Rat Hypothalamus Research Articles

Analysis of the finasteride treatment and its withdrawal in the rat hypothalamus and hippocampus at whole-transcriptome level

PurposeAs reported in patients treated for androgenetic alopecia with finasteride (i.e., a blocker of the enzyme 5 alpha-reductase) and in an animal model, side effects affecting sexual, psychiatric, neurological, and physical domains, may occur during the treatment and persist with drug suspension. The etiopathogenesis of these side effects has been poorly explored. Therefore, we performed a genome-wide analysis of finasteride effects in the brain of adult male rat.MethodsAnimals were treated (i.e., for 20 days) with finasteride (1mg/rat/day). 24 h after the last treatment and 1 month after drug suspension, RNA sequencing analysis was performed in hypothalamus and hippocampus. Data were analyzed by differential expression analysis and Gene-Set Enrichment Analyses (GSEA).ResultsData obtained after finasteride treatment showed that 186 genes (i.e., 171 up- and 15 downregulated) and 19 (i.e., 17 up- and 2 downregulated) were differentially expressed in the hypothalamus and hippocampus, respectively. Differential expression analysis at the drug withdrawal failed to identify dysregulated genes. Several gene-sets were enriched in these brain areas at both time points.ConclusionSome of the genes reported to be differentially expressed (i.e., TTR, DIO2, CLDN1, CLDN2, SLC4A5, KCNE2, CROT, HCRT, MARCKSL1, VGF, IRF2BPL) and GSEA, suggest a potential link with specific side effects previously observed in patients and in the animal model, such as depression, anxiety, disturbance in memory and attention, and sleep disturbance. These data may provide an important background for future experiments aimed at confirming the pathological role of these genes.

Decreased levels of hydrogen sulfide in the hypothalamic paraventricular nucleus contribute to sympathetic hyperactivity induced by cerebral infarction.

Paroxysmal sympathetic hyperactivity (PSH) is a common clinical feature secondary to ischemic stroke (IS), but its mechanism is poorly understood. We aimed to investigate the role of H2S in the pathogenesis of PSH. IS patients were divided into malignant (MCI) and non-malignant cerebral infarction (NMCI) group. IS in rats was induced by the right middle cerebral artery occlusion (MCAO). H2S donor (NaHS) or inhibitor (aminooxy-acetic acid, AOAA) were microinjected into the hypothalamic paraventricular nucleus (PVN). Compared with the NMCI group, patients in the MCI group showed PSH, including tachycardia, hypertension, and more plasma norepinephrine (NE) that was positively correlated with levels of creatine kinase, glutamate transaminase, and creatinine respectively. The 1-year survival rate of patients with high plasma NE levels was lower. The hypothalamus of rats with MCAO showed increased activity, especially in the PVN region. The levels of H2S in PVN of the rats with MCAO were reduced, while the blood pressure and renal sympathetic discharge were increased, which could be ameliorated by NaHS and exacerbated by AOAA. NaHS completely reduced the disulfide bond of NMDAR1 in PC12 cells. The inhibition of NMDAR by MK-801 microinjected in PVN of rats with MCAO also could lower blood pressure and renal sympathetic discharge. In conclusion, PSH may be associated with disease progression and survival in patients with IS. Decreased levels of H2S in PVN were involved in regulating sympathetic efferent activity after cerebral infarction. Our results might provide a new strategy and target for the prevention and treatment of PSH.

Functional Changes in Aqp4 Gene Expression in the Rat Hypothalamus under Different Drinking Regimens and Arterial Hypertension

Functional Changes in Aqp4 Gene Expression in the Rat Hypothalamus under Different Drinking Regimens and Arterial Hypertension

Sophora flavescens alcohol extract ameliorates insomnia and promotes PI3K/AKT/BDNF signaling transduction in insomnia model rats.

