Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1

CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.     

Quercetin attenuates oxidative stress-induced apoptosis via SIRT1/AMPK-mediated inhibition of ER stress in rat chondrocytes and prevents the progression of osteoarthritis in a rat model

Apoptosis of chondrocytes are the main initiator of osteoarthritis (OA) and can be explained by oxidative stress and endoplasmic reticulum (ER) stress, thus the pharmacological interventions aimed at inhibiting of these pathways may be a promising approach for the management of OA. Quercetin is a member of the flavonoid family and has antioxidant and anti-inflammatory properties in degenerative diseases. However, its effects and potential mechanisms on the pathological process of OA are not very clear. The present study aimed to investigate the protective effects of quercetin on OA and the underlying mechanisms. The tert-butyl hydroperoxide (TBHP)-stimulated rat chondrocytes and destabilization of the medial meniscus OA rat model was used to explore the protective effects of quercetin. Our results showed that quercetin treatment can attenuate oxidative stress, ER stress, and associated apoptosis. Moreover, quercetin inhibited ER stress through activating the sirtuin1/adenosine monophosphate-activated protein kinase (SIRT1/AMPK) signaling pathway. The protective effects of quercetin were also observed in OA rat model which is evidenced by abolished cartilage degeneration and decreased chondrocytes apoptosis in the knee joints. Our results suggested that quercetin is a promising treatment for OA.     

猪肌肉组织Col3a1基因表达的发育性变化及其对肌内胶原特性的影响(英文)

以30~90kg体重莱芜猪和40~100kg体重鲁莱黒猪共84头去势公猪为试验对象(每组6头),采用相对定量RT-PCR方法,以β-actin作为内标,研究肌肉中编码Ⅲ型胶原的Col3a1基因表达的发育性变化及其对肌肉中胶原蛋白含量和性质(溶解度)的影响。结果表明:研究的两个品种猪肌肉中Col3a1基因表达的发育性变化基本一致,即随体重的增加,肌肉中Col3a1 mRNA表达呈逐渐增加趋势,但莱芜猪和鲁莱黑猪分别在70kg和80kg体重组表达量略有下降。总体上莱芜猪肌肉组织Col3a1 mRNA表达丰度显著高于鲁莱黑猪(P<0.05)。相关分析表明,莱芜猪肌肉组织Col3a1 mRNA表达的发育性变化与总胶原和不溶性胶原含量呈极显著正相关(P<0.01),与胶原溶解度呈极显著负相关(P<0.01)。鲁莱黑猪肌肉组织Col3a1 mRNA表达的发育性变化与不溶性胶原和胶原溶解度分别呈显著正相关和负相关(P<0.05)。研究结果提示:猪肌肉组织中Col3a1基因表达具有明显的体重发育和品种特征,其mRNA表达对于肌内胶原的含量和性质有重要影响。     

Matrilin-3 Induction of IL-1 receptor antagonist Is required for up-regulating collagen II and aggrecan and down-regulating ADAMTS-5 gene expression

Introduction

Deletion or mutation of the gene encoding the cartilage extracellular matrix (ECM) protein matrilin-3 (MATN3) results in the early onset of osteoarthritis (OA), suggesting chondroprotective properties of MATN3. To understand the mechanisms underlying these properties, we determined the effects of MATN3 protein on the expression of several key anabolic and catabolic genes involved in chondrocyte homeostasis, and the dependence of such regulation on the anti-inflammatory cytokine: IL-1 receptor antagonist (IL-1Ra).

Methods

The effects of recombinant human (rh) MATN3 protein were examined in C28/I2 immortalized human chondrocytes, primary human chondrocytes (PHCs), and primary mouse chondrocytes (PMCs). Messenger RNA levels of IL-1Ra, COL2A1, ACAN, MMP-13, and ADAMTS-4 and -5 were determined using real-time RT-PCR. Knocking down IL-1Ra was achieved by siRNA gene silencing. IL-1Ra protein levels were quantified by ELISA and the Bio-Plex Suspension Array System. COL2A1 protein level was quantified using Western blot analysis. Statistic analysis was done using the two-tailed t-test or one-way ANOVA.

Results

rhMATN3 protein induced gene expression of IL-1Ra in C28/I2 cells, PHCs, and PMCs in a dose- and time-dependent manner. Treatment of C28/I2 cells and PHCs with MATN3 protein stimulated gene expression of COL2A1 and ACAN. Conversely, mRNA levels of COL2A1 and ACAN were decreased in MATN3 KO mice. MATN3 protein treatment inhibited IL-1β-induced MMP-13, ADAMTS-4 and ADAMTS-5 in C28/I2 cells and PHCs. Knocking down IL-1Ra abolished the MATN3-mediated stimulation of COL2A1 and ACAN and inhibition of ADAMTS-5, but had no effect on MATN3 inhibition of MMP-13 mRNA.

