Oroxylin A alleviates myocardial ischemia-reperfusion injury by quelling ferroptosis via activating the DUSP10/MAPK-Nrf2 pathway.

Reperfusion therapy is the primary treatment strategy for acute myocardial infarction (AMI). Paradoxically, it can lead to myocardial damage, namely myocardial ischemia/reperfusion injury (MIRI). This study explored whether oroxylin A (OA) protects the myocardium after MIRI by inhibiting ferroptosis and the underlying mechanism. In vivo, we established an MIRI model to investigate the protective effect of OA. In vitro, H9C2 cells were used to explore the regulation of ferroptosis by OA through immunofluorescence staining, western blotting, assay kits, etc. Additionally, RNA sequencing analysis (RNA-seq) and network pharmacology analyses were conducted to elucidate the molecular mechanisms. Our results showed that MIRI caused cardiac structural and functional damage in rats. MIRI promoted ferroptosis, which was consistently observed in vitro. However, pretreatment with OA reversed these effects. The mitogen-activated protein kinases (MAPK) signaling pathway participated in the MIRI process, with dual-specificity phosphatase 10 (DUSP10) found to regulate it. Further confirmation was provided by knocking down DUSP10 using small interfering RNA (siRNA), demonstrating the activation of the DUSP10/MAPK-Nrf2 pathway by OA to protect H9C2 cells from ferroptosis. Our research has demonstrated the mitigating effect of OA on MIRI and the improvement of myocardial function for the first time. The inhibition of ferroptosis has been identified as one of the mechanisms through which OA exerts its myocardial protective effects. Moreover, we have first unveiled that DUSP10 serves as an upstream target involved in mediating ferroptosis, and the regulation of the DUSP10/MAPK-Nrf2 pathway by OA is crucial in inhibiting ferroptosis to protect the myocardium.

Validation of simple indexes for nonalcoholic fatty liver disease in western China: a retrospective cross-sectional study.

Various noninvasive algorithms have been developed for predicting the presence of nonalcoholic fatty liver disease (NAFLD). The evaluation of the indexes' diagnostic performance has been reported in Europe and Asia over the past decade; however, external validation of them in China is rare. This study was aimed to evaluate various indexes for NAFLD in western China. It was a retrospective cross-sectional study, using data from a large-scale health check-up project at Sichuan provincial hospital. Receiver operating characteristic (ROC) curves of eight indexes, including the fatty liver index (FLI), the hepatic steatosis index, lipid accumulation product and etc., were developed to predict ultrasonographic NAFLD. There were 13,122 subjects in this study (2,692 NAFLD patients and 10,430 non-NAFLD participants). The area under ROC curve of FLI for predicting NAFLD was 0.880 (95% confidence interval, 0.874-0.886), which was significantly higher than other seven indexes. Accuracy, sensitivity and specificity of FLI for NAFLD were good (cut-off value = 30, 0.782, 0.832, 0.770 and cut-off value = 60, 0.838, 0.443, 0.940, respectively). Furthermore, FLI also presented advantages in expenditure and accessibility, compared with other indexes. It supports FLI as an easily accessible index for physicians and a reliable predictor for NAFLD screening in western China.

Upregulation of PLZF is Associated with Neuronal Injury in Lipopolysaccharide-Induced Neuroinflammation.

Promyelocytic leukemia zinc finger (PLZF) protein has been identified as a tumor suppressor in a variety of cancers, including leukemia, malignant mesothelioma, malignant melanoma, pancreatic cancer and prostate cancer. Studies have demonstrated that altered expression of PLZF affected its biological functions associated with tumorigenesis, such as proliferation, cell cycle, and apoptosis. However, information regarding its regulation and possible function in the central nervous system diseases is still limited. In this study, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral ventricle injection in adult rats and detected increased expression of PLZF in the brain cortex. Immunofluorescence assay indicated that PLZF was significantly increased in neurons 3 day after LPS injection, but not in astrocytes and microglia. Moreover, there was a concomitant upregulation of active caspase-3, cyclin D1, and CDK4 in vivo and vitro studies. In addition, the expression of these proteins in cortical primary neurons was inhibited after knocking down PLZF by siRNA. Collectively, all these results suggested that the upregulation of PLZF might be involved in neuronal apoptotic-like injury in neuroinflammation after LPS injection.

