IFN-β通过STAT1诱导SARI表达抑制AML细胞增殖并促进凋亡

目的:探讨IFN-β诱导SARI表达对急性粒细胞性白血病(AML)细胞增殖、凋亡的作用,并筛选其潜在的调控分子。方法:qPCR、Western blot筛选SARI低表达的AML细胞作为实验细胞株;不同浓度IFN-β干预AML细胞,于不同时间采用qPCR、Western blot检测SARI表达,选取IFN-β作用的适当浓度和时间;采用RNA-Seq转录组测序及KEGG富集分析初步筛选IFN-β诱导AML细胞SARI表达的潜在调控分子;通过相应分子抑制剂联合IFN-β处理AML细胞,MTS法检测细胞增殖,流式细胞术检测细胞凋亡;明确该分子参与IFN-β诱导SARI表达对AML细胞增殖及凋亡的作用。结果:HL60和NB4细胞SARI表达相对较低,选为实验细胞株;1 ng/ml IFN-β作用12 h后AML细胞SARI表达升高且细胞增殖被抑制,凋亡增多;筛选STAT1为IFN-β诱导SARI表达的潜在调控分子;抑制STAT1后,IFN-β对AML细胞SARI表达、增殖抑制、凋亡促进的作用被明显逆转。结论:IFN-β可通过STAT1诱导AML细胞SARI表达,抑制细胞增殖,促进细胞凋亡。

血清RAGE、HMGB1水平与重症肺炎急性呼吸窘迫综合征发病及IFN-γ/IL-4变化的关系

目的探究血清晚期糖基化终产物受体(RAGE)、高迁移率族蛋白B1(high mobility group protein B1,HMGB1)水平与重症肺炎(SP)急性呼吸窘迫综合征(ARDS)发病及γ-干扰素(IFN-γ)/白细胞介素4(IL-4)变化的关系。方法前瞻性选取2020年3月至2022年2月我院收治的100例SP患儿为研究对象,根据患儿是否发生继发性ARDS将患儿分为ARDS组(n=56)和对照组(n=44),收集患儿一般资料,采集外周血以酶联免疫吸附法进行RAGE、HMGB1、IFN-γ和IL-4表达水平检测,采用多因素logistic回归分析SP患儿继发ARDS的影响因素,采用Pearson相关性分析其与IFN-γ/IL-4的相关性,并采用受试者工作曲线(ROC)分析RAGE、HMGB1表达对SP患儿继发ARDS的预测价值。结果两组SP患儿性别、年龄、体温以及发病季节之间无显著差异,ARDS组致病菌种类多于对照组,PaO_(2)/FiO_(2)和APS评分、血清RAGE、HMGB1、IFN-γ和IL-4表达水平以及IFN-γ/IL-4比值均高于对照组(P<0.05)。经多因素logistic回归分析可知,致病菌种类、PaO_(2)/FiO_(2)、RAGE、HMGB1表达、IFN-γ、IL-4和IFN-γ/IL-4均为SP患儿继发ARDS的影响因素。经Pearson相关检验,SP患儿血清RAGE、HMGB1表达水平与IFN-γ、IL-4和IFN-γ/IL-4均呈正相关(P<0.05)。经ROC曲线分析可得,血清RAGE、HMGB1水平预测SP患儿发生ARDS的AUC分别为0.707和0.750,灵敏度分别为73.2%、64.3%,特异度分别为68.2%、77.3%,两者联合预测的AUC为0.848,灵敏度和特异度分别为80.4%和81.8%。结论SP继发ARDS患儿血清中RAGE、HMGB1表达水平较高,与IFN-γ/IL-4呈正相关,监测患儿血清RAGE、HMGB1表达对SP患儿继发ARDS的风险有一定的预测价值。

