Machine learning-derived immunosenescence index for predicting outcome and drug sensitivity in patients with skin cutaneous melanoma.

The functions of immunosenescence are closely related to skin cutaneous melanoma (SKCM). The aim of this study is to uncover the characteristics of immunosenescence index (ISI) to identify novel biomarkers and potential targets for treatment. Firstly, integrated bioinformatics analysis was carried out to identify risk prognostic genes, and their expression and prognostic value were evaluated. Then, we used the computational algorithm to estimate ISI. Finally, the distribution characteristics and clinical significance of ISI in SKCM by using multi-omics analysis. Patients with a lower ISI had a favorable survival rate, lower chromosomal instability, lower somatic copy-number alterations, lower somatic mutations, higher immune infiltration, and sensitive to immunotherapy. The ISI exhibited robust, which was validated in multiple datasets. Besides, the ISI is more effective than other published signatures in predicting survival outcomes for patients with SKCM. Single-cell analysis revealed higher ISI was specifically expressed in monocytes, and correlates with the differentiation fate of monocytes in SKCM. Besides, individuals exhibiting elevated ISI levels could potentially receive advantages from chemotherapy, and promising compounds with the potential to target high ISI were recognized. The ISI model is a valuable tool in categorizing SKCM patients based on their prognosis, gene mutation signatures, and response to immunotherapy.

Efficacy of psychosocial interventions to reduce affective symptoms in sexual and gender minorities: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Lesbian, gay, bisexual, transgender, and queer/questioning (LGBTQ) individuals are more likely than cisgender heterosexuals to experience mental, physical, and sexual health issues. A promising contemporary strategy to address the issue of affective symptoms in sexual and gender minorities (SGM) is psychosocial intervention. OBJECTIVE: To systematically evaluate the effect of psychosocial interventions on the improvement of affective symptoms in SGM, and to provide a reference for the implementation of effective psychological interventions for SGM with affective symptoms. METHODS: Between the date of database construction until December 10, 2022, a computerized search of the English-language literature published both nationally and worldwide was done. 8 literature databases and 3 additional gray databases were searched. We gathered randomized controlled trials that used psychological interventions for SGM. To evaluate risk bias in included papers in accordance with Cochrane cooperation criteria, we used Review Manager 5.4 software. In conjunction with post-test and follow-up data, mean differences were standardized using Stata 12.0 software. Subgroup analysis was used to investigate the cause of heterogeneity. The study was conducted strictly in accordance with PRISMA guidelines, and it was registered on the PROSPERO platform (CRD42023408610). RESULTS: This review covered 18 research, and 14 studies were included in the meta-analysis. A total of 1194 study cases, including 706 cases from the control group and 488 cases from the experimental group, were included in these investigations. Compared to the control group, the psychosocial intervention group had significantly lower levels of depression (standardized mean difference (SMD) = -0.17;95% CI = [-0.30, -0.04]; p = 0.012) and anxiety (SMD = -0.22; 95% CI = [-0.41, -0.04]; p = 0.01), but no significant differences were found for distress (SMD = -0.19; 95% CI = [-0.45,0.07]; p = 0.021). CONCLUSION: According to this study, psychosocial interventions helped lessen the symptoms of depression and anxiety in SGM but had no significant effect on their psychological distress. To assess the impact of psychological intervention on SGM, more randomized controlled trials with larger sample sizes and numerous follow-up times should be done.

The core role of macrophages in hepatocellular carcinoma: the definition of molecular subtypes and the prognostic risk system.