Active ingredient of Sophora flavescens is reported to promote non-rapid eye movement (NREM) sleep. However, the role of Sophora flavescens alcohol extract in insomnia is elusive, which is addressed in this study, together with the exploration on its potential mechanism. An insomnia model of rats was established by para-chlorophenylalanine induction and further treated with SFAE or Zaoren Anshen capsule (ZRAS; positive control drug). Sleep quality and sleep architecture of rats were evaluated by the sleep test, electroencephalogram and electromyogram. The levels of monoamine neurotransmitters in rat hypothalamus were determined using ELISA, and the transduction of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/brain-derived neurotrophic factor (BDNF) signaling in the brain tissues of rats was examined by Western blot. SFAE and ZRAS increased the sleeping time and decreased the sleep latency of insomnia rats. SFAE reduced waking time and increased NREM and REM time, while changing power density of wakefulness, NREM sleep, and REM sleep in insomnia rats. SFAE and ZRAS upregulated levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, and downregulated those of norepinephrine and dopamine in insomnia rats. Besides, SFAE and ZRAS elevated BDNF expression as well as the ratios of phosphorylated (p)-PI3K/PI3K and p-AKT/AKT. The role of SFAE in insomnia model rats was similar with that of ZRAS. SFAE reduces insomnia and enhances the PI3K/AKT/BDNF signaling transduction in insomnia model rats, which can function as a drug candidate for insomnia.

Network pharmacology combined with lipidomics to reveal the regulatory effects and mechanisms of Kangzao granules in the hypothalamus of rats with central precocious puberty

Central precocious puberty (CPP) is a prevalent endocrine disorder that primarily affects children, specifically females, and is associated with various physical and psychological complications. Although Kangzao granules (KZG) are efficacious in managing CPP, the underlying mechanisms remain unclear. Therefore, this study aimed to elucidate the therapeutic mechanisms of KZG using network pharmacology, molecular docking, pharmacodynamics, and pathway validation. A putative compound–target–pathway network was constructed using Cytoscape, before KEGG and Gene Ontology enrichment analyses were conducted. Moreover, molecular docking was performed using AutoDockTools. Quality control of the 10 key components of KZG was carried out using UHPLC-ESI/LTQ-Orbitrap-MS/MS, and hypothalamic lipids were analyzed using UHPLC-Q-Exactive Orbitrap MS/MS. In total, 87 bioactive compounds that targeting 110 core proteins to alleviate CPP were identified in KZG. Lipidomic analysis revealed 18 differential lipids among the CPP, KZG, and control groups, wherein fatty acids were significantly reduced in the model group; however, these changes were effectively counteracted by KZG treatment. Molecular docking analysis revealed a strong binding affinity between flavonoids and RAC-alpha serine/threonine-protein kinase (AKT) when docked into the crystal structure. Moreover, a substantial disruption in lipid metabolism was observed in the model group; however, treatment with KZG efficiently reversed these alterations. Furthermore, the phosphoinositide 3-kinase/AKT signaling pathway was identified as a pivotal regulator of hypothalamic lipid metabolism regulator. Overall, this study highlights the effectiveness of a multidisciplinary approach that combines network pharmacology, lipidomics, molecular docking, and experimental validation in the elucidation of the therapeutic mechanisms of KZG in CPP treatment.

Duodenal-jejunal bypass improves hypothalamic oxidative stress and inflammation in diabetic rats via glucagon-like peptide 1-mediated Nrf2/HO-1 signaling.

Type 2 diabetes mellitus (T2DM) is often accompanied by impaired glucose utilization in the brain, leading to oxidative stress, neuronal cell injury and infla-mmation. Previous studies have shown that duodenal jejunal bypass (DJB) surgery significantly improves brain glucose metabolism in T2DM rats, the role and the metabolism of DJB in improving brain oxidative stress and inflammation condition in T2DM rats remain unclear. To investigate the role and metabolism of DJB in improving hypothalamic oxidative stress and inflammation condition in T2DM rats. A T2DM rat model was induced via a high-glucose and high-fat diet, combined with a low-dose streptozotocin injection. T2DM rats were divided into DJB operation and Sham operation groups. DJB surgical intervention was carried out on T2DM rats. The differential expression of hypothalamic proteins was analyzed using quantitative proteomics analysis. Proteins related to oxidative stress, inflammation, and neuronal injury in the hypothalamus of T2DM rats were analyzed by flow cytometry, quantitative real-time PCR, Western blotting, and immunofluorescence. Quantitative proteomics analysis showed significant differences in proteins related to oxidative stress, inflammation, and neuronal injury in the hypothalamus of rats with T2DM-DJB after DJB surgery, compared to the T2DM-Sham groups of rats. Oxidative stress-related proteins (glucagon-like peptide 1 receptor, Nrf2, and HO-1) were significantly increased (P < 0.05) in the hypothalamus of rats with T2DM after DJB surgery. DJB surgery significantly reduced (P < 0.05) hypothalamic inflammation in T2DM rats by inhibiting the activation of NF-κB and decreasing the expression of interleukin (IL)-1β and IL-6. DJB surgery significantly reduced (P < 0.05) the expression of factors related to neuronal injury (glial fibrillary acidic protein and Caspase-3) in the hypothalamus of T2DM rats and upregulated (P < 0.05) the expression of neuroprotective factors (C-fos, Ki67, Bcl-2, and BDNF), thereby reducing hypothalamic injury in T2DM rats. DJB surgery improve oxidative stress and inflammation in the hypothalamus of T2DM rats and reduce neuronal cell injury by activating the glucagon-like peptide 1 receptor-mediated Nrf2/HO-1 signaling pathway.

