Study on mechanism of transdermal administration of eugenol for pain treatment by network pharmacology and molecular docking technology.

The objective of this study was to explore the pharmacological mechanism of transdermal administration of eugenol (EUG) for pain treatment. Firstly, network pharmacology techniques were employed to identify the potential targets responsible for the analgesic effect of EUG. Subsequently, molecular docking technology was used to validate interactions between EUG and the crystal structure of the core target protein. Finally, the impact of EUG on the expression and activation of TRPV1 receptors in HaCaT cells was evaluated through in vitro experiments, thus confirming the analysis of network pharmacology. The study suggested that the transdermal administration of EUG for pain treatment might target the TRPV1 receptor. Molecular docking revealed that EUG could spontaneously bind to the TRPV1 receptor with a high binding ability. The analysis of Western blot (WB) and intracellular Ca2+ levels demonstrated that EUG could increase the expression of TRPV1 in HaCaT cells, activating TRPV1 to induce intracellular Ca2+ influx (P < 0.05). These findings suggested that the initial application of EUG would cause a brief stimulation of TRPV1 receptors and upregulation of TRPV1 expression. Upon continued exposure, EUG would act as a TRPV1 agonist, increasing intracellular Ca2+ levels that might be associated with desensitization of pain sensations.

Biosynthesis of Enfumafungin-type Antibiotic Reveals an Unusual Enzymatic Fusion Pattern and Unprecedented C-C Bond Cleavage.

Enfumafungin-type antibiotics, represented by enfumafungin and fuscoatroside, belong to a distinct group of triterpenoids derived from fungi. These compounds exhibit significant antifungal properties with ibrexafungerp, a semisynthetic derivative of enfumafungin, recently gaining FDA's approval as the first oral antifungal drug for treating invasive vulvar candidiasis. Enfumafungin-type antibiotics possess a cleaved E-ring with an oxidized carboxyl group and a reduced methyl group at the break site, suggesting unprecedented C-C bond cleavage chemistry involved in their biosynthesis. Here, we show that a 4-gene (fsoA, fsoD, fsoE, fsoF) biosynthetic gene cluster is sufficient to yield fuscoatroside by heterologous expression in Aspergillus oryzae. Notably, FsoA is an unheard-of terpene cyclase-glycosyltransferase fusion enzyme, affording a triterpene glycoside product that relies on enzymatic fusion. FsoE is a P450 enzyme that catalyzes successive oxidation reactions at C19 to facilitate a C-C bond cleavage, producing an oxidized carboxyl group and a reduced methyl group that have never been observed in known P450 enzymes. Our study thus sets the important foundation for the manufacture of enfumafungin-type antibiotics using biosynthetic approaches.

MicroRNA let-7 Suppresses Influenza A Virus Infection by Targeting RPS16 and Enhancing Type I Interferon Response.

Given the frequent emergence of drug-resistant influenza virus strains and new highly pathogenic influenza virus strains, there is an urgent need to identify new antiviral drugs and targets. We found that influenza A virus (IAV) infection caused a significant decrease of microRNA let-7 expression in host cells; that overexpression of let-7 increased interferon expression and effectively inhibit IAV infection; and that let-7 targets the 3'-untranslated region (UTR) of the ribosomal protein 16 (RPS16) gene, decreasing its expression. Knocking down the expression of RPS16 increased the expression of type I interferon and inhibited viral replication. The present study uncovered the regulatory effect of let-7b and let-7f on influenza A infection, which is a potential biomarker of IAV infection. In addition, let-7 may be a promising therapeutic agent against influenza A.

Roles of corticotropin-releasing factor signaling in the lateral habenula in anxiety-like and alcohol drinking behaviors in male rats.