Conclusion

Our findings point to a novel regulatory role of MATN3 in cartilage homeostasis due to its capacity to induce IL-1Ra, to upregulate gene expression of the major cartilage matrix components, and to downregulate the expression of OA-associated matrix-degrading proteinases in chondrocytes. The chondroprotective properties of endogenous MATN3 depend partly on its induction of IL-1Ra. Our findings raise a possibility to use rhMATN3 protein for anti-inflammatory and chondroprotective therapy.     

A Novel Zinc Finger Protein 219-like (ZNF219L) is Involved in the Regulation of Collagen Type 2 Alpha 1a (col2a1a) Gene Expression in Zebrafish Notochord

The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly expressed in the zebrafish midbrain-hindbrain boundary, hindbrain, and notochord during development. The znf219L morpholino knockdown caused partial abnormal notochord phenotype and reduced expression of endogenous col2a1a in the notochord specifically. In addition, ZNF219L could recognize binding sites with GGGGG motifs and trigger augmented activity of the col2a1a promoter in a luciferase assay. Furthermore, in vitro binding experiments revealed that ZNF219L recognizes the GGGGG motifs in the promoter region of the zebrafish col2a1a gene through its sixth and ninth zinc finger domains. Taken together, our results reveal that ZNF219L is involved in regulating the expression of col2a1a in zebrafish notochord specifically.     

Human Immunodeficiency Virus Type 1 Enhancer-binding Protein 3 Is Essential for the Expression of Asparagine-linked Glycosylation 2 in the Regulation of Osteoblast and Chondrocyte Differentiation

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.     

Upregulation of Ras/Raf/ERK1/2 signaling and ERK5 in the brain of autistic subjects

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. A number of studies have shown that the Ras/Raf/ERK1/2 (extracellular signal-regulated kinase) signaling pathway plays important roles in the genesis of neural progenitors, learning and memory. Ras/Raf/ERK1/2 and ERK5 have also been shown to have death-promoting apoptotic roles in neural cells. Recent studies have shown a possible association between neural cell death and autism. In addition, two recent studies reported that a deletion of a locus on chromosome 16, which included the mitogen-activated protein kinase 3 (MAPK3) gene that encodes ERK1, is associated with autism. Most recently, our laboratory detected that Ras/Raf/ERK1/2 signaling activities were significantly enhanced in the brain of BTBR mice that model autism, as they exhibit many autism-like behaviors. We thus hypothesized that Ras/Raf/ERK1/2 signaling and ERK5 could be abnormally regulated in the brain of autistic subjects. In this study, we show that the expression of Ras protein was significantly elevated in the frontal cortex of autistic subjects. C-Raf phosphorylation was increased in the frontal cortex, while both C-Raf and A-Raf activities were enhanced in the cerebellum of autistic subjects. We also detected that both the protein expression and activities of ERK1/2 were significantly upregulated in the frontal cortex of autistic subjects, but not in the cerebellum. Furthermore, we showed that ERK5 protein expression is upregulated in both frontal cortex and cerebellum of autistic subjects. These results suggest that the upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities mainly found in the frontal cortex of autistic subjects may be critically involved in the pathogenesis of autism.     

Continuous high expression of XBP1 and GRP78 is important for the survival of bone marrow cells in CCl4-treated cirrhotic liver

We have previously shown that infusion of bone marrow cells (BMC) improves CCl₄-induced cirrhosis. However, it is unclear why the injected BMC are resistant to CCl₄ damage and subsequently improve the local microenvironment in damaged liver. To analyze the cellular phenomena involved in this process, we studied the damaged liver using electron microscopy. We found that CCl₄ caused rough endoplasmic reticula to swell in hepatocytes. To analyze the gene expression patterns associated with this process, we conducted PCR-selected suppressive subtractive hybridization. We found that expression levels of HSP84, HSP40, and XBP1 differed markedly between control liver and liver infused with BMC. Immunohistochemical staining revealed that expression levels of HSP84 and HSP40 were markedly higher in the early phase of differentiation immediately after BMC infusion, but decreased over time. XBP1 expression remained high during the late phase, and GRP78 expression increased with XBP1 activation. We also found that GFP-positive BMC expressed XBP1 and GRP78. XBP1 and GRP78 are associated with ER stress. Thus, continuous high XBP1 and GRP78 expression might be essential for the survival and proliferation of BMC in a CCl₄-induced persistent liver damage environment.     