补肾健脾方联合IFN-γ对Hepg2.2.15细胞IFN-γ/STAT1信号通路的影响

目的 研究补肾健脾方含药血清对γ干扰素(Interferon-gamma, IFN-γ)抗乙型肝炎病毒(Hepatitis b virus, HBV)的增效作用及可能机制。方法 常规制备大鼠含药血清,WST-1法筛选含药血清干预Hepg2.2.15细胞的安全剂量;以不同浓度IFN-γ加补肾健脾方含药血清加入Hepg2.2.15细胞系,检测培养上清中乙型肝炎表面抗原(Hepatitis B surface antigen, HBsAg)、乙型肝炎核心相关抗原(Hepatitis B e antigen, HBeAg)以及乙型肝炎病毒脱氧核糖核酸(Hepatitis B Virus DNA,HBV DNA)水平,筛选补肾健脾方联合IFN-γ给药剂量;筛选出IFN-γ干预剂量后,将Hepg2.2.15细胞分为对照组、IFN-γ单独处理组、IFN-γ联合空白血清组以及IFN-γ联合补肾健脾方含药血清组,干预24h后,收集上清和蛋白样本,检测上清中HBsAg、HBeAg以及HBV DNA水平,蛋白免疫印迹(Western blot, WB)技术检测Hepg2.2.15细胞中γ干扰素(Interferon-gamma, IFN-γ)/信号传导转录激活因子1(Signal transducer and activator of transcription 1,STAT1)信号通路关键蛋白蛋白,包括精氨酸甲基化转移酶1(Protein arginine methyltransferase 1,PRMT1)、STAT1、磷酸化信号传导转录激活因子1(p-signal transducer and activator of transcription 1,P-STAT1)以及抗病毒蛋白2',5'-寡腺苷酸合成酶3(2'-5'-oligoadenylate synthetase 3,OAS3)、双链RNA依赖的蛋白质激酶(Double-stranded RNA-dependent protein kinase, PKR)、黏病毒耐药蛋白1(Recombinant Myxovirus Resistance 1,Mx1)蛋白表达水平。结果 与IFN-γ单独处理组以及IFN-γ联合5%空白血清组相比,IFN-γ联合5%补肾健脾方含药血清干预Hepg2.2.15细胞24h后,细胞培养上清中HBV DNA、HBeAg水平显著降低(P<0.05),HBsAg未见明显变化(P>0.05);IFN-γ联合5%补肾健脾方含药血清组γ干扰素受体2(Interferon gamma receptor 2,IFNGR2)蛋白表达水平较IFN-γ组显著上调(P<0.05);IFN-γ联合5%补肾健脾方含药血清组γ干扰素受体1(Interferon gamma receptor 1,IFNGR1)、STAT1、P-STAT1、PRMT1、OAS3、Mx1蛋白表达水平较IFN-γ组和IFN-γ联合5%空白血清组显著升高(P<0.05)。结论 补肾健脾方含药血清对IFN-γ抗HBV产生增效作用,其作用机制可能与上调IFN-γ受体(IFNGR1),激活IFN-γ/STAT1信号通路,进而上调IFN-γ下游抗病毒蛋白OAS3、Mx1表达有关。

血红素加氧酶-1通过诱导抗病毒蛋白的表达增强IFN-α抗HBV效应

目的探讨血红素加氧酶-1(HO-1)对HBV复制的作用及HO-1联合α-干扰素(IFN-α)的抗病毒效应。方法以HepG2.2.15细胞和HBV 1.3质粒转染HepG2细胞即HepG2-HBV1.3为HBV复制细胞模型;血红素(Hemin)分别处理HepG2.2.15和HepG2-HBV1.3细胞,诱导HO-1表达;CCK-8评估Hemin对HepG2、HepG2.2.15的毒性作用;化学发光法分析Hemin处理组及si-HO-1等实验组上清液中HBsAg、HBeAg;RT-qPCR分析HO-1、IFN-β、HBV-DNA;Western blot分析IRF-3、JAK/STAT信号通路中相关分子的表达;Hemin联合IFN-α处理HepG2.2.15,监测HO-1是否具有协同IFN-α抗病毒效应。结果Hemin剂量依赖性诱导HO-1,HO-1被诱导后发挥显著的抗HBV效应,同时IFN-β、IRF-3及JAK/STAT信号通路中IRF-9、MxA的表达均增加。沉默HO-1表达能逆转Hemin诱导组的抗病毒效应,同时I型干扰素IFN-β也呈现低表达,JAK/STAT信号通路中的IRF-9、MxA的表达也被抑制。Hemin联合IFN-α发挥更强的抗病毒作用。结论HO-1能够发挥抗HBV效应,这种效应可能是增加IRF-3的磷酸化诱导I型干扰素表达来激活JAK/STAT信号通路发挥抗病毒效应;HO-1可以协同IFN-α发挥抗病毒作用。

