ObjectiveTo explore the mechanism by which Buyang Huanwutang regulates the glutathione peroxidase 4 （GPX4）-acyl-CoA synthetase long-chain family member 4 （ACSL4） axis to inhibit ferroptosis and promote neurological functional recovery after spinal cord injury （SCI）. MethodsNinety rats were randomly divided into five groups： sham operation group， model group， low-dose Buyang Huanwutang group （12.5 g·kg^-1）， high-dose Buyang Huanwutang group （25 g·kg^-1）， and Buyang Huanwutang + inhibitor group （25 g·kg^-1 + 5 g·kg^-1 RSL3）. The SCI model was established by using the allen method. Tissue was collected on the 7th and 28th days after operation. Motor function was assessed by using the Basso-Beattie-Bresnahan （BBB） scale. Hematoxylin-eosin （HE）， Nissl， and Luxol fast blue （LFB） staining were performed to observe spinal cord histopathology. Transmission electron microscopy was used to examine mitochondrial ultrastructure. Immunofluorescence staining was used to detect the number of NeuN-positive cells and the fluorescence intensity of myelin basic protein （MBP）， GPX4， and ACSL4. Real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） was used to analyze the mRNA expression of GPX4 and ACSL4. Enzyme linked immunosorbent assay （ELISA） was performed to measure the levels of reactive oxygen species （ROS）， malondialdehyde （MDA）， glutathione （GSH）， and superoxide dismutase （SOD）. Colorimetric assays were used to determine the iron content in spinal cord tissue. ResultsCompared to the sham operation group， the model group exhibited significantly reduced BBB scores （P<0.01）， severe pathological damage in spinal cord tissue， and marked mitochondrial ultrastructural disruption. In addition， the model group showed a decrease in the number of NeuN-positive cells （P<0.01）， reduced fluorescence intensity of MBP and GPX4 （P<0.01）， lower levels of GSH and SOD （P<0.01）， and downregulated mRNA expression of GPX4 （P<0.01）. Moreover， compared to the sham operation group， the model group had elevated levels of ROS， MDA， and tissue iron content （P<0.01）， along with increased fluorescence intensity and mRNA expression of ACSL4 （P<0.01）. Compared with the model group and Buyang Huanwutang + inhibitor group， the Buyang Huanwutang group showed significantly improved BBB scores （P<0.05， P<0.01） and exhibited less severe spinal cord tissue damage， reduced edema and inflammatory cell infiltration， increased neuronal survival， and more intact myelin structures. Additionally， mitochondrial ultrastructure was significantly improved in the Buyang Huanwutang group. Compared to the model group and Buyang Huanwutang + inhibitor group， the Buyang Huanwutang group significantly increased the number of NeuN-positive cells and the fluorescence intensity of MBP （P<0.05， P<0.01）. Furthermore， Buyang Huanwutang significantly increased the fluorescence intensity and mRNA expression of GPX4 （P<0.01） and decreased the fluorescence intensity and mRNA expression of ACSL4 （P<0.01） compared to the model group and Buyang Huanwutang + inhibitor group. Finally， the Buyang Huanwutang group significantly decreased ROS， MDA， and tissue iron content （P<0.01） and significantly increased GSH and SOD levels （P<0.01） compared to the model group and Buyang Huanwutang + inhibitor group. ConclusionBuyang Huanwutang inhibits ferroptosis through the GPX4/ACSL4 axis， reduces secondary neuronal and myelin injury and oxidative stress， and ultimately promotes the recovery of neurological function.

ObjectiveTo investigate the processing technology of calcined Haematitum based on the concept of quality by design（QbD） and to assess its health risk. MethodsTaking whole iron content， Fe²⁺ dissolution content and looseness as critical quality attributes（CQAs）， and calcination temperature， calcination time， spreading thickness and particle size as critical process parameters（CPPs） determined by the failure mode and effect analysis（FMEA）， the processing technology of calcined Haematitum was optimized by orthogonal test combined with analytic hierarchy process-criteria importance through intercriteria correlation（AHP-CRITIC） hybrid weighting method. The contents of heavy metals and harmful elements were determined by inductively coupled plasma mass spectrometry， and the health risk assessment was carried out by daily exposure（EXP）， target hazard quotient（THQ） and lifetime cancer risk（LCR）， and the theoretical value of the maximum limit was deduced. ResultsThe optimal processing technology for calcined Haematitum was calcination at 650 ℃， calcination time of 1 h， particle size of 0.2-0.5 cm， spreading thickness of 1 cm， and vinegar quenching for 1 time［Haematitum-vinegar（10：3）］. The contents of 5 heavy metals and harmful elements in 13 batches of calcined Haematitum were all decreased with reductions of up to 5-fold. The cumulative THQ of 2 batches of samples was>1， while the cumulative THQ of all batches of Haematitum was>1. The LCR of As in 1 batches of Haematitum was 1×10^-6-1×10^-4， and the LCR of the rest was<1×10^-6， and the LCRs of calcined Haematitum were all<1×10^-6， indicating that the carcinogenic risk of calcined Haematitum was low， but special attention should still be paid to Haematitum medicinal materials. Preliminary theoretical values of the maximum limits of Cu， As， Cd， Pb and Hg were formulated as 1 014， 25， 17， 27， 7 mg·kg^-1. ConclusionThe optimized processing technology of calcined Haematitum is stable and feasible， and the contents of heavy metals and harmful elements are reduced after processing. Preliminary theoretical values of the maximum limits of Cu， As， Cd， Pb and Hg are formulated to provide a scientific basis for the formulation of standards for the limits of harmful elements in Haematitum.