Increased nuclear receptor subfamily 2, group E, member 1 (NR2E1) concentrations of PBMCs are associated with chronic inflammation in overweight/obesity.

Background: Chronic inflammation plays a crucial role in the pathogenesis of overweight/obesity. Nuclear receptor subfamily 2, group E, member 1 (NR2E1) is one of the nuclear receptor family proteins that play crucial roles in regulating numerous life processes. In this study, we attempted to detect NR2E1 levels in peripheral blood mononuclear cells (PBMCs) of overweight/obese people and preliminarily elucidate the regulatory role of NR2E1 in obesity-related chronic inflammation. Methods: We conducted a cross-sectional analysis of the clinical and biochemical data from 62 overweight/obese people and 70 control subjects. PBMCs of the participants were collected for detection of NR2E1 levels. PBMCs isolated from the control subjects were treated with different concentrations of palmitic acid (PA). We also transfected p-EGFP-N1-NR2E1 plasmids into PBMCs and treated them with PA, then detected TNF-α and IL-6 concentrations in the supernatant of PBMCs. Results: The NR2E1 mRNA and protein levels in overweight/obese people were both significantly higher than those in normal-BMI people (p < 0.01). NR2E1 mRNA levels in PBMCs of overweight/obese people were positively related with TC, FFA, IL-6, TNF-α (r = 0.387, 0.440, 0.610, 0.530, p < 0.01) and LDL-c (r = 0.290, p < 0.05). A similar correlation was also found between NR2E1 protein levels and these parameters. The expression of NR2E1 in PBMCs from the control subjects increased apparently with the treatment of PA in a concentration-depend manner in vitro. Overexpression of NR2E1 in PBMCs decreased TNF-α and IL-6 expression induced by PA (p < 0.01). Conclusion: NR2E1 levels are increased in overweight/obese people and have a positive relationship with TC, FFA, LDL-C, TNF-α and IL-6. Overexpression of NR2E1 could alleviate PA-induced chronic inflammation. NR2E1 may be a potential target for regulating chronic inflammation in obesity.

Involvement of Plasma Melatonin in Medication-Overuse Headache: A Cross-Sectional Study.

OBJECTIVES: Patients with medication-overuse headache (MOH) are often complicated with anxiety, depression, and sleep disorders and are associated with dependence behavior and substance abuse. Melatonin has physiological properties including analgesia, regulation of circadian rhythms, soporific, and antidepressant and affects drug preference and addiction. This study aimed to investigate the role of melatonin in MOH compared with episodic migraine (EM) and healthy controls and to verify the relationship between plasma melatonin levels and psychiatric symptoms. METHODS: Thirty patients affected by MOH, 30 patients with EM, and 30 matched healthy controls were enrolled. All subjects completed a detailed headache questionnaire and scales including the Hospital Anxiety and Depression Scale (HADS), the Pittsburgh Sleep Quality Index, the Leeds Dependence Questionnaire. Melatonin levels in plasma samples were measured by enzyme immunoassay method. RESULTS: The levels of plasma melatonin were significantly different among 3 groups of subjects (MOH, 7.74 [5.40-9.89]; EM, 9.79 [8.23-10.62]; Control, 10.16 [8.60-17.57]; H = 13.433; P = 0.001). Significantly lower levels of melatonin were found in MOH patients compared with healthy controls ( P = 0.001). The level of plasma melatonin inversely correlated with the scores of HADS-Anxiety ( r = -0.318, P = 0.002), HADS-Depression ( r = -0.368, P < 0.001), Pittsburgh Sleep Quality Index ( r = -0.303, P = 0.004), and Leeds Dependence Questionnaire ( r = -0.312, P = 0.003). CONCLUSIONS: This study innovatively detects the plasma melatonin levels in MOH patients and explores the association between melatonin levels and psychiatric symptoms. Melatonin may be potential complementary therapy in the treatment of MOH considering its comprehensive role in multiple aspects of MOH.

