Effect Of Lipopolysaccharide-induced Expression Of CD14,P38 MAPK And INOS By Sophoridine In RAW264.7 Cells

Objective To observe the effects of sophoridine (SRI) on the expression of cluster of differentiation 14 (CD14), p38 mitogen-activated protein kinase (p38MAPK), inducible nitrogenoxide synthase (iNOS) , tumor necrosis factor-α(TNF-α) and nitric oxide (NO) in RAW264.7 cells stimulated by lipopolysaccharide ( LPS) ,and to explore the mechanism of sophoridine in anti-inflammation induced by LPS.Methods In this study, lipopolysaccharide (LPS)-activated macrophage cells (RAW264.7) was employed as an inflammation model. RAW264.7 cells were divided into eight groups (n = 6): control group, LPS1 group, LPS2 group, SRI (sophoridine) treating group, SRI pretreating group, SRI premixing group, SB203580 group, SB203580 + SRI group. RAW264.7 cells were stimulated with SRI (15.63 mg/L). After activation, the maximal atoxic concentration (TC0) was detected by MTT; The expression of CD14 mRNA, p38 mRNA, iNOS mRNA and expression of CD14 protein, p-p38 protein, iNOS protein were measured by using reverse transcription polymerase chain reaction (RT-PCR) and western blotting, respectively; The expression of NO and TNF-αwere measured by nitrate reductase method and radio immunoassay, respectively.Results (1)The maximal atoxic concentration(TCo) of sophoridine on RAW264.7 cells was 200 mg·L^-1 according to Reed Meuench.(2) Compared with the control group, expression of mRNA and protein of CD14, p38 and iNOS in LPS1 group were significantly increased (P<0.01). Compared with LPS1 group, expression of mRNA of CD14, p38 and iNOS and protein of CD14, p-p38 and iNOS in SRI treating group were significantly decreased (P<0.01 or P<0.05) , while greater than the control group(P<0.01). Compared with LPS1 group, expression of mRNA and protein of iNOS in SB203580 group were significantly decreased (P<0.01 or P<0.05). Compared with SB203580 group, expression of mRNA and protein of iNOS in SB203580 + SRI group were significantly decreased (P<0.01 or P<0.05).(3) Compared with the control group, expression of mRNA and protein of CD14, p38 and iNOS in LPS2 group were significantly increased (P<0.01), which reached a peak at 120 min (P<0.01). Compared with LPS2 group, expression of mRNA of CD14, p38 and iNOS and protein of p-p38 and iNOS in pretreating SRI group were decreased (P<0.01 or P<0.05), while greater than the control group(P<0.01). Compared with LPS2 group, expression of mRNA of CD14, p38 and iNOS and protein of CD14, p-p38 and iNOS in SRI premixing group were decreased (P<0.01 or P<0.05) , while greater than the control group(P<0.01).(4) Compared with the control group, expression of NO and TNF-αin LPS1 group were significantly increased (P<0.01). While compared with LPS1 group, expression of NO and TNF-αin treating SRI and SB203580 group were significantly decreased(P<0.01 or P<0.05), which decreased with time in treating SRI group and reached the level of the control group(P>0.05) at 120 min. Compared with SB203580 group expression of NO and TNF-αin SB203580 + SRI group were significantly decreased (P<0.01 or P<0.05), which reached the level of the control group (P>0.05) at 120 min and they were time-depended.(5) Compared with LPS1 group, expression of NO and TNF-αin treating SRI (31.25 mg·L^-1, 15.63 mg·L^-1) and SB203580 group were significantly decreased (P<0.01 or P<0.05), and the expression of NO and TNF-αin treating SRI group increased with the concentration of sophoridine (7.82 mg·L^-1, 3.91 mg·L^-1, 1.95 mg?L-1) down. Compared with SB203580 group, expression of NO and TNF-αin SB203580+SRI group (31.25 mg·L^-1, 15.63 mg·L^-1) were significantly decreased (P<0.01 or P<0.05), and the expression of NO and TNF-αin SB203580 + SRI group increased with the concentration of sophoridine down (7.82 mg·L^-1, 3.91 mg·L^-1, 1.95 mg·L^-1) and they were concentration-depended.Conclusion (1) According to Reed Meuench, the maximal atoxic concentration (TC0) of sophoridine on RAW264.7 cells was 200 mg·L^-1.(2) Sophoridine (SRI) played a role by decreasing the transcription, translation and protein phosphorylation of CD14, p38 and iNOS induced by LPS, in inflammatory . It is also directly related to the role of endotoxin inactivation.(3) Sophoridine inhibited the expression of NO and TNF-αin RAW264.7 cells induced by LPS which were time-depended and concentration-depended.(4) Sophoridine (SRI) can abolish inflammation by decreasing the expression of iNOS, NO and TNF-αand be synergistic effect with SB203580. It can also interfere with other signaling pathway except p38 MAPK pathway..