ObjectiveTo investigate the processing technology of calcined Haematitum based on the concept of quality by design（QbD） and to assess its health risk. MethodsTaking whole iron content， Fe²⁺ dissolution content and looseness as critical quality attributes（CQAs）， and calcination temperature， calcination time， spreading thickness and particle size as critical process parameters（CPPs） determined by the failure mode and effect analysis（FMEA）， the processing technology of calcined Haematitum was optimized by orthogonal test combined with analytic hierarchy process-criteria importance through intercriteria correlation（AHP-CRITIC） hybrid weighting method. The contents of heavy metals and harmful elements were determined by inductively coupled plasma mass spectrometry， and the health risk assessment was carried out by daily exposure（EXP）， target hazard quotient（THQ） and lifetime cancer risk（LCR）， and the theoretical value of the maximum limit was deduced. ResultsThe optimal processing technology for calcined Haematitum was calcination at 650 ℃， calcination time of 1 h， particle size of 0.2-0.5 cm， spreading thickness of 1 cm， and vinegar quenching for 1 time［Haematitum-vinegar（10：3）］. The contents of 5 heavy metals and harmful elements in 13 batches of calcined Haematitum were all decreased with reductions of up to 5-fold. The cumulative THQ of 2 batches of samples was>1， while the cumulative THQ of all batches of Haematitum was>1. The LCR of As in 1 batches of Haematitum was 1×10^-6-1×10^-4， and the LCR of the rest was<1×10^-6， and the LCRs of calcined Haematitum were all<1×10^-6， indicating that the carcinogenic risk of calcined Haematitum was low， but special attention should still be paid to Haematitum medicinal materials. Preliminary theoretical values of the maximum limits of Cu， As， Cd， Pb and Hg were formulated as 1 014， 25， 17， 27， 7 mg·kg^-1. ConclusionThe optimized processing technology of calcined Haematitum is stable and feasible， and the contents of heavy metals and harmful elements are reduced after processing. Preliminary theoretical values of the maximum limits of Cu， As， Cd， Pb and Hg are formulated to provide a scientific basis for the formulation of standards for the limits of harmful elements in Haematitum.

Esophageal carcinoma is a malignant disease with a high incidence rate and poor prognosis. The mitochondrial apoptosis pathway plays a pivotal role in the regulation of cell death and has become a focal point in current cancer therapeutics research. Various active components from traditional Chinese medicine （TCM） can target the mitochondrial apoptosis pathway to inhibit esophageal carcinoma， presenting as potential therapeutic agents for this disease. This paper summarizes relevant research on the inhibition of esophageal carcinoma by active components in TCM via targeting the mitochondrial apoptosis pathway. It has been found that flavonoids （casticin， icariin， luteolin， kaempferol， hesperetin， deguelin， etc.）， terpenoids （oridonin， Jaridonin， artesunate， ethyl acetate fraction of pleurotus ferulatus triterpenoid， etc.）， alkaloids （matrine， swainsonine， etc.）， polyphenols （curcumin， epigallocatechin-3-gallate， corilagin， etc.）， steroids （α-hederin， polyphyllin Ⅵ， etc.）， phenols （optimized scorpion venom peptide CT-K3K7， gecko active polypeptide， etc.）， volatile oils （cinnamaldehyde， α -asarone， etc.） and other active components from TCM can target the intrinsic mitochondrial apoptosis pathway， induce apoptosis in esophageal carcinoma cells， and inhibit their proliferation， invasion and migration by regulating oxidative stress， blocking the cell cycle， regulating signaling pathways such as PI3K/Akt and MAPK.