Linear brain measurement: a new screening method for cognitive impairment in elderly patients with cerebral small vessel disease.

Background: The old adults have high incidence of cognitive impairment, especially in patients with cerebral small vessel disease (CSVD). Cognitive impairment is not easy to be detected in such populations. We aimed to develop clinical prediction models for different degrees of cognitive impairments in elderly CSVD patients based on conventional imaging and clinical data to determine the better indicators for assessing cognitive function in the CSVD elderly. Methods: 210 CSVD patients were screened out by the evaluation of Magnetic Resonance Imaging (MRI). Then, participants were divided into the following three groups according to the cognitive assessment results: control, mild cognitive impairment (MCI), and dementia groups. Clinical data were collected from all patients, including demographic data, biochemical indicators, carotid ultrasound, transcranial Doppler (TCD) indicators, and linear measurement parameters based on MRI. Results: Our results showed that the brain atrophy and vascular lesions developed progressive worsening with increased degree of cognitive impairment. Crouse score and Interuncal distance/Bitemporal distance (IUD/BTD) were independent risk factors for MCI in CSVD patients, and independent risk factors for dementia in CSVD were Crouse Score, the pulsatility index of the middle cerebral artery (MCAPI), IUD/BTD, and Sylvian fissure ratio (SFR). Overall, the parameters with high performance were the IUD/BTD (OR 2.28; 95% CI 1.26-4.10) and SFR (OR 3.28; 95% CI 1.54-6.91), and the AUC (area under the curve) in distinguishing between CSVD older adults with MCI and with dementia was 0.675 and 0.724, respectively. Linear brain measurement parameters had larger observed effect than other indexes to identify cognitive impairments in CSVD patients. Conclusion: This study shows that IUD/BTD and SFR are good predictors of cognitive impairments in CSVD elderly. Linear brain measurement showed a good predictive power for identifying MCI and dementia in elderly subjects with CSVD. Linear brain measurement could be a more suitable and novel method for screening cognitive impairment in aged CSVD patients in primary healthcare facilities, and worth further promotion among the rural population.

Activation of chaperone-mediated autophagy exerting neuroprotection effect on intracerebral hemorrhage-induced neuronal injury by targeting Lamp2a.

Intracerebral hemorrhage (ICH) is a common and devastating type of stroke, marked by significant morbidity and a grim prognosis. The inflammation cascade triggered by astrocytes plays a critical role in secondary brain injury (SBI) following ICH, leading to detrimental effects such as cell death. However, effective intervention strategies are currently lacking. This study aims to investigate the role of the astrocyte cascade reaction following ICH and identify potential intervention targets. Utilizing the GSE216607 and GSE206971 databases for analysis, we established a mouse autologous blood model. Firstly, our research revealed a significant activation of the autophagy pathway following intracerebral hemorrhage (ICH), with a notable upregulation of Lamp2a, a key factor in chaperone-mediated autophagy (CMA), primarily localized in astrocytes. Additionally, the downregulation of Lamp2a resulted in a significant augmentation of A1 reactive astrocytes, concomitant with a reduction in myelin coverage area, heightened neuronal injury, exacerbated motor and sensory deficits, and diminished neurological scores after ICH in mice. Conversely, CA77.1, an activator of CMA, could reverse ICH-induced augmentation of A1 reactive astrocytes, myelin damage, neuronal death, and neurobehavioral disorders. In conclusion, the activation of astrocyte CMA following ICH can exert neuroprotective effects. Lamp2a represents a promising therapeutic target for post-ICH treatment.

Study on risk factors of impaired fasting glucose and development of a prediction model based on Extreme Gradient Boosting algorithm.