Corticotropin-releasing factor (CRF) signaling in the mesocorticolimbic system is known to modulate anxiety-like behavior and alcohol consumption, behaviors that also have been associated with the hyper-glutamatergic state of the lateral habenula (LHb) neurons in rats. However, the role of CRF signaling in the LHb on the glutamate transmission, anxiety-like behaviors and alcohol consumption is unknown. Here, we used male rats that had been consuming alcohol for three months to address this gap in the literature. First, using electrophysiological techniques, we evaluated CRF's effects on the glutamate transmission in LHb neurons in brain slices. CRF facilitated glutamate transmission. The facilitation was greater in neurons of alcohol-withdrawing rats than in those of naïve rats. The facilitation was mimicked by the activation of CRF receptor 1 (CRF1R) but attenuated by the activation of CRF receptor 2 (CRF2R). This facilitation was mediated by upregulating CRF1R-protein kinase A signaling. Conversely, protein kinase C blockade attenuated CRF's facilitation in neurons of naïve rats but promoted it in neurons of alcohol-withdrawing rats. Next, using site-direct pharmacology, we evaluated the role of CRF signaling in the LHb on anxiety-like behaviors and alcohol consumption. Intra-LHb inhibition of CRF1R or activation of CRF2R ameliorated the anxiety-like behaviors in alcohol-withdrawing rats and reduced their alcohol intake when drinking was resumed. These observations provide the first direct behavioral pharmacological and cellular evidence that CRF signaling in the LHb modulates glutamate transmission, anxiety-like behaviors and alcohol consumption, and that adaptation occurs in CRF signaling in the LHb after chronic alcohol consumption.

Salbutamol combined with budesonide in treatment of pediatric bronchial asthma and its effect on eosinophils.

BACKGROUND: The aim of this study was to investigate and discuss the salbutamol combined with budesonide in treatment of pediatric bronchial asthma (BA) and its effect on eosinophils (EOS). METHODS: Ninety-eight BA children admitted and treated in our hospital from July 2016 to June 2017 were collected and divided into control group (N.=49) and observation group (N.=49) according to random number table. The children in control group were treated with budesonide and those in observation group were treated with salbutamol combined with budesonide. The clinical efficacy, pulmonary functions and levels of T-lymphocyte subsets (including cluster of differentiation 3 (CD3)+, CD4+, CD8+ and CD4+/CD8+) in the immune system between two groups were compared after the treatment; the levels of eosinophil cationic protein (ECP) and eotaxin in the children were compared before the treatment and at 1, 4 and 8 weeks after the treatment; the changes in EOS counts in blood and induced sputum of the children before and after the treatment were compared, and the EOS apoptosis rate was compared at 1, 4 and 8 weeks after the treatment. RESULTS: The effective rate of treatment in observation group was significantly higher than that in control group (P<0.05). After the treatment, the indexes of pulmonary function in observation group were obviously better than those in control group (P<0.05). Compared with those in control group, the levels of CD3+, CD4+ and CD4+/CD8+ of the children in observation group were elevated remarkably, while the CD8+ level was lowered (P<0.05). The levels of ECP and eotaxin in the two groups were decreased after the treatment compared with those before the treatment, and the levels in observation group were superior to those in control group (P<0.05). After the treatment, the EOS counts of both groups of children were lower than those before the treatment, and the decrease in observation group was more notable than that in control group. At 1, 4 and 8 weeks after the treatment, the EOS apoptosis rate in observation group was obviously higher than that in control group (P<0.05). CONCLUSIONS: The treatment of salbutamol combined with budesonide for pediatric BA has significant therapeutic effects; it can restore the pulmonary functions rapidly and improve the immunity of the lung, reduce the levels of eotaxin, ECP and EOS of the child patients and promote EOS apoptosis.

Inhibition of the ROS-EGFR Pathway Mediates the Protective Action of Nox1/4 Inhibitor GKT137831 against Hypertensive Cardiac Hypertrophy via Suppressing Cardiac Inflammation and Activation of Akt and ERK1/2.

Oxidative stress, inflammation, and hypertension constitute a self-perpetuating vicious circle to exacerbate hypertension and subsequent hypertensive cardiac hypertrophy. NADPH oxidase (Nox) 1/4 inhibitor GKT137831 alleviates hypertensive cardiac hypertrophy in models of secondary hypertension; however, it remains unclear about its effect on hypertensive cardiac hypertrophy in models of essential hypertension. This study is aimed at determining the beneficial role of GKT137831 in hypertensive cardiac hypertrophy in spontaneously hypertensive rats (SHRs) and its mechanisms of action. Treating with GKT137831 prevented cardiac hypertrophy in SHRs. Likewise, decreasing production of reactive oxygen species (ROS) with GKT137831 reduced epidermal growth factor receptor (EGFR) activity in the left ventricle of SHRs. Additionally, EGFR inhibition also reduced ROS production in the left ventricle and blunted hypertensive cardiac hypertrophy in SHRs. Moreover, inhibition of the ROS-EGFR pathway with Nox1/4 inhibitor GKT137831 or selective EGFR inhibitor AG1478 reduced protein and mRNA levels of proinflammatory cytokines tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 1ß (IL-1ß), as well as the activities of Akt and extracellular signal-regulated kinase (ERK) 1/2 in the left ventricle of SHRs. In summary, GKT137831 prevents hypertensive cardiac hypertrophy in SHRs, Nox-deprived ROS regulated EGFR activation through positive feedback in the hypertrophic myocardium, and inhibition of the ROS-EGFR pathway mediates the protective role of GKT137831 in hypertensive cardiac hypertrophy via repressing cardiac inflammation and activation of Akt and ERK1/2. This research will provide additional details for GKT137831 to prevent hypertensive cardiac hypertrophy.

