Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA.

BACKGROUND: Total human immunodeficiency virus (HIV) DNA and integrated HIV DNA are widely used markers of HIV persistence. Droplet digital polymerase chain reaction (ddPCR) can be used for absolute quantification without needing a standard curve. Here, we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA. METHODS: The limit of detection, dynamic ranges, sensitivity, and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat (LTR) and human CD3 gene (for total HIV DNA) and ACH-2 cells (for integrated HIV DNA). Forty-two cases on stable suppressive antiretroviral therapy (ART) were assayed in total HIV DNA and integrated HIV DNA. Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive (CD4 + ) T-cell counts, CD8 + T-cell counts and CD4/CD8 T-cell ratio, respectively. The assay linear dynamic range and lower limit of detection (LLOD) were also assessed. RESULTS: The assay could detect the presence of HIV-1 copies 100% at concentrations of 6.3 copies/reaction, and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction (95% confidence intervals [CI]: 3.6-6.5 copies/reaction) with linearity over a 5-log 10 -unit range in total HIV DNA assay. For the integrated HIV DNA assay, the LLOD was 8.0 copies/reaction (95% CI: 5.8-16.6 copies/reaction) with linearity over a 3-log 10 -unit range. Total HIV DNA in CD4 + T cells was positively associated with integrated HIV DNA ( r = 0.76, P <0.0001). Meanwhile, both total HIV DNA and integrated HIV DNA in CD4 + T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8 + T-cell counts. CONCLUSIONS: This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity. It can be readily adapted for measuring HIV DNA with non-B clades, and it could be beneficial for testing in clinical trials.

Beyond the promise: Exploring the complex interactions of nanoparticles within biological systems.

The exploration of nanoparticle applications is filled with promise, but their impact on the environment and human health raises growing concerns. These tiny environmental particles can enter the human body through various routes, such as the respiratory system, digestive tract, skin absorption, intravenous injection, and implantation. Once inside, they can travel to distant organs via the bloodstream and lymphatic system. This journey often results in nanoparticles adhering to cell surfaces and being internalized. Upon entering cells, nanoparticles can provoke significant structural and functional changes. They can potentially disrupt critical cellular processes, including damaging cell membranes and cytoskeletons, impairing mitochondrial function, altering nuclear structures, and inhibiting ion channels. These disruptions can lead to widespread alterations by interfering with complex cellular signaling pathways, potentially causing cellular, organ, and systemic impairments. This article delves into the factors influencing how nanoparticles behave in biological systems. These factors include the nanoparticles' size, shape, charge, and chemical composition, as well as the characteristics of the cells and their surrounding environment. It also provides an overview of the impact of nanoparticles on cells, organs, and physiological systems and discusses possible mechanisms behind these adverse effects. Understanding the toxic effects of nanoparticles on physiological systems is crucial for developing safer, more effective nanoparticle-based technologies.

hucMSCs Treatment Ameliorated Pulmonary Fibrosis via Downregulating the circFOXP1-HuR-EZH2/STAT1/FOXK1 Autophagic Axis.

This study was performed to determine the effect of human umbilical cord mesenchymal stem cells (hucMSCs) treatment on pulmonary fibrosis and investigate the circFOXP1-mediated autophagic mechanism of hucMSCs treatment. Pulmonary fibrosis models were established by spraying bleomycin in mice and TGF-ß1 treatment of MRC-5 cells. Results showed that hucMSCs were retained in lung and hucMSCs treatment alleviated pulmonary fibrosis. Morphological staining indicated that hucMSCs-treated mice had thinner alveolar walls, effectively improved alveolar structure, significantly reduced alveolar inflammation, and decreased collagen deposition than control mice. Fibrotic proteins, including vimentin, α-SMA, collagens I and III, and the differentiation-related protein S100 calcium-binding protein A4 was reduced considerably in the hucMSCs-treated group. The mechanistic study revealed that the inhibition of hucMSCs treatment on pulmonary fibrogenesis depended on downregulating circFOXP1, in which hucMSCs treatment promoted circFOXP1-mediated autophagy process via blocking the nuclear human antigen R (HuR) translocation and promoting the HuR degradation, leading to a marked decrease in autophagy negative regulators EZH2, STAT1, and FOXK1. In conclusion, hucMSCs treatment significantly improved pulmonary fibrosis by downregulating the circFOXP1-HuR-EZH2/STAT1/FOXK1 autophagic axis. hucMSCs can act as an effective treatment for pulmonary fibrosis.