Objective: The aim of this study was to develop and validate a machine learning-based model to predict the development of impaired fasting glucose (IFG) in middle-aged and older elderly people over a 5-year period using data from a cohort study. Methods: This study was a retrospective cohort study. The study population was 1855 participants who underwent consecutive physical examinations at the First Affiliated Hospital of Soochow University between 2018 and 2022.The dataset included medical history, physical examination, and biochemical index test results. The cohort was randomly divided into a training dataset and a validation dataset in a ratio of 8:2. The machine learning algorithms used in this study include Extreme Gradient Boosting (XGBoost), Support Vector Machines (SVM), Naive Bayes, Decision Trees (DT), and traditional Logistic Regression (LR). Feature selection, parameter optimization, and model construction were performed in the training set, while the validation set was used to evaluate the predictive performance of the models. The performance of these models is evaluated by an area under the receiver operating characteristic (ROC) curves (AUC), calibration curves and decision curve analysis (DCA). To interpret the best-performing model, the Shapley Additive exPlanation (SHAP) Plots was used in this study. Results: The training/validation dataset consists of 1,855 individuals from the First Affiliated Hospital of Soochow University, yielded significant variables following selection by the Boruta algorithm and logistic multivariate regression analysis. These significant variables included systolic blood pressure (SBP), fatty liver, waist circumference (WC) and serum creatinine (Scr). The XGBoost model outperformed the other models, demonstrating an AUC of 0.7391 in the validation set. Conclusions: The XGBoost model was composed of SBP, fatty liver, WC and Scr may assist doctors with the early identification of IFG in middle-aged and elderly people.

Transcriptomic analysis reveals novel hub genes associated with astrocyte autophagy in intracerebral hemorrhage.

Introduction: Neuroinflammation serves as a critical local defense mechanism against secondary brain injury following intracerebral hemorrhage (ICH), and astrocytes play a prominent role in this process. In this study, we investigated astrocytic changes during the inflammatory state after ICH to identify new targets for improving the inflammatory response. Methods: We stimulated mouse astrocytes with lipopolysaccharide (LPS) in vitro and analyzed their transcriptomes via ribonucleic acid sequencing. We created an ICH model in living organisms by injecting autologous blood. Results: RNA sequencing revealed that 2,717 genes were differentially expressed in the LPS group compared to those in the saline group, with notable enrichment of the autophagic pathway. By intersecting the 2,717 differentially expressed genes (DEGs) with autophagy-related genes, we identified 36 autophagy-related DEGs and seven hub genes. Previous studies and quantitative reverse transcription-polymerase chain reaction results confirmed the increased expression of phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3), AKT serine/threonine kinase 1 (Akt1), and unc-51 like autophagy activating kinase 2 (Ulk2) in astrocytes after ICH. Transcription factors and target miRNAs were identified for the final three DEGs, and 3-methyladenine and leupeptin were identified as potential therapeutic agents for ICH. Conclusion: Our findings suggest that astrocyte autophagy plays a critical role in ICH complexity, and that Pik3c3, Akt1, and Ulk2 may be potential therapeutic targets.

CDK5 Mediates Proinflammatory Effects of Microglia through Activated DRP1 Phosphorylation in Rat Model of Intracerebral Hemorrhage.

Introduction: Cyclin-dependent kinase-5 (CDK5) is a key kinase involved in brain development and function and recently found to be involved in neuronal and astroglial apoptosis, neural stem/progenitor cell stemness, mitochondrial fission, and synaptic transmission. But the specific mechanism of CDK5-mediated anti-inflammatory remains unclear in ICH. The aim of the present study was to explore the role of CDK5 in mediating microglia activity through activated DRP1 phosphorylation in a rat ICH model. Methods: We measured behavioral change after ICH; detected the expression of CDK5 in the rat brain using immunohistochemistry; and measured the protein levels of CDK5, p35, p25, p-histone H1, and p-DRP1 using Western blot analysis. Coimmunoprecipitation analysis indicated interaction of CDK5 and DRP1. Tumor necrosis factor-α, interleukin- (IL-) 1ß, and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA). Results: After ICH, CDK5 protein level and kinase activity increased. Western blot data showed that CDK5 expression increased from 6 h and peaked at 2 d after ICH (p < 0.05), and the expression of p35 was lowest at 12 h, while the expression of p25 peaked at 2 d after ICH. Besides, p-DRP1 expression change follows with CDK5 kinase activity change. Coimmunoprecipitation showed that interaction between CDK5 and DRP1 certainly exists in microglia. Then, knockdown CDK5 or p35 expression by siRNA reduced the expression level of p-DRP1. ELISA data showed that the protein levels of proinflammatory mediators, such as TNF-α, IL-1ß, and IL-6, were decreased by knockdown of CDK5. Conclusion: CDK5 may regulate DRP1 by direct phosphorylation in microglia and further induce microglia secreting proinflammation factor.

