The TF/Nrf2/GSTP1 pathway is involved in stress-induced hepatocellular injury through ferroptosis.

Stress triggers a comprehensive pathophysiological cascade in organisms. However, there is a substantial gap in the research regarding the effects of stress on liver function. This study aimed to investigate the impact of restraint stress on hepatocellular damage and elucidate the underlying molecular mechanisms. An effective mouse restraint stress model was successfully developed, and liver function analysis was performed using laser speckle imaging, metabolomics and serum testing. Alterations in hepatocyte morphology were assessed using haematoxylin and eosin staining and transmission electron microscopy. Oxidative stress in hepatocytes was assessed using lipid reactive oxygen species and malondialdehyde. The methylation status and expression of GSTP1 were analysed using DNA sequencing and, real-time PCR, and the expression levels of GPX4, TF and Nrf2 were evaluated using real-time quantitative PCR, western blotting, and immunohistochemical staining. A stress-induced model was established in vitro by using dexamethasone-treated AML-12 cells. To investigate the underlying mechanisms, GSTP1 overexpression, small interfering RNA, ferroptosis and Nrf2 inhibitors were used. GSTP1 methylation contributes to stress-induced hepatocellular damage and dysfunction. GSTP1 is involved in ferroptosis-mediated hepatocellular injury induced by restraint stress via the TF/Nrf2 pathway. These findings suggest that stress-induced hepatocellular injury is associated with ferroptosis, which is regulated by TF/Nrf2/GSTP1.

The association between DNA methylation and human height and a prospective model of DNA methylation-based height prediction.

As a vital anthropometric characteristic, human height information not only helps to understand overall developmental status and genetic risk factors, but is also important for forensic DNA phenotyping. We utilized linear regression analysis to test the association between each CpG probe and the height phenotype. Next, we designed a methylation sequencing panel targeting 959 CpGs and subsequent height inference models were constructed for the Chinese population. A total of 11,730 height-associated sites were identified. By employing KPCA and deep neural networks, a prediction model was developed, of which the cross-validation RMSE, MAE and R2 were 5.62 cm, 4.45 cm and 0.64, respectively. Genetic factors could explain 39.4% of the methylation level variance of sites used in the height inference models. Collectively, we demonstrated an association between height and DNA methylation status through an EWAS analysis. Targeted methylation sequencing of only 959 CpGs combined with deep learning techniques could provide a model to estimate human height with higher accuracy than SNP-based prediction models.

Integrative lncRNA, circRNA, and mRNA analysis reveals expression profiles of six forensic body fluids/tissue.

RNAs have attracted much attention in forensic body fluid/tissue identification (BFID) due to their tissue-specific expression characteristics. Among RNAs, long RNAs (e.g., mRNA) have a higher probability of containing more polymorphic sites that can be used to assign the specific donor of the body fluid/tissue. However, few studies have characterized their overall profiles in forensic science. In this study, we sequenced the transcriptomes of 30 samples from venous blood, menstrual blood, semen, saliva, vaginal secretion, and skin tissue, obtaining a comprehensive picture of mRNA, lncRNA, and circRNA profiles. A total of 90,305 mRNAs, 102,906 lncRNAs (including 19,549 novel lncRNAs), and 40,204 circRNAs were detected. RNA type distribution, length distribution, and expression distribution were presented according to their annotation and expression level, and many novel body fluid/tissue-specific RNA markers were identified. Furthermore, the cognate relations among the three RNAs were analyzed according to gene annotations. Finally, SNPs and InDels from RNA transcripts were genotyped, and 21,611 multi-SNP and 4,471 multi-InDel transcriptomic microhaplotypes (tMHs) were identified. These results provide a comprehensive understanding of transcriptome profiles, which could provide new avenues for tracing the origin of the body fluid/tissue and identifying an individual.

Differential cannabinoid-like effects and pharmacokinetics of ADB-BICA, ADB-BINACA, ADB-4en-PINACA and MDMB-4en-PINACA in mice: A comparative study.

