The impact of noradrenergic neurotoxin DSP-4 and noradrenaline transporter knockout (NET-KO) on the activity of liver cytochrome P450 3A (CYP3A) in male and female mice

Background Our earlier studies have shown that the brain noradrenergic system regulates cytochrome P450 (CYP) in rat liver via neuroendocrine mechanism. In the present work, a comparative study on the effect of intraperitoneal administration of the noradrenergic neurotoxin DSP-4 and the knockout of noradrenaline transporter (NET-KO) on the CYP3A in the liver of male and female mice was performed. Methods The experiments were conducted on C57BL/6J WT and $NET^{–/–}$ male/female mice. DSP-4 was injected intraperitoneally as a single dose (50 mg/kg ip.) to WT mice. The activity of CYP3A was measured as the rate of 6β-hydroxylation of testosterone in liver microsomes. The CYP3A protein level was estimated by Western blotting. Results DSP-4 evoked a selective decrease in the noradrenaline level in the brain of male and female mice. At the same time, DSP-4 reduced the CYP3A activity in males, but not in females. The level of CYP3A protein was not changed. The NET knockout did not affect the CYP3A activity/protein in both sexes. Conclusions The results with DSP-4 treated mice showed sex-dependent differences in the regulation of liver CYP3A by the brain noradrenergic system (with only males being responsive), and revealed that the NET knockout did not affect CYP3A in both sexes. Further studies into the hypothalamic–pituitary–gonadal hormones in DSP-4 treated mice may explain sex-specific differences in CYP3A regulation, whereas investigation of monoaminergic receptor sensitivity in the hypothalamic/pituitary areas of $NET^{–/–}$ mice will allow for understanding a lack of changes in the CYP3A activity in the NET-KO animals

The miR151 and miR5100 Transfected Bone Marrow Stromal Cells Increase Myoblast Fusion in IGFBP2 Dependent Manner

BACKGROUND: Bone marrow stromal cells (BMSCs) form a perivascular cell population in the bone marrow. These cells do not present naïve myogenic potential. However, their myogenic identity could be induced experimentally in vitro or in vivo. In vivo, after transplantation into injured muscle, BMSCs rarely fused with myofibers. However, BMSC participation in myofiber reconstruction increased if they were modified by NICD or PAX3 overexpression. Nevertheless, BMSCs paracrine function could play a positive role in skeletal muscle regeneration. Previously, we showed that SDF-1 treatment and coculture with myofibers increased BMSC ability to reconstruct myofibers. We also noticed that SDF-1 treatment changed selected miRNAs expression, including miR151 and miR5100. METHODS: Mouse BMSCs were transfected with miR151 and miR5100 mimics and their proliferation, myogenic differentiation, and fusion with myoblasts were analyzed. RESULTS: We showed that miR151 and miR5100 played an important role in the regulation of BMSC proliferation and migration. Moreover, the presence of miR151 and miR5100 transfected BMSCs in co-cultures with human myoblasts increased their fusion. This effect was achieved in an IGFBP2 dependent manner. CONCLUSIONS: Mouse BMSCs did not present naïve myogenic potential but secreted proteins could impact myogenic cell differentiation. miR151 and miR5100 transfection changed BMSC migration and IGFBP2 and MMP12 expression in BMSCs. miR151 and miR5100 transfected BMSCs increased myoblast fusion in vitro. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12015-022-10350-y

Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

Background: T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. Objective: We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. Methods: T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. Results: Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD81 suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)–150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH2/2 or miRNA-1502/2 mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. Conclusions: This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains

