Deubiquitinase ubiquitin-specific protease 3 (USP3) inhibits HIV-1 replication via promoting APOBEC3G (A3G) expression in both enzyme activity-dependent and -independent manners

Background: Ubiquitination plays an essential role in many biological processes, including viral infection, and can be reversed by deubiquitinating enzymes (DUBs). Although some studies discovered that DUBs inhibit or enhance viral infection by various mechanisms, there is lack of information on the role of DUBs in virus regulation, which needs to be further investigated.Methods: Immunoblotting, real-time polymerase chain reaction,in vivo/in vitro deubiquitination, protein immunoprecipitation, immunofluorescence, and co-localization biological techniques were employed to examine the effect of ubiquitin-specific protease 3 (USP3) on APOBEC3G (A3G) stability and human immunodeficiency virus (HIV) replication. To analyse the relationship between USP3 and HIV disease progression, we recruited 20 HIV-infected patients to detect the levels of USP3 and A3G in peripheral blood and analysed their correlation with CD4^(+) T-cell counts. Correlation was estimated by Pearson correlation coefficients (for parametric data).Results: The results demonstrated that USP3 specifically inhibits HIV-1 replication in an A3G-dependent manner. Further investigation found that USP3 stabilized 90% to 95% of A3G expression by deubiquitinating Vif-mediated polyubiquitination and blocking its degradation in an enzyme-dependent manner. It also enhances the A3G messenger RNA (mRNA) level by binding to A3G mRNA and stabilizing it in an enzyme-independent manner. Moreover, USP3 expression was positively correlated with A3G expression (r= 0.5110) and CD4^(+) T-cell counts (r= 0.5083) in HIV-1-infected patients.Conclusions: USP3 restricts HIV-1 viral infections by increasing the expression of the antiviral factor A3G. Therefore, USP3 may be an important target for drug development and serve as a novel therapeutic strategy against viral infections.

Ubiquitin-specific protease 21 promotes tumorigenicity and stemness of colorectal cancer by deubiquitinating and stabilizing ZEB1

BACKGROUND Colorectal cancer(CRC)is one very usual tumor together with higher death rate.Ubiquitin-specific protease 21(USP21)has been confirmed to take part into the regulation of CRC progression through serving as a facilitator.Interestingly,the promotive function of USP21 has also discovered in the progression of CRC.ZEB1 has illustrated to be modulated by USP7,USP22 and USP51 in cancers.However,the regulatory functions of USP21 on ZEB1 in CRC progression need more invest-igations.AIM To investigate the relationship between USP21 and ZEB1 in CRC progression.METHODS The mRNA and protein expressions were assessed through RT-qPCR,western blot and IHC assay.The interaction between USP21 and ZEB1 was evaluated through Co-IP and GST pull down assays.The cell proliferation was detected through colony formation assay.The cell migration and invasion abilities were determined through Transwell assay.The stemness was tested through sphere formation assay.The tumor growth was evaluated through in vivo mice assay.RESULTS In this work,USP21 and ZEB1 exhibited higher expression in CRC,and resulted into poor prognosis.Moreover,the interaction between USP21 and ZEB1 was further investigated.It was demonstrated that USP21 contributed to the stability of ZEB1 through modulating ubiquitination level.In addition,USP21 streng-thened cell proliferation,migration and stemness through regulating ZEB1.At last,through in vivo assays,it was illustrated that USP21/ZEB1 axis aggravated tumor growth.CONCLUSION For the first time,these above findings manifested that USP21 promoted tumorigenicity and stemness of CRC by deubiquitinating and stabilizing ZEB1.This discovery suggested that USP21/ZEB1 axis may provide novel sights for the treatment of CRC.