Anxiety during alcohol withdrawal involves 5-HT2C receptors and M-channels in the lateral habenula.

Anxiety disorders often co-occur with alcohol use disorders, but the mechanisms underlying this comorbidity remain elusive. Previously, we reported that rats withdrawn from chronic alcohol consumption (Post-EtOH rats) exhibited robust anxiety-like behaviors (AB), which were accompanied by neuronal hyperexcitability, and the downregulation of M-type potassium channels (M-channels) in the lateral habenula (LHb); and that serotonin (5-HT) stimulated LHb neurons via type 2C receptors (5-HT2CRs). Also, 5-HT2CR activation is known to inhibit M-current in mouse hypothalamic neurons. The present study investigated whether LHb 5-HT2CRs and M-channels contribute to AB in adult male Long-Evans rats. We used the intermittent-access to 20% ethanol two-bottle free-choice drinking paradigm to induce dependence. We measured AB with the elevated plus-maze, open-field, and marble-burying tests at 24 h withdrawal. We found that intra-LHb infusion of SB242084, a selective 5-HT2CR antagonist alleviated AB and reduced the elevated c-Fos expression in the LHb of Post-EtOH rats. By contrast, intra-LHb infusion of the selective 5-HT2CR agonist WAY161503 induced AB and increased c-Fos expression in the LHb in alcohol-naive but not Post-EtOH rats. Also, intra-LHb SB242084 significantly reduced self-administration of alcohol intake in the operant chambers. Furthermore, both 5-HT2CR protein levels and 5-HIAA/5-HT ratio was increased in the LHb of Post-EtOH rats. Finally, intra-LHb SB242084 increased LHb KCNQ2/3 membrane protein expression in Post-EtOH rats. Collectively, these results suggest that enhanced LHb 5-HT2CR signaling that interacted with M-channels triggers AB in Post-EtOH rats and that 5-HT2CRs may be a promising target for treating comorbid anxiety disorders in alcoholics.

Differences between male and female rats in alcohol drinking, negative affects and neuronal activity after acute and prolonged abstinence.

Alcohol consumption afflicts men and women differently. However, the underlying neuronal mechanisms that contribute to the difference are mostly unexplored. Although more men suffer from alcohol use disorders (AUD), women more frequently accelerate to dependence and develop adverse consequences of alcoholism sooner than men. Women also exhibit more significant negative emotions that cues more reactivity and alcohol-craving than men. Despite ample evidence that women are vulnerable to AUD, results of preclinical studies on sex differences in alcohol consumption and withdrawal-related behaviors are inconclusive. In this study, we trained adult male and female Sprague-Dawley rats to drink alcohol in the intermittent access to 20% ethanol two-bottle free-choice paradigm for two months. Their behaviors and Fos expression in related brain regions were measured at acute (24 h) and after prolonged (28 days) abstinence. We found that female rats drank more alcohol than males. After acute abstinence, rats of both sexes showed higher sensitivity to depressive, thermal, and mechanical stimuli. Females also displayed higher anxiety levels. After prolonged abstinence, rats of both sexes displayed depressive-like behaviors; the males displayed allodynia; the females showed higher anxiety levels and drank more alcohol upon reaccess to alcohol. Furthermore, during acute withdrawal, Fos-positive nuclei were increased in the prefrontal cortex, anterior cingulate cortex (ACC), nucleus accumbens (NAc), amygdala and lateral habenula (LHb) in the females, versus only in the ACC, amygdala, and LHb in the males. Conversely, after prolonged abstinence, Fos-positive nuclei were decreased in the prefrontal cortex, ACC, and NAc in the females, but fell in the ACC, NAc, and LHb of the males. Thus, adaptations in diverse brain regions may contribute to the sex differences in behaviors in ethanol-withdrawn rats.

