The Effects Of Sinomenine On TLR4/MyD88/NF-κB Signaling Pathway In Peripheral Blood Monocytes Of Rheumatoid Arthritis Patients

Objective: Rheumatoid Arthritis(RA) is a chronic autoimmune disease, characterized by synovial inflammation and progressive disability. Toll-like receptors(TLRs) are the initial proteins in the chain of inflammatory response. On binding to their respective ligands, TLRs initiate inflammatory response. Upon activation by their respective ligands,TLRs initiate an intracellular pro-inflammatory/anti-inflammatory signaling cascade via recruitment of various adaptor proteins. TLR signaling cascade progression may be Myeloid differentiation factor 88(My D88) dependent or My D88 independent. TLRs active signaling pathway, which results in the activation of the nuclear factor kappa B predominate(NF-κB) and the production of pro-inflammatory cytokines such as tumour necrosis factor alpha(TNF-α), interleukin 1 beta(IL-1β),etc, causing the inflammatory response of the host. TLRs signaling pathways, especially TLR4/My D88/NF-κB pathway play an essential role in the pathogenesis of RA. The alkaloid sinomenine(SN) is an active compound isolated from the Chinese medicinal plant Sinoenium acutum, which has been widely recognized in the treatment of RA. The purpose of this study is to explore the therapeutic mechanism of SN in RA treatment via the influence of SN on the TLR4/My D88/NF-κB signaling pathway in peripheral blood monocytes of RA patients.Methods: First, 30 RA patients were recruited for our study. All patients’ basic information and clinical features including the levels of C-reactive protein(CRP), erythrocyte sedimentation rate(ESR) and the DAS28 scores were record at the same time of blood sample collection. According to DAS28 scores classification criteria, the RA patients were divided into two groups, that is, active RA group(n=17) and remissive RA group(n=13). Besides, 10 healthy volunteers in a healthy control group were also recruited.The plasma was isolated from the peripheral blood in vitro, the cytokine concentrations TNF-α and IL-1β were detected by Enzyme-linked immunosorbent assay( ELISA).Second, the active RA patients and healthy volunteers were selected for the further study. The peripheral blood mononuclear cells(PBMCs) were isolated from peripheral blood without plasma by centrifugation on Ficoll-Hypaque solution. Then, the PBMCs were cultured in RPMI-1640 medium in plates and maintained in a 5 % CO2 humidified atmosphere at 37 ℃ for 4 hours. After 4 hours, the cells culture medium was removed and then flushed thoroughly with warming PBS at 37 ℃ for two times to remove and collect any adherent monocytes. Following the second washes, the cells supernatant was discarded and the adherent monocytes were resuspended in RPMI-1640 complete medium and placed into 24-well plates. The monocytes of active RA patients were divided into five groups:(1)vcontrol group: monocytes were cultured in RPMI-1640;(2) lipopolysaccharide(LPS) group: monocytes were cultured with LPS(100 ng/ml) in RPMI-1640;(3) low dose SN group: monocytes were cultured with LPS(100 ng/ml) and SN(200 μmol/L) in RPMI-1640;(4) high dose SN group: monocytes were cultured with LPS(100 ng/ml) and SN(1000 μmol/L) in RPMI-1640;(5) TAK-242 group: monocytes were cultured with LPS(100 ng/ml) and TAK-242(1000 pg/ml) in RPMI-1640. Then maintained the plates in a 5 % CO2 humidified atmosphere at 37 ℃ for 24 hours. Besides, the monocytes of healthy volunteers as the healthy control group were cultured with RPMI-1640.Last, the expression of TLR4, My D88, NF-κB(p65) in monocytes were detected by the following methods:(1) Flow cytometry( FCM) was used to detect the expression of TLR4 positive rate on CD14+ monocytes;(2) Real-time fluorescent quantitative reverse transcription-polymerase chain reaction(FQ RT-PCR) was performed to determine TLR4, My D88, NF-κB(P65) m RNAs.(3) Western Blot was performed to determine TLR4, My D88, NF-κB(P65) proteins.(4) ELISA was used to detect the cytokine TNF-α in culture supernatant of monocytes.Results: 1. The levels of plasma TNF-α, IL-1β in RA patients and their relevance with the levels of ESR, CRP and DAS28. The levels of plasma TNF-α, IL-1β in RA active group were significantly higher than those in healthy control group and RA remissive group(P<0.001); the levels of ESR, CRP, DAS28 in the RA active group were significantly higher than those in RA remissive group(P<0.05); The levels of plasma TNF-α and IL-1β were positively correlated with the levels of ESR, CRP, DAS28(P<0.05).2. Flow cytometry(FCM) was used to detect the expression of TLR4 positive rate on CD14 + monocytes in peripheral blood of RA patients affected by SN. The expression of TLR4 positive rate on CD14 + monocytes were significantly increased in control group compared with that of healthy control group(P<0.05); the expression of TLR4 positive rate were significantly increased in LPS group compared with control group(P<0.05); the expression of TLR4 positive rate in low dose SN group, high dose SN group and TAK-242 group were significantly decreased, compared with LPS group(P<0.01), and the high dose SN group were significantly decreased compared with low dose SN group(P<0.001).3. The m RNA expression of TLR4, My D88, NF-κB(p65) in monocytes in RA patients affected by SN. The m RNA expression of TLR4, My D88, NF-κB(p65) in monocytes were significantly increased in control group compared with that of healthy control group(P<0.001), the m RNA expression of TLR4, My D88, NF-κB(p65) were significantly increased in LPS group, compared with control group(P<0.001). The m RNA expression of TLR4, NF-κB(p65) in monocytes were significantly decreased in low dose SN group, high dose SN group and TAK-242 group compared with LPS group(P<0.01, P<0.001, P<0.001); The m RNA expression of My D88 in monocytes were significantly decreased in low dose SN group, high dose SN group and TAK-242 group,compared with LPS group(P<0.01, P<0.001, P<0.01).4. The protein expression of TLR4, My D88, NF-κB(p65) in monocytes in RA patients affected by SN. The protein expression of TLR4, My D88, NF-κB(p65) in mon-ocytes were significantly increased in control group compared with that of healthy control group(P<0.001), the protein expression of TLR4, My D88, NF-κB(p65) were significantly increased in LPS group, compared with control group(P<0.001). The protein expression of TLR4, My D88, NF-κB( p65) were significantly decreased in low dose SN group, high dose SN group and TAK-242 group compared with LPS group(P<0.001), and the high dose SN group were significantly decreased compared with low dose group SN(P<0.001).5. The TNF-α expression in culture supernatant of monocytes in RA patients affected by SN. The TNF-α expression in culture supernatant of monocytes in control group were significantly higher than those in healthy control group(P<0.05);the TNF-α expression in LPS group were higher than those in control group(P<0.05);the TNF-α expression in low dose SN group, high dose SN group and TAK-242 group were significantly decreased, compared with LPS group(P<0.01), and the high dose SN group were significantly decreased compared with low dose SN group(P<0.001).Conclusion: 1. The levels of plasma TNF-α, IL-1β were significantly increased in active RA patients, the expression of TLR4, My D88, NF-κB(p65) in monocytes and the levels of TNF-α in culture supernatant were significantly increased in active RA patients.Active RA patients may exist the activation of TLR4/My D88/NF-κB signaling pathway.2. These results suggest that SN could inhibit the expression of TLR4, My D88, NF-κB(p65) and TNF-α in monocytes in RA patients. The effect may be related to inhibiting TLR4/My D88/NF-κB signaling pathway.