The Molecular Structure and Self-Assembly Behavior of Reductive Amination of Oxidized Alginate Derivative for Hydrophobic Drug Delivery

On account of the rigid structure of alginate chains, the oxidation-reductive amination reaction was performed to synthesize the reductive amination of oxidized alginate derivative (RAOA) that was systematically characterized for the development of pharmaceutical formulations. The molecular structure and self-assembly behavior of the resultant RAOA was evaluated by an FT-IR spectrometer, a 1H NMR spectrometer, X-ray diffraction (XRD), thermal gravimetric analysis (TGA), a fluorescence spectrophotometer, rheology, a transmission electron microscope (TEM) and dynamic light scattering (DLS). In addition, the loading and in vitro release of ibuprofen for the RAOA microcapsules prepared by the high-speed shearing method, and the cytotoxicity of the RAOA microcapsules against the murine macrophage RAW264.7 cell were also studied. The experimental results indicated that the hydrophobic octylamine was successfully grafted onto the alginate backbone through the oxidation-reductive amination reaction, which destroyed the intramolecular hydrogen bond of the raw sodium alginate (SA), thereby enhancing its molecular flexibility to achieve the self-assembly performance of RAOA. Consequently, the synthesized RAOA displayed good amphiphilic properties with a critical aggregation concentration (CAC) of 0.43 g/L in NaCl solution, which was significantly lower than that of SA, and formed regular self-assembled micelles with an average hydrodynamic diameter of 277 nm (PDI = 0.19) and a zeta potential of about −69.8 mV. Meanwhile, the drug-loaded RAOA microcapsules had a relatively high encapsulation efficiency (EE) of 87.6 % and good sustained-release properties in comparison to the drug-loaded SA aggregates, indicating the good affinity of RAOA to hydrophobic ibuprofen. The swelling and degradation of RAOA microcapsules and the diffusion of the loaded drug jointly controlled the release rate of ibuprofen. Moreover, it also displayed low cytotoxicity against the RAW264.7 cell, similar to the SA aggregates. In view of the excellent advantages of RAOA, it is expected to become the ideal candidate for hydrophobic drug delivery in the biomedical field.

RNA-seq-Based Screening in Coal Dust-Treated Cells Identified PHLDB2 as a Novel Lung Cancer-Related Molecular Marker

Lung cancer is one of the most serious leading cancers with high incidence globally. Identifying molecular markers is key for disease diagnosis and treatment. Coal dust might be important triggering factors in disease development. Here, we first performed RNA-seq-based screening in coal dust treated and nontreated RAW264.7 cell lines. PHLDB2 was found to be the top differentially expressed gene. By retrieving TCGA lung cancer dataset, we observed that PHLDB2 showed upregulations in males and smoker patients. Patients with lower PHLDB2 expression survived longer than those with higher expressions. Furthermore, PHLDB2 was negatively correlated with EMT makers, and a total of 2.74% mutation rate were observed in 1,059 patients. This finding highlights the critical role of PHLDB2 in lung cancer development. PHLDB2 might be a molecular maker for disease diagnosis or treatment.

Mechanism of Exosomes Involved in Osteoimmunity Promoting Osseointegration Around Titanium Implants With Small-Scale Topography

Exosomes are nanoscale extracellular vesicles. Several studies have shown that exosomes participate in intercellular communication and play a key role in osseointegration. However, it is unclear whether exosomes and their contents participate in the communication between the immune and skeletal systems in the process of osseointegration. In this study, we obtained smooth titanium disks by polishing and small-scale topography titanium disks by sandblasted large-grit acid-etched (SLA) technology combined with alkali thermal reaction. After stimulating mouse RAW264.7 cells with these two kinds of titanium disks, we co-cultured the MC3T3-E1 cells and the RAW264.7 cells, obtained and identified the exosomes derived from RAW264.7 cells, and studied the effect of the osteoimmune microenvironment and the exosomes on the osseointegration of mouse MC3T3-E1 cells. Cell counting kit-8 (CCK-8), real time quantitative PCR, western blotting, alizarin red staining, and quantitative and confocal fluorescence microscopy were used to study the effects of exosomes on MC3T3-E1 cells; RNA sequencing and correlation analysis were performed. We found that the osteoimmune microenvironment could promote the osseointegration of MC3T3-E1 cells. We successfully isolated exosomes and found that RAW264.7 cell-derived exosomes can promote osteogenic differentiation and mineralization of MC3T3-E1 cells. Through RNA sequencing and gene analysis, we found differentially expressed microRNAs that targeted the signal pathways that may be related, such as mTOR, AMPK, Wnt, etc., and thus provide a reference for the mechanism of osteoimmunue regulation of implant osseointegration. The study further elucidated the mechanism of implant osseointegration and provided new insights into the effect of exosomes on implant osseointegration, and provided reference for clinical improvement of implant osseointegration and implant success rate.