Association of 370-371insACA, 494T〉C, and 1423C〉T haplotype in ubiquitin-specific protease 26 gene and male infertility： a meta-analysis

Whether the 370-371insACA, 494T〉C, and 1423C〉T haplotype in ubiquitin-specific protease 26 （USP26） gene is associated with male infertility is controversial. To clarify this issue, we conducted a meta-analysis based on the most recent studies. Eligible studies were screened by using PubMed and Embase. Pooled odd ratio （OR） with 95% confidence interval （CI） was calculated with fixed effect models. Ten studies with 1603 patients and 2505 controls were included, Overall, the results indicated that there was an association between the haplotype and male infertile risk （OR = 1.74, 95% CI： 1.09-2.77）. The OR calculated based on the five studies in Asia and three in Europe was 1.96 （95% CI： 1,05-3.67） and 1.54 （95% Ch 0.75-3.16） respectively, however, the OR was 0.86 （95% Ch 0.05-15,29） based on the two investigations in America. In addition, the data from the patients with azoospermia （AZO） showed an increased pooled OR of 2.35 （95% Cl： 1.22-4.50）. In contrast, the studies with oligoasthenoteratozoospermia （OAT） exhibited that the pooled OR was 0,97 （95% Ch 0.43-2.16）. Our analyses indicate that there is an association of alteration in USP26 with male infertility, especially in AZO and Asian population.

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility

Aim： To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 （USP26） gene and its involvement in idiopathic male infertility in China. Methods： Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. Results： Of 41 infertile men, 9 （22.0%, P = 0.01） had changes in USP26 gene on the X chromosome. A compound mutation （364insACA; 460G→A） was detected in 8 patients （19.5%, P = 0.01） and a 1044T→A substitution was found in 1 patient （2.4%, P 〉 0.05）. All three variations led to changes in the coding amino acids. Two substitutions predict some changes： 460G→ A changes a valine into an isoleucine, and 1044T → A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. Conclusion： The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.

Ubiquitin-specific protease 22 enhances intestinal cell proliferation and tissue regeneration after intestinal ischemia reperfusion injury

BACKGROUND Intestinal ischemia reperfusion(I/R) injury is a serious but common pathophysiological process of many diseases, resulting in a high mortality rate in clinical practice. Ubiquitin-specific protease 22(USP22) acts as regulator of cell cycle progression, proliferation, and tumor invasion. Depleted USP22 expression has been reported to contribute to arrested cell cycle and disrupted generation of differentiated cell types in crypts and villi. However, the role of USP22 in intestinal damage recovery has not been investigated. Therefore, elucidation of the underlying mechanism of USP22 in intestinal I/R injury may help to improve the tissue repair and patient prognosis in clinical practice.AIM To investigate the role of USP22 in intestinal cell proliferation and regeneration after intestinal I/R injury.METHODS An animal model of intestinal I/R injury was generated in male Sprague-Dawley rats by occlusion of the superior mesenteric artery followed by reperfusion.Chiu's scoring system was used to grade the damage to the intestinal mucosa. An in vitro model was developed by incubating rat intestinal epithelial IEC-6 cells in hypoxia/reoxygenation conditions in order to simulate I/R in vivo. siRNA and overexpression plasmid were used to regulate the expression of USP22. USP22,Cyclin D1, and proliferating cell nuclear antigen(PCNA) expression levels were measured by Western blot analysis and immunohistochemistry staining. Cell survival(viability) and cell cycle were evaluated using the Cell Counting Kit-8and flow cytometry, respectively.RESULTS USP22 expression was positively correlated with the expression levels of PCNA and Cyclin D1 both in vivo and in vitro, which confirmed that USP22 was involved in cell proliferation and intestinal regeneration after intestinal I/R injury. Decreased levels of Cyclin D1 and cell cycle arrest were observed in the USP22 knockdown group(P < 0.05), while opposite results were observed in the USP22 overexpression group(P < 0.05). In addition, increased expression of USP22 was related to improved intestinal pathology or IEC-6 cell viability after I/R or hypoxia/reoxygenation. These results suggested that USP22 may exert a protective effect on intestinal I/R injury by regulating cell proliferation and facilitating tissue regeneration.CONCLUSION USP22 is correlated with promoting intestinal cell proliferation and accelerating intestinal tissue regeneration after intestinal I/R injury and may serve as a potential target for therapeutic development for tissue repair during intestinal I/R injury.

