Lung adenocarcinoma (LUAD) is a common subtype of lung cancer with a dismal prognosis and a lack of effective biomarkers at an early stage. Calcium plays a crucial role in immunomodulation and immunotherapy, and can effectively predict the prognosis of tumors. This study aimed to probe into the potential value of calcium-related genes (CRGs) in the prognosis of LUAD. RNA sequencing data, somatic mutation data, and demographic and clinical data of LUAD were collected from the TCGA database. GSE31210 data were collected from the GEO database, and data on CRGs were from GeneCards. Univariate, LASSO, multivariate Cox regression analyses, Kaplan-Meier survival analysis, ROC curve analysis, gene set enrichment analysis (GSEA), and other methods were employed to establish the prediction model and validate and annotate the functions. The tumor immune microenvironment in different risk groups was evaluated using ESTIMATE, single-sample GSEA, and CIBERSORT algorithms. The immunophenoscore and tumor immune dysfunction and exclusion scores were examined to predict immunotherapy response sensitivity. Anti-tumor drugs were screened through correlation analysis and differential comparison. Eight calcium-related biomarkers (GRIA1, BTK, CLCA1, PDGFB, S100P, TRPA1, F2RL1, FBN2) significantly associated with the prognosis of LUAD were identified. A reliable risk-scoring model was constructed and its capability was validated. Patients with LUAD with worse clinical features (advanced stage, higher tumor burden, and lymph node metastases) had higher riskscores and a worse prognosis. Patients in the low-risk (LR) group exhibited a strong immune response, especially significantly increased mast cells, B cells, and Tfh cells. The high-risk (HR) group exhibited enrichment of immunosuppressive cells (e.g., Tregs), suggesting that the LR group may benefit more from the immune checkpoint suppressive therapy. Moreover, we predicted the clinical potential of drug candidates such as Erlotinib, Afatinib, and Barasertib. We discovered that multiple CRGs were significantly associated with the survival of LUAD and were differentially expressed in LUAD. We created a risk model for prognosis prediction based on CRGs in LUAD, which can not only effectively predict prognosis but also reflect changes in the tumor immune microenvironment among different risk groups. These findings may be beneficial for clinical decision-making.

Introduction: Intervertebral disc degeneration (IVDD) is a primary cause of low back pain, with most research focusing on nucleus pulposus repair. However, the role of cartilage endplate (CEP) degeneration in IVDD progression has been largely overlooked in developing therapeutic strategies. Objectives: This study aimed to explore the relationship between serum ferritin levels and IVDD severity, emphasizing the role of ferroptosis-induced oxidative stress in accelerating CEP degeneration. Additionally, it sought to investigate the therapeutic potential of human decidual mesenchymal stem cell-derived extracellular vesicles (hDMSC-EVs) in reversing CEP damage. Methods: hDMSC-EVs were utilized to combat ferroptosis-related oxidative stress, inhibit osteogenic transdifferentiation, and promote extracellular matrix anabolism in damaged discs. Mechanistic studies focused on the delivery of miR-21-5p by hDMSC-EVs, which targets HIF1AN to activate the HIF-1 alpha signaling pathway. Results: The study established a correlation between serum ferritin levels and IVDD severity and demonstrated that hDMSC-EVs reversed CEP degeneration. Rescue experiments showed that knocking down HIF-1 alpha eliminated the protective effects of hDMSC-EVs on CEP ferroptosis via the miR-21-5p/HIF-1 alpha axis. Conclusion: hDMSC-EVs have significant therapeutic potential in managing IVDD by targeting ferroptosis through the miR-21-5p/HIF-1 alpha pathway, offering a promising strategy for future clinical applications in IVDD treatment.