非生物胁迫和植物激素对与水稻OsRac5结合的含CC域蛋白编码基因OsMY1和OsMY2表达的影响

ROP(Rho of plant)作为植物中唯一具有信号传递功能的小GTP结合蛋白,在极性生长、发育和环境应答等过程中具有重要作用.本文采用酵母双杂交技术,从水稻 幼穗筛选出2种与水稻ROP家族成员OsRac5互作蛋白的基因OsMY1和OsMY2.序列分析显示,OsMY1与OsMY2的核苷酸、氨基酸序列的相似性分别为14%和49%,均含有卷曲螺旋结构域;GST pull down分析显示,OsMY1可以和激活型OsRac5在体外结合;实时定量PCR分析显示,OsMY1和OsMY2在水稻生长发育时期的根、茎、叶中广泛表达,尤其在幼穗中的表达量显著高于其它组织.干旱、低温、高盐等非生物胁迫和植物激素吲哚乙酸(IAA)、脱落酸(ABA)、赤霉素（GA）处理能不同程度地上调OsMY1和OsMY2 基因在水稻幼苗中的表达,但油菜素内酯（BR）、水杨酸（SA）、6-苄氨基嘌呤 （6-BA）对2种基因的表达没有显著的影响.本文为进一步研究OsMY1和OsMY2基因在水稻生长发育、植物激素和胁迫应答中的功能提供了重要依据,为研究OsMY1、 OsMY2和OsRac5蛋白质相互作用的功能联系和作用机制奠定了基础.     

艾迪注射液联合XELOX方案对晚期结直肠癌患者Th1/Th2免疫平衡和血清肿瘤标志物的影响

摘要 目的：探讨艾迪注射液联合卡培他滨和奥沙利铂方案（XELOX方案）对晚期结直肠癌患者Th1/Th2免疫平衡和血清肿瘤标志物的影响。方法：病例选取自我院2018年7月~2021年6月期间收治的晚期结直肠癌患者80例,采用计算机随机生成的方式分为对照组（XELOX方案）和研究组（艾迪注射液联合XELOX方案）,各为40例。对比两组疗效、Karnofsky （KPS）评分、Th1/Th2免疫平衡和血清肿瘤标志物,观察两组不良反应发生率。结果：研究组的客观缓解率 （ORR）、疾病控制率（DCR）高于对照组（P<0.05）。两组治疗后KPS评分升高,且研究组高于对照组（P<0.05）。两组治疗后Th1、干扰素-γ（IFN-γ）、Th1/Th2降低,但研究组高于对照组（P<0.05）。两组治疗后Th2、白细胞介素-10（IL-10）升高,但研究组低于对照组（P<0.05）。两组治疗后癌胚抗原(CEA)、糖类抗原199 ( CA199) 、糖类抗原724( CA724)下降,且研究组低于对照组（P<0.05）。研究组不良反应发生率低于对照组（P<0.05）。结论：艾迪注射液联合XELOX方案治疗晚期结直肠癌患者,可在一定程度上阻止肿瘤疾病进展,减轻免疫抑制,提高近期疗效,减少因药物副作用引起的不良反应,进而改善患者生活质量。     

一种环境激素联合冷应激大鼠模型阴茎组织中 Sprouty 2、ERK1/2表达的研究

目的:研究Sprouty 2(Spry 2)和细胞外调节蛋白激酶(ERK1/2)在环境激素联合冷应激大鼠模型阴茎组织中的表达,并观察伊木萨克片对二者表达的影响。方法:选用30只正常的雄性SD大鼠,其中10只为正常对照组(N组),余20只为造模组,采用富含环境雌激素饲料+寒冷环境的干预条件建立复合性应激大鼠模型(20 w),其中10只大鼠采用伊木萨克片干预2 w(Y组),剩余10只为模型组(M组)。采用免疫组化方法检测各组大鼠阴茎组织中Spry 2、ERK1/2的表达。结果:M组大鼠阴茎组织中Spry2表达低于N组(P0.05),Y组大鼠阴茎组织中Spry 2表达明显高于M组(P0.05)。三组大鼠阴茎组织中总ERK1/2(t-ERK1/2)的表达比较差异均无统计学意义(P0.05)。M组大鼠阴茎组织中磷酸化ERK1/2(p-ERK1/2)表达高于N组(P0.05),Y组大鼠阴茎组织中p-ERK1/2表达明显低于M组(P0.05)。结论:Spry 2表达下调和ERK1/2活化可能促进应激反应的发生发展,伊木萨克片可能通过上调Spry 2的表达和减少ERK1/2的活化发挥抗应激作用。     

Retinoic Acid-Mediated G1 Arrest Is Associated with Induction of p27 and Inhibition of Cyclin-Dependent Kinase 3 in Human Lung Squamous Carcinoma CH27 Cells