Restraint stress-induced expression of &lt;i&gt;Fos&lt;/i&gt; and several related genes in the hypothalamus of the hypertensive ISIAH rats

Stress can play a significant role in the development of arterial hypertension and many other complications of cardiovascular diseases. Considerable attention is paid to the study of the molecular mechanisms involved in the body’s response to stressful influences, but there are still many blank spots in understanding the details. ISIAH rats model a stress-sensitive form of arterial hypertension. ISIAH rats are characterized by genetically determined enhanced hypothalamic-adrenal-cortical and sympathetic adrenomedullary systems activity, which suggests a functional state of increased stress reactivity. In the present study, for the first time, the time course of the Fos and several related genes’ expression was studied in the hypothalamus of adult male hypertensive ISIAH rats after exposure to a single restraint stress of different duration (30, 60, and 120 minutes). The results of the study showed the activation of Fos transcription with a peak 1 hour after the onset of restraint stress. The dynamics of Fos gene activation coincides with the dynamics of blood pressure increase after stress. Restraint stress also alters the transcription of several other genes encoding transcription factors (Jun, Nr4a3, Jdp2, Ppargc1a) associated with the development of cardiovascular diseases. Since Fos induction is a marker of brain neuron activation, we can conclude that increased stress reactivity of the hypothalamic-pituitary-adrenocortical and sympathoadrenal systems of hypertensive ISIAH rats during short-term restriction is accompanied by activation of hypothalamic neurons and increased blood pressure level.

A new ghrelin receptor antagonist, agrelax, reduces emotional overeating caused by the stimulation of the lateral hypothalamus reward zone in well-fed rats

BACKGROUND: Ghrelin receptor antagonists hold promise for the treatment of eating disorders. The reward zone of the lateral hypothalamus has been proposed as a target for mediating the effects of the ghrelin system in emotional overeating.&#x0D; AIM: This study aimed to evaluate the effects of a new ghrelin receptor antagonist, agrelax, on emotional overeating induced by the stimulation of the reward zone of the lateral hypothalamus in well-fed rats.&#x0D; MATERIALS AND METHODS: Male Wistar rats were trained to self-stimulate in a Skinner box. After training, a feeder was placed in the Skinner box, and a food-conditioned reflex was developed in the rats for 5 days. Then, the reaction of food self-deprivation, i.e., behavior under conditions of choice of self-stimulation or food intake, was assessed.&#x0D; RESULTS: The reaction of food self-deprivation when the animals did not approach the feeder was 10% of the threshold current. Self-stimulation of the lateral hypothalamus with a threshold current caused numerous approaches to the feeder and food intake. Sulpiride, a dopamine D2/D3 antagonist (5 and 20 mg/kg ip), administered to well-fed rats reduced both feeding behavior and the intensity of self-stimulation in the food self-deprivation test at threshold currents. The ghrelin receptor antagonists [D-LYS3]-GHRP-6 and the novel antagonist agrelax (1 µg/µL, 20 µL intranasally) also reduced both feeding behavior and the intensity of self-stimulation under these conditions.&#x0D; CONCLUSIONS: Ghrelin and dopamine receptors are involved in emotional overeating. Agrelax, a novel ghrelin receptor antagonist, reduces emotional overeating induced by the activation of the lateral hypothalamic reward system. Because emotional overeating is strongly associated with clinical eating disorders such as bulimia and binge-eating disorder, the use of ghrelin antagonists to treat and prevent this problem is promising.