二陈汤合三子养亲汤治疗慢性阻塞性肺疾病(痰湿蕴肺证)疗效及对肺功能、IFN-γ、ET-1的影响

目的探究二陈汤合三子养亲汤治疗慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)(痰湿蕴肺证)的疗效及对肺功能、伽马干扰素(interferon γ,IFN-γ)、内皮素(endothelin-1,ET-1)的影响。方法采用随机分组法将医院2019年2月—2022年3月收治的86例慢阻肺患者分为观察组与对照组,两组各43例。对照组采用西医常规治疗,观察组在此基础上联合使用二陈汤合三子养亲汤治疗。观察两组患者治疗前后肺功能、蛋白质羰基(protein carbonyl content,PC)、8-羟基脱氧鸟苷(8-hydroxy-2 deoxyguanosine,8-OHdG)、丙二醛(malonic aldehyde,MDA)含量和IFN-γ、ET-1。观察两组患者临床症状消失时间和临床疗效。结果两组患者治疗后FEV1、PEF和Ppeak水平显著高于治疗前(P<0.05)。治疗后,观察组FEV1、PEF和Ppeak水平显著高于对照组(P<0.05)。两组患者治疗后PC、8-OHdG和MDA水平显著低于治疗前(P<0.05)。治疗后,观察组PC、8-OHdG和MDA水平显著低于对照组(P<0.05)。两组患者治疗后IFN-γ和ET-1水平显著低于治疗前(P<0.05)。治疗后,观察组IFN-γ和ET-1水平显著低于对照组(P<0.05)。观察组临床症状消失时间显著快于对照组(P<0.05)。观察组总有效率为83.72%(36/43)显著高于对照组(65.12%,28/43)(χ^(2)=3.909,P=0.048)。结论二陈汤合三子养亲汤能有效改善慢阻肺患者肺功能,降低氧化应激相关产物蛋白质的含量,减少临床症状持续时间,利于降低FN-γ、ET-1水平,临床疗效得到显著提升。

Humanβ-defensin-1 affects the mammalian target of rapamycin pathway and autophagy in colon cancer cells through long noncoding RNA TCONS_00014506

BACKGROUND Colorectal cancer has a low 5-year survival rate and high mortality.Humanβ-defensin-1(hBD-1)may play an integral function in the innate immune system,contributing to the recognition and destruction of cancer cells.Long non-coding RNAs(lncRNAs)are involved in the process of cell differentiation and growth.AIM To investigate the effect of hBD-1 on the mammalian target of rapamycin(mTOR)pathway and autophagy in human colon cancer SW620 cells.METHODS CCK8 assay was utilized for the detection of cell proliferation and determination of the optimal drug concentration.Colony formation assay was employed to assess the effect of hBD-1 on SW620 cell proliferation.Bioinformatics was used to screen potentially biologically significant lncRNAs related to the mTOR pathway.Additionally,p-mTOR(Ser2448),Beclin1,and LC3II/I expression levels in SW620 cells were assessed through Western blot analysis.RESULTS hBD-1 inhibited the proliferative ability of SW620 cells,as evidenced by the reduction in the colony formation capacity of SW620 cells upon exposure to hBD-1.hBD-1 decreased the expression of p-mTOR(Ser2448)protein and increased the expression of Beclin1 and LC3II/I protein.Furthermore,bioinformatics analysis identified seven lncRNAs(2 upregulated and 5 downregulated)related to the mTOR pathway.The lncRNA TCONS_00014506 was ultimately selected.Following the inhibition of the lncRNA TCONS_00014506,exposure to hBD-1 inhibited p-mTOR(Ser2448)and promoted Beclin1 and LC3II/I protein expression.CONCLUSION hBD-1 inhibits the mTOR pathway and promotes autophagy by upregulating the expression of the lncRNA TCONS_00014506 in SW620 cells.

Development of RPA-Cas12a-fluorescence assay for rapid and reliable detection of human bocavirus 1

Human bocavirus(HBoV)1 is considered an important pathogen that mainly affects infants aged 6–24 months,but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging.Herein,we present a novel faster,lower cost,reliable method for the detection of HBoV1,which integrates a recombinase polymerase amplification(RPA)assay with the CRISPR/Cas12a system,designated the RPA-Cas12a-fluorescence assay.The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37℃without the need for sophisticated instruments.The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens.Furthermore,the method was appraised using 28 clinical samples,and displayed high accuracy with positive and negative predictive agreement of 90.9%and 100%,respectively.Therefore,our proposed rapid and sensitive HBoV1 detection method,the RPA-Cas12a-fluorescence assay,shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care.The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection.The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/μl.

