Metabolic profiling in human SH-SY5Y neuronal cells exposed to perfluorooctanoic acid (PFOA)

Perfluorooctanoic acid (PFOA) is an abundant per- and polyfluoroalkyl substance (PFAS) detected in both indoor and outdoor environments. While studies suggest exposure concerns for humans, studies investigating PFOA-induced neurotoxicity are lacking. To address this gap, we exposed differentiated human SH-SY5Y cells to PFOA (0.1 μM up to 500 μM) at different time points (4, 24, 48, and 72 h) and measured cell viability, Casp3/7 activity, ATP levels, ATP synthase enzyme activity, mitochondrial membrane potential, reactive oxygen species (ROS), oxygen consumption rates for mitochondrial stress test (XFe24 Flux analyzer), glucose utilization, and global metabolome profiles to assess the potential for PFOA-induced neurotoxicity. Treatment with 10 or 100 μM PFOA did not compromise cell viability nor induce cytotoxicity to SH-SY5Y cells over a 48-hour exposure period. However, >250 μM PFOA compromised cell viability, induced cytotoxicity, and induced caspase 3/7 activity at 48 h. ATP levels were reduced in cells treated with 400 μM PFOA for 24 and 48 h, and with 100 μM PFOA and higher at 72 h. ATP synthase activity was inhibited by 250 μM PFOA but was unchanged by PFOA treatment at 200 μM or less. Conversely, mitochondrial membrane potential was reduced by >10 μM PFOA after 24 h. Total ROS was increased with 100 μM PFOA and higher after 4 h of exposure. Several mitochondria-related endpoints (basal respiration, ATP production, maximum respiration) were negatively affected at 250 μM PFOA at both 24- and 48-hour exposure, but were unaltered at concentrations of 100 μM PFOA or less. One exception was mitochondrial spare capacity, which was reduced by 100 μM PFOA after 24-hour exposure. Similarly, glycolysis, glycolytic capacity, and glycolytic reserve of SH-SY5Y cells were not altered by 10 nor 100 μM PFOA. Nontargeted metabolomics was conducted in cells treated with either 10 or 100 μM PFOA for 48 h, as these two concentrations were not cytotoxic and 28 metabolites differed among treatments. Notable was that 10 μM PFOA had little effect on the SH-SY5Y metabolome, and the metabolic profile was not statistically different from media nor solvent controls. On the other hand, 100 μM PFOA shifted the metabolic signature of the neuronal cells, leading to reduced abundance of ATP-related metabolites (adenine, nicotinamide), neurotransmitter precursors (DL-tryptophan, l-tyrosine), and metabolites that protect mitochondria during oxidative stress (betaine, orotic acid, and l-acetyl carnitine). We hypothesize that this metabolic signature may be associated with the reduced mitochondrial membrane potential observed at lower PFOA concentrations. Metabolic shifts appear to precede compromised cell viability, cytotoxicity, and apoptosis. This study generates mechanistic knowledge regarding PFOA-induced neurotoxicity, focusing on mitochondrial oxidative respiration and the neuronal metabolome.     

Hax-1对胶质瘤细胞增殖、迁移和侵袭的作用

目的 探讨造血干细胞特异性相关结合蛋白-1（Hax-1）对胶质瘤细胞增殖、迁移和侵袭的影响。方法 选择2018年9月至2019年6月手术切除并得到术后病理证实的胶质瘤组织35例和颅脑损伤内减压术切除的正常脑组织35例,采用qRT-PCR检测Hax-1 mRNA水平;同时检测胶质瘤细胞系（U87、A172、T98及U343）和人星形胶质细胞（NHAs）Hax-1 mRAN水平。使用Lipofectamine 2000法将Hax-1 siRNAs和阴性对照siRNA转染U87细胞构建Hax-1低表达细胞株,将Hax-1高表达质粒和PCDNA3.1空载质粒转染U343细胞构建Hax-1过表达细胞株,使用蛋白印迹法验证转染效果,采用CCK-8法检测细胞增殖,采用Transwell小室实验检测细胞迁移和侵袭,免疫印迹法检测NF-κB信号通路相关蛋白表达水平（NF-κB、CCND1、C-myc、MMP-2、MMP-9）。结果 胶质瘤组织Hax-1 mRNA水平明显高于正常脑组织（P<0.05）;胶质瘤细胞系（U87、A172、T98及U343）Hax-1 mRNA水平明显高于人星形胶质细胞（P<0.05）,其中U87细胞Hax-1 mRNA水平最高,U343细胞最低。U343细胞转染Hax-1过表达质粒后,Hax-1蛋白水平显著增高（P<0.05）,细胞增殖、侵袭、迁移能力均明显增强（P<0.05）,NF-κB p65及IκBα蛋白磷酸化水平以及CCND1、C-Myc、MMP-2、MMP-9蛋白表达水平明显增高（P<0.05）。U87细胞转染Hax-1 siRNA后Hax-1蛋白水平显著降低（P<0.05）,细胞增殖、侵袭、迁移能力均明显降低（P<0.05）,NF-κB p65及IκBα蛋白磷酸化水平以及CCND1、C-Myc、MMP-2、MMP-9蛋白表达水平明显降低（P<0.05）。结论 胶质瘤组织Hax-1呈高表达,明显促进肿瘤细胞增殖、侵袭和迁移,其机制可能与激活NF-κB信号通路有关。     

