Protective effects of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja on STZ-stimulated INS-1 cells through regulating of autophagy and apoptosis

OBJECTIVE To explore the protective effects of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja on STZ-stimulated INS-1 cells through regulating of autophagy and apoptosis.METHODS INS-1cells were cultured in media containing 3n M STZ and different doses of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja.The proliferation of cells was examined by MTT assay,ROS content were detected by fluorescence enzyme label.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),hydrogen peroxidase(CAT),malondialdehyde(MDA)were also measured by colorimetry.The activities of caspase-3,9 were also observed by Caspase colorimetric assay kit in each group.The expressions of Bcl-2 m RNA and Bax m RNA were detected by Real time PCR,protein expression of LC3-Ⅱand PARP were detected by Western blotting.RESULTS Compared with the control group,total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja could promote the proliferation of INS-1 cells no matter with STZ or not when its concentration lower than 25μg·m L-1;but when its concentration higher than 100μg·m L-(1use individually)or 50μg·m L-1(combined use),total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja might significantly inhibite the growth of the cells whether STZ existed or not.Total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja(6.25,12.5,25μg·m L-1)might inhibit INS-1cell apoptosis,decrease intra-cellular ROS contents,improve the s upernatant liquid SOD,GSH-PX,CAT activities,decrease MDA level,promote INS-1 cell secreting insulin,decrease the protein expressions of autophagy protein LC3Ⅱand apoptosis regulating protein cleaved PARP protein,up-regulate the anti-apoptotic protein Bcl-2 m RNA expression,down-regulate the pro-apoptotic protein Bax m RNA expression and Bcl-2and Bax,reduce the activity of caspase-9 and caspase-3(P<0.05 or P<0.01).CONCLUSION Total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja has good protective effect on STZ-stimulated INS-1cells.It can inhibit STZ injured INS-1 cells to overproduce ROS production,enhance endogenous antioxidant enzymes(GSH-Px,SOD,CAT activities),reduce the expression of autophagy protein LC3Ⅱand apoptosisregulating protein cleaved PARP,up-reglute the antiapoptosis protein Bcl-2 expression,down-regulate the pro-apoptotic protein Bax expression,decrease the caspase-9 and caspase-3 activities,and improve the INS-1cell survival rate,and then play a protective effect on damaged INS-1 cells.

Impact of Bisphenol A (BPA) and Free Fatty Acids (FFA) on Th2 Cytokine Secretion from INS-1 Cells

Bisphenol A (BPA) is used in huge amounts for many plastic products and is a hormone (estrogen) disrupting agent. BPA as well as FFAs may be deleterious for the immune system. The aim was to identify Th2 cytokines and some of their signal transduction mechanisms in INS-1 cells, an insulin secreting cell line. Screening using a proteome profile indicated an increase of IL-1, IL-2, IL-4, IL-6, IL-10, IL-13 and IL-17 by BPA. Also FFAs (in combination with LPS) were positive. In detailed quantitative measurements, these results were confirmedly indicating a complex array of pro-and anti-inflammatory potential. The interaction of BPA with 17β-estradiol was non-additive with respect to IL-4 and IL-6 release and additive with respect to FFA interaction indicating same and different mechanisms of action, respecttively. As signal transduction PI3K (Wortmannin-sensitive) and STAT-3/6 (Tofacitinib-sensitive) are involved in various effects, INS-1 cells release several cytokines due to BPA and FFA attack which may be involved in disturbance of glucose homoeostasis and type 1 diabetes.