Ubiquitin-specific protease 15 contributes to gastric cancer progression by regulating the Wnt/β-catenin signaling pathway

BACKGROUND Ubiquitin-specific protease 15(USP15)is an important member of the ubiquitinspecific protease family,the largest deubiquitinase subfamily,whose expression is dysregulated in many types of cancer.However,the biological function and the underlying mechanisms of USP15 in gastric cancer(GC)progression have not been elucidated.AIM To explore the biological role and underlying mechanisms of USP15 in GC progression.METHODS Bioinformatics databases and western blot analysis were utilized to determine the expression of USP15 in GC.Immunohistochemistry was performed to evaluate the correlation between USP15 expression and clinicopathological characteristics of patients with GC.A loss-and gain-of-function experiment was used to investigate the biological effects of USP15 on GC carcinogenesis.RNA sequencing,immunofluorescence,and western blotting were performed to explore the potential mechanism by which USP15 exerts its oncogenic functions.RESULTS USP15 was up-regulated in GC tissue and cell lines.The expression level of USP15 was positively correlated with clinical characteristics(tumor size,depth of invasion,lymph node involvement,tumor-node-metastasis stage,perineural invasion,and vascular invasion),and was related to poor prognosis.USP15 knockdown significantly inhibited cell proliferation,invasion and epithelialmesenchymal transition(EMT)of GC in vitro,while overexpression of USP15 promoted these processes.Knockdown of USP15 inhibited tumor growth in vivo.Mechanistically,RNA sequencing analysis showed that USP15 regulated the Wnt signaling pathway in GC.Western blotting confirmed that USP15 silencing led to significant down-regulation ofβ-catenin and Wnt/β-catenin downstream genes(c-myc and cyclin D1),while overexpression of USP15 yielded an opposite result and USP15 mutation had no change.Immunofluorescence indicated that USP15 promoted nuclear translocation ofβ-catenin,suggesting activation of the Wnt/β-catenin signaling pathway,which may be the critical mechanism promoting GC progression.Finally,rescue experiments showed that the effect of USP15 on gastric cancer progression was dependent on Wnt/β-catenin pathway.CONCLUSION USP15 promotes cell proliferation,invasion and EMT progression of GC via regulating the Wnt/β-catenin pathway,which suggests that USP15 is a novel potential therapeutic target for GC.

Emerging potential of ubiquitin-specific proteases and ubiquitinspecific proteases inhibitors in breast cancer treatment

Breast cancer is the most frequently diagnosed cancer in women,accounting for 30%of new diagnosing female cancers.Emerging evidence suggests that ubiquitin and ubiquitination played a role in a number of breast cancer etiology and progression processes.As the primary deubiquitinases in the family,ubiquitin-specific peptidases(USPs)are thought to represent potential therapeutic targets.The role of ubiquitin and ubiquitination in breast cancer,as well as the classification and involvement of USPs are discussed in this review,such as USP1,USP4,USP7,USP9X,USP14,USP18,USP20,USP22,USP25,USP37,and USP39.The reported USPs inhibitors investigated in breast cancer were also summarized,along with the signaling pathways involved in the investigation and its study phase.Despite no USP inhibitor has yet been approved for clinical use,the biological efficacy indicated their potential in breast cancer treatment.With the improvements in phenotypic discovery,we will know more about USPs and USPs inhibitors,developing more potent and selective clinical candidates for breast cancer.

Inhibition of Ubiquitin-specific Protease 4 Attenuates Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells via Transforming Growth Factor Beta Receptor Type Ⅰ

Objective Ubiquitin-specific protease 4(USP4)facilitates the development of transforming growth factor-beta 1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)in various cancer cells.Moreover,EMT of renal tubular epithelial cells(RTECs)is required for the progression of renal interstitial fibrosis.However,the role of USP4 in EMT of RTECs remains unknown.The present study aimed to explore the effect of USP4 on the EMT of RTECs as well as the involved mechanism.Methods In established unilateral ureteral obstruction(UUO)rats and NRK-52E cells,immunohistochemistry and Western blot assays were performed.Results USP4 expression was increased significantly with obstruction time.In NRK-52E cells stimulated by TGF-β1,USP4 expression was increased in a time-dependent manner.In addition,USP4 silencing with specific siRNA indicated that USP4 protein was suppressed effectively.Meanwhile,USP4 siRNA treatment restored E-cadherin and weakened alpha smooth muscle actin(α-SMA)expression,indicating that USP4 may promote EMT.After treatment with USP4 siRNA and TGF-β1 for 24 h,the expression of TGF-β1 receptor type I(TβRI)was decreased.Conclusion USP4 promotes the EMT of RTECs through upregulating TβRI,thereby facilitating renal interstitial fibrosis.These findings may provide a potential target of USP4 in the treatment of renal fibrosis.

