circNOX4 activates an inflammatory fibroblast niche to promote tumor growth and metastasis in NSCLC via FAP/IL-6 axis.

BACKGROUND: Cancer-associated fibroblasts (CAFs) orchestrate a supportive niche that fuels cancer metastatic development in non-small cell lung cancer (NSCLC). Due to the heterogeneity and plasticity of CAFs, manipulating the activated phenotype of fibroblasts is a promising strategy for cancer therapy. However, the underlying mechanisms of fibroblast activation and phenotype switching that drive metastasis remain elusive. METHODS: The clinical implications of fibroblast activation protein (FAP)-positive CAFs (FAP+CAFs) were evaluated based on tumor specimens from NSCLC patients and bioinformatic analysis of online databases. CAF-specific circular RNAs (circRNAs) were screened by circRNA microarrays of primary human CAFs and matched normal fibroblasts (NFs). Survival analyses were performed to assess the prognostic value of circNOX4 in NSCLC clinical samples. The biological effects of circNOX4 were investigated by gain- and loss-of-function experiments in vitro and in vivo. Fluorescence in situ hybridization, luciferase reporter assays, RNA immunoprecipitation, and miRNA rescue experiments were conducted to elucidate the underlying mechanisms of fibroblast activation. Cytokine antibody array, transwell coculture system, and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the downstream effectors that promote cancer metastasis. RESULTS: FAP+CAFs were significantly enriched in metastatic cancer samples, and their higher abundance was correlated with the worse overall survival in NSCLC patients. A novel CAF-specific circRNA, circNOX4 (hsa_circ_0023988), evoked the phenotypic transition from NFs into CAFs and promoted the migration and invasion of NSCLC in vitro and in vivo. Clinically, circNOX4 correlated with the poor prognosis of advanced NSCLC patients. Mechanistically, circNOX4 upregulated FAP by sponging miR-329-5p, which led to fibroblast activation. Furthermore, the circNOX4/miR-329-5p/FAP axis activated an inflammatory fibroblast niche by preferentially inducing interleukin-6 (IL-6) and eventually promoting NSCLC progression. Disruption of the intercellular circNOX4/IL-6 axis significantly suppressed tumor growth and metastatic colonization in vivo. CONCLUSIONS: Our study reveals a role of the circRNA-induced fibroblast niche in tumor metastasis and highlights that targeting the circNOX4/FAP/IL-6 axis is a promising strategy for the intervention of NSCLC metastasis.

Danshensu alleviates pseudo-typed SARS-CoV-2 induced mouse acute lung inflammation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can induce acute inflammatory response like acute lung inflammation (ALI) or acute respiratory distress syndrome, leading to severe progression and mortality. Therapeutics for treatment of SARS-CoV-2-triggered respiratory inflammation are urgent to be discovered. Our previous study shows that Salvianolic acid C potently inhibits SARS-CoV-2 infection. In this study, we investigated the antiviral effects of a Salvia miltiorrhiza compound, Danshensu, in vitro and in vivo, including the mechanism of S protein-mediated virus attachment and entry into target cells. In authentic and pseudo-typed virus assays in vitro, Danshensu displayed a potent antiviral activity against SARS-CoV-2 with EC50 of 0.97 µM, and potently inhibited the entry of SARS-CoV-2 S protein-pseudo-typed virus (SARS-CoV-2 S) into ACE2-overexpressed HEK-293T cells (IC50 = 0.31 µM) and Vero-E6 cell (IC50 = 4.97 µM). Mice received SARS-CoV-2 S via trachea to induce ALI, while the VSV-G treated mice served as controls. The mice were administered Danshensu (25, 50, 100 mg/kg, i.v., once) or Danshensu (25, 50, 100 mg·kg-1·d-1, oral administration, for 7 days) before SARS-CoV-2 S infection. We showed that SARS-CoV-2 S infection induced severe inflammatory cell infiltration, severely damaged lung tissue structure, highly expressed levels of inflammatory cytokines, and activated TLR4 and hyperphosphorylation of the NF-κB p65; the high expression of angiotensinogen (AGT) and low expression of ACE2 at the mRNA level in the lung tissue were also observed. Both oral and intravenous pretreatment with Danshensu dose-dependently alleviated the pathological alterations in mice infected with SARS-CoV-2 S. This study not only establishes a mouse model of pseudo-typed SARS-CoV-2 (SARS-CoV-2 S) induced ALI, but also demonstrates that Danshensu is a potential treatment for COVID-19 patients to inhibit the lung inflammatory response.

