Rat Chondrocyte Proliferation Research Articles

Harpagide inhibits the TNF-α-induced inflammatory response in rat articular chondrocytes by the glycolytic pathways for alleviating osteoarthritis

Osteoarthritis (OA) causes severe and functional dysfunction due to abnormal inflammation. The objective of this study was to evaluate the effect of Harpagide (HPG) on TNF-α-induced inflammation in vitro and in vivo. The effect of HPG on the proliferation of rat chondrocytes was studied. The anti-inflammatory effect of HPG and its molecular mechanisms were elucidated by qPCR, Western blotting, flow cytometry, metabolome analysis in vitro. In addition, the OA rat model was established, and the effect of HPG on OA was verified in vivo. We revealed 10 μM HPG demonstrated biocompatibility. The results demonstrated that HPG restored the upregulation of MMP-13, COX2, IL-1β and IL-6 induced by TNF-α. Moreover, HPG reversed TNF-α induced degradation of the extracellular matrix of chondrocytes. TNF-α treatment induced down-regulation of the mRNA/protein levels of proliferative markers Bcl2, CDK1 and Cyclin D1 were also recovered. HPG can inhibit TNF-α-induced inflammatory response through glycolytic metabolic pathways. HPG can restore TNF-α-induced upregulation of GRP78/IRE1α, and downregulation of AMPK proteins. In vivo experiments demonstrated that after HPG treatment, the appearance and physiological structure of articular cartilage were more integrated with highly organized chondrocytes and rich cartilage matrix compared with OA group. Finally, the molecular docking of HPG and selected key factors in glycolysis results showed that HPG had good binding potential with PFKM, PFKP, PFKFB3, PKM, HK2, and PFKL. In conclusion, the results shown HPG protects and activates chondrocytes, inhibits TNF-α-induced inflammatory response by glycolysis pathway in rat articular chondrocytes, and plays a role in the treatment of OA.

Albiflorin alleviation efficacy in osteoarthritis injury using in-vivo and in-vitro models.

Osteoarthritis seriously affects the daily life of people. Albiflorin (AF) has anti-inflammatory and antioxidant functions in various human diseases. This study aimed to clarify the function and mechanism of AF in osteoarthritis. The functions of AF on rat chondrocyte proliferation and apoptosis, inflammatory response, oxidative stress and extracellular matrix (ECM) degradation in rat chondrocytes induced by interleukin-1beta (IL-1β) were evaluated by Western blot, immunofluorescence, flow cytometry and enzyme-linked immunosorbent assay. The mechanism of AF on the IL-1β induced rat chondrocyte injury was investigated by multiple experiments in vitro. Meanwhile, the AF function in vivo was assessed using haematoxylin-eosin staining, Alcian blue, Safranin O/Fast green staining, immunohistochemical analysis and TUNEL assay. Functionally, AF accelerated the rat chondrocyte proliferation and repressed cell apoptosis. Meanwhile, AF reduced the inflammatory response, oxidative stress and ECM degradation in rat chondrocytes caused by IL-1β. Mechanistically, the receptor activator of the NF-kappaB ligand (RANKL), an activator for the NF-κB signalling pathway, partially reversed the alleviating effect of AF on IL-1β-induced chondrocyte injury. Furthermore, the in-vitro results confirmed that AF exerted protective properties against osteoarthritis injury in vivo. Albiflorin relieved osteoarthritis injury in rats by inactivating the NF-κB pathway.

