BACKGROUND:Ferroptosis is closely associated with the pathogenesis of ischemic stroke,and targeting ferroptosis is a promising regimen for the treatment of ischemic stroke,but the specific regulatory targets are unclear. OBJECTIVE:To screen ferroptosis-related characterized genes in ischemic stroke by bioinformatics and machine learning methods and validate them by cellular experiments to investigate the role of ferroptosis in ischemic stroke. METHODS:Eligible ischemic stroke-related datasets and ferroptosis expression datasets were selected based on GEO database and FerrDb database,and ferroptosis-related differential genes were screened by t-test.GO functional enrichment analysis with KEGG signaling pathway enrichment analysis was performed for ferroptosis-related differential genes.Characterized genes for ferroptosis in ischemic stroke were screened by PPI network analysis and machine learning.The reliability and biological functions of the characterized genes were explored using ROC analysis and GSEA analysis,followed by cell experiment.HT22 cells were divided into control and ischemic stroke groups.No intervention was made in the control group,and 0.1 mM H2O2 was added to the ischemic stroke group for 24 hours to simulate cellular oxidative stress injury and ferroptosis.The ferroptosis and the expression of characterized genes were verified by real-time fluorescence quantitative polymerase chain reaction(RT-PCR)and western blot assay. RESULTS AND CONCLUSION:(1)Forty-five ferroptosis-associated differential genes were obtained,and GO and KEGG enrichment analyses revealed that the differential genes were closely associated with oxidative stress,autophagy,ferroptosis,adipocytokine signaling pathway,and mitochondrial metabolism.(2)A total of one ferroptosis characterized gene,nuclear factor erythroid 2-related factor 2(NFE2L2),was identified by the MCODE plugin and cytoHubba plugin in the PPI network with the LASSO algorithm and SVM-RFE algorithm in machine learning.(3)Receiver operating characteristic curve analysis of NFE2L2 revealed that the diagnostic prediction models constructed in the training and validation sets had good accuracy and specificity.GSEA analysis of NFE2L2 revealed that the characterized gene was involved in the regulation of ischemic stroke pathogenesis through immunity,inflammatory response,amino acid metabolism,and neurofactor regulation.(4)RT-PCR and western blot analyses showed that the acyl coenzyme A synthetase long chain family,member 4(ACSL4)mRNA and protein expression levels were significantly higher in the ischemic stroke group compared with the control group(P<0.05),and the glutathione peroxidase 4(GPX4)mRNA and protein expression levels were significantly lower in the ischemic stroke group(P<0.05).Compared with the control group,the mRNA and protein expression levels of the characterized gene NFE2L2 were significantly higher in the ischemic stroke group(P<0.05).(5)It suggests that ischemic stroke is closely related to ferroptosis,and targeting the characterized gene NFE2L2 may provide certain ideas and directions for the study and treatment of ischemic stroke.

Objective To investigate the role of Toll-like receptor 4(TLR4)in the regulation of homocys-teine(Hcy)-induced ferroptosis in macrophages.Methods Mouse macrophage cells RAW264.7 were cultured and divided into control group,Hcy intervention group(Hcy group),and Hcy plus ferroptosis inhibitor group(Hcy+Fer-1 group).After transfection with interference fragments,macrophages were treated with Hcy,and then divided into control group,Hcy intervention group(Hcy group),TLR4 interference negative control plus Hcy intervention group(si-NC+Hcy group),and TLR4 interference plus Hcy intervention group(si-TLR4+Hcy group).Macrophages were transfected with overexpression lentivirus and treated with Hcy,then were divided into control group,Hcy intervention group(Hcy group),a TLR4 overexpression negative control plus Hcy intervention group(OE-NC+Hcy group),and a TLR4 overexpression plus Hcy intervention group(OE-TLR4+Hcy group).After 48 hours of intervention,real-time fluorescent quantitative PCR and western blot were used to detect the expression levels of TLR4 in macrophages treated with Hcy;western blot was used to detect the expression levels of ferroptosis-related proteins ACSL4,GPX4,and FTH1 in macrophages,and ferrous ion assay kit to detect the concentration of Fe2+in macrophages;reactive oxygen species(ROS)assay kit and laser confocal microscopy were used to detect the content of intracellular reactive oxygen species.Results Compared with those in the control group,the expression level of the pro-ferroptosis protein ACSL4 was increased in the Hcy group(P<0.05),while the expression levels of anti-ferroptosis proteins GPX4 and FTH1 were decreased(P<0.05);the concentration of Fe2+was increased(P<0.05),and the content of ROS was increased.Meanwhile,the protein and mRNA expres-sion levels of TLR4 were both increased in the Hcy group(P<0.05).After macrophages were transfected with TLR4 interference fragments,compared with those in the si-NC+Hcy group,the expression levels of GPX4 and FTH1 were increased(P<0.05);the expression level of ACSL4 was decreased(P<0.05);the concentration of Fe2+was decreased(P<0.05),and the content of ROS was reduced in the si-TLR4+Hcy group.After macro-phages were transfected with TLR4 overexpression lentivirus,compared with those in the OE-NC+Hcy group,the expression levels of GPX4 and FTH1 were decreased(P<0.05),and the expression level of ACSL4 was increased(P<0.05)in the OE-TLR4+Hcy group.Conclusion Hcy induces the occurrence of ferroptosis in macrophages,and Toll-like receptor 4 has a positive feedback regulatory effect on ferroptosis in macrophages.

