OBJECTIVE To explore the mechanism of action of Qingre huatan huoxue decoction against atherosclerosis （AS）based on macrophage polarization. METHODS Using atorvastatin served as the positive control， the drug-containing serum of the Qingre huatan huoxue decoction was prepared to treat RAW264.7 macrophages. Macrophage viability， apoptosis rate， and the fluorescence intensities of CD86 and CD206 were measured， along with the levels of tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β）. Apolipoprotei n E-deficient （ApoE －/－ ） mice （AS model mice） fed with a high-fat diet were randomly assigned to model group， atorvastatin group （2.6 mg/kg）， and low-， medium- and high-dose groups of Qingre huatan huoxue decoction （90， 180， 360 mg/kg）， respectively. C57BL/6J mice fed with a standard diet served as the normal control group， with 10 mice per group. The treatment group mice were administered the corresponding drugs intragastrically， once daily， for 8 consecutive weeks. Serum levels of TNF-α and IL-1β were measured in all groups. Lipid deposition in the aorta （assessed by the percentage of plaque in the entire aorta and aortic root） and morphological changes in the aortic root were observed. Expression levels of CD86 and CD206 in aortic tissue， as well as the protein expression levels of inducible nitric oxide synthase （iNOS）， arginase-1 （Arg-1）， AMP-activated protein kinase （AMPK）， phosphorylated AMPK （p-AMPK）， and peroxisome proliferator-activated receptor γ （PPAR-γ） in aortic tissues were all detected. RESULTS Cell experiment results showed that， at concentrations of 5-100 μg/mL， the drug-containing serum of the Qingre huatan huoxue decoction significantly increased RAW264.7 cell viability （ P ＜0.05）. The drug-containing serum of the Qingre huatan huoxue decoction at concentrations of 10， 50， and 100 μg/mL， along with atorvastatin， significantly reduced apoptosis rates， CD86 fluorescence intensity， and TNF-α and IL-1β levels in RAW264.7 cells， while markedly enhancing CD206 fluorescence intensity （ P ＜0.05）. Animal experiment results showed that， compared with the model group， all dosage groups of Qingre huatan huoxue decoction and the atorvastatin group showed significantly reduced/down-regulated levels of TNF-α and IL-1β in serum， along with decreased aortic total and root plaque percentages， CD86 expression， and iNOS protein expression. CD206 expression and Arg-1， p-AMPK/AMPK， PPAR-γ protein expression were significantly up-regulated （ P ＜0.05）. Pathological morphology of the aorta showed varying degrees of improvement. CONCLUSIONS The formula of Qingre huatan huoxue decoction exerts its anti-AS effects by regulating macrophage polarization， increasing the proportion of M2 macrophages， thereby effectively inhibiting AS plaque formation and reducing inflammatory responses.

Aim To investigate the effect of hydrogen sulfide on macrophage calcification and its underlying mo-lecular mechanisms.Methods Oil red O staining was used to observe intracellular lipid accumulation,and von Kossa staining and atomic absorption spectroscopy were used for morphological and quantitative analysis of calcium deposition and intracellular calcium content in a mononuclear macrophage calcification model.Western blot and RT-PCR were used to detect the mRNA and protein expression of osteopontin(OPN)at different doses and treatment times of hydrogen sulfide.At the same time,Western blot was used to detect the expression changes of early growth response factor 1(EGR1),endo-plasmic reticulum stress-related markers C/EBP homologous protein(CHOP)and glucose-regulated protein 78(GRP78).Reactive oxygen species levels were evaluated by fluorescence probe staining,and the effect of hydrogen sulfide on macro-phage calcification was evaluated by combining von Kossa staining and calcium ion fluorescence probe staining.The mo-lecular mechanisms of hydrogen sulfide affecting macrophage calcification were explored by interfering with EGR1 expression and using endoplasmic reticulum stress inhibitor 4-phenylbutyric acid(4-PBA).Results Compared with oxidized low density lipoprotein(ox-LDL)group,β-glycerophosphate(β-GP)+40 g/L ox-LDL group showed a significant increase in intracellular lipid accumulation,while hydrogen sulfide significantly inhibited macrophage calcification in a con-centration-and time-dependent manner.Compared with the β-GP+ox LDL group,the most significant effect was observed after incubation with 100 μmol/L NaHS for 4 days.The hydrogen sulfide group showed a 66％decrease in intracellular calcium content(P＜0.01),a 71％decrease in intercellular calcium deposition(P＜0.01),and a 50％and 48％decrease in OPN mRNA and protein expression,respectively(P＜0.05).Hydrogen sulfide treatment upregulated the ex-pression of EGR1 by 21％,while downregulating the expression of CHOP and GRP78 by 58％and 59％,respectively(P＜0.01).The endoplasmic reticulum stress inhibitor 4-PBA could downregulate OPN expression by 73％(P＜0.01),while interfering with EGR1 expression completely counteracts the inhibitory effect of hydrogen sulfide on OPN expression and calcium deposition(P＜0.01).Conclusion Hydrogen sulfide significantly inhibits macrophage calcification by upregulating EGR1 expression and suppressing endoplasmic reticulum stress.

