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.

Objective To screen for differentially expressed apoptosis-related circular RNAs(circRNAs)in osteoblasts from steroid-induced osteonecrosis of the femoral head(SONFH)and to investigate their roles and mechanisms in osteoblast apoptosis.Methods MC3T3-E1 cells were cultured and divided into two groups:Control and DEX-treated.Western blot analysis was employed to evaluate the expression levels of BCL2-Associated X protein(Bax)and B-cell lymphoma 2(Bcl-2).Cell apoptosis was assessed using TUNEL staining and flow cytometry.RNA was extracted from both normal and DEX-treated MC3T3-E1 cells,followed by RNA-seq to identify differen-tially expressed circular RNAs(circRNAs).The functions and pathways of these circRNAs were analyzed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).The differentially expressed mmu_circ_0000818 was selected for further verification at the cellular level.Its chromosomal location and conservation were examined using the UCSC Genome Browser Gateway and circBase.Overexpression plasmids and small interfering RNAs(siRNAs)targeting mmu_circ_0000818 were constructed and transfected into the cells.Subsequently,apop-tosis in MC3T3-E1 cells from each group was evaluated by flow cytometry.Results Compared with the control group,the apoptosis rate of MC3T3-E1 cells was significantly increased in the DEX group(P<0.01).Differen-tially expressed circRNAs were identified based on log2foldchange(≥2)and P value(P<0.05).Relative to the control group,there were 234 differentially expressed circRNAs in the DEX group,including 138 up-regulated and 96 down-regulated circRNAs.GO and KEGG enrichment analyses of the target genes of these differentially expressed circRNAs revealed significant associations with apoptosis and the PI3K-Akt signaling pathway.qRT-PCR results demonstrated that the expression level of mmu_circ_0000818 was markedly higher in the DEX group com-pared to the control group(P<0.01).Analysis using the UCSC Genome Browser and CircBase indicated that mmu_circ_0000818,located at chromosome 17:78712463-78715086,is formed by the cyclization of exons 6-7 of the Crim1 gene and exhibits high conservation across species.Flow cytometry results indicated that knockdown of mmu_circ_0000818 attenuated DEX-induced apoptosis in MC3T3-E1 cells,while overexpression of mmu_circ_0000818 exacerbated apoptosis.Conclusions CircRNA mmu_circ_0000818 was significantly upregulated in DEX-treated MC3T3-E1 cells,and its downregulation mitigated DEX-induced apoptosis.Consequently,mmu_circ_0000818 may represent a promising therapeutic target for the prevention and treatment of SONFH.

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.

Objective To screen for differentially expressed apoptosis-related circular RNAs(circRNAs)in osteoblasts from steroid-induced osteonecrosis of the femoral head(SONFH)and to investigate their roles and mechanisms in osteoblast apoptosis.Methods MC3T3-E1 cells were cultured and divided into two groups:Control and DEX-treated.Western blot analysis was employed to evaluate the expression levels of BCL2-Associated X protein(Bax)and B-cell lymphoma 2(Bcl-2).Cell apoptosis was assessed using TUNEL staining and flow cytometry.RNA was extracted from both normal and DEX-treated MC3T3-E1 cells,followed by RNA-seq to identify differen-tially expressed circular RNAs(circRNAs).The functions and pathways of these circRNAs were analyzed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).The differentially expressed mmu_circ_0000818 was selected for further verification at the cellular level.Its chromosomal location and conservation were examined using the UCSC Genome Browser Gateway and circBase.Overexpression plasmids and small interfering RNAs(siRNAs)targeting mmu_circ_0000818 were constructed and transfected into the cells.Subsequently,apop-tosis in MC3T3-E1 cells from each group was evaluated by flow cytometry.Results Compared with the control group,the apoptosis rate of MC3T3-E1 cells was significantly increased in the DEX group(P<0.01).Differen-tially expressed circRNAs were identified based on log2foldchange(≥2)and P value(P<0.05).Relative to the control group,there were 234 differentially expressed circRNAs in the DEX group,including 138 up-regulated and 96 down-regulated circRNAs.GO and KEGG enrichment analyses of the target genes of these differentially expressed circRNAs revealed significant associations with apoptosis and the PI3K-Akt signaling pathway.qRT-PCR results demonstrated that the expression level of mmu_circ_0000818 was markedly higher in the DEX group com-pared to the control group(P<0.01).Analysis using the UCSC Genome Browser and CircBase indicated that mmu_circ_0000818,located at chromosome 17:78712463-78715086,is formed by the cyclization of exons 6-7 of the Crim1 gene and exhibits high conservation across species.Flow cytometry results indicated that knockdown of mmu_circ_0000818 attenuated DEX-induced apoptosis in MC3T3-E1 cells,while overexpression of mmu_circ_0000818 exacerbated apoptosis.Conclusions CircRNA mmu_circ_0000818 was significantly upregulated in DEX-treated MC3T3-E1 cells,and its downregulation mitigated DEX-induced apoptosis.Consequently,mmu_circ_0000818 may represent a promising therapeutic target for the prevention and treatment of SONFH.

