Periodontitis is a chronic inflammatory disease, and its occurrence and development are closely related to the imbalance of local innate immune responses. The caspase family plays a crucial role in regulating inflammatory responses and cell death pathways in periodontal innate immune cells (such as gingival epithelial cells, neutrophils, macrophages, dendritic cells, and natural killer cells). These proteases exhibit a dual regulatory effect on cellular functions. On one hand, apoptotic pathways mediated by caspase-3/7/9 enable the programmed clearance of senescent or damaged cells, while pyroptosis pathways mediated by caspase-1/4 contribute to immune defense and pathogen elimination, collectively helping to maintain tissue homeostasis. On the other hand, excessive activation of the caspase-1/gasdermin D pathway, as well as inflammatory amplification pathways involving caspase-4/6/8, promotes the release of inflammatory cytokines such as IL-1β and IL-18, leading to the disruption of the epithelial barrier and exacerbation of periodontal tissue damage. Caspase regulation exhibits both commonality and cell specificity. In gingival epithelial cells, caspase-1 mediates pyroptosis and inflammation activation, caspase-3 regulates apoptosis and proliferation signaling, and caspase-4 participates in differentiation regulation and pathogen-selective immune responses, collectively adapting to physiological and pathological changes. Neutrophils can utilize the caspase-1/gasdermin D signaling pathway to drive the release of neutrophil extracellular traps without triggering typical pyroptosis. In macrophages, caspase-1 and caspase-8 synergistically promote polarization toward the M1 phenotype, while caspase-3 acts as an apoptosis executor to facilitate macrophage transition to the M2 phenotype in specific microenvironments. This article reviews caspase’s specific mechanism of action in periodontal innate immune-related cells, aiming to provide a new theoretical basis for targeted regulation of caspase in the treatment of periodontitis.

AIM:To explore suitable methods for the induction and cryopreservation of mouse bone marrow-derived macrophages(BMDMs).METHODS:Mouse fibroblasts(L929 cells)were cultured under varying conditions of temperature,seeding density,and serum concentration.The concentration of macrophage colony-stimulating factor(M-CSF)in the cell supernatant was measured using ELISA to determine the optimal conditions.Mouse bone marrow cells were extracted,and a differential adherence method was employed to pre-culture the bone marrow cells for 4 h,followed by flow cytometry to assess the proportion of resident macrophages.Flow cytometry was utilized to assess the ratio of F4/80 positive cells among the suspended and adherent cells.Induction of BMDMs was conducted using L929 cell supernatant or recombinant M-CSF for 7 d,and flow cytometry was applied to evaluate the proportion of F4/80 and CD11b double-positive cells.The morphologic changes during cell induction were observed under an inverted microscope,and the phagocytic ca-pacity and inflammatory response levels of BMDMs derived from C57BL/6N and C57BL/6J mice were evaluated using neu-tral red and ELISA methods.The cells were immediately cryopreserved after extraction,and then induced after recovery,or cryopreserved after successful induction and recovered.The cell morphology was observed under an inverted micro-scope,cell viability was assessed using the CCK-8 method,and phagocytic ability was measured using the neutral red method.RESULTS:The M-CSF concentration in the supernatant from L929 cells cultured at 33℃,10%fetal bovine se-rum(FBS)for 7 d was rich.Following 4 h of pre-culture,the proportion of F4/80 positive cells in adherent cells was sig-nificantly higher than that in suspended cells(P＜0.01).After 7 d of induction with L929 cell supernatant or recombinant M-CSF,the proportions of F4/80+CD11b+cells showed no significant difference(P＞0.05).Compared with the BMDMs derived from C57BL/6J mice,those from C57BL/6N mice exhibited stronger phagocytic capacity(P＜0.01),and released lower levels of TNF-α(P＜0.01)and IL-6(P＜0.05),and higher levels of IL-1β(P＜0.05).Compared with the BMDMs that were induced after recovery from initial cryopreservation,those cryopreserved immediately after extraction and in-duced upon recovery exhibited better macrophage morphology,higher cell viability(P＜0.01),and enhanced phagocytic ability(P＜0.01).CONCLUSION:The supernatant from L929 cells cultured at 33℃,10%FBS for 7 d is rich in M-CSF,successfully inducing bone marrow cells to differentiate into mouse BMDMs.The differential adherence method for pre-culturing can eliminate resident macrophages from the original bone marrow.The phagocytic capacity and inflammato-ry response levels differ between BMDMs derived from the C57BL/6N and C57BL/6J mouse subtypes.Cryopreserving bone marrow cells immediately after extraction and subsequently inducing them upon recovery is a preferable method for BMDM cryopreservation.