玄参水提取物对高糖暴露条件下INS-1细胞AMPK的激活作用

目的研究玄参水提取物(AESN)对高糖(HG)条件下INS-1细胞腺苷酸活化蛋白激酶(AMPK)活性的影响。方法将INS-1细胞培养于HG培养基中,并给予不同浓度AESN共孵育处理;利用细胞计数试剂-8(CCK-8)法检测AESN干预对细胞增殖/活力和焦亡小体形成的影响,使用Western blotting法观察AESN对细胞内AMPK表达及磷酸化水平的影响;利用时间分辨-荧光共振能量转移(TR-FRET)实验检测AESN对AMPK激酶活性的影响。结果CCK-8检测结果显示同正常培养条件相比,HG暴露显著降低INS-1细胞增殖/活力并增加焦亡小体形成数量,Western blotting检测结果表明HG暴露可导致细胞内AMPK磷酸化水平下降;而AESN共孵育可呈浓度依赖性地增加INS-1细胞增殖/活力、抑制焦亡小体形成并激活AMPK。TR-FRET实验结果表明,AESN可浓度依赖性增加AMPK激酶活性。结论AESN对HG暴露条件下INS-1细胞AMPK具有激活作用。

Efficacy and predictive factors of transarterial chemoembolization combined with lenvatinib plus programmed cell death protein-1 inhibition for unresectable hepatocellular carcinoma

BACKGROUND The efficacy and safety of transarterial chemoembolization(TACE)combined with lenvatinib plus programmed cell death protein-1(PD-1)for unresectable hepato-cellular carcinoma(HCC)have rarely been evaluated and it is unknown which factors are related to efficacy.AIM To evaluate the efficacy and independent predictive factors of TACE combined with lenvatinib plus PD-1 inhibitors for unresectable HCC.METHODS This study retrospectively enrolled patients with unresectable HCC who received TACE/lenvatinib/PD-1 treatment between March 2019 and April 2022.Overall survival(OS)and progression-free survival(PFS)were determined.The objective response rate(ORR)and disease control rate(DCR)were evaluated in accordance with the modified Response Evaluation Criteria in Solid Tumors.Additionally,the prognostic factors affecting the clinical outcome were assessed.RESULTS One hundred and two patients were enrolled with a median follow-up duration of 12.63 months.The median OS was 26.43 months(95%CI:17.00-35.87),and the median PFS was 10.07 months(95%CI:8.50-11.65).The ORR and DCR were 61.76%and 81.37%,respectively.The patients with Barcelona Clinic Liver Cancer Classification(BCLC)B stage,early neutrophil-to-lymphocyte ratio(NLR)response(decrease),or early alpha-fetoprotein(AFP)response(decrease>20%)had superior OS and PFS than their counterparts.CONCLUSION This study showed that TACE/lenvatinib/PD-1 treatment was well tolerated with encouraging efficacy in patients with unresectable HCC.The patients with BCLC B-stage disease with early NLR response(decrease)and early AFP response(decrease>20%)may achieve better clinical outcomes with this triple therapy.

TM9SF1 promotes bladder cancer cell growth and infiltration

BACKGROUND Bladder cancer(BC)is the most common urological tumor.It has a high recur-rence rate,displays tutor heterogeneity,and resists chemotherapy.Furthermore,the long-term survival rate of BC patients has remained unchanged for decades,which seriously affects the quality of patient survival.To improve the survival rate and prognosis of BC patients,it is necessary to explore the molecular mechanisms of BC development and progression and identify targets for treatment and intervention.Transmembrane 9 superfamily member 1(TM9SF1),also known as MP70 and HMP70,is a member of a family of nine transmembrane superfamily proteins,which was first identified in 1997.TM9SF1 can be expressed in BC,but its biological function and mechanism in BC are not clear.AIM To investigate the biological function and mechanism of TM9SF1 in BC.Overexpression of TM9SF1 increased the in vitro proliferation,migration,and invasion of BC cells by promoting the entry of BC cells into the G2/M phase.Silencing of TM9SF1 inhibited in vitro proliferation,migration,and invasion of BC cells and blocked BC cells in the G1 phase.CONCLUSION TM9SF1 may be an oncogene in BC.

