Adipocyte-secreted microvesicle-derived miR-148a regulates adipogenic and osteogenic differentiation by targeting Wnt5a/Ror2 pathway.

AIMS: Adipocyte-secreted microvesicles (MVs)-derived microRNAs (miRNAs) are relevant to adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteonecrosis of the femoral head (ONFH). Our aims are to investigate the mechanism of adipocyte-derived MVs-miR-148a in ONFH. MATERIALS AND METHODS: Adipocyte-derived MVs were identified via transmission electron microscopy and specific markers expression. The adipogenic and osteogenic differentiation were investigated by Oil-Red O staining, alkaline phosphatase (ALP) activity, Alizarin Red S (ARS) staining and osteogenic or adipogenic factors levels. Genes and proteins expression were detected by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The relationship between miR-148a and Wnt5a was tested via dual-luciferase reporter analysis. The adipogenic differentiation and osteogenic differentiation in methylprednisolone (MPS)-induced ONFH rat model were assessed via hematoxylin-eosin (HE) staining, and immunohistochemical staining of collagen I (COL I). KEY FINDINGS: Adipocyte-derived MVs promoted adipogenic differentiation via increasing Oil-Red O staining positive cells, adiponectin (Adipoq), acid-binding protein 2 (aP2) and peroxisome proliferator-activated receptor γ (PPAR-γ) levels, and repressed osteogenic differentiation of BMSCs via decreasing ARS staining positive cells, ALP, Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) levels. MiR-148a was present in adipocyte-derived MVs, and miR-148a knockdown inhibited adipogenic differentiation and promoted osteogenic differentiation. Furthermore, Wnt5a expression was regulated by miR-148a. MiR-148a overexpression facilitated adipogenic differentiation and suppressed osteogenic differentiation via regulating the Wnt5a/Ror2 pathway. Adipocyte-derived MVs promoted adipogenic differentiation and inhibited osteogenic differentiation in MPS-induced ONFH rat model. SIGNIFICANCE: Adipocyte-derived MVs-miR-148a promoted adipogenic differentiation and suppressed osteogenic differentiation via targeting the Wnt5a/Ror2 pathway.

Application of an optimized pharyngeal swab-assisted device in pharyngeal sampling for COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a global pandemic. Healthcare workers are placed at an elevated risk of nosocomial cross-infection from clinical exposure. One diagnostic criterion for COVID-19 is a positive result from a real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay of pharyngeal swab specimens, which has been a routine procedure for healthcare workers during the outbreak. In the context of a global shortage of personal protective equipment (PPE), we aimed to lower the probability of clinical cross-infection without impacting the results of pharynx sampling through an optimized pharyngeal swab assisted device (OPAD). METHODS: To evaluate the efficacy and feasibility of an OPAD for the detection of SARS-CoV-2, 22 confirmed COVID-19 cases were enrolled in our self-controlled study. The results of two pharyngeal sampling qRT-PCR tests using the OPAD or the traditional method were recorded each. Clinical data including baseline characteristics, laboratory tests, and computed tomography (CT) results were also collected. The procedure duration and levels of pharynx exposure with the OPAD, and the diagnostic consistency between the OPAD and the traditional method for pharyngeal sampling qRT-PCR, were evaluated individually. Additionally, a questionnaire was designed for healthcare workers who had performed the pharyngeal swab to deepen our understanding of their attitude during their service on the frontline. RESULTS: In all 44 samplings (22 samples with each method), the qRT-PCR results of 18 pairs (81.82%) were consistent, while 3 (13.64%) were single positive with the OPAD. The positive rate was slightly higher with the OPAD (54.55%, 12/22) than with the traditional method (45.45%, 10/22). Using the OPAD, the average procedure duration of sampling was 30 s (30±13 s). Pharynx exposure was excellent in 21 subjects (95.45%, 21/22), which meant that the operator could acquire the swabs without difficulty. CONCLUSIONS: As the COVID-19 pandemic escalates, our OPAD has identical efficacy compared to the traditional method for pharyngeal swabs, and it can also contribute to protecting the safety of healthcare workers.

Effects of isoimperatorin on proliferation and apoptosis of human gastric carcinoma cells.

