一种快速mRNA差异显示技术及用于分离辐射诱导转录子

介绍一种从不同类型细胞或不同生长状态细胞中分离差异表达基因的快速ｍＲＮＡ差异显示技术．其特点是不用同位素标记,操作简便,在普通琼脂糖凝胶电泳中就能分辨差异显示的ｃＤＮＡ带,便于ＤＮＡ回收和进一步重组克隆．用此方法成功地分离到电离辐射诱导转录子．     

Phosphorylation of CLK2 at Serine 34 and Threonine 127 by AKT Controls Cell Survival after Ionizing Radiation

AKT phosphorylates components of the intrinsic cell survival machinery and promotes survival to various stimuli. In the present study, we identified CDC-like kinase 2 (CLK2) as a new substrate of AKT activation and elucidated its role in cell survival to ionizing radiation. AKT directly binds to and phosphorylates CLK2 on serine 34 and threonine 127, in vitro and in vivo. CLK2 phosphorylation was detected in HeLa cells overexpressing active AKT. In addition, we demonstrated that ionizing radiation induces CLK2 phosphorylation via AKT activation. In contrast, the suppression of endogenous AKT expression by siRNA inhibited CLK2 phosphorylation in response to 2 gray of γ-ray or insulin. Furthermore, we examined the effect of CLK2 on the survival of irradiated CCD-18Lu cells overexpressing Myc-CLK2. CLK2 overexpression significantly increased cell growth and inhibited cell death induced by 2 gray. The role of CLK2 in cell survival to ionizing radiation was dependent on the phosphorylation of serine 34 and threonine 127. Our results suggest that AKT activation controls cell survival to ionizing radiation by phosphorylating CLK2, revealing an important regulatory mechanism required for promoting cell survival.     

Ameliorative Effect of Quercetin on Neurochemical and Behavioral Deficits in Rotenone Rat Model of Parkinson's Disease: Modulating Autophagy (Quercetin on Experimental Parkinson's Disease)

Autophagy is necessary for neuronal homeostasis and its dysfunction has been implicated in Parkinson's disease (PD) as it can exacerbate endoplasmic reticulum (ER) stress and ER stress‐induced apoptosis. Quercetin is a flavonoid known for its neuroprotective and antioxidant effects. The present study investigated the protective, autophagy‐modulating effects of quercetin in the rotenone rat model of PD. Rotenone was intraperitoneally injected at dose of 2 ml/kg/day for 4 weeks. Simultaneous intraperitoneal injection of quercetin was given at a dose of 50 mg/kg/day also for 4 weeks. Neurobehavioral changes were studied. Oxidative/antioxidant status, C/EBP homologous protein (CHOP), Beclin‐1, and dopamine levels were assessed. DNA fragmentation and histopathological changes were evaluated. This research work revealed that quercetin significantly attenuated rotenone‐induced behavioral impairment, augmented autophagy, ameliorated ER stress‐ induced apoptosis with attenuated oxidative stress. From the current study, quercetin can act as an autophagy enhancer in PD rat model and modulates the microenvironment that leads to neuronal death.     

Activation of ER Stress and Autophagy Induced by TDP-43 A315T as Pathogenic Mechanism and the Corresponding Histological Changes in Skin as Potential Biomarker for ALS with the Mutation

TAR DNA binding protein 43 (TDP-43) A315T mutation (TDP-43^A315T) has been found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) as a disease causing mutation with enhanced protein aggregation, formation of protease-resistant fragments, and neurotoxicity. However, the molecular mechanisms for its pathogenic effects are largely unknown. In this study, we demonstrate that TDP-43^A315T enhanced neuronal toxicity via activating endoplasmic reticulum (ER) stress-mediated apoptosis in SH-SY5Y cells. Moreover, autophagy was activated by overexpression of TDP-43^A315T in a self-defensive manner to decrease neuronal toxicity. Inhibition of autophagy attenuates TDP-43^A315T induced neuronal cell death. Furthermore, the expression levels of TDP-43, ER chaperone 78 kDa glucose-regulated protein (GRP-78), and autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) in the skin tissues from ALS patients with TDP-43^A315T mutation were markedly higher than those from the healthy control. Thus, our findings provide new molecular evidence for TDP-43^A315T neuropathology. In addition, the pathological change in the skin tissues of the patients with TDP-43^A315T mutation can be used as a quick diagnostic biomarker.     

