Objective To establish a flow cytometry method for detecting C1s protein on platelet surface and preliminarily explore its potential application value in the auxiliary diagnosis of primary immune thrombocytopenia(ITP).Methods C1s-conjugated 2 μm car-boxylated magnetic beads(C1s beads)were prepared and used as quality control particles.Fluorescein isothiocyanate(FITC)-labeled anti-C1s antibody was employed as the detection antibody to develop a flow cytometric assay for detecting C1s protein expression on platelets.The intra-assay and inter-assay precision,as well as the dilution linearity of the method,were evaluated.Subsequently,the expression levels of C1 s protein on the surface of platelets were compared among the ITP group,the non-ITP thrombocytopenia group,and the healthy control group.Results Light microscopy showed that both unconjugated carboxylated magnetic beads(blank beads)and C1s-conjugated beads were uniformly dispersed without aggregation.Under fluorescence microscopy,C1s beads exhibited strong yellow-green fluorescence,whereas the blank beads showed no fluorescence signal.The established flow cytometry assay exhibited ac-ceptable precision,with intra-assay coefficient of variation(CV)values of 7.02％,7.12％,and 3.91％for low,medium,and high con-centrations of C1s beads,respectively,and inter-assay CV values of 13.49％,6.15％,and 0.78％,respectively.The dilution linearity was satisfactory,coefficient of determination(R2)=0.998 8.Clinical sample testing revealed that the proportion of C1s-positive plate-lets in ITP group(2.56±0.79)％was significantly higher than that in healthy control group(0.23±0.18)％and the non-ITP thrombo-cytopenia control group(0.22±0.10)％,with statistically significant differences(both P＜0.05).Conclusion This study successfully established a stable and reliable flow-cytometry method for quantifying C1s expression on platelet surface and preliminarily demonstrated that C1s expression is significantly elevated on platelets of ITP patients,suggesting that C1s could serve as a potential auxiliary diag-nostic marker for ITP.

Aim To investigate the effect of ACSL4 on the malignant biological behavior of esophageal cancer cells and gefitinib resistance by regulating FASN,and to explore the related mechanism.Methods Thirty-five fresh esophageal cancer tissues and adjacent nor-mal tissues,and 30 esophageal cancer tissues with ge-fitinib resistance were collected.The expressions of ACSL4 and FASN were detected by qRT-PCR and im-munohistochemistry.The expression levels of ACSL4 and FASN in human normal esophageal cells HET-1 A,esophageal cancer cell lines ECA109,EC9706,TE-1 and TE-1/GR were detected by qRT-PCR.Cells in each group were constructed by liposome transfection technique,and the drug resistance and proliferation a-bility of cells were detected by cloning and CCK-8 as-say,cell apoptosis was detected by flow cytometry,cell invasion ability was detected by Transwell,and EMT pathway protein expression was detected by Western blot.Results Compared with adjacent normal tis-sues,the expression of ACSL4 and FASN genes in cancer tissues increased,and there was a positive corre-lation.The expression of ACSL4 significantly increased in ECA109,EC9706 and TE-1 cells compared with HET-1 A cells.With the increase of gefitinib concen-tration,the expression of ACSL4 in TE-1 cells gradually increased,and the expression of ACSL4 in TE-1/GR cells was higher than that of TE-1.Compared with the control group and the si-NC group,the cell proliferation and invasion ability of si-ACSL4 group decreased,the number of apoptosis increased,the expression of E-Cadherin increased,and the expression of N-Cadherin,Vimentin and β-catenin decreased.The response ex-periment showed that compared with the si-ACSL4 group and the si-ACSL4+oe-NC group,the cells in the si-ACSL4+oe-FASN group increased drug resistance,increased proliferation and invasion ability,decreased apoptosis number and decreased expression of E-Cad-herin.The expressions of N-Cadherin,Vimentin and β-catenin increased.Conclusions By down-regulating the expression of FASN,ACSL4 reverses the resistance of esophageal cancer TE-1/GR cells to gefitinib and in-hibits the proliferation,invasion and accelerates apopto-sis of TE-1/GR cells,which may be related to the regu-lation of EMT signaling pathway.

