Pelargonidin ameliorates inflammatory response and cartilage degeneration in osteoarthritis via suppressing the NF-κB pathway.

Pelargonidin (PG), a derivative of anthocyanins, has anti-oxidant and anti-inflammatory properties. Herein, the protective effect and the mechanism of PG in counteract the osteoarthritis (OA) progression were needed to further evaluate. In the current study, C57BL/6 mice was induced by destabilization of medial meniscus (DMM) surgery to establish the OA model. Primary chondrocytes were acquired from the knee cartilage of newborn mice. Then, PG was administrated to OA mice and IL-1ß-stimulated chondrocytes to evaluate its protective effects, respectively. Results uncovered that no conspicuous cytotoxic effects were observed when chondrocytes were treated with PG at a concentration lower than 40 µM for 24-72 h. Thus, 10 µM, 20 µM, and 40 µM PG were chosen for subsequent experiments in vitro. Then, we observed that 10, 20, and 40 µM PG reduced the levels of IL-6, TNF-α, COX-2 and iNOS in chondrocytes. In line, PG inhibited the IL-1ß-induced ECM catabolism in chondrocytes, as evidenced by deepening toluidine blue staining, increased expression of Collagen II, and decreased expressions of ADAMTS5 and MMP13. Moreover, PG also reduced the IL-1ß-stimulated p-p65 overexpression and nuclear translocation of p65 in chondrocytes. In vivo, Safranin O/Fast green and HE staining showed that articular cartilage surface morphology was basically smooth and complete after PG treatment for 8 weeks. Similarly, OARSI scores and MMP13 expression were apparently decreased, whereas Aggrecan expression was elevated in PG-treated mice 8 weeks after DMM surgery. In conclusion, PG can effectively ameliorate inflammatory reactions and cartilage degeneration via suppressing the NF-κB pathway, thereby restraining the OA progression.

[A gain-of-function mutation in FGFR2 influences mandibular condylar development on mice].

The development of the skeleton is regulated by numerous signaling molecules expressed in epiphyseal cartilage controlling both chondrogenesis and osteogenesis such as fibroblast growth factor receptors (FGFRs). In order to explore the important effect of fibroblast growth factor receptor 2 (FGFR2) in the process of mandibular condylar growth, we introduced gain-of-function Fgfr2(+/S252W) mice, and investigated mandibular condylar morphology by means of safranin-o/fast green staining at the stage of 1 week, 3 weeks and 6 weeks. The mutant mice displayed narrower width of the mandibular condylar growth plate, stronger stainings of trabecular bone at the stage of 1 week, 3 weeks and 6 weeks and faster degradation of the calcified cartilage cell layer at the stage of 6 weeks. We also assessed the expression of type X collagen (Col X) in mandibular condyle at the stage of 3 weeks by immunohistochemical staining and real-time PCR. The results showed that Col X was increased in the mutant mice. In conclusion, the gain-of-function mutation in FGFR2 resulted in histopathological abnormalities and development deformity of mandibular condyle cartilage in mice, which inhibited endochondral bone formation.

[Hematopoiesis is normally maintained in osteoblast-specific Smad4 gene knockout mice].

It was recently discovered that a subset of osteoblasts functions as a key component of the hematopoietic stem cells (HSC) niche in vivo, controlling HSC self-renewal and multi-lineage differentiation. Disruption of Smad4 gene specifically in osteoblasts leads to a remarkable decrease of osteoblast number and endosteal surface area. In order to elucidate if the osteoblast loss has any effect on hematopoietic activity, the bone marrow (BM) and extramedullary hematopoiesis in the osteoblast-specific Smad4 knockout mice were systematically analyzed, the proportions of mature hematocytes in BM, liver and spleen were detected by flow cytometry, the hematopoietic progenitor number in different stages was measured by colong-forming assay, CFU-S and analysis of LSK cells. The results indicated that the conditional mutant mice demonstrated normal BM hematopoiesis without sign of extramedullary hematopoiesis. Furthermore, the proportion of hematopoietic progenitor cells was normal, while cell number/body weight of the conditional knockout mice increased. It is concluded that hematopoiesis is normally maintained in osteoblast-specific Smad4 knockout mice, and osteoblast loss does not of necessity result in the decrease in BM hematopoiesis.

[Establishment of osteoblast-specific Cre transgenic mice].

An osteoblast-specific Cre transgenic construct (pOC-Cre) containing the osteocalcin promoter, Cre recombinase gene and polyA of human growth hormone gene was generated. The 4.6 kb DNA fragment of OC-Cre was introduced into fertilized zygotes by microinjection. 2 out of 16 offspring were identified as founders carrying the transgene by PCR and Southern blot analysis, and the integration efficiency is 12.5 %. To check the tissue distribution of the OC-Cre, the OC-Cre transgenic founder mice were bred with the mice carrying Smad4 conditional alleles. PCR results showed that the genomic DNA fragments after Cre mediated recombination could be only amplified from bone tissues of the transgenic mice. LacZ staining of OC-Cre; ROSA26 double transgenic mice revealed that Cre recombinase expressed in osteoblasts and mediated DNA recombination between LoxP sites at the ROSA26 locus. All these data indicated that the Cre recombinase was ex-pressed in the osteoblasts of the OC-Cre transgenic mice and could mediate DNA recombination between LoxP sites. The OC-Cre transgenic mice we generated in this study could serve as a useful tool for generating osteoblast specific gene-knockout mice.