Serum cytoskeleton-associated protein 4 as a biomarker for the diagnosis of hepatocellular carcinoma.

BACKGROUND: Alpha-fetoprotein (AFP) is the most commonly applied biomarker for diagnosis of hepatocellular carcinoma (HCC), but the low sensitivity and specificity limit its clinical application. Cytoskeleton-associated protein 4 (CKAP4) is a novel oncogenic protein involved in the development and progression of HCC. This study aimed to evaluate whether measurement of circulating CKAP4 could improve diagnostic accuracy for HCC. METHODS: We analyzed data for patients with HCC, chronic hepatitis B infection, and cirrhosis and healthy controls (n=100 in each group), recruited from two centers between July 2013 and December 2015. Circulating levels of CKAP4 were measured with commercial enzyme-linked immunosorbent assay kits. Receiver operating characteristics were used to evaluate diagnostic accuracy. RESULTS: Serum concentrations of CKAP4 were significantly elevated in the HCC group, in comparison with the three control groups (all P<0.001). The combined biomarker panel (AFP and CKAP4), created by binary logistic regression, presented better performance (area under the curve [AUC] 0.936, 95% CI [0.908-0.965], sensitivity 0.800, specificity 0.963) than AFP (AUC 0.875 [0.835-0.914], sensitivity 0.930, specificity 0.430, P=0.001) or CKAP4 (AUC 0.821 [0.776-0.866], sensitivity 0.790, specificity 0.670, P<0.001) alone to identify HCC, even though CKAP4 alone was not better than AFP (P=0.093). Furthermore, the combined panel also presented a better performance even in identifying early HCC (AUC 0.922 [0.833-0.961]). CONCLUSION: Serum CKAP4 is a novel biomarker for HCC, and it could complement AFP in improving diagnostic accuracy.

Bex1 attenuates neuronal apoptosis in rat intracerebral hemorrhage model.

Brain expressed x-linked gene 1 (Bex1) which is at high levels in several populations of central nervous system (CNS) neurons, belongs to a family of small proteins of unknown function, playing roles as adaptors or modulators of intracellular signaling pathways. But its distribution and function in CNS remains unclear. Neuronal apoptosis is the major pathogenesis in secondary brain injury of intracerebral hemorrhage (ICH). In this study, the roles of Bex1 were explored in the pathophysiology of ICH. Western blot, immunohistochemistry, and immunofluorescence showed that obvious up-regulation of Bex1 in neurons adjacent to the hematoma after ICH. Furthermore, the increase of Bex1 expression was accompanied by the enhanced expression of Bax and active caspase-3, and decreased expression of B-cell lymphoma 2 (Bcl-2) following ICH. The in vitro study using Bex1 siRNA transfection in hemin-exposed PC12 cells suggested that Bex1 exerted anti-apoptotic function. Therefore, Bex1 may play the neuronal anti-apoptosis role following ICH, implying a novel molecular target for the therapy of ICH.

KLF15 suppresses cell growth and predicts prognosis in lung adenocarcinoma.

