BACKGROUND:The mesenchymal stem cell secretome contains bioactive substances,cytokines,and growth factors.Three-dimensional cell culture can regulate the secretion of these components,potentially enhancing the ability to promote injury repair.OBJECTIVE:To investigate the repair effect of three-dimensional cultured human umbilical cord mesenchymal stem cell secretome on skin injuries in mice.METHODS:Human umbilical cord mesenchymal stem cells were cultured in conventional two-dimensional culture dishes and 96-well U-bottom cell culture plates,from which their secretory components were subsequently collected.The expression of skin damage repair related secretory factors in umbilical cord mesenchymal stem cells was analyzed using RT-qPCR.The protein expression level of skin damage repair related factors in umbilical cord mesenchymal stem cell secretome was detected using enzyme-linked immunosorbent assay.The potential of human umbilical cord mesenchymal stem cell secretome to repair vascular injuries was evaluated using an immortalized human umbilical vein endothelial cell migration model.A mouse skin injury model was established,and the human umbilical cord mesenchymal stem cell secretome was injected subcutaneously.Repair effects on skin injury were assessed through wound healing rates and histopathological analysis.RESULTS AND CONCLUSION:(1)After three days of cultivation,human umbilical cord mesenchymal stem cells cultured in two dimensions exhibited a fibroblast-like,swirling growth pattern,whereas three-dimensional culture led to the formation of uniform microspheres.(2)Compared with two-dimensional culture,three-dimensional culture significantly increased the mRNA expression of transforming growth factor β and basic fibroblast growth factor in human umbilical cord mesenchymal stem cells.(3)Compared with two-dimensional culture,three-dimensional cultured human umbilical cord mesenchymal stem cell secretome significantly enhanced the protein expression of vascular endothelial growth factor,interleukin-10,and granulocyte-macrophage colony-stimulating factor in the human umbilical cord mesenchymal stem cell secretome.(4)Compared with two-dimensional culture,three-dimensional cultured human umbilical cord mesenchymal stem cell secretome significantly promoted the migration of immortalized human umbilical cord mesenchymal stem cells.(5)Compared with the untreated control group and the two-dimensional cultured human umbilical cord mesenchymal stem cell secretome,the three-dimensional cultured human umbilical cord mesenchymal stem cell secretome can significantly accelerate the skin wound healing rate and wound skin structure remodeling in mice.These results indicate that three-dimensional culture can enhance the expression of paracrine factors of human umbilical cord mesenchymal stem cells,and their secretome can significantly promote the repair of mouse skin damage.

BACKGROUND:The mesenchymal stem cell secretome contains bioactive substances,cytokines,and growth factors.Three-dimensional cell culture can regulate the secretion of these components,potentially enhancing the ability to promote injury repair.OBJECTIVE:To investigate the repair effect of three-dimensional cultured human umbilical cord mesenchymal stem cell secretome on skin injuries in mice.METHODS:Human umbilical cord mesenchymal stem cells were cultured in conventional two-dimensional culture dishes and 96-well U-bottom cell culture plates,from which their secretory components were subsequently collected.The expression of skin damage repair related secretory factors in umbilical cord mesenchymal stem cells was analyzed using RT-qPCR.The protein expression level of skin damage repair related factors in umbilical cord mesenchymal stem cell secretome was detected using enzyme-linked immunosorbent assay.The potential of human umbilical cord mesenchymal stem cell secretome to repair vascular injuries was evaluated using an immortalized human umbilical vein endothelial cell migration model.A mouse skin injury model was established,and the human umbilical cord mesenchymal stem cell secretome was injected subcutaneously.Repair effects on skin injury were assessed through wound healing rates and histopathological analysis.RESULTS AND CONCLUSION:(1)After three days of cultivation,human umbilical cord mesenchymal stem cells cultured in two dimensions exhibited a fibroblast-like,swirling growth pattern,whereas three-dimensional culture led to the formation of uniform microspheres.(2)Compared with two-dimensional culture,three-dimensional culture significantly increased the mRNA expression of transforming growth factor β and basic fibroblast growth factor in human umbilical cord mesenchymal stem cells.(3)Compared with two-dimensional culture,three-dimensional cultured human umbilical cord mesenchymal stem cell secretome significantly enhanced the protein expression of vascular endothelial growth factor,interleukin-10,and granulocyte-macrophage colony-stimulating factor in the human umbilical cord mesenchymal stem cell secretome.(4)Compared with two-dimensional culture,three-dimensional cultured human umbilical cord mesenchymal stem cell secretome significantly promoted the migration of immortalized human umbilical cord mesenchymal stem cells.(5)Compared with the untreated control group and the two-dimensional cultured human umbilical cord mesenchymal stem cell secretome,the three-dimensional cultured human umbilical cord mesenchymal stem cell secretome can significantly accelerate the skin wound healing rate and wound skin structure remodeling in mice.These results indicate that three-dimensional culture can enhance the expression of paracrine factors of human umbilical cord mesenchymal stem cells,and their secretome can significantly promote the repair of mouse skin damage.

