Effect fraction of Bletilla striata (Thunb.) Reichb.f. alleviates LPS-induced acute lung injury by inhibiting p47phox/NOX2 and promoting the Nrf2/HO-1 signaling pathway.

BACKGROUND & AIMS: The effect fraction of Bletilla striata (Thunb.) Reichb.f. (EFBS), a phenolic-rich extract, has significant protective effects on lipopolysaccharide (LPS)-induced acute lung injury (ALI), but its composition and molecular mechanisms are unclear. This study elucidated its chemical composition and possible protective mechanisms against LPS-induced ALI from an antioxidant perspective. METHODS: EFBS was prepared by ethanol extraction, enriched by polyamide column chromatography, and characterized using ultra-performance liquid chromatography/time-of-flight mass spectrometry. The LPS-induced ALI model and the RAW264.7 model were used to evaluate the regulatory effects of EFBS on oxidative stress, and transcriptome analysis was performed to explore its possible molecular mechanism. Then, the pathway by which EFBS regulates oxidative stress was validated through inhibitor intervention, flow cytometry, quantitative PCR, western blotting, and immunofluorescence techniques. RESULTS: A total of 22 compounds in EFBS were identified. The transcriptome analyses of RAW264.7 cells indicated that EFBS might reduce reactive oxygen species (ROS) production by inhibiting the p47phox/NADPH oxidase 2 (NOX2) pathway and upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Both in vitro and in vivo data confirmed that EFBS significantly inhibited the expression and phosphorylation of p47phox protein, thereby weakening the p47phox/NOX2 pathway and reducing ROS production. EFBS significantly increased the expression of Nrf2 in primary peritoneal macrophages and lung tissue and promoted its nuclear translocation, dose-dependent increase in HO-1 levels, and enhancement of antioxidant activity. In vitro, both Nrf2 and HO-1 inhibitors significantly reduced the scavenging effects of EFBS on ROS, further confirming that EFBS exerts antioxidant effects at least partially by upregulating the Nrf2/HO-1 pathway. CONCLUSIONS: EFBS contains abundant phenanthrenes and dibenzyl polyphenols, which can reduce ROS production by inhibiting the p47phox/NOX2 pathway and enhance ROS clearance activity by upregulating the Nrf2/HO-1 pathway, thereby exerting regulatory effects on oxidative stress and improving LPS-induced ALI.

A novel approach to improving colonoscopy learning efficiency through a colonoscope roaming system: randomized controlled trial.

Background: Colonoscopy is indispensable in the diagnosis and treatment of lower digestive tract (LDT) diseases. Skilled colonoscopists are in great demand, but it takes considerable time for beginners to become experts. In addition, patients may refuse to permit primary learners to practise colonoscopy on them. Thus, improving the instructional programmes and models for primary learners is a key issue in endoscopy training. Convenience and a self-paced, learner-centred approach make e-learning an excellent instructional prospect. Therefore, we created the Colonoscope Roaming System (CRS) to assist in colonoscopy teaching procedures. We aimed to develop the e-learning software, test it with beginner colonoscopists and evaluate its effectiveness via subjective and objective methods. Methods: Through a randomized controlled trial, participants were randomly allocated to an e-learning group (EG) or a control group (CG) after a pretest evaluation. The CG learned through the traditional colonoscopy teaching mode, while the EG used CRS in addition to the traditional teaching mode. Subsequent to the training, the participants completed a posttest and colonoscopy examination. The EG also completed a satisfaction questionnaire. Results: Of the 84 participants, 81 (96%) finished the colonoscopy learning and evaluation modules of the CRS. No conspicuous differences in the pretest scores were found between the EG and CG (p > 0.05). Two months later, the posttest scores for the EG were higher than those of the CG (p < 0.001), and the EG had better performance on the colonoscopy examination (p < 0.01). Overall, 86.25% of questions raised in Q1-Q20 were satisfied with the CRS and considered it successful. Conclusions: The use of CRS may be an effective approach to educate beginner colonoscopists to attain skills.

Chemical Composition, Antitumor Properties, and Mechanism of the Essential Oil from Plagiomnium acutum T. Kop.

Plagiomnium acutum T. Kop. (P. acutum) has been used as a traditional Chinese medicine for thousands of years to treat cancer but lacks evidence. The objective of this work was to reveal the chemical composition of P. acutum essential oil (PEO) and explore its potential antitumor activity and molecular mechanism. PEO was prepared by the simultaneous distillation-extraction method and characterized by gas chromatography/mass spectroscopy. CCK8 assay, flow cytometry, western blot, and immunofluorescence techniques were used to analyze the effects and mechanism of PEO against cancer cells. A total of 74 constituents of PEO were identified, with diterpenes (26.5%), sesquiterpenes (23.89%), and alcohols (21.81%) being the major constituents. Two terpenoids, selina-6-en-4-ol and dolabella-3,7-dien-18-ol, were detected in PEO for the first time. PEO showed significant cell growth inhibitory activity on HepG2 and A549 cells by blocking the G1 phase and inducing apoptosis, which may be attributed to its upregulation of p21Cip1 and p27Kip1 proteins and interference with mitochondrial membrane potential effect. Dolabella-3,7-dien-18-ol accounts for 25.5% of PEO and is one of the main active components of PEO, with IC50 values in HepG2 and A549 cells of (25.820 ± 0.216) µg/mL and (23.597 ± 1.207) µg/mL, respectively. These results confirmed the antitumor medicinal value of P. acutum and showed great application potential in the pharmaceutical industry.

