the control group; Ct the Cross Threshold. The significance of comparison between the experimental group and the control group is analyzed by one-way ANOV A. The analysis of results of RT-PCR is carried out by randomization test using REST software specially made for quantitive PRC analysis [29]. P < 0.05 was considered to be statistically significant.3.Results3.1.The effect of Evo on food intakeAfter treatment with Evo, the food intake of the rats decreased (Fig. 1). The group treated with Evo 40 mg/kg had a significant decrease in food intake from the 12th day compared with the control. The group treated with Evo 4 mg/kg had a decrease in food intake during the middle and the last medication period but with no significant difference. The result was shown once every three days according to food intake.3.2.The effect of Evo on body weightAfter treatment with Evo, the body weight of the rats decreased (Fig. 2). The group treated with Evo 40 mg/kg had a noticeable fall in the body weight from the 15th day. The group treated with Evo 4 mg/kg had no change in body weights during the early medication period, however as the medication period extended, the body weight of the rats has some decrease but with no significant difference. The result was shown once every five days according to body weight.3.3.The effect of Evo on mRNA expressionThe results are shown in Fig. 3. After treatment with Evo 40 mg/kg, the expression of hypothalamic orexigenic peptides, NPY and AGRP mRNA had a noticeable fall in comparison with the control group (P < 0.05, P < 0.01 respectively). Expression of Pmch mRNA has a slight fall but it was not significant. The expression of anorexigenic peptide POMC mRNA increased and there was no significant difference in comparison with the control group (P > 0.05). The expression of anorexigenic factors CART and MC4R mRNA decreased and there was no significant difference in comparison with the control group (P > 0.05).After treatment with Evo 4 mg/kg, the expression of POMC mRNA increased remarkably in comparison with the control group (P < 0.05). The expression of hypothalamic NPY, AGRP and Pmch mRNA decreases and there was no remarkable difference in comparison with the control group (P >0.05). The expression of hypothalamic CART and MC4R mRNA dropped and there was no remarkable difference in comparison with the control group (P > 0.05).3.4.The effect of Evo on serum leptin levelsAfter treatment with Evo 40 mg/kg, the serum leptin level of the rats increased dramatically in comparison with the control group (P < 0.05) and the group treated with 4 mg/kg (P < 0.01). The administration of Evo 4 mg/kg down-regulated the serum leptin levels and indicated no significant difference compared with the control group (P > 0.05). The result is illustrated in Fig. 4.4.DiscussionEvodiamine (Evo) is an alkaloidal component extracted from the fruit of Evodiae fructus (Evodia rutaecarpa Benth., Rutaceae). Evo decreases body weight through a poorly defined mechanism. The hypothalamus is the center that controls food intake and energy expenditure. It is reasonable to postulate that the hypothalamus is the main site through which Evo mediates its body weight decreasing activity.Our results showed that treatment with Evo 40 mg/kg significantly reduced food intake, body weight and gene expression of NPY and AGRP in the hypothalamus, while it significantly increased the circulating leptin level compared with the control group. In the Evo 40mg/kg group, food intake significantly reduced from the 12th day and body weight significantly decreased from the 15th day. Evo suppressing hypothalamic NPY and AGRP mRNA expression plays a key role in inhibiting food intake. Although Evo up-regulated POMC mRNA and down-regulated CART and MC4R mRNA, there was no significant difference compared with the control group. The anorexic effect is stronger than the orexic effect when they interact with each other. In the end, food intake was reduced and therefore body weight decreased. This correlates with previous investigations [32,33,34].Treatment with Evo 4mg/kg significantly increased the gene expression of POMC in the hypothalamus. NPY and AGRP mRNA expression decreased but there was no significant difference compared with the control group. Other anorexic factors, CART and MC4R mRNA expression and the circulating leptin level, decreased without significant difference. We thought that the effects of up-regulated POMC mRNA might have been counteracted with other orexic peptides. The decreased serum leptin level did not efficiently depress the hypothalamic mRNA expression of NPY and AGRP. The interaction between orexic peptides and anorexic peptides did not affect food intake and body weight. Therefore, we indicated that Evo must have some dosage-pharmacodynamic action in regulating feeding. The dosage and time course of Evo in decreasing body weight needs further investigation. Evo significantly reduced gene expression of NPY and AGRP in the hypothalamus, while significantly increasing the circulating leptin level. Leptin