猪腺病毒3型Protease蛋白在大肠杆菌中的表达及其抗体制备

试验旨在研究猪腺病毒3型(PADV3)Protease蛋白在大肠杆菌中的表达,并制备该蛋白的多克隆抗体。利用PCR扩增PADV3 Protease基因,构建重组原核表达载体pET28a-PADV3-Protease和真核表达载体pEGFP-PADV3-Protease,采用双酶切和测序鉴定;将原核表达载体pET28a-PADV3-Protease转化大肠杆菌BL21(DE3)感受态细胞得到重组原核表达菌株,经IPTG诱导收集蛋白,采用SDS-PAGE和Western blotting鉴定,将目的蛋白纯化后与佐剂乳化制备免疫原免疫家兔,制备Protease蛋白多克隆抗体;将真核表达载体pEGFP-PADV3-Protease转染HEK293细胞经G418筛选建立稳定表达EGFP-Protease融合蛋白的细胞系,以该稳定表达细胞系为包被抗原,采用免疫过氧化物酶单层细胞染色法(IPMA)检测抗体的免疫活性与抗体滴度。结果显示,Protease基因开放阅读框(ORF)为615 bp,原核表达系统中Protease蛋白以包涵体形式存在,分子质量大小为23 ku,与真核细胞表达的Protease蛋白分子质量一致,该蛋白在细胞核与细胞质中均有分布,制备的Protease蛋白多克隆抗体能与EGFP-Protease融合蛋白稳定表达真核细胞系发生特异性的反应,与对照细胞无反应。本试验构建了Protease蛋白原核表达菌株和真核表达细胞株,制备的PADV3-Protease蛋白多克隆抗体免疫活性良好,为进一步研究Protease蛋白的生物学功能和PADV3的血清学诊断提供了基础材料。

Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry

BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.

Synthesis of N1-Substituted-3-aryl-4-alkyl-4, 5-dihydro-1H-1-pyra-zolethiocarboxamide as Novel Small Molecule Inhibitors of Cysteine Protease of T.cruzi

A series of N1-substituted-3-aryl-4-alkyl-4, 5-dihydro-1H-1-pyrazolethiocarboxamide were prepared from the Mannich bases of aryl ketones in good yields. Some derivatives were found to be active against the cysteine protease of T.cruzi..

Chlorogenic acid may be a potent inhibitor of dimeric SARS-CoV-2 main protease 3CLpro: an in silico study

Background:Since the emergence of coronavirus disease 2019 to date,there is no available approved drug or definitive treatment for coronavirus disease 2019 viral infection,and the identification of novel hits against therapeutic targets has become a global emergency.Echinacea purpurea is a traditional herb utilized to treat cough,fever,sore throat,respiratory tract infection,and so on as an immune stimulant.In this study,in silico molecular docking approach was used to screen phytocompounds from E.purpurea against severe acute respiratory syndrome coronavirus 2 main protease 3C-like protease(3CLpro)and severe acute respiratory syndrome coronavirus main peptidase(96%sequence similarity)to blunt the viral gene expression and viral replication.Methods:Initially,we screened phytocompounds for their druggability and ADMET property.Furthermore,x-ray crystallographic structures of main proteases 3CLpro and main peptidase having Protein Data Bank ID 6LU7 and 2GTB were used as protein targets for the identification of potential drug candidates.We performed docking using AutoDock Vina by PyRx 0.8 software.BIOVIA Discovery Studio Visualizer v2019 was used to analyze ligand-protein complex.The probable protein targets of the selected compound were predicted by BindingDB(P≥0.7).STRING and Kyoto Encyclopedia of Genes and Genomes pathways are utilized to identify the molecular pathways modulated by the predicted targets(FDR≤0.05),and the network interaction between compounds and protein pathways was constructed by Cytoscape 3.6.1.Results:Among all the compounds,chlorogenic acid showed druggable characteristics and scored the lowest binding energy with main protease and main peptidase via interacting with active site 1 domain amino acid residues.Interestingly,chlorogenic acid interacted with Phe140 main protease 3CLpro,which is potentially involved in the dimerization.Enrichment analysis identified chlorogenic acid to modulate insulin resistance,necroptosis,interleukin-17,tumor necrosis factor signaling pathway,legionellosis,T helper 17 cell differentiation,advanced glycation end products and receptor for advanced glycation end products,mitogen-activated protein kinase,Ras,estrogen,vascular endothelial growth factor,B-cell receptor,nuclear factor kappa B,Rap1,hypoxia inducible factor-1,phosphatidylinositide 3-kinase-Akt,insulin,mechanistic target of rapamycin,p53,retinoic acid inducible gene I like receptor,and ErbB signaling pathways.Conclusion:Chlorogenic acid may act as a potent main protease 3CLpro inhibitor and may also inhibit the severe acute respiratory syndrome coronavirus 2 dimerization,viral gene expression,and replication within the lung epithelium.Chlorogenic acid may go a long way in finding one of the multipronged solutions to tackle coronavirus disease 2019 viral infection in the future.