Objective To investigate the potential mechanism by which dihydromyricetin(DHM)ameliorates diabetic nephropathy(DN),and to screen and validate its possible key molecular targets.Methods A DN model was established using db/db mice,and 100 mg/(kg·d)DHM was administered via gavage 5 d per week for totally 10 weeks.Renal morphological changes were observed after staining to evaluate the effects of DHM.GSE161885 and GSE270526 datasets were obtained from the Gene Expression Omnibus(GEO)database and analyzed in combination with the GeneCards database to screen for DN-related differentially expressed genes(DEGs).Protein-protein interaction(PPI)network and molecular docking were employed to predict potential DHM targets.Western blotting and immunofluorescence staining were performed to detect the effects of DHM on pyroptosis-related pathways in the renal tissues of db/db mice and in high glucose(HG)-induced human renal tubular epithelial cells(HK-2).The specific NLR family pyrin domain containing protein 3(NLRP3)inhibitor MCC950 was also used to validate the predicted mechanism.Results In vivo experiments showed that DHM significantly ameliorated renal pathological damage in db/db mice,alleviated glomerular hypertrophy and mesangial expansion,and markedly reduced Paller scores(P<0.001).Immunofluorescence staining revealed significantly weakened fluorescence signals for α-smooth muscle actin(α-SMA),fibronectin,and collagen Ⅰ in renal tissues.Western blot results showed that the expression levels of collagen Ⅰ,collagen Ⅲ,α-SMA,and transforming growth factor beta 1(TGF-β1)were significantly decreased(P<0.05).A total of 16 DN-related DEGs were identified.Enrichment analysis revealed that these genes were primarily enriched in pathways such as viral protein interactions,cytokine-cytokine receptor interaction,and the AGE-RAGE signaling pathway in diabetic complications,and were primarily involved in gene functions such as the positive regulation of lymphocyte-mediated immunity,positive regulation of adaptive immune response,and chemokine activity.Molecular docking confirmed NLRP3 as a potential target of DHM.In vivo validation showed that DHM significantly down-regulated gasdermin-D(GSDMD)fluorescence signals and inhibited the expression of pyroptosis-related proteins including NLRP3,Caspase 1,Cleaved-Caspase 1,interleukin 18(IL-18),and GSDMD(P<0.05).In vitro studies further confirmed that both DHM and the specific NLRP3 inhibitor MCC950 alleviate high glucose-induced fibrosis and pyroptosis in HIC-2 cells.Conclusion DHM can ameliorate the progression of DN,and its mechanism is related to inhibiting NLRP3 inflammasome-mediated pyroptosis,thereby alleviating renal inflammation and fibrosis.

OBJECTIVE To design and synthesize rifamycin S derivatives and load them into milk exosomes to evaluate their in vitro antimicrobial activity.METHODS Rifamycin S derivatives were synthe-sized and characterized by mass spectrometry and NMR.Using the dilution assay method,the inhibitory activity of each rifamycin S derivatives molecule against Staphylococcus aureus and Pseudomonas aerugi-nosa was determined,and the IC50 was calculated.Derivatives molecules with excellent antimicrobial activity were selected and loaded into milk exosomes using the ultrasonication method,resulting in the preparation of milk exosome-loaded rifamycin S derivatives.The antimicrobial activity against Staphylo-coccus aureus was determined using the dilution assay method.The inhibitory effect of the exosome-loaded rifamycin S derivatives on Staphylococcus aureus residing within macrophages was detected using the plate colony counting method.RESULTS Three rifamycin S derivatives were successfully designed and synthesized,which demonstrated superior antimicrobial activity against Staphylococcus aureus(the parent compound's antimicrobial activity is merely from 1/20 to 1/80 of that of the three rifamycin S derivatives)and Pseudomonas aeruginosa(the parent compound's antimicrobial activity is only 1/14 and 1/9 of that of compound 1 and compound 3)compared to the parent compound.The loading of milk exosomes with the rifamycin S derivatives compound 3 was successfully achieved,with a loading efficiency of 10.9％.The antimicrobial activity of the compound after exosome loading was significantly enhanced against Staphylococcus aureus in vitro and against Staphylococcus aureus residing within macrophages(P＜0.01).CONCLUSION The designed and synthesized derivatives of rifamycin S possess stronger anti-microbial activity,and their antibacterial efficacy against both extracellular and intracellular bacteria can be further enhanced after loading into exosomes.