Aim To investigate the effect of ACSL4 on the malignant biological behavior of esophageal cancer cells and gefitinib resistance by regulating FASN,and to explore the related mechanism.Methods Thirty-five fresh esophageal cancer tissues and adjacent nor-mal tissues,and 30 esophageal cancer tissues with ge-fitinib resistance were collected.The expressions of ACSL4 and FASN were detected by qRT-PCR and im-munohistochemistry.The expression levels of ACSL4 and FASN in human normal esophageal cells HET-1 A,esophageal cancer cell lines ECA109,EC9706,TE-1 and TE-1/GR were detected by qRT-PCR.Cells in each group were constructed by liposome transfection technique,and the drug resistance and proliferation a-bility of cells were detected by cloning and CCK-8 as-say,cell apoptosis was detected by flow cytometry,cell invasion ability was detected by Transwell,and EMT pathway protein expression was detected by Western blot.Results Compared with adjacent normal tis-sues,the expression of ACSL4 and FASN genes in cancer tissues increased,and there was a positive corre-lation.The expression of ACSL4 significantly increased in ECA109,EC9706 and TE-1 cells compared with HET-1 A cells.With the increase of gefitinib concen-tration,the expression of ACSL4 in TE-1 cells gradually increased,and the expression of ACSL4 in TE-1/GR cells was higher than that of TE-1.Compared with the control group and the si-NC group,the cell proliferation and invasion ability of si-ACSL4 group decreased,the number of apoptosis increased,the expression of E-Cadherin increased,and the expression of N-Cadherin,Vimentin and β-catenin decreased.The response ex-periment showed that compared with the si-ACSL4 group and the si-ACSL4+oe-NC group,the cells in the si-ACSL4+oe-FASN group increased drug resistance,increased proliferation and invasion ability,decreased apoptosis number and decreased expression of E-Cad-herin.The expressions of N-Cadherin,Vimentin and β-catenin increased.Conclusions By down-regulating the expression of FASN,ACSL4 reverses the resistance of esophageal cancer TE-1/GR cells to gefitinib and in-hibits the proliferation,invasion and accelerates apopto-sis of TE-1/GR cells,which may be related to the regu-lation of EMT signaling pathway.

Objective To establish a flow cytometry method for detecting C1s protein on platelet surface and preliminarily explore its potential application value in the auxiliary diagnosis of primary immune thrombocytopenia(ITP).Methods C1s-conjugated 2 μm car-boxylated magnetic beads(C1s beads)were prepared and used as quality control particles.Fluorescein isothiocyanate(FITC)-labeled anti-C1s antibody was employed as the detection antibody to develop a flow cytometric assay for detecting C1s protein expression on platelets.The intra-assay and inter-assay precision,as well as the dilution linearity of the method,were evaluated.Subsequently,the expression levels of C1 s protein on the surface of platelets were compared among the ITP group,the non-ITP thrombocytopenia group,and the healthy control group.Results Light microscopy showed that both unconjugated carboxylated magnetic beads(blank beads)and C1s-conjugated beads were uniformly dispersed without aggregation.Under fluorescence microscopy,C1s beads exhibited strong yellow-green fluorescence,whereas the blank beads showed no fluorescence signal.The established flow cytometry assay exhibited ac-ceptable precision,with intra-assay coefficient of variation(CV)values of 7.02％,7.12％,and 3.91％for low,medium,and high con-centrations of C1s beads,respectively,and inter-assay CV values of 13.49％,6.15％,and 0.78％,respectively.The dilution linearity was satisfactory,coefficient of determination(R2)=0.998 8.Clinical sample testing revealed that the proportion of C1s-positive plate-lets in ITP group(2.56±0.79)％was significantly higher than that in healthy control group(0.23±0.18)％and the non-ITP thrombo-cytopenia control group(0.22±0.10)％,with statistically significant differences(both P＜0.05).Conclusion This study successfully established a stable and reliable flow-cytometry method for quantifying C1s expression on platelet surface and preliminarily demonstrated that C1s expression is significantly elevated on platelets of ITP patients,suggesting that C1s could serve as a potential auxiliary diag-nostic marker for ITP.

High mobility group box protein 1(HMGB1)is one of the most widely expressed protein member in the HMGs family,which is well known for its involvement in the body inflammatory response.Previous researches have found that it plays a significant role in cell migration,immune identification and neuroprotection.Spinal cord injury is a disease that causes severe damage to the nervous system,and neural circuits are disrupted after a spinal cord injury,which leads to many conditions including ischemia and hypoxia,inflammatory responses,demyelinating lesions,and glial scar formation that are detrimental to nerve regeneration and repair,making it one of the most difficult diseases to treat in the modern spinal surgery field.HMGB1 is upregulated after spinal cord injury,thereby regulating neuroinflam-matory responses,and participating in the neuronal apoptosis,promoting neuronal regeneration,and inducing neural stem cell differentiation and migration,which plays an important role in the process of neural function recovery.This paper summarizes the structure and function of HMGB1,as well as its role in spinal cord injury,in order to provide direction for founding therapeutic target for neurological function recovery after spinal cord injury.

