OBJECTIVE: To explore the neuroprotective effect and underlying mechanism of Xingnao Kaiqiao acupuncture (acupuncture for regaining consciousness and opening orifices) in the rat models of cerebral ischemia-reperfusion injury (CIRI) based on the p53 protein (p53)/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling pathway. METHODS: Of 102 male Wistar rats, 20 rats were randomly collected as a sham-operation group. Using a modified external carotid artery filament insertion method, CIRI models were prepared by occluding the middle cerebral artery in the rest rats. After modeling and excluding 1 non-successfully modeled rat and 1 dead one, the other modeled rats were randomized into a model group, an agonist group, an acupuncture group, and an acupuncture + agonist group, 20 rats in each one. Xingnao Kaiqiao acupuncture therapy was delivered in the rats of the acupuncture group and the acupuncture + agonist group. The acupoints included "Shuigou" (GV26), bilateral "Neiguan" (PC6), and "Sanyinjiao" (SP6) on the affected side. Electroacupuncture was attached to "Neiguan" (PC6) and "Sanyinjiao" (SP6) on the affected side, with dense-disperse wave, a frequency of 2 Hz/15 Hz and intensity of 1 mA. The intervention was delivered twice daily, 20 min each time and for 7 consecutive days. In the agonist group and acupuncture+agonist group, p53 agonist, COTI-2 was intraperitoneally injected (15 mg/kg), once daily for 7 consecutive days. Neurological deficit was evaluated using Zausinger's six-point scale. Cerebral infarction volume was quantified by triphenyl tetrazolium chloride (TTC) staining. Histopathological changes were observed using hematoxylin-eosin (HE) staining. Iron deposition was assessed by Prussian blue staining. Mitochondrial ultrastructure in the ischemic cortex was examined under transmission electron microscopy (TEM). Serum iron (Fe²⁺) was measured with chromometry. Malondialdehyde (MDA) and glutathione (GSH) levels in the ischemic hippocampus were determined using thiobarbituric acid and microplate assays, respectively. The mean fluorescence intensity of reactive oxygen species (ROS) in the ischemic cortex was analyzed by flow cytometry. The mRNA and protein expression of GPX4, SLC7A11, and p53 in the ischemic hippocampus were evaluated using quantitative real-time PCR (qRT-PCR) and Western blotting, respectively. RESULTS: Compared with the sham-operated group, the model group exhibited the decrease in neurological deficit score (P<0.01), and the increase in cerebral infarction volume percentage (P<0.01). The changes of brain tissue were presented in extensive cellular necrosis, pyknotic and deeply-stained nuclei, and vacuolar degeneration. The iron deposition was elevated in cortex and hippocampus (P<0.01), mitochondrial membrane density increased, the cristae was broken or reduced, and the outer membrane ruptured. The levels of Fe²⁺ and MDA, as well as the mean flourscence intensity of ROS were elevated (P<0.01) and the level of GSH was reduced (P<0.01). The mRNA and protein expression of GPX4 and SLC7A11 was reduced (P<0.01), while that of p53 rose (P<0.01). When compared with the model group, in the agonist group, the neurological deficit score was reduced (P<0.05), the percentage of infarction volume was higher (P<0.01), the histopathological damage was further exacerbated, and the percentage of iron deposition increased in the cortex and hippocampus (P<0.01). The mitochondrial quantity decreased, the membrane density increased, the mitochondrial cristae were broken or reduced, and the outer membrane was ruptured. The levels of Fe²⁺ and MDA, as well as the mean flourscence intensity of ROS were higher (P<0.01, P<0.05) and the level of GSH was reduced (P<0.05). The mRNA and protein expression of GPX4 and SLC7A11 decreased (P<0.01, P<0.05), while that of p53 was elevated (P<0.01). Besides, in comparison with the model group, the neurological deficit score was higher in the acupuncture group and the acupuncture + agonist group (P<0.01, P<0.05), the percentage of cerebral infarction volume was lower in the acupuncture group (P<0.01), the pathological damage of brain tissue was alleviated in the acupuncture group and the acupuncture + agonist group, and the percentage of iron depositiondecreased in the cortex and hippocampus (P<0.01). The mitochondrial structure was relatively clear, the mitochondrial cristae were fractured or reduced mildly in the acupuncture group and the acupuncture + agonist group. The levels of Fe²⁺ and MDA, as well as the mean flourscence intensity of ROS were lower (P<0.01) and the level of GSH was higher (P<0.01) in the acupuncture group. The mean fluorescence intensity of ROS were dropped (P<0.01) in the acupuncture + agonist group. The mRNA expression of GPX4 and SLC7A11 was elevated (P<0.01) and that of p53 was reduced (P<0.01, P<0.05) in either the acupuncture group or the acupuncture + agonist group; the protein expression of GPX4 and SLC7A11 rose (P<0.05, P<0.01) and that of p53 was dropped (P<0.01) in the acupuncture group; and the protein expression of p53 was also lower in the acupuncture + agonist group (P<0.05). When