Objective:To explore the relationship between trait anger and depression among college students, as well as the mediating roles of perceived stress and emotion-oriented coping strategies.Methods:From March to April 2022, a total of 2 037 college students were surveyed by the trait anger scale, the perceived stress scale, the coping inventory for stressful situations, and the 9-item patient health questionnaire.The collected data were analyzed by SPSS 27.0 software and AMOS 24.0 statistical software packages.Results:The score of trait anger among college students was (14.78±3.56), the score of perceived stress was (10.02±4.77), the score of emotion-oriented coping was (14.43±3.38), and the score of depression was 2.00(0, 9.00). Correlation analysis showed that depression was significantly and positively correlated with temperamental anger, reactive anger, perceived stress, and emotion-oriented coping ( r=0.303-0.467, all P<0.01). Temperamental anger and reactive anger were significantly and positively correlated with both perceived stress and emotion-oriented coping ( r=0.279-0.332, all P<0.01). Emotion-oriented coping was significantly and positively correlated with perceived stress ( r=0.397, P<0.01). Mediation effect analysis showed that the total effect of trait anger on depression was 0.420. The direct effect of trait anger on depression was 0.241, accounting for 57.38% of the total effect. The mediating effects of perceived stress and emotion-oriented coping were 0.145 and 0.024, accounting for 34.52% and 5.72% of the total effect, respectively. The chain mediating effect of perceived stress and emotion-oriented coping was 0.010, accounting for 2.38% of the total effect. Conclusion:Trait anger can significantly and positively predict depression among college students, and it can also positively predict the level of depression among college students through the chain mediation effect of perceived stress and emotion orientation.

OBJECTIVE To design and synthesize rifamycin S derivatives and load them into milk exosomes to evaluate their in vitro antimicrobial activity.METHODS Rifamycin S derivatives were synthe-sized and characterized by mass spectrometry and NMR.Using the dilution assay method,the inhibitory activity of each rifamycin S derivatives molecule against Staphylococcus aureus and Pseudomonas aerugi-nosa was determined,and the IC50 was calculated.Derivatives molecules with excellent antimicrobial activity were selected and loaded into milk exosomes using the ultrasonication method,resulting in the preparation of milk exosome-loaded rifamycin S derivatives.The antimicrobial activity against Staphylo-coccus aureus was determined using the dilution assay method.The inhibitory effect of the exosome-loaded rifamycin S derivatives on Staphylococcus aureus residing within macrophages was detected using the plate colony counting method.RESULTS Three rifamycin S derivatives were successfully designed and synthesized,which demonstrated superior antimicrobial activity against Staphylococcus aureus(the parent compound's antimicrobial activity is merely from 1/20 to 1/80 of that of the three rifamycin S derivatives)and Pseudomonas aeruginosa(the parent compound's antimicrobial activity is only 1/14 and 1/9 of that of compound 1 and compound 3)compared to the parent compound.The loading of milk exosomes with the rifamycin S derivatives compound 3 was successfully achieved,with a loading efficiency of 10.9％.The antimicrobial activity of the compound after exosome loading was significantly enhanced against Staphylococcus aureus in vitro and against Staphylococcus aureus residing within macrophages(P＜0.01).CONCLUSION The designed and synthesized derivatives of rifamycin S possess stronger anti-microbial activity,and their antibacterial efficacy against both extracellular and intracellular bacteria can be further enhanced after loading into exosomes.

