Aim To reveal the mechanism of CIP4 homologs protein 1(RICH1)are involved in the regu-lation of myocardial fibrosis.Methods Mouse cardiac fibroblasts(MCFs)cells were treated with transforming growth factor-β(TGF-β1)to induce the formation of a myocardial fibrosis cell model;the level of the target protein was detected by Western blotting;and the RICH1 gene was detected by transfection of the cells with plasmid.The RICH1 gene was overexpressed(RICH 1 OE)using plasmid transfection;the RICH1 gene was silenced using siRNA fragment(siRICH1);and the expression levels of myocardial fibrosis marker genes,such as Col1 a1,Col3 a1,and Acta2,were de-tected using RT-qPCR.Results RICH1 was signifi-cantly down-regulated in TGF-β1-treated MCFs;the expression levels of myocardial fibrosis marker genes,such as Col1 a1,Col3a1,and Acta2,were down-regu-lated in the RICH1 OE+TGF-β1 group;and in the siRICH1+TGF-β1 group,myocardial fibrosis marker genes,such as Col1 a1,Col3a1 and Acta2 were up-regulated at the expression level;phosphorylated SMAD2(p-SMAD2)and phosphorylated SMAD3(p-SMAD3)levels were down-regulated in the siRICH1 OE+TGF-β1 group.p-SMAD2 and P-SMAD3 levels were upregulated in the siRICH1+TGF-β1 group.Conclusion RICH1 inhibits TGF-β1-induced myo-cardial fibrosis;RICH1 inhibits TGF-β1-induced myo-cardial fibrosis by negatively regulating the SMAD2/3 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.

Objective:To isolate Yersinia enterocolitica phage and study its biological and genomic characteristics, and explore its application value. Methods:Phages were isolated from cecum of Apodemus chevrieri in the plague focus of wild rodent in Lijiang employing Yersinia enterocolitica (CMCC52301) as the host strain. After purification, proliferation and concentration, the morphology was observed by transmission electron microscope, and the host spectrum of the phage was determined by double-layer plate method. Phage DNA was extracted and sequenced, and the raw sequences data were assembled and visually analyzed, also performed genome functional annotation, phage classification status, phylogenetic tree, genome collinearity analysis, etc. Results:One strain of Yersinia enterocolitica phage was isolated and named vB_Yen_YN301-151, which had a head and a contractile tail. The phage only lysed Yersinia enterocolitica, and could not lyse other tested strains. And at a lysis temperature of 37 ℃, more transparent plaques were observed compared to at a lysis temperature of 21 ℃. The gene structure of this phage was double-stranded DNA, with a genome length of 51 176 bp and a GC content of 46.96%, and 95 genes were predicted to encode 15 structural morphological related proteins, 9 proteins involved in replication and metabolism, 3 DNA assembly functional proteins, 1 phage lysis related protein, and 67 hypothetical proteins. It was identified that the phage belong to the Drexlerviridae family of the Caudoviricetes class, clustered with vB_YenM_534, vB_Yen_X1, vB_YenM_281, and vB_YenM_531 in the phylogenetic tree and exhibited good collinearity with PY100, vB_YenM_ 25, and vB_Yen_X1. Conclusions:The successfully isolated vB_Yen_YN301-151 is a novel phage belonging to the Drexlerviridae family, and its gene encoded proteins are mostly hypothetical proteins. It only lyses Yersinia enterocolitica, with host specificity and has the potential to be used as diagnostic phage for screening Yersinia enterocolitica.