Despite synthetic cannabinoids' (SCs) prevalent use among humans, these substances often lack comprehensive pharmacological data, primarily due to their rapid emergence in the market. This study aimed to discern differences and causal factors among four SCs (ADB-BICA, ADB-BINACA, ADB-4en-PINACA and MDMB-4en-PINACA), with respect to locomotor activity, body temperature and nociception threshold. Adult male C57BL/6 mice received intraperitoneal injections of varying doses (0.5, 0.1 and 0.02 mg/kg) of these compounds. Three substances (including ADB-BINACA, ADB-4en-PINACA and MDMB-4en-PINACA) demonstrated dose- and time-dependent hypolocomotive and hypothermic effects. Notably, 0.1 mg/kg MDMB-4en-PINACA exhibited analgesic properties. However, ADB-BICA did not cause any effects. MDMB-4en-PINACA manifested the most potent and sustained effects, followed by ADB-4en-PINACA, ADB-BINACA and ADB-BICA. Additionally, the cannabinoid receptor 1 (CB1R) antagonist AM251 suppressed the effects induced by acute administration of the substances. Analysis of molecular binding configurations revealed that the four SCs adopted a congruent C-shaped geometry, with shared linker binding pockets conducive to robust steric interaction with CB1R. Essential residues PHE268 , PHE200 and SER173 within CB1R were identified as pivotal contributors to enhancing receptor-ligand associations. During LC-MS/MS analysis, 0.5 mg/kg MDMB-4en-PINACA exhibited the highest plasma concentration and most prolonged detection window post-administration. The study of SCs' pharmacological and pharmacokinetic profiles is crucial for better understanding the main mechanisms of cannabinoid-like effects induced by SCs, interpreting clinical findings related to SC uses and enhancing SCs risk awareness.

Roles of lncLingo2 and its derived miR-876-5p in the acquisition of opioid reinforcement.

Recent studies found that non-coding RNAs (ncRNAs) played crucial roles in drug addiction through epigenetic regulation of gene expression and underlying drug-induced neuroadaptations. In this study, we characterized lncRNA transcriptome profiles in the nucleus accumbens (NAc) of mice exhibiting morphine-conditioned place preference (CPP) and explored the prospective roles of novel differentially expressed lncRNA, lncLingo2 and its derived miR-876-5p in the acquisition of opioids-associated behaviours. We found that the lncLingo2 was downregulated within the NAc core (NAcC) but not in the NAc shell (NAcS). This downregulation was found to be associated with the development of morphine CPP and heroin intravenous self-administration (IVSA). As Mfold software revealed that the secondary structures of lncLingo2 contained the sequence of pre-miR-876, transfection of LV-lncLingo2 into HEK293 cells significantly upregulated miR-876 expression and the changes of mature miR-876 are positively correlated with lncLingo2 expression in NAcC of morphine CPP trained mice. Delivering miR-876-5p mimics into NAcC also inhibited the acquisition of morphine CPP. Furthermore, bioinformatics analysis and dual-luciferase assay confirmed that miR-876-5p binds to its target gene, Kcnn3, selectively and regulates morphine CPP training-induced alteration of Kcnn3 expression. Lastly, the electrophysiological analysis indicated that the currents of small conductance calcium-activated potassium (SK) channel was increased, which led to low neuronal excitability in NAcC after CPP training, and these changes were reversed by lncLingo2 overexpression. Collectively, lncLingo2 may function as a precursor of miR-876-5p in NAcC, hence modulating the development of opioid-associated behaviours in mice, which may serve as an underlying biomarker and therapeutic target of opioid addiction.

The effects of restraint stress on ceramide metabolism disorders in the rat liver: the role of CerS6 in hepatocyte injury.