Aim To explore the impact and mechanism of proprotein convertase subtilisin kexin 9(PCSK9)on the progression of abdominal aortic aneurysm(AAA).Methods 6～8 week old ApoE-/-mice were selected to estab-lish the AAA model.Angiotensin Ⅱ(Ang Ⅱ)was continuously infused through subcutaneous implantation of a micro-os-motic pump.The mice were fed with high-fat diet and killed after 28 days.The expression of PCSK9 in abdominal aor-tic smooth muscle cells was detected by immunohistochemistry and immunofluorescence in normal abdominal aortic blood vessels and AAA samples in human and mice.Primary cultured murine vascular smooth muscle cells(mVSMC)of C57BL/6 mice were treated with different concentrations of AngⅡ for 24 h,and the expression of PCSK9 mRNA and pro-tein was detected.PCSK9 overexpression and knockdown cell models were established,and mitochondrial reactive oxygen species(mtROS),mitochondrial membrane potential(MMP),mitochondrial permeability transition pore(MPTP)open-ing,and Z-DNA binding protein 1(ZBP1)protein expression were detected.Bioinformatics was used to analyze the dif-ferential expression of multiple single-cell sequencing datasets to obtain the key differentially expressed genes,and to study their expression and role in AAA.Results Immunohistochemistry and immunofluorescence results showed that PCSK9 expression in human and mouse AAA increased(P＜0.01),and co-localized with smooth muscle.Ang Ⅱ promoted PCSK9 expression in mVSMC in a concentration-dependent manner,the 2.0 μmol/L Ang Ⅱ group showed a 2.9-fold and 1.1-fold increase in the expression of PCSK9 mRNA and protein,respectively(P＜0.01),with the most significant effect observed.After successfully constructing PCSK9 overexpression and PCSK9 interference mVSMC models,PCSK9 overex-pression led to an increase in intracellular mtROS,a decrease in MMP,an increase in MPTP opening,and a decrease in cellular activity(P＜0.01);PCSK9 knockdown could reduce Ang Ⅱ induced increase in mtROS,decrease in MMP and MPTP opening;compared with the siNC+Ang Ⅱ group,the siPCSK9+Ang Ⅱ group showed a decrease in mtROS and an in-crease in the fluorescence brightness of MMP and MPTP(P＜0.05).Bioinformatics analysis revealed that ZBP1 was a core differentially expressed gene in AAA.Immunohistochemistry and immunofluorescence results showed that ZBP1 ex-pression in human and mouse AAA tissues increased,and co-localized with smooth muscle.Western blot results showed that PCSK9 overexpression or treatment with 2.0 μmol/L Ang Ⅱ could increase ZBP1 protein expression(P＜0.01),while PCSK9 knockdown could alleviate the increased ZBP1 expression caused by AngⅡ(P＜0.05).Conclusion PCSK9 may induce mitochondrial damage in smooth muscle cells,activate downstream molecule ZBP1 to cause cell damage,and promote the development of AAA.

N6-methyladenosine(m6A)is the most common mRNA modification in eukaryotes,and fat mass and obesity-related protein(FTO),are its demethylases,which efficiently remove the modification of m6A mRNA,and is strongly associated with obesity.Atherosclerosis is a chronic inflammatory lesion of the blood vessel wall driven by lipids.It was found that FTO-mediated m6A may influence the process of atherosclerosis through lipid metabolism,oxidative stress,mitochondrial dysfunction,and macrophage foaminess.