Establishment of a humanized ST6GAL1 mouse model for influenza research

Background:This study aimed to construct and characterize a humanized influenza mouse model expressing hST6GAL1.Methods:Humanized fragments,consisting of the endothelial cell-specific K18 promoter,human ST6GAL1-encoding gene,and luciferase gene,were microinjected into the fertilized eggs of mice.The manipulated embryos were transferred into the oviducts of pseudopregnant female mice.The offspring were identified using PCR.Mice exhibiting elevated expression of the hST6GAL1 gene were selectively bred for propagation,and in vivo analysis was performed for screening.Expression of the humanized gene was tested by performing immunohistochemical(IHC)analysis.Hematologic and biochemical analyses using the whole blood and serum of humanized hST6GAL1 mice were performed.Results:Successful integration of the human ST6GAL1 gene into the mouse genome led to the overexpression of human SiaT ST6GAL1.Seven mice were identified as carrying copies of the humanized gene,and the in vivo analysis indicated that hST6GAL1gene expression in positive mice mirrored influenza virus infection characteristics.The IHC results revealed that hST6GAL1 was expressed in the lungs of humanized mice.Moreover,the hematologic and biochemical parameters of the positive mice were within the normal range.Conclusion:A humanized influenza mouse model expressing the hST6GAL1 gene was successfully established and characterized.

MicroRNA-298 determines the radio-resistance of colorectal cancer cells by directly targeting human dual-specificity tyrosine(Y)-regulated kinase 1A

BACKGROUND Radiotherapy stands as a promising therapeutic modality for colorectal cancer(CRC);yet,the formidable challenge posed by radio-resistance significantly undermines its efficacy in achieving CRC remission.AIM To elucidate the role played by microRNA-298(miR-298)in CRC radio-resistance.METHODS To establish a radio-resistant CRC cell line,HT-29 cells underwent exposure to 5 gray ionizing radiation that was followed by a 7-d recovery period.The quantification of miR-298 levels within CRC cells was conducted through quantitative RT-PCR,and protein expression determination was realized through Western blotting.Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and proliferation by clonogenic assay.Radio-induced apoptosis was discerned through flow cytometry analysis.RESULTS We observed a marked upregulation of miR-298 in radio-resistant CRC cells.MiR-298 emerged as a key determinant of cell survival following radiation exposure,as its overexpression led to a notable reduction in radiation-induced apoptosis.Intriguingly,miR-298 expression exhibited a strong correlation with CRC cell viability.Further investigation unveiled human dual-specificity tyrosine(Y)-regulated kinase 1A(DYRK1A)as miR-298’s direct target.CONCLUSION Taken together,our findings underline the role played by miR-298 in bolstering radio-resistance in CRC cells by means of DYRK1A downregulation,thereby positioning miR-298 as a promising candidate for mitigating radioresistance in CRC.

Subclinical hepatitis E virus genotype 1 infection:The concept of“dynamic human reservoir”

Hepatitis E virus(HEV)is hyperendemic in South Asia and Africa accounting for half of total Global HEV burden.There are eight genotypes of HEV.Among them,the four common ones known to infect humans,genotypes 1 and 2 are prevalent in the developing world and genotypes 3 and 4 are causing challenge in the industrialized world.Asymptomatic HEV viremia in the general population,especially among blood donors,has been reported in the literature worldwide.The clinical implications related to this asymptomatic viremia are unclear and need further exploration.Detection of viremia due to HEV genotype 1 infection,apparently among healthy blood donors is also reported without much knowledge about its infection rate.Similarly,while HEV genotype 3 is known to be transmitted via blood transfusion in humans and has been subjected to screening in many European nations,instances of transmission have also been documented albeit without significant clinical consequences.Epidemiology of HEV genotype 1 in endemic areas often show waxing and waning pattern.Occasional sporadic occurrence of HEV infection interrupted by outbreaks have been frequently seen.In absence of known animal reservoir,where HEV exists in between outbreak is a mystery that needs further exploration.However,occurrence of asymptomatic HEV viremia due to HEV genotype 1 during epidemiologically quiescent period may explain that this phenomenon may act as a dynamic reservoir.Since HEV genotype 1 infection cannot cause chronicity,subclinical transient infection and transmission of virus might be the reason it sustains in interepidemic period.This might be the similar phenomenon with SARS COVID-19 corona virus infection which is circulating worldwide in distinct phases with peaks and plateaus despite vaccination against it.In view of existing evidence,we propose the concept of“Dynamic Human Reservoir.”Quiescent subclinical infection of HEV without any clinical consequences and subsequent transmission may contribute to the existence of the virus in a community.The potential for transmitting HEV infection by asymptomatic HEV infected individuals by fecal shedding of virus has not been reported in literature.This missing link may be a key to Pandora's box in understanding epidemiology of HEV infection in genotype 1 predominant region.