Kv1.3,a voltage-gated potassium channel,is highly expressed in T lymphocytes,the nervous system,and vascular smooth muscle cells.It plays a critical role in membrane excitability and electrical signal transduction,serving as an important target for studying T-cell function and providing a promising direction for developing therapeutics against autoimmune and inflammatory diseases.Therefore,the de-velopment of specific inhibitors of Kv1.3 channel has emerged as a novel therapeutic strategy for these disorders.In this study,we isolated and purified a novel Kv1.3-inhibitory peptide toxin,LmKTx13,from the venom of the scorpion Lychas mucronatus using reversed-phase high-performance liquid chroma-tography(RP-HPLC).LmKTx13 consists of 38 amino acid residues,including six cysteines that form three disulfide bonds.Whole-cell patch-clamp recordings revealed that LmKTx13 potently inhibited Kv1.3 with an IC50 of 7.92±3.0 nmol/L.Selectivity analysis showed that 2 μmol/L LmKTx13 also in-hibited Kv1.2 and Kv1.7,but exhibited no significant effects on other potassium channel subtypes or voltage-gated sodium channels.Further investigation into the mechanism demonstrated that LmKTx13 acts as a pore-blocking inhibitor of Kv1.3.By analyzing the effects of LmKTx13 on Kv1.3 channel gating ki-netics and performing sequence alignment of the pore regions of Kv1.3 and Kv1.5,we constructed site-directed mutants and identified the pore region of Kv1.3 as the critical binding site for LmKTx13.Key residues involved in the interaction included T425,G427,and H451.In summary,we discovered a no-vel pore-blocking Kv1.3 inhibitor,LmKTx13,from L.mucronatus venom,which exhibits high affinity and selectivity for Kv1.3.These findings highlight its potential as a potential lead molecule for developing Kv1.3-targeted therapeutics.

Objective To analyze the clinical characteristics and prognostic risk factors of patients with atrial fibrillation(AF)related to septic shock admitted to the intensive care unit(ICU).Methods This retrospective cohort study included 152 patients with septic shock who were admitted to the ICU between January 2020 and December 2023.Patients were categorized into AF(n=48)and non-AF(n=104)groups based on the occurrence of AF.Clinical parameters were compared between the two groups,and variables with statistically significant differences(P<0.05)were entered into a multivariate logistic regression analysis to identify clinical features associated with AF.Patients were also stratified into survival(n=103)and death(n=49)groups based on 28-day outcomes.Variables showing signifi-cant intergroup differences(P<0.05)were further included in a multivariate model to identify independent risk factors for mortality.Kaplan-Meier survival analysis was performed using AF as a binary variable,and log-rank tests were applied to compare survival curves.Results The incidence of AF was 31.58%among patients with septic shock.Compared with the non-AF group,the AF group was older,received higher cumulative doses of vasoactive agents,and had elevated levels of lactate(Lac),N-terminal brain natriuretic peptide precursor(NT-proBNP),troponin I(cTnI),tumor necrosis factorα(TNF-α),and C-reactive protein(CRP).The AF group also had higher E/e'ratios,left ventricular ejection fraction(LVEF),Acute Physiology and Chronic Health Evaluation-Ⅱ(APACHEⅡ)scores,Sequen-tial Organ Failure Assessment(SOFA)scores,and 28-day mortality rates(all P<0.05).Multivariate logistic regression identified age(odds ratio[OR]=1.060,P=0.015),SOFA score(OR=1.525,P=0.001),TNF-α(OR=1.009,P=0.026),CRP(OR=1.008,P=0.002),and E/e'(OR=1.363,P=0.023)as independent risk factors for AF.The 28-day mortality rate was 32.24%.Compared with survivors,non-survivors had significantly higher Lac,cTnI,E/e',LVEF,and TNF-α levels,higher APACHEⅡand SOFA scores,longer ICU stays,and a higher incidence of AF(all P<0.05).Multivariate analysis identified SOFA score,cTnI,E/e',and AF as independent predictors of 28-day mortality.Kaplan-Meier analysis revealed significantly poorer survival in patients with AF(χ2=6.747,P=0.009).Conclusion AF is common in ICU patients with septic shock.Advanced age,greater organ dysfunction,elevated inflammatory markers,and diastolic dysfunction are associated with AF occurrence.Organ dysfunction,myocardial injury,diastolic dysfunction,and AF are inde-pendent predictors of mortality in this population.Greater clinical attention is warranted in patients with AF and septic shock due to their elevated risk of poor outcomes.