Aim To investigate the effect and mechanism of angiotensin Ⅱ(Ang Ⅱ)on ferroptosis in white adi-pocytes.Methods The 3T3-L1 preadipocytes were differentiated into white adipocytes by inducer stimulation.The experiment was divided into control group,Ang Ⅱ group,Ang Ⅱ+Fer-1(ferroptosis inhibitor)group and Ang Ⅱ+PFT-α(p53 inhibitor)group.Ang Ⅱ was used to treat cells.RT-qPCR and Western blot were used to detect the expression levels of ferroptosis factors and adipokines.JC-1 kit was used to detect mitochondrial membrane potential(MMP)level.Iron ion kit was used to detect intracellular iron content.Glutathione(GSH)kit was used to detect GSH content.Fer-1 and Ang Ⅱ were added to treat cells to detect the the changes of ferroptosis level.The expression of p53 and spermidine/spermine N1-acetyltransferase 1(SAT1)protein was detected.Subsequently,PFT-α and Ang Ⅱ were added to co-treat cells to detect the changes of p53 and SAT1 protein expression,and to observe the effect of inhibiting p53 expression on the expression levels of ferroptosis factors and adipokines.Results 3T3-L1 cells were successfully differentiated into white adipocytes by stimulator-induced differentiation.Ang Ⅱ induced ferroptosis in white adipocytes.RT-qPCR results showed that compared with control group,the mRNA expression of anti-ferroptosis factor glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11)and iron regulatory protein 1(IRP-1)was down-regulated in Ang Ⅱ group,and the mRNA expression of pro-ferroptosis factor acyl-CoA synthetase of long-chain family member 4(ACSL4)was up-regulated.Western blot results showed that compared with control group,the protein expression of SLC7A11 and GPX4 was down-regulated in Ang Ⅱ group,and the protein expression of ACSL4 was up-regulated.Ang Ⅱ treatment increased the content of intracellular iron ions and decreased the levels of GSH and MMP.Compared with Ang Ⅱ group,the mRNA expression of IRP-1 and SLC7A11 was up-regulated in Ang Ⅱ+Fer-1 group.Ang Ⅱ induced changes in the expression profile of adipokines in white adipocytes.Western blot results showed that compared with control group,the protein ex-pression of pro-inflammatory adipokine leptin(LEP),resistin(RETN),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)was up-regulated in Ang Ⅱ group,and the protein expression of anti-inflammatory adipokine adiponectin(AD-PN)and omentin 1(ITLN1)was down-regulated.In addition,Ang Ⅱ increased the protein expression of p53 and SAT1.Inhibition of p53 expression can improve the level of ferroptosis and adipokine expression in white adipocytes trea-ted with Ang Ⅱ.Western blot results showed that compared with Ang Ⅱ group,the protein expression of p53 and SAT1 was down-regulated in Ang Ⅱ+PFT-α group,the protein expression of SLC7A11 and GPX4 was up-regulated,and the protein expression of ACSL4 was down-regulated.The protein expression of ADPN was up-regulated in Ang Ⅱ+PFT-αgroup,and the protein expression of TNF-α,LEP and RETN was down-regulated.Conclusion Ang Ⅱ induces fer-roptosis in white adipocytes through activating the p53/SAT1 signaling pathway.