Regulation role of miR-204 on SIRT1/VEGF in metabolic memory induced by high glucose in human retinal pigment epithelial cells

AIM:To examine the regulatory role of microRNA-204(miR-204)on silent information regulator 1(SIRT1)and vascular endothelial growth factor(VEGF)under highglucose-induced metabolic memory in human retinal pigment epithelial(hRPE)cells.METHODS:Cells were cultured with either normal(5 mmol/L)or high D-glucose(25 mmol/L)concentrations for 8d to establish control and high-glucose groups,respectively.To induce metabolic memory,cells were cultured with 25 mmol/L D-glucose for 4d followed by culture with 5 mmol/L D-glucose for 4d.In addition,exposed in 25 mmol/L D-glucose for 4d and then transfected with 100 nmol/L miR-204 control,miR-204 inhibitor or miR-204 mimic in 5 mmol/L D-glucose for 4d.Quantitative reverse transcription-polymerase chain reaction(RT-qPCR)was used to detect miR-204 mRNA levels.SIRT1 and VEGF protein levels were assessed by immunohistochemical and Western blot.Flow cytometry was used to investigate apoptosis rate.RESULTS:It was found that high glucose promoted miR-204 and VEGF expression,and inhibited SIRT1 activity,even after the return to normal glucose culture conditions.Upregulation of miR-204 promoted apoptosis inhibiting SIRT1 and increasing VEGF expression.However,downregulation of miR-204 produced the opposite effects.CONCLUSION:The study identifies that miR-204 is the upstream target of SIRT1and VEGF,and that miR-204 can protect hRPE cells from the damage caused by metabolic memory through increasing SIRT1 and inhibiting VEGF expression.

SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo

AIM:To explore the effect of silent information regulator factor 2-related enzyme 1(SIRT1)on modulating apoptosis of human lens epithelial cells(HLECs)and alleviating lens opacification of rats through suppressing endoplasmic reticulum(ER)stress.METHODS:HLECs(SRA01/04)were treated with varying concentrations of tunicamycin(TM)for 24h,and the expression of SIRT1 and C/EBP homologous protein(CHOP)was assessed using real-time quantitative polymerase chain reaction(RT-PCR),Western blotting,and immunofluorescence.Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8(CCK-8)assay,respectively.In the SRA01/04 cell apoptosis model,which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation,the expression levels of SIRT1,CHOP,glucose regulated protein 78(GRP78),and activating transcription factor 4(ATF4)were examined.The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid(4-PBA;an ER stress inhibitor)was investigated.In vivo,age-related cataract(ARC)rat models were induced by sodium selenite injection,and the protective role of SIRT1,activated by SRT1720 intraperitoneal injections,was evaluated through morphology observation,hematoxylin and eosin(H&E)staining,Western blotting,and RT-PCR.RESULTS:SIRT1 expression was downregulated in TMinduced SRA01/04 cells.Besides,in SRA01/04 cells,both cell apoptosis and CHOP expression increased with the rising doses of TM.ER stress was stimulated by TM,as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model.Inhibition of SIRT1 by siRNA knockdown increased ER stress activation,whereas SRT1720 treatment had opposite results.4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis.In vivo,SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models.CONCLUSION:SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress.These findings suggest a novel strategy for cataract treatment focused on targeting ER stress,highlighting the therapeutic potential of SIRT1 modulation in ARC development.

IFN-γ/sPD-1联合过表达的小鼠骨髓间充质干细胞具有较高生物活性和遗传稳定性

目的研究慢病毒介导的γ干扰素(IFN-γ)及可溶性程序性细胞死亡蛋白1(sPD-1)过表达对骨髓间充质干细胞(BMSC)生物活性及遗传稳定性的影响。方法全骨髓贴壁法分离培养BMSC;流式细胞术鉴定BMSC表面标志物的表达;扩增分别带有绿色荧光蛋白(GFP)和红色荧光蛋白(RFP)的IFN-γ及sPD-1基因序列,通过慢病毒转染分别构建IFN-γ、sPD-1和IFN-γ/sPD-1双基因过表达的BMSC,采用倒置显微镜观察细胞形态,实时荧光定量PCR和Western blot法分别检测IFN-γ和sPD-1的mRNA和蛋白表达;CCK-8法、BMSC端粒酶活性检测法分析IFN-γ和sPD-1过表达BMSC的生物活性和遗传稳定性。结果成功分离培养了CD44、CD105阳性,CD34、CD11b、CD45阴性的BMSC。与空载体组相比,IFN-γ/sPD-1联合组的IFN-γ、sPD-1 mRNA表达水平显著提高,但各组细胞生长情况类似。与空载体组相比,IFN-γ组端粒酶活性显著降低,但sPD-1组和IFN-γ/sPD-1联合组端粒酶活性变化不明显。结论IFN-γ和sPD-1联合过表达的BMSC具有较高生物活性和遗传稳定性。