The engineering application of microbially induced carbonate precipitation(MICP)is limited by pH-dependent conformational dynamics of urease.Focusing on the α-subunit urease from Sporosarcina pasteurii,this study integrated conductivity experiments and constant-pH molecular dynamics simulations to analyze active site conformational dynamics and catalytic function across pH 3-11.Results showed that under neutral conditions(pH 7-8),key histidine residues(HIS139/HIS249)exhibited minimal dis-placement(＜0.5 ?),the longest hydrogen bond lifetime(＞8 ps),highest conformational stability(root mean square deviation,RMSD:0.15-0.18 nm),and optimal catalytic activity(conductivity change rate:0.03 mS/cm·min-1,CaCO3 precipitation:3.84 g).Extreme pH(pH 3/11)induced structural collapse(displacement up to 1.8 ?)and complete activity loss.Simulations revealed that neutral pH sta-bilizes a protonation-dependent cooperative allosteric network by maintaining active site cavity volume(～120 ?3)and moderate conformational coherence(correlation coefficient～0.8).This work deciphers the molecular mechanism of pH-regulated urease dynamics through protonation states,providing theoreti-cal support for MICP applications in acidic mine tailing remediation and alkaline soil stabilization.

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 explore the effects of apolipoprotein A Ⅰ(ApoA Ⅰ)and apolipoprotein A Ⅰ binding protein(AIBP)on THP-1-derived macrophage pyroptosis.Methods The lactate dehydrogenase(LDH)detection kit was used to evaluate cell membrane integrity,Hoechst33342/PI staining was used to observe cell membrane permeability,ELISA was used to detect the levels of inflammatory factors such as interleukin-1 β(IL-1β)and interleukin-18(IL-18),Western blot was used to detect the expression of pyroptosis-related protein nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3(NLRP3),gasdermin D(GSDMD),cleaved Caspase-1,IL-1β and IL-18.Results Oxidized low density lipoprotein(ox-LDL)upregulated the expression of NLRP3,GSDMD-N,cleaved Caspase-1,IL-1β and IL-18 in THP-1-derived macrophages in a concentration-dependent manner,and promoted the release of IL-1β,IL-18 and LDH(P＜0.05 or P＜0.01),indicating that ox-LDL induced pyroptosis in THP-1-derived macrophages in a concentration-dependent manner.Co-treatment of macrophages with ApoA Ⅰ and AIBP significantly downregulated the ex-pression of NLRP3,GSDMD-N,cleaved Caspase-1,IL-1β and IL-18,reduced the release of IL-1 β,IL-18 and LDH,and inhibited ox-LDL induced pyroptosis(P＜0.05 or P＜0.01).After ATP-binding cassette transporter A1(ABCA1)siRNA transfection,co-treatment with ApoA Ⅰ and AIBP had no significant effect on the expression of pyroptosis-related proteins and secretion of inflammatory factors(P＞0.05).Co-treatment of macrophages with ApoA Ⅰ and AIBP significantly re-duced the expression of purinergic 2X7R receptor(P2X7R)on the cell membrane,inhibited P2X7R mediated protein ki-nase R(PKR)phosphorylation and NLRP3 inflammasome assembly(P＜0.05 or P＜0.01).After P2X7R siRNA trans-fection,co-treatment with ApoA Ⅰ and AIBP had no significant effect on the expression of pyroptosis-related proteins and secretion of inflammatory factors(P＞0.05).Conclusion ApoA Ⅰ and AIBP reduce the expression of P2X7R on the cell membrane through ABCA1,inhibiting P2X7R/PKR/NLRP3 mediated macrophage pyroptosis.