抗阻运动对高脂饮食诱导的肥胖大鼠脂肪组织自噬和炎症因子的影响

为探讨运动对肥胖患者自噬活性和内脏脂肪组织炎症反应的影响,本研究将60只肥胖小鼠随机分为高脂饮食组(B)、正常饮食组(C)、正常饮食加耐力运动干预组(D)、正常饮食加耐力运动干预组(E)。D组和E组分别进行10周的耐力和抗阻运动,然后用RT-PCR检测自噬、炎症的基因和蛋白表达。结果显示,三个干预组的Lee指数和BFI均显著降低,2个运动组的Lee指数和BFI均显著降低,但差异无显著性;D组和E组Beclin 1表达较C组显著降低,p62表达明显升高;与C组相比,D组p62显著升高,E组无明显升高;与D组相比,E组Beclin 1基因表达增加,p62蛋白表达降低;与C组相比,D组和E组IL-6和IL-0蛋白表达均显著升高;10周不同运动干预对大鼠减脂效果无差异。运动后内脏脂肪组织的自噬活性受到抑制,尤其是无氧运动;肥胖患者运动干预前后内脏脂肪组织自噬和炎症的变化趋势一致,其中IL-10的变化最为明显。     

Heparin Prevents Intracellular Hyaluronan Synthesis and Autophagy Responses in Hyperglycemic Dividing Mesangial Cells and Activates Synthesis of an Extensive Extracellular Monocyte-adhesive Hyaluronan Matrix after Completing Cell Division

Growth-arrested rat mesangial cells (RMCs) at a G₀/G₁ interphase stimulated to divide in hyperglycemic medium initiate intracellular hyaluronan synthesis that induces autophagy/cyclin D3-induced formation of a monocyte-adhesive extracellular hyaluronan matrix after completing cell division. This study shows that heparin inhibits the intracellular hyaluronan synthesis and autophagy responses, but at the end of cell division it induces synthesis of a much larger extracellular monocyte-adhesive hyaluronan matrix. Heparin bound to RMC surfaces by 1 h, internalizes into the Golgi/endoplasmic reticulum region by 2 h, and was nearly gone by 4 h. Treatment by heparin for only the first 4 h was sufficient for its function. Streptozotocin diabetic rats treated daily with heparin showed similar results. Glomeruli in sections of diabetic kidneys showed extensive accumulation of autophagic RMCs, increased hyaluronan matrix, and influx of macrophages over 6 weeks. Hyaluronan staining in the glomeruli of heparin-treated diabetic rats was very high at week 1 and decreased to near control level by 6 weeks without any RMC autophagy. However, the influx of macrophages by 6 weeks was as pronounced as in diabetic glomeruli. The results are as follows: 1) heparin blocks synthesis of hyaluronan in intracellular compartments, which prevents the autophagy and cyclin D3 responses thereby allowing RMCs to complete cell division and sustain function; 2) interaction of heparin with RMCs in early G₁ phase is sufficient to induce signaling pathway(s) for its functions; and 3) influxed macrophages effectively remove the hyaluronan matrix without inducing pro-fibrotic responses that lead to nephropathy and proteinurea in diabetic kidneys.     

Caspase 1 Activation Is Protective against Hepatocyte Cell Death by Up-regulating Beclin 1 Protein and Mitochondrial Autophagy in the Setting of Redox Stress

Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed.     