Background: In patients with hepatocellular carcinoma (HCC), the tumor microenvironment (TME) is resistant to immunotherapy because of its specificity. It is meaningful to explore the role of macrophage, which is one of the most abundant immune cells in the TME, in cellular communication and its effect on the prognosis and immunotherapy of HCC. Methods: Dimensionality reduction and clustering of the single-cell RNA-seq data from the GSE149614 dataset were carried out to identify the cellular composition of HCC. CellChat was used to analyze the communication between different cells. The specifically highly expressed genes of macrophages were extracted for univariate Cox regression analysis to obtain prognostic genes for HCC cluster analysis, and the risk system of macrophage-specifically highly expressed genes was developed by random forest analysis and multivariate Cox regression analysis. Prognosis, TME infiltration, potential responses to immunotherapy, and antineoplastic drugs were compared among molecular subtypes and between risk groups. Results: We found that HCC included nine identifiable cell types, of which macrophages had the highest communication intensity with each of the other eight cell types. Of the 179 specifically highly expressed genes of macrophage, 56 were significantly correlated with the prognosis of HCC, which classified HCC into three subtypes, which were reproducible and produced different survival outcomes, TME infiltration, and immunotherapy responses among the subtypes. In the integration of four macrophage-specifically highly expressed genes for the development of a risk system, the risk score was significantly involved in higher immune cell infiltration, poor prognosis, immunotherapy response rate, and sensitivity of six drugs. Conclusion: In this study, through single-cell RNA-seq data, we identified nine cell types, among which macrophage had the highest communication intensity with the rest of the cell types. Based on specifically highly expressed genes of macrophage, we successfully divided HCC patients into three clusters with distinct prognosis, TME, and therapeutic response. Additionally, a risk system was constructed, which provided a potential reference index for the prognostic target and preclinical individualized treatment of HCC.

Efficacy and safety of modified fasting therapy for weight loss in 2054 hospitalized patients.

OBJECTIVE: The aim of this study was to evaluate the efficacy and safety of modified fasting therapy, and a retrospective study was conducted to analyze changes in clinical indicators of hospitalized fasting patients. METHODS: A total of 2054 hospitalized fasting patients were enrolled in this observational study. All participants underwent 7 days of modified fasting therapy. The clinical efficacy biomarkers, safety indicators, and body composition were measured before and after fasting. RESULTS: The modified fasting therapy reduced body weight, BMI, abdominal circumference, systolic blood pressure, and diastolic blood pressure significantly. Blood glucose and indicators of body composition were improved to various extents (all p < 0.05). There was a small increase in liver function, kidney function, uric acid, electrolytes, blood count, coagulation, and uric biomarkers. Subgroup analysis results showed that cardiovascular diseases benefited from modified fasting therapy. CONCLUSIONS: At present this study is the largest retrospective population-based study about modified fasting therapy. The results from 2054 patients showed that the modified fasting therapy lasting 7 days was efficient and safe. It led to improvements in physical health and body weight-associated indicators, as well as body composition and relevant cardiovascular risk factors.

Understanding MMPI-2 response structure between schizophrenia and healthy individuals.

Background: Using Minnesota Multiphasic Personality Inventory-2 (MMPI-2) clinical scales to evaluate clinical symptoms in schizophrenia is a well-studied topic. Nonetheless, research focuses less on how these clinical scales interact with each other. Aims: Investigates the network structure and interaction of the MMPI-2 clinical scales between healthy individuals and patients with schizophrenia through the Bayesian network. Method: Data was collected from Wuhan Psychiatric Hospital from March 2008 to May 2018. A total of 714 patients with schizophrenia and 714 healthy subjects were identified through propensity score matching according to the criteria of the International Classification of Diseases (ICD-11). Separated MMPI-2 clinical scales Bayesian networks were built for healthy subjects and patients with schizophrenia, respectively. Results: The Bayesian network showed that the lower 7 scale was a consequence of the correlation between the lower 2 scale and the greater 8 scale. A solely lower 7 scale does yield neither a lower 2 scale nor a higher 8 scale. The proposed method showed 72% of accuracy with 78% area under the ROC curve (AUC), similar to the previous studies. Limitations: The proposed method simplified the continuous Bayesian network to predict binary outcomes, including other categorical data is not explored. Besides, the participants might only represent an endemic as they come from a single hospital. Conclusion: This study identified MMPI-2 clinical scales correlation and built separated Bayesian networks to investigate the difference between patients with schizophrenia and healthy people. These differences may contribute to a better understanding of the clinical symptoms of schizophrenia and provide medical professionals with new perspectives for diagnosis.

Online psychosocial interventions for improving mental health in people during the COVID-19 pandemic: A systematic review and meta-analysis.