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective:To explore the effect of the protease inhibitor from Agaricus bisporus(J.E.Lange)Imbach(AbPI)on glucose uptake and oxidative stress in 3 T3-L1 adipocytes.Methods:Adipocytes were differentiated and stained with OilRed-O staining to confirm adipogenesis.The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay,intracellular reactive oxygen species generation through flow cytometry,and morphologically through confocal microscopy using propidium iodide,4,6-diamino-2-phenylindol dihydrochloride,and 2’,7’-dichlorofluorescein diacetate dyes.The uptake of fluorescent glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy.Results:MTT assay showed that the cell survival rate was(28.00±3.00)%,(92.33±2.60)%,and(71.34±2.10)%in the presence of 2 mM H2O2,AbPI alone,and AbPI and H2O2 both,respectively,in comparison to the control.Oil-Red-O staining indicated that Ab PI enhanced adipogenesis.AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone,and showed insulinsensitizing effect in the presence of insulin,but failed to stimulate the uptake in the absence of insulin.Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon Ab PI treatment.Confocal microscopy showed that the damaged cell population rose to 3.50%,117.84%,and 261.50%in the presence of Ab PI alone,AbPI with H2O2,and H2O2 alone,respectively.Conclusions:The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.

耐盐碱性蛋白酶菌株LK-3的筛选及酶学性质

为了寻找产碱性蛋白酶活性高且稳定性良好的野生菌株,作者从上海临港区域的东海海水中筛选出一株高产稳定且耐盐的产碱性蛋白酶菌株。对该菌株的形态学特性、生理特性以及16S rDNA基因序列进行测序和分析,确定该菌株为同温层芽孢杆菌(Bacillus stratosphericus),并命名为B. stratosphericus LK-3。酶学性质研究结果显示,该酶的最适作用条件为温度35℃、p H 8.5、最适接种体积分数7%,该酶在上述条件下发酵72 h的酶活力为(581.74±0.81) U/mL。此外该酶具有较好的耐盐性,即使在40 g/dL的高饱和盐质量浓度下仍然保持22.03%的相对原始酶活力。蛋白酶的耐盐性是一个有价值的特性,可将其纯化后作为洗涤剂的生物添加剂应用于工业生产。