Upregulation of cystic fibrosis transmembrane conductance regulator expression by oestrogen and Bak Foong Pill in mouse uteri

Although cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to be expressed in the female reproductive tract, its functional role in the uterus is not fully understood. The present study investigated a possible physiological role of CFTR by comparing the effects of 17beta-oestradiol and Bak Foong Pill (BFP), an over-the-counter Chinese medicine used for centuries for the treatment of various gynaecological disorders, on uterus size and the expression of CFTR in the uterus of ovariectomised mice using RT-PCR. Treatment of ovariectomised mice with 17beta-oestradiol (0.2 mg/kg, p.o.) for 12 days caused a significant increase in uterine wet weight compared to vehicle. However, treatment with BFP (3 g/kg, p.o.) for the same period failed to increase uterine wet weight, indicating a lack of direct oestrogen-like activity of BFP. Analysis of CFTR mRNA expression in the harvested uteri using RT-PCR showed that both 17beta-oestradiol and BFP induced an increase in CFTR mRNA expression in mouse uteri compared to levels observed in vehicle-treated animals. These results suggest that CFTR can be upregulated by oestrogen and BFP, however, the effect exerted by BFP does not seem to be mediated by direct oestrogen-like activity. Regulation of CFTR expression by both oestrogen and gynaecological medication BFP indicates an important role of CFTR in reproductive functions.     

Induced systemic resistance responses in perennial ryegrass against Magnaporthe oryzae elicited by semi‐purified surfactin lipopeptides and live cells of Bacillus amyloliquefaciens

The suppressive ability of several strains of cyclic lipopeptide‐producing Bacillus rhizobacteria to grey leaf spot disease caused by Magnaporthe oryzae has been documented previously; however, the underlying mechanism(s) involved in the induced systemic resistance (ISR) activity in perennial ryegrass (Lolium perenne L.) remains unknown. Root‐drench application of solid‐phase extraction (SPE)‐enriched surfactin and live cells of mutant Bacillus amyloliquefaciens strain FZB42‐AK3 (produces surfactin, but not bacillomycin D and fengycin) significantly reduced disease incidence and severity on perennial ryegrass. The application of the treatments revealed a pronounced multilayered ISR defence response activation via timely and enhanced accumulation of hydrogen peroxide (H₂O₂), elevated cell wall/apoplastic peroxidase activity, and deposition of callose and phenolic/polyphenolic compounds underneath the fungal appressoria in naïve leaves, which was significantly more intense in treated plants than in mock‐treated controls. Moreover, a hypersensitive response (HR)‐type reaction and enhanced expression of LpPrx (Prx, peroxidase), LpOXO4 (OXO, oxalate oxidase), LpPAL (PAL, phenylalanine ammonia lyase), LpLOXa (LOX, lipoxygenase), LpTHb (putative defensin) and LpDEFa (DEFa, putative defensin) in perennial ryegrass were associated with SPE‐enriched surfactin and live AK3 cell treatments, acting as a second layer of defence when pre‐invasive defence responses failed. The results indicate that ISR activity following surfactin perception may sensitize H₂O₂‐mediated defence responses, thereby providing perennial ryegrass with enhanced protection against M. oryzae.     

硒性白内障大鼠模型晶状体中GR和GSH-Px的表达

 为探讨硒性白内障大鼠晶状体中谷胱甘肽过氧化物酶 (GSH Px)和谷胱甘肽还原酶 (GR)的活性调节在硒性白内障形成中的作用及调节方式 ,采用半定量RT PCR方法 ,比较正常晶状体、核中心混浊晶状体 (核白 )和完全混浊晶状体 (全白 )中GSH Px和GR的mRNA水平及酶活性的变化 .研究发现 ,核白晶状体中 2种酶的活性和mRNA水平均升高 ,其中酶活性的升高幅度小于mRNA水平 .随着白内障的发展 ,2种酶的活性和mRNA水平均逐渐下降 .至晶状体全白时 ,2种酶的活性均显著低于正常 ;全白时GR的mRNA水平降至正常 ,GSH Px的mRNA水平则仍高于正常 .结果表明 ,硒性白内障形成与细胞内GSH Px和GR的活性调节密切相关 ,GSH Px和GR的活性调节可能主要发生在转录水平     

Comparative Cytotoxicity,Oxidative Stress,and Cytokine Secretion Induced by Two Cyanotoxin Variants,Microcystin LR and RR,in Human Intestinal Caco‐2 Cells