hucMSCs treatment prevents pulmonary fibrosis by reducing circANKRD42-YAP1-mediated mechanical stiffness.

Idiopathic pulmonary fibrosis (IPF) is a fibrosing interstitial pneumonia of unknown cause. The most typical characteristic of IPF is gradual weakening of pulmonary elasticity and increase in hardness/rigidity with aging. This study aims to identify a novel treatment approach for IPF and explore mechanism of mechanical stiffness underlying human umbilical cord mesenchymal stem cells (hucMSCs) therapy. Target ability of hucMSCs was examined by labeling with cell membrane dye Dil. Anti-pulmonary fibrosis effect of hucMSCs therapy by reducing mechanical stiffness was evaluated by lung function analysis and MicroCT imaging system and atomic force microscope in vivo and in vitro. Results showed that stiff environment of fibrogenesis caused cells to establish a mechanical connection between cytoplasm and nucleus, initiating expression of related mechanical genes such as Myo1c and F-actin. HucMSCs treatment blocked force transmission and reduced mechanical force. For further exploration of mechanism, ATGGAG was mutated to CTTGCG (the binding site of miR-136-5p) in the full-length sequence of circANKRD42. Wildtype and mutant plasmids of circANKRD42 were packaged into adenovirus vectors and sprayed into lungs of mice. Mechanistic dissection revealed that hucMSCs treatment repressed circANKRD42 reverse splicing biogenesis by inhibiting hnRNP L, which in turn promoted miR-136-5p binds to 3'-Untranslated Region (3'-UTR) of YAP1 mRNA directly, thus inhibiting translation of YAP1 and reducing YAP1 protein entering nucleus. The condition repressed expression of related mechanical genes to block force transmission and reduce mechanical forces. The mechanosensing mechanism mediated directly by circANKRD42-YAP1 axis in hucMSCs treatment, which has potential general applicability in IPF treatment.

Dissolved oxygen gradient on three dimensionally printed microfluidic platform for studying its effect on fish at three levels: cell, embryo, and larva.

A simple and low-cost dissolved oxygen gradient platform of three dimensionally (3D) printed microfluidic chip was developed for cultivating cells, embryos, and larvae of fish. "Christmas tree" structure channel networks generated a dissolved oxygen gradient out of two fluids fed to the device. Polydimethylsiloxane (PDMS) membrane with high biocompatibility was used as the substrate for cell culture in the 3D-printed microfluidic chip, which made the cell analysis easy. The embryos and larvae of fish could be cultured directly in the chip, and their development can be observed in real time with a microscope. Using zebrafish as a model, we assessed the effect of different dissolved oxygen on its cells, embryos, and larvae. Hypoxia induced production of reactive oxygen species (ROS) in zebrafish cells, embryos, and larvae, eventually leading to cell apoptosis and developmental impairment. Hypoxia also increased nitric oxide content in zebrafish cells, which might be a defensive strategy to overcome the adverse effect of hypoxia in fish cells. This is the first platform that could comprehensively investigate the effects of different dissolved oxygen on fish at the cell, embryo, and larva levels, which has great potential in studying the responses of aquatic organisms under different oxygen concentrations.

Diesel exhaust PM2.5 greatly deteriorates fibrosis process in pre-existing pulmonary fibrosis via ferroptosis.