Notch Signaling Regulates MMP-13 Expression via Runx2 in Chondrocytes

Notch signaling is involved in the early onset of osteoarthritis. The aim of this study was to investigate the role of Notch signaling changes during proliferation and differentiation of chondrocyte, and to testify the mechanism of MMP-13 regulation by Notch and Runx2 expression changes during osteoarthritis. In this study, Chondrocytes were isolated from rat knee cartilages. Notch signaling was activated/inhibited by Jagged-1/DAPT. Proliferative capacity of Chondrocytes was analyzed by CCK-8 staining and EdU labeling. ColX, Runx2 and MMP-13 expressions were analyzed as cell differentiation makers. Then, Runx2 gene expression was interfered using lentivirus transfection (RNAi) and was over-expressed by plasmids transfected siRNA in chondrocytes, and MMP-13 expression was analyzed after Jagged-1/DAPT treatment. In vivo, an intra-articular injection of shRunx2 lentivirus followed with Jagged1/DAPT treatments was performed in rats. MMP-13 expression in articular cartilage was detected by immunohistochemistry. Finally, MMP-13 expression changes were analyzed in chondrocytes under IL-1β stimulation. Our findings showed that, CCK-8 staining and EdU labeling revealed suppression of cell proliferation by Notch signaling activation after Jagged-1 treatment in chondrocytes. Promoted differentiation was also observed, characterized by increased expressions of Col X, MMP-13 and Runx2. Meanwhile, Sox9, aggrecan and Col II expressions were down-regulated. The opposite results were observed in Notch signaling inhibited cells by DAPT treatment. In addition, Runx2 RNAi significantly attenuated the ‘regulatory sensitivity’ of Notch signaling on MMP-13 expression both in vitro and in vivo. However, we found there wasn’t significant changes of this ‘regulatory sensitivity’ of Notch signaling after Runx2 over-expression. Under IL-1β circumstance, MMP-13 expression could be reduced by both DAPT treatment and Runx2 RNAi, while Runx2 interference also attenuated the ‘regulatory sensitivity’ of Notch in MMP-13 under IL-1β stimulation. In conclusion, Notch signaling is an important regulator on rat chondrocyte proliferation and differentiation, and this regulatory effect was partially mediated by proper Runx2 expression under both normal and IL-1β circumstances. In the meanwhile, DAPT treatment could effectively suppress expression of MMP-13 stimulated by IL-1 β.

Effects of Bakuchiol on chondrocyte proliferation via the PI3K-Akt and ERK1/2 pathways mediated by the estrogen receptor for promotion of the regeneration of knee articular cartilage defects.

ObjectivesCartilaginous tissue degradation occurs because of the lack of survival of chondrocytes. Here, we ascertained whether bakuchiol (BAK) has the capability of activating chondrocyte proliferation.Materials and methodsThe effect of BAK on the proliferation of rat chondrocytes at a concentration of 10 and 20 µmol/L was investigated. The molecular mechanisms involving target binding and signalling pathways were elucidated by RNA‐sequencing, qPCR, molecular docking and Western blotting. Matrigel mixed with bakuchiol was implanted locally into rat knee articular cartilage defects to verify the activation of chondrocytes due to bakuchiol in vivo.ResultsBakuchiol implantation resulted in the activation of rat chondrocyte proliferation in a dose‐dependent manner. RNA‐sequencing revealed 107 differentially expressed genes (DEGs) with 75 that were up‐regulated and 32 that were down‐regulated, indicating increased activation of the PI3K‐Akt and cell cycle pathways. Activation of the phosphorylation of Akt, ERK1/2 and their inhibitors blocked the proliferative effect of bakuchiol treatment, confirming its direct involvement in these signal transduction pathways. Molecular docking and siRNA silencing revealed that estrogen receptor‐α (ERα) was the target of bakuchiol in terms of its cell proliferative effect via PI3K activation. Two weeks after implantation of bakuchiol, the appearance and physiological structure of the articular cartilage was more integrated with abundant chondrocytes and cartilage matrix compared to that of the control.ConclusionsBakuchiol demonstrated significant bioactivity towards chondrocyte proliferation via the PI3K‐Akt and ERK1/2 pathways mediated by estrogen receptor activation and exhibited enhanced promotion of the remodelling of injured cartilage.

A study on the effect of icaritin on rat chondrocytes

To investigate the eff ect of icaritin on proliferation and apoptosis in rat chondrocytes and to provide new theory for osteochondropathy treatment. Icaritin (with a purity of 99%) at different concentrations (0, 4, 8, 16, 32, 64 μmol/L) was incubated with rat chondrocytes for different time. The cell proliferation and apoptosis was assayed by MTT and fl ow cytometry, respectively. Compared with the control group, the cell proliferation was increased in the groups with icaritin at 4 or 8 μmol/L (P<0.05), whereas the proliferation was decreased in the groups with icaritin at 16, 32 or 64 μmol/L groups compared to the control group (P<0.05); the cell apoptosis ratio in the group with icaritin at 4 μmol/L was obviously lower than that in the control group aft er incubation of icaritin for 24 h and 48 h. Beyond 4 μmol/L, the higher concentration of icaritin, the higher apoptosis ratio of cell. However, it did not show a time-dependent manner at a same concentration of icaritin. The icaritin at low concentration (4 or 8 μmol/L) can promote rat chondrocyte proliferation and inhibit cell apoptosis, while the effect of icaritin on rat chondrocyte at high concentration was reversed.