Restraint Stress-Induced Expression of Fos and Several Related Genes in the Hypothalamus of Hypertensive ISIAH Rats

Stress can play a significant role in arterial hypertension and many other complications of cardiovascular diseases. Considerable attention is paid to the study of the molecular mechanisms involved in the body response to stressful influences, but there are still many blank spots in understanding the details. ISIAH rats model the stress-sensitive form of arterial hypertension. ISIAH rats are characterized by genetically determined enhanced activities of the hypothalamic-pituitary-adrenocortical and sympathetic-adrenomedullary systems, suggesting a functional state of increased stress reactivity. For the first time, the temporal expression patterns of Fos and several related genes were studied in the hypothalamus of adult male hypertensive ISIAH rats after a single exposure to restraint stress for 30, 60, or 120 min. Fos transcription was activated and peaked 1 h after the start of restraint stress. The time course of Fos activation coincided with that of blood pressure increase after stress. Activation of hypothalamic neurons also alters the transcription levels of several transcription factor genes (Jun, Nr4a3, Jdp2, and Ppargc1a), which are associated with the development of cardiovascular diseases. Because Fos induction is a marker of brain neuron activation, activation of hypothalamic neurons and an increase in blood pressure were concluded to accompany increased stress reactivity of the hypothalamic-pituitary-adrenocortical and sympathoadrenal systems in hypertensive ISIAH rats during short-term restraint.

Restraint Stress-Induced Expression of Fos and Several Related Genes in the Hypothalamus of Hypertensive ISIAH Rats

Restraint Stress-Induced Expression of Fos and Several Related Genes in the Hypothalamus of Hypertensive ISIAH Rats

Methyl Jasmonate Modulates Feeding Behaviors and Hypothalamic Expression of the Orexin 1 Receptor in Rats.

Active plant ingredients have been successfully used in modern medicine to control appetite and energy hemostasis. This study was designed to evaluate the efficacy of the phytohormone methyl jasmonate (MJ) on food-related behaviors in rats. Adult male Wistar rats were randomly divided into different groups (7 rats) and infused intracerebroventricularly (i.c.v.) with MJ vehicle (DMSO) or MJ (2.5, 5 and 10 μg/rat). Then, the individual rats were placed in an automated open field-like apparatus to assess a 12-h food-related activity in light and dark times. After behavioral tests, immunofluorescence staining of the orexin 1 receptor (Orx1R) was studied in the hypothalamus of rats. MJ (2.5, 5, and 10 μg/rat) administration significantly decreased food intake in the light and dark phases compared with the control group. Moreover, all the MJ-treated groups exhibited a decrease in visits to food containers at the light and dark times (p < 0.001). In addition, rats infused with MJ at 5 μg and 10 μg spent less time in the ports of food containers in the light and dark phases in comparison with control rats. Time in zone-related to food and locomotor activity was significantly decreased in the MJ (5 μg) groups during the light time and in all MJ-injected groups in the dark time. Moreover, hypothalamic expression of Orx1R in rats treated with MJ (5 μg) was significantly lower as compared to the control group. Overall, the results indicated the potential of MJ to modulate feeding-related behavior and Orx1R expression in the hypothalamus of rats.

Investigation of Melatonin Receptors Gene Expression Levels in the Hippocampus and Hypothalamus in Rats with an Experimental Morphine Dependence Model

Objective: Morphine is one of the important opioids used in chronic and acute pain management. However, it has many side effects, including the development of addiction, which seriously limits its use. Melatonin shows its physiological effects through melatonin receptor 1 (MT1), melatonin receptor 2 (MT2) and heterodimer receptors (MT1/MT2). No study has examined the presence of MT1/MT2 mRNA in the hypothalamus and hippocampus and its relationship with the addiction process. The aim of this study was to investigate the gene expression levels of MT1, MT2 and MT1/MT2 in rat hippocampus and hypothalamus during morphine addiction and morphine withdrawal. Materials and Methods: A total of 36 male Wistar rats were divided into 3 groups (n=12). Control (C) group received saline subcutaneously for 6 days. Morphine (M) and morphine+naloxone (M+N) groups received 10 mg/kg/day morphine subcutaneously for 6 days. On the seventh day, saline was injected intraperitoneally into C and M groups and 1 mg/kg naloxone was injected intraperitoneally into M+N group. 30 minutes later, hippocampus and hypothalamus tissues of rats were dissected. Melatonin receptor gene expression level was analysed by quantitative qPCR. Results: Both MT1 and MT1/MT2 gene expression levels in the hypothalamus were in the M+N group than in the C group (p&lt;0.05). There was no difference between the expression levels of MT2 receptors in the hypothalamus (p&gt;0.05). There was no difference in MT1, MT2 and MT1/MT2 gene expression in the hippocampus (p&gt;0.05). Conclusion: This is the first study to show the presence of MT1/MT2 in the hypothalamus and hippocampus, and it is possible that MT1 and MT1/MT2 receptors, especially in the hypothalamus, play a role in the addiction process.