Surface-enhanced Raman spectroscopy (SERS) is a significant analytical technique based on the interaction between light, nanostructures, and molecules. Its enhancement efficiency relies on the “hot spots” generated by the synergistic action of localized electromagnetic fields and charge transfer at the SERS substrate surface. However, due to the uneven enhancement characteristics and distribution of these hot spots, only a limited number of target molecules can be effectively enhanced. The “confined space effect” of the nanopore structure further enhances the localization accuracy of hotspots and increases molecular enrichment in hotspot regions, thereby providing new opportunities for single-molecule (SM) analysis. First, this review outlines the development of SERS technology and summarizes the research directions since the turn of the century. It then discusses the enhancement mechanisms of SERS and the key factors influencing the hotspots in nanopore-based SERS. From the perspectives of material systems and pore morphology, this article focuses on two types of SERS substrates: porous pure metals and porous metal-nonmetal composites. It systematically explores the preparation strategies and performance of porous SERS platforms from the viewpoint of two types of pore structures: random disorder and regular order. Furthermore, the paper analyzes the significant progress of nanopore structures in SM SERS-based quantitative analysis. The review also covers the application of porous SERS platforms in healthcare, particularly in sensing research related to prevention, screening, and prognosis, including areas such as health prevention detection, gas molecule detection, liquid biopsy, and tumor imaging and therapy. Finally, based on current research, the review presents key insights on nanopore SERS substrates.

Congenital human cytomegalovirus (HCMV) infection is the leading cause of neurodevelopmental disorders in children, including nongenetic sensorineural hearing loss. Previous studies have shown that HCMV immediate early 1 (IE1) protein, also known as IE72, contributes to brain maldevelopment. However, the underlying mechanisms are unclear due to the strict species specificity of cytomegaloviruses (CMVs), limiting animal model study. In the current study, we used CRISPR/Cas9 technology to construct a transgenic mouse model (Rosa26-LSL-IE1+/−, Camk2ɑ-Cre) specifically and stably expressing IE1 protein in brain. These transgenic mice exhibited impaired spatial working memory, hippocampal neurodegeneration, and proinflammatory activation of brain microglia and astrocytes. Transcriptome sequencing revealed that IE1 protein upregulated genes linked to metabolism and downregulated genes implicated in nervous system development. Furthermore, IE1 alters the lactate production pathway in astrocytes, thereby reducing the energy supply available to neurons. These findings suggest that long-term IE1 protein expression disrupts neurodevelopment by inducing neuroinflammation and uncoupling neurons from metabolic support by astrocytes. These results provide a clear molecular mechanism for neurodevelopmental disorders in infants with congenital HCMV infection.

Objective Sepsis-induced cardiomyopathy (SICM) is a life-threatening complication. Phospholipase D2 (PLD2) is crucial in mediating inflammatory reactions and is associated with the prognosis of patients with sepsis. Whether PLD2 is involved in the pathophysiology of SICM remains unknown. This study aimed to investigate the effect of PLD2 knockout on SICM and to explore potential mechanisms.Methods The SICM model was established using cecal ligation and puncture in wild-type and PLD2-knockout mice and lipopolysaccharide (LPS)-induced H9C2 cardiomyocytes. Transfection with PLD2-shRNA lentivirus and a PLD2 overexpression plasmid were used to interfere with PLD2 expression in H9C2 cells. Cardiac pathological alterations, cardiac function, markers of myocardial injury, and inflammatory factors were used to evaluate the SICM model. The expression of pyroptosis-related proteins (NLRP3, cleaved caspase 1, and GSDMD-N) was assessed using western blotting, immunofluorescence, and immunohistochemistry.Results SICM mice had myocardial tissue damage, increased inflammatory response, and impaired heart function, accompanied by elevated PLD2 expression. PLD2 deletion improved cardiac histological changes, mitigated cTNI production, and enhanced the survival of the SICM mice. Compared with controls, PLD2-knockdown H9C2 exhibits a decrease in inflammatory markers and lactate dehydrogenase production, and scanning electron microscopy results suggest that pyroptosis may be involved. The overexpression of PLD2 increased the expression of NLRP3 in cardiomyocytes. In addition, PLD2 deletion decreased the expression of pyroptosis-related proteins in SICM mice and LPS-induced H9C2 cells.Conclusion PLD2 deletion is involved in SICM pathogenesis and is associated with the inhibition of the myocardial inflammatory response and pyroptosis through the NLRP3/caspase 1/GSDMD pathway.