Objective To analyze the characteristics,causes,influencing factors of compensation,and appraisal features of second-instance medical malpractice cases involving cardiovascular diseases in Henan Province from 2017 to 2022,and to provide reference for forensic appraisal and judicial trial.Methods Cases were retrieved from China Judgments Online between 2017 and 2022.A total of 1,957 documents were reviewed,including 1,397 medical malpractice cases and 130 cardiovascular disease cases.Results The total compensation awarded in these second-instance cases was 27.04 million yuan,with a median of 158,600 yuan.Cases involving patient death accounted for 80.00％(104/130).Among 107 cases with first-instance appraisals,55.14％(59/107)raised objections,while 74.62％(97/130)of the second-instance trials upheld the original judgment.The most common degree of responsibility borne by medical institutions was secondary responsibility(41.54％,54/130).The top three medical faults were:inadequate observation and management of patient conditions(46.15％),omission of auxiliary examinations(37.50％),and insufficient notification(36.54％).In terms of violations-including medical record documentation,inappropriate treatment,misdiagnosis or missed diagnosis,out-of-scope practice,and improper medication use-the actual proportions in judgments(24/113,34/113,12/113,7/113,14/113)were all significantly lower than the patients' claims(all P＜0.05).Conclusion Cardiovascular medical malpractice cases in second-instance trials involve substantial compensation and a high proportion of death outcomes.Both medical institutions and patients should pay greater attention to first-instance trials.Appraisal organizations should proactively provide explanation and education regarding issues likely to raise patient doubts,thereby reducing unnecessary appeals.They should also avoid hasty revisions of appraisal opinions when faced with objections and instead focus on improving the quality and credibility of appraisals.In determining medical faults,emphasis should be placed on evaluating whether there was inadequate patient monitoring and management,omission of auxiliary examinations,and insufficient notification.

Objective:To explore the relationship between trait anger and depression among college students, as well as the mediating roles of perceived stress and emotion-oriented coping strategies.Methods:From March to April 2022, a total of 2 037 college students were surveyed by the trait anger scale, the perceived stress scale, the coping inventory for stressful situations, and the 9-item patient health questionnaire.The collected data were analyzed by SPSS 27.0 software and AMOS 24.0 statistical software packages.Results:The score of trait anger among college students was (14.78±3.56), the score of perceived stress was (10.02±4.77), the score of emotion-oriented coping was (14.43±3.38), and the score of depression was 2.00(0, 9.00). Correlation analysis showed that depression was significantly and positively correlated with temperamental anger, reactive anger, perceived stress, and emotion-oriented coping ( r=0.303-0.467, all P<0.01). Temperamental anger and reactive anger were significantly and positively correlated with both perceived stress and emotion-oriented coping ( r=0.279-0.332, all P<0.01). Emotion-oriented coping was significantly and positively correlated with perceived stress ( r=0.397, P<0.01). Mediation effect analysis showed that the total effect of trait anger on depression was 0.420. The direct effect of trait anger on depression was 0.241, accounting for 57.38% of the total effect. The mediating effects of perceived stress and emotion-oriented coping were 0.145 and 0.024, accounting for 34.52% and 5.72% of the total effect, respectively. The chain mediating effect of perceived stress and emotion-oriented coping was 0.010, accounting for 2.38% of the total effect. Conclusion:Trait anger can significantly and positively predict depression among college students, and it can also positively predict the level of depression among college students through the chain mediation effect of perceived stress and emotion orientation.