Up-Regulation of p53/miR-628-3p Pathway, a Novel Mechanism of Shikonin on Inhibiting Proliferation and Inducing Apoptosis of A549 and PC-9 Non-Small Cell Lung Cancer Cell Lines.

Shikonin (SHK) is a pleiotropic agent with remarkable cell growth inhibition activity against various cancer types, especially non-small cell lung cancer (NSCLC), but its molecular mechanism is still unclear. Our previous study found that miR-628-3p could inhibit the growth of A549 cells and induce its apoptosis. Bioinformatics analysis predicted that miR-628-3p promoter sequence contained p53 binding sites. Considering the regulatory effect of SHK on p53, we speculate that SHK may inhibit the growth and induce apoptosis of NSCLC cells by up-regulating miR-628-3p. CCK-8 and EdU assay confirmed the inhibitory effect of SHK on A549 and PC-9 cells. Meanwhile, quantitative reverse transcription-polymerase chain reaction and Western blot showed that SHK could promote the expression of p53 and miR-628-3p in a dose-dependent manner. Overexpression of p53 or miR-628-3p can inhibit the growth and promote apoptosis of A549 and PC-9 cells, while silencing p53 or miR-628-3p has the opposite effect. Dual luciferase reporting assay and ChIP (chromatin immunoprecipitation) assay further verified the direct interaction between p53 and the promoter of miR-628-3p. Gene knockdown for p53 or miR-628-3p confirmed that SHK inhibits the growth and induces apoptosis of A549 and PC-9 cells at least partly by up-regulating p53/miR-628-3p signaling pathway. Therefore, these novel findings provide an alternative approach to target p53/miR-628-3p axis and could be used for the development of new treatment strategies for NSCLC.

Corrigendum: Downregulation of miR-135b-5p Suppresses Progression of Esophageal Cancer and Contributes to the Effect of Cisplatin.

[This corrects the article DOI: 10.3389/fonc.2021.679348.].

Downregulation of miR-135b-5p Suppresses Progression of Esophageal Cancer and Contributes to the Effect of Cisplatin.

Esophageal cancer (EC) is one of the commonest human cancers, which accompany high morbidity. MicroRNAs (miRNAs) play a pivotal role in various cancers, including EC. Our research aimed to reveal the function and mechanism of miR-135b-5p. Our research identified that miR-135b-5p was elevated in EC samples from TCGA database. Correspondingly real-time PCR assay also showed the miR-135b-5p is also higher expressed in Eca109, EC9706, KYSE150 cells than normal esophageal epithelial cells (Het-1A). CCK8, Edu, wound healing, Transwell assay, and western blot demonstrated miR-135b-5p inhibition suppresses proliferation, invasion, migration and promoted the apoptosis in Eca109 and EC9706 cells. Moreover, the miR-135b-5p inhibition also inhibited xenograft lump growth. We then predicted the complementary gene of miR-135b-5p using miRTarBase, TargetScan, and DIANA-microT. TXNIP was estimated as a complementary gene for miR-135b-5p. Luciferase report assay verified the direct binding site for miR-135b-5p and TXNIP. Real-time PCR and western blot assays showed that the inhibition of miR-135b-5p remarkably enhanced the levels of TXNIP in Eca109 and EC9706 cells. Furthermore, cisplatin (cis-diamminedichloroplatinum II, DDP) decreased miR-135b-5p expression and increased TXNIP expression. Enhanced expression of miR-135b-5p attenuated the inhibitory ability of cisplatin (cis-diamminedichloroplatinum II, DDP) in Eca109 cells, accompanied by TXNIP downregulation. In conclusion, the downregulation of miR-135b-5p suppresses the progression of EC through targeting TXNIP. MiR-135b-5p/TXNIP pathway contributes to the anti-tumor effect of DDP. These findings may provide new insight into the treatment of EC.

Identification of a Novel Prognostic Signature for Gastric Cancer Based on Multiple Level Integration and Global Network Optimization.

Gastric Cancer (GC) is a common cancer worldwide with a high morbidity and mortality rate in Asia. Many prognostic signatures from genes and non-coding RNA (ncRNA) levels have been identified by high-throughput expression profiling for GC. To date, there have been no reports on integrated optimization analysis based on the GC global lncRNA-miRNA-mRNA network and the prognostic mechanism has not been studied. In the present work, a Gastric Cancer specific lncRNA-miRNA-mRNA regulatory network (GCsLMM) was constructed based on the ceRNA hypothesis by combining miRNA-target interactions and data on the expression of GC. To mine for novel prognostic signatures associated with GC, we performed topological analysis, a random walk with restart algorithm, in the GCsLMM from three levels, miRNA-, mRNA-, and lncRNA-levels. We further obtained candidate prognostic signatures by calculating the integrated score and analyzed the robustness of these signatures by combination strategy. The biological roles of key candidate signatures were also explored. Finally, we targeted the PHF10 gene and analyzed the expression patterns of PHF10 in independent datasets. The findings of this study will improve our understanding of the competing endogenous RNA (ceRNA) regulatory mechanisms and further facilitate the discovery of novel prognostic biomarkers for GC clinical guidelines.