Cocaine-Induced Time-Dependent Alterations in Cytochrome P450 and Liver Function

Cytochrome P450 is responsible for the metabolism of endogenous substrates, drugs and substances of abuse. The brain and nervous system regulate liver cytochrome P450 via neuroendocrine mechanisms, as shown in rodents. Cocaine exerts its addictive effects through the dopaminergic system, the functioning of which undergoes changes during its continuous use. Therefore, it can be hypothesized that the regulation of cytochrome P450 by cocaine may also alter during the addiction process, cessation and relapse. We analyzed preclinical studies on the mechanisms of the pharmacological action of cocaine, the role of the brain’s dopaminergic system in the neuroendocrine regulation of cytochrome P450 and the in vitro and in vivo effects of cocaine on the cytochrome P450 expression/activity and hepatotoxicity. The results of passive cocaine administration indicate that cocaine affects liver cytochrome P450 enzymes (including those engaged in its own metabolism) via different mechanisms involving the expression of genes encoding cytochrome P450 enzymes and interaction with enzyme proteins. Thus, it may affect its own oxidative metabolism and the metabolism of endogenous substrates and other co-administered drugs and may lead to hepatotoxicity. Its effect depends on the specific cytochrome P450 enzyme affected, cocaine dosage, treatment duration and animal species. However, further complementary studies are needed to find out whether cocaine affects cytochrome P450 via the brain’s dopaminergic system. The knowledge of cocaine’s effect on cytochrome P450 function during the entire addiction process is still incomplete. There is a lack of information on the enzyme expression/activity in animals self-administering cocaine (addicted), in those withdrawn after cocaine self-administration, and during relapse in animals previously addicted; furthermore, there is no such information concerning humans. The subject of cytochrome P450 regulation by cocaine during the addiction process is an open issue, and addressing this topic may help in the treatment of drug abuse patients

Serotonin Receptors of 5-HT; 2; Type in the Hypothalamic Arcuate Nuclei Positively Regulate Liver Cytochrome P450 via Stimulation of the Growth Hormone-Releasing Hormone/Growth Hormone Hormonal Pathway

Our recent study carried out after local injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine into the arcuate nucleus (ARC) of the hypothalamus suggested a positive influence of the serotonergic innervation of the ARC on growth hormone (GH) secretion and GH-dependent expression of cytochrome P450. The aim of our present study was to determine the effect of the activation of the serotonin (5-HT)-type receptors, 5-HT; 1; or 5-HT; 2; , in the ARC on the expression and activity of cytochrome P450 in the liver of male rats. The serotonergic agonist 5-carboxyamidotryptamine [(5-CT), a 5-HT; 1; -type receptor agonist] or 2,5-dimethoxy-4-iodoamphetamine [(DOI), a 5-HT; 2; -type receptor agonist] was injected into the ARC for 5 days. The activity and expression of cytochrome P450 isoenzymes and the levels of serum and pituitary hormones were estimated. DOI significantly increased the activity and expression (both mRNA and protein levels) of; CYP2C11; ,; CYP3A1/23; , and; CYP3A2; , which positively correlated with an increase in the pituitary growth hormone-releasing hormone (GHRH) and serum GH level. The injection of 5-CT into the ARC did not affect the activity of liver P450 enzymes or hormone levels. The obtained results indicate that 5-HT; 2; , but not the 5-HT; 1; -type receptors in the ARC, are engaged in the positive neuroendocrine regulation of cytochrome P450, possibly by the stimulation of hypothalamic GHRH release and pituitary GH secretion, and an increase in the serum GH concentration. Further studies are going to identify which of the 5-HT; 2; receptor subtypes (5-HT; 2A; , 5-HT; 2B; , or 5-HT; 2C; ) is responsible for the observed neuroendocrine regulation of cytochrome P450

Stimulation of 5-HT; 2C; serotonin receptor subtype in the hypothalamic arcuate nuclei (ARC) increases the cytochrome P450 activity in the liver