Milk-Derived Extracellular Vesicles Suppress Inflammatory Cytokine Expression and Nuclear Factor-κB Activation in Lipopolysaccharide-Stimulated Macrophages

In milk and milk products, small membrane-enclosed vesicles can be found, commonly termed extracellular vesicles (EVs). Milk-derived EVs have previously been suggested to have immunoregulatory properties, especially important for infants without a fully functioning immune system. In the present study, EV fractions were isolated from human milk, mature and colostrum bovine milk, and two dairy fractions, and successively surveyed for their immunomodulating effects on lipopolysaccharide (LPS)-stimulated macrophages (RAW264.7). RAW264.7 cell material and supernatant were evaluated by monitoring degradation of IκBα in the NF-κB pathway, and IL-6 and IL-1β cytokine production, using Western blotting and enzyme-linked immunosorbent assaying, respectively. The results revealed that preincubation with EVs derived from raw human and bovine milk lowered the LPS-activated response of the NF-κB pathway. Additionally, it was found that preincubation with EVs, from human and bovine milk as well as dairy whey or skim milk-derived fractions, decreased secretion of proinflammatory cytokines from LPS-activated RAW264.7 cells. The findings that milk-derived EVs can change the inflammatory response in macrophages support the notion that milk EVs have an important role in mother-to-infant communication and protection of a newborn.

Research on Effect of Exosome Excreted by RAW264.7 Cell on Damage of Mice Lung Function and Its Mechanism

The exosome excreted by RAW264.7 cell was found to participate in the damage of lung function. Our purpose was to assess the exact mechanism. The animal experiment was adopted in our study. It was provided reference for clinical experiment. 30 healthy male rats were selected and assigned into normal control group, normal exosome group and LPS with exosome group by random number table followed by analysis of the size and form of exosome under electron microscope, the lung structural change by hematoxylin and eosin staining, TNF-α and IL-1β secretion by immunohistochemistry, and the expression of tightly bound protein one (ZO-1), closed protein, β-chain protein and two groups of exosome-marked protein (CD9 and CD63) by western blot. In normal control group, the lung structure was very integral with clear alveolar space. The cell wall was smooth relatively without exudation substance in alveolar space and mesenchyme. Normal exosome group showed relatively integral lung structure and thicker cell wall. The inside of alveolar space was very clear without exudation substance in alveolar space and mesenchyme. LPS + exosome group presented abundant inflammatory cells in alveolar space and mesenchyme with obvious bleeding. Inflammatory factor secretion such as TNF-α (1.334 ± 0.205%) and IL-1β (0.803 ± 0.097%)was significantly higher and the content of closed protein (0.203 ±0.017%) was lower in LPS with exosome group than the other two groups (P < 0.05). The exosome secreted by RAW264.7 cell after stimulation by lipopolysaccharide could impair the lung function in rats and is related with the decline of closed protein.

Integrative analyses reveal RNA regulatory network in Ti particles induced inflammation

Introduction Wear particles induced inflammatory osteolysis is the most important initiating factors in the mechanism of aseptic loosening. However, the molecular network changes in this process remain largely elusive. Methods Here, we performed whole transcriptome analysis using Ti particles induced RAW264.7 cell model to identify specific genes and pathways. Results Sequencing results totally identified 159 mRNAs, 96 lncRNAs, 31 circRNAs, and 12 miRNAs were significantly differently expressed. Of these, we selected two of each RNA for qRT-PCR validation and the results were highly consistent with the RNA-seq data. GSEA analysis shows that upregulated gene sets were related to the three classical inflammation pathway, cytokine–cytokine receptor interaction, TNF, and NF-kappa B signaling pathway. The enriched genes included not only IL-1β and TNF- α, which were independently verified before sequencing, but also other inflammatory osteolysis-related genes such as Mmp9, Fas, and Ccl2. Co-differentially expressed RNAs were employed to construct the ceRNA co-regulatory network. Conclusion: The results revealed that 4 lncRNAs and 2 circRNAs formed a regulatory network to simultaneously regulate miR-3065-3p targeting Myo18a. The present study helps to comprehensively understand the molecular mechanisms and regulatory interaction networks during early inflammatory response.