Fine particulate matter (PM2.5) has been widely reported to contribute to the pathogenesis of pulmonary diseases. The direct hazardous effect of PM2.5 on the respiratory system at high concentrations in vitro and in vivo have been well identified. However, its effect on the pre-existing respiratory diseases of patients at environment-related concentrations remains unclear. Diesel exhaust PM2.5 as a primary representative of ambient PM2.5 fine particles were used to investigated the effect of PM2.5 on the fibrosis progression of existing pulmonary fibrosis disease models. This study reported that PM2.5 could result in the enhanced sensitivity to fibrotic response, which may be ascribed to ferroptosis induced by PM2.5 in damaged lung areas. Proteomic analysis revealed that the upregulation of HO-1 as a key mechanism in the ferroptosis and exacerbation of pulmonary fibrosis induced by PM2.5. As a result, HO-1 degraded heme-containing protein and released iron in fibrotic cells, leading to generation of mitochondrial ROS and impaired mitochondrial function. Transmission electron microscopic assay verified that PM2.5 entered the mitochondria of fibrotic cells and was accompanied by significant mitochondrial morphological changes characterized by increased mitochondrial membrane density and reduced mitochondrial size. The HO-1 inhibitor zinc protoporphyrin and mitochondrion-targeted antioxidant Mito-TEMPO significantly attenuated PM2.5-induced ferroptosis and exacerbation of fibrosis. In addition, AMPK-ULK1 axis-triggered autophagy activation and NCOA4-mediated degradation of ferritin by autophagy were found to be related to the PM2.5-induced ferroptosis of fibrotic cells. As evidenced by the inhibition of autophagy with 3-methyladenine or AMPK inhibitor, NCOA4 knockdown decreased intracellular iron accumulation and lipid peroxidation, thereby relieving PM2.5-induced epithelial-mesenchymal transition and cell death in fibrotic cells. Overall, this study provided experimental support for the idea that PM2.5 greatly deteriorates fibrosis process in pre-existing pulmonary fibrosis, and HO-1-mediated mitochondrial dysfunction and NCOA4-mediated ferritinophagy are jointly required for the PM2.5-induced ferroptosis and enhanced fibrosis effects.

Corrigendum: Danshensu methyl ester enhances autophagy to attenuate pulmonary fibrosis by targeting lncIAPF-HuR complex.

[This corrects the article DOI: 10.3389/fphar.2022.1013098.].

Danshensu methyl ester enhances autophagy to attenuate pulmonary fibrosis by targeting lncIAPF-HuR complex.

Pulmonary fibrosis is an irreversible fibrotic process that has a high mortality rate and limited treatment options; thus, developing a novel therapeutic drug is critical. In this study, we synthesized danshensu methyl ester (DME) and explored its anti-pulmonary fibrotic ability on TGF-ß1-stimulated lung fibroblast in vitro and on bleomycin-induced pulmonary fibrosis in vivo. Results showed that DME decreased the expression of differentiation-related proteins, including fibroblast activation protein 1 (FAP1) and S100 calcium-binding protein A4 (S100A4), and fibrotic markers, such as a-SMA, vimentin, and collagen in vivo and in vitro. In addition, DME markedly repressed myofibroblast proliferation and migration. Mechanistically, chromatin immunoprecipitation-PCR, RNA immunoprecipitation, half-life, and other experiments revealed that DME inhibited activating transcription factor 3 expression via TGF-ß1 signal transduction leading to a decrease in lncIAPF transcription and stability. Moreover, DME blocked human antigen R (HuR) nucleocytoplasmic translocation and promoted its degradation via downregulating lncIAPF, which markedly decreased the expression of HuR target genes such as negative autophagic regulators (EZH2, STAT1, and FOXK1). Collectively, our results demonstrated that DME enhanced autophagy to attenuate pulmonary fibrosis via downregulating the lncIAPF-HuR-mediated autophagic axis and the lncIAPF-HuR complex can be the target for drug action.

The combined toxic effects of polyvinyl chloride microplastics and di(2-ethylhexyl) phthalate on the juvenile zebrafish (Danio rerio).