While MC‐LR and MC‐RR share significant structural similarity, MC‐RR is less cytotoxic than MC‐LR. In the current study, we have compared the effects of MC‐LR and MC‐RR in Caco‐2 cells by evaluating cytotoxicity, oxidative stress (reactive oxygen species production), and the cellular proinflammatory response (IL‐6 and IL‐8 production). Following treatment with 100 µM microcystins (MC), cytotoxicity was two‐fold greater with MC‐LR as compared to MC‐RR after 24 h exposure. Whereas the reactive oxygen species production and IL‐6 secretion were similar following a 24‐h treatment with either MC, 100 µM MC‐LR induced a five‐fold greater IL‐8 secretion when compared to MC‐RR. Our study has demonstrated that, although both MC‐LR and MC‐RR induced some cytotoxicity in human intestinal cells, a major difference in IL‐8 production was observed between the two variants. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:253‐258, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21482     

Oxidative Stress and Hepatocellular Injury Induced by Oral Administration of Cr3+ in Chicken

This study aims to investigate the oxidative stress and hepatocellular injury induced by Cr³⁺ in chicken. Different doses of CrCl₃ solutions (50% LD₅₀, 25% LD₅₀, and 12.5% LD₅₀) and equivalent water were orally administered to chicken. Chicken liver samples were measured for the activities of antioxidant enzymes, the contents of glutathione, total antioxidant capacity (T‐AOC), malondialdehyde (MDA), and hydrogen peroxide to indirectly evaluate the oxidative stress in chicken liver. Results indicated that the oral administration of Cr³⁺ at high dose significantly increased (P < 0.05) the MDA levels after 28 days of exposure, with decreased T‐AOC, glutathione, and antioxidant enzymes activities. Low and medium doses groups show that T‐AOC, glutathione, and antioxidant enzymes activities increased after 14 days, then decreased gradually, but low and medium groups higher than control group, only high group lower than control group finally. These statistics and histopathological analysis suggest that high dose and long‐term exposure of Cr³⁺ induce oxidative stress and hepatocellular injury.     

Alantolactone promotes ER stress‐mediated apoptosis by inhibition of TrxR1 in triple‐negative breast cancer cell lines and in a mouse model

Triple‐negative breast cancer (TNBC) is a subtype of breast cancer with poor clinical outcome and currently no effective targeted therapies are available. Alantolactone (ATL), a sesquiterpene lactone, has been shown to have potential anti‐tumour activity against various cancer cells. However, the underlying mechanism and therapeutic effect of ATL in the TNBC are largely unknown. In the present study, we found that ATL suppresses TNBC cell viability by reactive oxygen species (ROS) accumulation and subsequent ROS‐dependent endoplasmic reticulum (ER) stress both in vitro and in vivo. Thioredoxin reductase 1 (TrxR1) expression and activity of were significantly up‐regulated in the TNBC tissue specimens compare to the normal adjacent tissues. Further analyses showed that ATL inhibits the activity of TrxR1 both in vitro and in vivo in TNBC and knockdown of TrxR1 in TNBC cells sensitized ATL‐induced cell apoptosis and ROS increase. These results will provide pre‐clinical evidences that ATL could be a potential therapeutic agent against TNBC by promoting ROS‐ER stress‐mediated apoptosis through partly targeting TrxR1.     

Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.     

Inhibition of deubiquitination by PR‐619 induces apoptosis and autophagy via ubi‐protein aggregation‐activated ER stress in oesophageal squamous cell carcinoma

ObjectivesTargeting the deubiquitinases (DUBs) has become a promising avenue for anti‐cancer drug development. However, the effect and mechanism of pan‐DUB inhibitor, PR‐619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated.Materials and MethodsThe effect of PR‐619 on ESCC cell growth and cell cycle was evaluated by CCK‐8 and PI staining. Annexin V‐FITC/PI double staining was performed to detect apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine autophagy. Intercellular Ca²⁺ concentration was monitored by Fluo‐3AM fluorescence. The accumulation of ubi‐proteins and the expression of the endoplasmic reticulum (ER) stress‐related protein and CaMKKβ‐AMPK signalling were determined by immunoblotting.ResultsPR‐619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR‐619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered apoptosis by the ATF4‐Noxa axis. Moreover, the ER stress increased cytoplasmic Ca²⁺ and then stimulated autophagy through Ca²⁺‐CaMKKβ‐AMPK signalling pathway. Ubiquitin E1 inhibitor, PYR‐41, could reduce the accumulation of ubi‐proteins and alleviate ER stress, G2/M cell cycle arrest, apoptosis and autophagy in PR‐619‐treated ESCC cells. Furthermore, blocking autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and apoptosis induced by PR‐619.ConclusionsOur findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR‐619) and imply that targeting DUBs may be a potential anti‐ESCC strategy.     