Aim To investigate the effect of hydrogen sulfide on macrophage calcification and its underlying mo-lecular mechanisms.Methods Oil red O staining was used to observe intracellular lipid accumulation,and von Kossa staining and atomic absorption spectroscopy were used for morphological and quantitative analysis of calcium deposition and intracellular calcium content in a mononuclear macrophage calcification model.Western blot and RT-PCR were used to detect the mRNA and protein expression of osteopontin(OPN)at different doses and treatment times of hydrogen sulfide.At the same time,Western blot was used to detect the expression changes of early growth response factor 1(EGR1),endo-plasmic reticulum stress-related markers C/EBP homologous protein(CHOP)and glucose-regulated protein 78(GRP78).Reactive oxygen species levels were evaluated by fluorescence probe staining,and the effect of hydrogen sulfide on macro-phage calcification was evaluated by combining von Kossa staining and calcium ion fluorescence probe staining.The mo-lecular mechanisms of hydrogen sulfide affecting macrophage calcification were explored by interfering with EGR1 expression and using endoplasmic reticulum stress inhibitor 4-phenylbutyric acid(4-PBA).Results Compared with oxidized low density lipoprotein(ox-LDL)group,β-glycerophosphate(β-GP)+40 g/L ox-LDL group showed a significant increase in intracellular lipid accumulation,while hydrogen sulfide significantly inhibited macrophage calcification in a con-centration-and time-dependent manner.Compared with the β-GP+ox LDL group,the most significant effect was observed after incubation with 100 μmol/L NaHS for 4 days.The hydrogen sulfide group showed a 66％decrease in intracellular calcium content(P＜0.01),a 71％decrease in intercellular calcium deposition(P＜0.01),and a 50％and 48％decrease in OPN mRNA and protein expression,respectively(P＜0.05).Hydrogen sulfide treatment upregulated the ex-pression of EGR1 by 21％,while downregulating the expression of CHOP and GRP78 by 58％and 59％,respectively(P＜0.01).The endoplasmic reticulum stress inhibitor 4-PBA could downregulate OPN expression by 73％(P＜0.01),while interfering with EGR1 expression completely counteracts the inhibitory effect of hydrogen sulfide on OPN expression and calcium deposition(P＜0.01).Conclusion Hydrogen sulfide significantly inhibits macrophage calcification by upregulating EGR1 expression and suppressing endoplasmic reticulum stress.

Aim To investigate the effect and mechanism of angiotensin Ⅱ(Ang Ⅱ)on ferroptosis in white adi-pocytes.Methods The 3T3-L1 preadipocytes were differentiated into white adipocytes by inducer stimulation.The experiment was divided into control group,Ang Ⅱ group,Ang Ⅱ+Fer-1(ferroptosis inhibitor)group and Ang Ⅱ+PFT-α(p53 inhibitor)group.Ang Ⅱ was used to treat cells.RT-qPCR and Western blot were used to detect the expression levels of ferroptosis factors and adipokines.JC-1 kit was used to detect mitochondrial membrane potential(MMP)level.Iron ion kit was used to detect intracellular iron content.Glutathione(GSH)kit was used to detect GSH content.Fer-1 and Ang Ⅱ were added to treat cells to detect the the changes of ferroptosis level.The expression of p53 and spermidine/spermine N1-acetyltransferase 1(SAT1)protein was detected.Subsequently,PFT-α and Ang Ⅱ were added to co-treat cells to detect the changes of p53 and SAT1 protein expression,and to observe the effect of inhibiting p53 expression on the expression levels of ferroptosis factors and adipokines.Results 3T3-L1 cells were successfully differentiated into white adipocytes by stimulator-induced differentiation.Ang Ⅱ induced ferroptosis in white adipocytes.RT-qPCR results showed that compared with control group,the mRNA expression of anti-ferroptosis factor glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11)and iron regulatory protein 1(IRP-1)was down-regulated in Ang Ⅱ group,and the mRNA expression of pro-ferroptosis factor acyl-CoA synthetase of long-chain family member 4(ACSL4)was up-regulated.Western blot results showed that compared with control group,the protein expression of SLC7A11 and GPX4 was down-regulated in Ang Ⅱ group,and the protein expression of ACSL4 was up-regulated.Ang Ⅱ treatment increased the content of intracellular iron ions and decreased the levels of GSH and MMP.Compared with Ang Ⅱ group,the mRNA expression of IRP-1 and SLC7A11 was up-regulated in Ang Ⅱ+Fer-1 group.Ang Ⅱ induced changes in the expression profile of adipokines in white adipocytes.Western blot results showed that compared with control group,the protein ex-pression of pro-inflammatory adipokine leptin(LEP),resistin(RETN),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)was up-regulated in Ang Ⅱ group,and the protein expression of anti-inflammatory adipokine adiponectin(AD-PN)and omentin 1(ITLN1)was down-regulated.In addition,Ang Ⅱ increased the protein expression of p53 and SAT1.Inhibition of p53 expression can improve the level of ferroptosis and adipokine expression in white adipocytes trea-ted with Ang Ⅱ.Western blot results showed that compared with Ang Ⅱ group,the protein expression of p53 and SAT1 was down-regulated in Ang Ⅱ+PFT-α group,the protein expression of SLC7A11 and GPX4 was up-regulated,and the protein expression of ACSL4 was down-regulated.The protein expression of ADPN was up-regulated in Ang Ⅱ+PFT-αgroup,and the protein expression of TNF-α,LEP and RETN was down-regulated.Conclusion Ang Ⅱ induces fer-roptosis in white adipocytes through activating the p53/SAT1 signaling pathway.