BACKGROUND: Stress is implicated in various pathological conditions leading to liver injury. Existing evidence suggests that excessive stress can induce mitochondrial damage in hepatocytes, yet the underlying mechanism remains unclear. Ceramide synthase 6 (CerS6)-derived C16:0 ceramide is recognised as a lipotoxic substance capable of causing mitochondrial damage. However, the role of CerS6 in stress has received insufficient attention. This study aimed to explore the involvement of CerS6 in stress-induced hepatic damage and its associated mechanisms. METHODS: The rat restraint stress model and a corticosterone (CORT)-induced hepatocyte stress model were employed for in vivo and in vitro experimental analyses, respectively. Changes in mitochondrial damage and ceramide metabolism in hepatocytes induced by stress were evaluated. The impact of CORT on mitochondrial damage and ceramide metabolism in hepatocytes was assessed following CerS6 knockdown. Mitochondria were isolated using a commercial kit, and ceramides in liver tissue and hepatocytes were detected by LC-MS/MS. RESULTS: In comparison to the control group, rats subjected to one week of restraint exhibited elevated serum CORT levels. The liver displayed significant signs of mitochondrial damage, accompanied by increased CerS6 and mitochondrial C16:0 ceramide, along with activation of the AMPK/p38 MAPK pathway. In vitro studies demonstrated that CORT treatment of hepatocytes resulted in mitochondrial damage, concomitant with elevated CerS6 and mitochondrial C16:0 ceramide. Furthermore, CORT induced sequential phosphorylation of AMPK and p38 MAPK proteins, and inhibition of the p38 MAPK pathway using SB203580 mitigated the CORT-induced elevation in CerS6 protein. Knocking down CerS6 in hepatocytes inhibited both the increase in C16:0 ceramide and the release of mitochondrial cytochrome c induced by CORT. CONCLUSIONS: CerS6-associated C16:0 ceramide plays a mediating role in stress-induced mitochondrial damage in hepatocytes. The molecular mechanism is linked to CORT-induced activation of the AMPK/p38 MAPK pathway, leading to upregulated CerS6.

Advances and Strategies towards Synthesis of Aspidosperma Indole Alkaloids Goniomitine.

Goniomitine is of the aspidosperma alkaloid family, with an angularly fused tetracyclic skeleton housing an all-carbon quaternary carbon chiral center alongside an aminal functional group. This natural product has garnered attention as a synthetic target due to its intriguing molecular architecture and anti-proliferative activity in recent years. Following the first synthesis of (-)-goniomitine by Takano in 1991, synthetic chemists have developed various methods. This review provides an overview of the methodologies used in the synthesis of goniomitine in racemic and enantiopure forms via divergent construction indole framework, indole functionalization, and the integrated oxidation/reduction/cyclization (iORC) sequence from 1991 to 2023.

Region-Specific Effects of Metformin on Gut Microbiome and Metabolome in High-Fat Diet-Induced Type 2 Diabetes Mouse Model.

The glucose-lowering drug metformin alters the composition of the gut microbiome in patients with type 2 diabetes mellitus (T2DM) and other diseases. Nevertheless, most studies on the effects of this drug have relied on fecal samples, which provide limited insights into its local effects on different regions of the gut. Using a high-fat diet (HFD)-induced mouse model of T2DM, we characterize the spatial variability of the gut microbiome and associated metabolome in response to metformin treatment. Four parts of the gut as well as the feces were analyzed using full-length sequencing of 16S rRNA genes and targeted metabolomic analyses, thus providing insights into the composition of the microbiome and associated metabolome. We found significant differences in the gut microbiome and metabolome in each gut region, with the most pronounced effects on the microbiomes of the cecum, colon, and feces, with a significant increase in a variety of species belonging to Akkermansiaceae, Lactobacillaceae, Tannerellaceae, and Erysipelotrichaceae. Metabolomics analysis showed that metformin had the most pronounced effect on microbiome-derived metabolites in the cecum and colon, with several metabolites, such as carbohydrates, fatty acids, and benzenoids, having elevated levels in the colon; however, most of the metabolites were reduced in the cecum. Thus, a wide range of beneficial metabolites derived from the microbiome after metformin treatment were produced mainly in the colon. Our study highlights the importance of considering gut regions when understanding the effects of metformin on the gut microbiome and metabolome.

Severe Hypothermia Induces Ferroptosis in Cerebral Cortical Nerve Cells.