Protective Effects of 5,7,3’,4’-Tetramethoxyflavone on Proliferation of Rat Chondrocytes Induced by IL-1β

Osteoarthritis (OA) is a degenerative joint disease that affects millions of people. Currently, there is no effective drug treatment for it. The purpose of this study is to investigate the chondroprotective effects of 5,7,3’,4’- tetramethoxyflavone in isolated cartilage chondrocytes cultured pre-treatment by IL-1β. The results showed that 5,7,3’,4’- tetramethoxyflavone could significantly down regulate the concentration of MMP-13, but up regulate the expression of TIMP-1, increase the proliferative activity of rat chondrocytes.

Inhibition effect of osthole on proliferation of rat chondrocytes

To investigate the effects of osthole on chondrocyte proliferation in vitro. Primary rat chondrocytes were isolated from the femoral head of newborn rats using collagenase digestion and cultured in Dulbecco's modified Eagle's medium. The proliferation of primary chondrocytes was assessed in second-passage cultures using cell counting kit-8 and the growth curve was drawn. Type II procollagen gene (Col2a1) expression in chondrocytes was also identified using cell immunofluorescence assay. The second-passage chondrocytes were divided into five groups, including control group and osthole groups at 6.25, 12.5, 25 and 50 μmol/L. The growth property of rat chondrocytes was observed after 24, 48 and 72 h of culture with osthole at corresponding dose. Both protein and mRNA expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 was measured by Western blot and polymerase chain reaction methods. The second-passage chondrocytes were viable and showed Col2a1 expression in the cytoplasm. The proliferation of rat chondrocytes was inhibited by osthole in a dose-dependent manner. Meanwhile, there were significant decreases in both protein and mRNA expression of PCNA and cyclin D1 in the osthole groups compared with the control group. Osthole exhibits inhibitory effect on proliferation of rat chondrocytes by down-regulating PCNA and cyclin D1 expression.

Periodic mechanical stress activates MEK1/2-ERK1/2 mitogenic signals in rat chondrocytes through Src and PLCγ1

The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively but the mechanisms whereby chondrocytes sense and respond to periodic mechanical stress remain a matter of debate. We explored the signal transduction pathways of chondrocyte proliferation and matrix synthesis under periodic mechanical stress. In particular, we sought to identify the role of the MEK1/2-ERK1/2 signaling pathway in chondrocyte proliferation and matrix synthesis following cyclic physiologic mechanical compression. Under periodic mechanical stress, both rat chondrocyte proliferation and matrix synthesis were significantly increased (P < 0.05) and were associated with increases in the phosphorylation of Src, PLCγ1, MEK1/2, and ERK1/2 (P < 0.05). Pretreatment with the MEK1/2-ERK1/2 selective inhibitor, PD98059, and shRNA targeted to ERK1/2 reduced periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis (P < 0.05), while the phosphorylation levels of Src-Tyr418 and PLCγ1-Tyr783 were not inhibited. Proliferation, matrix synthesis and phosphorylation of MEK1/2-Ser217/221 and ERK1/2-Thr202/Tyr204 were inhibited after pretreatment with the PLCγ1 inhibitor U73122 in chondrocytes in response to periodic mechanical stress (P < 0.05), while the phosphorylation site of Src-Tyr418 was not affected. Inhibition of Src activity with PP2 and shRNA targeted to Src abrogated chondrocyte proliferation and matrix synthesis (P < 0.05) and attenuated PLCγ1, MEK1/2 and ERK1/2 activation in chondrocytes subjected to periodic mechanical stress (P < 0.05). These findings suggest that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis in part through the Src-PLCγ1-MEK1/2-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade.

Ontogeny of rat chondrocyte proliferation: studies in embryo, adult and osteoarthritic (OA) cartilage

The aim of this work was to study the ontogeny of chondrocyte cell division using embryo, adult and osteoarthritic (OA) cartilage. We searched for mitosis phases and performed a comparative evaluation of mitotic index, basic fibroblast growth factor b (FGFb), transforming growth factor beta1 (TGF-beta1) receptors, cyclin dependent kinase (CDK1) and Cyclin-B expression in fetal, neonate, 3, 5, 8 weeks old rats and experimental OA. Our results showed that mitosis phases were observed in all normal cartilage studied, although, we found a decrease in mitotic index in relation to tissue development. No mitosis was detected in OA cartilage. We also found a statistical significant reduction in cell number in OA cartilage, compared with the normal tissue. Furthermore, FGFb and TGF-beta1 receptors diminished in relation to tissue development, and were very scarce in experimental OA. Western blot assays showed CDK-1 expression in all cases, including human-OA cartilage. Similar results were observed for Cyclin-B, except for 8 weeks, when it was not expressed. Our results suggest that cell division seems to be scarce, if not absent within the OA cartilage studied. Nevertheless, the existence of factors essential for cell division leaves open the question concerning chondrocyte proliferation in OA cartilage, which is likely to be present in the early stages of the disease.

