Background and aims The mechanism of cholestatic liver injury (CLI) is unclear, and effective therapies are lacking. While peroxisome proliferator-activated receptor alpha (PPARα) agonists show potential hepatoprotective effect and pyroptosis is implicated in hepatocellular damage, how PPARα activation mitigates lithocholic acid (LCA)-induced pyroptosis remains unknown. Methods The hepatoprotective effect of PPARα agonists was evaluated in a mouse model of intrahepatic cholestasis induced by LCA. Liver injury was assessed via serum biochemistry, hematoxylin and eosin and TUNEL staining, and electron microscopy. Pyroptosis pathways were analyzed using real-time quantitative polymerase chain reaction, Western blot, and co-immunoprecipitation. Results Combined morphological, histopathological, and biochemical analyses confirmed that PPARα activation protects against CLI. Compared with LCA treatment alone, PPARα activation significantly attenuated the elevation of serum lactate dehydrogenase (LDH), the increased TUNEL-positive cells, and the formation of hepatocyte membrane pores. Mechanistically, PPARα activation suppressed both NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis and apoptosis protease-activating factor-1 (APAF-1)/CASPASE-3/GSDME-mediated pyroptosis. Furthermore, PPARα agonist pretreatment inhibited activation of the nuclear factor-kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways. Conclusions PPARα protects against LCA-induced CLI by inhibiting both NLRP3 inflammasome-mediated pyroptosis associated with NF-κB and APAF-1/CASPASE-3/GSDME-mediated pyroptosis associated with the FOXO1 signaling pathway.

Aim To investigate the effect of high-fat diet on the tight junction function injury of Sertoli cells through the NF-κB/NLRP3 signaling pathway in mice and to explore the underlying mechanism.Methods Male C57BL/6J mice were fed with high-fat or normal diet for five months.The body and gonadal organ weight of mice were measured,and their indices were calculated.The sperm concentration,the sperm viabili-ty,the testicular histomorphology and the expression levels of tight junction proteins ZO-1,Occludin and Claudin-11 were measured.TM4 cells were treated with palmitic acid(PA)for 24 h.Cell viability was detected by CCK-8 method.Then,TM4 cells were di-vided into different groups treated with PA(0,50,100,200 and 300 μmnol·L-1),and the expression lev-els of tight junction proteins ZO-1,Occludin and Clau-din-11 were detected by Western blot.The tight junc-tion permeability of TM4 cells were detected by transepithelial electrical resistance(TEER)and FITC-dextran.The expression levels of mRNA and proteins for the NF-κB/NLRP3 pathway-related factors were de-tected by RT-qPCR and Western blot.Results The results from animal experiments showed that high-fat diet increased body weight and seminal vesicle weight of mice,and decreased testicular index,epididymal in-dex,sperm concentration and sperm motility of mice.High-fat diet also caused testicular tissue structure damage and down-regulated the expression levels of tight junction proteins ZO-1 and Occludin,without af-fecting the expression of Claudin-11.In vitro,PA sig-nificantly down-regulated the expression levels of ZO-1,Occludin and Claudin-11 in TM4 cells,increased the cell permeability,as well as up-regulated the mRNA and protein expression levels of NLRP3/NF-κB signa-ling pathway-related factors in TM4 cells.Conclusions High-fat diet can impair the function of tight junction of testicualr Sertoli cells,and the machanism may be related to the activation of the NF-κB/NLRP3 signaling pathway,resulting in Sertoli cell inflammation in mice.