The endothelial glycocalyx is damaged in postcardiac arrest syndrome (PCAS), but the prognostic value is unknown. We aimed to observe the expression and prognostic value of glycocalyx shedding products, including syndecan-1 (SDC-1), hyaluronan (HA), and heparan sulfate (HS) in PCAS. Data on clinical and 28-day outcomes of seventy-one consecutive patients with out-of-hospital cardiac arrest (OHCA) after the return of spontaneous circulation (ROSC) were collected. SDC-1, HA, and HS were measured on days 0, 1, and 3 after ROSC. Thirty healthy individuals were controls. Glycocalyx shedding was observed in human umbilical vein endothelial cells (HUVECs) stimulated during hypoxia and reoxygenation in vitro. Within 4 h of ROSC, SDC-1 and HA levels, significantly increased. In the 28-day non-survivors, HA levels showed a gradual upward trend, SDC-1 remained at a high level, and HS levels first increased, then decreased. Kaplan-Meier curves and binary logistic regression analysis showed the prognostic value of SDC-1 levels on days 0, 1, and 3, HA levels on days 1 and 3, and HS levels on day 1. Only HS levels on day 1 showed a prognostic value for 28-day neurological outcomes. SDC-1 and HA levels were positively correlated with the no-flow time. In vitro, HUVECs showed shedding of SDC-1 and HS during a prolonged duration of hypoxia. After ROSC, SDC-1, HA, and HS levels may predict the 28-day survival after PCAS, and HS levels are associated with functional outcomes.

Copper cell death (copper proptosis) is a new chronic disease mainly caused by exposure to copper. To date, cuproptosis as a new target for the treatment of patients with chronic obstructive pulmonary disease (COPD) has not been studied, and the gene expression of cuproptosis in patients with COPD is still unclear. Based on this, this study aimed to use the NCBI Gene Expression Omnibus (GEO) database to screen differentially expressed genes related to COPD and analyze their functional enrichment. As a result, several potential therapeutic targets are proposed for reducing the incidence of cuproptosis in patients with COPD.

Epstein-Barr virus (EBV), a ubiquitous oncogenic herpesvirus, infects more than 90% of the adult population worldwide. The long noncoding RNA H19 is downregulated in EBV-positive gastric cancer (EBVaGC) and nasopharyngeal cancer (NPC). In this study, we found that loss of H19 is caused by hypermethylation status of the H19 promoter in EBV-positive GC and NPC cell lines. Furthermore, latent membrane protein 1 (LMP1), encoded by EBV, induced H19 promoter hypermethylation and deregulated the expression of H19 by upregulating DNMT1 expression. Transwell assays showed that H19 promoted cell migration. Furthermore, H19 promoted cell proliferation and inhibited apoptosis in CCK-8 and flow cytometry assays, respectively. p53, a well-known tumor suppressor, was upregulated in EBVaGC and NPC cell lines. miR-675-5p derived from H19 inhibited p53 protein expression by targeting the 3' untranslated region of the gene. Overall, we found that LMP1 induced p53 protein expression via the H19/miR-675-5p axis in EBVaGC and NPC. LMP1 induced H19 promoter hypermethylation, which repressed the expression of H19 and miR-675-5p and caused p53 protein overexpression in EBVaGC and NPC cells. IMPORTANCE Epstein-Barr virus (EBV) is the first virus to be known to have direct association with human cancer and to be considered as an important DNA tumor virus. The EBV life cycle consists of both latent and lytic modes of infection in B lymphocytes and epithelial cells. The persistence of EBV genomes in malignant cells promoted cell growth. p53, acting as a critical gatekeeper tumor suppressor, is involved in multiple virus-mediated tumorigeneses. Overexpression of p53 inhibits the ability of BZLF1 (EBV-encoded immediate early gene) to disrupt viral latency. In our study, we found LMP1 induces H19 promoter hypermethylation, which represses the expression of H19 and miR-675-5p and results in p53 protein overexpression in EBVaGC and NPC cells. These observations suggest a new mechanism of aberrant expression of p53 by LMP1, which facilitates EBV latency.

