Molecular Mechanisms Of The Inhibitory Effects Of Curcumin On The Osteoclastogenesis Of The PBMC From RA Patients

Objective:The present study was performed to investigate the effects of curcumin on the osteoclastogenesis fromperipheral blood mononuclear cells (PBMCs) from rheumatoid arthritis (RA) patientsin vitro, the expressionof osteoclast-relatedtranscription factorsc-Fos、 NFATcl and the activation of MAPKs signaling pathways. We next investigated whether TNF-a can indunce osteoclastogenesis from PBMCswhen RANKL was absent in vitro, and the effects of curcumin on the osteoclastogenesisunder the inflammatory conditions.Probes into the effects of curcuminon osteoclastogenic potential ofPBMC from patients of RAand the underlying molecular mechanisms.Provides the new laboratory basis for the clinical application of curcumin, and the new way for the treatment of RA by traditional Chinese medicine.Methods:1. PBMCs of patients with RAseparated by density centrifugationwere cultured in the medium containing 50ng/mL M-CSF and 100ng/mL RANKL for 14 days or 21 days.The cells were identified by TRAP staining and bone resorption pit assay, and the cells with positive TRAP and >3 nuclei were counted as osteoclasts.PBMCswere incubated and treatedwith differentconcentrations of curcumin (0-40μM), CCK8was used to measure the cell viability at 24h、48h and 72h,to detect the cytotoxicity ofcurcumin on PBMCs.There were 4 groups namedblank control group, curcumin 2.5μmol/1 group, curcumin 5μmol/L group and curcumin 10μmol/l group.PBMCs of patients with RAwere incubated, addingdifferent concentrationsof curcumin in the process. After cultured for 14 days, TRAP staining, acid phosphatase activity test and CatK、MMP-9 by ELISA method were used to evaluate the inhibitory effects of curcumin on the osteoclastogenesis. The Protein expression of c-Fos> NFAT1、ERK1/2、p-ERK1/2、p38、p-p38、JNK、p-JNK were detected by western blotting.2. According to the source of PBMC and the different medium, the groups were divided intoN0(healthy controls, R+M), N1(healthy controls,2.5ng/ml TNF-a+M), N2(healthy controls,5ng/ml TNF-a+M), N3(healthy controls,10 ng/ml TNF-a+M), N4(healthy controls, 5ng/ml TNF-a+R+M), R0(RA, R+M), R1(RA,2.5ng/ml TNF-a+M), R2(RA,5ng/ml TNF-a+M), R3(RA, 10ng/ml TNF-a+M), R4(RA,5ng/ml TNF-a+R+M). After cultured for 14 days, TRAP staining and cell counting were performed。 PBMCs of patients with RAwere cultured in the medium containing different concentrations of recombinanthuman TNF-aas well as 50ng/mL M-CSF and 100ng/mL RANKL. After cultured for 14 days, TRAP-positive cells counted, the protein expression of RNAK、TRAP6 were detected by western blotting. Furthermore,PBMCs of patients with RAwere culturedby 5ng/ml TNF-a,50 ng/mL M-CSF and 100 ng/mL RANKL, and different concentrations of curcumin (0,2.5μmol/L,5 μmol/L, 10μmol/L). After incubated for 14 days, TRAP-positive cells counted, the expressionof RANK was measured by real-time PCR and western blotting.Results:1. After cultured for 14 days, the number of TRAP-positive osteoclasts were decreased significantly, comparedcurcumin 2.5umol/l group, curcumin 5μmol/l group, curcumin 10μmol/l group with the blank control group, the difference had statistical significance (P<0.05). Acid phosphatase activity test and ELISA test showed the different levels of curcumin could decrease the secretion of TRAP, CatK and MMP9 by TRAP-positive osteoclasts after culture 14 days(P<0.05). Western blotting results showed that compared with the blank control group, the protein expression of c-Fos、NFATcl in curcumin 2.5 μmol/L group,5 μmol/l group and 10 μmol/groupwere significantly decreased (P<0.05). In the blank control group, the phosphorylation rate of ERK1/2 was0.758±0.016,p38 was0.759 ±0.054 and JNK was0.734±0.032,were significantly higher than those of all curcumin groups (P< 0.05).2. The number of TRAP-positive osteoclasts differentiated from PBMCs ofpatients with RA was significantly higher than that of the healthy control,and the difference were remarkable (?<0.05).When RANKL was absent,2.5ng/ml,5ng/ml,7.5ng/ml TNF-a couldn’tindunce PBMCs ofpatients with RA and of the healthy control differentiate into TRAP-positive osteoclastsin vitro. But after culturedin the medium containing different concentrations of recombinanthuman TNF-aas well as 50ng/mL M-CSF and 100ng/mL RANKL for 14 days,TNF-a could increase the number of TRAP-positive osteoclastsdifferentiated from PBMCs ofpatients with RAin a concentration dependentmanner (P<0.05).At the same time, TNF-acould increase the protein expression of RNAK、TRAP6 (P<0.05). After culturedby 5ng/ml TNF-α,50ng/mL M-CSF,100ng/mL RANKL, and different concentrations of curcumin for 14 days,The number of TRAP-positive of the blank control group was137.11±6.49, curcumin 2.5 μmol/L group was130.56± 3.32,5μmol/l group was112.33±14.64 and 10μmol/groupwas88.44±1.81, the difference significantly (P<0.05). RT-PCR and Western blottingresults showed that compared with the blank control group, the expression of RANK mRNA andproteinin curcumin 2.5μmol/L group, 5μmol/L group and 10μmol/L group, were significantly decreased (P<0.05).Conclusions:1. This studydemonstrated that Curcumin caninhibit osteoclastogenic potential of PBMCs from RA patients through the suppression of the activationof MAPKs pathways. Curcumin can decreasethe number of TRAP-positive osteoclasts and the secretion of TRAP, CatK and MMP-9 as well as inhibit c-Fos and NFATcl expression.2.The osteoclastogenic potential of PBMCs from RA patientsis significantly higher than that of the healthy persons3. TNF-a can’t indunce PBMCs differentiate into osteoclasts without RANKL in vitro, but can increase the ability of RANKL through increase the expression of RNAK and TRAP6.4.Curcumin can decreasethe cell’s number, inhibitRANK mRNA andprotein expression of the TRAP-positive osteoclasts from RA patients’PBMCs induncedby TNF-a, M-CSF andRANKL.5.Curcumin might be usefulas a new therapeutic agent for bone deteriorations in inflammatory diseases such as RA.