Effect of acetyl L-carnitine on human retinal pigment epithelium-19 cells in hypoxic conditions

AIM:To investigate the effect of acetyl-L-carnitine(ALCAR)on cell viability,morphological integrity,and vascular endothelial growth factor(VEGF)expression in human retinal pigment epithelium(ARPE-19)cells using a hypoxic model.METHODS:In the first set of experiments,the optimal CoCl_(2) dose was determined by exposing ARPE-19 cell cultures to different concentrations.To evaluate the effect of ALCAR on cell viability,five groups of ARPE-19 cell culture were established that included a control group,a sham group(200μM CoCl_(2)),and groups that received 1,10 and 100 mM doses of ALCAR combined with 200μM CoCl_(2),respectively.The cell viability was measured by MTT assay.The morphological characteristics of cells were observed by an inverted phase contrast microscope.The levels of VEGF and HIF-1α secretion by ARPE-19 cells were detected by enzyme linked immunosorbent assay(ELISA)assay.RESULTS:ARPE-19 cells were exposed to different doses of CoCl_(2) in order to create a hypoxia model.Nevertheless,when exposed to a concentration of 200μM CoCl_(2),a notable decrease in viability to 83% was noted.ALCAR was found to increase the cell viability at 1 mM and 10 mM concentrations,while the highest concentration(100 mM)did not have an added effect.The cell viability was found to be significantly higher in the groups treated with a concentration of 1 mM and 10 mM ALCAR compared to the Sham group(P=0.041,P=0.019,respectively).The cell viability and morphology remained unaffected by the greatest dose of ALCAR(100 mM).The administration of 10 mM ALCAR demonstrated a statistically significant reduction in the levels of VEGF and HIF-1α compared with the Sham group(P=0.013,P=0.033,respectively).CONCLUSION:The findings from the current study indicate that ALCAR could represent a viable therapeutic option with the potential to open up novel treatment pathways for retinal diseases,particular relevance for age-related macular degeneration(AMD).However,to fully elucidate ALCAR’s application potential in retinal diseases,additional investigation is necessary to clearly define the exact mechanisms involved.

MicroRNA-451 from Human Umbilical Cord-Derived Mesenchymal Stem Cell Exosomes Inhibits Alveolar Macrophage Autophagy via Tuberous Sclerosis Complex 1/Mammalian Target of Rapamycin Pathway to Attenuate Burn-Induced Acute Lung Injury in Rats

Objective Our previous studies established that microRNA(miR)-451 from human umbilical cord mesenchymal stem cell-derived exosomes(hUC-MSC-Exos)alleviates acute lung injury(ALI).This study aims to elucidate the mechanisms by which miR-451 in hUC-MSC-Exos reduces ALI by modulating macrophage autophagy.Methods Exosomes were isolated from hUC-MSCs.Severe burn-induced ALI rat models were treated with hUC-MSC-Exos carrying the miR-451 inhibitor.Hematoxylin-eosin staining evaluated inflammatory injury.Enzyme-linked immunosorbnent assay measured lipopolysaccharide(LPS),tumor necrosis factor-α,and interleukin-1βlevels.qRT-PCR detected miR-451 and tuberous sclerosis complex 1(TSC1)expressions.The regulatory role of miR-451 on TSC1 was determined using a dual-luciferase reporter system.Western blotting determined TSC1 and proteins related to the mammalian target of rapamycin(mTOR)pathway and autophagy.Immunofluorescence analysis was conducted to examine exosomes phagocytosis in alveolar macrophages and autophagy level.Results hUC-MSC-Exos with miR-451 inhibitor reduced burn-induced ALI and promoted macrophage autophagy.MiR-451 could be transferred from hUC-MSCs to alveolar macrophages via exosomes and directly targeted TSC1.Inhibiting miR-451 in hUC-MSC-Exos elevated TSC1 expression and inactivated the mTOR pathway in alveolar macrophages.Silencing TSC1 activated mTOR signaling and inhibited autophagy,while TSC1 knockdown reversed the autophagy from the miR-451 inhibitor-induced.Conclusion miR-451 from hUC-MSC exosomes improves ALI by suppressing alveolar macrophage autophagy through modulation of the TSC1/mTOR pathway,providing a potential therapeutic strategy for ALI.