Krüppel-like factors (KLFs) are transcription factors containing three different C2H2-type zinc finger domains in their carboxy-terminal regions which have been identified to play important roles in a variety of cancers. However, little is known about KLF15 in lung adenocarcinoma (LAUD). Our study demonstrated that the expression levels of KLF15 were observably down-regulated in LAUD tissues compared to paired adjacent normal tissues. LUAD patients with low expression levels of KLF15 have worse prognosis than those with high expression levels of KLF15. KLF15 could suppress cell growth, which was partly via up-regulating CDKN1 A/p21 and CDKN2A/p15. Our findings suggested that KLF15 showed a significant role in LAUD progression and may shed light on a promising novel therapeutic target for blocking progression of LAUD.

Up-regulation of Dyrk1b promote astrocyte activation following lipopolysaccharide-induced neuroinflammation.

Astrocytes become activated in response to different stimulation. Dyrk1b is an arginine-directed serine/threonineprotein kinase that is expressed at elevated levels in many cancers but remains unknown in the pathologies of neuroinflammation. In this study, in vivo, we demonstrated that Dyrk1b expression was significantly increased and reached a peak at 12 h after LPS injection via Western blot. Double immunofluorescence staining showed that Dyrk1b co-located with GFAP and Ki67. In vitro, the expression of Dyrk1b, Ki67 and cyclinD1 was gradually increased and reached a peak at 12 h in a time-dependent manner after 1 µg/mL LPS stimulation. Knockdown of Dyrk1b significantly reduced the expression of Ki67 and cyclinD1. In addition, the data exhibited that silenced Dyrk1b decreased the expression of p-STAT3 in primary astrocyte cells, and Dyrk1b interacted with STAT3 in LPS-induced neuroinflammation. In conclusion, these results suggested that Dyrk1b is increased and may play a crucial role in regulating astrocyte cell activation via interact with STAT3 in LPS-induced neuroinflammation.

Up-regulation of TNF Receptor-associated Factor 7 after spinal cord injury in rats may have implication for neuronal apoptosis.

TNF receptor-associated factor 7 (TRAF7), is an E3 ubiquitin ligase for several proteins involved in the activation of TLR-dependent NF-kappaB signaling. TRAF7 links TNF receptor family proteins to signaling pathways, thus participates in regulating cell death and survival mediated by TNF family ligands. To date, the biological function of TRAF7 after spinal cord injury (SCI) is still with limited acquaintance. In this study, we have performed an acute SCI model in adult rats and investigated the dynamic changes of TRAF7 expression in the spinal cord. Our results showed that TRAF7 was up-regulated significantly after SCI, which was paralleled with the levels of the apoptotic protein active caspase-3. Immunofluorescent labeling showed that TRAF7 was co-localizated with active caspase-3 in neurons. To further investigate the function of TRAF7, an apoptosis model was established in primary neuronal cells. When TRAF7 was knocked down by specific short interfering RNA (siRNA), the protein levels of active caspase-3 and the number of apoptotic primary neurons were significantly decreased in our study. Taken together, our findings suggest that the change of TRAF7 protein expression plays a key role in neuronal apoptosis after SCI.

An Up-regulation of IRF-1 After a Spinal Cord Injury: Implications for Neuronal Apoptosis.

IRF-1, a kind of transcription factor, is expressed in many cell types, except in early embryonal cells. IRF-1 has played an essential role in various physiological and pathological processes, including tumor immune surveillance, viral infection, development of immunity system and pro-inflammatory injury. However, the expression and function of IRF-1 in spinal cord injury (SCI) are still unknown. In this study, we have performed an acute SCI model in adult rats and investigated the dynamic changes of IRF-1 expression in the spinal cord. Western blot have shown that IRF-1 protein levels gradually increased, reaching a peak at day 3 and then gradually declined to a normal level at day 14 after SCI. Double immunofluorescence staining showed that IRF-1 immunoreactivity was found in neurons, but not in astrocytes and microglia. Additionally, colocalization of IRF-1/active caspase-3 was detected in neurons. In vitro, IRF-1 depletion, by short interfering RNA, obviously decreases neuronal apoptosis. In conclusion, this is the first description of IRF-1 expression in spinal cord injury. Our results suggested that IRF-1 might play crucial roles in CNS pathophysiology after SCI.