compared with the agonist group, in the acupuncture + agonist group, neurological deficit score increased (P<0.01), the percentage of cerebral infarction volume decreased (P<0.01), the pathological brain tissue damage was reduced, the percentage of iron deposition in cortex and hippocampus decreased (P<0.01), the mitochondrial structure was relatively clear and the cristae broken or reduced slightly; the levels of Fe²⁺ and MDA, as well as the mean fluorescence intensity of ROS were dropped (P<0.01), while the level of GSH increased (P<0.05); the mRNA and protein expression of GPX4 and SLC7411 was elevated (P<0.01, P<0.05), and that of p53 reduced (P<0.01). In comparison with the acupuncture + agonist group, in the acupuncture group, the neurological deficit score increased (P<0.05), the percentage of cerebral infarction volume decreased (P<0.05), the pathological brain tissue damage was alleviated, the percentage of iron deposition in cortex and hippocampus decreased (P<0.01), the mitochondrial structure was normal in tendency; the levels of Fe²⁺ and MDA, as well as the mean fluorescence intensity of ROS were reduced (P<0.05), while the level of GSH rose (P<0.01); the mRNA and protein expression of GPX4 and SLC7411 was elevated (P<0.01, P<0.05), and that of p53 reduced (P<0.01, P<0.05). CONCLUSION Xingnao Kaiqiao acupuncture can alleviate neurological damage in CIRI rats, which is obtained probably by inhibiting ferroptosis through p53/SLC7A11/GPX4 pathway.
Animals ; Reperfusion Injury/metabolism* ; Male ; Acupuncture Therapy ; Rats ; Tumor Suppressor Protein p53/genetics* ; Brain Ischemia/metabolism* ; Rats, Wistar ; Signal Transduction ; Humans ; Phospholipid Hydroperoxide Glutathione Peroxidase/genetics* ; Disease Models, Animal ; Acupuncture Points ; Amino Acid Transport System y+/genetics*

This study aims to explore the effects and action mechanisms of the active ingredients in Buyang Huanwu Decoction(BYHWD), namely tetramethylpyrazine(TMP) and hydroxy-safflor yellow A(HSYA), on oxygen-glucose deprivation/reglucose-reoxygenation(OGD/R)-induced inflammation and oxidative stress of microglia(MG). Network pharmacology was used to screen the effective monomer ingredients of BYHWD and determine the safe concentration range for each component. Inflammation and oxidative stress models were established to further screen the best ingredient combination and optimal concentration ratio with the most effective anti-inflammatory and antioxidant effects. OGD/R BV2 cell models were constructed, and BV2 cells in the logarithmic growth phase were divided into a normal group, a model group, an HSYA group, a TMP group, and an HSYA + TMP group. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of inflammatory cytokines such as interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6). Oxidative stress markers, including superoxide dismutase(SOD), nitric oxide(NO), and malondialdehyde(MDA), were also measured. Western blot was used to analyze the protein expression of both inflammation-related pathway [Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)] and oxidative stress-related pathway [nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)]. Immunofluorescence was used to assess the expression of proteins such as inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1). The most effective ingredients for anti-inflammatory and antioxidant effects in BYHWD were TMP and HSYA. Compared to the normal group, the model group showed significantly increased levels of IL-1β, TNF-α, IL-6, NO, and MDA, along with significantly higher protein expression of NF-κB, TLR4, Nrf2, and HO-1 and significantly lower SOD levels. The differences between the two groups were statistically significant. Compared to the model group, both the HSYA group and the TMP group showed significantly reduced levels of IL-1β, TNF-α, IL-6, NO, and MDA, lower expression of NF-κB and TLR4 proteins, higher levels of SOD, and significantly increased protein expression of Nrf2 and HO-1. Additionally, the expression of the M1-type MG marker iNOS was significantly reduced, while the expression of the M2-type MG marker Arg-1 was significantly increased. The results of the HSYA group and the TMP group had statistically significant differences from those of the model group. Compared to the HSYA group and the TMP group, the HSYA + TMP group showed further significant reductions in IL-1β, TNF-α, IL-6, NO, and MDA levels, along with significant reductions in NF-κB and TLR4 protein expression, an increase in SOD levels, and elevated Nrf2 and HO-1 protein expression. Additionally, the expression of the M1-type MG marker iNOS was reduced, while the M2-type MG marker Arg-1 expression increased significantly in the HSYA + TMP group compared to the TMP or HSYA group. The differences in the results were statistically significant between the HSYA + TMP group and the TMP or HSYA group. The findings indicated that the combined use of HSYA and TMP, the active ingredients of BYHWD, can effectively inhibit OGD/R-induced inflammation and oxidative stress of MG, showing superior effects compared to the individual use of either component.