Our previous study has demonstrated that activation of the 5-HT; 2; , but not 5-HT; 1; serotonin receptor type in the hypothalamic arcuate nucleus (ARC) is responsible for the neuroendocrine regulation of liver cytochrome P450. The goal of these studies was to determine whether 5-HT; 2C; serotonin receptor subtype in the ARC is engaged in the regulation of liver cytochrome P450.; The 5-HT; 2C; serotonin receptor agonist CP-809,101 was injected into the ARC for 5 days. The liver cytochrome P450 activity and protein level were measured.; In rats receiving an injection of the 5-HT; 2C; serotonin receptor agonist CP-809,101 into the ARC (1 μg/side) for five days, the activities of CYP2B, CYP2C11 and CYP3A significantly increased corresponding with the elevated enzyme protein level.; The obtained results suggest that the 5-HT; 2C; serotonin receptor subtype in the ARC is involved in the positive neuroendocrine regulation of cytochrome P450. Further studies are in progress to explain the physiological mechanism which is responsible for the observed regulation of cytochrome P450 by 5-HT; 2C; receptor present in the ARC

The activity of brain and liver cytochrome P450 2D (CYP2D) is differently affected by antidepressants in the chronic mild stress (CMS) model of depression in the rat

The effect of two second-generation antidepressants escitalopram and venlafaxine on the activity of brain and liver cytochrome P450 2D (CYP2D) involved in the metabolism of psychotropics and neurotransmitters was determined in the chronic mild stress (CMS) model of depression. Escitalopram or venlafaxine (10 mg/kg ip/day each) were administered to control and CMS rats for 5 weeks. The activity of CYP2D was studied by measurement of the rate of bufuralol 1'-hydroxylation in microsomes derived from the liver or different brain structures. The obtained results indicate that CMS and the studied antidepressants had different effects on the CYP2D activity depending on the location of the enzyme. In the brain, CMS produced an increase in the CYP2D activity in the hippocampus. Chronic escitalopram or venlafaxine had no effect on the CYP2D activity in the brain of nonstressed rats, however, the antidepressants increased the enzyme activity in the frontal cortex, hypothalamus and cerebellum of stressed animals. In the liver, CMS did not affect the CYP2D activity, while chronic escitalopram or venlafaxine significantly decreased the CYP2D activity and protein level in nonstressed and stressed rats. We conclude that: 1) CMS stimulates the CYP2D activity in the hippocampus and triggers the stimulatory effect of antidepressants on CYP2D in other brain structures; 2) the local brain metabolism of CYP2D substrates (neurosteroids, neurotransmitters, psychotropics) may be enhanced by CMS and/or antidepressants; 3) in contrast to the brain, the liver metabolism of CYP2D substrates may be slower during long-term treatment with escitalopram or venlafaxine

Damage to the Brain Serotonergic System Increases the Expression of Liver Cytochrome P450

Genes coding for cytochrome P450 are regulated by endogenous hormones such as the growth hormone, corticosteroids, thyroid, and sex hormones. Secretion of these hormones is regulated by the respective hypothalamus-pituitary-secretory organ axes. Since the brain sends its serotonergic projections from the raphe nuclei to the hypothalamus, we have assumed that damage to these nuclei may affect the neuroendocrine regulation of cytochrome P450 expression in the liver. Thereby, 5,7-dihydroxytryptamine (5,7-DHT), a serotonergic neurotoxin, was injected into the dorsal and median raphe nuclei of male Wistar rats. Ten days after the neurotoxin injections, the brain concentrations of neurotransmitters, serum hormone, and cytokine levels, as well as the expression of cytochrome P450 in the liver were measured. Injection of 5,7-DHT decreased serotonin concentration in the brain followed by a significant rise in the levels of the growth hormone, corticosterone, and testosterone, and a drop in triiodothyronine concentration in the serum. No changes in interleukin (IL) levels (IL-2 and IL-6) were observed. Simultaneously, the activity and protein level of liver CYP1A, CYP3A1, and CYP2C11 rose (the activity of CYP2A/2B/2C6/2D was not significantly changed). Similarly, the mRNA levels of CYP1A1, CYP1A2, CYP2C11, and CYP3A1 were elevated. This is the first report demonstrating the effect of intracerebral administration of serotonergic neurotoxin on liver cytochrome P450. The obtained results indicate involvement of the brain serotonergic system in the neuroendocrine regulation of liver cytochrome P450 expression. The physiologic and pharmacological significance of the findings is discussed