Objective To compare the effects of different exercise acclimatization(EA)durations on liver injury and inflammatory response in mice with exertional heatstroke(EHS).Methods A total of 168 male C57BL/6 mice were randomly assigned to four groups using a random number table:no exercise acclimation group(EA0W,n=54),1-week exercise acclimation group(EA1W,n=54),2-week exercise acclimation group(EA2W,n=54),and blank control group(n=6).The blank control group did not undergo acclimatization training or EHS modeling.The EA1W and EA2W groups underwent daily 2-hour exercise training at a speed of 10 m/min in an environment maintained at(26.0±0.5)℃for 1 and 2 weeks,respectively,followed by a 2-day rest after training completion.EHS modeling was performed in mice of EA0W,EA1W,and EA2W groups through running at 10 m/min under controlled environmental conditions(39.5℃ambient temperature,65%relative humidity).The modeling endpoint was defined as loss of consciousness accompanied by a core body temperature≥42.7℃.All modeling procedures were systematically documented.Following modeling,18 mice from EA0W,EA1W,and EA2W groups underwent 24-hour survival analysis.Blood samples from the abdominal aorta and liver tissues were collected at 6,12 and 24 hours post-modeling(6 mice per time point for each group).Plasma levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and creatine kinase(CK)were quantified.Interleukin(IL)-1β and IL-6 concentrations were determined using enzyme-linked immunosorbent assay(ELISA).Liver tissue specimens underwent hematoxylin-eosin(HE)staining and pathological scoring.Results The EHS model was successfully established in all EA groups.When all mice in EA0W group developed EHS(65 min after the modeling initiation),the incidence rates in EA1W and EA2W groups were 50.0%and 22.2%,respectively,with a statistically significant difference between EA0W group and the latter two groups(P<0.05).When all mice in the three groups developed EHS,the time to EHS onset was significantly longer in both EA1W and EA2W groups compared to EA0W group,with EA2W group showing a longer onset time than EA1W group(P<0.05).Survival analysis revealed a significantly higher 24-hour survival rate in EA2W group(61.1%)compared to EA0W group(33.3%)(P<0.05),while no significant difference was observed between EA1W group and the other two groups(P>0.05).The levels of IL-1β,IL-6,and CK were highest at 6 h post-modeling in all EA groups(P<0.05),and liver injury was most severe at 12 h post-modeling(P<0.05).Compared to EA0W group,the levels of ALT,AST,and IL-1β,as well as liver pathology scores,were significantly lower at 12 h post-modeling in both EA1W and EA2W groups(P<0.05),with EA2W group showing significantly lower ALT and AST levels,as well as liver pathology scores than EA1W group(P<0.05).At 6 h post-modeling,CK levels were significantly higher in EA1W and EA2W groups compared to EA0W group(P<0.05),with EA2W group exhibiting higher CK levels than in EA1W group(P<0.05).Conclusions Exercise acclimation helps reduce the incidence of EHS.Following EHS onset,the survival rate of exercise-acclimated mice is higher than that non-acclimated mice,with a significantly higher survival rate in mice acclimated for 2 weeks compared to non-acclimated mice.However,no significant difference in survival rate is observed between mice acclimated for 1 week and non-acclimated mice.Additionally,exercise acclimation for 2 weeks is more effective in reducing liver injury and inflammatory responses compared to 1-week acclimation.

Aim To reveal the mechanism of CIP4 homologs protein 1(RICH1)are involved in the regu-lation of myocardial fibrosis.Methods Mouse cardiac fibroblasts(MCFs)cells were treated with transforming growth factor-β(TGF-β1)to induce the formation of a myocardial fibrosis cell model;the level of the target protein was detected by Western blotting;and the RICH1 gene was detected by transfection of the cells with plasmid.The RICH1 gene was overexpressed(RICH 1 OE)using plasmid transfection;the RICH1 gene was silenced using siRNA fragment(siRICH1);and the expression levels of myocardial fibrosis marker genes,such as Col1 a1,Col3 a1,and Acta2,were de-tected using RT-qPCR.Results RICH1 was signifi-cantly down-regulated in TGF-β1-treated MCFs;the expression levels of myocardial fibrosis marker genes,such as Col1 a1,Col3a1,and Acta2,were down-regu-lated in the RICH1 OE+TGF-β1 group;and in the siRICH1+TGF-β1 group,myocardial fibrosis marker genes,such as Col1 a1,Col3a1 and Acta2 were up-regulated at the expression level;phosphorylated SMAD2(p-SMAD2)and phosphorylated SMAD3(p-SMAD3)levels were down-regulated in the siRICH1 OE+TGF-β1 group.p-SMAD2 and P-SMAD3 levels were upregulated in the siRICH1+TGF-β1 group.Conclusion RICH1 inhibits TGF-β1-induced myo-cardial fibrosis;RICH1 inhibits TGF-β1-induced myo-cardial fibrosis by negatively regulating the SMAD2/3 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.