X线引导关节腔内脉冲射频联合玻璃酸钠注射对慢性难治性膝骨关节炎的疗效观察

【摘要】 目的 评估X线引导膝关节腔内脉冲射频联合玻璃酸钠注射对慢性难治性膝骨关节炎的疗效。方法 2017年6月至2019年6月门诊收治保守治疗无效的慢性难治性膝关节骨性关节炎患者62 例（72侧膝关节）,经X线引导行膝关节腔内脉冲射频联合玻璃酸钠注射治疗。通过视觉模拟评分量表（VAS）评估治疗前和治疗后1周、1个月、6个月和12个月的疼痛变化并观察膝关节总体活动能力的改变。结果 治疗之前的平均疼痛评分为8.22±1.32,治疗1周后,该评分降低为2.31±2.11,随访1个月降低为2.61±2.43,随访6个月为2.99±2.67,随访12个月降低为4.96±2.97,与治疗前相比疼痛评分显著降低（P＜0.01）。63/72（87.5％）膝关节总体活动能力改善。未观察到与此类治疗相关的并发症发生。结论 膝关节腔内脉冲射频联合玻璃酸钠注射是一种有效、安全的治疗慢性难治性膝骨性关节炎的技术。     

Endovascular treatment of symptomatic intracranial atherosclerotic stenosis with low profile visualized intraluminal support stent

ObjectiveSevere intracranial atherosclerotic stenosis has become one of the main causes resulting transient ischemic attack and stroke. This study aimed to evaluate the efficacy and safety of low profile visualized intraluminal support (LVIS) stent in treating symptomatic intracranial atherosclerotic stenosis.MethodsData of 31 patients with at least 70% stenosis treated with LVIS stent in our center were retrospectively collected between July 2017 and November 2020. Further evaluation of lesion characteristics, technical success rate, preoperative complication, clinical and angiographic follow-up outcome, delayed in-stent stenosis were conducted.ResultsStent delivery and deployment were successfully achieved in all 31 patients (100%). 22 cases (71%) were located in anterior circulation and 9 cases (29%) were located in posterior circulation. The mean degree of stenosis lesion before stent deployment was 85.6 ± 9.4%, while after stenting was 11.2 ± 11.8%. One patient suffered from ischemic complication in stenting procedure, and timely delivery of rt-PA successfully recanalized the artery. Clinical follow-up was available in all 31patients (100%) with mean follow-up time 15.0 ± 12.1(3–45) months. No patients experienced the recurrence of stroke or TIA or death after discharge. Angiographic follow-up was available in 21patients (67.7%) with mean follow-up time 11.43 ± 6.8 (6–36) months. 19 patients (90.5%) were stable while 2 patients (9.5%) developed ISR in their last angiographic follow-up. The 2 patients received balloon angioplasty and reached satisfactory results after retreatment.ConclusionThis preliminary study suggests that LVIS stent deployment was a feasible approach in treating intracranial atherosclerotic stenosis with satisfactory procedure success rate, low complication rate and favorable long-term outcome.     

Polymeric immunoglobulin receptor (PIGR) exerts oncogenic functions via activating ribosome pathway in hepatocellular carcinoma

