Abstract. We investigated the effects of mechanical strain on the progressive ankylosis (ANK) gene and extracellular nucleotide phosphatase/phosphodiesterase (ENPP)1 mRNA expression and TGF-β1 protein expression in rat endplate chondrocytes in vitro. Endplate chondrocytes were isolated and cultured in vitro. Following identification with toluidine blue and immunocytochemical staining, chondrocytes were subjected to 10% elongation with various frequencies (0.5, 1, 1.5 and 2 Hz) using a Flexercell Tension Plus system at various intervals (3, 6, 12, 24, 36 and 48 h). As a control, cells that had been cultured statically on the same type of plate but were not subjected to stretch were also observed. Real-time reverse transcription-polymerase chain reaction and the enzyme-linked immunosorbent assay were used to study the effects of mechanical strain on ANK and ENPP1 mRNA expression and TGF-β1 concentration in the supernatant, respectively. Following treatment, the shape of the chondrocytes displayed a significant change from the original polygon to a typical spindle cell morphology; and the arrangement of the cells exhibited a change from a haphazard arrangement to an alignment with a certain direction. In the 0.5 Hz, 24-h group, the ANK gene expression was significantly increased compared to the control group (P<0.05); whereas in the other groups, the ANK and ENPP1 expression levels were reduced. With the increased frequencies in the 24-h group, the ANK gene expression gradually reduced. Changes in the expression of ANK and ENPP1 followed similar trends. TGF-β1 in the supernatant increased gradually in each frequency group, with a clear increase in the 0.5 Hz group. We conclude that various frequencies of mechanical strain can affect the expression of ANK, ENPP1 and endogenous TGF-β1 in endplate chondrocytes. Our results indicate that 0.5 Hz, 24 h may be the optimal stimulation condition to prevent calcification occurrence and to maintain the function of endplate chondrocytes.
Our results directly showed that ICMT induced the calcification and downregulation of ankh gene expression of end plate chondrocytes, which may be caused by the endogenous TGF-β1.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic, which has resulted in more than two million deaths at 2021 February . There is currently no approved therapeutics for treating COVID-19. The SARS-CoV-2 Spike protein is considered a key therapeutic target by many researchers. Here we describe the identification of several monoclonal antibodies that target SARS-CoV-2 Spike protein. One human antibody, CA521FALA, demonstrated neutralization potential by immunizing human antibody transgenic mice. CA521FALA showed potent SARS-CoV-2-specific neutralization activity against SARS-CoV-2 pseudovirus and authentic SARS-CoV-2 infection in vitro. CA521FALA also demonstrated having a long half-life of 9.5 days in mice and 9.3 days in rhesus monkeys. CA521FALA inhibited SARS-CoV-2 infection in SARS-CoV-2 susceptible mice at a therapeutic setting with virus titer of the lung reduced by 4.5 logs. Structural analysis by cryo-EM revealed that CA521FALA recognizes an epitope overlapping with angiotensin converting enzyme 2 (ACE2)-binding sites in SARS-CoV-2 RBD in the Spike protein. CA521FALA blocks the interaction by binding all three RBDs of one SARS-CoV-2 spike trimer simultaneously. These results demonstrate the importance for antibody-based therapeutic interventions against COVID-19 and identifies CA521FALA a promising antibody that reacts with SARS-CoV-2 Spike protein to strongly neutralize its activity.
SARS-CoV-2 Omicron subvariants have demonstrated extensive evasion from monoclonal antibodies (mAbs) developed for clinical use, which raises an urgent need to develop new broad-spectrum mAbs. Here, we report the isolation and analysis of two anti-RBD neutralizing antibodies BA7208 and BA7125 from mice engineered to produce human antibodies. While BA7125 showed broadly neutralizing activity against all variants except the Omicron sublineages, BA7208 was potently neutralizing against all tested SARS-CoV-2 variants (including Omicron BA.1–BA.5) except Mu. By combining BA7208 and BA7125 through the knobs-into-holes technology, we generated a biparatopic antibody BA7208/7125 that was able to neutralize all tested circulating SARS-CoV-2 variants. Cryo-electron microscopy structure of these broad-spectrum antibodies in complex with trimeric Delta and Omicron spike indicated that the contact residues are highly conserved and had minimal interactions with mutational residues in RBD of current variants. In addition, we showed that administration of BA7208/7125 via the intraperitoneal, intranasal, or aerosol inhalation route showed potent therapeutic efficacy against Omicron BA.1 and BA.2 in hACE2-transgenic and wild-type mice and, separately, effective prophylaxis. BA7208/7125 thus has the potential to be an effective candidate as an intervention against COVID-19.
Objective The purpose was to observe whether systemic administration with silibinin(SIL) have an positive effect on bone defect regeneration through HIF-1α/VEGF and Notch signaling pathway in an ovariectomized(OVX) rat model. Methods The MC3T3-E1 cells were co-cultured with lower SIL and higher SIL and induced to osteogenesis, and the cell viability, osteogenic activity were observed by Cell Count Kit-8(CCK-8), Alkaline phosphatase (ALP) staining, Alizarin Red(RES) staining and Western blotting(WB). After the drilling defect model was established, the OVX rats were treated with SIL for 12 weeks. Micro-CT, histology and Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis were used to observe the therapeutic effect and explore the possible mechanism. Results CCK-8, ALP and ARS staining results show that the cell mineralization and osteogenic activity of LSIL and HSIL group is significantly higher than the Con group. Protein expressions show that related regulatory proteins such as ALP, OPN, RUNX-2, OC, VEGFA, HIF-1α, Notch 1, JAG 1, HEY 1 and HES 1 of LSIL and HSIL group are significantly higher than Con group. Micro-CT and Histological analysis evaluation show that group SIL + OVX presented the stronger effect on bone regeneration, bone mineralization, higher expression of VEGFA and HIF-1α, when compared with OVX group. RT-qPCR analysis shows that SIL + OVX group showed increased Notch 1, HES1, HEY1 and JAG1 than the OVX group(p < 0.05). Conclusions Our current study demonstrated that systemic administration with SIL is a scheme for rapid repair of femoral condylar defects, and these effects may be achieved by activating HIF-1α/VEGF and Notch signaling pathway.
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