restrains the ARC-NPY/AGRP neurons which are consistent with known roles of leptin [35,42].It has been demonstrated that NPY plays a pivotal role in the control of food intake and body weight within the hypothalamus. NPY is a potent orexigenic factor which stimulates feeding and reduces energy expenditure. Infusion of NPY into the brain of satiated rats may elicit a ravenous food intake and repeated infusions may lead to obesity [30,31]. Therefore, NPY over expression results in obesity. Knocking out NPY gene in ob/ob mice effectively inhibits hyperphagia and obesity by reducing food intake and increasing energy expenditure [32]. A primary central nervous site of leptin for control of energy balance is the hypothalamus. Ob-Rb has been co-localized with NPY/AGRP and POMC/CART in the ARC neurons and with MCH and orexins in the lateral hypothalamus neurons [33-36]. Leptin inhibits expression of orexigenic NPY/AGRP hypothalamic neurons, while stimulating anorexigenic POMC/CART hypothalamic neurons in ARC. The gene mutation of leptin, or its receptor, defects inhibition of ARC-NPY neurons through which NPY activity over expression results in hyperphagia and obesity [37,38]. AGRP over expression also results in obesity [39] and AGRP isup-regulated in diabetic mutant mice [40]. Leptin decreases AGRP mRNA levels in the hypothalamus [41-44]. These findings suggest that AGRP inhibition may be an important mechanism by which leptin can enhance its anorectic effect in the hypothalamus. We indicted that Evo down regulated hypothalamic NPY and AGRP mRNA levels by enhancing the inhibition of leptin, therefore causing decreases in food intake and body weight.Although NPY immunoreactivity is widely distributed throughout the hypothalamus [45,46], in situ hybridization histochemistry revealed that NPY expressing neurons are primarily localized in the arcuate nucleus (ARC) and the dorsomedial hypothalamus (DMH) [47-49]. Leptin directly acts on ARC NPY neurons but not on DMH NPY neurons. NPY and OB-Rb are co-expressed in ARC neurons, but NPY and OB-Rb gene are expressed in different neuronal populations to DMH [47]. DMH NPY may play an important role in maintaining energy homeostasis in response to long-term alterations in energy balance. DMH was the brain site most sensitive to the feeding inhibitory effects of administration of exogenous CCK [50]. Double immunostaining experiments revealed that NPY and CCK1 receptors were co-localized and CCK1 receptor staining was found on the surface of NPY neurons in DMH neurons [51]. DMH parenchymal injections of CCK significantly reduced NPY mRNA levels and 30 min food intake [51]. OLETF rats lacking CCK1 receptors become hyperphagic and obese mediated by DMH NPY over expression [52]. These data suggested that CCK acting through DMH CCK1 receptors regulates DMH NPY gene expression to affect feeding behavior. Evo induces CCK release in a dose-dependent manner and inhibits both gastric emptying and gastrointestinal transit [7]. The selective CCK1 receptor antagonists, devazepide and lorglumide, effectively attenuated the Evo-induced inhibition of gastric emptying and gastrointestinal transit [7]. CCK is a well known feeding inhibitor. We thought that because Evo depresses DMH NPY gene expression by inducing CCK releasing, this partly causes decreased food intake and body weight in the Evo-treated rats.Leptin and cholecystokinin (CCK) have a synergistic interaction in the suppression of food intake and body weight [53-57]. Retrospective administration of subthreshold doses of leptin and CCK did not reduce food intake, whereas central or peripheral administration of leptin effectively inhibited food intake in combination with peripheral injections of CCK. Koletsky (fak/fak) rats, which develop severe obesity due to a leptin receptor–deficiency, have markedly increased meal sizes and reduced satiety in response to CCK. This suggests a critical role for leptin signaling in response to endogenous signals that promote meal termination [58]. Synergistic interaction between CCK and leptin to induce satiety is mediated by intracelluar crosstalk between SRC and STAT3 pathways [59]. CCK co-injected with leptin, potentiated Fos expression selectively in the areas of the postrema, NTS and paraventricular nucleus of the hypothalamus (PVN). This suggests that the PVN, as part of the afferent and efferent limbs of the circuitry, is involved in the synergistic interaction between leptin and CCK [60]. The synergistic interaction between leptin and CCK may integrate short-term meal-related input signals into long-term control of energy balance and persist in reducing food intake.Our research proves that the hypothalamus is the primary central nervous site where Evo inhibits food intake and body weight of rats. Evo performs its function of regulating and controlling food intake and reducing body weight through significantly increasing the serum leptin level and decreasing the mRNA expression of hypothalamic feeding-related peptides NPY and AGRP. 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