AIM:To investigate the potential impact of mitoNEET[mitochondrial protein containing Asn-Glu-Glu-Thr(NEET)sequence]on mitochondrial metabolism in brown adipocytes,and to elucidate its underlying mecha-nism.METHODS:An in vitro model of primary mouse brown adipocytes was established.Western blot were utilized to detect relevant proteins,and iron ion and ATP content was measured using kits.Mitochondrial membrane potential and re-active oxygen species(ROS)were assessed by fluorescence microscopy and flow cytometry.RESULTS:The expression of the ferroptosis-related protein ACSL4 increased by 1.13 times in ferroptosis inducer erastin treatment group,whereas the expression of SLC7A11 and GPX4 decreased by 27.33%and 25.33%,respectively,compared with control group(P<0.05).The expression of Nrf1,PGC-1α,MFN2 and UCP1 proteins,related to mitochondrial energy metabolism,de-creased by 20.98%,15.17%,15.03%and 34.22%,respectively(P<0.05).Additionally,the mitoNEET protein con-tent was significantly reduced by 42.14%(P<0.05).The iron ion content in erastin group was substantially increased by 1.80 times compared with control group.However,a notable decrease in ATP content of 14.95%was seen(P<0.05).The results obtained from fluorescence microscopy and flow cytometry demonstrated a significant decrease in the mitochon-drial membrane potential of brown adipocytes in erastin group,with reductions of 52.18%and 61.31%(P<0.05),re-spectively.A substantial increase in mitochondrial ROS content of 80.97%was seen(P<0.05).Western blot analysis of overexpressed stable strains revealed a significant elevation in mitoNEET levels in brown adipocytes following lentivirus transfection,exhibiting an increase of 11.19 times(P<0.05),thus confirming successful transfection.The LV-mitoNEET group exhibited a significant decrease of 37.95%in the expression of ferroptosis-related protein ACSL4 in brown adipose cells compared with control group.Additionally,there was a notable increase of 77.82%and 66.3%in the expression of SLC7A11 and GPX4,respectively(P<0.05).Up-regulation was observed in the expression of MFN2(79.06%),PGC-1α(72.89%),Nrf1(40.14%),and UCP1(31.68%)(P<0.05).The test results demonstrated that the LV-mitoNEET group experienced a reduction of 43.5%in iron ion content compared with control group while exhibiting an increase of 33.5%in ATP content(P<0.05).The results obtained from fluorescence microscopy and flow cytometry demonstrated that mitoNEET overexpression led to a significant increase in the mitochondrial membrane potential of erastin-induced brown adipocytes,with increments of 17.61%and 96.05%,respectively.Additionally,mitoNEET overexpression effec-tively reduced the production of mitochondrial ROS by 24.48%(P<0.05).CONCLUSION:Our findings suggest that mitoNEET overexpression can effectively inhibit the disruption of mitochondrial energy metabolism caused by ferroptosis-induced death of brown adipocytes.

In traditional clinical teaching of impacted tooth extraction, there are problems including limited observation fields for students, insufficient doctor-patient communication training, inadequate clinical thinking development, and low levels of clinical participation. Based on the core elements of visualizing jaw structure with perspective, formulating clinical strategy, immersing in clinical participation, and cultivating the spirit of caregiving, we developed a virtual reality simulation teaching platform for the clinical decision analysis of impacted tooth extraction. The virtual simulation-based platform can create three different impacted tooth scenarios in 3D, which demonstrates the process of clinical decision analysis of impacted tooth extraction through virtuality-reality interaction, allowing students to immerse in the discovery, analysis, and resolution of the medical and humanistic problems in the process of impacted tooth extraction. The questionnaire survey showed that 81.36% (48/59) of the students believed that the software could improve their clinical thinking ability; 76.27% (45/59) of the students believed that it could help them master the basic process of impacted tooth extraction; and 62.71% (37/59) of the students believed that it could improve their skills of impacted tooth extraction. By providing immersive learning experience, constructive teaching design, and multi-dimensional teaching evaluation, the software achieved the goals of cultivating students' clinical thinking ability and professional literacy in oral and maxillofacial surgery.