Bone marrow-derived mesenchymal stem cell-derived exosomeloaded miR-129-5p targets high-mobility group box 1 attenuates neurological-impairment after diabetic cerebral hemorrhage

BACKGROUND Diabetic intracerebral hemorrhage(ICH)is a serious complication of diabetes.The role and mechanism of bone marrow mesenchymal stem cell(BMSC)-derived exosomes(BMSC-exo)in neuroinflammation post-ICH in patients with diabetes are unknown.In this study,we investigated the regulation of BMSC-exo on hyperglycemia-induced neuroinflammation.AIM To study the mechanism of BMSC-exo on nerve function damage after diabetes complicated with cerebral hemorrhage.METHODS BMSC-exo were isolated from mouse BMSC media.This was followed by transfection with microRNA-129-5p(miR-129-5p).BMSC-exo or miR-129-5poverexpressing BMSC-exo were intravitreally injected into a diabetes mouse model with ICH for in vivo analyses and were cocultured with high glucoseaffected BV2 cells for in vitro analyses.The dual luciferase test and RNA immunoprecipitation test verified the targeted binding relationship between miR-129-5p and high-mobility group box 1(HMGB1).Quantitative polymerase chain reaction,western blotting,and enzyme-linked immunosorbent assay were conducted to assess the levels of some inflammation factors,such as HMGB1,interleukin 6,interleukin 1β,toll-like receptor 4,and tumor necrosis factorα.Brain water content,neural function deficit score,and Evans blue were used to measure the neural function of mice.RESULTS Our findings indicated that BMSC-exo can promote neuroinflammation and functional recovery.MicroRNA chip analysis of BMSC-exo identified miR-129-5p as the specific microRNA with a protective role in neuroinflammation.Overexpression of miR-129-5p in BMSC-exo reduced the inflammatory response and neurological impairment in comorbid diabetes and ICH cases.Furthermore,we found that miR-129-5p had a targeted binding relationship with HMGB1 mRNA.CONCLUSION We demonstrated that BMSC-exo can reduce the inflammatory response after ICH with diabetes,thereby improving the neurological function of the brain.

UCHL1 promotes the proliferation of porcine granulosa cells by stabilizing CCNB1

Background The proliferation of porcine ovarian granulosa cells(GCs)is essential to follicular development and the ubiquitin–proteasome system is necessary for maintaining cell cycle homeostasis.Previous studies found that the deubiquitinase ubiquitin carboxyl-terminal hydrolase 1(UCHL1)regulates female reproduction,especially in ovarian development.However,the mechanism by which UCHL1 regulates porcine GC proliferation remains unclear.Results UCHL1 overexpression promoted GC proliferation,and knockdown had the opposite effect.UCHL1 is directly bound to cyclin B1(CCNB1),prolonging the half-life of CCNB1 and inhibiting its degradation,thereby promoting GC proliferation.What's more,a flavonoid compound-isovitexin improved the enzyme activity of UCHL1 and promoted the proliferation of porcine GCs.Conclusions UCHL1 promoted the proliferation of porcine GCs by stabilizing CCNB1,and isovitexin enhanced the enzyme activity of UCHL1.These findings reveal the role of UCHL1 and the potential of isovitexin in regulating proliferation and provide insights into identifying molecular markers and nutrients that affect follicle development.

PbGIF1 promoting cell-proliferation in pear fruit is transcriptionally activated by Pb RR1

As a cell proliferation regulator involved in wide biological processes in plants,GRF-INTERACTING FACTOR(GIF)controls different tissues development.However,whether GIF participates in fruit development remains unclear.According to transcriptome data,we identified PbGIF1was highly expressed during fruit development in cytokinins induced parthenocarpy pear.In the present study,the biofunction of PbGIF1 was initially verified.Overexpression of PbGIF1 promoted fruit size of transgenic tomato.The size of flesh fruit was not affected by cell expansion but the cell proliferation was promoted by overexpressing Pb GIF1.The accelerated cell proliferation process was also observed in PbGIF1-overexpressed transgenic pear fruit calli.The transcriptional regulation of cytokinins on PbGIF1 was further confirmed by exogenous CPPU treatments in pear fruitlets.To investigate the underlying mechanism,the cytokinins-responded factor,PbRR1,was further focused on.The results of Yeast-one-hybrid assay suggested that PbRR1 can bind to the promoter sequence of PbGIF1.The transcriptional activation of PbRR1 on PbGIF1 was also confirmed by Dual-Luciferase assays.Taken together,the results showed that cytokinins control pear fruit development via the transcriptional activation of PbGIF1 by PbRR1.