BACKGROUND: During the COVID-19 pandemic, interventions for public mental health were mostly delivered through online modalities. Although many studies have explored the effectiveness of online psychosocial interventions through randomized controlled trials, there is a lack of quantitative synthesis of the effectiveness of online psychosocial interventions and an examination of their overall application. OBJECTIVE: To understand the commonly used psychosocial interventions during the COVID-19 pandemic and the effectiveness of the interventions. METHODS: Risk bias was assessed in the included studies according to the Cochrane Collaboration criteria, and data from post-test and follow-up were combined for standardized mean differences using Stata 16.0 software, and sources of heterogeneity were explored by subgroup analysis, and risk bias was assessed in the included studies using Review Manager 5.4 software. The study was written in strict accordance with PRISMA specifications, and registration was completed on the PROSPERO platform (CRD42022302917). RESULTS: The online psychosocial intervention had an ameliorating effect on anxiety (SMD = -0.78), depression (SMD = -0.80), and insomnia (SMD = -0.19) in the public during the COVID-19 pandemic, was ineffective for the intervention on stress, and the effectiveness of the intervention on depression continued at follow-up. Subgroup analyses showed that the type of intervention, intervention form, Duration of intervention, and setting of the control group influenced the trial results to some extent, with cognitive behavioral therapy being the most effective intervention for anxiety and depressive symptoms, self-help interventions being more effective than interventions with therapist interventions, and 1-4 week interventions being more effective than 5-8 week interventions. Due to the limited number of studies included in the analysis and variability in quality, more randomized controlled trials are needed to test the findings. CONCLUSION: Online psychosocial interventions can be effective in improving symptoms of anxiety, depression, stress, and insomnia in the public during the COVID-19 pandemic. However, additional randomized controlled trial studies are needed to determine which types of interventions are more appropriate for which populations and how they can be implemented to achieve better intervention outcomes.

A meta-analysis of co-occurrence of non-suicidal self-injury and suicide attempt: Implications for clinical intervention and future diagnosis.

Background: Non-suicidal self-injury (NSSI) and suicide attempt (SA) are risk behaviors that lead to physical injury and even death in individuals, and are a very powerful risk factor when both occur together, with individuals presenting with more severe psychological and behavioral problems. Due to the different demographic characteristics of different study subjects, an overall understanding of the incidence and occurrence of this co-occurrence is lacking to clarify the focus of clinical interventions and future research directions. Methods: A systematic search was conducted for relevant studies in English and Chinese that reported data on co-occurring non-suicidal self-injury and suicide attempts as of May 2022. The incidence of co-occurrence of non-suicidal self-injury and suicide attempt (NSSI + SA) was calculated using Stata version 16.0 software based on a random-effects model, and the differences in incidence in different populations were compared by subgroups of age group, comorbidity, and time of occurrence. The study was written in strict accordance with PRISMA norms and registration was completed on the PROSPERO platform (CRD42022329095). Results: A total of 37 studies (139,573 individuals) were included for meta-analysis, and the combined incidence of non-suicidal self-injury and suicide attempt co-occurrence was 9.6%. Among different groups, the prevalence of NSSI + SA was 10, 11, 6, and 26% in adolescents and young adults, adults, the general population, and people with mental illness, respectively, and the co-occurrence of NSSI + SA within 12 months was 17%. Conclusion: There is a significant group with a history of both non-suicidal self-injury and suicide attempts and presenting with more severe symptoms clinically. Targeted prevention and intervention are urgently needed, but the direction of intervention needs further research on the occurrence trajectory of this co-occurrence. Systematic Review Registration: http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42022329095, identifier: CRD42022329095.

LINC00922 promotes the proliferation, migration, invasion and EMT process of liver cancer cells by regulating miR-424-5p/ARK5.

AMPK-related protein kinase 5 (ARK5) promotes the deterioration of hepatocellular carcinoma (HCC). From the perspective of lncRNA-miRNA-mRNA, this study explored in-depth the intervention mechanism of ARK5. The binding relationship between miR-424-5p and two genes (LINC00922 and ARK5) were analyzed by Bioinformatics and dual-luciferase experiments. After clinical sample collection, the expressions of miR-424-5p, LINC00922 and ARK5 in HCC tissues were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The correlation between LINC00922, miR-424-5p, and ARK5 in HCC tissues was analyzed by Pearson correlation. The influences of miR-424-5p, LINC00922 and ARK5 on the basic functions (viability, migration and invasion) of cancer cells were detected by cell counting kit-8, wound healing, and Transwell experiments, and their regulatory effects on related genes, as well as their relationship, were tested by qRT-PCR and Western blot. MiR-424-5p was low expressed, whereas LINC00922 and ARK5 were high expressed in HCC tissues. MiR-424-5p was negatively associated with LINC00922 and ARK5 that was positively associated with LINC00922. Interestingly, LINC00922 partially shared an identical binding site of miR-424-5p with ARK5. LINC00922 its overexpression partially offset the inhibitory effect of miR-424-5p on cancer cell functions. ARK5 silencing repressed the malignant phenotype of cancer cells and inhibited the expressions of epithelial-to-mesenchymal transition (EMT)-related molecules (Vimentin, Snail and N-Cadherin). However, these effects were partially neutralized by miR-424-5p inhibitors. LINC00922 increases the cell viability, migration, invasion and EMT process of HCC cells by regulating the miR-424-5p/ARK5 axis, and thus may serve as a potential target for targeted therapy.