Objective:To isolate Yersinia enterocolitica phage and study its biological and genomic characteristics, and explore its application value. Methods:Phages were isolated from cecum of Apodemus chevrieri in the plague focus of wild rodent in Lijiang employing Yersinia enterocolitica (CMCC52301) as the host strain. After purification, proliferation and concentration, the morphology was observed by transmission electron microscope, and the host spectrum of the phage was determined by double-layer plate method. Phage DNA was extracted and sequenced, and the raw sequences data were assembled and visually analyzed, also performed genome functional annotation, phage classification status, phylogenetic tree, genome collinearity analysis, etc. Results:One strain of Yersinia enterocolitica phage was isolated and named vB_Yen_YN301-151, which had a head and a contractile tail. The phage only lysed Yersinia enterocolitica, and could not lyse other tested strains. And at a lysis temperature of 37 ℃, more transparent plaques were observed compared to at a lysis temperature of 21 ℃. The gene structure of this phage was double-stranded DNA, with a genome length of 51 176 bp and a GC content of 46.96%, and 95 genes were predicted to encode 15 structural morphological related proteins, 9 proteins involved in replication and metabolism, 3 DNA assembly functional proteins, 1 phage lysis related protein, and 67 hypothetical proteins. It was identified that the phage belong to the Drexlerviridae family of the Caudoviricetes class, clustered with vB_YenM_534, vB_Yen_X1, vB_YenM_281, and vB_YenM_531 in the phylogenetic tree and exhibited good collinearity with PY100, vB_YenM_ 25, and vB_Yen_X1. Conclusions:The successfully isolated vB_Yen_YN301-151 is a novel phage belonging to the Drexlerviridae family, and its gene encoded proteins are mostly hypothetical proteins. It only lyses Yersinia enterocolitica, with host specificity and has the potential to be used as diagnostic phage for screening Yersinia enterocolitica.

Objective To investigate the effect and mechanism of methyl oxofulnonone A(META)on high glucose(HG)-induced H9c2 cell injury.Methods H9c2 cells were divided into control group(normal culture),model group(55 mmol·L-1 glucose)and experimental-L,-M,-H groups(55 mmol·L-1 glucose+12.5,25.0,50.0 μmol·L-1 META).Cell viability was detected by cell counting kit-8;intracellular reactive oxygen species(ROS)level was detected by DCFH-DA fluorescent probe;intracellular adenosine triphosphate(ATP)content was detected by luciferase;and autophagy-related protein expression was detected by Western blotting.Results The optical density values of 72-hour cells in the control group,model group and experimental-M,-H groups were 0.91±0.03,0.61±0.01,0.69±0.02 and 0.72±0.03;the ROS levels were(40.75±1.53)％,(43.73±1.30)％,(30.87±1.27)％and(28.28±1.43)％;the ATP contents were(8.16±0.71),(4.03±0.29),(5.29±0.31)and(5.83±0.31)nmol·mg-1;the relative expression levels of autophagy-related gene 5 protein were 1.05±0.06,1.46±0.09,0.98±0.11 and 0.89±0.09;the relative expression levels of ubiquitin-binding protein were 1.05±0.10,0.55±0.13,0.89±0.04 and 0.98±0.04;the ratios of microtubule-associated protein 1 light chain 3 11/Ⅰ protein were 1.09±0.09,1.82±0.05,1.67±0.29 and 1.09±0.15,respectively.Among the above indicators,there were statistically significant differences between the model group and the control and experimental-M,-H groups(P＜0.05,P＜0.01).Conclusion META significantly ameliorates H9c2 cardiomyocyte damage caused by high glucose,ameliorates oxidative stress,protects mitochondrial respiration and inhibits autophagy.