Abstract Background: Pulmonary fibrosis is a common pathogenic change of COPD and associated with worse outcome, however, there is a lack of research on mechanisms of COPD with pulmonary fibrosis. In our study, we studied the influence of TGF-β1 and its single nucleotide polymorphism (SNP) on COPD complicated with pulmonary fibrosis. Methods: In this research, six GEO datasets were included to screen dysregulated genes in COPD patients. The dysregulated genes were detected by PCR-DNA sequencing based on 98 COPD patients and 90 healthy volunteers. Results: Five genes were upregulated in COPD patients including CDCP1, CYP1B1, PELO, RNF24 and TGF-β1. However, of the five genes, only TGF-β and CYP1B1 expression were significantly increased in COPD compared with normal group validated by R2 database. And only TGF-β1 highly expressed in 1-4 stage of COPD versus 0 stage. The expression of TGF-β in COPD patients with fibrosis was significantly higher than normal and COPD patients. Moreover, allele C of TGF-β1 +869 locus was associated with the susceptibility of COPD and airflow restriction. The genotype frequency of CC in patients with severe airflow restriction (23.1%) was significantly higher than that in patients with mild and moderate airflow restriction (6.5%). Conclusion: TGF-β1 was upregulated in the COPD patients with fibrosis, especially in patients with pulmonary fibrosis. The SNP at +869 allele C in TGF-β1 may be a genetic locus and therapeutic target for COPD with fibrosis.

Background and aims: Gastric cancer (GC) is a malignant tumor that seriously affects human health and Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a molecular subtype of GC. This study aims to determine the relationship between the methylation status of the TMEM130 gene and GC, and to explore the influence of EBV infection.Methods: qRT-PCR was conducted to investigate the transcriptional expression of TMEM130 in GC. BSP and MSP assays were used to detect the methylation level of the TMEM130 promoter. The cell migration ability was detected by Transwell and western blot after transfection of TMEM130 plasmids in GC cells.Results: The transcriptional expression of TMEM130 decreased in GC with hypermethylation of the promoter region. The DNA methyltransferase inhibitor could increase the mRNA expression of TMEM130. Moreover, hypermethylation of the TMEM130 promoter in GC tissues was associated with EBV infection. Overexpression of TMEM130 in GC cell lines suppresses cell migration ability.Conclusion: This study was the first to research the expression and function of TMEM130 and found that TMEM130 gene hypermethylation might contribute to GC migration and EBV infection as a cause of hypermethylation of the TMEM130 gene. TMEM130 is a promising biomarker for the diagnosis of GC. (c) 2021 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Increasing evidence shows that Epstein-Barr virus (EBV) infection is closely related to various lymphoid and epithelioid malignancies. However, the underlying mechanisms are unclear. GCNT3 (core 2β-1,6-acetylglucosaminyltransferase) is a new type of core mucin synthase, and its expression in EBV-associated gastric cancer (EBVaGC) is lower than that in EBV-negative gastric cancer (EBVnGC). EBV-encoded latent membrane protein 2A (LMP2A) is a transmembrane protein with tumorigenic transformation properties. Here, we demonstrated that LMP2A inhibited the transcription of GCNT3 by inhibiting Smad2/3 and Smad4. LMP2A restrained the activation of the mTORC1 pathway by inactivating the TGF-β1/Smad pathway and then downregulated GCNT3 expression. The mTORC1-GCNT3 pathway promoted cell proliferation and migration and inhibited G0/G1 cell arrest. Related proteins involved in epithelial-mesenchymal transition (EMT) were downstream molecules of the TGF-β1/Smad-mTORC1-GCNT3 pathway. GCNT3 inhibited autophagy by inducing mTORC1 phosphorylation. These findings indicate that targeting the TGF-β1/Smad-mTORC1-GCNT3 axis may represent a novel therapeutic target in GC.ImportanceEpstein-Barr virus (EBV) is an opportunistic pathogen, and the latent membrane protein 2A (LMP2A) encoded by EBV plays a key role in ensuring the incubation period of EBV. Glycosylation modification is an important marker of cancer cells, and recent studies have reported that it is related to EBV. Our conclusions provide deeper theoretical support for the role of LMP2A and TGF/Smad-mTORC1-GCNT3 in EBVaGC and help to understand glycosylation abnormalities in cancer. Our results may provide novel therapeutic targets for the treatment of gastric cancer against the TGF/Smad-mTORC1-GCNT3 signaling cascade.