Microplastics (MPs) have the characteristics of large specific surface area, high hydrophobicity and surface charge, so they are easy to combine with other pollutants and cause toxic effects on aquatic organisms. Here, we prepared a polyvinyl chloride-microplastics (PVC-MPs) fragmentation model to simulate the real microplastic state, and characterized its composition, morphology, particle size and zeta potential. On this basis, we used single and compound exposure of PVC and di(2-ethylhexyl) phthalate (DEHP) to explore their effects on hatchability and mortality of zebrafish (Danio rerio) embryos and toxicity to oxidative stress and cardiac development in zebrafish larvae. Herein, PVC-MPs slowed down the hatching rate of zebrafish embryos and induced the death of zebrafish, while DEHP could slow down the induced of death, it had no effect on hatching rate. The PVC-MPs/DEHP single pollution could induce the reactive oxygen species (ROS) and activated the antioxidant defense signaling pathway, while the compound group showed the level of feedback autoregulation of NF-E2-related factor 2 (Nrf2) signaling pathway. The single pollution also could inhibit the expression of genes related to cardiac development, while the combined pollution showed an antagonistic effect. This study provided a theoretical basis for the ecotoxicology and biomonitoring of MPs in the natural state.

Pimozide inhibits the growth of breast cancer cells by alleviating the Warburg effect through the P53 signaling pathway.

The Warburg effect is a promising target for the diagnosis and treatment of cancer, referring to the ability of cancer cells to generate energy through high levels of glycolysis even in the presence of oxygen, allowing them to grow and proliferate rapidly. The antipsychotic Pimozide has strong anti-breast cancer effects both in vivo and in vitro, whether Pimozide has an inhibitory effect on aerobic glycolysis has not been elucidated. In this study, Pimozide inhibited the Warburg effect of breast cancer cells by hindering glucose uptake, ATP level and lactate production; reducing the extracellular acidification rate (ECAR); suppressing the expression of PKM2, a rate-limiting enzyme in glycolysis. Intriguingly, Pimozide was significantly involved in reprogramming glucose metabolism in breast cancer cells through a p53-dependent manner. Mechanistic studies demonstrated Pimozide increased the expression of p53 through inhibition of the PI3K/Akt/MDM2 signaling pathway, which in turn downregulated the expression of PKM2. In sum, our results suggest that Pimozide mediates the p53 signaling pathway through PI3K/AKT/MDM2 to inhibit the Warburg effect and breast cancer growth, and it may be a potential aerobic glycolysis inhibitor for the treatment of breast cancer.

Quantitative assessment of in vivo distribution of nanoplastics in bivalve Ruditapes philippinarum using reliable SERS tag-labeled nanoplastic models.

Nanoplastics (NPs) as emerging marine pollutants can be taken up by seafood organisms. It is crucial to quantitatively assess NP's distribution behavior in organisms to elucidate concentration dependent biological effects. Such a knowledge gap has remained due to the lack of reliable NP models and analytical methods. Herein, surface enhanced Raman scattering (SERS)-labeled NP models were developed and their bioavailability, distribution and accumulation in Ruditapes philippinarum, a typical marine bivalve, were quantitatively studied. Taking advantage of the sensitive and characteristic SERS signals of the NP models, distribution could be quickly and accurately obtained by the Raman imaging technique. Moreover, quantitative analysis of NPs could be performed by the detection of gold element contents via inductively coupled plasma mass spectroscopy (ICP-MS) detection. ICP-MS results revealed that after 3 days exposure of monodispersed NPs (100 nm, 0.2 mg L-1), the digestive gland accumulated 86.7% of whole-body NPs followed by gill (5.2%), mantle (5.1%), foot (1.3%), exhalant siphon (1.1%), and adductor (0.6%). Upon 11 days depuration, 98.7% of NPs in the digestive gland were excreted, whereas the clearance ratios in other organs were much lower. NP aggregates (around 1.5 µm) demonstrated similar distribution and clearance trends to the monodispersed ones. However, the accumulation amount in each organ was 15.2% to 77.6% lower. Surface adherence and passive ingestion routes resulted in NP accumulation, which contributed to the comparable NP abundance in these organs. Additionally, boiling treatment (mimicking a cooking process) did not decrease the NP amount in these organs. This work provided a dual-mode and quantitative analysis protocol for NPs for the first time, and suggested the risk of NP uptake by humans via bivalve seafood diets.