Severe open tibiofibular fractures account for approximately 28.1% of all open fractures. Among them, Gustilo-Anderson type IIIB/C fractures present significant clinical challenges due to associated bone and soft tissue defects, high infection rates, and risk of amputation. Inadequate preoperative assessment may lead to suboptimal emergency surgical planning or intraoperative complications. Historically, external fixation was often preferred, but this approach has been associated with limitations such as restricted joint mobility, delayed bone union, joint stiffness, and disuse osteoporosis, resulting in poor functional recovery. With advancements of debridement techniques, standardization of antibiotic use, and popularization of early soft tissue coverage, early internal fixation has gained broader acceptance. Nevertheless, controversies persist regarding the choice of fixation method, timing of definitive fixation, use of reamed versus unreamed intramedullary nailing, and necessity of fibular fixation. To standardize the diagnosis and early management of severe open tibiofibular fractures, reduce complication rates, and improve functional recovery, the Society of Microsurgery of the Chinese Medical Association organized a panel of domestic experts to develop the Evidence-based guideline for the diagnosis and early fixation of severe open tibiofibular fractures ( version 2025), using evidence-based methodology. The guidelines provided 12 recommendations covering diagnostic and early fixation strategies of severe open tibiofibular fractures, aiming to provide clinicians with scientifically grounded and standardized guidance.

Cemental tear is a rare and indetectable condition unless obvious clinical signs present with the involvement of surrounding periodontal and periapical tissues. Due to its clinical manifestations similar to common dental issues, such as vertical root fracture, primary endodontic diseases, and periodontal diseases, as well as the low awareness of cemental tear for clinicians, misdiagnosis often occurs. The critical principle for cemental tear treatment is to remove torn fragments, and overlooking fragments leads to futile therapy, which could deteriorate the conditions of the affected teeth. Therefore, accurate diagnosis and subsequent appropriate interventions are vital for managing cemental tear. Novel diagnostic tools, including cone-beam computed tomography (CBCT), microscopes, and enamel matrix derivatives, have improved early detection and management, enhancing tooth retention. The implementation of standardized diagnostic criteria and treatment protocols, combined with improved clinical awareness among dental professionals, serves to mitigate risks of diagnostic errors and suboptimal therapeutic interventions. This expert consensus reviewed the epidemiology, pathogenesis, potential predisposing factors, clinical manifestations, diagnosis, differential diagnosis, treatment, and prognosis of cemental tear, aiming to provide a clinical guideline and facilitate clinicians to have a better understanding of cemental tear.
Humans ; Dental Cementum/injuries* ; Consensus ; Diagnosis, Differential ; Cone-Beam Computed Tomography ; Tooth Fractures/therapy*