[Dihydroartemisinin alleviates atopic dermatitis in mice by inhibiting mast cell infiltration].

OBJECTIVE: To observe the therapeutic effect of different doses of dihydroartemisinin (DHA) on atopic dermatitis (AD) in mice and explore the mechanism. METHODS: Forty-two C57BL/6 mice were randomly divided into 7 groups (n=6), including a blank control group, a 2, 4-dinitrochlorobenzene (DNCB)-induced AD model group, a solvent-treated group, 3 DHA treatment groups treated with 25, 75, and 125 mg/kg DHA, and a dexamethasone treatment group. The counts of skin scratches were recorded and the lesion scores were evaluated on a daily basis. After 7 consecutive days of treatment, skin tissues were sampled from the lesions on the back and ear of the mice for pathological examination with HE staining, Masson staining and toluidine blue staining. RESULTS: Treatment with 25, 75, and 125 mg/kg DHA and dexamethasone all alleviated AD symptoms of mice, reduced the severity scores of skin lesions, and ameliorated pathological changes of the skin tissue. DHA at 125 mg/kg produced the most obvious therapeutic effect and significantly alleviated mast cell infiltration in the lesions as compared with the other treatment groups (P < 0.05). CONCLUSIONS: DHA is effective for the treatment of AD in mice with an optimal dose of 125 mg/kg. The therapeutic effect of DHA is achieved probably through regulation of local immunity by inhibiting mast cell infiltration in the lesions.

Effect of Intratracheal Instillation of ZnO Nanoparticles on Acute Lung Inflammation Induced by Lipopolysaccharides in Mice.

Although studies have shown toxic effects of zinc oxide (ZnO) particles following inhalation, additional effects on injured lungs, which are characterized by dysfunction of the alveolar-capillary barriers, remain uncharacterized. To explore these additional effects, nano-sized ZnO (nZnO) and bulk-sized ZnO were applied to lipopolysaccharide (LPS)-challenged mouse lungs, which were used as a disease model of acute lung inflammation. An elevated Zn2+ concentration was detected in lung tissue after LPS plus nZnO exposure. Exposure to nZnO in LPS-challenged mice resulted in higher total cell number, proportion of neutrophils, and total protein level in bronchoalveolar lavage fluid. Intratracheal instillation of nZnO intensively aggravated LPS-induced lung inflammation that was accompanied by enhanced expression of interleukin-1ß, interleukin-6, monocyte chemotactic protein-1α, and granulocyte-macrophage colony stimulating factor. Catalase, glutathione, and total superoxide dismutase levels were significantly decreased, and the malondialdehyde level was obviously increased in the LPS plus nZnO group. 8-Hydroxyguanosine, a marker for DNA damage, was highly concentrated in the lungs from the LPS plus nZnO group. Furthermore, nZnO increased lung apoptosis in an acute lung inflammation model. Taken together, this evidence indicates that nZnO aggravates lung inflammation related to LPS. This enhancement effect may be mediated via oxidative stress, which can lead to DNA damage and apoptosis. This work is important because of the ever-increasing exposure of people to ZnO nanoparticles in industry. The identification of the toxic effects of nZnO and possible mechanisms revealed in this study provide valuable information for future studies.

The expression and construction of engineering Escherichia coli producing humanized AluY RNAs.