The occurrence and development of atherosclerotic cardiovascular diseases is closely related to abnormally elevated plasma low density lipoprotein cholesterol(LDLC)level.Low density lipoprotein receptor(LDLR)plays a central role in the maintenance of cholesterol homeostasis by mediating the endocytotic clearance of LDLC,and the abundance of LDLR on the surface of the cell membrane is closely related to the expression level and recirculation of LD-LR.Recent studies have found that RING-E3 ubiquitin ligase can regulate LDLR levels through a dual mechanism:on the one hand,it directly ubiquitinates and modifies LDLR to promote its degradation via the endosome-lysosome pathway;on the other hand,it reduces LDLR synthesis through activation of the liver X receptor(LXR)pathway or inhibition of the nuclear translocation of sterol regulatory element-binding protein(SREBP).Together,these two mechanisms lead to a decrease in cell membrane LDLR abundance,impairing cholesterol metabolic homeostasis and exacerbating LDLC accumu-lation.Therefore,targeted inhibition of RING-E3 ubiquitin ligase activity may be a novel strategy to regulate LDLR ex-pression,reduce plasma LDLC levels,and combat cardiovascular disease.This article reviews the mechanism of action of RING-E3 ubiquitin ligase in regulating LDLR and its related research progress.

Aim To investigate the biological function and molecular mechanisms of piRNA-823 in the phenotypic transformation of human umbilical vein endothelial cells(HUVEC)induced by high glucose.Methods HUVEC were incubated in high glucose(33.3 mmol/L)culture medium for 72 h.The relative expression levels of piR-NA-823 were detected by RT-qPCR,the expression changes of endothelial cell markers,mesenchymal cell markers and proteins related to transforming growth factor-β1(TGF-β1)signaling pathway were detected by Western blot,the changes of cell migration ability were evaluated by scratch and Transwell assays,the formation of new angiogenesis were assessed through angiogenesis experiments.piRNA-823 mimic(overexpression of piRNA-823)were transfected into HUVEC to analyze their effects on high glucose induced endothelial-mesenchymal transition(EndMT)and angiogenesis.Further in-tervention was performed using TGF-β1 activator(SRI011381)and inhibitor(SB525334)to verify whether piRNA-823 ex-erts its effect by regulating the TGF-β1/Smad2/3 signaling pathway.Results piRNA-823 mimic significantly inhibited the viability,proliferation,migration and angiogenesis of HUVEC induced by high glucose.The piRNA-823 mimic inhibited high glucose induced EndMT in HUVEC,characterized by upregulation of endothelial cell markers and downregulation of mesenchymal cell markers.Scratch experiments,Transwell experiments and angiogenesis experiments further confirmed that piRNA-823 mimic could effectively reverse high glucose induced HUVEC proliferation,migration a-bility enhancement,and increase in the number of new angiogenesis.Mechanistic studies revealed that the TGF-β1 acti-vator partially reversed the protective effect of piRNA-823 mimic,whereas the TGF-β1 inhibitor enhanced its effect,sug-gesting that piRNA-823 exerts its regulatory role by suppressing the activation of the TGF-β1/Smad2/3 signaling pathway.Conclusion piRNA-823 significantly inhibits high glucose induced EndMT,proliferation,migration and angiogenesis in HUVEC by suppressing the activation of TGF-β1/Smad2/3 signaling pathway.

Diabetic cardiomyopathy(DCM)is a prevalent cardiovascular complication associated with diabe-tes,which is characterized by abnormalities in heart structure and function.Chronic inflammation,which is mediated by the nuclear factor-κB(NF-κB)signaling pathway,is pivotal in the onset and progression of DCM.NF-κB can be activated by diabetes-related factors,including hyperglycemia,oxidative stress,and endoplasmic reticulum stress,which in turn promote the expression of inflammatory cytokines,chemokines,and adhesion molecules.This cascade leads to cardiac in-flammation,fibrosis,and apoptosis.Therefore,we summarized the role of the NF-κB signaling pathway in DCM,particu-larly its activation mechanisms and downstream reactions.The regulatory effects of cytokines,receptors,reactive oxygen species,and other factors involved in NF-κB activation were also analyzed.In addition,potential therapeutic strategies to modulate NF-κB activity,including natural compounds,synthetic inhibitors,gene therapy,and monoclonal antibodies,were summarized in this study.Targeting the NF-κB signaling pathway can mitigate the inflammatory response and en-hance cardiac function in patients with DCM.However,the development of selective and effective NF-κB inhibitors,along with the validation of their safety and efficacy through clinical trials,remains a critical focus for future research.