Study On The Effect Of Luteolin On Macropahge Associated Inflammation And Angiogenesis Factors In Osteoarthritis

Purpose:Osteoarthritis (OA) is a common degenerative disease of the elderly, which can cause the articular cartilage defects and degeneration, reactive hyperplasia of joint edge and subchondral bone and then affecting the joint function. As one of the important immune cells, macrophages play a certain role in the progression of OA by promoting inflammation, angiogenesis, and tissue repair. In this study, we simulated inflammatory environment by stimulating macrophage, and then treated them with luteolin. By observing the alteration of cytokines secreted by macrophage, the effect of luteolin on macrophage associated inflammation and angiogenesis is confirmed.Methods:The experiment was divided into 4 groups. Mouse macrophage cell line RAW264.7 is cultured as control group. The RAW264.7 cell stimulated by Lipopolysaccharides (Lps) for 2 hours (h) in order to induce the M1 macrophage polarization, as LPS group. The RAW264.7 cell was stimulated by interleukin-4 (IL-4) for 2h in order to induce the M2 macrophage polarization, as the IL-4 group. In luteolin group, we stimulate RAW264.7 cell lines by Lps/or IL-4, then treated cells with luteolin for 24h. By real-time PCR, western blot, and immunofluorescence, we observed the different expression of inflammatory factor (TNF-a iNOS, IL-1β, and IL-6) and angiogenesis factors (VEGF, HIF-la, PDGF, TF, and TGF-β) among groups.Results:After 5 μg·mL-1 LPS stimulate, the results of real-time PCR showed that the expression of inflammatory factors TNF-a, iNOS, IL-1β, and IL-6 were significantly higher in the Lps group than in the control group (P<0.05). The expression of those inflammatory factors were significantly reduced in the luteolin treatment group than in the Lps group (P<0.05). Immunofluorescence results showed high expression of IL-1β induced by Lps could be inhibited by luteolin. While the results of real-time PCR, western blot, and immunofluorescence revealed the high expression of VEGF, HIF-la, PDGF, TF, and TGF-β in Lps/IL-4 group was reduced by 60μmol·mL-1 luteolin (P<0.05). The expression of PDGF, VEGF, and HIF-1 in treatment group was lower than that in Lps group (P<0.05). But the expression of TF and TGF-p was not affected (P>0.05). In M2 polarization experiment, after 30μg·mL-1 IL-4 stimulated, we observed the high expression of TGF-P in IL-4 group, but there was no significant difference between the luteolin group and the IL-4 group (P>0.05). While the results of real-time PCR, western blot, and immunofluorescence revealed the higher expression of PDGF, VEGF, TF, HIF-1α in 5μg·mL-1 Lps group (P<0.05). The expression of PDGF, VEGF and HIF-1 in Luteolin group was lower than the Lps group (P<0.05). But the expression of TF was not affected (P>0.05). In M2 polarization experiment, after 30 ng·mL-1 IL-4 stimulated, we observed the high expression of TGF-β in IL-4 group, but there was no significant difference between the luteolin group and the IL-4 group (P>0.05).Conclusions:Luteolin can inhibit the expression of inflammatory factor TNF-α, iNOS, IL-1β, and IL-6, as wells as angiogenic factors PDGF, VEGF, and HIF-la in macrophage, but there was no effect on the expression of TF and TGF-β.