Aim To explore the mechanism of ligustil-ide(LIG)improving demyelination by inhibiting in-flammatory response in mice with distal middle cerebral artery occlusion(dMCAO)through AIM2/caspase-1 signal pathway.Methods Thirty C57BL/6N male mice were randomly divided into three groups:sham operation group(Sham group,n=10),model group(dMCAO group,n=10)and treatment group(LIG group,n=10).The dMCAO mouse model was estab-lished by electrocoagulation in dMCAO group and LIG group.The mice were scored by Longa after waking up,and the changes of cerebral blood flow were moni-tored by laser speckle blood flow imaging system after dMCAO.One hour after modeling,LIG(30 mg·kg-1·d-1)was injected intraperitoneally in LIG group,and the same amount of normal saline was injected in sham group and dMCAO group for one week until the end of the experiment.The mice in each group were stained with TTC,and the brain injury was observed pathologically.Fatigue turning bar test and open field test were used to evaluate the motor function and anxie-ty degree of mice,and then the brain tissues of mice were taken for black gold staining to compare the chan-ges of myelin sheath in each group.Immunofluores-cence staining was used to detect the average fluores-cence intensity of MBP,IBA1 and GFAP in CC,CPU and CX regions of mouse brains.ELISAwas used to de-tect the contents of TNF-α,IL-6,IL-1 β,IL-17A and BDNF in brain tissue proteins of mice.Western blot-ting was used to detect the protein expressions of AIM2,caspase-1 and ASC-in each group.Results Compared with the dMCAO group,the infarct area was reduced,the behavior was significantly improved and the demyelination was reduced in the LIG group.The expression of MBP protein in CC,CPU and CX regions increased(P＜0.05),the expression of IBA1 in CX decreased(P＜0.01),and the expression of GFAP in-creased in CC,CPU and CX regions(P＜0.01).The results of ELISA showed that the levels ofTNF-α(P＜0.01),IL-6(P＜0.01),IL-1β(P＜0.05)and IL-17A(P＜0.01)significantly decreased,while the ex-pression of BDNF increased(P＜0.05).The protein expression levels of AIM2,caspase-1 and ASC in mouse brain decreased after treatment(P＜0.01).Conclusion LIG has a protective effect on demyelina-tion in dMCAO mice,which may be related to the inhi-bition of AIM2/caspase-1 signaling pathway and in-flammation and to the promotion of BDNF secretion.

Aim To investigate the effect of neogam-bogic acid(NGA)on inducing ferroptosis in osteosar-coma K7M2 cells and subcutaneous transplanted tumor mice and explore the underlying mechanism.Methods MTT assay was employed to detect the effect of NGA(1,2,4,8,16,32,64,128 μmol·L-1)on cell prolif-eration,and the IC50 value was calculated.Calcein AM assay was used to detect cell viability.Transwell was applied to detect cell invasion.TEM was utilized to ob-serve the mitochondria morphology.K7M2 cells were subjected to treat with ferroptosis inducers erastin(Era)and inhibitors ferrostatin-1(Fer-1)to assess the levels of MDA,GSH,Fe2+,and LDH.RT-qPCR and Western blot were used to detect the mRNA and protein expression of STAT3,GPX4,and SLC7A11.A transplanted tumor model was established and treated with NGA to assess the impact of it on tumor growth and ferroptosis in vivo.HE staining was applied to ana-lyze the pathological status of tumor tissues.Nile red fluorescence staining was applied to detect the level of lipid components in tumor tissues.Results The pro-liferation,viability and invasion ability of K7M2 cells were significantly reduced after treatment with NGA at different concentrations(P＜0.05),and typical fea-tures of ferroptosis such as decreased mitochondrial vol-ume and reduced mitochondrial spine were observed.Compared to the control,the expression of MDA,Fe2+and LDH significantly increased(P＜0.01),while the content of GSH significantly decreased(P＜0.01).The ferroptosis in osteosarcoma was enhanced by the erastin,while inhibited by ferrostatin-1.In terms of mechanism,NGA inhibited the mRNA and protein ex-pression levels of STAT3,GPX4 and SLC7A11(P＜0.05).In vivo experiments confirmed that NGA signif-icantly improved the pathological state of tumor tissues,inhibited tumor growth,and induced ferroptosis in os-teosarcoma tissue cells.Conclusion NGA induces ferroptosis in osteosarcoma cells both in vitro and in vi-vo by inhibiting the STAT3/GPX4/SLC7A11 signaling axis,thereby exerting an anti-osteosarcoma effect.

