Role Of Pyrroloquinoline Quinine In Chondrocyte Proliferation And IL-1β Induced Chondrocyte Apoptosis

ObjectiveTo investigate the role of PQQ(Pyrroloquinoline quinine) in chondrocyte proliferation and IL-1βinduced chondrocyte apoptosis in articμlar cartilage of knee joints and verify the protective mechanism involved. Methods1. Take knee cartilage tissue from New Zealand white rabbit(one month-old) in a sterile environment, adding 0.2% collagenase Ⅱ. After filtration and centrifugation, chondrocytes were resuspended in DMEM/F-12 medium with FBS and 1% anti-double(1x105U / L penicillin, 100 mg / L streptomycin)。The isolated chondrocytes were cultured in the environment of 37℃and 5% CO2.2. Observing the forms of chondrocytes in different periods from being seeded in culture dishes until the the bottom almostly covered under the inverted phase contrast microscope.3. After adhering to microslides,the second generation chondrocytes were cultured in serum-free medium with PQQ at the concentration of 0, 6.25, 12.5, 25.0, 50.0, 100.0 μmol for 48 hours, then we evaluated the cell viability using MTT methods.4. The second generation chondrocytes at logarithmic growth were seeded in culture dish, adhering after 24 hours, then cultured in serum-free medium with PQQ at the concentration of 0, 6.25, 12.5, 25.0, 50.0, 100.0 μmol / L for 30 hours. Chondrocytes were collected and, then with 70% cold ethanol overnight at 4℃ environment. Washed by PBS solution, put into 100μl RNase A 37℃ water bathing for 30 minutes, then added to 400μl Propidium Iodide for staining,chondrocytes were checked with flow cytometry for cell cycle and proliferation index.5. Chondrocytes were seeded in culture dish,then cultured in serum-free medium with PQQ at the concentration of 0, 6.25, 12.5, 25.0, 50.0, 100.0 μmol / L for 24 hours. Then IL-1β was added to a final concentration of 10ng/ml, lasting for 15 hours. The supernatant was collected and mixed with suspension digested with 0.25% trypsin(without EDTA). After centrifugation, the cells were washed with PBS pre-cooling, then added into the Binding Buffer suspension cells, and then adding 5μl of the Annexin V-FITC(AV) and 5μl Propidium Iodide(PI) without light, incubated at room temperature for 15 minutes. Binding Buffer was added and mixed gently. Apoptosis rate was measured by flow cytometry. The results1.After adhesion, the chondrocytes showed triangular, polygonal or irregular in shape, while the cells connecting visible films showed "cobblestone".2. MTT results showed that, chondrocyte proliferation activity increased with the increasing of PQQ concentration, and it had statistically significant differences(P <0.05), compared with the control group. It reached the maximum in the PQQ concentration of 25.0 μmol/L, and in the concentration of 50.0μmol/L and 100.0 μmol/L it decreased.3. PQQ can significantly improve the proliferative activity of chondrocytes in S(%) phase, G2/M(%) ratio and the proliferation index(P<0.05), and it reached the maximum at the concentration of 12.5μmol/L and 25.0μmol/L.4 PQQ inhibited the early and late chondrocytes apoptosis(P<0.05) mediated by IL-1β, and it had the most effective roles in the PQQ concentration of 25.0μmol/L. ConclusionWe suggest PQQ can promote chondrocyte proliferation and protect the cell from IL-1β induced apoptosis.