Abnormal shifts in global climate, leading to extreme weather, significantly threaten the safety of individuals involved in outdoor activities. Hypothermia-induced coma or death frequently occurs in clinical and forensic settings. Despite this, the precise mechanism of central nervous system injury due to hypothermia remains unclear, hindering the development of targeted clinical treatments and specific forensic diagnostic indicators. The GEO database was searched to identify datasets related to hypothermia. Post-bioinformatics analyses, DEGs, and ferroptosis-related DEGs (FerrDEGs) were intersected. GSEA was then conducted to elucidate the functions of the Ferr-related genes. Animal experiments conducted in this study demonstrated that hypothermia, compared to the control treatment, can induce significant alterations in iron death-related genes such as PPARG, SCD, ADIPOQ, SAT1, EGR1, and HMOX1 in cerebral cortex nerve cells. These changes lead to iron ion accumulation, lipid peroxidation, and marked expression of iron death-related proteins. The application of the iron death inhibitor Ferrostatin-1 (Fer-1) effectively modulates the expression of these genes, reduces lipid peroxidation, and improves the expression of iron death-related proteins. Severe hypothermia disrupts the metabolism of cerebral cortex nerve cells, causing significant alterations in ferroptosis-related genes. These genetic changes promote ferroptosis through multiple pathways.

Combined Diagnostic Value of Hsa-miR-592 and Hsa-miR-9-3p in Plasma for Methamphetamine Addicts.

A number of studies have reported that drug addiction is associated with microRNAs (miRNAs). However, the roles of plasma miRNAs in methamphetamine (METH) addicts have not been clearly explained. This study aimed to profile a panel of miRNAs as non-invasive predictive biomarkers and therapeutic targets for METH addiction. Differentially expressed miRNAs were derived from next-generation sequencing technology (NGS) and were validated by quantitative real-time PCR (RT-qPCR). The diagnostic value of specific altered miRNAs was evaluated by receiver operating characteristic (ROC) analysis and area under the curve (AUC). NGS results revealed that 63 miRNAs were significantly altered in the METH-exposed paradigm. The levels of hsa-miR-592, hsa-miR-9-3p, hsa-miR-206 and hsa-let-7b-3p were significantly elevated in the plasma of METH addicts. Hsa-miR-9-3p was a useful biomarker discriminating METH addicts from normal (AUC was 0.756). Importantly, combining detection of hsa-miR-592 and hsa-miR-9-3p achieved the highest AUC of 0.87, with a sensitivity and specificity of 82.7% and 78.9%, respectively. Target gene BDNF decreased significantly in METH addicts. Although METH addicts showed significant depressive symptoms, there was no correlation between the expression level of miR-592 and miR-9-3p and the degree of depression. Our findings suggested that hsa-miR-592, hsa-miR-9-3p, hsa-miR-206, and hsa-let-7b-3p may play a potential role in the pathology of METH addiction, and a combination of hsa-miR-592 and hsa-miR-9-3p could serve as potential peripheral biomarker and therapeutic target for METH addiction.

The Regulation of Frontal Cortex Cholesterol Metabolism Abnormalities by NR3C1/NRIP1/NR1H2 Is Involved in the Occurrence of Stress-Induced Depression.

Stress-induced alterations in central neuron metabolism and function are crucial contributors to depression onset. However, the metabolic dysfunctions of the neurons associated with depression and specific molecular mechanisms remain unclear. This study initially analyzed the relationship between cholesterol and depression using the NHANES database. We then induced depressive-like behaviors in mice via restraint stress. Applying bioinformatics, pathology, and molecular biology, we observed the pathological characteristics of brain cholesterol homeostasis and investigated the regulatory mechanisms of brain cholesterol metabolism disorders. Through the NHANES database, we initially confirmed a significant correlation between cholesterol metabolism abnormalities and depression. Furthermore, based on successful stress mouse model establishment, we discovered the number of cholesterol-related DEGs significantly increased in the brain due to stress, and exhibited regional heterogeneity. Further investigation of the frontal cortex, a brain region closely related to depression, revealed stress caused significant disruption to key genes related to cholesterol metabolism, including HMGCR, CYP46A1, ACAT1, APOE, ABCA1, and LDLR, leading to an increase in total cholesterol content and a significant decrease in synaptic proteins PSD-95 and SYN. This indicates cholesterol metabolism affects neuronal synaptic plasticity and is associated with stress-induced depressive-like behavior in mice. Adeno-associated virus interference with NR3C1 in the prefrontal cortex of mice subjected to short-term stress resulted in reduced protein levels of NRIP1, NR1H2, ABCA1, and total cholesterol content. At the same time, it increased synaptic proteins PSD95 and SYN, effectively alleviating depressive-like behavior. Therefore, these results suggest that short-term stress may induce cholesterol metabolism disorders by activating the NR3C1/NRIP1/NR1H2 signaling pathway. This impairs neuronal synaptic plasticity and consequently participates in depressive-like behavior in mice. These findings suggest that abnormal cholesterol metabolism in the brain induced by stress is a significant contributor to depression onset.