Background Oxidative stress is an important pathogenic factor in influenza A virus infection. It has been found that reactive oxygen species induced by the H9N2 influenza virus is associated with viral replication. However, the mechanisms involved remain to be elucidated. Methods In this study, the role of autophagy was investigated in H9N2 influenza virus-induced oxidative stress and viral replication in A549 cells. Autophagy induced by H9N2 was inhibited by an autophagy inhibitor or RNA interference, the autophagy level, viral replication and the presence of oxidative stress were detected by western blot, TCID50 assay, and Real-time PCR. Then autophagy and oxidative stress were regulated, and viral replication was determined. At last, the Akt/TSC2/mTOR signaling pathways was detected by western blot. Results Autophagy was induced by the H9N2 influenza virus and the inhibition of autophagy reduced the viral titer and the expression of nucleoprotein and matrix protein. The blockage of autophagy suppressed the H9N2 virus-induced increase in the presence of oxidative stress, as evidenced by decreased reactive oxygen species production and malonaldehyde generation, and increased superoxide dismutase 1 levels. The changes in the viral titer and NP mRNA level caused by the antioxidant, N -acetyl-cysteine (NAC), and the oxidizing agent, H 2 O 2 , confirmed the involvement of oxidative stress in the control of viral replication. NAC plus transfection with Atg5 siRNA significantly reduced the viral titer and oxidative stress compared with NAC treatment alone, which confirmed that autophagy was involved in the replication of H9N2 influenza virus by regulating oxidative stress. Our data also revealed that autophagy was induced by the H9N2 influenza virus through the Akt/TSC2/mTOR pathway. The activation of Akt or the inhibition of TSC2 suppressed the H9N2 virus-induced increase in the level of LC3-II, restored the decrease in the expression of phospho-pAkt, phospho-mTOR and phospho-pS6 caused by H9N2 infection, suppressed the H9N2-induced increase in the presence of oxidative stress, and resulted in a decrease in the viral titer. Conclusion A utophagy is involved in H9N2 virus replication by regulating oxidative stress via the Akt/TSC2/mTOR signaling pathway. Thus, autophagy maybe a target which may be used to improve antiviral therapeutics.

Epstein-Barr virus (EBV) was the first oncovirus found to encode microRNAs. In EBV-associated gastric cancer (EBVaGC), EBV-encoded BamHI-A rightward transcript microRNAs (BARTs) are highly expressed. However, the role of BARTs in EBVaGC remains obscure. In this study, we found that EBV-miR-BART12 (miR-BART12) inhibits cell proliferation and migration. Zinc finger protein SNAI1 (Snail) is an important epithelial-mesenchymal transition (EMT) inducer, and overexpression of Snail is closely associated with cancer metastasis. Here, we report that Snail expression in EBVaGC cells is lower than in EBV-negative gastric cancer (EBVnGC) cells. A dual luciferase reporter assay showed that miR-BART12 targets Snail directly by interacting with its 3ʹ-UTR. A CHX chase assay revealed that miR-BART12 accelerates the degradation of Snail. Furthermore, we found that miR-BART12 can regulate the expression of EMT-related genes. Flow cytometry analysis showed that transfection with miR-BART12 induced G2/M phase arrest and promoted cell apoptosis. In summary, the results of our study have suggested a new mechanism by which BARTs can repress cell proliferation and migration in gastric cancer.

Pulmonary hypertension (PH) is a proliferative disease characterized by pulmonary arterial remodeling (PAR). SAM and SH3 domain containing 1 (SASH1) is a novel tumor suppressor gene whose biological function in PH is unclear. In this study, a hypoxia-induced pulmonary hypertension (HPH) rat model was constructed to explore the role of SASH1 in PAR. Histopathological changes in the lung tissue and hemodynamic alteration were detected in SASH1-knockdown rats through adeno-associated virus type-1 (AAV1) infection. In vitro, primary human pulmonary arterial smooth muscle cells (HPASMCs) were transfected with SASH1siRNA to investigate the effects of SASH1 on hypoxia-induced proliferation and migration. The molecular mechanisms associated with SASH1 were explored through knockdown and overexpression approaches. We found that SASH1 expression was significantly increased in rat pulmonary arteries and HPASMCs after hypoxia exposure. In vivo, silencing the SASH1 gene expression improved HPH in rats. The SASH1 downregulation inhibited proliferation and migration of hypoxia-induced HPASMCs. The protein expression of phospho-AKT (known as protein kinase B), proliferating cell nuclear antigen, and matrix metalloproteinase 9 (MMP9) in HPASMCs were increased after SASH1 overexpression, whereas these effects were inhibited by SASH1 knockdown. In conclusion, SASH1 downregulation improved hypoxia-induced PAR and PH. SASH1 may be a novel target for PH gene therapy in the era of precision medicine.