依托咪酯对视神经损伤成年大鼠视网膜神经节细胞的保护作用及对半胱氨酸天冬氨酸蛋白酶3、脑源性神经营养因子蛋白表达的影响

目的:探讨依托咪酯对视神经损伤成年大鼠视网膜神经节细胞的保护作用及半胱氨酸天冬氨酸蛋白酶3(Caspase-3)、脑源性神经营养因子(BDNF)蛋白表达的影响。方法:选择成年雄性SD大鼠40只,随机选8只为正常照组,其余32只采用动脉夹夹持法损伤视神经建立视神经损伤模型并分组,即模型组(视神经损伤大鼠),依托咪酯低、中、高剂量组(依托咪酯腹腔注射),剂量分别为2、4、6 mg/kg。分析比较干预后各组大鼠眼压变化。并行HE染色观察视网膜组织结构,比较各组大鼠视网膜神经节细胞(RCGs)存活数目及存活率,Western blot法检测视网膜组织中Caspase-3、BDNF蛋白表达。结果:模型组、依托咪酯各组眼压高于正常组,依托咪酯各组末次给药后眼压降低,且呈剂量依赖性(均P<0.05)。模型组大鼠视网膜水肿增厚,以神经纤维层最为明显,且有空泡,RGC细胞肿胀,内、外核层细胞数量减少,排列紊乱。依托咪酯各组视网膜病理损伤均有改善,高剂量组好于中剂量组,中剂量组好于低剂量组。与正常组比较,模型组大鼠RCGs存活数目减少(P<0.05),与模型组比较,依托咪酯各组RCGs存活数目增多(P<0.05)。依托咪酯高剂量组RCGs存活数目、相对存活率高于中、低剂量组(均P<0.05)。正常组大鼠视网膜组织中BDNF蛋白表达高于模型组,Caspase-3低于模型组,依托咪酯各组视网膜组织中BDNF升高,Caspase-3下降,均呈剂量依赖性(均P<0.05)。结论:Caspase-3蛋白在大鼠视神经损伤中表达升高,BDNF蛋白表达降低,依托咪酯干预能够促进视网膜RGCs存活,对视神经损伤具有保护作用。

基于Caspase-3/PARP通路探究龟鹿二仙胶及拆方对IL-1β诱导的退变软骨细胞凋亡及细胞外基质的影响

目的 基于半胱氨酸蛋白酶3/多聚腺苷二磷酸核糖聚合酶(Caspase-3/PARP)通路探究龟鹿二仙胶及拆方对白细胞介素-1β(IL-1β)诱导的退变软骨细胞凋亡及细胞外基质(ECM)的影响。方法 采用SD大鼠制备空白血清、龟鹿二仙胶含药血清、龟甲胶含药血清及鹿角胶含药血清,采用酶消法体外培养C57BL/6小鼠软骨细胞。将软骨细胞分为5组,空白组加入空白血清培养,模型组加入IL-1β和空白血清培养,龟鹿组加入IL-1β和龟鹿二仙胶含药血清培养,龟板组加入IL-1β和龟甲胶含药血清培养,鹿角组加入IL-1β和鹿角胶含药血清培养,干预24 h后,采用CCK-8法检测软骨细胞活性,采用TUNEL法检测软骨细胞凋亡情况,采用免疫荧光染色法检测Caspase-3、PARP-1表达情况,分别采用Western blot和qRT-PCR法检测细胞中Caspase-3、PARP-1、基质金属蛋白酶-13(MMP-13)、聚集蛋白聚糖(Aggrecan)蛋白及mRNA表达情况。结果 与空白组比较,模型组软骨细胞活性明显降低(P<0.05),软骨细胞凋亡率明显升高(P<0.05);细胞中Caspase-3、PARP-1平均荧光强度和Caspase-3、PARP-1、MMP-13蛋白及mRNA相对表达量均明显升高(P均<0.05),细胞中Aggrecan蛋白及mRNA相对表达量均明显降低(P均<0.05)。与模型组比较,龟鹿组、龟板组及鹿角组软骨细胞活性均明显升高(P均<0.05),软骨细胞凋亡率均明显降低(P均<0.05);细胞中Caspase-3、PARP-1平均荧光强度和Caspase-3、PARP-1、MMP-13蛋白及mRNA相对表达量均明显降低(P均<0.05),细胞中Aggrecan蛋白及mRNA相对表达量均明显升高(P均<0.05)。与龟板组、鹿角组比较,龟鹿组软骨细胞活性更高(P均<0.05),软骨细胞凋亡率更低(P均<0.05);细胞中Caspase-3、PARP-1平均荧光强度和Caspase-3、PARP-1、MMP-13蛋白及mRNA相对表达量均更低(P均<0.05),细胞中Aggrecan蛋白及mRNA相对表达量均更高(P均<0.05)。与龟板组比较,鹿角组软骨细胞活性,软骨细胞凋亡率,细胞中Caspase-3、PARP-1平均荧光强度,细胞中Caspase-3、PARP-1、MMP-13、Aggrecan蛋白及mRNA相对表达量高均更高(P均<0.05)。结论 龟板胶可抑制软骨细胞凋亡及ECM降解,鹿角胶可促进软骨细胞增殖和ECM合成代谢,龟鹿二仙胶作用效果优于龟板胶及鹿角胶,作用机制可能与激活Caspase-3/PARP信号通路有关。