Phosphorylated CPI-17 and MLC2 as Biomarkers of Coronary Artery Spasm-Induced Sudden Cardiac Death.

Coronary artery spasm (CAS) plays an important role in the pathogeneses of various ischemic heart diseases and has gradually become a common cause of life-threatening arrhythmia. The specific molecular mechanism of CAS has not been fully elucidated, nor are there any specific diagnostic markers for the condition. Therefore, this study aimed to examine the specific molecular mechanism underlying CAS, and screen for potential diagnostic markers. To this end, we successfully constructed a rat CAS model and achieved in vitro culture of a human coronary-artery smooth-muscle cell (hCASMC) contraction model. Possible molecular mechanisms by which protein kinase C (PKC) regulated CAS through the C kinase-potentiated protein phosphatase 1 inhibitor of 17 kDa (CPI-17)/myosin II regulatory light chain (MLC2) pathway were studied in vivo and in vitro to screen for potential molecular markers of CAS. We performed hematoxylin and eosin staining, myocardial zymogram, and transmission electron microscopy to determine myocardial and coronary artery injury in CAS rats. Then, using immunohistochemical staining, immunofluorescence staining, and Western blotting, we further demonstrated a potential molecular mechanism by which PKC regulated CAS via the CPI-17/MLC2 pathway. The results showed that membrane translocation of PKCα occurred in the coronary arteries of CAS rats. CPI-17/MLC2 signaling was observably activated in coronary arteries undergoing CAS. In addition, in vitro treatment of hCASMCs with angiotensin II (Ang II) increased PKCα membrane translocation while consistently activating CPI-17/MLC2 signaling. Conversely, GF-109203X and calphostin C, specific inhibitors of PKC, inactivated CPI-17/MLC2 signaling. We also collected the coronary artery tissues from deceased subjects suspected to have died of CAS and measured their levels of phosphorylated CPI-17 (p-CPI-17) and MLC2 (p-MLC2). Immunohistochemical staining was positive for p-CPI-17 and p-MLC2 in the tissues of these subjects. These findings suggest that PKCα induced CAS through the CPI-17/MLC2 pathway; therefore, p-CPI-17 and p-MLC2 could be used as potential markers for CAS. Our data provide novel evidence that therapeutic strategies against PKC or CPI-17/MLC2 signaling might be promising in the treatment of CAS.

The application of vibrational spectroscopy in forensic analysis of biological evidence.

Vibrational spectroscopy is a powerful analytical domain, within which Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy stand as exemplars, offering high chemical specificity and sensitivity. These methodologies have been instrumental in the characterization of chemical compounds for an extensive period. They are particularly adept at the identification and analysis of minute sample quantities. Both FTIR and Raman spectroscopy are proficient in elucidating small liquid samples and detecting nuanced molecular alterations. The application of chemometrics further augments their analytical prowess. Currently, these techniques are in the research phase within forensic medicine and have yet to be broadly implemented in examination and identification processes. Nonetheless, studies have indicated that a combined classification model utilizing FTIR and Raman spectroscopy yields exceptional results for the identification of biological fluid-related information and the determination of causes of death. The objective of this review is to delineate the current research trajectory and potential applications of these two vibrational spectroscopic techniques in the detection of body fluids and the ascertainment of causes of death within the context of forensic medicine.