Polystyrene nanoplastics demonstrate high structural stability in vivo: A comparative study with silica nanoparticles via SERS tag labeling.

Nanoplastics are regarded as inert particulate pollutants pose potential threat to organisms. It has been verified that they can penetrate biological barriers and accumulate in organisms; however, there is still a knowledge gap on the in vivo stability and degradation behaviors due to the lack of ideal analytical methods. Herein, a surface-enhanced Raman scattering (SERS) tag labeling technique was developed to study the in vivo behaviors of polystyrene (PS) nanoplastics by comparison with silica (SiO2) nanoparticles (NPs). The labeled NPs were composed of gold NP core, attached Raman reporters as well as PS and silica shell, respectively, demonstrating strong SERS signals which were responsive to the compactness of the shells. The labeled NPs enabled the probing of in vivo structural stability of PS and silica in the liver, spleen and lung of mice after intravenous injection via the time-dependent evolution of SERS signal intensity and gold element content in the organs. The results indicated that both PS and silica model NPs retained in these organs without apparent excretion within 28 d. However, the structural stabilities of PS and silica differed dramatically as reflected by the SERS signal and tissue slice characterization. The silica shell completely degraded whereas the PS shell was still compact. Our results verified the long-term accumulation and in vivo inert property of nanoplastics, hinting that they were distinct from natural NPs and probably induce higher health risks from the aspect of the non-degradation property.

Abnormal Shift in B Memory Cell Profile Is Associated With the Expansion of Circulating T Follicular Helper Cells via ICOS Signaling During Acute HIV-1 Infection.

Interactions between T follicular helper (Tfh) cells and germinal center B cells are essential for the differentiation of B cells and specific antibody responses against HIV-1 infection. However, the extent to which HIV-1 infection affects the dynamic interplay between these two cell populations in the bloodstream remains unclear. In this study, the dynamics of circulating Tfh (cTfh) and B cells and their relationship in individuals with acute and chronic HIV-1 infection were investigated. Twenty-five study subjects were enrolled from the Beijing PRIMO clinical cohort, a prospective cohort of HIV-1-negative men who have sex with men (MSM) for the identification of cases of acute HIV-1 infection (AHI) at Beijing Youan Hospital, Capital Medical University. Individuals with AHI were selected at random. Matched samples were also collected and analyzed from the same patients with chronic HIV-1 infection. None of the study subjects received antiretroviral therapy during acute or chronic infection. Multicolor flow cytometry was used for the immunophenotypic and functional characterization of cTfh cell and B cell subsets. AHI resulted in increased proportions in bulk cTfh, ICOS+cTfh or IL-21+ICOS+cTfh cells. In both acute and chronic infections, activated memory (AM), tissue-like memory (TLM), and plasmablast (PB) B cell levels were increased whilst resting memory (RM) and naïve mature (NM) B cell levels were decreased. Classical memory (CM) B cells were unaffected during infection. Association analyses showed that the levels of ICOS+cTfh and IL-21+ICOS+cTfh cells were negatively correlated with those of AM, CM, RM cells, and positively correlated with those of NM cells in AHI but not chronic HIV-1 infection stage (CHI). Moreover, the frequency of IL-21+ICOS+cTfh cells was also positively correlated with plasma HIV-1 viral load, and had an opposite association trend with CD4+T cell count in AHI. Our data suggests that HIV-1 infection drives the expansion of cTfh cells, which in turn leads to perturbations of B cell differentiation through ICOS signaling during acute infection stage. These findings provide insight on the role of ICOS in the regulation of cTfh/B cell interaction during AHI and may potentially guide the design of effective strategies for restoring anti-HIV-1 immunity in the infected patients.