Rosuvastatin(RVS)is an excellent drug with anti-inflammatory and lipid-lowering properties in the aca-demic and medical fields.However,this drug faces a series of challenges when used to treat atherosclerosis caused by hyperhomocysteinemia(HHcy),including high oral dosage,poor targeting,and long-term toxic side effects.In this study,we applied nanotechnology to construct a biomimetic nano-delivery system,macrophage membrane(M?m)-coated RVS-loaded Prussian blue(PB)nanoparticles(MPR NPs),for improving the bioavailability and targeting capacity of RVS,specifically to the plaque lesions associated with HHcy-induced atherosclerosis.In vitro assays demonstrated that MPR NPs effectively inhibited the Toll-like receptor 4(TLR4)/hypoxia-inducible factor-1α(HIF-1α)/nucleotide-binding and oligomerization domain(NOD)-like receptor thermal protein domain associated protein 3(NLRP3)signaling pathways,reducing pyroptosis and inflammatory response in macrophages.Additionally,MPR NPs reversed the abnormal distribution of adenosine triphosphate(ATP)-binding cassette transporter A1(ABCA1)/ATP binding cassette transporter G1(ABCA1)/ATP binding cassette transporter G1(ABCG1)caused by HIF-1α,promoting cholesterol efflux and reducing lipid deposition.In vivo studies using apolipoprotein E knockout(ApoE-/-)mice confirmed the strong efficacy of MPR NPs in treating atherosclerosis with favorable bio-security,and the mechanism behind this efficacy is believed to involve the regulation of serum metabolism and the remodeling of gut microbes.These findings suggest that the synthesis of MPR NPs provides a promising nanosystem for the targeted therapy of HHcy-induced atherosclerosis.