AIM:To explore suitable methods for the induction and cryopreservation of mouse bone marrow-derived macrophages(BMDMs).METHODS:Mouse fibroblasts(L929 cells)were cultured under varying conditions of temperature,seeding density,and serum concentration.The concentration of macrophage colony-stimulating factor(M-CSF)in the cell supernatant was measured using ELISA to determine the optimal conditions.Mouse bone marrow cells were extracted,and a differential adherence method was employed to pre-culture the bone marrow cells for 4 h,followed by flow cytometry to assess the proportion of resident macrophages.Flow cytometry was utilized to assess the ratio of F4/80 positive cells among the suspended and adherent cells.Induction of BMDMs was conducted using L929 cell supernatant or recombinant M-CSF for 7 d,and flow cytometry was applied to evaluate the proportion of F4/80 and CD11b double-positive cells.The morphologic changes during cell induction were observed under an inverted microscope,and the phagocytic ca-pacity and inflammatory response levels of BMDMs derived from C57BL/6N and C57BL/6J mice were evaluated using neu-tral red and ELISA methods.The cells were immediately cryopreserved after extraction,and then induced after recovery,or cryopreserved after successful induction and recovered.The cell morphology was observed under an inverted micro-scope,cell viability was assessed using the CCK-8 method,and phagocytic ability was measured using the neutral red method.RESULTS:The M-CSF concentration in the supernatant from L929 cells cultured at 33℃,10%fetal bovine se-rum(FBS)for 7 d was rich.Following 4 h of pre-culture,the proportion of F4/80 positive cells in adherent cells was sig-nificantly higher than that in suspended cells(P＜0.01).After 7 d of induction with L929 cell supernatant or recombinant M-CSF,the proportions of F4/80+CD11b+cells showed no significant difference(P＞0.05).Compared with the BMDMs derived from C57BL/6J mice,those from C57BL/6N mice exhibited stronger phagocytic capacity(P＜0.01),and released lower levels of TNF-α(P＜0.01)and IL-6(P＜0.05),and higher levels of IL-1β(P＜0.05).Compared with the BMDMs that were induced after recovery from initial cryopreservation,those cryopreserved immediately after extraction and in-duced upon recovery exhibited better macrophage morphology,higher cell viability(P＜0.01),and enhanced phagocytic ability(P＜0.01).CONCLUSION:The supernatant from L929 cells cultured at 33℃,10%FBS for 7 d is rich in M-CSF,successfully inducing bone marrow cells to differentiate into mouse BMDMs.The differential adherence method for pre-culturing can eliminate resident macrophages from the original bone marrow.The phagocytic capacity and inflammato-ry response levels differ between BMDMs derived from the C57BL/6N and C57BL/6J mouse subtypes.Cryopreserving bone marrow cells immediately after extraction and subsequently inducing them upon recovery is a preferable method for BMDM cryopreservation.

Objective To analyze the characteristics,causes,influencing factors of compensation,and appraisal features of second-instance medical malpractice cases involving cardiovascular diseases in Henan Province from 2017 to 2022,and to provide reference for forensic appraisal and judicial trial.Methods Cases were retrieved from China Judgments Online between 2017 and 2022.A total of 1,957 documents were reviewed,including 1,397 medical malpractice cases and 130 cardiovascular disease cases.Results The total compensation awarded in these second-instance cases was 27.04 million yuan,with a median of 158,600 yuan.Cases involving patient death accounted for 80.00％(104/130).Among 107 cases with first-instance appraisals,55.14％(59/107)raised objections,while 74.62％(97/130)of the second-instance trials upheld the original judgment.The most common degree of responsibility borne by medical institutions was secondary responsibility(41.54％,54/130).The top three medical faults were:inadequate observation and management of patient conditions(46.15％),omission of auxiliary examinations(37.50％),and insufficient notification(36.54％).In terms of violations-including medical record documentation,inappropriate treatment,misdiagnosis or missed diagnosis,out-of-scope practice,and improper medication use-the actual proportions in judgments(24/113,34/113,12/113,7/113,14/113)were all significantly lower than the patients' claims(all P＜0.05).Conclusion Cardiovascular medical malpractice cases in second-instance trials involve substantial compensation and a high proportion of death outcomes.Both medical institutions and patients should pay greater attention to first-instance trials.Appraisal organizations should proactively provide explanation and education regarding issues likely to raise patient doubts,thereby reducing unnecessary appeals.They should also avoid hasty revisions of appraisal opinions when faced with objections and instead focus on improving the quality and credibility of appraisals.In determining medical faults,emphasis should be placed on evaluating whether there was inadequate patient monitoring and management,omission of auxiliary examinations,and insufficient notification.