Surface-enhanced Raman scattering labeled nanoplastic models for reliable bio-nano interaction investigations.

Nanoplastics (NPs) have attracted great attention as an emerging pollution. To date, their interaction with biological systems has been studied mostly by using fluorescent-labeled NPs, which suffered from serious drawbacks such as biological autofluorescence interference and false-positive results. Reliable optically labeled NP models are eagerly desired until now. Herein, a novel near-infrared (NIR) surface-enhanced Raman scattering (SERS) labeled NP model was proposed, which gained single-particle ultra-sensitivity, deep tissue detection, multiplex labeling ability, and anti-interference property. More importantly, the NP demonstrated satisfactory in vivo signal stability which completely prevented the positive-false problems. The advantages of the NPs enabled direct, dynamic in vivo behavior imaging study in living zebrafish embryo, adult zebrafish and green vegetable Brassica rapa. It was found for the first time that NPs entered blood circulation system of zebrafish larva via dermal uptake route, which only occurred in a short 48 h-window post-hatch. NPs widely distributed in roots, shoots and leaves of Brassica rapa seedlings germinating and growing in the NP-containing hydroponic culture. Different depths of one root showed varied adsorption capabilities towards NPs with fulvic acid, lipid and sodium dodecyl sulfate eco-coronas. This work provided an ideal tool for reliable bio-NP interaction study for a variety of organisms, which could promote the research of NPs.

Tracking of realistic nanoplastics in complicated matrices by iridium element labeling and inductively coupled plasma mass spectroscopy.

Herein, we proposed a protocol to track realistic nanoplastics (NPs) by labeling them with an iridium-containing organic molecular agent (denoted as Ir) followed by inductively coupled plasma mass spectroscopy detection, as exemplified by polyethylene terephthalate (PET) NPs prepared from water bottles. The Ir showed satisfactory labeling stability in typical environmental and biological matrices. After 3d's incubation, the leaching ratios were less than 3% in water, phosphate buffered saline, sea water, cell culture medium, artificial gastric juice, artificial intestinal fluid, sediment resuspension, and around 5% in fetal bovine serum. On this basis, in vivo distribution of PET NPs in mice was analyzed. The intravenously injected NPs widely distributed in liver, spleen, lung and kidney. Comparatively, NPs could hardly be detected in these organs after intragastric administration, suggesting that they could not penetrate the intestinal barriers. The temporal and spatial distribution of the NPs in an intertidal zone sediment resuspension model was also investigated. The NPs mostly deposited at the overlying deposit, implying the absorption-driven sinking behavior of NPs with natural organic matters. This work provided an effective way to quantitatively track realistic NPs, which could promote the understanding of the fate and effect of NPs in natural environments and organisms.

Revisiting the cellular toxicity of benzo[a]pyrene from the view of nanoclusters: size- and nanoplastic adsorption-dependent bioavailability.

Benzo[a]pyrene (Bap) is one of the main organic pollutants in the atmospheric haze that is rich in fine water drops and particulate matters. The understanding of the Bap's form in water is of great importance to unveil its real biological effects toward the respiratory system. To date, various reports have documented its toxicological effects in the molecular form. Herein, we found that Bap existed as self-aggregated nanoclusters of tunable sizes rather than as dissolved molecules in water and different sized nanoclusters illustrated varied cytotoxicity. These findings indicated that the size, which has been ignored in previous studies, is also a dominant parameter similar to the molecular concentration for determining Bap's cytotoxicity. Polystyrene (PS) nanoparticles, as a model for nanoplastics, could adsorb Bap nanoclusters and serve as carriers that enter the cells. The combination effect interestingly altered the cytotoxicity distinction of Bap of different sizes. The intracellular fate of the nanoparticles and subcellular organelle damages were studied to unveil the mechanisms of cytotoxic distinction. Small Bap nanoclusters entered cells faster than their large counterparts. The Bap of the PS@Bap complex was stably adsorbed on PS at the early stages of endocytosis until it was detached during the lysosomal transport and maturation process. The dissociated Bap may bypass the lysosome pathway and be released into the cytosol with a nanocluster structure or relocate into the endoplasmic reticulum. On the other hand, the detached PS preferred to bind to the mitochondria or be excreted out of the cell via the lysosomal pathway. Moreover, the PS@Bap complex resulted in a significant loss of the mitochondrial membrane potential and induced apoptosis through the mitochondria-involved apoptosis pathway. This study provides a new perspective towards the toxicological mechanism of insoluble hydrophobic organic compounds and reveals the environmental significance of nanoplastics for regulating the biological effects of conventional pollutants.