BACKGROUND: Asthma is a common chronic inflammatory airway disease and intermittent hypoxia is increasingly recognized as a factor that may impact disease progression. The present study investigated whether intermittent hypoxia (IH) could aggravate asthma by promoting hypoxia-inducible factor-1α (HIF-1α)/nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain-containing protein 3 (NLRP3)/interleukin (IL)-1β-dependent pyroptosis and the inflammatory response and further elucidated the underlying molecular mechanisms involved. METHODS: A total of 49 patients diagnosed with severe bronchial asthma and diagnosed by polysomnography were enrolled at Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, between January 2022 and December 2022, and their general data and induced sputum were collected. BEAS-2B cells were treated with IL-13 and subjected to IH. An ovalbumin (OVA)-treated mouse model was also used to assess the effects of chronic intermittent hypoxia (CIH) on asthma. Pyroptosis, the inflammatory response, and related signalling pathways were assessed in vivo and in vitro . RESULTS: In this study, as the apnoea and hypopnea index (AHI) increased, the proportion of patients with uncontrolled asthma increased. The proportions of neutrophils and the levels of IL-6, IL-8, HIF-1α and NLRP3 in induced sputum were related to the AHI. NLRP3-mediated pyroptosis, which could be mediated by the HIF-1α signalling pathway, was activated in IL-13 plus IH-treated BEAS-2B cells and in the lungs of OVA/CIH mice. HIF-1α downregulation significantly reduced lung pyroptosis and ameliorated neutrophil inflammation by modulating the NLRP3/IL-1β pathway both in vitro and in vivo . Similarly, pretreatment with LW6, an inhibitor of HIF-1α, effectively blocked the generation of inflammatory cytokines in neutrophils. In addition, administration of the NLRP3 activator nigericin obviously increased lung neutrophil inflammation. CONCLUSIONS Obstructive sleep apnoea-hypopnea syndrome (OSAHS) is a risk factor for asthma exacerbation. IH aggravates neutrophil inflammation in asthma via NLRP3/IL-1β-dependent pyroptosis mediated by the HIF-1α signalling pathway, which should be considered a potential therapeutic target for the treatment of asthma with OSAHS.
NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Humans ; Asthma/metabolism* ; Animals ; Pyroptosis/physiology* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Mice ; Signal Transduction/physiology* ; Male ; Hypoxia/metabolism* ; Female ; Interleukin-1beta/metabolism* ; Adult ; Inflammation/metabolism* ; Middle Aged ; Mice, Inbred C57BL

Objective To investigate the effect and mechanism of methyl oxofulnonone A(META)on high glucose(HG)-induced H9c2 cell injury.Methods H9c2 cells were divided into control group(normal culture),model group(55 mmol·L-1 glucose)and experimental-L,-M,-H groups(55 mmol·L-1 glucose+12.5,25.0,50.0 μmol·L-1 META).Cell viability was detected by cell counting kit-8;intracellular reactive oxygen species(ROS)level was detected by DCFH-DA fluorescent probe;intracellular adenosine triphosphate(ATP)content was detected by luciferase;and autophagy-related protein expression was detected by Western blotting.Results The optical density values of 72-hour cells in the control group,model group and experimental-M,-H groups were 0.91±0.03,0.61±0.01,0.69±0.02 and 0.72±0.03;the ROS levels were(40.75±1.53)％,(43.73±1.30)％,(30.87±1.27)％and(28.28±1.43)％;the ATP contents were(8.16±0.71),(4.03±0.29),(5.29±0.31)and(5.83±0.31)nmol·mg-1;the relative expression levels of autophagy-related gene 5 protein were 1.05±0.06,1.46±0.09,0.98±0.11 and 0.89±0.09;the relative expression levels of ubiquitin-binding protein were 1.05±0.10,0.55±0.13,0.89±0.04 and 0.98±0.04;the ratios of microtubule-associated protein 1 light chain 3 11/Ⅰ protein were 1.09±0.09,1.82±0.05,1.67±0.29 and 1.09±0.15,respectively.Among the above indicators,there were statistically significant differences between the model group and the control and experimental-M,-H groups(P＜0.05,P＜0.01).Conclusion META significantly ameliorates H9c2 cardiomyocyte damage caused by high glucose,ameliorates oxidative stress,protects mitochondrial respiration and inhibits autophagy.