Anti-Inflammatory Effect Fraction of Bletilla striata and Its Protective Effect on LPS-Induced Acute Lung Injury.

Bletilla striata is a well-known traditional Chinese herb with anti-inflammatory properties that is widely used in the treatment of lung conditions such as silicosis, tuberculosis, and pneumogastric hemorrhage. However, little information on the anti-inflammatory ingredients and their activities is available. In this study, an effect fraction of Bletilla striata (EFBS) was enriched, and its anti-inflammatory activities and underlying mechanisms were investigated. EFBS was enriched by polyamide column chromatography and characterized by HPLC; an LPS-induced acute lung injury model was used to evaluate the anti-inflammatory activities of EFBS. Meanwhile, the main anti-inflammation-contributing ingredients and possible molecular mechanism of anti-inflammatory activity in EFBS were verified by component-knockout method combined with LPS-induced RAW264.7 cell model. The EFBS mainly consisted of coelonin (15.88%), batatasin III (32.49%), 3'-O-methylbatatasin III (6.96%), and 3-hydroxy-5-methoxy bibenzyl (2.51%). Pretreatment with the EFBS (20 mg/kg and 60 mg/kg) for five days prior to the administration of LPS resulted in decreases in wet-to-dry lung weight ratio, neutrophil number, MPO activity, total protein concentration, NO level, and MDA level, as well as IL-1ß, IL-6, MCP-1, and TNF-α concentrations in the bronchoalveolar lavage fluid. Western blot analysis demonstrated the increased expressions of iNOS, COX-2, and NF-κB p65 in the LPS treatment group, all of which were ameliorated by EFBS pretreatment. Histological examination confirmed the protective effect of the EFBS. Additionally, component-knockout assay confirmed that these four quantitative components contributed significantly to the anti-inflammatory effect of EFBS. Coelonin, batatasin III, 3'-O-methylbatatasin III and 3-hydroxy-5-methoxy bibenzyl were the main anti-inflammatory components of EFBS and could regulate the expression of downstream inflammatory cytokines by inhibiting p65 nuclear translocation. These findings uncover, in part, the molecular basis underlying the anti-inflammatory activity of Bletilla striata.

The potential role of TNFAIP3 in malignant transformation of gastric carcinoma.

Tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a deubiquitinating enzyme, plays an essential regulatory role in inflammation, immune responses and tumorigenesis. Our present study indicates that TNFAIP3 is required for the ubiquitination degradation of epithelial-mesenchymal transition (EMT) related transcription factors Snail and ZEB1, which further altered the expression of EMT-related marker proteins and eventually contributing to the malignant phenotype and poorer prognosis of gastric carcinoma. Depletion of TNFAIP3 attenuated the capacity of proliferation, migration and invasion of gastric cancer cells in vitro. Taken together, these findings propose a pathway linking the TNFAIP3 to the EMT-mediated metastatic process in gastric cancer, which provides a viable strategy regarding the interventions and prognostic analysis of gastric carcinoma in clinical practice.

Three-dimensional microtissues as an in vitro model for personalized radiation therapy.

This paper describes the use of 3D microtissues as an intermediate model between the 2D cell culture and the animal model to assess radiation-induced cellular and DNA damage in the context of personalized radiation therapy. An agarose microwell array was used to generate 3D microtissues with controlled size and shape. The microtissues were exposed to X-ray radiation of various doses, and the radiation damage to cells was examined using a variety of techniques with different end points. Damage to cell membranes and reduction in metabolic activity were examined with the MTT assay and dye inclusion assay. DNA damage was tested with the micronucleus assay, γ-H2AX immunostaining, and HaloChip assay. 3D microtissues exposed to X-rays are smaller compared to unexposed ones in extended cultures, indicating that X-ray radiation can retard the growth of cells in 3D microtissues, where cells at the outer shells of microtissues can prevent further damage to those inside.

Mechanical and Morphological Analysis of Cancer Cells on Nanostructured Substrates.

Cancer metastasis is a major cause of cancer-induced deaths in patients. Mimicking nanostructures of an extracellular matrix surrounding cancer cells can provide useful clues for metastasis. This paper compares the morphology, proliferation, spreading, and stiffness of highly aggressive glioblastoma multiforme cancer cells and normal fibroblast cells seeded on a variety of ordered polymeric nanostructures (nanopillars and nanochannels). Both cell lines survive and proliferate on the nanostructured surface and show more similarity on nanostructured surfaces than on flat surfaces. Although both show similar stiffness on the nanochannel surface, glioblastomas are softer, spread to a larger area, and elongate less than fibroblasts. The nanostructured surfaces are useful for in vitro model of an extracellular matrix to study the cancer cell migratory phenotype.