Tumor-derived DEFB1 induces immune tolerance by inhibiting maturation of dendritic cell and impairing CD8+T cell function in esophageal squamous cell carcinoma

Objective:CD8+T cells are the key effector cells in the anti-tumor immune response.The mechanism underlying the infiltration of CD8+T cells in esophageal squamous cell carcinoma(ESCC)has not been clearly elucidated.Methods:Fresh ESCC tissues were collected and grouped according to the infiltration density of CD8+T cells.After the transcriptome sequencing on these samples and the combined analyses with The Cancer Genome Atlas(TCGA)ESCC data,a secreted protein DEFB1 was selected to explore its potential role in the infiltration of CD8+T cells.Bioinformatics analyses,histological verification and in vitro experiments were then performed.Results:DEFB1 was highly expressed in ESCC,and the high expression of DEFB1 was an independent risk factor for overall survival.Since the up-regulation or down-regulation of DEFB1 did not affect the proliferation,migration and apoptosis of ESCC cells,we speculated that the oncogenic effect of DEFB1 was achieved by regulating microenvironmental characteristics.Bioinformatics analyses suggested that DEFB1 might play a major role in the inflammatory response and anti-tumor immune response,and correlate to the infiltration of immature dendritic cell(imDC)in ESCC.Histological analyses further confirmed that there were less CD8+T cells infiltrated,less CD83+mature DC(mDC)infiltrated and more CD1a+imDC infiltrated in those ESCC samples with high expression of DEFB1.After the treatment with recombinant DEFB1 protein,the maturation of DC was hindered significantly,followed by the impairment of the killing effects of T cells in both 2D and 3D culture in vitro.Conclusions:Tumor-derived DEFB1 can inhibit the maturation of DC and weaken the function of CD8+T cells,accounting for the immune tolerance in ESCC.The role of DEFB1 in ESCC deserves further exploration.

Neural stem cell-derived exosomes regulate cell proliferation,migration,and cell death of brain microvascular endothelial cells via the miR-9/Hes1 axis under hypoxia

Background:Our previous study found that mouse embryonic neural stem cell(NSC)-derived exosomes(EXOs)regulated NSC differentiation via the miR-9/Hes1 axis.However,the effects of EXOs on brain microvascular endothelial cell(BMEC)dysfunction via the miR-9/Hes1 axis remain unknown.Therefore,the current study aimed to determine the effects of EXOs on BMEC proliferation,migration,and death via the miR-9/Hes1 axis.Methods:Immunofluorescence,quantitative real-time polymerase chain reaction,cell counting kit-8 assay,wound healing assay,calcein-acetoxymethyl/propidium iodide staining,and hematoxylin and eosin staining were used to determine the role and mechanism of EXOs on BMECs.Results:EXOs promoted BMEC proliferation and migration and reduced cell death under hypoxic conditions.The overexpression of miR-9 promoted BMEC prolifera-tion and migration and reduced cell death under hypoxic conditions.Moreover,miR-9 downregulation inhibited BMEC proliferation and migration and also promoted cell death.Hes1 silencing ameliorated the effect of amtagomiR-9 on BMEC proliferation and migration and cell death.Hyperemic structures were observed in the regions of the hippocampus and cortex in hypoxia-induced mice.Meanwhile,EXO treatment improved cerebrovascular alterations.Conclusion:NSC-derived EXOs can promote BMEC proliferation and migra-tion and reduce cell death via the miR-9/Hes1 axis under hypoxic conditions.Therefore,EXO therapeutic strategies could be considered for hypoxia-induced vascular injury.

Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

TuBG1 promotes hepatocellular carcinoma via ATR/P53-apoptosis and cycling pathways

Background:As reported,γ-tubulin(TuBG1)is related to the occurrence and development of various types of malignant tumors.However,its role in hepatocellular cancer(HCC)is not clear.The present study was to investigate the relationship between TuBG1 and clinical parameters and survival in HCC patients.Methods:The correlation between TuBG1 and clinical parameters and survival in HCC patients was ex-plored by bioinformatics analysis.Immunohistochemistry was used for the verification.The molecular function of TuBG1 was measured using colony formation,scratch assay,trans-well assay and flow cytometry.Gene set enrichment analysis(GSEA)was used to pick up the enriched pathways,followed by investigating the target pathways using Western blotting.The tumor-immune system interactions and drug bank database(TISIDB)was used to evaluate TuBG1 and immunity.Based on the TuBG1-related immune genes,a prognostic model was constructed and was further validated internally and externally.Results:The bioinformatic analysis found high expressed TuBG1 in HCC tissue,which was confirmed us-ing immunohistochemistry and Western blotting.After silencing the TuBG1 in HCC cell lines,more G1 arrested cells were found,cell proliferation and invasion were inhibited,and apoptosis was promoted.Furthermore,the silence of TuBG1 increased the expressions of Ataxia-Telangiectasia and Rad-3(ATR),phospho-P38 mitogen-activated protein kinase(P-P38MAPK),phospho-P53(P-P53),B-cell lymphoma-2 associated X protein(Bax),cleaved caspase 3 and P21;decreased the expressions of B-cell lymphoma-2(Bcl-2),cyclin D1,cyclin E2,cyclin-dependent kinase 2(CDK2)and CDK4.The correlation analysis of immunohistochemistry and clinical parameters and survival data revealed that TuBG1 was negatively corre-lated with the overall survival.The constructed immune prognosis model could effectively evaluate the prognosis.Conclusions:The increased expression of TuBG1 in HCC is associated with poor prognosis,which might be involved in the occurrence and development of HCC.

Prohibitin 1 inhibits cell proliferation and induces apoptosis via the p53-mediated mitochondrial pathway in vitro

BACKGROUND Prohibitin 1(PHB1)has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed,and it participates in a variety of essential cellular functions,including apoptosis,cell cycle regulation,prolifera-tion,and survival.Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma(HCC).However,the role of PHB1 in HCC is controversial.AIM To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro.METHODS HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria;then,PHB1 levels in the sera and liver tissues of these participates were determined using ELISA,RT-PCR,and immunohistoche-mistry.Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA(shRNA-PHB1)for 24-72 h.Cell prolif-eration was analysed with an MTT assay.Cell cycle progression and apoptosis were analysed using flow cytometry(FACS).The mRNA and protein expression levels of the cell cycle-related molecules p21,Cyclin A2,Cyclin E1,and CDK2 and the cell apoptosis-related molecules cytochrome C(Cyt C),p53,Bcl-2,Bax,caspase 3,and caspase 9 were measured by real-time PCR and Western blot,respectively.RESULTS Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals,and decreased PHB1 was positively correlated with low differentiation,TNM stage III-IV,and alpha-fetoprotein≥400μg/L.Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner.FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis.The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells.The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41%±1.06%,which was significantly greater than that of apoptotic control cells(3.65%±0.85%,P<0.01)and empty vector-transfected cells(4.21%±0.52%,P<0.01).Similar results were obtained with SMMC-7721 cells.Furthermore,the mRNA and protein expression levels of p53,p21,Bax,caspase 3,and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2,Cy-clin E1,CDK2,and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells.However,when PHB1 was upregulated in human HCC cells,Cyt C expression levels were increased in the cytosol and decreased in the mitochondria,which indicated that Cyt C had been released into the cytosol.Conversely,these effects were reversed when PHB1 was knocked down.CONCLUSION PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.