OBJECTIVE Epileptic networks are char-acterized as two states,seizures or more prolonged inter-ictal periods.However,cellular mechanisms underlying the contribution of interictal periods to ictal events remain unclear.METHODS Here,we present the procedure for labeling seizure-activated and interictal-activated neuro-nal ensembles in mouse hippocampal kindling model using an enhanced-synaptic-activity-responsive element.This technique is combined with genetically encoded effectors to characterize and manipulate neuronal ensembles recruited by focal seizures(FS-Ens)and interictal periods(IP-Ens)in piriform cortex,a region that plays a key role in seizure generation.RESULTS Ca2+ activities and histo-logical evidence reveal a disjointed correlation between the two ensembles during FS dynamics.Optogenetic acti-vation of FS-Ens promotes further seizure development,while IP-Ens protects against it.Interestingly,both ensem-bles are functionally involved in generalized seizures(GS)due to circuit rearrangement.IP-Ens bidirectionally modulates FS but not GS by controlling coherence with hippocampus.CONCLUSION This study indicates that the interictal state may represent a seizure-preventing environment,and the interictal-activated ensemble may serve as a potential therapeutic target for epilepsy.

Anti-seizure medications (ASMs) are the main therapy for epilepsy.There are many kinds of ASMs with complex mechanism of action, so it is difficult for pharmacists to examine prescriptions.This paper put forward some suggestions on the indications, dosage forms/routes of administration, appropriateness of usage and dosage, combined medication and drug interaction, long-term prescription review, individual differences in pathophysiology of children, and drug selection when complicated with common epilepsy, for the reference of doctors and pharmacists.

Objective:To investigate the effect of 1 064-nm Q-switched Nd:YAG laser at different energy settings on cell viability, protease activity and structures of Malassezia furfur. Methods:Cultured standard strains of Malassezia furfur were divided into several groups to be irradiated with 1 064-nm Q-switched Nd:YAG laser at different energies of 0 (control group) , 500, 600, 700, 800 and 900 mJ, respectively. Then, fungal suspensions in the above groups were inoculated onto the Leeming & Notman medium separately. After 7-day culture, the diameter and number of colonies were measured to evaluate the fungal cell viability, the protease activity was measured by using the whole-milk plate medium, and the ultrastructure of Malassezia furfur in each group was observed by transmission electron microscopy. One-way analysis of variance was used for comparisons among multiple groups, least significant difference- t test for multiple comparisons, and Pearson correlation analysis for analyzing correlations of laser energy with colony diameter, number and protease activity. Results:The colony diameter and number both significantly differed among the control group, 500-, 600-, 700-, 800- and 900-mJ groups (colony diameter: 4.05 ± 0.69, 3.76 ± 0.51, 3.28 ± 0.41, 3.09 ± 0.72, 2.54 ± 0.64 and 2.43 ± 0.41 mm, respectively; colony number: 4 787 ± 597, 4 287 ± 761, 1 879 ± 275, 1 082 ± 248 and 209 ± 42, 72 ± 31 colony-forming units, respectively; F = 14.83, 231.85, respectively, both P < 0.05) , and were significantly decreased in the 600-, 700-, 800- and 900-mJ groups compared with the control group (all P < 0.05) . The laser energy was negatively correlated with the colony diameter and number ( r = -0.67, -0.91, respectively, both P < 0.05) . The protease activity significantly differed among the control group, 500-, 700- and 900-mJ groups ( F = 346.60, P < 0.05) , and was significantly lower in the 700- and 900-mJ groups than in the control group (both P < 0.05) . There was a negative correlation between the laser energy and protease activity ( r = -0.94, P < 0.05) . Transmission electron microscopy showed intact fungal structures in the control group, relatively intact fungal structures in the 500-mJ group, and obviously damaged fungal structures in the 600- to 900-mJ groups, and the greater the laser energy, the more severely the fungal structures were damaged. Conclusion:The 1 064-nm Q-switched Nd:YAG laser could affect the cell viability of and protease activity in Malassezia furfur, and damage its structures.