Objective:To explore the medication law of national TCM master Lu Fang in the treatment of primary trigeminal neuralgia (PTN) based on data mining.Methods:With the prescription of the outpatient patients of Harbin Traditional Chinese Medicine Hospital of Professor Lu Fang from September 2014 to September 2022 as the data source, the frequency, property and taste, and meridian tropism of the prescribed drugs were analyzed using Excel 2022 software. R 4.2.1 was used for mining analysis on Chinese materia medica, including correlation, relevance, and clustering,and the medication law in the treatment of PTN was discussed.Results:A total of 300 prescriptions were analyzed, involving 177 kinds of Chinese materia medica, with a frequency of 3 120 times, and 34 kinds of of high-frequency Chinese materia medica. The high frequently Chinese materia medica included Chuanxiong Rhizoma, Angelicae Dahuricae Radix, Puerariae Lobatae Radix, Ligustici Rhizoma et Radix, and Viticis Fructus. The main properties were warm, slightly cold, and neutral, while the main tastes were pungent, bitter, and sweet. The meridian tropism analysis ranked the liver, lung, spleen, and stomach meridians in descending order. Analysis yielded 21 strong association rules, and the association analysis formed a core prescription group based on Chuanxiong Rhizoma, Angelicae Dahuricae Radix, and Ligustici Rhizoma et Radix. The analysis obtained 5 types of clustering combinations.Conclusion:Professor Lu Fang's the medication law to treat primary trigeminal neuralgia is mainly dispelling wind and alleviating pain, which is often combined with the methods, such as searching and dredging collaterals, clearing and dispelling the stagnated heat, calming the liver and subduing yang, soothing the liver and invigorating the spleen.

Objective To assess the impact of freeze-drying and dry heat virus inactivation processes on the activity ofα2-macroglobulin(A2M)derived from human plasma Cohn fraction Ⅳ.Methods A2M derived from human plasma Cohn fraction Ⅳ was prepared and subjected to programmed freeze-drying with dry heat virus inactivation.The lyophilized products were evaluated for their appearance,water content,and validation of the viral inactivation process.The bioactivity of the products before and after lyophilization as well as before and after dry heat inactivation was determined via trypsin inhibition,and the comparisons were studied.Results The appearance of the lyophilized product was fluffy,and the water content was(5.83±0.45)％.The specific activities of the samples before and after lyophilization were(10.199±0.137)and(10.033±0.201)μg/mg,respectively,with no statistically significantdifference between the two groups(P＞0.05).The viral inactivation of the samples was carried out by using dry heat inactivation conditions at 100 ℃ for 30 min.After inactivation,the reduction was ≥5.125 LgTCID50/0.1 mL in Pseudorabies virus(PRV)titers,≥4.500 LgTCID50/0.1 mL in Sindbis virus(SinV)titers,≥6.375 LgTCID50/0.1 mL in encephalomyocarditis virus(EMCV)titers,and≥4.500 LgTCID50/0.1 mL in porcine parvovirus(PPV)titers.The specific activities of the samples before and after dry heat were(9.921±0.292)and(10.091±0.278)μ g/mg,respectively,with no statistically significant difference between the two groups.Conclusion A2M derived from human plasma Cohn fraction Ⅳ,when subjected to freeze-drying followed by dry heat inactivation at 100 ℃ for 30 minutes,can effectively inactivate viruses without altering the biological activity of the product.