Aim To investigate the effect of high-fat diet on the tight junction function injury of Sertoli cells through the NF-κB/NLRP3 signaling pathway in mice and to explore the underlying mechanism.Methods Male C57BL/6J mice were fed with high-fat or normal diet for five months.The body and gonadal organ weight of mice were measured,and their indices were calculated.The sperm concentration,the sperm viabili-ty,the testicular histomorphology and the expression levels of tight junction proteins ZO-1,Occludin and Claudin-11 were measured.TM4 cells were treated with palmitic acid(PA)for 24 h.Cell viability was detected by CCK-8 method.Then,TM4 cells were di-vided into different groups treated with PA(0,50,100,200 and 300 μmnol·L-1),and the expression lev-els of tight junction proteins ZO-1,Occludin and Clau-din-11 were detected by Western blot.The tight junc-tion permeability of TM4 cells were detected by transepithelial electrical resistance(TEER)and FITC-dextran.The expression levels of mRNA and proteins for the NF-κB/NLRP3 pathway-related factors were de-tected by RT-qPCR and Western blot.Results The results from animal experiments showed that high-fat diet increased body weight and seminal vesicle weight of mice,and decreased testicular index,epididymal in-dex,sperm concentration and sperm motility of mice.High-fat diet also caused testicular tissue structure damage and down-regulated the expression levels of tight junction proteins ZO-1 and Occludin,without af-fecting the expression of Claudin-11.In vitro,PA sig-nificantly down-regulated the expression levels of ZO-1,Occludin and Claudin-11 in TM4 cells,increased the cell permeability,as well as up-regulated the mRNA and protein expression levels of NLRP3/NF-κB signa-ling pathway-related factors in TM4 cells.Conclusions High-fat diet can impair the function of tight junction of testicualr Sertoli cells,and the machanism may be related to the activation of the NF-κB/NLRP3 signaling pathway,resulting in Sertoli cell inflammation in mice.

Aim To explore the mechanism of ligustil-ide(LIG)improving demyelination by inhibiting in-flammatory response in mice with distal middle cerebral artery occlusion(dMCAO)through AIM2/caspase-1 signal pathway.Methods Thirty C57BL/6N male mice were randomly divided into three groups:sham operation group(Sham group,n=10),model group(dMCAO group,n=10)and treatment group(LIG group,n=10).The dMCAO mouse model was estab-lished by electrocoagulation in dMCAO group and LIG group.The mice were scored by Longa after waking up,and the changes of cerebral blood flow were moni-tored by laser speckle blood flow imaging system after dMCAO.One hour after modeling,LIG(30 mg·kg-1·d-1)was injected intraperitoneally in LIG group,and the same amount of normal saline was injected in sham group and dMCAO group for one week until the end of the experiment.The mice in each group were stained with TTC,and the brain injury was observed pathologically.Fatigue turning bar test and open field test were used to evaluate the motor function and anxie-ty degree of mice,and then the brain tissues of mice were taken for black gold staining to compare the chan-ges of myelin sheath in each group.Immunofluores-cence staining was used to detect the average fluores-cence intensity of MBP,IBA1 and GFAP in CC,CPU and CX regions of mouse brains.ELISAwas used to de-tect the contents of TNF-α,IL-6,IL-1 β,IL-17A and BDNF in brain tissue proteins of mice.Western blot-ting was used to detect the protein expressions of AIM2,caspase-1 and ASC-in each group.Results Compared with the dMCAO group,the infarct area was reduced,the behavior was significantly improved and the demyelination was reduced in the LIG group.The expression of MBP protein in CC,CPU and CX regions increased(P＜0.05),the expression of IBA1 in CX decreased(P＜0.01),and the expression of GFAP in-creased in CC,CPU and CX regions(P＜0.01).The results of ELISA showed that the levels ofTNF-α(P＜0.01),IL-6(P＜0.01),IL-1β(P＜0.05)and IL-17A(P＜0.01)significantly decreased,while the ex-pression of BDNF increased(P＜0.05).The protein expression levels of AIM2,caspase-1 and ASC in mouse brain decreased after treatment(P＜0.01).Conclusion LIG has a protective effect on demyelina-tion in dMCAO mice,which may be related to the inhi-bition of AIM2/caspase-1 signaling pathway and in-flammation and to the promotion of BDNF secretion.