Oxidative Stress/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Animals ; Mice ; Glucose/metabolism* ; Cell Line ; Inflammation/genetics* ; Oxygen/metabolism* ; Pyrazines/pharmacology* ; Microglia/metabolism* ; NF-E2-Related Factor 2/immunology* ; NF-kappa B/immunology* ; Toll-Like Receptor 4/immunology* ; Anti-Inflammatory Agents/pharmacology* ; Humans

Spermatogenesis is a fundamental process that requires a tightly controlled epigenetic event in spermatogonial stem cells (SSCs). The mechanisms underlying the transition from SSCs to sperm are largely unknown. Most studies utilize gene knockout mice to explain the mechanisms. However, the production of genetically engineered mice is costly and time-consuming. In this study, we presented a convenient research strategy using an RNA interference (RNAi) and testicular transplantation approach. Histone H3 lysine 9 (H3K9) methylation was dynamically regulated during spermatogenesis. As Jumonji domain-containing protein 1A (JMJD1A) and Jumonji domain-containing protein 2C (JMJD2C) demethylases catalyze histone H3 lysine 9 dimethylation (H3K9me2), we firstly analyzed the expression profile of the two demethylases and then investigated their function. Using the convenient research strategy, we showed that normal spermatogenesis is disrupted due to the downregulated expression of both demethylases. These results suggest that this strategy might be a simple and alternative approach for analyzing spermatogenesis relative to the gene knockout mice strategy.
Spermatogenesis/physiology* ; Animals ; Male ; Mice ; Epigenesis, Genetic ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Histones/metabolism* ; RNA Interference ; Testis/metabolism* ; Methylation ; Mice, Knockout ; Histone Demethylases

OBJECTIVES: To evaluate the inhibitory effect of oridonin against proliferation of pancreatic cancer cells and the mechanism underlying the synergistic effect of low-intensity pulsed ultrasound (LIPUS). METHODS: PANC-1 cells treated with different concentrations of oridonin were examined for changes in cell proliferation using CCK-8 assay and in MDA, GSH and ATP levels using flow cytometry. The protein expressions of GPX4, Nrf2 and HO-1 in the treated cells were detected with Western blotting. The effect of Fer-1, a ferroptosis inhibitor, on proliferation of oridonin-treated cells were assessed, and the effects of oridonin combined with LIPUS on PIEZO1 protein expression was evalauted using Western blotting. A C57BL/6J mouse model bearing pancreatic cancer cell xenograft was established and treated with oridonin, LIPUS, or both, and the histological changes in the tumor tissues and tumor cell proliferation were examined with HE staining and immunohistochemistry for Ki67; the changes in GPX4 expression in the tumor tissues were detected using Western blotting and immunofluorescence staining. RESULTS: In PANC-1 cells, oridonin treatment significantly inhibited cell proliferation, increased intracellular Fe²⁺, ROS, and MDA levels, and decreased GSH and ATP levels. Oridonin also resulted in lowered GPX4 and increased HO-1 and Nrf2 protein expression levels in the cells. The combined treatment with LIPUS signficiantly enhanced the inhibitory effect of oridonin on PANC-1 cell viability in vitro and on xenograft growth in the mouse models, resulting also in more obvious reduction of the intensity of Ki67 staining and GPX4 protein expression and more pronounced increase of PIEZO1 protein expression in the tumor tissues in the mouse models. CONCLUSIONS LIPUS enhances the effect of oridonin to promote ferroptosis of pancreatic cancer cells by activating PIEZO1 through the Nrf2/HO-1/GPX4 pathway.