The role of brain noradrenergic system in the regulation of liver cytochrome P450 expression

The aim of the present study was to examine the effect of the brain noradrenergic system on the expression of cytochrome P450 in the liver. The experiment was carried out on male Wistar rats. Intracerebroventricular injection of the noradrenergic neurotoxin DSP-4 diminished noradrenaline level in the brain. Simultaneously, significant decreases in the serum concentration of the growth hormone, testosterone and the thyroid hormone thyroxine, as well as an increase in corticosterone level were observed. The concentrations of triiodothyronine and the cytokines interleukine 2 (IL-2) and 6 (IL-6) were not changed by DSP-4. The neurotoxin produced complex changes in the functioning of cytochrome P450. Significant decreases in the activity of liver CYP2C11 (measured as a rate of the 2α- and 16α-hydroxylation of testosterone) and CYP3A (measured as a rate of the 2β- and 6β-hydroxylation of testosterone) were found. In contrast, the activity of CYP1A (measured as a rate of caffeine metabolism) rose, while that of CYP2A (measured as a rate of the 7α-hydroxylation of testosterone), CYP2C6 (measured as a rate of the 7-hydroxylation of warfarin) and CYP2D (the 1'-hydroxylation of bufuralol) remained unchanged. The changes in the activity of CYP1A, CYP2C11 and CYP3A correlated positively with those in CYP protein levels and with the CYP mRNA levels of CYP1A1, CYP2C11 and CYP3A1/2 genes, respectively. The obtained results indicate an important role of the brain noradrenergic system in the neuroendocrine regulation of liver cytochrome P450 expression, which may be of significance in pathological states involving this system, or during pharmacotherapy with drugs affecting noradrenergic transmission

The Effect of the Selective N-methyl-D-aspartate (NMDA) Receptor GluN2B Subunit Antagonist CP-101,606 on Cytochrome P450 2D (CYP2D) Expression and Activity in the Rat Liver and Brain

The CYP2D enzymes of the cytochrome P450 superfamily play an important role in psychopharmacology, since they are engaged in the metabolism of psychotropic drugs and endogenous neuroactive substrates, which mediate brain neurotransmission and the therapeutic action of those drugs. The aim of this work was to study the effect of short- and long-term treatment with the selective antagonist of the GluN2B subunit of the NMDA receptor, the compound CP-101,606, which possesses antidepressant properties, on CYP2D expression and activity in the liver and brain of male rats. The presented work shows time-, organ- and brain-structure-dependent effects of 5-day and 3-week treatment with CP-101,606 on CYP2D. Five-day treatment with CP-101,606 increased the activity and protein level of CYP2D in the hippocampus. That effect was maintained after the 3-week treatment and was accompanied by enhancement in the CYP2D activity/protein level in the cortex and cerebellum. In contrast, a 3-week treatment with CP-101,606 diminished the CYP2D activity/protein level in the hypothalamus and striatum. In the liver, CP-101,606 decreased CYP2D activity, but not the protein or mRNA level, after 5-day or 3-week treatment. When added in vitro to liver microsomes, CP-101,606 diminished the CYP2D activity during prolonged incubation. While in the brain, the observed decrease in the CYP2D activity after short- and long-term treatment with CP-101,606 seems to be a consequence of the drug effect on enzyme regulation. In the liver, the direct inhibitory effect of reactive metabolites formed from CP-101,606 on the CYP2D activity may be considered. Since CYP2Ds are engaged in the metabolism of endogenous neuroactive substances, it can be assumed that apart from antagonizing the NMDA receptor, CP-101,606 may modify its own pharmacological effect by affecting brain cytochrome P450. On the other hand, an inhibition of the activity of liver CYP2D may slow down the metabolism of co-administered substrates and lead to pharmacokinetic drug–drug interactions