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.

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。

黄精皂苷对慢性应激抑郁大鼠大脑皮层5-HT_(1A) R-β-arrestin2-akt信号通路的影响

目的探讨黄精皂苷(SRP)对慢性应激抑郁模型大鼠行为学的影响及部分机制。方法 60只SD大鼠随机均分为正常组、模型组、SRP(400、200、100 mg/kg)组和氟西汀(2 mg/kg)组。采用7种不同应激方法建立大鼠慢性应激抑郁模型,观察大鼠行为学指标变化;免疫组化法测定大脑皮层区5-羟色胺1A受体(5-HT1AR)的表达;Western blot法分析大脑皮层区5-HT1AR、β-arrestin2、akt蛋白表达水平。结果应激刺激后,与正常组相比,模型组大鼠体重降低、自主活动次数减少,处于抑郁状态。免疫组化法及Western blot法结果显示,与正常组相比,模型组大脑皮层5-HT1AR表达降低,β-arrestin2、akt表达升高(P<0.05),而SRP组可以逆转这种现象(P<0.05,P<0.01)。结论中药SRP的抗抑郁作用可能与调节5-HT1AR/β-arrestin2/akt信号通路有关。

复方苦参汤对溃疡性结肠炎DOR-β-arrestin1-Bcl-2信号转导通路的干预作用

目的:探讨复方苦参汤对溃疡性结肠炎大鼠DOR-β-arrestin1-Bcl-2信号转导通路的干预作用.方法:SD♂大鼠84只(体质量200g±20g)随机分为空白对照组、模型组、美沙拉嗪组、复方苦参汤大剂量组、复方苦参汤中剂量组和复方苦参汤小剂量组,每组14只.除对照组外,其余5组均依据Morris等三硝基苯磺酸(trinitrobenzene sulfonic acid,TNBS)建立大鼠溃疡性结肠炎模型.造模后观察记录大鼠的大便和精神状态,并于第3天随机处死2只造模大鼠,取结肠镜下观察其病理组织学变化发现:大鼠结肠糜烂、充血及溃疡,证明造模成功.建立大鼠溃疡性结肠炎模型后给予美沙拉嗪组大鼠3mL/d(0.5g/L)美沙拉嗪混悬液灌胃,复方苦参汤大、中、小剂量组分别按不同含生药浓度的复方苦参汤液3mL/d(0.67、0.34、0.17g/L)灌胃,对照组和模型组给予等量的生理盐水3mL/d灌胃,连续灌胃15d后,禁食24h,处死大鼠,取结肠组织石蜡包埋切片,HE染色比较各组结肠组织病理组织学改变,Realtime-PCR和免疫组织化学技术检测实验大鼠结肠组织的Bcl-2、β-arrestin1及δ阿片受体(delta opioid receptor,DOR)mRNA和蛋白表达表达变化.结果:各组大鼠结肠组织中Bcl-2、β-arrestin1和DOR表达有显著差异(P<0.05).与对照组比较,模型组的大鼠结肠黏膜组织Bcl-2、β-arrestin1和DOR表达明显升高(24.11±12.61vs11.88±5.90,38.90±5.30vs14.34±8.97,23.57±9.96vs9.68±3.94,均P<0.05);与模型组比较,美沙拉嗪组和复方苦参汤大、中、小剂量组的大鼠结肠黏膜组织Bcl-2、β-arrestin1、DOR表达均显著下降,但美沙拉嗪组和复方苦参汤大、中、小剂量组之间比较Bcl-2、β-arrestin1、DOR表达无显著差异.结论:在实验性溃疡性结肠炎大鼠结肠组织中DOR、β-arrestin1和Bcl-2的表达升高,DOR-β-arrestin1-Bcl-2信号转导通路可能参与了溃疡性结肠炎的病理过程,复方苦参汤可改善溃疡性结肠炎大鼠的结肠组织病理学改变,机制可能与调节该信号通路有关.

