Effects Of Curcumin On Differentiation And Maturation Of Osteoclasts From Rheumatoid Arthritis Patients’ Peripheral Blood And The Expression Of RANK MRNA

Objective: In vitro culture of peripheral blood mononuclear cells(PBMCs) will induction training as osteoclasts. To investigate osteoclast activity and the effects of different concentrations of curcumin peripheral blood in RA patients with osteoclast cell differentiation and maturation and receptor activator of nuclear factor kappa B(RANK)affect the level of expression, to explore the role of osteoclasts bone injury in RA and curcumin possible molecular mechanisms of prevention and cure, provides a new experimental basis for traditional Chinese medicine monomer of curcumin in the treatment of RA.Methods:(1) ① The peripheral blood mononuclear cells from 12 cases of RA patients and 10 healthy controls were cultured in the medium containing receptor activator of nuclear factor κB-ligand(RANKL) and macrophage colony-stimulating factor(M-CSF).After being cultured for 14 days, cytochemistry was applied to detect tartrate-resistant acid phosphatase(TRAP) expression, and the cells with positive TRAP and ≥3 nuclei were counted and defined as osteoclasts, count osteoclasts. After being cultured for 21 days, absorption experiments on cultured cell lines were investigated osteoclast function Comparison of RA patients and healthy controls were detected in peripheral blood osteoclasts. ② To evaluate the clinical indexes in RA patients with bone destruction including hands joint X-ray examination of Sharp score, dual-energy X-ray absorptiometry in bone density test(BMD) values; and the number of osteoclasts in peripheral blood and bone destruction of the above clinical indicators were analyzed.(2) ① PBMCs from 3 healthy controls were cultured in the medium containing different concentration of curcumi(0, 2.5, 5, 10, 20, 40 μmol/L), were cultured in 24, 48, 72 hours, CCK-8 was used to evaluate the cell viability. ② PBMCs from 10 cases of RA patients, separation of PBMCs, adding RANKL(100 nmol/L) and M-CSF(50nmol/L PBMCs) induced to osteoclast differentiation. Adding different concentrations of curcumin in the cell culture process for intervention, were divided into 4groups, namely blank control group, curcumin 2.5 μmol/l group, curcumin 5 μmol/L curcumin and 10 μmol/l group. After being cultured for 14 days, cytochemistry was applied to detect TRAP expression and the cells with positive TRAP and ≥3 nuclei were counted and defined as osteoclasts, comparing each group number of osteoclasts. ③PBMCs from 5 cases of RA patients, separation of PBMCs, under the intervention of RANKL and M-CSF were induced in vitro culture, adding different concentrations of curcumin in the training process to intervene, experimental groups ibid. After being cultured for 10 days, RT-PCR technology were used to detect the expression of RANK m RNA in osteoclast precursor cell. Western-blot method to detect osteoclast precursor cells the expression of RANK protein.Results:(1) ① RA group and healthy control group with similar cell morphology, but the number of osteoclasts in RA group(128.92 ± 6.999 cells/10 vision) are significantly higher than that in healthy control group(79 ± 3.887 cells/10 vision)(P<0.05). Bone resorption in RA group was significantly higher than that in healthy control group. ②The number of osteoclasts and Sharp score was a significant positive correlation(r=0.810, P=0.001) in RA group, while the BMD(T value) was a significant negative correlation(r=-0.685, P=0.014)(2) ① CCK-8 method test show that low concentration of curcumin(2.5 μmol/l, 5μmol/l, 10 μmol/l) had no significant effect on PBMCs cell activity, higher concentration of curcumin(20 μmol/l and 40 μmol/l) significantly reduced cell vitality.② After being cultured for 14 days, the blank control group(179.9±3.8 cells/10 vision),compared with curcumin 2.5 μmol/l group(1.8±3.5 cells/10 vision), curcumin 5μmol/l group(79.9±3.8 cells/10 vision), curcumin 10 μmol/l group(60.6±4.4 cells/10 vision)the number of osteoclast decreased significantly, the difference had statistical significance(P<0.05). ③ RT-PCR results showed that compared with the blank controlgroup, the expression of RANK m RNA in curcumin 2.5 μmol/ group, 5 μmol/l group and 10 μmol/group, curcumin osteoclast precursor cells were significantly decreased, and in a concentration dependent manner(P<0.05). Western-blot results showed that compared with the blank control group, the expression of RANK m RNA in curcumin 2.5 μmol/ group, 5 μmol/ groupand, 10μmol/group,curcumin osteoclast pr-ecursor cells were significantly decreased, and in a concentration dependent manner(P<0.05).Conclusion:(1)RA patients PBMCs osteoclast differentiation ability increased significantly, and closely related with bone destruction RA clinical indicators. This may be the important mechanism of RA bone damage.(2)The curcumin have this ability that it was able to inhibits the PBMCs differentiated into osteoclasts in vitro, and to inhibits the expression of Osteoclast precursor cells in RANK m RNA and protein, All show that in a dose-dependent manner. This suggests we that curcumin may by acting on RANK-mediated signaling pathway inhibits osteoclast differentiation and maturation.This may be the important role of curcumin to treat RA bone destruction mechanism.