Mucin corona delays intracellular trafficking and alleviates cytotoxicity of nanoplastic-benzopyrene combined contaminant.

Nanoplastics have recently become a worldwide concern as newly emerging airborne pollutants, which can associate with polycyclic aromatic hydrocarbons (PAHs) and form combined contaminant nanoparticles (CCNPs). After being inhaled in the respiratory system, the CCNPs would first encounter the mucous gel layer being rich in mucin. Herein, polystyrene-benzopyrene (PS@Bap) NPs were prepared as CCNPs model and their interaction with mucin and the resultant biological responses were studied. It was observed that mucin corona stably attached to the CCNPs surface, which significantly altered the fate of the CCNPs in lung epithelial cells (A 549 cell line). The mucin corona would 1) stably adsorbed on PS@Bap at the early stages of endocytosis until degraded during the lysosomal transport and maturation process, 2) delay intracellular trafficking of PS@Bap and the progress of Bap detached from PS, 3) enhance uptake of PS@Bap but reduce the cytotoxicity elicited by PS@Bap, as indicated by cell viability, generation of reactive oxygen species, impairment on mitochondrial function, and further cell apoptosis. In addition, in vivo study also verified the enhanced effect of PS on the development of an acute lung inflammatory response induced by Bap. This study highlights the significance of incorporating the effects of mucin for precisely assessing the respiratory system toxicity of nanoplastics based CCNPs in atmospheric environments.

Saquinavir plus methylprednisolone ameliorates experimental acute lung injury.

Glucocorticoid insensitivity is an important barrier to the treatment of several inflammatory diseases, including acute lung injury (ALI). Saquinavir (SQV) is an inhibitor of the human immunodeficiency virus protease, and the therapeutic effects of SQV in ALI accompanied with glucocorticoid insensitivity have not been previously investigated. In this study, the effects of SQV on lipopolysaccharide (LPS)-mediated injury in human pulmonary microvascular endothelial cells (HPMECs), human type I alveolar epithelial cells (AT I), and alveolar macrophages were determined. In addition, the effects of SQV on an LPS-induced ALI model with or without methylprednisolone (MPS) were studied. In LPS-stimulated HPMECs, SQV treatment resulted in a decrease of high mobility group box 1 (HMGB1), phospho-NF-κB (p-NF-κB), and toll-like receptor 4 (TLR4), and an increase of VE-cadherin. Compared to MPS alone, MPS plus SQV attenuated the decrease of glucocorticoid receptor alpha (GRα) and IκBα in LPS-stimulated HPMECs. HMGB1, TLR4, and p-NF-κB expression were also lessened in LPS-stimulated alveolar macrophages with SQV treatment. In addition, SQV reduced the injury in human AT I with a decrease of HMGB1 and p-NF-κB, and with an increase of aquaporin 5 (AQP 5). SQV ameliorated the lung injury caused by LPS in rats with reductions in vascular permeability, myeloperoxidase activity (MPO) and histopathological scores, and with lowered HMGB1, TLR4, and p-NF-κB expression, but with enhanced VE-cadherin expression. By comparison, SQV plus MPS increased GRα and IκBα in lung tissues of rats with ALI. This study demonstrated that SQV prevented experimental ALI and improved glucocorticoid insensitivity by modulating the HMGB1/TLR4 pathway.