Articular chondrocytes and synoviocytes in culture: influence of antioxidants on lipid peroxidation and proliferation

Chondrocytes and synoviocytes are the main cell types in articular joints. Articular cartilage is fed by synoviocytes via synovial fluid and has a low partial oxygen pressure. Thus, chondrocytes show oxygen radical protective mechanisms in vivo and are unprotected against these factors under common culture conditions. We investigated the influence of ascorbic acid, Fe2+, glutathione and alpha-tocopherol on lipid peroxidation and proliferation of rat articular chondrocytes and rabbit synoviocytes (HIG-82) in vitro. A combination of ascorbic acid and Fe2+ induced the production of thiobarbituric acid-reactive material as a marker of radical-mediated lipid peroxidation in homogenates and/or supernatants of cultured chondrocytes and synoviocytes. The amount of lipid peroxidation of chondrocytes was about 3-fold higher than that of synoviocytes. Ascorbic acid or Fe2+ alone had no significant influence on the production of thiobarbituric acid-reactive material. Lipid peroxidation could be abolished by addition of the radical scavenger alpha-tocopherol, whereas glutathione had no effect. 25-50 microM alpha-tocopherol decreased the ascorbic acid-(100 micrograms/ml) and Fe(2+)-(3 microM) induced lipid peroxidation to a basal level. Moreover, ascorbic acid inhibited the proliferation of rat chondrocytes and rabbit synoviocytes measured by [3H]-thymidine incorporation. Alpha-tocopherol and glutathione had no influence on the proliferation of chondrocytes but alpha-tocopherol decreased the growth of synoviocytes and increased the anti-proliferative effect of ascorbic acid on these cells. The importance of these findings for the use of ascorbic acid, glutathione and alpha-tocopherol in chondrocyte and synoviocyte cultures, or the influence of these molecules on the etiology and treatment of articular diseases will be discussed.

Rat chondrocyte proliferation was regulated by natriuretic peptide C and its receptor, natriuretic peptide receptor-B, in autocrine manner

Rat chondrocyte proliferation was regulated by natriuretic peptide C and its receptor, natriuretic peptide receptor-B, in autocrine manner

Autocrine regulation of rat chondrocyte proliferation by natriuretic peptide C and its receptor, natriuretic peptide receptor-B.

Natriuretic peptide receptor-B (NPR-B) was identified in rat chondrocytes, and its physiological functions were investigated. Rat tissues, including the xiphoid cartilage, brain, lung, liver, adrenal gland, and kidney, were screened for NPR-B activity, which we assayed by receptor guanylate cyclase activity specifically stimulated by C-type natriuretic peptide (CNP), a known selective activator of NPR-B. Cartilage showed distinctly higher NPR-B activity. Furthermore, exposure of cultured rat chondrocytes to CNP (10(-6) M) resulted in a large increase in intracellular cGMP production (376 +/- 38 pmol/well), with threshold responses occurring between 10(-10) and 10(-9) M CNP. Atrial natriuretic peptide and brain natriuretic peptide also stimulated cGMP production in rat chondrocytes but with a potency that was at least 10 times less than that of CNP. Polymerase chain reaction analysis also demonstrated NPR-B gene expression in adult rat xiphisternum and cultured chondrocytes. These findings indicate that NPR-B is present in rat chondrocytes. In rat chondrocytes exposed to CNP, [3H]thymidine incorporation was inhibited in a dose-dependent manner (half-maximal response, 10(-11)M). However, much higher concentrations of atrial natriuretic peptide were required to induce the inhibition of thymidine incorporation. Interestingly, CNP-like immunoreactivity was detected in the conditioned medium from chondrocyte cultures. In addition, TGF-beta 1, a multifunctional cytokine, induced a marked increase in CNP secretion and CNP mRNA levels in chondrocytes. These results indicate that autocrine CNP inhibits mitogenesis in chondrocytes via NPR-B under the control of TGF-beta 1.