乌梅丸对TNBS诱导的溃疡性结肠炎大鼠脾脏组织β-arrestin1表达的影响

目的观察乌梅丸对2,4,6-三硝基苯磺酸(TNBS)诱导的溃疡性结肠炎大鼠脾脏组织β-arrestin1表达的影响。方法将56只SD大鼠随机分为空白对照组、模型组、美沙拉嗪组、乌梅丸组各14只,除空白对照组外,其余三组均应用TNBS灌肠;溃疡性结肠炎模型建成2 d后,空白对照组和模型组以蒸馏水、美沙拉嗪组以美沙拉嗪混悬液(50 g/L)、乌梅丸组以乌梅丸液(0.51 g/L)灌胃,均为每只3 mL,连续灌胃15 d后取脾脏组织,采用RT-PCR法检测β-arrestin1 mRNA,Western blot法检测β-arrestin1蛋白。结果与空白对照组比较,模型组β-arrestin1 mRNA、蛋白表达均升高(P均<0.05);与模型组比较,乌梅丸组、美沙拉嗪组β-arrestin1 mRNA、蛋白表达均下降(P均<0.05)。结论乌梅丸可下调TNBS诱导的溃疡性结肠炎大鼠脾脏组织β-arrestin1 mRNA、蛋白表达。

β-arrestin1激活JNK信号通路促进慢性髓细胞白血病细胞K562增殖

目的以CML K562细胞为研究对象,探索β-arrestin1促进CML细胞增殖的相关信号通路。方法以β-arrestin1慢病毒载体感染CML K562细胞,形成稳定的K562-siβ1和K562-β1细胞,及非特异性si RNA对照K562-Ctrl细胞。以此为研究对象,利用细胞计数与CCK-8实验检测细胞增殖能力;Western blot检测蛋白表达;免疫共沉淀(Co-IP)实验检测蛋白间的相互作用。结果细胞计数与CCK-8实验结果显示K562-β1细胞增殖与细胞存活率能力显著高于K562-Ctrl,而K562-siβ1显著低于K562-Ctrl。Western blot结果表明β-arrestin1特异性增强磷酸化JNK表达,JNK抑制剂SP600125能抑制p-JNK表达和K562细胞增殖;免疫共沉淀实验表明β-arrestin1能与Src结合。结论 CML K562细胞中β-arrestin1与Src结合,促进JNK信号通路激活,从而促进细胞增殖。

β-arrestin1激活STAT3信号通路影响白血病细胞K562体外迁移侵袭能力

目的:探讨β-arrestin1对白血病细胞生物学功能的影响及相关机制的研究。方法:特异性的β-Arrestin1-siRNA和阴性对照siRNA转染K562细胞,形成β-Arrestin1-siRNA组、阴性对照siRNA组和空白组对照组。以此为实验分组,应用transwell小室法检测各组细胞的迁移侵袭能力变化,Western blot检测p STAT3蛋白的表达。结果:β-Arrestin1-siRNA处理组细胞的迁移和侵袭能力较对照组分别下降43%及52%(P<0.05);且p STAT3蛋白表达减少;STAT3抑制剂能抑制K562细胞体外迁移侵袭能力。结论:白血病细胞K562中β-arrestin1蛋白高表达激活STAT3信号通路,从而促进细胞的迁移侵袭能力。

天芪平颤颗粒通过β-arrestin1预防PD运动并发症的机制研究

目的:观察使用天芪平颤颗粒后,大鼠左旋多巴诱发的运动并发症的行为学及纹状体β-arrestin1表达的变化。方法:通过脑立体定向仪定向至大鼠前脑内侧束,注射6-OHDA建立帕金森病模型(n=25),并随机分为五组。PD对照组(n=5,腹腔注射0.2%维生素C液29 d),西药组(连续腹腔注射50 mg.kg-1左旋多巴甲酯和25 mg.kg-1苄丝肼29 d),中药大剂量组、中药中剂量组、中药小剂量组(在给予左旋多巴甲酯/苄丝肼的基础上分别加用中药(天芪平颤颗粒)29 d)。观察不同剂量天芪平颤颗粒对PD模型大鼠运动并发症的行为学影响,通过免疫组织化学方法和Western blotting法检测不同处理组大鼠纹状体区β-arrestin1蛋白的表达情况。结果:PD大鼠长期使用左旋多巴出现类似于人类LID行为表现。免疫组化结果显示长期左旋多巴的使用使β-arrestin1表达进行性减少,天芪平颤颗粒能抑制β-arrestin1阳性细胞指数的减少,其中中剂量组β-arrestin1阳性细胞指数为(3.67±0.56)×104比PD大鼠增多(P<0.01)。Western blot结果显示,与长期使用左旋多巴的大鼠(65.44±6.68)%比较,中剂量组和大剂量组β-arrestin1表达量分别为(73.62±3.82)%和(72.56±6.66)%,明显升高,差异有统计学意义(P<0.05)。结论:天芪平颤颗粒通过增加β-arrestin1蛋白表达量,减少了左旋多巴长期用药引起的PD异动症的发生。