OBJECTIVE To screen endogenous differential metabolites in mice that die from chlor-promazine(CPZ)poisoning and investigate the detoxification mechanism of triheptanoin(TriHep)against CPZ-induced lethality.METHODS Mice were randomly divided into the following groups(half male and half female):normal control,CPZ 2.5LD50,CPZ LD50 intoxication(CPZI),CPZ LD50 death(CPZD),TriHep-control,and TriHep-intervention(TriHep+CPZ LD50).The CPZ 2.5LD50,CPZI and CPZD groups were intragastrically given a corresponding dose of CPZ,respectively.The TriHep-control group and the TriHep-intervention group were intragastrically given saline and CPZ LD50 respectively before being intragastrically given TriHep(3 μL·g-1)10 min later.Plasma samples from the CPZ 2.5LD50 group and normal control group were analyzed using liquid chromatography-tandem mass spectrometry(LC-MS/MS)for metabolite identification and quantification.MetaboAnalyst 5.0 was employed to perform principal component analysis(PCA),orthogonal partial least squares-discriminant analysis(OPLS-DA),and metabolic pathway analysis to screen and identify differential metabolites.More comparisons were made of the levels of differential metabolites in plasma between the normal control,CPZI,CPZD,TriHep-intervention,and TriHep-control groups.RESULTS In the PCA score plot,metabolomic samples from the CPZ 2.5LD50 group and normal control group showed clear separation,indicating distinct clus-tering patterns.Primary screening under three conditions,including P＜0.05,variable importance in projec-tion(VIP)score≥ 1 and fold change(FC)≥1.5 or ≤0.67 for a comparison of CPZ 2.5LD50 group with normal control group 28 metabolites were identified.Following quantitative enrichment and structural identifica-tion,three significantly differential metabolites were confirmed:acetylcarnitine,propionylcarnitine,and succinic acid.Compared with the normal control group,both CPZI and CPZD groups showed signifi-cantly decreased plasma levels of acetylcarnitine and propionylcarnitine,while the succinic acid content was markedly increased in the CPZD group.In the TriHep control group,levels of acetylcarnitine and succinic acid were significantly elevated,with no significant change in propionylcarnitine levels.Com-pared with the CPZI group,the CPZD group showed a significant increase in plasma succinic acid levels,but no significant change was observed in the acetylcarnitine content.The TriHep-intervention group demonstrated metabolite profiles(all the three differential metabolites)similar to those in the CPZI group,with significantly reduced propionylcarnitine and succinic acid concentrations compared to the CPZD group.CONCLUSION In the early stage of CPZ intoxication,TriHep can alleviate CPZ poisoning via acetylcarnitine,which can stabilize the level of succinic acid in plasma via indirect succinic acid replenishment.

OBJECTIVE To screen endogenous differential metabolites in mice that die from chlor-promazine(CPZ)poisoning and investigate the detoxification mechanism of triheptanoin(TriHep)against CPZ-induced lethality.METHODS Mice were randomly divided into the following groups(half male and half female):normal control,CPZ 2.5LD50,CPZ LD50 intoxication(CPZI),CPZ LD50 death(CPZD),TriHep-control,and TriHep-intervention(TriHep+CPZ LD50).The CPZ 2.5LD50,CPZI and CPZD groups were intragastrically given a corresponding dose of CPZ,respectively.The TriHep-control group and the TriHep-intervention group were intragastrically given saline and CPZ LD50 respectively before being intragastrically given TriHep(3 μL·g-1)10 min later.Plasma samples from the CPZ 2.5LD50 group and normal control group were analyzed using liquid chromatography-tandem mass spectrometry(LC-MS/MS)for metabolite identification and quantification.MetaboAnalyst 5.0 was employed to perform principal component analysis(PCA),orthogonal partial least squares-discriminant analysis(OPLS-DA),and metabolic pathway analysis to screen and identify differential metabolites.More comparisons were made of the levels of differential metabolites in plasma between the normal control,CPZI,CPZD,TriHep-intervention,and TriHep-control groups.RESULTS In the PCA score plot,metabolomic samples from the CPZ 2.5LD50 group and normal control group showed clear separation,indicating distinct clus-tering patterns.Primary screening under three conditions,including P＜0.05,variable importance in projec-tion(VIP)score≥ 1 and fold change(FC)≥1.5 or ≤0.67 for a comparison of CPZ 2.5LD50 group with normal control group 28 metabolites were identified.Following quantitative enrichment and structural identifica-tion,three significantly differential metabolites were confirmed:acetylcarnitine,propionylcarnitine,and succinic acid.Compared with the normal control group,both CPZI and CPZD groups showed signifi-cantly decreased plasma levels of acetylcarnitine and propionylcarnitine,while the succinic acid content was markedly increased in the CPZD group.In the TriHep control group,levels of acetylcarnitine and succinic acid were significantly elevated,with no significant change in propionylcarnitine levels.Com-pared with the CPZI group,the CPZD group showed a significant increase in plasma succinic acid levels,but no significant change was observed in the acetylcarnitine content.The TriHep-intervention group demonstrated metabolite profiles(all the three differential metabolites)similar to those in the CPZI group,with significantly reduced propionylcarnitine and succinic acid concentrations compared to the CPZD group.CONCLUSION In the early stage of CPZ intoxication,TriHep can alleviate CPZ poisoning via acetylcarnitine,which can stabilize the level of succinic acid in plasma via indirect succinic acid replenishment.