Resistance to apoptosis is an characteristic of cancer cells that serves a critical function in tumor development and represents a target for antitumor therapy. Isoimperatorin (ISOIM), a coumarin compound, exhibits antitumor functions in multiple types of tumor cells. However, its antitumor effects and molecular mechanisms with respect to gastric cancer have not been elucidated. The present study assessed the anti-proliferative and apoptotic effects of ISOIM on human BGC-823 gastric cancer cells and elucidated its underlying molecular mechanisms. Cell proliferation was evaluated using MTT assays. Analysis of cell morphology was performed by hematoxylin and eosin, Hoechst 33258 and acridine orange/ethidium bromide staining. In addition, cell cycle and apoptosis was evaluated using flow cytometry analysis; expression of apoptosis-associated proteins was studied by western blotting. The results of the present study revealed that ISOIM significantly inhibited cell proliferation by arresting the cell cycle at the G2/M phase and induced apoptosis by increasing Bcl-2-associated X (Bax) expression with a concomitant decrease in Bcl-2 expression, resulting in a decreased Bcl-2/Bax ratio compared with the control. In addition, ISOIM treatment also resulted in cytochrome c translocating from the mitochondria to the cytosol. Furthermore, caspase-3 was significantly activated in response to treatment with ISOIM, suggesting that apoptosis in BGC-823 cells is induced in the mitochondrial pathway. Taken together, the results of the present study indicate that ISOIM may significantly induce apoptosis in BGC-823 cells and that the pro-apoptotic mechanisms of ISOIM could be associated with the mitochondrial pathway.

Isolation and Purification of Condensed Tannin from the Leaves and Branches of Prunus cerasifera and Its Structure and Bioactivities.

Prunus cerasifera has a rich resource and a weak utilization rate and its biological functions have been investigated. We found that the contents of total phenol (TP) in leaves and branches of Prunus cerasifera were 117.8 ± 8.8 and 100.04 ± 0.9 mg/g, respectively; the contents of soluble condensed tannin (SCT) were 73.95 ± 0.9 and 78.65 ± 4.1 mg/g, respectively; the structure of SCT containing afzelechin/epiafzelechin, catechin/epicatechin, and atechin/epicatechin as the main units and the SCT from leaves and branches exhibited better anti-tyrosinase and antioxidant activities. This study could clarify Prunus cerasifera condensed tannin resource availability and lay a theoretical foundation for its development as a natural antioxidant and tyrosinase inhibitor.

Construction of self-assembled cartilage tissue from bone marrow mesenchymal stem cells induced by hypoxia combined with GDF-5.

It is widely known that hypoxia can promote chondrogenesis of human bone marrow derived mesenchymal stem cells (hMSCs) in monolayer cultures. However, the direct impact of oxygen tension on hMSC differentiation in three-dimensional cultures is still unknown. This research was designed to observe the direct impact of oxygen tension on the ability of hMSCs to "self assemble" into tissue-engineered cartilage constructs. hMSCs were cultured in chondrogenic medium (CM) containing 100 ng/mL growth differentiation factor 5 (GDF-5) at 5% (hypoxia) and 21% (normoxia) O2 levels in monolayer cultures for 3 weeks. After differentiation, the cells were digested and employed in a self-assembly process to produce tissue-engineered constructs under hypoxic and normoxic conditions in vitro. The aggrecan and type II collagen expression, and type X collagen in the self-assembled constructs were assessed by using immunofluorescent and immunochemical staining respectively. The methods of dimethylmethylene blue (DMMB), hydroxyproline and PicoGreen were used to measure the total collagen content, glycosaminoglycan (GAG) content and the number of viable cells in each construct, respectively. The expression of type II collagen and aggrecan under hypoxic conditions was increased significantly as compared with that under normoxic conditions. In contrast, type X collagen expression was down-regulated in the hypoxic group. Moreover, the constructs in hypoxic group showed more significantly increased total collagen and GAG than in normoxic group, which were more close to those of the natural cartilage. These findings demonstrated that hypoxia enhanced chondrogenesis of in vitro, scaffold-free, tissue-engineered constructs generated using hMSCs induced by GDF-5. In hypoxic environments, the self-assembled constructs have a Thistological appearance and biochemical parameters similar to those of the natural cartilage.

Early total hip arthroplasty for severe displaced acetabular fractures.

OBJECTIVE: To investigate the effect of early total hip arthroplasty for severe displaced acetabular fractures. METHODS: Total hip arthroplasty was performed on 17 cases of severe fracture of the acetabulum from 1997 to 2003. The mean follow-up was 2.1 years (1-6 years) and the average period from fracture to operation was 8 days (5-21 day). The average age of the patients was 53 years (26-69 years). RESULTS: At the final follow-up the Harris hip score averaged 82 (69-100) points and 15 cases have got a good outcome. There was one case of heterotopic bone formation. There were no radiographic evidences of late loosening of the prosthesis. One patient had severe central displacement of the cup. CONCLUSIONS: In patients with severe displaced acetabular fractures, particularly in elderly patients, early total hip arthroplasty is probably an alternative efficient way to achieve a painless and stable hip.