Epstein‐Barr virus (EBV) infection is one of the causes of gastric cancer (GC). Besides, previous studies have demonstrated that EBV‐encoded latent membrane protein 2A (LMP2A) influences the pathogenesis of EBV‐associated gastric cancer (EBVaGC) through regulating several key pathways. In this study, the expression level of Smad2 was observed, which was reduced in EBVaGC cell lines, especially in the presence of LMP2A. Meanwhile, we found that LMP2A promoted the expression of miR‐155‐5p by activated nuclear factor‐κB (NF‐κB) signaling. After being treated with NF‐κB inhibitor (BAY 11‐7082), miR‐155‐5p sharply decreased. Western blot analysis proved that the overexpression of miR‐155‐5p could inhibit Smad2. Functional studies showed that the role of miR‐155‐5p might lead to good prognosis in EBV‐positive GC through promoting cell apoptosis and cell cycle arrest, as well as inhibiting tumor proliferation. In addition, p‐Smad2 protein was also reduced or induced by overexpression or knockdown, respectively, of miR‐155‐5p. Immunofluorescence analysis further indicated that LMP2A prevented p‐Smad2 from transferring to the nucleus, which played a crucial role in transforming growth factor‐β (TGF‐β) signaling. In summary, our findings confirmed the relationship between LMP2A and Smad2 and provided a potential regulation of the TGF‐β pathway in EBVaGC.

Lung cancer is a common malignant tumor in clinic and easy to have bone metastases. Vascular endothelial growth factor (VEGF) is an angiogenic factor. Matrix metalloproteinases (MMPs) is associated with invasion and metastases. This study was to detect VEGF, microvessel density (MVD), MMP-2 and MMP-9 (MMP-2/9) levels in the serum and tissues of lung cancer patients with bone metastases to analyze the relaitonship between these changes and lung cancer with bone metastases. 60 patients with metastatic tumor of lung cancer were selected as experimental group, primary malignant tumor of bone and benign osteoma were regarded as control group, the contents of VEGF, MMP-2/9 in blood and tissues were analyzed by ELISA and IHC, respectively. VEGF, MVD, MMP-2/9 levels in serum and tissues of lung cancer bone metastasis group were significantly higher than that of primary bone tumor group and benign bone tumor group and their changes were significantly associated with pathological types of lung cancer, numbers and sizes of primary foci, numbers of bone metastases, other organ metastases and whether or not receiving radiotherapy and chemotherapy (P < 0.05). In conclusion, the level of VEGF, MVD, MMP-2/9 is significantly elevated in lung cancer patients with bone metastases, suggesting that these molecules might be involved in the pathogenesis of lung cancer with bone metastases.

应用短发卡RNA(shRNA)慢病毒表达载体感染小鼠肺微血管内皮细胞(PMVEC),对其M2型瞬时受体电位(TRPM2)基因进行干扰,以建立稳定shRNA TRPM2 PMVEC细胞株.结果 表明,正常PMVEC对胰酶的最大耐受浓度和嘌呤霉素最小致死剂量分别为0.4 μg/mL和0.6μg/mL;然后加入0.6、2.0、4.0、8.0 μg/mL嘌呤霉素筛选稳定抑制TRPM2表达的shRNA TRPM2 PMVEC株,结果在嘌呤霉素达到8 μg/mL时该细胞株仍细胞生长良好;Semi-quantitative PCR和Western blot对获得阳性细胞株进行TRPM2基因和蛋白表达的检测显示,shRNA TRPM2阳性PMVEC的TRPM2基因和蛋白表达相对量显著低于阴性对照和正常对照组(P＜0.01).该研究通过shRNA慢病毒载体成功建立了稳定shRNA TRPM2 PMVEC细胞株,为进一步开展TRPM2在流感病毒诱导肺微血管内皮细胞损伤的作用机制奠定了基础.