Aim To investigate the effect of neogam-bogic acid(NGA)on inducing ferroptosis in osteosar-coma K7M2 cells and subcutaneous transplanted tumor mice and explore the underlying mechanism.Methods MTT assay was employed to detect the effect of NGA(1,2,4,8,16,32,64,128 μmol·L-1)on cell prolif-eration,and the IC50 value was calculated.Calcein AM assay was used to detect cell viability.Transwell was applied to detect cell invasion.TEM was utilized to ob-serve the mitochondria morphology.K7M2 cells were subjected to treat with ferroptosis inducers erastin(Era)and inhibitors ferrostatin-1(Fer-1)to assess the levels of MDA,GSH,Fe2+,and LDH.RT-qPCR and Western blot were used to detect the mRNA and protein expression of STAT3,GPX4,and SLC7A11.A transplanted tumor model was established and treated with NGA to assess the impact of it on tumor growth and ferroptosis in vivo.HE staining was applied to ana-lyze the pathological status of tumor tissues.Nile red fluorescence staining was applied to detect the level of lipid components in tumor tissues.Results The pro-liferation,viability and invasion ability of K7M2 cells were significantly reduced after treatment with NGA at different concentrations(P＜0.05),and typical fea-tures of ferroptosis such as decreased mitochondrial vol-ume and reduced mitochondrial spine were observed.Compared to the control,the expression of MDA,Fe2+and LDH significantly increased(P＜0.01),while the content of GSH significantly decreased(P＜0.01).The ferroptosis in osteosarcoma was enhanced by the erastin,while inhibited by ferrostatin-1.In terms of mechanism,NGA inhibited the mRNA and protein ex-pression levels of STAT3,GPX4 and SLC7A11(P＜0.05).In vivo experiments confirmed that NGA signif-icantly improved the pathological state of tumor tissues,inhibited tumor growth,and induced ferroptosis in os-teosarcoma tissue cells.Conclusion NGA induces ferroptosis in osteosarcoma cells both in vitro and in vi-vo by inhibiting the STAT3/GPX4/SLC7A11 signaling axis,thereby exerting an anti-osteosarcoma effect.

Objective To investigate the clinical effects of different bone graft materials in internal fixation surgery for Schatzker type Ⅱ tibial plateau fracture(TPF).Methods A total of 108 patients with Schatzker type Ⅱ TPF in Lianyungang Municipal Oriental Hospital were selected and randomly assigned to the allogeneic cancellous bone and the artificial bone groups,with 54 cases in each group.The perioperative indicators,four coagulation items[thrombin time(TT),fibrinogen(FIB),activated partial thromboplastin time(APTT),prothrombin time(PT)],bone metabolic factors[bone gla protein(BGP),alkaline phosphatase(ALP),precollagen type Ⅰ N-terminal peptide(PINP)],knee motion,and numerical rating scale(NRS)score before and after operation,as well as complications,prognosis,bone graft stability were compared in the two groups.Results The hospital stay and fracture healing time in the artificial bone group were shorter than those in the allogeneic cancellous bone group(P<0.05).On the 7th day after surgery,the artificial bone group had longer peripheral blood APTT,TT and PT than the allogeneic cancellous bone group,and lower FIB level than the allogeneic cancellous bone group(P<0.05).The serum ALP,BGP and PINP levels 3 months after surgery in the two groups were higher than those before surgery(P<0.05).At 3 months after surgery,the ranges of knee extension and flexion in the artificial bone group were greater than those in the allogeneic cancellous bone group(P<0.05),and the NRS score was lower than that in the allogeneic cancellous bone group(P<0.05).The incidence of complications in the artificial bone group was lower than that in the allogeneic cancellous bone group(P<0.05).The excellent and good rate of knee joint function recovery 6 months after surgery in the artificial bone group was higher than that in the allogeneic cancellous bone group(P<0.05).The overall collapse height of the tibial lateral condylar articular surface,overall increase of lateral compartment angle,and overall loss height of lateral compartment 12 months after surgery in the artificial bone group were lower than those in the allogeneic cancellous bone group(P<0.05).Conclusion Compared with allogeneic cancellous bone,the use of artificial bone as the graft material during internal fixation in patients with Schatzker type Ⅱ TPF can more effectively ameliorate the postoperative coagulation status,reduce the incidence of complications,and enhance the graft stability,which is conducive to better prognosis.