过表达神经调节蛋白1的人羊膜间充质干细胞促进小鼠皮肤创面愈合

背景:神经调节蛋白1具有促进细胞增殖、分化以及血管生长等特性。人羊膜间充质干细胞是组织工程领域重要的种子细胞,已被证实参与组织修复及再生过程。目的:构建过表达神经调节蛋白1的人羊膜间充质干细胞,探究其增殖、迁移能力以及对创面愈合的影响。方法:(1)体外分离培养人羊膜间充质干细胞并对其进行鉴定;(2)构建神经调节蛋白1过表达慢病毒,将人羊膜间充质干细胞分为空载组、神经调节蛋白1组、对照组,分别转染空载慢病毒、过表达神经调节蛋白1慢病毒,对照组不进行转染;(3)EdU实验检测各组细胞增殖能力,Transwell实验检测各组细胞迁移能力;(4)构建C57BL/6小鼠创面损伤模型,随机分成对照组、空载组和神经调节蛋白1组,每组8只,分别在创面局部多点均匀注射1 mL转染空载慢病毒或转染过表达神经调节蛋白1慢病毒的人羊膜间充质干细胞,对照组注射等量的生理盐水;(5)造模后1,7,14 d观察创面愈合情况,苏木精-伊红染色观察创面愈合组织学变化,免疫组化观察创面CD31的表达。结果与结论:(1)成功构建过表达神经调节蛋白1的人羊膜间充质干细胞,细胞内神经调节蛋白1的mRNA、蛋白表达较空载组明显上调(P<0.05);(2)过表达神经调节蛋白1促进了人羊膜间充质干细胞的迁移(P<0.01)和增殖(P<0.05);(3)过表达神经调节蛋白1的人羊膜间充质干细胞促进了小鼠创面愈合(P<0.05)和创面的血管生成(P<0.05)。结果表明,过表达神经调节蛋白1提高了人羊膜间充质干细胞的增殖和迁移能力,以及增强了促进创面愈合和创面血管生成的能力。

血清HE4、IFN-α、CCL19、OAS1与系统性红斑狼疮患者疾病活动度的相关性分析

目的分析血清人附睾蛋白4(HE4)、干扰素-α(IFN-α)、趋化因子配体19(CCL19)、寡聚腺苷酸合成酶1(OAS1)与系统性红斑狼疮(SLE)患者疾病活动度的相关性。方法选取2022年5月至2023年5月该院收治的95例SLE患者为SLE组,根据SLE疾病活动度评分(SLEDAI)将患者分成轻度组(SLEDAI=0~<10分,40例)、中度组(SLEDAI=10~<15分,30例)和重度组(SLEDAI≥15分,25例),另选取同期100例体检健康者作为健康对照组。采用酶联免疫吸附试验检测血清HE4、IFN-α、CCL19、OAS1水平,并比较SLE组和健康对照组HE4、IFN-α、CCL19、OAS1水平,以及不同疾病活动度患者HE4、IFN-α、CCL19、OAS1水平;采用Spearman相关分析HE4、IFN-α、CCL19、OAS1与SLE疾病活动度的相关性,采用受试者工作特征(ROC)曲线分析HE4、IFN-α、CCL19、OAS1对重度SLE的诊断价值。结果SLE组HE4、IFN-α、CCL19、OAS1水平明显高于健康对照组,差异均有统计学意义(P<0.05)。血清HE4、IFN-α、CCL19、OAS1水平在健康对照组、轻度组、中度组、重度组中依次升高,两两组间比较,差异均有统计学意义(P<0.05)。HE4、IFN-α、CCL19、OAS1水平与SLE疾病活动度均呈正相关(r_(s)=0.598、0.838、0.562、0.917,P<0.05)。血清HE4、IFN-α、CCL19、OAS1单独诊断重度SLE的曲线下面积(AUC)分别为0.849、0.991、0.806、0.974,4项指标联合诊断重度SLE的AUC为0.998,大于单独检测的AUC(P<0.05)。结论血清HE4、IFN-α、CCL19、OAS1与SLE患者疾病活动度关系密切,可作为SLE疾病活动度评估的辅助指标,同时对SLE临床诊治具有重要指导意义。