Indinavir Plus Methylprednisolone Ameliorates Experimental Acute Lung Injury In Vitro and In Vivo.

BACKGROUND: An abnormal HMGB1 activation plays a key role in the pathogenesis of ALI. METHODS: In this study, the effects of Indinavir plus methylprednisolone on the LPS-mediated activation in human pulmonary microvascular endothelial cells (HPMECs), on the injury of AT I in vitro, and on rats with LPS-induced two-hit model with or without methylprednisolone were investigated. RESULTS: Indinavir treatment resulted in a reduction of HMGB1, its receptor TLR-4, and HMGB1's downstream p-NF-κB, attenuating a decrease of VE-cadherin in LPS-stimulated HPMECs. Apoptosis of AT I was attenuated with an increase of RAGE and aquaporin 5. Compared to methylprednisolone alone, methylprednisolone plus Indinavir attenuated the decrease of GRα and IκB-α in cytoplasm and avoid GRα deficiency in LPS-stimulated HPMECs for 96 h, attenuated the increase of p-NF-κB in nucleus. Indinavir ameliorated histopathological changes of two-hit ALI model of rats with reductions in microvascular permeability, lower HMGB1, TLR4, p-NF-κB, and MPO expression, whereas higher RAGE, aquaporin 5, and VE-cadherin in LPS-instilled lungs. Compared to methylprednisolone alone, methylprednisolone plus Indinavir attenuated the decrease of GRα and IκB-α in cytoplasm, decreased p-NF-κB in nucleus of lung tissue of two-hit ALI rats, and enhanced the anti-inflammatory effect of methylprednisolone for avoiding GRα deficiency. CONCLUSION: It demonstrated that Indinavir prevented experimental ALI model of rats by modulating the HMGB1/TLR-4 pathway to resolve systemic inflammation response in a greater degree with methylprednisolone, reduced the use time and dose of methylprednisolone, and avoided GRα deficiency in ALI and ARDS.

Protective Effect of Atazanavir Sulphate Against Pulmonary Fibrosis In Vivo and In Vitro.

Atazanavir sulphate, an antiretroviral protease inhibitor, has been used to treat HIV/AIDS, but its ability to serve as an antipulmonary fibrosis (PF) agent remains unknown. In this study, the effects of atazanavir sulphate on various aspects of PF were examined and CoCl2 was used to induce the hypoxia-mimicking condition in vitro, including epithelial-mesenchymal transition (EMT) in A549 cells, endothelial-mesenchymal transition (EndMT) in human pulmonary microvascular endothelial cells (HPMECs), proliferation in human lung fibroblasts (HLF-1) and potential protective effects in human type I alveolar epithelial cells (AT I). Additionally, the effects of atazanavir sulphate were examined using a bleomycin (BLM)-induced pulmonary fibrosis model. After atazanavir sulphate treatment, in A549 cells and HPMECs, the expression of vimentin, HMGB1, Toll-like receptor 4 (TLR-4) and p-NF-κB decreased, while the expression of E-cadherin and VE-cadherin increased. In AT I cells, the expression of aquaporin 5 and RAGE were increased after atazanavir treatment. Proliferation of HLF-1 was reduced after atazanavir treatment, meanwhile the expression of hypoxia-inducible factor-1α (HIF-1α), prolyl hydroxylase domain protein 2 (PHD-2), HMGB1, TLR-9, p-NF-κB, collagen I and collagen III was decreased. In the BLM-induced pulmonary fibrosis rat model, atazanavir sulphate ameliorated PF by reducing pathological score, collagen deposition and the expression of α-SMA, HIF-1α, PHD-2, HMGB1, TLR-4, TLR-9 and p-NF-κB. In summary, our study supports the proposal that atazanavir sulphate may have a therapeutic potential in reducing the progression of pulmonary fibrosis by suppressing HMGB1/TLR signalling.