| Objective To investigate the intervention effect of sinomenine(SIN)on the differentiation of myeloid derived suppressor cell(MDSC)into osteoclast(OC)in mice with collagen induced arthritis(CIA),and to further investigate its possible molecular mechanisms involved,so as to provide a new idea for the prevention and treatment of rheumatoid arthritis.Methods There were 60 C57BL/6 mice,8 of which were randomly selected to the normal group,others were ready to be injected with adjuvant.Briefly,bovine native collagenⅡ(C Ⅱ)was emulsified in equal volumes of complete Freund ’s adjuvant(CFA).Mice were injected intradermally with the CⅡ/ CFA emulsion,using the loose skin at the base of the tail on day 0.On day 21 after the primary immunization,an intradermal booster injection was administered with CⅡ / incomplete Freund ’s adjuvant.There were 8 normal mice were used as control group,and the other successfully established CIA mice were randomly divided into CIA group,sinomenine 30 mg/kg group,sinomenine 30 mg/kg group,and anti Gr-1 m Ab group.SIN and Anti-Gr-1 m Ab were gavaged to mice at the corresponding dose once daily,while the control group and the CIA group were gavaged with phosphate buffer saline,starting on the 24 th day after the first immunization and ending on the 36 th day.24 hours after the last admininstration,mice were sacrificed and the samples and peripheral blood sampled from the eyeballs of mice were prepared for the following detections:(1)Arthritis indexes were observed and recorded.(2)Flow cytometry was used to detect the expression of MDSC in the peripheral blood.(3)Hematoxylin-eosin(H&E)staining was used to observe the infiltration of inflammatory cells and the degree of cartilage and bone destruction.(4)Safranin-O(S-O)staining was used to detect the degree of cartilage and bone destruction.(5)The morphology and quantity of OC were observed by tartrate-resistant acid phospholipase(TRAP)staining.(6)The expression of TRAP and matrix metalloproteinase-9(MMP-9)were detected by enzyme-linked immunosorbent assay(ELISA).(7)The expression of nuclear factor-κB(NF-κB)and phosphorylated κB inhibitor protein(p-IκB)were observed by immunohistochemistry.(8)Western blot was used to detect the expression of NF-κB and p-IκB.Results(1)The CIA model was successfully established and the redness or swelling of the joints appeared 22 days after the initial immunization,mainly from the interphalangeal joint,and then spread to the foot skin pad and ankle joint.The redness or swelling of the joints and polyarthritic lesions reached their peak 32 days after the initial immunization.(2)Compared with the control group,the number of MDSC in the peripheral blood of the mice in the CIA group was significantly increased(P<0.01);Compared with the CIA group,the number of MDSC in the peripheral blood of the mice in anti Gr-1 m Ab,SIN 30 mg/kg and SIN 300 mg/kg groups were significantly decreased(P<0.05 or P<0.01);Compared with the SIN 30 mg/kg group,the number of MDSC in the peripheral blood of mice in SIN 300 mg/kg group was significantly decreased(P<0.01).(3)Compared with the control group,the number of inflammatory cell infiltration,the degree of cartilage damage,and bone destruction in the CIA group was significantly increased(P<0.01);Compared with the CIA group,the inflammatory cell infiltration,cartilage damage,and bone destruction in the anti Gr-1 m Ab,SIN 30 mg/kg and SIN 300 mg/kg groups were significantly decreased(P<0.05 or P<0.01).(4)Compared with the control group,the degree of cartilage damage and bone destruction in the CIA group was significantly increased(P<0.01);Compared with the CIA group,the degree of cartilage damage and bone destruction in the anti Gr-1 m Ab,SIN 30 mg/kg and SIN300 mg/kg groups were significantly decreased(P<0.05 or P<0.01);Compared with the SIN30 mg/kg group,the degree of cartilage damage and bone destruction in the SIN 300 mg/kg group was significantly decreased(P<0.01).(5)Compared with the control group,the expression of TRAP positive cells in the CIA group was significantly increased(P<0.01);Compared with the CIA group,the expression of TRAP positive cells in the anti Gr-1 m Ab,SIN 30 mg/kg,and SIN 300 mg/kg groups were significantly decreased(P<0.05 or P<0.01);Compared with the SIN 30 mg/kg group,the expression of TRAP positive cells in the SIN300 mg/kg group was significantly decreased(P<0.01).(6)Compared with the control group,the expressions of TRAP and MMP-9 in the serum of the CIA group were significantly increased(P<0.01);Compared with the CIA group,the expressions of TRAP and MMP-9 in the serum in anti Gr-1 m Ab,SIN 30 mg/kg,and SIN 300 mg/kg groups were significantly decreased(P<0.05 or P<0.01);Compared with the SIN 30 mg/kg group,the expressions of MMP-9 in the serum of mice in the SIN 300 mg/kg group were significantly decreased(P<0.01).(7)Compared with the control group,the expression of NF-κB and p-IκB in the knee joint of mice in the control group was significantly increased(P<0.01);Compared with the CIA group,the expression of NF-κB and p-IκB in the knee joint of mice in the anti Gr-1m Ab,SIN 30 mg/kg,and SIN 300 mg/kg groups were significantly decreased(P<0.05 or<0.01);Compared with the SIN 30 mg/kg group,the expressions of NF-κB and p-IκB in the knee joint of mice in the SIN 300 mg/kg group were significantly decreased(P<0.01).(8)Compared with the control group,the expression of NF-κB and P-IκB in knee joint of mice in the CIA group was significantly increased(P<0.01);Compared with the CIA group,the expression of NF-κB and p-IκB in knee joint of mice in the anti Gr-1 m Ab,SIN 30 mg/kg and SIN 300 mg/kg groups were significantly decreased(P<0.05 or <0.01);Compared with the SIN 30 mg/kg group,the expressions of NF-κB and p-IκB in the knee joint of mice in the SIN300 mg/kg group were significantly decreased(P<0.01).Conclusion SIN can significantly improve bone destruction in CIA mice,the mechanism of which may be related to the inhibition of MDSC differentiation into OC and the inhibition of NF-κB signaling pathway. |
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