β-arrestin1在实验性大鼠结肠炎发生机制中的作用及氧化苦参碱的干预作用

目的研究β-arrestin1在实验性大鼠结肠炎发生机制中的作用,δ阿片受体-β-arrestin1-Bcl-2信号转导通路是否参与实验性大鼠结肠炎的病理过程,氧化苦参碱(OMT)能否通过干预δ阿片受体-β-ar-restin1-Bcl2信号转导通路减轻实验性大鼠结肠炎。方法将26只SD大鼠随机分为4组:分别为正常对照组6只、模型组6只、美沙拉嗪组6只和OMT组8只。正常对照组未行造模,其余3组大鼠均采用三硝基苯磺酸(TNBS)造模。OMT组大鼠给予苦参素注射液肌肉注射,美沙拉嗪组大鼠给予美沙拉嗪混悬液灌胃,模型组和正常组大鼠均以蒸馏水3mL灌胃。治疗15天,观察实验大鼠结肠病理组织学改变,并分别用免疫组化和Western免疫印迹技术分别检测实验大鼠结肠黏膜组织和脾脏T淋巴细胞δ阿片受体、β-arrestin1和Bcl-2表达变化。结果与正常对照组比较,模型组δ阿片受体、β-arrestin1和Bcl-2表达明显增高(P<0.01)。与模型组比较,美沙拉嗪组和OMT组δ阿片受体、β-arrestin1、Bcl-2表达均显著下降(P<0.01)。结论δ阿片受体-β-arrestin1-Bcl-2信号转导通路参与TNBS诱导的实验性大鼠结肠炎的发病机制;OMT抑制δ阿片受体-β-arrestin1-Bcl-2信号转导通路,是OMT改善TNBS诱导的实验性大鼠结肠炎的机制之一。