Prox1 Suppresses Proliferation and Drug Resistance of Retinoblastoma Cells via Targeting Notch1

Objective Retinoblastoma(RB)is a prevalent type of eye cancer in youngsters.Prospero homeobox 1(Prox1)is a homeobox transcriptional repressor and downstream target of the proneural gene that is relevant in lymphatic,hepatocyte,pancreatic,heart,lens,retinal,and cancer cells.The goal of this study was to investigate the role of Prox1 in RB cell proliferation and drug resistance,as well as to explore the underlying Notch1 mechanism.Methods Human RB cell lines(SO-RB50 and Y79)and a primary human retinal microvascular endothelial cell line(ACBRI-181)were used in this study.The expression of Prox1 and Notch1 mRNA and protein in RB cells was detected using quantitative real time-polymerase chain reaction(RT-qPCR)and Western blotting.Cell proliferation was assessed after Prox1 overexpression using the Cell Counting Kit-8 and the MTS assay.Drug-resistant cell lines(SO-RB50/vincristine)were generated and treated with Prox1 to investigate the role of Prox1 in drug resistance.We employed pcDNA-Notch1 to overexpress Notch1 to confirm the role of Notch1 in the protective function of Prox1.Finally,a xenograft model was constructed to assess the effect of Prox1 on RB in vivo.Results Prox1 was significantly downregulated in RB cells.Overexpression of Prox1 effectively decreased RB cell growth while increasing the sensitivity of drug-resistant cells to vincristine.Notch1 was involved in Prox1’s regulatory effects.Notch1 was identified as a target gene of Prox1,which was found to be upregulated in RB cells and repressed by increased Prox1 expression.When pcDNA-Notch1 was transfected,the effect of Prox1 overexpression on RB was removed.Furthermore,by downregulating Notch1,Prox1 overexpression slowed tumor development and increased vincristine sensitivity in vivo.Conclusion These data show that Prox1 decreased RB cell proliferation and drug resistance by targeting Notch1,implying that Prox1 could be a potential therapeutic target for RB.

TAX1BP1 and FIP200 orchestrate non-canonical autophagy of p62 aggregates for mouse neural stem cell maintenance

Autophagy plays a pivotal role in diverse biological processes,including the maintenance and differentiation of neural stem cells(NSCs).Interestingly,while complete deletion of Fip200 severely impairs NSC maintenance and differentiation,inhibiting canonical autophagy via deletion of core genes,such as Atg5,Atg16l1,and Atg7,or blockade of canonical interactions between FIP200 and ATG13(designated as FIP200-4A mutant or FIP200 KI)does not produce comparable detrimental effects.This highlights the likely critical involvement of the non-canonical functions of FIP200,the mechanisms of which have remained elusive.Here,utilizing genetic mouse models,we demonstrated that FIP200 mediates non-canonical autophagic degradation of p62/sequestome1,primarily via TAX1BP1 in NSCs.Conditional deletion of Tax1bp1 in fip200hGFAP conditional knock-in(cKI)mice led to NSC deficiency,resembling the fip200hGFAP conditional knockout(cKO)mouse phenotype.Notably,reintroducing wild-type TAX1BP1 not only restored the maintenance of NSCs derived from tax1bp1-knockout fip200hGFAP cKI mice but also led to a marked reduction in p62 aggregate accumulation.Conversely,a TAX1BP1 mutant incapable of binding to FIP200 or NBR1/p62 failed to achieve this restoration.Furthermore,conditional deletion of Tax1bp1 in fip200hGFAP cKO mice exacerbated NSC deficiency and p62 aggregate accumulation compared to fip200hGFAP cKO mice.Collectively,these findings illustrate the essential role of the FIP200-TAX1BP1 axis in mediating the non-canonical autophagic degradation of p62 aggregates towards NSC maintenance and function,presenting novel therapeutic targets for neurodegenerative diseases.