Ferroptosis/drug effects* ; Animals ; Pancreatic Neoplasms/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Humans ; Cell Line, Tumor ; Mice ; Heme Oxygenase-1/metabolism* ; Diterpenes, Kaurane/pharmacology* ; Cell Proliferation/drug effects* ; Mice, Inbred C57BL ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Ion Channels/metabolism* ; Ultrasonic Waves ; Signal Transduction

The prelimbic cortex (PL) plays a critical role in processing both the sensory and affective components of pain. However, the underlying molecular mechanisms remain poorly understood. In this study, we observed a reduction in hyperpolarization-activated cation current (I_h) in layer V pyramidal neurons of the contralateral PL in a mouse model of spared nerve injury (SNI). The expression of hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) channels was also decreased in the contralateral PL. Conversely, microinjection of fisetin, a partial agonist of HCN2, produced both analgesic and anxiolytic effects. Additionally, we found that cyclin-dependent kinase 5 (CDK5) was activated in the contralateral PL, where it formed a complex with HCN2 and phosphorylated its C-terminus. Knockdown of CDK5 restored HCN2 expression and alleviated both pain hypersensitivity and anxiety-like behaviors. Collectively, these results indicate that CDK5-mediated dysfunction of HCN2 in the PL underlies nerve injury-induced mechanical hypersensitivity and anxiety.
Animals ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism* ; Hyperalgesia/metabolism* ; Cyclin-Dependent Kinase 5/metabolism* ; Neuralgia/metabolism* ; Male ; Anxiety/metabolism* ; Mice ; Potassium Channels/metabolism* ; Mice, Inbred C57BL ; Disease Models, Animal ; Pyramidal Cells/metabolism*

OBJECTIVE: To investigate the relationship between physical activity and genetic risk and their combined effects on the risk of developing chronic obstructive pulmonary disease. METHODS: This prospective cohort study included 318,085 biobank participants from the UK. Physical activity was assessed using the short form of the International Physical Activity Questionnaire. The participants were stratified into low-, intermediate-, and high-genetic-risk groups based on their polygenic risk scores. Multivariate Cox regression models and multiplicative interaction analyses were used. RESULTS: During a median follow-up period of 13 years, 9,209 participants were diagnosed with chronic obstructive pulmonary disease. For low genetic risk, compared to low physical activity, the hazard ratios ( HRs) for moderate and high physical activity were 0.853 (95% confidence interval [ CI]: 0.748-0.972) and 0.831 (95% CI: 0.727-0.950), respectively. For intermediate genetic risk, the HRs were 0.829 (95% CI: 0.758-0.905) and 0.835 (95% CI: 0.764-0.914), respectively. For participants with high genetic risk, the HRs were 0.809 (95% CI: 0.746-0.877) and 0.818 (95% CI: 0.754-0.888), respectively. A significant interaction was observed between genetic risk and physical activity. CONCLUSION Moderate or high levels of physical activity were associated with a lower risk of developing chronic obstructive pulmonary disease across all genetic risk groups, highlighting the need to tailor activity interventions for genetically susceptible individuals.