Protective effects of calorie restriction on insulin resistance and islet function in STZ-induced type 2 diabetes rats.

BACKGROUND: Caloric restriction (CR) has become increasingly attractive in the treatment of type 2 diabetes mellitus (T2DM) because of the increasingly common high-calorie diet and sedentary lifestyle. This study aimed to evaluate the role of CR in T2DM treatment and further explore its potential molecular mechanisms. METHODS: Sixty male Sprague-Dawley rats were used in this study. The diabetes model was induced by 8 weeks of high-fat diet (HFD) followed by a single dose of streptozotocin injection (30 mg/kg). Subsequently, the diabetic rats were fed HFD at 28 g/day (diabetic control) or 20 g/day (30% CR regimen) for 20 weeks. Meanwhile, normal rats fed a free standard chow diet served as the vehicle control. Body mass, plasma glucose levels, and lipid profiles were monitored. After diabetes-related functional tests were performed, the rats were sacrificed at 10 and 20 weeks, and glucose uptake in fresh muscle was determined. In addition, western blotting and immunofluorescence were used to detect alterations in AKT/AS160/GLUT4 signaling. RESULTS: We found that 30% CR significantly attenuated hyperglycemia and dyslipidemia, leading to alleviation of glucolipotoxicity and thus protection of islet function. Insulin resistance was also markedly ameliorated, as indicated by notably improved insulin tolerance and homeostatic model assessment for insulin resistance (HOMA-IR). However, the improvement in glucose uptake in skeletal muscle was not significant. The upregulation of AKT/AS160/GLUT4 signaling in muscle induced by 30% CR also attenuated gradually over time. Interestingly, the consecutive decrease in AKT/AS160/GLUT4 signaling in white adipose tissue was significantly reversed by 30% CR. CONCLUSION: CR (30%) could protect islet function from hyperglycemia and dyslipidemia, and improve insulin resistance. The mechanism by which these effects occurred is likely related to the upregulation of AKT/AS160/GLUT4 signaling.

Rural-urban disparities in knowledge, behaviors, and mental health during COVID-19 pandemic: A community-based cross-sectional survey.