ZmCER1,a putative ECERIFERUM 1 protein in maize,functions in cuticular wax biosynthesis and bulliform cell development

The cuticular wax,acting as the ultimate defense barrier,is essential for the normal morphogenesis of plant organs.Despite this importance,the connection between wax composition and leaf development has not been thoroughly explored.In this study,we characterized a new maize mutant,ragged leaf4(rgd4),which exhibits crinkled and ragged leaves starting from the sixth leaf stage.The phenotype of rgd4 is conferred by ZmCER1,which encoding an aldehyde decarbonylase involved in wax biosynthesis.ZmCER1 function deficient mutant displayed reduced cuticular wax density and disordered bulliform cells(BCs),while ZmCER1 overexpressing plants exhibited the opposite effects,indicating that ZmCER1 regulates cuticular wax biosynthesis and BCs development.Additionally,as the density of cuticular wax increased,the water loss rate of detached leaf decreases,suggesting that ZmCER1 is positively correlated with plant drought tolerance.

丝胶靶向Akt1调控PI3K/Akt信号通路促进STZ致损伤INS-1细胞增殖

目的探讨丝胶是否通过靶向Akt1调控磷酸肌醇-3-激酶(PI3K)/蛋白激酶B(protein kinase B,Akt)信号通路促进链脲佐菌素(STZ)致损伤大鼠胰岛素瘤细胞(INS-1细胞)增殖。方法INS-1细胞随机分为4组:对照组(不予任何处理)、模型组(给予10 mmol/L STZ处理细胞)、丝胶组(给予10 mmol/L STZ+600μg/mL丝胶处理细胞)、抑制剂组(给予10 mmol/LSTZ+600μg/mL丝胶+0.3 mmol/L的Akt1抑制剂A-674563处理细胞)。药物作用时间均为24 h。CCK-8法检测各组INS-1细胞存活率。免疫蛋白印迹法(western blot)检测PI3K/Akt信号通路相关蛋白PI3K、p-Akt和增殖相关蛋白增殖细胞核抗原(PCNA)、细胞核增殖相关抗原Ki67(Ki67)的表达情况。结果与对照组相比,模型组INS-1细胞存活率下降(P<0.05),PI3K、p-Akt1、PCNA和Ki67的蛋白表达显著下降(P<0.05);与模型组相比,丝胶组INS-1细胞存活率上升(P<0.05),PI3K、p-Akt1、PCNA和Ki67的蛋白表达显著增多(P<0.05);与丝胶组相比,抑制剂组INS-1细胞存活率下降(P<0.05),p-Akt1、PCNA和Ki67的蛋白表达显著下降(P<0.05)。结论丝胶具有促进STZ致损伤INS-1细胞增殖的作用,其作用机制与丝胶通过靶向Akt1调控PI3K/Akt信号通路有关。

Ginsenoside Rb1 induces hepatic stellate cell ferroptosis to alleviate liver fibrosis via the BECN1/SLC7A11 axis

Liver fibrosis is primarily driven by the activation of hepatic stellate cells(HSCs),a process associated with ferroptosis.Ginsenoside Rb1(GRb1),a major active component extracted from Panax ginseng,inhibits HSC activation.However,the potential role of GRb1 in mediating HSC ferroptosis remains unclear.This study examined the effect of GRb1 on liver fibrosis both in vivo and in vitro,using CCl4-induced liver fibrosis mouse model and primary HSCs,LX-2 cells.The findings revealed that GRb1 effectively inactivated HSCs in vitro,reducing alpha-smooth muscle actin(a-SMA)and type I collagen(Col1A1)levels.Moreover,GRb1 significantly alleviated CCl4-induced liver fibrosis in vivo.From a mechanistic standpoint,the ferroptosis pathway appeared to be central to the antifibrotic effects of GRb1.Specifically,GRb1 promoted HSC ferroptosis both in vivo and in vitro,characterized by increased glutathione depletion,malondialdehyde production,iron overload,and accumulation of reactive oxygen species(ROS).Intriguingly,GRb1 increased Beclin 1(BECN1)levels and decreased the System Xc-key subunit SLC7A11.Further experiments showed that BECN1 silencing inhibited GRb1-induced effects on HSC ferroptosis and mitigated the reduction of SLC7A11 caused by GRb1.Moreover,BECN1 could directly interact with SLC7A11,initiating HSC ferroptosis.In conclusion,the suppression of BECN1 counteracted the effects of GRb1 on HSC inactivation both in vivo and in vitro.Overall,this study highlights the novel role of GRb1 in inducing HSC ferroptosis and promoting HSC inactivation,at least partly through its modulation of BECN1 and SLC7A11.