Capase-12、Capase-3、DAPK1对重症脓毒症患者感染性休克的预测价值分析

目的探讨半胱氨酸天冬氨酸蛋白酶12(Capase-12)、半胱氨酸天冬氨酸蛋白酶3(Capase-3)、死亡相关蛋白激酶1(DAPK1)对重症脓毒症患者感染性休克的预测价值。方法选取2020年4月—2023年4月本院收治的90例重症脓毒症患者设为观察组,同期本院90例体检健康者设为对照组。比较两组血清Capase-12、Capase-3、DAPK1水平,观察组患者根据是否发生感染性休克将其分为感染性休克组(n=23)、非感染性休克组(n=67),比较两组血清Capase-12、Capase-3、DAPK1水平,绘制受试者工作曲线(ROC),计算曲线下面积(AUC),分析血清Capase-12、Capase-3、DAPK1对感染性休克的预测效能。单因素、多因素Logistic回归分析感染性休克发生的危险因素。结果观察组血清Capase-12、Capase-3、DAPK1水平均高于对照组(P<0.05)。感染性休克组血清Capase-12、Capase-3、DAPK1水平均高于非感染性休克组(P<0.05)。血清Capase-12、Capase-3、DAPK1联合检测诊断感染性休克的AUC是0.863,95%CI 0.768~0.952,灵敏度(95.28%)、特异度(90.76%)均高于单一检测(78.19%、72.82%)、(75.12%、70.34%)、(72.67%、68.19%)(P<0.05)。Logistic回归分析显示,血清Capase-12、Capase-3、DAPK1、器官衰竭数量、低蛋白血症、血容量不足、血清PCT是诱发感染性休克的危险因素(P<0.05)。结论血清Capase-12、Capase-3、DAPK1水平异常高表达与重症脓毒症患者感染性休克的发生有着极为密切的联系,联合检测可提高对感染性休克的预测效能,感染性休克的发生与多种因素有关,应当引起临床重视与关注。

老年乳腺癌组织中PTBP3、USP28表达与术后复发转移的关系

目的分析老年乳腺癌组织中多聚嘧啶结合蛋白3(PTBP3)、泛素特异性蛋白酶28(USP28)表达与术后复发转移的关系。方法选择2017年2月—2020年2月辽宁省肿瘤医院乳腺外科诊治老年乳腺癌患者178例。以免疫组化法检测组织中PTBP3、USP28表达。随访3年,Kaplan-Meier生存分析不同PTBP3、USP28表达对老年乳腺癌术后复发转移的影响。Cox回归分析影响老年乳腺癌术后复发转移的因素。结果乳腺癌组织中PTBP3、USP28阳性率为65.17%(116/178)、67.42%(120/178),高于癌旁组织12.92%(23/178)、11.24%(20/178),差异有统计学意义(χ^(2)=102.080、117.725,P均<0.001)。TNM分期Ⅲ期、中低分化程度乳腺癌组织中PTBP3、USP28阳性率高于TNM分期Ⅱ期、高分化程度癌组织,差异均有统计学意义(χ^(2)/P=9.822/0.002,14.606/0.001,8.337/0.004,28.925/<0.001)。3年无复发转移率PTBP3阳性组为70.43%(81/115),低于阴性组的93.55%(58/62)(Log Rankχ^(2)=12.521,P<0.001);3年无复发转移率USP28阳性组为72.27%(86/119),低于阴性组的91.38%(53/58)(Log Rankχ^(2)=8.511,P=0.003)。Cox回归分析结果表明,PTBP3阳性、USP28阳性、肿瘤分期Ⅲ期、中低分化程度是影响乳腺癌患者术后复发转移的独立危险因素[OR(95%CI)=1.502(1.054~2.142),1.642(1.107~2.435),1.523(1.147~2.024),1.513(1.159~1.975),P均<0.05]。结论老年乳腺癌组织中PTBP3、USP28表达升高,PTBP3阳性、USP28阳性是影响老年乳腺癌患者术后复发转移的危险因素。