Retinoids are promising agents for the prevention and treatment of several human malignancies including lung cancer. In this study, the effect of retinoic acid (RA) on cell growth and the mechanism of growth modulation were examined in human lung squamous carcinoma CH27 cells. Here we report that RA mediated the dose- and time-dependent growth arrest in G1 phase, accompanied by the up-regulation of p27^Kip1 and the down-regulation of the cyclin-dependent kinase 3 (Cdk3) and p21^CIP1/Waf1 proteins. Furthermore, RA-induced growth arrest of CH27 cells was also associated with increased retinoic acid receptor β (RARβ) and reduced c-Myc expression. However, RA had no effect on the levels of cyclins A, D1, D3, E, or H, or on Cdk2, Cdk4, Cdk5, CDk6, Cdk7, p16^Ink4A, p15^Ink4B, p53, or pRb proteins in CH27 cells. Evaluation of the kinase activity of cyclin–Cdk complexes showed that RA increases p27^Kip1 expression in CH27 cells leading to markedly reduced cyclin A/Cdk2 kinase activity and slightly reduced cyclin E/Cdk2 kinase activity, with no effect on cyclin D/Cdk4 and cyclin D/Cdk6 activities. Moreover, coincident with the decrease in kinase activity was a drastic increase in cyclin A-bound p27^Kip1. These results suggest that increases in the levels of p27^Kip1 and its binding to cyclin A, as well as reduction of Cdk3 protein expression, are strong candidates for the cell cycle regulator that prevents the entry into the S phase in RA-treated CH27 cells, with prolongation of G1 phase and inhibition of DNA synthesis.     

Variant rs2237892 of KCNQ1 Is Potentially Associated with Hypertension and Macrovascular Complications in Type 2 Diabetes Mellitus in A Chinese Han Population

KCNQ1 has been identified as a susceptibility gene of type 2 diabetes mellitus(T2DM) in Asian populations through genome-wide association studies. However, studies on the association between gene polymorphism of KCNQ1 and T2 DM complications remain unclear. To further analyze the association between different alleles at the single nucleotide polymorphism(SNP) rs2237892 within KCNQ1 and TD2 M and its complications, we conducted a case-control study in a Chinese Han population. The C allele of rs2237892 variant contributed to susceptibility to T2DM(odds ratio [OR], 1.45; 95% confidence interval [CI], 1.20–1.75). Genotypes CT(OR, 1.97; 95% CI,1.24–3.15) and CC(OR, 2.49; 95% CI, 1.57–3.95) were associated with an increased risk of T2 DM. Multivariate regression analysis was performed with adjustment of age, gender, and body mass index. We found that systolic blood pressure(P = 0.015), prevalence of hypertension(P = 0.037), and risk of macrovascular disease(OR, 2.10; CI, 1.00–4.45) were significantly higher in subjects with the CC genotype than in the combined population with genotype either CT or     

Modulation of endoplasmic reticulum chaperone GRP78 by high glucose in hippocampus of streptozotocin-induced diabetic mice and C6 astrocytic cells

Diabetes mellitus is known to increase the risk of neurodegeneration, and both diseases are reported to be linked to dysfunction of endoplasmic reticulum (ER). Astrocytes are important in the defense mechanism of central nervous system (CNS), with great ability of tolerating accumulation of toxic substances and sensitivity in Ca²⁺ homeostasis which are two key functions of ER. Here, we investigated the modulation of the glucose-regulated protein 78 (GRP78) in streptozotocin (STZ)-induced diabetic mice and C6 cells cultured in high glucose condition. Our results showed that more reactive astrocytes were presented in the hippocampus of STZ-induced diabetic mice. Simultaneously, decrease of GRP78 expression was found in the astrocytes of diabetic mice hippocampus.     

吸烟对大鼠肺组织B7-1/B7-2及其相关配体表达的影响

目的通过研究吸烟对大鼠肺组织B7-1/B7-2及其相关配体表达的影响,探讨专职抗原提呈细胞（APC）在吸烟所致肺部慢性炎症发生发展中的作用。方法将30只健康雄性Wistar大鼠随机分为不吸烟组、吸烟6周组和吸烟12周组,每组10只。采用免疫组化半定量法测定大鼠气道周围肺间质中慢性炎症细胞胞膜B7-1、B7-2、CD28和CTLA-4的表达水平。结果吸烟6周组与吸烟12周组大鼠肺组织B7-1、B7-2、CD28和CTLA-4表达量较不吸烟组均显著增高（P〈0.01）,吸烟12周组较吸烟6周组表达量也均增高（P〈0.01）,随吸烟时间的延长各指标表达量均呈上升趋势。结论吸烟可引起大鼠肺组织B7/CD28/CTLA-4表达水平的增高,提示APC可能在吸烟所致肺部慢性炎症发生发展中起重要作用。