旨在探讨瞬时电位受体M2离子通道(TRPM2)在H9N2流感病毒感染小鼠肺微血管内皮细胞(PMVEC)导致线粒体损伤过程中的作用.在已建立TRPM2 shRNA PMVEC的基础上,采用5MOI H9N2猪流感病毒感染细胞,在病毒作用后24和48 h提取各组细胞的线粒体进行蛋白定量,检测各组细胞线粒体超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、一氧化氮合成酶(NOS)、线粒体呼吸链复合物Ⅳ活性和ATP酶水平;利用激光共聚焦显微镜观察线粒体膜电位变化(JC-1染色)及细胞凋亡(Annexin V-FITC/PI双染色法)情况.结果 表明:H9N2-SIV感染后TRPM2 shRNA PMVEC内SOD活性、GSH水平和Na+-K+-ATP、线粒体呼吸链复合物Ⅳ活性显著高于对照shRNA PMVEC(P＜0.05或P＜0.01);mtNOS活性则极显著低于shRNA PMVEC(P＜0.01).JC-1染色显示TRPM2-siRNA PMVEC线粒体膜电位水平高于对照shRNA PMVEC,但是AnnexinV-FITC/PI染色显示细胞凋亡则低于H9N2感染对照shRNA PMVEC.结果 提示:TRPM2基因沉默有效减缓H9N2感染导致NOS产生,显著降低SOD、GSH、线粒体呼吸链复合物Ⅳ、Na+-K+-ATP的消耗以及线粒体膜电位的下降程度,进而有效缓解PMVEC线粒体损伤及细胞凋亡.

试验旨在确定H9N2亚型猪源性流感病毒(SIV)在小鼠肺微血管内皮细胞(PMVEC)中增殖的最佳条件.将PMVEC解冻、复苏、培养,取长成单层的PMVEC,在不同浓度TPCK-胰蛋白酶维持液(0.1、0.2、0.3、0.4、0.6、0.8、1.0μg/mL)、不同H9N2亚型SIV (A/swine/HeBei/012/2008/(H9N2)接种剂量(1∶10、1∶100、1∶1 000、1∶10 000、1∶100 000、1∶1 000 000和1∶10000 000)及不同病毒吸附时间(0.5、1.0、2.0和3.0h)条件下观察PMVEC形态变化,并测定细胞上清液中H9N2亚型SIV的HA滴度.在未加病毒的情况下,低于0.6 μg/mL的TPCK胰蛋白酶对PMVEC的生长未造成任何影响,但随着TPCK-胰蛋白酶浓度的增加,PMVEC开始出现肿胀、变圆,甚至脱落;采用含有0.6 μg/mL TPCK-胰蛋白酶维持液将H9N2亚型SIV稀释为不同的浓度感染PMVEC,在0.3 μg/mL TPCK-胰蛋白酶维持液、10-2病毒稀释倍数感染条件下48和72 h HA滴度分别为4.61og2和6.41og2;病毒吸附时间为2h且中间震荡20 s的条件下H9N2 HA滴度最佳.结果表明,当TPCK-胰蛋白酶维持液浓度为0.3 μg/mL、病毒接种浓度为10-2、吸附时间为2h且中间震荡20 s时,H9N2亚型SIV在PMVEC中增殖最佳,达到6.8log2.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal).This article has been retracted at the request of the Editor-in-Chief as there are concerns about the reliability of the results included in the article. The journal was initially contacted by the first author to request the retraction as they reported that results were not reproducible post publication. Also, the author acknowledged that the corresponding authors were not aware of the submission of this article.Given the comments of Dr Elisabeth Bik https://scienceintegritydigest.com/2020/02/21/the-tadpole-paper-mill/ regarding this article, the journal requested the author to provide the raw data. However, the author was not able to fulfil this request.