Kv1.3,a voltage-gated potassium channel,is highly expressed in T lymphocytes,the nervous system,and vascular smooth muscle cells.It plays a critical role in membrane excitability and electrical signal transduction,serving as an important target for studying T-cell function and providing a promising direction for developing therapeutics against autoimmune and inflammatory diseases.Therefore,the de-velopment of specific inhibitors of Kv1.3 channel has emerged as a novel therapeutic strategy for these disorders.In this study,we isolated and purified a novel Kv1.3-inhibitory peptide toxin,LmKTx13,from the venom of the scorpion Lychas mucronatus using reversed-phase high-performance liquid chroma-tography(RP-HPLC).LmKTx13 consists of 38 amino acid residues,including six cysteines that form three disulfide bonds.Whole-cell patch-clamp recordings revealed that LmKTx13 potently inhibited Kv1.3 with an IC50 of 7.92±3.0 nmol/L.Selectivity analysis showed that 2 μmol/L LmKTx13 also in-hibited Kv1.2 and Kv1.7,but exhibited no significant effects on other potassium channel subtypes or voltage-gated sodium channels.Further investigation into the mechanism demonstrated that LmKTx13 acts as a pore-blocking inhibitor of Kv1.3.By analyzing the effects of LmKTx13 on Kv1.3 channel gating ki-netics and performing sequence alignment of the pore regions of Kv1.3 and Kv1.5,we constructed site-directed mutants and identified the pore region of Kv1.3 as the critical binding site for LmKTx13.Key residues involved in the interaction included T425,G427,and H451.In summary,we discovered a no-vel pore-blocking Kv1.3 inhibitor,LmKTx13,from L.mucronatus venom,which exhibits high affinity and selectivity for Kv1.3.These findings highlight its potential as a potential lead molecule for developing Kv1.3-targeted therapeutics.

High mobility group box protein 1(HMGB1)is one of the most widely expressed protein member in the HMGs family,which is well known for its involvement in the body inflammatory response.Previous researches have found that it plays a significant role in cell migration,immune identification and neuroprotection.Spinal cord injury is a disease that causes severe damage to the nervous system,and neural circuits are disrupted after a spinal cord injury,which leads to many conditions including ischemia and hypoxia,inflammatory responses,demyelinating lesions,and glial scar formation that are detrimental to nerve regeneration and repair,making it one of the most difficult diseases to treat in the modern spinal surgery field.HMGB1 is upregulated after spinal cord injury,thereby regulating neuroinflam-matory responses,and participating in the neuronal apoptosis,promoting neuronal regeneration,and inducing neural stem cell differentiation and migration,which plays an important role in the process of neural function recovery.This paper summarizes the structure and function of HMGB1,as well as its role in spinal cord injury,in order to provide direction for founding therapeutic target for neurological function recovery after spinal cord injury.

Objective To investigate the effects of peripheral blood neutrophil infiltration on the polarization regulation of cerebral resident microglia under a permanent ischemic stroke model.Methods Fifty-eight C57BL/6 mice were divided into two groups.One group was sham group,and the other group of mice was subjected to permanent middle cerebral artery occlusion surgery.Mice were euthanized 48 hours,7 days,14 days,and 30 days after surgery for tissue collection.Western blotting was used to detect expression levels of M1 microglia markers CD 16,M2 microglia marker arginase 1(Arg1),inflammatory cytokine interleukin-1 β(IL-1β),and neutrophil marker myeloperoxidase(MPO)in brain tissue.Immunofluorescence histochemical staining was used to assess neutrophil infiltration and M2 microglial distribution around the infarct area in brain sections.In vitro,purified neutrophils were co-cultured with BV2 microglial cells.After lipopolysaccharide stimulation,the phagocytosis of neutrophils by BV2 cells was observed,and the expression levels of CD16 and Arg1 proteins in BV2 cells were detected.Results Western blotting showed that the levels of CD16(P＜0.05),IL-1β(P＜0.001),and MPO(P＜0.05)in brain tissue increased significantly 48 hours and 7 days after surgery,then decreased,with MPO expression returning to normal levels 30 days after surgery.Immunofluorescence showed a significant increase of MPO-positive cells around the infarct area of the mouse cerebral cortex 48 hours after surgery(P＜0.001),followed by a decrease(P＜0.05).The number of ionized calcium binding adapter molecule 1(Iba1)and MPO double-positive cells gradually increased after surgery,and reached their peak at 14 days(P＜0.05).Iba1 and Arg1 double-positive cells also increased significantly 7 days(P＜0.05)and 14 days(P＜0.01)after surgery.In vitro,co-culture experiments showed that after BV2 phagocytosing neutrophils,CD 16(P＜0.05)significantly decreased and Arg1 significantly upregulated(P＜0.05).Conclusion In a permanent ischemic stroke model,microglia transition from M1 to M2 type after phagocytosing neutrophils,and the injured brain area changes from pro-inflammatory state to anti-inflammatory state.