A method for determination and targeted screening of diflorasone components in health products using ultra performance liquid chromatography-quadrupole time of flight mass spectrometry(UPLC-Q-TOF/MS)was established.Four representative diflorasone and esters(diflorasone,diflorasone diacetate,diflorasone-17-propionate,and diflorasone-21-propionate)were selected to optimize the pretreatment conditions,and 10 mL of extraction solvent dosage,15 min of extraction time and 5 g of salting-out agent as the optimal conditions were selected by response surface methodology.The results showed that the four analytes exhibited good linearity within the concentration range of 2.0?100 μg/L with the chromatographic peak area,and the correlation coefficients(R2)were all greater than 0.9990,while the results of recovery and relative standard deviation could satisfy the requirements of determination.The common characteristic ions of diflorasone and esters werem/z121 andm/z335,and their specific structures were obtained by analyzing the cleavage pathway based on the optimized determination conditions.A targeted screening method for other esters of diflorasone based on characteristic ions guidance strategy was established.This method had many advantages such as high efficiency,high sensitivity and good reproducibility,and could be used for targeted screening of diflorasone and esters in health products.The developed characteristic ion guided strategy could be employed to construct mass spectral databases for various glucocorticoids,enabling comprehensive targeted screening across a broad range of compounds.

Nitrofurazone(NFZ)is an antibiotic that is used as a veterinary drug in aquaculture.NFZ abuse can lead to a series of environmental and health issues,making it crucial to establish a rapid and highly sensitive method for NFZ detection.In this study,platinum nanoparticle(PtNPs)-loaded zeolitic imidazolate framework(ZIF-8)was used as a precursor,and PtNPs functionalized nitrogen doped porous carbon(NC)was obtained through pyrolysis.Pt@NC was combined with multi-walled carbon nanotubes(MWCNTs)and cast onto a glassy carbon electrode(GCE)surface to construct an electroch-emical sensor.Electrochemical tests revealed that Pt@NC/WCNT/GCE exhibited an electrochemical active area of 0.066 cm2 and a heterogeneous electron transfer rate constant(k0)of 2.03×10-3 cm/s,which were higher than other materials.Compared with the electrodes modified by other materials,the NFZ generated the highest peak current of irreversible reduction peak on the Pt@NC/WCNT/GCE electrode.In comparison with Pt@ZIF-8/WCNT/GCE,after pyrolysis and carbonization treatment,the reduction current of NFZ increased by 2.19 times,and the reduction peak potential shifted positively by 19 mV simultaneously.When compared with NC/WCNT/GCE,the PtNPs in the composite material enhanced the NFZ current by 4.25 times.Additionally,the experimental conditions for detecting NFZ using the sensor were optimized,including the carbonization temperature of Pt@ZIF-8,ratio of Pt@NC to CNT,loading amount of the modified material,and electrolyte pH.Under the optimized conditions,the sensor demonstrated a linear detection range for NFZ of 0.20-240 μmol/L,a sensitivity of 9.995 μA/((μmol/L)?cm2)and a limit of detection(LOD)of 0.06 μmol/L.The sensor exhibited excellent anti-interference capability,good reproducibility,and stability,with spiked recoveries for NFZ in water samples ranging from 94.6%to 105.6%.This study provided a novel electrochemical sensing approach for NFZ detection.