Grid1 regulates the onset of puberty in female rats.

The study aimed to investigate the effect of Grid1, encoding the glutamate ionotropic receptor delta type subunit 1 (GluD1), on puberty onset in female rats. Grid1 mRNA and protein expression was detected in the hypothalamus of female rats at prepuberty and puberty. The levels of Grid1 mRNA in the hypothalamus, the fluorescence intensity in the arcuate nucleus and paraventricular nucleus of the prepubertal rats was significantly lower than pubertal. Additionally, the expression of Grid1 was suppressed in primary hypothalamus cells and prepubertal rat. Finally, investigated the effect of Grid1 knockdown on puberty onset and reproductive performance. Treatment of hypothalamic neurons with LV-Grid1 decreased the level of Grid1 and Rfrp-3 (encoding RFamide-related peptide 3) mRNA expression, but increased the Gnrh (encoding gonadotropin-releasing hormone) mRNA levels. After an ICV injection, the time for the rat vaginal opening occurred earlier. Moreover, Gnrh mRNA expression was increased, whereas Rfrp-3 mRNA expression was decreased in the hypothalamus. The concentration of progesterone (P4) in the serum was significantly decreased compare with control group. Ovary hematoxylin-eosin staining revealed that the LV-Grid1 group mainly contained primary and secondary follicles. The reproductive performance of the rats was not affected by the Grid1 knockdown. Therefore, Grid1 may affect the onset of puberty in female rats by regulating the levels of Gnrh, and Rfrp-3 in the hypothalamus, as well as the concentrations of P4, but not reproduction performance.

Stress Response of Neurotransmitters and Micro-RNAs 34c and 7a in Serum and Hypothalamus of Rats after Exposure to Different Powers of Noise

Stress Response of Neurotransmitters and Micro-RNAs 34c and 7a in Serum and Hypothalamus of Rats after Exposure to Different Powers of Noise

Mechanism of GW117 antidepressant action: Melatonin receptor-mediated regulation of sleep rhythm

Alterations in circadian sleep patterns constitute a salient manifestation in major depressive disorder. GW117, an emergent antidepressant, functions as an agonist for melatonin 1 and melatonin 2 (MT1/MT2) receptors, in tandem with antagonism of the serotonin (5-HT) 2C receptor. The present investigation is dedicated to elucidating the role and underlying mechanisms by which GW117 ameliorates circadian sleep disruptions. Utilizing an adapted chronic unpredictable mild stress protocol, we induced a depressive-like phenotype and perturbed circadian rhythms in rodent models. Our methodological approach integrated quantitative polymerase chain reaction (qPCR) in real-time, enzyme-linked immunosorbent assay (ELISA), and immunoblotting techniques to probe alterations in the expression of core circadian genes and homeostatic sleep markers. The impact of GW117 was assessed across various dosages (10, 20, and 40 mg/kg) on these molecular signatures. In a parallel examination, we evaluated the influence of GW117 (administered at 15, 40, and 60 mg/kg) on the sleep patterns of healthy mice. The results showed that GW117 significantly improved sleep-wake circadian rhythms, altered sleep architecture, and shortened sleep latency. Furthermore, GW117 increased the expression of several clock genes in the hypothalamus of chronic unpredictable mild stress model rats and normal mice. It also regulated circadian biomarkers, including melatonin and cortisol. Based on our findings, we propose that the beneficial effects of GW117 on sleep rhythms may be due to the melatonin system-mediated activation of the Wnt/β-catenin signaling pathway.