Endoplasmic reticulum stress induced by turbulence of mitochondrial fusion and fission was involved in stressed cardiomyocyte injury.

Mitochondria are sensitive organelles that sense intrinsic and extrinsic stressors and maintain cellular physiological functions through the dynamic homeostasis of mitochondrial fusion and fission. Numerous pathological processes are associated with mitochondrial fusion and fission disorders. However, the molecular mechanism by which stress induces cardiac pathophysiological changes through destabilising mitochondrial fusion and fission is unclear. Therefore, this study aimed to investigate whether the endoplasmic reticulum stress signalling pathway initiated by the turbulence of mitochondrial fusion and fission under stressful circumstances is involved in cardiomyocyte damage. Based on the successful establishment of the classical stress rat model of restraint plus ice water swimming, we measured the content of serum lactate dehydrogenase. We used haematoxylin-eosin staining, special histochemical staining, RT-qPCR and western blotting to clarify the cardiac pathology, ultrastructural changes and expression patterns of mitochondrial fusion and fission marker proteins and endoplasmic reticulum stress signalling pathway proteins. The results indicated that mitochondrial fusion and fission markers and proteins of the endoplasmic reticulum stress JNK signalling pathway showed significant abnormal dynamic changes with the prolongation of stress, and stabilisation of mitochondrial fusion and fission using Mdivi-1 could effectively improve these abnormal expressions and ameliorate cardiomyocyte injury. These findings suggest that stress could contribute to pathological cardiac injury, closely linked to the endoplasmic reticulum stress JNK signalling pathway induced by mitochondrial fusion and fission turbulence.

CCK1R2R-/- ameliorates myocardial damage caused by unpredictable stress via altering fatty acid metabolism.

The heart is the main organ of the circulatory system and requires fatty acids to maintain its activity. Stress is a contributor to aggravating cardiovascular diseases and even death, and exacerbates the abnormal lipid metabolism. The cardiac metabolism may be disturbed by stress. Cholecystokinin (CCK), which is a classical peptide hormone, and its receptor (CCKR) are expressed in myocardial cells and affect cardiovascular function. Nevertheless, under stress, the exact role of CCKR on cardiac function and cardiac metabolism is unknown and the mechanism is worth exploring. After unpredictable stress, a common stress-inducing model that induces the development of mood disorders such as anxiety and reduces motivated behavior, we found that the abnormal contraction and diastole of the heart, myocardial injury, oxidative stress and inflammation of mice were aggravated. Cholecystokinin A receptor and cholecystokinin B receptor knockout (CCK1R2R-/-) significantly reversed these changes. Mechanistically, fatty acid metabolism was found to be altered in CCK1R2R-/- mice. Differential metabolites, especially L-tryptophan, L-aspartic acid, cholesterol, taurocholic acid, ADP, oxoglutaric acid, arachidonic acid and 17-Hydroxyprogesterone, influenced cardiac function after CCK1R2R knockout and unpredictable stress. We conclude that CCK1R2R-/- ameliorated myocardial damage caused by unpredictable stress via altering fatty acid metabolism.

Prediction of BMI traits in the Chinese population based on the gut metagenome.