ObjectiveTo investigate the triglyceride-glucose (TyG) index and the level of serum homocysteine (Hcy) in association with the incidence of stroke in type 2 diabetes mellitus (T2DM) patients. MethodsBased on the chronic disease risk factor surveillance cohort in Pudong New Area, Shanghai, excluding those with stroke in baseline survey, T2DM patients who joined the cohort from January 2016 to October 2020 were selected as the research subjects. During the follow-up period, a total of 318 new-onset ischemic stroke patients were selected as the case group, and a total of 318 individuals matched by gender without stroke were selected as the control group. The Cox proportional hazards regression model was used to adjust for confounding factors and explore the serum TyG index and the Hcy biochemical indicator in association with the risk of stroke. ResultsThe Cox proportional hazards regression results showed that after adjusting for confounding factors, the risk of stroke in T2DM patients with 10 μmol·L⁻¹-¹ and Hcy>15 μmol·L⁻¹ was 1.532 times (95%CI: 1.017‒2.309 times, P=0.041) and 1.738 times (95%CI: 1.119‒2.699 times, P=0.014) higher respectively, compared to T2DM patients with Hcy≤10 μmol·L⁻¹. T2DM patients with TyG>9.074 had 1.396 times higher risk of stroke (95%CI: 1.091‒1.787, P=0.008) compared to those with TyG≤9.074. The study found that urea and α1-antitrypsin (α1-AT) were independent risk factors for the incidence of stroke in T2DM patients. For every unit increase in urea andα1-AT, the risk of stroke in T2DM patients increased by 233.6% (HR=3.336, 95%CI: 1.588‒7.009, P=0.001) and 11.3% (HR=1.113, 95%CI: 1.028‒1.205, P=0.008), respectively. Cumulative risk curves showed that Hcy>10 μmol·L⁻¹ and TyG>9.074 accelerated the time to stroke occurrence in T2DM patients. ConclusionElevated levels of Hcy>10 μmol·L⁻¹ and a higher TyG index are risk factors for stroke in T2DM patients. Effective interventions for Hcy and TyG should be conducted for diabetic patients to reduce the incidence of stroke.

BACKGROUND: Doxorubicin hydrochloride (DOX) is extensively used in the treatment of various tumors. However, its clinical application is limited due to dose-dependent cardiotoxicity. Currently, few effective strategies exist to mitigate or eliminate DOX-induced cardiomyopathy (DIC). Although ferroptosis is implicated in DIC and its inhibition partially alleviates the condition, the direct targets of DOX in the progression of cardiotoxicity remain unclear. This study aimed to discover the direct targets of DOX in ferroptosis-mediated DIC. METHODS: A DOX pulldown assay was performed to identify proteins specifically binding to DOX in murine hearts, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify candidate proteins. A cardiac injury mouse model was established by DOX treatment. Based on this, multiple ferroptosis biomarkers were detected by flow cytometry, quantitative real-time polymerase chain reaction, western blotting, immunochemistry, etc. Besides, specific activator and inhibitor of signaling pathways were applied to illuminate molecular mechanisms. RESULTS: Glutathione S-transferase P1 (GSTP1) was identified as a DOX target. GSTP1 activity was inhibited in DOX-treated cardiomyocytes, while its overexpression significantly alleviated DIC. Moreover, GSTP1 overexpression inhibited acyl-CoA synthetase long-chain family member 4 (ACSL4)-dependent ferroptosis. Mechanistically, GSTP1 overexpression suppressed c-Jun N-terminal kinase (JNK) phosphorylation, thereby reducing reactive oxygen species (ROS) production and inhibiting ferroptosis in DIC. CONCLUSIONS This study identifies the DOX/GSTP1/JNK axis as a critical pathway mediating ACSL4-dependent ferroptosis in DIC. GSTP1 is highlighted as a potential key mediator of ferroptosis and a promising therapeutic target for DIC.

Abdominal aortic aneurysm (AAA) is a deadly condition of the aorta, carrying a significant risk of death upon rupture. Currently, there is a dearth of efficacious pharmaceutical interventions to impede the advancement of AAA and avert it from rupturing. Here, we investigated dihydromyricetin (DHM), one of the predominant bioactive flavonoids in Ampelopsis grossedentata (A. grossedentata), as a potential agent for inhibiting AAA. DHM effectively blocked the formation of AAA in angiotensin II-infused apolipoprotein E-deficient (ApoE^-/-) mice. A combination of network pharmacology and whole transcriptome sequencing analysis revealed that DHM's anti-AAA action is linked to heme oxygenase (HO)-1 (Hmox-1 for the rodent gene) and hypoxia-inducible factor (HIF)-1α in vascular smooth muscle cells (VSMCs). Remarkably, DHM caused a robust rise (∼10-fold) of HO-1 protein expression in VSMCs, thereby suppressing VSMC inflammation and oxidative stress and preserving the VSMC contractile phenotype. Intriguingly, the therapeutic effect of DHM on AAA was largely abrogated by VSMC-specific Hmox1 knockdown in mice. Mechanistically, on one hand, DHM increased the transcription of Hmox-1 by triggering the nuclear translocation and activation of HIF-1α, but not nuclear factor erythroid 2-related factor 2 (NRF2). On the other hand, molecular docking, combined with cellular thermal shift assay (CETSA), isothermal titration calorimetry (ITC), drug affinity responsive target stability (DARTS), co-immunoprecipitation (Co-IP), and site mutant experiments revealed that DHM bonded to HO-1 at Lys243 and prevented its degradation, thereby resulting in considerable HO-1 buildup. In summary, our findings suggest that naturally derived DHM has the capacity to markedly enhance HO-1 expression in VSMCs, which may hold promise as a therapeutic strategy for AAA.

