耐力运动对关节炎大鼠氧化应激与软骨炎性表达及CHRNA7信号的影响

目的 探讨耐力运动对关节炎大鼠氧化应激、软骨炎性表达及CHRNA7信号表达的影响。方法 将50只SD大鼠随机分为健康组、关节炎组、低耐力组、中耐力组、高耐力组，每组10只。检测各组大鼠行为学及氧化应激水平差异性；采用热板致痛阈值（PWTL）及机械性撤足阈值（PWT）；酶联免疫法检测各组大鼠炎性因子水平；采用Western Blot法检测滑膜组织中a7nAChR、STAT3表达情况；HE染色观察大鼠膝关节软骨组织的形态。结果 HE染色结果显示，健康组大鼠膝关节软骨表面平整光滑，无裂缝或缺损；关节炎组大鼠膝关节软骨的表面不平整，有缺损；低耐力组大鼠膝关节软骨的表面比较光滑、平整；中、高耐力组大鼠膝关节软骨的表面平整，软骨细胞排列较为整齐。 与健康组相比，关节炎组TNF-α、IL-1水平升高（P＜0.05）；与关节炎组相比，低耐力组、中耐力组和高耐力组TNF-α、IL-1水平降低明显（P＜0.05）；与低耐力相比，中耐力组TNF-α、IL-1降低明显（P＜0.05）；与中耐力组相比，高耐力组TNF-α、IL-1水平升高（P＜0.05）。与健康组相比，关节炎组大鼠PWTL、PWT、SOD、GSH-Px、α7nAChR、STAT3表达水平明显降低，MDA水平升高(P＜0.05)；与关节炎组大鼠相比，低、中和高耐力组大鼠PWTL、PWT、SOD、GSH-Px、α7nAChR、STAT3水平升高，MDA水平降低(P＜0.05)；与低耐力组相比，中耐力组PWTL、PWT、SOD、GSH-Px、α7nAChR、STAT3水平升高，MDA水平降低(P＜0.05)；与中耐力组相比，高耐力组PWTL、PWT、SOD、GSH-Px、α7nAChR、STAT3水平下降，MDA水平升高(P＜0.05)。结论 中强度耐力运动可抑制TNF-α、IL-1炎性水平，改善疼痛水平及氧化应激反应，对关节起到保护作用，其机制可能与调控α7nAChR/STAT3蛋白表达有关。     

Oncogenic mutations of thyroid hormone receptor β

The C-terminal frame-shift mutant of the thyroid hormone receptor TRβ1, PV, functions as an oncogene. An important question is whether the oncogenic activity of mutated TRβ1 is uniquely dependent on the PV mutated sequence. Using four C-terminal frame-shift mutants—PV, Mkar, Mdbs, and AM—we examined that region in the oncogenic actions of TRβ1 mutants. Remarkably, these C-terminal mutants induced similar growth of tumors in mouse xenograft models. Molecular analyses showed that they physically interacted with the p85α regulatory subunit of PI3K similarly in cells. In vitro GST-binding assay showed that they bound to the C-terminal Src-homology 2 (CSH2) of p85α with markedly higher avidity. The sustained association of mutants with p85α led to activation of the common PI3K-AKT-ERK/STAT3 signaling to promote cell proliferation and invasion and to inhibit apoptosis. Thus, these results argue against the oncogenic activity of PV being uniquely dependent on the PV mutated sequence. Rather, these four mutants could favor a C-terminal conformation that interacted with the CSH2 domain of p85α to initiate activation of PI3K to relay downstream signaling to promote tumorigenesis. Thus, we propose that the mutated C-terminal region of TRβ1 could function as an “onco-domain” and TRβ1 is a potential therapeutic target.     