ABSTRACT: To examine the knowledge level, behaviors, and psychological status of the Chinese population during the COVID-19 pandemic, and to explore the differences between urban and rural areas.We carried out a cross-sectional survey of the knowledge, behaviors related to COVID-19, and mental health in a probability sample of 3001 community residents in 30 provinces or districts across China from February 16-23, 2020. Convenience sampling and a snowball sampling were adopted. We used General Anxiety Disorder (GAD), the 9-item Patient Health Questionnaire (PHQ-9), and knowledge and behaviors questionnaire of community residents regarding COVID-19 designed by us to investigate the psychological status, disease-related knowledge, and the behavior of Chinese urban and rural residents during the pandemic.The average score of anxiety and depression among urban residents was 9.15 and 11.25, respectively, while the figures in rural areas were 8.69 and 10.57, respectively. There was a statistically significant difference in the levels of anxiety (P < .01) and depression (P < .01). Urban participants reported significantly higher levels of knowledge regarding COVID-19 in all aspects (transmission, prevention measures, symptoms of infection, treatment, and prognosis) (P < .01), compared to their rural counterparts. While a majority of respondents in urban areas obtained knowledge through WeChat, other apps, and the Internet (P < .01), residents in rural areas accessed information through interactions with the community (P < .01). Urban residents fared well in exchanging knowledge about COVID-19 and advising others to take preventive measures (P < .01), but fared poorly in advising people to visit a hospital if they displayed symptoms of the disease, compared to rural residents (P < .01). Regression analysis with behavior showed that being female (OR = 2.106, 95%CI = 1.259-3.522), aged 18 ≤ age < 65 (OR = 4.059, 95%CI = 2.166-7.607), being satisfied with the precautions taken by the community (OR = 2.594, 95%CI = 1.485-4.530), disinfecting public facilities in the community (OR = 2.342, 95%CI = 1.206-4.547), having knowledge of transmission modes (OR = 3.987, 95%CI: 2.039, 7.798), symptoms (OR = 2.045, 95%CI = 1.054-4.003), and outcomes (OR = 2.740, 95%CI = 1.513-4.962) of COVID-19, and not having anxiety symptoms (OR = 2.578, 95%CI = 1.127-5.901) were positively associated with affirmative behavior in urban areas. Being married (OR = 4.960, 95%CI = 2.608-9.434), being satisfied with the precautions taken by the community (OR = 2.484, 95%CI = 1.315-4.691), screening to ensure face mask wearing before entering the community (OR = 8.809, 95%CI = 2.649-19.294), and having knowledge about precautions (OR = 4.886, 95%CI = 2.604-9.167) and outcomes (OR = 2.657, 95%CI = 1.309-5.391) were positively associated with acceptable conduct in rural areas.The status of anxiety and depression among urban residents was more severe compared to those living in rural areas. There was a difference in being positively associated with constructive behaviors between rural and urban areas.

The Biological Characteristics of Novel H5N6 Highly Pathogenic Avian Influenza Virus and Its Pathogenesis in Ducks.

Clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses (HPAIVs) have caused outbreaks in poultry in the world. Some of these viruses acquired internal genes from other subtype avian influenza viruses (AIVs) such as H9 and H6 for the generation of novel reassortant viruses and continually circulated in poultry. Here, we applied a duck-origin virus DK87 and a chicken-origin virus CK66 to assess the biological characteristics of novel reassortant H5N6 HPAIVs and its pathogenesis in ducks. A genetic analysis indicated that the HA genes of the two H5N6 HPAIVs were closely related to the H5 viruses of clade 2.3.4.4 circulating in Eastern Asia and classified into H5 AIV/Eastern Asia (EA)-like lineage. Their NA genes fell into Eurasian lineage had close relationship with those of H5N6 viruses circulating in China, Laos, Vietnam, Japan, and Korea. All internal genes of DK87 were aggregated closely with H5 AIV/EA-like viruses. The internal genes (PB1, PA, NP, M, and NS) of CK66 were derived from H9N2 AIV/SH98-like viruses and the PB2 were derived from H5 AIV/EA-like viruses. These results indicate that clade 2.3.4.4 H5N6 AIVs have continually evolved and recombined with the H9N2 viruses circulating in Southern China. Pathogenicity test showed that the two viruses displayed a broader tissue distribution in ducks and caused no clinical signs. These results indicated that ducks were permissive for the replication of the chicken-origin reassortant virus CK66 without prior adaptation, but the duck-origin virus DK87-inoculated ducks showed significantly higher viral titers in some organs than the CK66-inoculated ducks at 5 day post-inoculated (DPI). The recovery of viruses from oropharyngea and cloacal swabs of contacted ducks indicated that they transmitted in native ducks by direct contact. Quantitative reverse transcription PCR (qRT-PCR) results revealed that the immune-relative genes (PRRs, IFNs, Mx-1, IL-6, and IL-8) in the lungs of inoculated ducks were expressed regardless of virus origin, but the expression of these genes was significantly higher in response to infection with the DK87 virus compared to the CK66 virus at 3 DPI. Overall, we should provide further insights into how clade 2.3.4.4 H5N6 AIVs undergo genetic and pathogenic variations to prevent outbreaks of this disease.

The preparation of bifunctional hybrid nano-flowers and their application in the enzyme-linked immunosorbent assay for Helicobacter pylori detection.