Objective To study the effect of DNA methyltransferase 1(DNMT1)on sm-like protein-4(LSM4)in hepatocyte apoptosis in mice induced with Hcy.Methods 12 ApoE-/-mice were divided into two groups:normal diet(ND,n=6)and high methionine diet(HMD,n=6)groups.Normal hepatocytes of NCTC1469 were divided into a normal group(control,0 μL/L Hcy),Hcy intervention group(Hcy,100 μL/L Hcy),NC siRNA-transfected control group(si-NC group,0 μmol/L Hcy),LSM4 siRNA-transfected group(si-LSM4 group,0 μmol/L Hcy),DNMT1 siRNA-transfected group(si-DNMT1 group,0 μmol/L Hcy),NC siRNA-transfected Hcy intervention group(Hcy+si-NC group,100 μmol/L Hcy),LSM4 siRNA-transfected Hcy intervention group(Hcy+si-LSM4 group,100 μmol/L Hcy),and DNMT1 siRNA-transfected Hcy intervention group(Hcy+si-DNMT1 group,100 μmol/L Hcy).Analysis of the expression of LSM4 in various tissues was conducted using the NCBI database.Quantitative real-time PCR(qRT-PCR)and Western blot were used to detect differences in LSM4 protein expression in mouse tissues(HMD and ND)and hepatocytes(control and Hcy).Western blot was used to detect the expression of Bcl2-associated X(Bax)and B-cell lymphoma-2(Bcl-2).The cell apoptosis rate in the Control,Hcy,Hcy+si-NC,and Hcy+si-LSM4 groups were detected by flow cytometry.MethPrimer online software was used to analyze the CpG islands of LSM4 promoter region.The expression of LSM4 in the Hcy+si-DNMT1 group was detected by qRT-PCR and Western blot.Results The expression of LSM4 in HMD,Hcy group was higher than that in the ND and Control group(P＜0.05).Bax protein expression was significantly higher,but Bcl-2 was significantly lower in Hcy group compared with those of the Control group(P＜0.05).The expression of Bax protein was significantly lower,but the level of Bcl-2 was significantly higher in the Hcy+si-LSM4 group compared with those in the Hcy+si-NC group(P＜0.05).The cell apoptosis rate in the Hcy group was higher than that in the Control group(P＜0.05),while the apoptosis rate in the Hcy+si-LSM4 group was lower than that in the Hcy+si-NC group(P＜0.05).MethPrimer database analysis showed that the promoter region of LSM4 was GC-rich,and there was one CpG island.Compared with the Hcy+si-NC group,the Hcy+si-DNMT1 group's expression of LSM4 protein was increased(P＜0.05).Conclusions DNMT1 regulates LSM4 hypomethylation to increase its expression,thereby promoting Hcy-induced apoptosis of mouse hepatocytes.

Objective To study the effect of DNA methyltransferase 1(DNMT1)on sm-like protein-4(LSM4)in hepatocyte apoptosis in mice induced with Hcy.Methods 12 ApoE-/-mice were divided into two groups:normal diet(ND,n=6)and high methionine diet(HMD,n=6)groups.Normal hepatocytes of NCTC1469 were divided into a normal group(control,0 μL/L Hcy),Hcy intervention group(Hcy,100 μL/L Hcy),NC siRNA-transfected control group(si-NC group,0 μmol/L Hcy),LSM4 siRNA-transfected group(si-LSM4 group,0 μmol/L Hcy),DNMT1 siRNA-transfected group(si-DNMT1 group,0 μmol/L Hcy),NC siRNA-transfected Hcy intervention group(Hcy+si-NC group,100 μmol/L Hcy),LSM4 siRNA-transfected Hcy intervention group(Hcy+si-LSM4 group,100 μmol/L Hcy),and DNMT1 siRNA-transfected Hcy intervention group(Hcy+si-DNMT1 group,100 μmol/L Hcy).Analysis of the expression of LSM4 in various tissues was conducted using the NCBI database.Quantitative real-time PCR(qRT-PCR)and Western blot were used to detect differences in LSM4 protein expression in mouse tissues(HMD and ND)and hepatocytes(control and Hcy).Western blot was used to detect the expression of Bcl2-associated X(Bax)and B-cell lymphoma-2(Bcl-2).The cell apoptosis rate in the Control,Hcy,Hcy+si-NC,and Hcy+si-LSM4 groups were detected by flow cytometry.MethPrimer online software was used to analyze the CpG islands of LSM4 promoter region.The expression of LSM4 in the Hcy+si-DNMT1 group was detected by qRT-PCR and Western blot.Results The expression of LSM4 in HMD,Hcy group was higher than that in the ND and Control group(P＜0.05).Bax protein expression was significantly higher,but Bcl-2 was significantly lower in Hcy group compared with those of the Control group(P＜0.05).The expression of Bax protein was significantly lower,but the level of Bcl-2 was significantly higher in the Hcy+si-LSM4 group compared with those in the Hcy+si-NC group(P＜0.05).The cell apoptosis rate in the Hcy group was higher than that in the Control group(P＜0.05),while the apoptosis rate in the Hcy+si-LSM4 group was lower than that in the Hcy+si-NC group(P＜0.05).MethPrimer database analysis showed that the promoter region of LSM4 was GC-rich,and there was one CpG island.Compared with the Hcy+si-NC group,the Hcy+si-DNMT1 group's expression of LSM4 protein was increased(P＜0.05).Conclusions DNMT1 regulates LSM4 hypomethylation to increase its expression,thereby promoting Hcy-induced apoptosis of mouse hepatocytes.