人脐带间充质干细胞对放射线照射后人肺成纤维细胞中经典WNT/β-catenin通路的影响

目的探讨人脐带间充质干细胞(HUMSCs)对放射线照射后的人肺成纤维细胞(HLFs)影响的机制。方法 HLFs分为照射组(A)、照射+共培养组(B)和正常对照组(C),A组和B组在5Gy X射线照射后,B组与HUMSCs通过Transwell系统共培养。照射12、24、36h后western blot检测HLFs中α-SMA、GSK-3β、p-GSK-3β和胞核内β-catenin蛋白表达,ELISA法检测细胞培养液中WISP-1、SFRP-1蛋白表达。Real time PCR检测正常和B组第36h HUMSCs中SFRP-1 mRNA表达。结果放射线照射后第24h、36h,HLFs中α-SMA、p-GSK-3β、p-GSK-3β/GSK-3β、胞核内β-catenin,以及培养液中WISP-1蛋白表达水平,A组较C组明显升高,而B组较A组明显下降,差异有统计学意义(P0.01);B组培养液中SFRP-1蛋白水平较A组、C组各时间点均显著升高,差异有统计学意义(P0.05);B组第36h的HUMSCs中SFRP-1 mRNA表达明显高于正常HUMSCs,差异有统计学意义(P0.01)。结论 HUMSCs可抑制放射线导致的HLFs中经典WNT/β-catenin通路的活化,并有可能通过该途径影响肺成纤维细胞的分化。     

Fendiline inhibits proliferation and invasion of pancreatic cancer cells by interfering with ADAM10 activation and β-catenin signaling

ADAM10 (A Disintegrin and Metalloprotease Domain 10) affects the pathophysiology of various cancers, and we had shown that inhibition of ADAM10 sensitizes pancreatic cancer cells to gemcitabine. ADAM10 is activated in response to calcium influx, and here we examined if calcium channel blockers (CCB) would impede ADAM10 activation and affect biology of pancreatic cancer cells. We find that the CCB, fendiline, significantly reduces proliferation, migration, invasion, and anchorage independent growth of pancreatic cancer cells. This was associated with ADAM10 inhibition and its localization at the actin-rich membrane protrusions. Further, fendiline-treated cells formed cadherin-catenin positive tight adherens junctions and elicited defective protein trafficking and recycling. Furthermore, the expression of β-catenin target genes, cyclinD1, c-Myc and CD44, were significantly decreased, suggesting that fendiline might prevent cell proliferation and migration by inhibiting ADAM10 function, cadherin proteolysis and stabilization of cadherin-catenin interaction at the plasma membrane. This will subsequently diminish β-catenin intracellular signaling and repress TCF/LEF target gene expression. Supporting this notion, RNAi-directed downregulation of ADAM10 in cancer cells decreased the expression of cyclinD1, c-Myc and CD44. Furthermore, analysis of human pancreatic tumor tissue microarrays and lysates showed elevated levels of ADAM10, suggesting that aberrant activation of ADAM10 plays a fundamental role in growth and metastasis of PDACs and inhibiting this pathway might be a viable strategy to combat PDACs.     

Reconstruction of an active SOCS3-based E3 ubiquitin ligase complex in vitro: identification of the active components and JAK2 and gp130 as substrates

Abstract

SOCS3 (suppressor of cytokine signaling 3) inhibits the intracellular signaling cascade initiated by exposure of cells to cytokines. SOCS3 regulates signaling via two distinct mechanisms: directly inhibiting the catalytic activity of Janus kinases (JAKs) that initiate the intracellular signaling cascade and catalysing the ubiquitination of signaling components by recruiting components of an E3 ubiquitin ligase complex. Here we investigate the latter mode-of-action biochemically by reconstructing a SOCS3-based E3 ubiquitin ligase complex in vitro using fully purified, recombinant components and examining its ability to promote the ubiquitination of molecules involved in the cytokine signaling cascade. We show that SOCS3 is an active substrate recruitment module for a Cullin5-based E3 ligase and have defined the core protein components required for ubiquitination. SOCS3-induced polyubiquitination was rapid and could proceed through a number of different ubiquitin lysines. SOCS3 catalyzed the ubiquitination of both the IL-6 receptor common chain (gp130) and JAK2.     