Quantitative analysis of microglia morphological changes in the hypothalamus of chronically stressed rats

Based on the successful establishment of a rat model of chronic restraint stress, we used multiple algorithms to quantify the morphological changes of rat hypothalamic microglia from various perspectives, providing a pathomorphological basis for the subsequent study of molecular mechanisms of hypothalamic stress injury, such as neuroinflammation. To verify the successful establishment of the chronic stress model, an enzyme-linked immunosorbent assay was performed to detect serum glucocorticoid levels. Microglia labeled with Iba1 in frozen sections of rat hypothalamus were scanned and photographed at multiple levels using confocal microscopy. Subsequently, images were processed for external contouring and skeletonization, and morphological indices of microglia were calculated and analyzed using fractal, skeleton, and Sholl analysis. In addition, the co-expression of CD68 (a marker that can reflect phagocytic activity) and Iba1 was observed by immunofluorescence technique. Compared with the control group, microglia in the chronic stress group displayed reduced fractal dimension and lacunarity, increased density and circularity, enlarged soma areas, and shortened and reduced branches. Sholl analysis confirmed the reduced complexity of microglia following chronic stress. Meanwhile, microglia CD68 increased significantly, indicating that the microglia in the chronic stress group have greater phagocytosis activity. In summary, chronic restraint stress promoted the conversion of microglia in the rat hypothalamus to a less complex form, manifested as larger soma, shorter and fewer branches, more uniform and dense texture, and increased circularity; indeed, the shape of these microglia resembled that of amoeba and they displayed strong phagocytosis activity.

FTO-mediated m6A demethylation regulates GnRH expression in the hypothalamus via the PLCβ3/Ca2+/CAMK signalling pathway

N6-methyladenosine (m6A) plays a crucial role in the development and functional homeostasis of the central nervous system. The fat mass and obesity-associated (FTO) gene, which is highly expressed in the hypothalamus, is closely related to female pubertal development. In this study, we found that m6A methylation decreased in the hypothalamus gradually with puberty and decreased in female rats with precocious puberty. FTO expression was increased at the same time. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that the m6A methylation of PLCβ3, a key enzyme of the Ca2+ signalling pathway, was decreased significantly in the hypothalamus in precocious rats. Upregulating FTO increased PLCβ3 expression and activated the Ca2+ signalling pathway, which promoted GnRH expression. Dual-luciferase reporter and MeRIP-qPCR assays confirmed that FTO regulated m6A demethylation of PLCβ3 and promoted PLCβ3 expression. Upon overexpressing FTO in the hypothalamic arcuate nucleus (ARC) in female rats, we observed advanced puberty onset. Meanwhile, PLCβ3 and GnRH expression in the hypothalamus increased significantly, and the Ca2+ signalling pathway was activated. Our study demonstrates that FTO enhances GnRH expression, which promotes puberty onset, by regulating m6A demethylation of PLCβ3 and activating the Ca2+ signalling pathway.

Troxerutin dampened hypothalamic neuroinflammation via microglial IL-22/IL-22R1/IRF3 activation in dihydrotestosterone-induced polycystic ovary syndrome rats

BackgroundPolycystic ovary syndrome (PCOS) is the most common reproductive-endocrine condition in premenopausal women. Troxerutin, a common clinical anti-coagulant agent, was shown to work as a strong IL-22 boosting agent counteracting the hyperactivated gonadotrophin releasing hormone (GnRH) neurons and heightened GnRH release, the neuroendocrine origin of PCOS with unknown mechanism in rats. Exploring the off-label use of troxerutin medication for PCOS is thus sorely needed. MethodsSerum IL-22 content and hypothalamic IL-22 protein were detected. Inflammatory factor levels in hypothalamo-pituitary were evaluated. Immunofluorescence staining was employed to determine the activation and M1/M2-prone polarization of microglia in arcuate hypothalamus and median eminence. RNA-sequencing and transcriptome analysis were applied to explore the potential driver of microglia M2-polarization in response to IL-22 bolstering effect. The function of microglial IL-22/IL-22R1/IRF3 system was further verified using in vivo knockdown of IL-22R1 and a potent IRF3 inhibitor in BV2 microglial cell lines in vitro. ResultsTroxerutin augmented serum IL-22 content, and its consequent spillover into the hypothalamus led to the direct activation of IL-22R1/IRF3 system on microglia, thereby promoted microglia M2 polarization in arcuate hypothalamus and median eminence, dampened hypothalamic neuroinflammation, inhibited hyperactive GnRH and rescued a breadth of PCOS-like traits in dihydrotestosterone (DHT) rats. The salutary effects of troxerutin treatment on hypothalamic neuroinflammation, microglial M1/2 polarization, GnRH secretion and numerous PCOS-like features were blocked by in vivo knockdown of IL-22R1. Moreover, evidence in vitro illustrated that IL-22 supplement to BV-2 microglia cell lines promoted M2 polarization, overproduction of anti-inflammatory marker and limitation of pro-inflammatory factors, whereas these IL-22 effects were blunted by geldanamycin, a potent IRF3 inhibitor. ConclusionHere, the present study reported the potential off-label use of troxerutin medication, a common clinical anti-coagulant agent and an endogenous IL-22 enhancer, for multiple purposes in PCOS. The rational underlying the application of troxerutin as a therapeutic choice in PCOS derived from its activity as an IL-22 memetic agent targeting the neuro-endocrine origin of PCOS, and its promotive impact on microglia M2 polarization via activating microglial IL-22R1/IRF3 system in the arcuate hypothalamus and median eminence of DHT female rats.