As the infection by Helicobacter pylori (H. pylori, HP) remains for a lifetime and may induce diseases such as gastric cancer, it is vital to detect and diagnose it. A new non-invasive indirect enzyme-linked immunosorbent assay (iELISA) method based on nano-flowers (NFs) is very advantageous for the sensitive detection of HP. Furthermore, the established iELISA method based on the organic-inorganic bifunctional hybrid nano-flowers including rabbit polyclonal antibody of HP labeled with peroxidase from horseradish (R-HP-Ab-HRP@Cu2+ NFs) showed linearity with HP at a concentration of 0-105 CFU mL-1 (R2 = 0.9997). Moreover, the limit of detection (LOD) reached 50 CFU mL-1, and not only was the detection sensitivity 20 times higher than that based on rabbit polyclonal antibody of HP labeled with peroxidase from horseradish (R-HP-Ab-HRP) but also the stability of R-HP-Ab-HRP in NFs was improved. In addition, the OD450 nm value was still linearly related to the concentration of HP at a range of 0-105 CFU mL-1 (R2 = 0.9952) with a LOD of 50 CFU mL-1 in an artificial saliva system. This study provided a sensitive, low-cost and convenient method for the non-invasive detection of HP.

The residents' mental health status and community's role during the COVID-19 pandemic: a community-based cross-sectional study in China.

BACKGROUND: The related research of coronavirus disease 2019 (COVID-19) pandemic on the mental health of community residents is still lacking. Here we reported the mental health status of Chinese residents as well as the community's prevention and control during the pandemic period of COVID-19 and further explored the influencing factors of mental status. METHODS: In this cross-sectional study, convenience sampling and snowball sampling methods were adopted from February 16 to February 23, 2020, and Chinese community residents were included according to the inclusion and exclusion criteria. Three questionnaires, including General Anxiety Disorder 7 (GAD-7), Patient Health Questionnaire 9 (PHQ-9), and a self-designed "Community prevention and control questionnaire", were used. A multivariate linear regression analysis was conducted to analyze the impact factors of anxiety and depression. RESULTS: A total of 3,001 community residents were included in this study. In total, 85.6% and 83.7% of participants had minimal anxiety and depression, respectively. Among all the community residents, 16.6% of participants proved that the communities they lived in had confirmed cases. 95.3% of participants reported that the residents were screened for mobility and contact history. 97.8% of participants reported the entrance and exit of the community were managed in their communities. There were 97.5% and 99% of participants needed to take body temperature and wear masks in their communities. Ninety-two percent (92%) of communities have their public areas and facilities disinfected daily, and 95.4% of communities have conducted health education about COVID-19. Factors including gender, education level, chronic illness, the frequency of going out, achieving information about COVID-19 by community and newspaper, and confirmed cases in the community, show association with community residents' anxiety and depression. CONCLUSIONS: The vast majority of Chinese residents have little anxiety and depression, and most communities have adopted standardized control measures following the government's regulations and policy, which plays a vital role in the control of COVID-19 and improving residents' anxiety and depression.

ARK5 promotes invasion and migration in hepatocellular carcinoma cells by regulating epithelial-mesenchymal transition.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated mortality worldwide. The highly invasive nature of HCC leads to poor prognosis in patients with malignant HCC. AMPK-related protein kinase 5 (ARK5) is a key mediator of migratory activity in human cancer cells. However, the role of ARK5 in invasion and metastasis of HCC cells remains unclear. The present study attempted to determine whether ARK5 is involved in invasion and migration via regulation of epithelial-mesenchymal transition (EMT). Wound healing and Transwell Matrigel invasion assays were utilized to detect the ability of the epithelial Huh7 and mesenchymal SNU387 HCC cells to migrate and invade. Next, the expression of ARK5 and EMT markers, E-cadherin and vimentin, were examined by western blot analysis. Inhibition of ARK5 was able to significantly reduce the ability HCC cells to invade and metastasize. Furthermore, the knockdown of ARK5 was able to reverse the process of EMT in HCC cells. These data suggested that ARK5 may serve an important role in regulating EMT in HCC cells. Taken together, these findings indicate that ARK5 is a potential molecular target for the development of novel HCC therapeutics, which focus on cell invasion and EMT regulation.

Disturbances in behavior and cortical enkephalin gene expression during the anticipation of ethanol in rats characterized as high drinkers.