BACKGROUND: Identifying individual characteristics based on trace evidence left at a crime scene is crucial in forensic identification. Microbial communities found in fecal traces have high individual specificity and could serve as potential markers for forensic characterization. Previous research has established that predicting body type based on the relative abundance of the gut microbiome is relatively accurate. However, the long-term stability and high individual specificity of the gut microbiome are closely linked to changes at the genome level of the microbiome. No studies have been conducted to deduce body shape from genetic traits. Therefore, in this study, the vital role of gut bacterial community characteristics and genetic traits in predicting body mass index (BMI) was investigated using gut metagenomic data from a healthy Chinese population. RESULTS: Regarding the gut microbial community, the underweight group displayed increased α-diversity in comparison to the other BMI groups. There were significant differences in the relative abundances of 19 species among these three BMI groups. The BMI prediction model, based on the 31 most significant species, showed a goodness of fit (R2) of 0.56 and a mean absolute error (MAE) of 2.09 kg/m2. The overweight group exhibited significantly higher α-diversity than the other BMI groups at the level of gut microbial genes. Furthermore, there were significant variations observed in the single-nucleotide polymorphism (SNP) density of 732 contigs between these three BMI groups. The BMI prediction model, reliant on the 62 most contributing contigs, exhibited a model R2 of 0.72 and an MAE of 1.56 kg/m2. The model predicting body type from 44 contigs correctly identified the body type of 93.55% of the study participants. CONCLUSION: Based on metagenomic data from a healthy Chinese population, we demonstrated the potential of genetic traits of gut bacteria to predict an individual's BMI. The findings of this study suggest the effectiveness of a novel method for determining the body type of suspects in forensic applications using the genetic traits of the gut microbiome and holds great promise for forensic individual identification.

Interleukin 6 (IL-6) Regulates GABAA Receptors in the Dorsomedial Hypothalamus Nucleus (DMH) through Activation of the JAK/STAT Pathway to Affect Heart Rate Variability in Stressed Rats.

The dorsomedial hypothalamus nucleus (DMH) is an important component of the autonomic nervous system and plays a critical role in regulating the sympathetic outputs of the heart. Stress alters the neuronal activity of the DMH, affecting sympathetic outputs and triggering heart rate variability. However, the specific molecular mechanisms behind stress leading to abnormal DMH neuronal activity have still not been fully elucidated. Therefore, in the present study, we successfully constructed a stressed rat model and used it to investigate the potential molecular mechanisms by which IL-6 regulates GABAA receptors in the DMH through activation of the JAK/STAT pathway and thus affects heart rate variability in rats. By detecting the c-Fos expression of neurons in the DMH and electrocardiogram (ECG) changes in rats, we clarified the relationship between abnormal DMH neuronal activity and heart rate variability in stressed rats. Then, using ELISA, immunohistochemical staining, Western blotting, RT-qPCR, and RNAscope, we further explored the correlation between the IL-6/JAK/STAT signaling pathway and GABAA receptors. The data showed that an increase in IL-6 induced by stress inhibited GABAA receptors in DMH neurons by activating the JAK/STAT signaling pathway, while specific inhibition of the JAK/STAT signaling pathway using AG490 obviously reduced DMH neuronal activity and improved heart rate variability in rats. These findings suggest that IL-6 regulates the expression of GABAA receptors via the activation of the JAK/STAT pathway in the DMH, which may be an important cause of heart rate variability in stressed rats.

[Analysis and evaluation on Paeoniae Radix Alba from different cultivars by UPLC-Q-TOF-MS and HPLC].

In this study, an established ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) method was combined with multivariate statistical analysis to investigate the commonality and difference of main chemical components in the medicinal parts of Paeonia lactiflora from different cultivars; in addition, a high performance liquid chromatography(HPLC) method was established to simultaneously determine the content of eight active components in Paeoniae Radix Alba. Non-targeted analysis was carried out by UPLC-Q-TOF-MS on a Waters ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm, 1.7 µm) column with a gradient elution of 0.1% aqueous formic acid(A)-acetonitrile(B) as the mobile phase at a flow rate of 0.2 mL·min~(-1). The column temperature was 30 ℃, and an electrospray ionization source was used to acquire mass spectrometry data in positive and negative ion modes. According to the accurate molecular weight and fragment ion information provided by multi-stage mass spectrometry and by comparison with reference substances and literature reports, thirty-six identical components were identified in Paeoniae Radix Alba from different cultivars with positive and negative ion modes. In the negative ion mode, two groups of samples were well separated; specifically, seventeen components with significant differences in content were screened and identified, and one component unique in "Bobaishao" was obtained. Quantitative analysis was conducted by high-performance liquid chromatography(HPLC) on an Agilent HC-C_(18)(4.6 mm×250 mm, 5 µm) column with a gradient elution of 0.1% aqueous phosphoric acid(A)-acetonitrile(B) as the mobile phase at a flow rate of 1.0 mL·min~(-1). The column temperature was 30 ℃ and the detection wavelength was at 230 nm. An HPLC method was developed for the simultaneous determination of eight active components(gallic acid, oxypaeoniflorin, catechin, albiflorin, paeoniflorin, galloylpaeoniflorin, 1,2,3,4,6-O-pentagalloylglucose, benzoyl-paeoniflorin) in Paeoniae Radix Albaa from different cultivars. Satisfactory linearity was achieved within the investigated linear ranges and with fine coefficients(r>0.999 0), and the methodological investigation showed that the method had good precision, repeatability and stability. The mean recoveries were 90.61% to 101.7% with RSD of 0.12% to 3.6%(n=6). UPLC-Q-OF-MS provided a rapid and efficient qualitative analytical method for the identification of the chemical components in Paeoniae Radix Alba, and the developed HPLC method was simple, rapid and accurate, which could provide a scientific basis for the evaluation of the germplasm resources and herbal quality of Paeoniae Radix Alba from different cultivars.