Objective: This report aimed to investigate the potential mechanism of polymeric immunoglobulin receptor (PIGR) in promoting cancer development in hepatocellular carcinoma (HCC).Methods: PIGR expression was investigated in Gene Expression Omnibus (GEO), Oncomine, The Cancer Genome Atlas (TCGA) and The Human Protein Atlas (HPA) databases. Relationships between PIGR and HCC survival and clinico-pathological features were conducted in TCGA. RNAseq of PIGR overexpression and knockdown samples in Bel-7404 cells were performed for identifying potential mechanisms.Results: PIGR was significantly overexpressed in tumors compared to nontumors and in HCC serum peripheral blood mononuclear cells (PBMC) than in healthy individuals (all p < 0.05). In TCGA, PIGR was highly altered in 14% HCC patients. PIGR upregulation was significantly associated with poor disease-free survival (p < 0.05). More patients recurred/progressed in PIGR altered group compared to unaltered group (p < 0.01). PIGR was significantly higher in HCC patients with incomplete cirrhosis (p < 0.001) and established cirrhosis (p < 0.05). Fewer patients had N₀ lymph node stage in PIGR altered group than those in the unaltered group (p < 0.05). PIGR RNAseq revealed that ribosome signaling was the common pathway in PIGR overexpression and PIGR knockdown samples. RNAseq analysis indicated that RPL10, RPL10A, RPL12, RPL19, RPL36, RPL38, RPL41, RPL6, RPL8, RPS12, RPS14, RPS15A, RPS2, RPS27A and RPSA were significantly upregulated in PIGR overexpression group and downregulated in PIGR underexpression group (all p < 0.05).Conclusions: Aberrant PIGR was associated with HCC recurrence, and PIGR stimulated ribosome pathway might be a potential mechanism.     

PFNA与InterTan治疗老年股骨转子间骨折的临床效果比较

目的比较股骨近端防旋髓内钉(PFNA)与InterTan治疗老年股骨转子间骨折的临床效果。方法回顾性分析2017年1月~2018年8月南通大学附属南京江北医院骨科中心符合标准的147例老年(≥65岁)股骨转子间骨折患者的临床资料,其中采用PFNA治疗的75例,采用InterTan治疗的72例。比较两组患者的一般资料(年龄、基础疾病、骨折分型等)、临床资料(手术时间、术中出血量、术中透视次数等)、髋关节并发症(再次骨折、髋内翻等)、内固定相关并发症(如头钉切割、内固定断裂、松动等)及髋关节Harris评分。结果两组患者均顺利手术,均获得随访12~18个月,平均随访(13.54±1.68)个月。在术中出血量、术中透视次数、手术时间、开始负重时间、住院时间、骨折愈合时间及围手术期并发症方面,两组差异无统计学意义(P>0.05);两组在髋关节及内固定相关并发症方面差异无统计学意义(P>0.05),但PFNA组发生率明显高于InterTan组;术后随访1、3、6及12个月髋关节Harris评分,两组差异无统计学意义(P>>0.05),但InterTan组Harris评分高于PFNA组。结论两组治疗老年股骨转子间骨折都具有比较好的疗效,均具有手术时间短、术中出血量少、生物力学优势及稳定性的优点,但InterTan术后髋关节及内固定并发症的发生率低及术后髋关节功能恢复良好。     

Ultrastructural studies of the neurovascular unit reveal enhanced endothelial transcytosis in hyperglycemia-enhanced hemorrhagic transformation after stroke

AimsPre‐existing hyperglycemia (HG) aggravates the breakdown of blood–brain barrier (BBB) and increases the risk of hemorrhagic transformation (HT) after acute ischemic stroke in both animal models and patients. To date, HG‐induced ultrastructural changes of brain microvascular endothelial cells (BMECs) and the mechanisms underlying HG‐enhanced HT after ischemic stroke are poorly understood.MethodsWe used a mouse model of mild brain ischemia/reperfusion to investigate HG‐induced ultrastructural changes of BMECs that contribute to the impairment of BBB integrity after stroke. Adult male mice received systemic glucose administration 15 min before middle cerebral artery occlusion (MCAO) for 20 min. Ultrastructural characteristics of BMECs were evaluated using two‐dimensional and three‐dimensional electron microscopy and quantitatively analyzed.ResultsMice with acute HG had exacerbated BBB disruption and larger brain infarcts compared to mice with normoglycemia (NG) after MCAO and 4 h of reperfusion, as assessed by brain extravasation of the Evans blue dye and microtubule‐associated protein 2 immunostaining. Electron microscopy further revealed that HG mice had more endothelial vesicles in the striatal neurovascular unit than NG mice, which may account for their deterioration of BBB impairment. In contrast with enhanced endothelial transcytosis, paracellular tight junction ultrastructure was not disrupted after this mild ischemia/reperfusion insult or altered upon HG. Consistent with the observed increase of endothelial vesicles, transcytosis‐related proteins caveolin‐1, clathrin, and hypoxia‐inducible factor (HIF)‐1α were upregulated by HG after MCAO and reperfusion.ConclusionOur study provides solid structural evidence to understand the role of endothelial transcytosis in HG‐elicited BBB hyperpermeability. Enhanced transcytosis occurs prior to the physical breakdown of BMECs and is a promising therapeutic target to preserve BBB integrity.