Adaptation in 5-HT2 receptors-CaMKII signaling in lateral habenula underlies increased nociceptive-sensitivity in ethanol-withdrawn rats.

Alcoholics often experience hyperalgesia, especially during abstinence, yet the underlying cellular and molecular bases are unclear. Recent evidence suggests that 5-HT type 2 receptors (5-HT2Rs) at glutamatergic synapses on lateral habenula (LHb) neurons may play a critical role. We, therefore, measured paw withdrawal responses to thermal and mechanical stimuli, and alcohol intake in a rat model of intermittent drinking paradigm, as well as spontaneous glutamatergic transmission (sEPSCs), and firing of LHb neurons in brain slices. Here, we report that nociceptive sensitivity was higher in rats at 24 h withdrawal from chronic alcohol consumption than that of alcohol-naive counterparts. The basal frequency of sEPSCs and firings was higher in slices of withdrawn rats than that of Naïve rats, and 5-HT2R antagonists attenuated the enhancement. Also, an acute ethanol-induced increase of sEPSCs and firings was smaller in withdrawal than in Naïve rats; it was attenuated by 5-HT2R antagonists but mimicked by 5-HT2R agonists. Importantly, intra-LHb infusion of 5-HT2R agonists increased nociceptive sensitivity in Naïve rats, while antagonists or 5-HT reuptake blocker decreased nociceptive sensitivity and alcohol intake in withdrawn rats. Additionally, KN-62, a CaMKII inhibitor, attenuated the enhancement of EPSCs and firing induced by acute alcohol and by 5-HT2R agonist. Furthermore, intra-LHb KN-62 reduced nociceptive sensitivity and alcohol intake. Quantitative real-time PCR assay detected mRNA of 5-HT2A and 2C in the LHb. Thus adaptation in 5-HT2R-CaMKII signaling pathway contributes to the hyper-glutamatergic state, the hyperactivity of LHb neurons as well as the higher nociceptive sensitivity in rats withdrawn from chronic alcohol consumption.

Alcohol withdrawal drives depressive behaviors by activating neurons in the rostromedial tegmental nucleus.

Rostromedial tegmental nucleus (RMTg) GABA neurons exert a primary inhibitory drive onto midbrain dopamine neurons and are excited by a variety of aversive stimuli. There is, however, little evidence that the RMTg-ventral tegmental area (VTA)-nucleus accumbens shell (Acb) circuit plays a role in the aversive consequences of alcohol withdrawal. This study was performed in adult male Long-Evans rats at 48-h withdrawal from chronic alcohol drinking in the intermittent schedule. These rats displayed clear anhedonia and depression-like behaviors, as measured with the sucrose preference, and forced swimming tests. These aberrant behaviors were accompanied by a substantial increase in cFos expression in the VTA-projecting RMTg neurons, identified by a combination of immunohistochemistry and retrograde-tracing techniques. Pharmacological or chemogenetic inhibition of RMTg neurons mitigated the anhedonia and depression-like behaviors. Ex vivo electrophysiological data showed that chemogenetic inactivation of RMTg neurons reduced GABA release and accelerated spontaneous firings of VTA dopamine neurons. Finally, using a functional hemispheric disconnection procedure, we demonstrated that inhibition of unilateral RMTg, when combined with activation of D1 and D2 dopamine receptors in the contralateral (but not ipsilateral) Acb, mitigated the anhedonia and depression-like behaviors in alcohol-withdrawal rats. These data show that the integrity in the RMTg-VTA-Acb pathway in a single hemisphere is sufficient to elicit depression-like behavior during ethanol-withdrawal. Overall, the present results reveal that the RMTg-VTA-Acb pathway plays a crucial role in the depression-like behavior in animals undergoing alcohol withdrawal, further advocating the RMTg as a potential therapeutic target for alcoholism.

Neoproterozoic rifting in the Upper Yangtze Continental Block: Constraints from granites in the Well W117 borehole, South China.