Humans ; Pulmonary Disease, Chronic Obstructive/epidemiology* ; Exercise ; Male ; Female ; Middle Aged ; Prospective Studies ; Aged ; Genetic Predisposition to Disease ; Risk Factors ; United Kingdom/epidemiology* ; Incidence ; Adult

Objective : To investigate the role of enhancer of zeste homolog 2(EZH2)-trimethylated lysine 27 of histone H3(H3K27me3) axis in the dedifferentiation of thyroid cancer and its clinical value as a potential target for the treatment of anaplastic thyroid cancer(ATC). Methods : Immunohistochemical SP method was used to detect the expression of EZH2, H3K27me3, paired box gene 8(PAX8), thyroglobulin(TG) and thyroid transcription factor 1(TTF1) in ATC and papillary thyroid carcinoma(PTC) and their adjacent tissues. The relationship between EZH2 and thyroid differentiation markers(PAX8, TTF1, TG) was further analyzed by gene expression omnibus(GEO) database. ATC cell lines 8305C and BHT-101 were culturedin vitro. Real-time reverse transcription PCR(RT-qPCR) was used to detect the expression of thyroid differentiation markers(TTF1, PAX8) mRNA in ATC cell lines treated with EZH2 inhibitor(GSK126), and evaluate the potential therapeutic effect of GSK126in vitro. The effects of GSK126 and BRAF inhibitor vemurafenib on the proliferation of ATC cell lines were observed by cell proliferation assay. Results : The expression of EZH2 in ATC tissues was significantly higher than that in papillary thyroid carcinoma and adjacent tissues(P<0.05). The expression of H3K37me3 in ATC tissues was significantly lower than that in PTC tissues(P<0.05). EZH2 was negatively correlated with PAX8 and TG expression levels, but not with TTF1 expression level.In vitroexperiments, GSK126 could reverse the expression of thyroid differentiation markers PAX8 and TTF1 in ATC cell lines. GSK126 combined with BRAF inhibitor vemurafenib could significantly inhibit the growth of ATC cell lines. Conclusion The EZH2-H3K27me3 axis plays an important role in regulating thyroid specific markers, and the inhibition of EZH2 by small molecular compounds is a promising target for ATC treatment in the future.

Objective To propose an evaluation and screening indicator for the reliability of emergency ventilators.Methods Firstly,a regression model was used to clean the data and remove noise to ensure the accuracy of regression analysis.Then,four groups of highly correlated data combinations,including inspiratory tidal volume-minute expiratory volume,peak airway pressure-minute expiratory volume,peak airway pressure-inspiratory tidal volume and positive end-expiratory pressure(PEEP)-mean airway pressure,were determined with the methods of curve fitting and transfer function,and the difference between the theoretical output and the actual output of the data combinations was regarded as an indicator to judge whether the ventilator functioned well or not;finally,the indicator proposed was applied to single and multiple ventilators,and the feasibility of the indicator was determined by the proportion of the ventilators functioning well.Results The evaluation results with a single ventilator showed the four groups of data combinations had the actual output fitted well with the theoretical output,and all the differences between the theoretical output and the actual output were lower than the threshold;the results with multiple ventilators indicated there were 71.49％ventilators functioning well,which was very close to the actual result that 72％ventilators behaved well.Conclusion The evaluation and screening indicator for emergency ventilators has high feasibility,and theoretical support is provided for reliability assessment and selection of series of emergency treatment equipment.[Chinese Medical Equipment Journal,2024,45(7):8-16]

Ion mobility spectroscopy(IMS)has many advantages such as fast detection speed,high sensitivity,and portability,and thus plays an important role in on-site detection of chemical agents,explosives,drugs,environmental pollutants,and other pollutants.As a key component of IMS,the ion number density and temporal width of the injected ion packets directly determine the IMS detection sensitivity and resolution.In yhis study,the dual-parallel-grid structure of Tyndall-Powell gate(TPG),which could effectively isolate the electric fields within the ionization region,gate region,and drift region,was utilized to develop a dual effect TPG gating method for simultaneously enhancing the mobility discrimination reduction and ion packet temporal width compression,thereby improving the IMS performance.A TPG-IMS platform was thus built up and the effects of parameters such as gate opening pulsed width,gate penetration voltage,and ion injection voltage on the sensitivity and resolving power of IMS were systematically investigated using the dual-effect TPG gating method.The results demonstrated that,when detecting diethyl phosphate(DEP)and diethyl methylphosphate(DEMP)mixture using the dual effect TPG gating method,the peak currents of(DEP)2H+and(DEP·DEMP)H+ions with low K0 values were increased by 18 and 45 times respectively,while maintaining a high resolution of about 90.The limits of detection for DEP and DEMP were decreased from 4 ppb(10-9)to 235 ppt(10-12)and from 5 ppb to 156 ppt,respectively.This gating method only regulated the potential of the TPG grid adjacent to the drift region,without changing the structure of the ion mobility tube,making it convenient to apply on existing commercial instruments.