The process of ethanol anticipation is a particularly important phenomenon that can determine subsequent drug-taking behavior. Recent studies suggest that systems within the medial prefrontal cortex (mPFC), during anticipation, may contribute to the goal-directed seeking of ethanol. The current investigation examined the possibility that the opioid peptide enkephalin (ENK), known to mediate some of the reinforcing properties of ethanol, may function in the mPFC during the anticipation of ethanol access. Using a limited access (3 h/d) paradigm for 10 days with 20% ethanol, Sprague-Dawley rats were first identified either as low drinkers (LD, <1.0 g/kg/3 h) or as high drinkers (HD, >2.0 g/kg/3 h) that exhibited a long-term phenotype of high ethanol consumption and a significant ethanol deprivation effect. During the anticipation period immediately preceding daily ethanol access, the HD rats compared to LD or Control animals with ad libitum ethanol access exhibited increased anticipatory behaviors, including greater exploratory behavior in a novel open field as revealed by significantly more time spent in the rearing position (+53-65%, p < 0.05) and increased number of rears made (+33-44%, p < 0.05) and greater novelty-seeking behavior in a hole-board apparatus revealed by an increase in total (+50-52%, p < 0.05) and novel nose pokes (+45-48%, p < 0.05). In the HD rats, analysis of the mPFC using real-time quantitative PCR showed significantly greater mRNA levels of ENK (p < 0.05) and the mu-opioid receptor (MOR) (p < 0.05), but not delta-opioid receptor (DOR), and this increase in ENK expression was found, using in situ hybridization, to occur specifically in the prelimbic (PrL) subregion of the mPFC. When injected into the PrL during the anticipation period, a MOR agonist but not DOR agonist significantly increased consumption of 20% ethanol (p < 0.05). These findings support the role of ENK, acting through MOR within the PrL to promote the anticipation and excessive consumption of ethanol.

Differential effects of acute and chronic ethanol exposure on orexin expression in the perifornical lateral hypothalamus.

BACKGROUND: Recent reports support the involvement of hypothalamic orexigenic peptides in stimulating ethanol intake. Our previous studies have examined the effects of ethanol on hypothalamic peptide systems of the paraventricular nucleus (PVN) and identified a positive feedback loop in which PVN peptides, such as enkephalin and galanin, stimulate ethanol intake and ethanol, in turn, stimulates the expression of these peptides. Recently, orexin (OX), a peptide produced mainly by cells in the perifornical lateral hypothalamus (PFLH), has been shown to play an important role in mediating the rewarding aspects of ethanol intake. However, there is little evidence showing the effects that ethanol itself may have on the OX peptide system. In order to understand the feedback relationship between ethanol and the OX system, the current investigation was designed to measure OX gene expression in the PFLH following acute as well as chronic ethanol intake. METHODS: In the first experiment, Sprague-Dawley rats were trained to voluntarily consume a 2 or 9% concentration of ethanol, and the expression of OX mRNA in the PFLH was measured using quantitative real-time polymerase chain reaction (qRT-PCR). The second set of experiments tested the impact of acute oral gavage of 0.75 and 2.5 g/kg ethanol solution on OX expression in the PFLH using qRT-PCR, as well as radiolabeled in situ hybridization. Further tests using digoxigenin-labeled in situ hybridization and immunofluorescence histochemistry allowed us to more clearly distinguish the effects of acute ethanol on OX cells in the lateral hypothalamic (LH) versus perifornical (PF) regions. RESULTS: The results showed chronic consumption of ethanol versus water to dose-dependently reduce OX mRNA in the PFLH, with a larger effect observed in rats consuming 2.5 g/kg/d (-70%) or 1.0 g/kg/d (-50%) compared to animals consuming 0.75 g/kg/d (-40%). In contrast to chronic intake, acute oral ethanol compared to water significantly enhanced OX expression in the PFLH, and this effect occurred at the lower (0.75 g/kg) but not higher (2.5 g/kg) dose of ethanol. Additional analyses of the OX cells in the LH versus PF regions identified the former as the primary site of ethanol's stimulatory effect on the OX system. In the LH but not the PF, acute ethanol increased the density of OX-expressing and OX-immunoreactive neurons. The increase in gene expression was detected only at the lower dose of ethanol (0.75 g/kg), whereas the increase in OX peptide was seen only at the higher dose of ethanol (2.5 g/kg). CONCLUSION: These results lead us to propose that OX neurons, while responsive to negative feedback signals from chronic ethanol consumption, are stimulated by acute ethanol administration, most potently in the LH where OX may trigger central reward mechanisms that promote further ethanol consumption.