基于NLRP3/Caspase-1/GSDMD信号通路探讨清化饮对慢性萎缩性胃炎大鼠胃上皮细胞焦亡的影响

目的观察清化饮对慢性萎缩性胃炎(chronic atrophic gastritis,CAG)大鼠胃上皮NOD样受体蛋白3(NLRP3)/胱天蛋白酶-1(Caspase-1)/消皮素D(GSDMD)细胞焦亡通路的影响,探讨清化饮治疗CAG的可能机制。方法随机将大鼠分为空白对照组(n=8)及造模组(n=28),采用“氨水+去氧胆酸钠+乙醇法灌胃+高脂高糖饮食+人工气候箱饲养”复合法建立病证结合CAG大鼠模型,造模成功后将其随机分为模型组、维酶素治疗组和清化饮治疗组(n=8)。采用HE染色观察大鼠胃黏膜病变情况,Real-time PCR法检测胃组织NLRP3、Caspase-1、β-catenin、GSDMD mRNA表达情况,ELISA法检测血清IL-1β、IL-6、IL-18水平。结果与空白对照组相比,模型组大鼠炎症细胞浸润明显,胃黏膜固有腺体萎缩,胃黏膜组织中NLRP3、Caspase-1、β-catenin mRNA表达水平显著升高(P<0.01),GSDMD mRNA表达水平显著降低(P<0.01);血清IL-1β、IL-6、IL-18含量显著上升(P<0.01)。与模型组比较,清化饮治疗组胃黏膜固有腺体萎缩情况及炎症程度明显改善,NLRP3、Caspase-1、β-catenin mRNA表达蛋白表达水平均显著下降(P<0.01),GSDMD mRNA表达水平上升(P<0.01);血清IL-1β、IL-6、IL-18含量显著下降(P<0.01)。结论清化饮可有效改善CAG大鼠胃黏膜组织病理改变,其机制可能与调控NLRP3/Caspase-1/GSDMD信号通路,抑制细胞焦亡,从而降低胃黏膜炎症水平有关。

迷迭香吸嗅通过SIRT1、NLRP3、Caspase-1信号通路介导细胞焦亡改善血管性认知障碍作用机制

目的:探讨迷迭香吸嗅调控沉默信息调节因子1(SIRT1)、Nod样受体蛋白3(NLRP3)和半胱氨酸天门冬氨酸蛋白酶-1(Caspase-1)信号通路介导细胞焦亡改善血管性认知障碍(VCI)的作用机制。方法:选取雄性SD大鼠50只,随机分为正常组、假手术组、模型组、迷迭香组和迷迭香+SIRT1抑制剂组,每组10只。给予正常组、假手术组、模型组蒸馏水吸嗅,给予迷迭香组迷迭香精油吸嗅,迷迭香+SIRT1抑制剂组在迷迭香组基础上腹腔注射SIRT1抑制剂EX527。评价各组大鼠的学习记忆能力,海马神经元中SIRT1表达,海马组织中SIRT1、NLRP3和Caspase-1通路相关蛋白[剪切型半胱氨酸天冬氨酸蛋白酶-3(cleaved Caspase-3)]表达情况。结果:相较于正常组和假手术组,模型组逃避潜伏期更长、过站台次数更少(P<0.05);相较于模型组,迷迭香组和迷迭香+SIRT1抑制剂组逃避潜伏期缩短,过站台次数增加,但迷迭香组变化更为显著(P<0.05)。相较于正常组和假手术组,模型组海马神经元中SIRT1水平更低(P<0.05);相较于模型组,迷迭香组和迷迭香+SIRT1抑制剂组海马神经元中SIRT1有所升高,且迷迭香组升高更明显(P<0.05)。与正常组和假手术组比较,模型组海马组织中剪切型半胱氨酸天冬氨酸蛋白酶-3(cleaved Caspase-3)、NLRP3、凋亡相关斑点样蛋白(ASC)、白细胞介素(IL)-18和IL-1β明显升高,SIRT1明显降低(P<0.05);相较于模型组,迷迭香组、迷迭香+SIRT1抑制剂组海马组织中cleaved Caspase-3、NLRP3、ASC、IL-18和IL-1β有所降低,SIRT1有所升高,其中迷迭香组变化更大(P<0.05)。结论:迷迭香吸嗅可改善VCI大鼠的空间记忆和学习能力,保护海马组织,减少神经元凋亡,其作用机制与SIRT1、NLRP3和Caspase-1信号通路介导的细胞焦亡有关。