Sigma-1 receptor (σ ₁R) has become a focus point of drug discovery for central nervous system (CNS) diseases. A series of novel 1-phenylethan-1-one O-(2-aminoethyl) oxime derivatives were synthesized. In vitro biological evaluation led to the identification of 1a, 14a, 15d and 16d as the most high-affinity (K _i < 4 nmol/L) and selective σ ₁R agonists. Among these, 15d, the most metabolically stable derivative exhibited high selectivity for σ ₁R in relation to σ ₂R and 52 other human targets. In addition to low CYP450 inhibition and induction, 15d also exhibited high brain permeability and excellent oral bioavailability. Importantly, 15d demonstrated effective antipsychotic potency, particularly for alleviating negative symptoms and improving cognitive impairment in experimental animal models, both of which are major challenges for schizophrenia treatment. Moreover, 15d produced no significant extrapyramidal symptoms, exhibiting superior pharmacological profiles in relation to current antipsychotic drugs. Mechanistically, 15d inhibited GSK3β and enhanced prefrontal BDNF expression and excitatory synaptic transmission in pyramidal neurons. Collectively, these in vivo proof-of-concept findings provide substantial experimental evidence to demonstrate that modulating σ ₁R represents a potential new therapeutic approach for schizophrenia. The novel chemical entity along with its favorable drug-like and pharmacological profile of 15d renders it a promising candidate for treating schizophrenia.

OBJECTIVES: To investigate the subcellular localization and biological functions of transmembrane protein 240 (TMEM240). METHODS: NCBI BLAST and TMHMM bioinformatics software were used for protein sequence analysis and prediction of transmembrane domain of TMEM240. Brain tissues from male C57BL/6 mice (18-20 days old) were examined for distribution of TMEM240 using in situ hybridization, and qPCR and Western blotting were used to detect TMEM240 expression in different mouse tissues and in cortical neurons at different time points (n=3). In the in vitro experiment, HepG2 and Neuro-2a cells were transfected with plasmids for overexpression of TMEM240, and subcellular localization of TMEM240 was analyzed using cell imaging. In primary cultures of cortical neurons isolated from C57BL/6 mice, TMEM240 expression and its biological functions were investigated using qPCR, Western blotting, and immunofluorescence staining. RESULTS: Human and mouse TMEM240 proteins share a 97.69% similarity in the protein sequences, and both are transmembrane proteins with two transmembrane domains. TMEM240 mRNA and protein were highly expressed in mouse brain tissues and cortical neurons. In isolated mouse cortical neurons, TMEM240 expression reached the peak level after primary culture for 9 days and distributed in scattered spots within the cells. In HepG2 cells, TMEM240 was characterized as intracellular membrane structures and showed 80% colocalization with peroxisomes. In Neuro-2a cells, TMEM240 overexpression caused significant enhancement of the expressions of Rho GDP dissociation inhibitor β (ARHGDIB) at both the mRNA and protein levels. CONCLUSIONS TMEM240 is a novel intracellular subcellular structure specifically expressed in neurons with significant potential for targeted cellular function regulation.
Animals ; Humans ; Mice ; Peroxisomes/metabolism* ; Membrane Proteins/genetics* ; Mice, Inbred C57BL ; Neurons/metabolism* ; Male ; rho-Specific Guanine Nucleotide Dissociation Inhibitors ; Hep G2 Cells ; Brain/metabolism*

Negative logarithm of the acid dissociation constant (pK _a) significantly influences the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of molecules and is a crucial indicator in drug research. Given the rapid and accurate characteristics of computational methods, their role in predicting drug properties is increasingly important. Although many pK _a prediction models currently exist, they often focus on enhancing model precision while neglecting interpretability. In this study, we present GraFpK _a, a pK _a prediction model using graph neural networks (GNNs) and molecular fingerprints. The results show that our acidic and basic models achieved mean absolute errors (MAEs) of 0.621 and 0.402, respectively, on the test set, demonstrating good predictive performance. Notably, to improve interpretability, GraFpK _a also incorporates Integrated Gradients (IGs), providing a clearer visual description of the atoms significantly affecting the pK _a values. The high reliability and interpretability of GraFpK _a ensure accurate pK _a predictions while also facilitating a deeper understanding of the relationship between molecular structure and pK _a values, making it a valuable tool in the field of pK _a prediction.