Objective To analyze the biological characteristics of a mutant strain of Salmonella ty-phimurium SL1344 with sseK2-deletion (SL1344△sseK2) in order to provide reference for further study of safe and effective live vaccines. Methods The mutant strain SL1344△sseK2 with a deletion of 1047 bp in sseK2 gene was constructed through a two-step allelic exchange using recombinant suicide plasmid. Its com-plemented strain, SL1344C△sseK2, was also constructed. Biological and immunological characteristics of the mutant strain were detected. Results PCR, double-enzyme digestion and sequencing analysis showed that the mutant strain SL1344△sseK2 and the complemented strain SL1344C△sseK2 were successfully con-structed. The serotype of the mutant strain was 1,4,[5],12:i:1,2, identical to the parent strain SL1344. In addition, the mutant strain showed no significant change in biochemical characteristics or growth rate and was genetically stable in vitro. Compared with the parent strain SL1344, the virulence of SL1344△sseK2 was attenuated in BALB/ c mice. The median lethal dose of SL1344△sseK2 for 6-week-old BALB/ c mice was 3. 44×108 colony-forming units (CFU), which was 1620 times lower than that of SL1344. Oral immuniza-tion with SL1344△sseK2 protected 62. 5％ of the mice against challenge with wild Salmonella typhimurium strains on 17 d after vaccination. The levels of serum IgG antibody peaked on 14 d after immunization. No significant difference in biological characteristics was observed between the complemented and the parent strains, indicating that the mutant strain was basically complemented to the wild-type strain.Conclusions The mutant strain SL1344△sseK2 was constructed successfully and genetically stable with sig-nificantly attenuated virulence and good immunogenicity. This study suggested that sseK2 gene played an im-portant role in regulating the virulence of SL1344, which might provide reference for further study of its func-tion and for assessing its potential as a candidate live attenuated vaccine.

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Objective:To discuss the effects of recombinant IFN-α-IL-18 fusion protein on chicken lymphocyte histamine induced and the NF-κB p65 activation and nucleo-cytoplasmic transport.Methods: The healthy chickens blood was sterile adopted with anticoagulant,then separation of the chicken peripheral blood lymphocyte and divided into 10 groups:The yeast expression and purification protein IFN-α-IL-18,IL-18,IFN-α were added with 250 ng/ml,500 ng/ml and 1 000 ng/ml respectively while the control was only added RPMI1640 with 3 repetitions for each group.Then the histidine decarboxylase activity,histamine,IFN-γ,PI3K,MAPK and NF-κB p65 in cell nucleus were detected.Results: The recombinant IFN-α-IL-18 and IL-18 could significantly promote the activity of histidine decarboxylase (P<0.01),increase the contents of histamine (P<0.01),induce IFN-γ (P<0.01),improve the contents of PI3K (P<0.01) and the NF-κB p65 levels in nucleus (P<0.01),and the higher concentration of IFN-α had a similar effect to lymphocytes.The effects of IFN-α-IL-18,IL-18 and IFN-α on MAPK was acratia.Conclusion: IFN-α-IL-18 and IL-18 can stimulate chicken peripheral blood lymphocyte populations increased histamine contents significantly and promote the induction of IFN-γ.IFN-α-IL-18,IL-18 and IFN-α increase PI3K expression in lymphocyte associated with the NF-κB activation and NF-κB p65 nucleo-cytoplasmic transport.The study built foundation for the function of IFN-α-IL-18 and the exploration of the mechanism of controlling epidemic diseases.