丝胶通过PI3K/Akt信号通路对受损INS-1细胞糖酵解的调控作用

目的观察丝胶对链脲佐菌素(STZ)致损伤大鼠胰岛素瘤细胞(INS-1细胞)PI3K/Akt信号通路和糖酵解的调控作用。方法实验以体外培养的INS-1细胞为研究对象随机分为五组,正常对照组、模型组、丝胶组、Akt1抑制剂组、Akt1激动剂组。运用蛋白印迹和实时荧光定量PCR法检测各分组细胞的磷脂酰肌醇-3-激酶(PI3K),蛋白激酶B(Akt),磷酸果糖激酶-1(PFK1),6-磷酸果糖-2,6-二磷酸酶(PFKFB2)的蛋白及mRNA的表达情况。结果与正常对照组相比,模型组PI3K,p-Akt,PFK1,PFKFB2的蛋白表达显著下降(P<0.05);丝胶组与模型组相比PI3K,p-Akt,PFK1,PFKFB2的蛋白表达显著增高(P<0.05);Akt1抑制剂组与丝胶组相比,p-Akt,PFK1,PFKFB2的蛋白表达显著降低(P<0.05);Akt1激动剂组与丝胶组相比,p-Akt,PFK1,PFKFB2的蛋白呈现上升的趋势。各组INS-1细胞中PI3K,Akt,PFK1,PFKFB2 mRNA的变化趋势与蛋白一致。结论丝胶对STZ致损伤INS-1细胞发挥保护作用的机制可能是,靶向Akt1影响PI3K/Akt信号通路增强糖酵解。

CircPMS1 promotes proliferation of pulmonary artery smooth muscle cells,pulmonary microvascular endothelial cells,and pericytes under hypoxia

Background:Circular RNAs(circRNAs)have been recognized as significant regulators of pulmonary hypertension(PH);however,the differential expression and function of circRNAs in different vascular cells under hypoxia remain unknown.Here,we identified co-differentially expressed circRNAs and determined their putative roles in the proliferation of pulmonary artery smooth muscle cells(PASMCs),pulmonary microvascular endothelial cells(PMECs),and pericytes(PCs)under hypoxia.Methods:Whole transcriptome sequencing was performed to analyze the differential expression of circRNAs in three different vascular cell types.Bioinformatic analysis was used to predict their putative biological function.Quantitative real-time polymerase chain reaction,Cell Counting Kit-8,and EdU Cell Proliferation assays were carried out to determine the role of circular postmeiotic segregation 1(circPMS1)as well as its potential sponge mechanism in PASMCs,PMECs,and PCs.Results:PASMCs,PMECs,and PCs exhibited 16,99,and 31 differentially expressed circRNAs under hypoxia,respectively.CircPMS1 was upregulated in PASMCs,PMECs,and PCs under hypoxia and enhanced the proliferation of vascular cells.CircPMS1may upregulate DEP domain containing 1(DEPDC1)and RNA polymerase II subunit D expression by targeting microRNA-432-5p(miR-432-5p)in PASMCs,upregulate MAX interactor 1(MXI1)expression by targeting miR-433-3p in PMECs,and upregulate zinc finger AN1-type containing 5(ZFAND5)expression by targeting miR-3613-5p in PCs.Conclusions:Our results suggest that circPMS1 promotes cell proliferation through the miR-432-5p/DEPDC1 or miR-432-5p/POL2D axis in PASMCs,through the miR-433-3p/MXI1 axis in PMECs,and through the miR-3613-5p/ZFAND5 axis in PCs,which provides putative targets for the early diagnosis and treatment of PH.