Roles of miR-592-3p and Its Target Gene, TMEFF1, in the Nucleus Accumbens During Incubation of Morphine Craving.

BACKGROUND: Prolonged forced abstinence from morphine can increase cue-induced cravings for the drug, contributing to a persistent vulnerability to relapse. Previous studies have identified the implications of aberrant microRNA (miRNA) regulation in the pathogenesis of morphine addiction, but the changes in miRNA expression during the incubation of morphine craving are still unknown. METHODS: Nucleus accumbens (NAc)-specific altered miRNA transcriptomics was determined in a mouse model of cue-induced incubation of morphine craving following a next-generation sequencing method and verified by RT-qPCR. Bioinformatics analysis was performed to predict the target gene of selected miRNA, and the protein expression of the target gene was detected by western blot. A dual-luciferase assay was performed to confirm the binding sites, and gain- and loss-of-function strategy was applied to understand the mechanism of miRNA and its target gene. RESULTS: The miR-592-3p observed to be downregulated in the NAc core was linked to the incubation of morphine craving, and a dual-luciferase assay was performed to confirm the binding sites of miR-592-3p in its target gene, tomoregulin-1 (TMEFF1). Also, gain- and loss-of-function analyses revealed that the inhibition of miR-592-3p expression in the NAc core negatively regulated TMEFF1 expression, thereby enhancing the incubation of morphine craving; however, the overexpression of miR-592-3p in the NAc core resulted in a decreased expression of TMEFF1, thereby reducing the incubation of morphine craving. CONCLUSION: Our findings demonstrated that miR-592-3p can improve the incubation of morphine craving by targeting TMEFF1, and thus, it holds a therapeutic potential to inhibit opioid craving.

A fatal case of accidental oral formaldehyde poisoning and its pathomorphological characteristics.

Formaldehyde is a colourless irritating gas at room temperature, which, therefore, is usually stored in liquid form. This compound is often used as an antiseptic, disinfectant and fumigant in biology and medicine. Formaldehyde, as a potential carcinogen confirmed by the World Health Organization (WHO), is seriously harmful to human systems, such as the respiratory system, immune system and reproductive system. This article reports a case of a 50-year-old woman who died after accidentally drinking 25% formaldehyde solution in a transparent plastic bottle. Anatomical examination revealed fixed tissue morphology of the stomach and adjacent organs. The toxicity test results showed that the concentrations of formaldehyde in the blood and gastric tissue were 36.56 mg/kg and 274.48 mg/kg, respectively, which was consistent with death from formaldehyde poisoning. Due to the particular smell of formaldehyde, poisoning by accidentally drinking formaldehyde solution is rare. Of late, the mechanism of death from formaldehyde poisoning is that it rapidly causes coagulation of tissue cell protein, which may lose its normal function. Based on the pathological characteristics of the case, we put forward a new viewpoint on the mechanism of death from formaldehyde poisoning in which formaldehyde causes rapid fixation of blood in the tissue, thus leading to acute circulatory disturbance.