Thymosin β10 promotes tumor-associated macrophages M2 conversion and proliferation via the PI3K/Akt pathway in lung adenocarcinoma

Background Thymosin β10 (TMSB10) has been reported to play a protumorigenic role in a majority of solid cancers. However, the existence of TMSB10 in immune microenvironment may contribute to the pathogenesis of lung adenocarcinoma has not been previously explored. Method TAMs-associated TMSB10 expression was evaluated by immunohistochemistry (IHC) in 184 lung adenocarcinomas. Xenograft mice model was established to investigate the effect of TMSB10 shRNA on TAMs phenotypes. The macrophages phenotype associated cytokines IL-6, IL-8, IL-12 and TNF-α were detected by ELISA after treated with TMSB10 shRNA or scramble. Furthermore, the target proteins were detected by immunoblotting. Results We found that high TAMs-associated TMSB10 expression was significantly correlated with the advanced TNM stage and T3/T4 tumor size. And high TAMs-associated TMSB10 expression was significantly correlated with poor overall and progression-free survival of lung adenocarcinoma, acting as an independent prognostic factor for lung adenocarcinoma. Furthermore, we investigated the biological functions of TMSB10 in macrophages in vivo and in vitro. TMSB10 knockdown dramatically reduced TAMs, THP-1 and RAW264.7 cell proliferation, and promoted macrophages phenotype conversion of M2 to M1, and TMSB10 knockdown reduced the levels of p-Akt (Sec473), p-mTOR (Sec2448) and p-p70S6K (Thr389) without effect on Akt, mTOR and p70S6K expression. Conclusions These results demonstrate that TAMs-associated TMSB10 promotes tumor growth through increasing TAMs M2 conversion and proliferation via PI3K/Akt signaling pathway, providing a promising tumor biomarker for predicting prognosis and a potential therapeutic target for lung adenocarcinoma.

Thymosin β10 promotes tumor-associated macrophages M2 conversion and proliferation via the PI3K/Akt pathway in lung adenocarcinoma

Background: Thymosin β10 (TMSB10) has been reported to play a protumorigenic role in a majority of solid cancers. However, the existence of TMSB10 in immune microenvironment may contribute to the pathogenesis of lung adenocarcinoma has not been previously explored. Method: TAMs-associated TMSB10 expression was evaluated by immunohistochemistry (IHC) in 184 lung adenocarcinomas. Xenograft mice model was established to investigate the effect of TMSB10 shRNA on TAMs phenotypes. The macrophages phenotype associated cytokines IL-6, IL-8, IL-12 and TNF-α were detected by ELISA after treated with TMSB10 shRNA or scramble. Furthermore, the target proteins were detected by immunoblot. Results: We found that high TAMs-associated TMSB10 expression was significantly correlated with the advanced TNM stage and T3/T4 tumor size. And high TAMs-associated TMSB10 expression was significantly correlated with poor overall and progression-free survival of lung adenocarcinoma, acting as an independent prognostic factor for lung adenocarcinoma. Furthermore, we investigated the biological functions of TMSB10 in macrophages in vivo and in vitro. TMSB10 knockdown dramatically reduced TAMs, THP-1 and RAW264.7 cell proliferation, and promoted macrophages phenotype conversion of M2 to M1, and TMSB10 knockdown reduced the levels of p-Akt (Sec473), p-mTOR (Sec2448) and p-p70S6K (Thr389) without effect on Akt, mTOR and p70S6K expression. Conclusions: These results demonstrate that TAMs-associated TMSB10 promotes tumor growth through increasing TAMs M2 conversion and proliferation via PI3K/Akt signaling pathway, providing a promising tumor biomarker for predicting prognosis and a potential therapeutic target for lung adenocarcinoma.