Well W117 in the Sichuan Basin reveals a suite of ~814 Ma quartz monzonites, unconformably overlain by Sinian clastic and carbonate sediments. The quartz monzonites contain no muscovite and amphibole, and are characterized by high SiO2 (72.26-77.93%), total alkali, and TFe2O3/MgO content, and low P2O5 and CaO abundance, with variable A/CNK ratio (0.93-1.19), classified as metaluminous to weakly aluminous highly fractionated I-type granites. They are preserved in the Neoproterozoic rift and exhibit restricted negative εNd(t) values (-7.0 to -5.2) and variable zircon εHf(t) values (-13.9 to 2.3), suggesting their generation via melting of both ancient and juvenile crustal materials in an extensional setting. Their parent magmas were formed in a low-temperature condition (831-650 °C) and finally emplaced at ca. 9-10 km below the surface, indicating that the intrusion underwent exhumation before the deposition of Sinian sag basin. Such geological processes, together with evidence for Neoproterozoic structures in the surrounding area, support that the Upper Yangtze craton experienced two main phases of rifting from 830-635 Ma. The Well W117 granites and its overlying sediments record a geodynamic evolution from orogenic collapse to continental rifting, and to thermal subsidence, probably related to the Rodinia supercontinent breakup.

Autoantibodies to NR2A Peptide of the Glutamate/NMDA Receptor in Patients with Seizure Disorders in Neuropsychiatric Systemic Lupus Erythematosus.

Objective. Seizure disorders are one of the most disabling, life-threatening, and the least understood syndromes associated with neuropsychiatric SLE (NPSLE). N-Methyl-D-aspartate (NMDA) receptors are a subgroup of the glutamate receptor family, whose NR2A subunit was found on neuronal cells (anti-NR2A) in NPSLE patients with different types of epilepsy. The present study was conducted to determine the serum levels of anti-NR2A antibodies in a large group of SLE patients, to investigate the possible correlation between the presence of the NR2A specific antibodies and NPSLE-related seizure disorders. Methods and Results. The study population consisted of 107 SLE patients and 43 age- and sex-matched healthy controls. 73 SLE patients had active disease. 36 of these had NPSLE. NMDA levels were measured by ELISA. Clinical and serological parameters were assessed according to routine procedures. The levels of anti-NR2A antibodies were significantly higher in NPSLE patients, compared with non-NPSLE patients and healthy controls. Furthermore, the levels of NPSLE in patients with seizure disorders were shown to be higher than in those with cognitive dysfunction and other CNS symptoms, however, without significance. Increase in serum anti-NR2A antibodies levels correlated to anti-dsDNA antibody and SLEDAI as well as complement levels. Conclusion. We suggest that anti-NR2A antibodies play a role in the pathogenesis of NPSLE with seizure disorders.

Low-dose ethanol excites lateral habenula neurons projecting to VTA, RMTg, and raphe.

It is unclear how social drinking can contribute to the development of addiction in susceptible individuals. However, alcohol's aversive properties are a well-known factor contributing to its abuse. The lateral habenula (LHb) is a key brain structure responding to various aversive stimuli, including those related to alcohol. We recently reported that ethanol at 10 mM or less that can be achieved by social drinking activates many LHb neurons and drives aversive conditioning. The current study sought to identify LHb circuits that are activated by a low-dose of ethanol using immunohistochemistry and anatomic tracing techniques on adult Sprague-Dawley rats. We showed here that an intraperitoneal injection of ethanol (0.25 g/kg), resulting in a blood ethanol concentration of 5.6 mM, significantly increased the number of cFos immunoreactive (IR) neurons in the LHb. Most of the ethanol-activated cFos-IR LHb neurons expressed vGluT2 (vesicular glutamate transporters 2, a marker of a glutamatergic phenotype). These LHb neurons projected to the ventral tegmental area (VTA), rostromedial tegmental nucleus (RMTg), and dorsal raphe. Moreover, injections of the anterograde tracer AAV-CaMKIIa-eGFP into the lateral hypothalamus produced a significant amount of labeled fibers with vGluT2 positive terminals on the ethanol-activated LHb cells. These results indicate that the LHb neurons stimulated by a low-dose of ethanol project to the VTA, RMTg, and dorsal raphe, and receive excitatory projections from the lateral hypothalamus. These neurocircuits may play a crucial role in mediating the initial aversive effects produced by a low-dose of ethanol.