Expression of GAD65/67 and VGLUT2 in Mediobasal Nuclei of Rat Hypothalamus during Aging.

The expression of glutamate decarboxylase GAD65/67, an enzyme of GABA synthesis, and vesicular glutamate transporter 2 (VGLUT2) in the arcuate, dorsomedial, and ventromedial nuclei of the hypothalamus of young (3 months), adult (12 months), and old male rats (24 months) was studied by Western blotting. In old rats, an increase in the expression of GAD65/67 in the arcuate and dorsomedial, VGLUT2 in the arcuate, dorsomedial, and ventromedial nuclei was observed. Thus, an increase in opposite processes of inhibition and excitation is observed in the hypothalamic nuclei during aging.

Effect of acupuncture on the expression of neuropeptides and related inflammatory factors in rats with diarrhea-predominant irritable bowel syndrome.

To observe the effect of acupuncture on the expressions of neuropeptides and related inflammatory factors in rats with diarrhea-predominant irritable bowel syndrome(IBS-D), so as to explore the mechanism of acupuncture in the treatment of IBS-D. Male Wistar rats were randomly divided into blank group, model group, medication group, and acupuncture group, with 6 rats in each group. Except for the blank group, the other groups were subjected to 14-day "acetic acid enema + restraint stress" to establish the IBS-D rat model. After successful modeling, the medication group received gavage of pinaverium bromide(15 mg/kg) once a day, and the acupuncture group received acupuncture at "Baihui"(GV20) and bilateral "Tianshu"(ST25), "Shangjuxu"(ST37), "Zusanli"(ST36), and "Taichong"(LR3) for 20 min every day, both groups were treated continuously for 14 days. The general state of the rats in each group was observed, and the body weight of the rats was measured. The open-field experiment was conducted to measure the horizontal and vertical movements, and the number of fecal pellets of rats. The histopathological morphology of hypothalamus and colon of rats was observed by HE staining. Toluidine blue staining was used to observe and count the mast cells(MCs) in the colon tissue of rats. ELISA was used to detect the serum contents of tumor necrosis factor-α(TNF-α) and interleukin(IL)-10. Real-time fluorescence quantitative PCR was performed to detect the mRNA expressions of calcitonin gene-related peptide(CGRP) in the hypothalamus and colon tissue. Western blot was used to detect the expressions of corticotropin-releasing factor(CRF) in the hypothalamus and colon tissue. HE staining showed that there was inflammatory cell infiltration in the lamina propria of colon in the model group, and it was reduced in the other groups. Compared with the blank group, the model group showed significantly decreased body weight, decreased walking distance and upright times in open field experiment, decreased serum IL-10 contents(P<0.05, P<0.01), increased fecal pellet number (P<0.01), increased MC number in the colon tissue, serum TNF-α contents, and CGRP mRNA expressions and CRF expressions in the hypothalamus and colon tissue(P<0.01). Compared with the model group, both medication and acupuncture groups showed significantly increased body weight, walking distance and upright times in the open-field experiment, and serum IL-10 contents(P<0.01, P<0.05), significantly decreased fecal pellet number (P<0.05), significantly decreased MC number in the colon tissue, serum TNF-α contents, and CGRP mRNA expressions in the hypothalamus and colon tissue(P<0.01)；at the same time, the acupuncture group showed significantly decreased CRF expressions in the hypothalamus and colon tissue(P<0.01, P<0.05). There was no significant difference in the above indicators between the medication group and the acupuncture group. Acupuncture can improve the general and emotional state, inflammatory response, and neuropeptide expression in rats with IBS-D, and alleviate the symptoms of IBS-D, which may be related to the regulation of neuropeptides and inflammatory factors levels.