Delta阿片受体、β-arrestin1、Bcl-2在溃疡性结肠炎大鼠脾脏组织中的表达及乌梅丸的干预作用

目的:观察溃疡性结肠炎(ulcerative colitis,UC)大鼠脾脏组织Delta阿片受体、β-arrestin1、Bcl-2 mRNA和蛋白表达及乌梅丸的干预作用,旨在研究乌梅丸治疗结肠炎的机制.方法:56只SD大鼠随机分成空白对照组、结肠炎模型组、美沙拉嗪组、乌梅丸组(每组14只).除空白对照组外,其他3组均应用2,4,6-三硝基苯磺酸灌肠.模型建成2 d后,空白对照组和结肠炎模型组分别以蒸馏水3 mL/只灌胃,美莎拉嗪组用美莎拉嗪混悬液(浓度50 g/L)、乌梅丸组用乌梅丸液(浓度0.51 g/L)以3 mL/只灌胃,连续给药15 d后取脾脏组织,应用Realtime-PCR检测Delta阿片受体、β-arrestin1、Bcl-2 mRNA的表达,应用Western blot技术检测Delta阿片受体、β-arrestin1、Bcl-2蛋白的表达.结果:空白对照组Delta阿片受体、β-arrestin1及Bcl-2 mRNA的相对表达分别为1.11±0.10、1.05±0.06及1.06±0.04;蛋白的相对表达分别为0.48±0.08、0.62±0.07及0.76±0.10.结肠炎模型组Delta阿片受体、β-arrestin1及Bcl-2的mRNA的相对表达分别为2.50±0.25、3.27±0.41及2.48±0.43;蛋白的相对表达分别为1.04±0.17、1.50±0.15及1.26±0.20.美沙拉嗪组Delta阿片受体、β-arrestin1及Bcl-2 mRNA的相对表达分别为1.52±0.09、1.63±0.27及1.56±0.09;蛋白的相对表达分别为0.68±0.17、0.77±0.15及0.96±0.16.乌梅丸组Delta阿片受体、β-arrestin1及Bcl-2 mRNA的相对表达分别为1.54±0.13、1.54±0.14及1.57±0.15;蛋白的相对表达分别为0.68±0.14、0.74±0.19及0.93±0.11.与空白对照组相比,结肠炎模型组Delta阿片受体、β-arrestin1、Bcl-2 mRNA和蛋白表达显著升高(P<0.05);与结肠炎模型组相比,乌梅丸组、美沙拉嗪组Delta阿片受体、β-arrestin1、Bcl-2 mRNA和蛋白表达显著下降(P<0.05),且乌梅丸组和美沙拉嗪组Delta阿片受体、β-arrestin1、Bcl-2 mRNA和蛋白表达均无显著差异(P>0.05).结论:乌梅丸下调UC大鼠脾脏组织Delta阿片受体、β-arrestin1、Bcl-2 mRNA和蛋白表达发挥治疗结肠炎的作用.

β-arrestin1过表达抑制Molt-4细胞异种移植小鼠急性T淋巴细胞白血病进展

目的探讨β-arrestin1对Molt-4细胞异种移植急性T淋巴细胞白血病(T-ALL)NCG小鼠模型疾病进展的影响。方法建立稳定表达荧光素酶的人Molt-4细胞系,酶标仪检测其荧光素酶强度;构建稳定过表达或敲低β-arrestin1和空载体的Molt-4细胞系,设为β1组、Siβ1组和Scram组并分别接种于亚致死计量辐射后NCG小鼠体内;记录小鼠移植后体质量及生存时间;利用活体荧光成像系统检测移植小鼠模型体内肿瘤进展;移植后16 d处死小鼠,取肝和脾做切片染色,检测肿瘤细胞浸润。结果成功构建稳定表达荧光素酶Molt-4细胞系(Molt4-Fluc)。在Molt4-Fluc基础上成功构建过表达β-arrestin1(β1)、敲低β-arrestin1(Siβ1)或空载体(Scram)的Molt-4细胞系。NCG小鼠T-ALL异种移植模型显示,β1组、Siβ1组及Scram组Molt4-Fluc细胞在受体NCG小鼠中进展,导致NCG小鼠在移植后体质量降低。小鼠生存时间显示,β1组细胞受体小鼠生存时间(43 d)显著长于Scram组细胞受体小鼠(33 d),P<0.001;Siβ1组细胞受体小鼠生存时间(20 d)显著短于Scram组细胞受体小鼠(33 d),P<0.001;活体荧光成像结果显示,β1组细胞受体小鼠体内T-ALL细胞荧光强度显著弱于Scram组细胞受体小鼠,Siβ1组细胞受体小鼠体内T-ALL细胞荧光强度强于Scram组细胞受体小鼠;处死移植后16 d小鼠,组织染色结果显示,β1组细胞受体小鼠肝、脾内肿瘤细胞浸润较Scram组细胞受体小鼠少。结论β-arrestin1过表达可抑制Molt-4细胞异种移植后小鼠T-ALL进展。

新辅助化疗对卵巢癌患者TLR2、ERCC1、β-arrestin 2的影响

原发性上皮性卵巢癌是常见的妇科恶性肿瘤,占卵巢肿瘤的50%~70%,死亡率高。目前临床治疗主采用初次肿瘤细胞减灭术以及术后予铂类＋紫杉醇为基础的化疗,初次手术是否彻底为患者预后的重影响因素之一。近年来,新辅助化疗在晚期卵巢癌的应用逐渐成为国内外学者关注的焦点。