Regulation of SOBIR1 accumulation and activation of defense responses in bir1–1 by specific components of ER quality control

Receptor‐like kinases play diverse roles in plant biology. Arabidopsis BAK1‐INTERACTING RECEPTOR‐LIKE KINASE 1 (BIR1) functions as a negative regulator of plant immunity. bir1‐1 mutant plants display spontaneous cell death and constitutive defense responses that are dependent on SUPPRESSOR OF BIR1,1 (SOBIR1) and PHYTOALEXIN DEFICIENT4 (PAD4). Here we report that mutations in three components of ER quality control, CALRETICULIN3 (CRT3), ER‐LOCALIZED DnaJ‐LIKE PROTEIN 3b (ERdj3b) and STROMAL‐DERIVED FACTOR‐2 (SDF2), also suppress the spontaneous cell death and constitutive defense responses in bir1‐1. Further analysis revealed that accumulation of the SOBIR1 protein is reduced in crt3‐1 and erdj3b‐1 mutant plants. These data suggest that ER quality control plays important roles in the biogenesis of SOBIR1, and is required for cell death and defense responses in bir1‐1.     

Thymol and Carvacrol Prevent Cisplatin‐Induced Nephrotoxicity by Abrogation of Oxidative Stress,Inflammation, and Apoptosis in Rats

The aim of the present study is to assess the possible protective effects of thymol and carvacrol against cisplatin (CP)‐induced nephrotoxicity. A single dose of CP {6 mg/kg, intraperitoneally (i.p.)} injected to male rats revealed significant increases in serum urea, creatinine, and tumor necrosis factor alpha levels. It also increased kidney contents of malondialdehyde and caspase‐3 activity with significant reduction in serum albumin, kidney content of reduced glutathione as well as catalase, and superoxide dismutase activity as compared to that of the control group. In contrast, administration of thymol {20 mg/kg, orally (p.o.)} and/or carvacrol (15 mg/kg, p.o.) for 14 days before CP injection and for 7 days after CP administration restored the kidney function and examined oxidative stress parameters. In conclusion, thymol was more effective nephroprotective than carvacrol. Moreover, a combination of thymol and carvacrol had a synergistic nephroprotective effect that might be attributed to antioxidant, anti‐inflammatory, and antiapoptotic activities.     

调强放射治疗对食管癌患者外周血Treg细胞、血清肿瘤标志物及应激激素水平的影响

摘要 目的：观察调强放射（IMRT）治疗对食管癌患者外周血调节性T细胞（Treg）细胞、血清肿瘤标志物及应激激素水平的影响。方法：选取2019年9月~2021年3月期间自贡市第一人民医院收治的食管癌患者80例,按照随机数字表法将患者分为对照组（普通适形放疗,40例）和研究组（IMRT治疗,40例）。对比两组临床总有效率、外周血Treg细胞比例、血清肿瘤标志物及应激激素水平,观察放疗期间出现的不良反应。结果：研究组的临床总有效率高于对照组（P<0.05）。两组放疗后癌胚抗原（CEA）、糖类抗原 199（CA199）、细胞角蛋白19片段（CYFRA21-1）均下降,且研究组低于对照组（P<0.05）。两组放疗后生长激素（GH）、催乳素（PRL）均下降,且研究组低于对照组（P<0.05）。两组放疗后外周血Treg细胞占CD4⁺T细胞的比例下降,且研究组较对照组低（P<0.05）。不良反应发生率两组组间对比,未见显著性差异（P>0.05）。结论：食管癌患者采用IMRT治疗,可有效降低外周血Treg细胞比例、血清肿瘤标志物及应激激素水平,疗效较好,具有一定的临床应用价值。