Objective To investigate the role of Toll-like receptor 4(TLR4)in the regulation of homocys-teine(Hcy)-induced ferroptosis in macrophages.Methods Mouse macrophage cells RAW264.7 were cultured and divided into control group,Hcy intervention group(Hcy group),and Hcy plus ferroptosis inhibitor group(Hcy+Fer-1 group).After transfection with interference fragments,macrophages were treated with Hcy,and then divided into control group,Hcy intervention group(Hcy group),TLR4 interference negative control plus Hcy intervention group(si-NC+Hcy group),and TLR4 interference plus Hcy intervention group(si-TLR4+Hcy group).Macrophages were transfected with overexpression lentivirus and treated with Hcy,then were divided into control group,Hcy intervention group(Hcy group),a TLR4 overexpression negative control plus Hcy intervention group(OE-NC+Hcy group),and a TLR4 overexpression plus Hcy intervention group(OE-TLR4+Hcy group).After 48 hours of intervention,real-time fluorescent quantitative PCR and western blot were used to detect the expression levels of TLR4 in macrophages treated with Hcy;western blot was used to detect the expression levels of ferroptosis-related proteins ACSL4,GPX4,and FTH1 in macrophages,and ferrous ion assay kit to detect the concentration of Fe2+in macrophages;reactive oxygen species(ROS)assay kit and laser confocal microscopy were used to detect the content of intracellular reactive oxygen species.Results Compared with those in the control group,the expression level of the pro-ferroptosis protein ACSL4 was increased in the Hcy group(P<0.05),while the expression levels of anti-ferroptosis proteins GPX4 and FTH1 were decreased(P<0.05);the concentration of Fe2+was increased(P<0.05),and the content of ROS was increased.Meanwhile,the protein and mRNA expres-sion levels of TLR4 were both increased in the Hcy group(P<0.05).After macrophages were transfected with TLR4 interference fragments,compared with those in the si-NC+Hcy group,the expression levels of GPX4 and FTH1 were increased(P<0.05);the expression level of ACSL4 was decreased(P<0.05);the concentration of Fe2+was decreased(P<0.05),and the content of ROS was reduced in the si-TLR4+Hcy group.After macro-phages were transfected with TLR4 overexpression lentivirus,compared with those in the OE-NC+Hcy group,the expression levels of GPX4 and FTH1 were decreased(P<0.05),and the expression level of ACSL4 was increased(P<0.05)in the OE-TLR4+Hcy group.Conclusion Hcy induces the occurrence of ferroptosis in macrophages,and Toll-like receptor 4 has a positive feedback regulatory effect on ferroptosis in macrophages.

Severe open tibiofibular fractures account for approximately 28.1% of all open fractures. Among them, Gustilo-Anderson type IIIB/C fractures present significant clinical challenges due to associated bone and soft tissue defects, high infection rates, and risk of amputation. Inadequate preoperative assessment may lead to suboptimal emergency surgical planning or intraoperative complications. Historically, external fixation was often preferred, but this approach has been associated with limitations such as restricted joint mobility, delayed bone union, joint stiffness, and disuse osteoporosis, resulting in poor functional recovery. With advancements of debridement techniques, standardization of antibiotic use, and popularization of early soft tissue coverage, early internal fixation has gained broader acceptance. Nevertheless, controversies persist regarding the choice of fixation method, timing of definitive fixation, use of reamed versus unreamed intramedullary nailing, and necessity of fibular fixation. To standardize the diagnosis and early management of severe open tibiofibular fractures, reduce complication rates, and improve functional recovery, the Society of Microsurgery of the Chinese Medical Association organized a panel of domestic experts to develop the Evidence-based guideline for the diagnosis and early fixation of severe open tibiofibular fractures ( version 2025), using evidence-based methodology. The guidelines provided 12 recommendations covering diagnostic and early fixation strategies of severe open tibiofibular fractures, aiming to provide clinicians with scientifically grounded and standardized guidance.