连翘酯苷对IFN-α和Mx1表达的影响

【目的】探讨连翘酯苷作用于感染PCV2的小鼠模型后,对IFN-α和Mx1表达的影响,以评价连翘酯苷的抗病毒作用。【方法】复制PCV2感染小鼠模型。以(2、4、10mg·mL-1)连翘酯苷分为高、中、低3个剂量组,并设空白对照组和病毒对照组。给药12、24、36h后,分别提取小鼠肺组织总RNA和总蛋白,应用实时荧光定量PCR检测IFN-α、Mx1 mRNA含量,采用Western blotting检测Mx1蛋白的表达水平。【结果】空白对照组小鼠肺组织中Mx1和IFN-α无表达,病毒对照组少量表达。随着连翘酯苷浓度的升高,IFN-α和Mx1 mRNA水平逐步升高,两者的表达量呈现平行相关;高剂量组中IFN-α和Mx1表达量最高(P<0.001);随着药物作用时间的延长表达量降低,12h时达到最高(P<0.001),其后逐渐下降。【结论】昆明小鼠适于PCV2感染模型的建立。中药连翘酯苷能显著上调IFN-α和Mx1的表达,并在给予高剂量药物后12h发挥显著的抗病毒作用。Mx1蛋白与病毒的感染密切相关,可用于病毒感染的早期诊断,及具有较好的应用前景。

电针对帕金森病模型大鼠黑质区c-Jun氨基末端激酶和TNF-α、IFN-γ、IL-1β蛋白表达的影响

目的:观察电针对帕金森病模型大鼠黑质区c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)信号通路及炎症因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、γ-干扰素(interferon-γ,IFN-γ)、白细胞介素1β(interleukin-1β,IL-1β)的作用,探讨针刺治疗帕金森病的作用机制。方法:健康雄性SPF级SD大鼠32只,随机平均分为正常组、假手术组、模型组、电针组。模型组和电针组经颈背部注射鱼藤酮(1 mg/kg,溶于一定比例二甲亚砜和生理盐水中,浓度0.25 mg/m L)造模14 d。电针组给予电针"风府"(GV16)和"太冲"(LR3)两穴治疗,连续治疗14 d,其余各组不做治疗。假手术组给予相同剂量的二甲亚砜(Dimethyl sulfoxide,DMSO)和生理盐水混合液。采用免疫蛋白印迹法检测大鼠黑质区酪氨酸羟化酶(Tyrosine hydroxylase,TH)、磷酸化的c-Jun和TNF-α、IFN-γ、IL-1β蛋白表达情况。结果:与正常组、假手术组比较,模型组大鼠黑质区TH蛋白表达明显减少(P<0.05),p-c-Jun、TNF-α、IFN-γ、IL-1β蛋白表达明显增加(P<0.05);与模型组比较,电针组大鼠黑质区TH蛋白表达明显增加(P<0.05),p-c-Jun、TNF-α、IFN-γ、IL-1β蛋白表达明显减少(P<0.05)。结论:电针可以通过调节帕金森病模型大鼠体内MAPK/JNK信号通路,降低p-c-Jun在帕金森病模型大鼠黑质区的表达,进而减少炎症因子TNF-α、IFN-γ、IL-1β的表达,对帕金森病的发生发展起到一定的调节作用。

TLR4mAb对急性期溃疡性结肠炎小鼠结肠黏膜中促炎因子TNF-α、IFN-γ、IL-1β的影响

目的探讨TLR4单克隆抗体(toll like receptor4monoclonal antibodies,TLR4mAb)对葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导的急性期溃疡性结肠炎(ulcerative colitis,UC)小鼠肠黏膜的促炎因子TNF-α、IFN-γ、IL-1β的影响。方法30只BALB/c小鼠分为A-E组,分别为对照组、模型组以及低、中、高剂量干预组。A组小鼠饮用蒸馏水7d;B-E组小鼠仅饮用5%DSS水溶液7d以产生急性期溃疡性结肠炎模型。造模开始的同时,3组干预组小鼠分别给予低、中、高剂量TLR4mAb腹腔内注射,对照组及模型组给予腹腔内注射生理盐水,以观察TLR4mAb的干预作用。观察指标包括疾病活动指数(disease activity index,DAI)、结肠组织病理学评分(histopathological score,HPS)。造模及干预7d后处死小鼠,RT-PCR法检测各组肠黏膜TNF-α、IFN-γ、IL-1β的mRNA表达。结果(1)与对照组相比,模型组小鼠结肠黏膜DAI及HPS明显增高(P<0.01)。与模型组相比,在使用TLR4mAb干预后低、中、高剂量组DAI和HPS均有不同程度的缓解,但无明显剂量依赖关系。(2)与模型组相比,在使用TLR4mAb后炎症因子TNF-α、IFN-γ、IL-1β在小鼠结肠黏膜中的表达均有不同程度的降低,并呈剂量依赖关系。结论TLR4mAb可能通过下调小鼠结肠黏膜促炎因子TNF-α、IFN-γ、IL-1β的mRNA表达而发挥其干预作用。