慢性内脏痛及其信号转导通路的研究进展

背景 慢性内脏痛是功能性胃肠病(functional gastrointestinal disorders,FGID)和慢性胰腺炎(chronic pancreatitis,CP)的主要症状,这种疼痛不但降低患者的生活质量也是其寻求医疗帮助的主要原因,但目前有关内脏痛的确切机制仍不清楚,因而其治疗效果不佳. 目的 就当前慢性内脏痛及其信号转导通路的研究进展进行综述. 内容 介绍内脏痛及其信号转导通路、神经胶质细胞特别是脊髓小胶质细胞在慢性内脏痛中的作用. 趋向 脊髓小胶质细胞可能是未来慢性内脏痛治疗的新靶点.     

STANDARDIZED CLINICAL PATHWAYS FOR ESOPHAGECTOMY ARE NOT A REALITY IN BRAZIL,EVEN WITH A HIGH PREVALENCE OF ESOPHAGEAL CANCER AND ACHALASIA

Background:

The adoption of standardized protocols and specialized multidisciplinary teams for esophagectomy involve changes in routines with the implantation of expensive clinical practices and deviations from ingrained treatment philosophies.

Aim:

To evaluate the prevalence of standardized protocols and specialized multidisciplinary teams in São Paulo state, Brazil.

Methods:

Institutions that routinely perform esophagectomies in São Paulo were contacted and questioned about the work team involved in the procedure and the presence of standardized routines in the preoperatory care.

Results:

Fifteen centers answered the questionnaire: 10 (67%) public institutions and five (33%) private. There were seven (47%) medical schools, six (40%) with a residency program and two (13%) nonacademic institutions. The mean number of esophagectomies per year was 23. There was a multidisciplinary pre-operative team in nine (60%). There was a multidisciplinary postoperative team in 11 (73%). Early mobilization protocol was adopted in 12 (80%) institutions, early feeding in 13 (87%), routinely epidural in seven (47%), analgesia protocol in seven (47%), hydric restriction in six (40%), early extubation in six (40%), standardized hospitalization time in four (27%) and standardized intensive care time in two (13%).

Conclusion:

The prevalence of standardized protocols and specialized teams is very low in Sao Paulo state, Brazil. The presence of specialized surgeons is a reality and standardized protocols related directly to surgeons have higher frequency than those related to other professionals in the multidisciplinary team.     

Wnt/β‐catenin signaling of cartilage canal and osteochondral junction chondrocytes and full thickness cartilage in early equine osteochondrosis

The objective of this study was to elucidate gene and protein expression of Wnt signaling molecules in chondrocytes of foals having early osteochondrosis (OC) versus normal controls. The hypothesis was that increased expression of components of Wnt signaling pathway in osteochondral junction (OCJ) and cartilage canal (CC) chondrocytes would be found in early OC when compared to controls. Paraffin‐embedded osteochondral samples (7 OC, 8 normal) and cDNA from whole cartilage (7 OC, 10 normal) and chondrocytes surrounding cartilage canals and osteochondral junctions captured with laser capture microdissection (4 OC, 6 normal) were obtained from femoropatellar joints of 17 immature horses. Equine‐specific Wnt signaling molecule mRNA expression levels were evaluated by two‐step real‐time qPCR. Spatial tissue protein expression of β‐catenin, Wnt‐11, Wnt‐4, and Dkk‐1 was determined by immunohistochemistry. There was significantly decreased Wnt‐11 and increased β‐catenin, Wnt‐5b, Dkk‐1, Lrp6, Wif‐1, Axin1, and SC‐PEP gene expression in early OC cartilage canal chondrocytes compared to controls. There was also significantly increased β‐catenin gene expression in early OC osteochondral junction chondrocytes compared to controls. Based on this study, abundant gene expression differences in OC chondrocytes surrounding cartilage canals suggest pathways associated with catabolism and inhibition of chondrocyte maturation are targeted in early OC pathogenesis. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1433–1438, 2015.