BACKGROUND: Exogenous RNAs can specifically up-regulate or down-regulate gene expression after they enter into cells. Alu RNAs are the main constituent of human transcriptome and participate in gene expression regulation. AluY elements belong to a subfamily of Alus and are the youngest Alus. In this paper, we established the technology method of preparing genetically engineered humanized AluY RNAs (AluY RNAs) from Escherichia coli (E. coli) strains. This technology method also can be used to prepare other genetically engineered humanized RNAs that can be used for cytology experiments. RESULTS: Different copies of human AluY elements were inserted into pET-28α plasmid (pET) to construct pET-AluY plasmids that were transformed into BMBL21-DE3 (DE3) E. coli. Isopropylthio-ß-D-galactoside (IPTG) induction inhibited transformed bacterial growth after DE3 E. coli were transformed by pET-AluY × 8 plasmid (8 copies of AluYs were inserted into pET); northern blotting was used to detect the amount of AluY RNAs after 2, 4, 6, 8, 10, 12, 14 and 16 h inducing with IPTG. The results showed that the amount of AluY RNAs was the highest at 4 h; 1, 2, 4, 8 or 14 copies of AluY elements were inserted into the pET to construct pET-AluY plasmids that were transformed into DE3 bacteria, the northern blotting results showed that AluY RNAs production amount increased with the increase of AluY copy number; pET-AluY × 8 DE3 bacteria did not produce AluY RNAs without IPTG induction, AluY RNA production kept similar when inducing by 0.1-0.4 mg/ml IPTG induction, however, AluY RNA production slightly decreased if deviating from the above concentration range; pET-AluY × 8 DE3 bacteria were cultured at 34, 37 or 40 °C and the results showed that AluY RNA production was the highest under 37 °C cultivation; pET-AluY × 8 plasmid was transformed into three kinds of BL21 bacteria, including DE3, BMBL21-DE3-pLysS (pLysS) and Trans BL 21 (TransBL), the results showed that AluY RNA production was the highest when using DE3 bacteria. CONCLUSIONS: The optimal conditions of producing AluY RNAs were: a kind of host bacteria of DE3, an engineering bacteria concentration of OD600 1.0, an IPTG concentration of 0.2 mg/ml, a culturing temperature of 37 °C and a culturing time of 4 h. Pure AluY RNAs occupied 15.8% of extractive total RNAs and the mean yield of pure AluY RNAs in 100 ml bacteria solution was 0.46 mg.

Combined sense-antisense Alu elements activate the EGFP reporter gene when stable transfection.

Alu elements in the human genome are present in more than one million copies, accounting for 10% of the genome. However, the biological functions of most Alu repeats are unknown. In this present study, we detected the effects of Alu elements on EGFP gene expression using a plasmid system to find the roles of Alu elements in human genome. We inserted 5'-4TMI-Alus-CMV promoter-4TMI-Alus (or antisense Alus)-3' sequences into the pEGFP-C1 vector to construct expression vectors. We altered the copy number of Alus, the orientation of the Alus, and the presence of an enhancer (4TMI) in the inserted 5'-4TMI-Alus-CMV promoter-4TMI-Alus (or antisense Alus)-3' sequences. These expression vectors were stably transfected into HeLa cells, and EGFP reporter gene expression was determined. Our results showed that combined sense-antisense Alu elements activated the EGFP reporter gene in the presence of enhancers and stable transfection. The combined sense-antisense Alu vectors carrying four copies of Alus downstream of inserted CMV induced much stronger EGFP gene expression than two copies. Alus downstream of inserted CMV were replaced to AluJBs (having 76% homology with Alu) to construct expression vectors. We found that combined sense-antisense Alu (or antisense AluJB) vectors induced strong EGFP gene expression after stable transfection and heat shock. To further explore combined sense-antisense Alus activating EGFP gene expression, we constructed Tet-on system vectors, mini-C1-Alu-sense-sense and mini-C1-Alu-sense-antisense (EGFP gene was driven by mini-CMV). We found that combined sense-antisense Alus activated EGFP gene in the presence of reverse tetracycline repressor (rTetR) and doxycycline (Dox). Clone experiments showed that Mini-C1-Alu-sense-antisense vector had more positive cells than that of Mini-C1-Alu-sense-sense vector. The results in this paper proved that Alu repetitive sequences inhibited gene expression and combined sense-antisense Alus activated EGFP reporter gene when Alu transcribes, which suggests that Alus play roles in maintaining gene expression (silencing genes or activating genes) in human genome.