Induction Of Macrophage Apoptosis By Sinomenine And Its Related Mechanisms

Sinomenine (SN) is an immunnosuppressive compound derived from the Chinese medicinal plant Sinomenium acutum. SN has been used in China for many centuries to treat various rheumatic diseases successfully. It is reported that SN is an analogue of morphine in structure and SN can effectively inhibit inflammation reaction, immune response and relieve pain, etc. Some studies have shown that SN can inhibit the proliferation of murine spleen cells and monocytes stimulated by mitogen, the production of antibody by B cells, and the production of pro-inflammatory factors such as tumour necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), prostaglandin E2 {PGE2), nitric oxide (NO) produced by macrophages. Additionally, SN can protect mice from fulminate hepatitis induced by endotoxin, and exerts synergistic effects when combined with suboptimal doses of CsA, inhibiting immune responses and prolonging cardiac allograft survival in rats.It is macrophage, T, B cell infiltration, the pro-inflammation cytokines secreted by macrophage, interaction between macrophage and immune cell, fibroblast that contribute to pathogenesis, progression, prognosis of rheumatoid arthritis. SN has been shown to be effective in treating rheumatoid arthritis. Macrophage activation has been demonstrated to play important roles in the innate immunity and even in the pathogenesis of some autoimmune diseases. So, macrophages are the powerful target immune cells for some immunosuppressive drugs. In another hand, induction of immune cell apoptosis has been proposed as one of the mechanisms by whichimmunosuppressive drugs exert their functions. For examples, D-penicillamine, triptolides, cyclophosphamide has been usually administered as immunosuppressive drug to inhibit inflammation and treat rheumatic diseases, partially because they can induce apoptosis of immune cells. Up to now, little is known about the mechanisms by which SN inhibits macrophage functions. Therefore, the primary aim of this study is to examine the inhibitory effects of SN on murine macrophages by investigating whether SN can induce the apoptosis of macrophages, and if so, what's the related mechanism.We selected macrophage cell line RAW264.7 as a macrophage model and then examined the effect of SN on the proliferation of RAW264.7 by MMT, and the apoptosis of RAW264.7 cells after SN treatment. We examined the apoptosis induction by several methods: Annexin V and PI staining to indicate the change of cell membrane, Rhodamine 123 staining to show the change of mitochondria membrane and Hoechst 33258 to show that of nuclei, since the apoptosis represents that the externalization of phosphatidyl serine, increased membrane permeability, destruction of mitochondria membrane, condensation, fragmentation of nuclei.We demonstrated that SN can inhibit the proliferation of murine macrophage RAW264.7 by inducing apoptosis in a dose- and time-dependent manner. We found activation of extracellular signal-regulated protein kinase (ERK) in SN-treated macrophages, and requirement for ERK activation in SN-induced apoptosis of macrophages. Contemporarily the expression of p27/KIPl, proapoptotic factor Bax increased, and expression of Bcl-2 decreased, which may cooperate to induce apoptosis. Inhibition of ERK activation reduced the increased expression of p27, Bax but had no effect on the decreased expression of Bcl-2, suggesting the involvement of ERK activation in the increased expression of p27 and Bax induced by SN. These results demonstrate that SN can induce apoptosis of macrophages through activation of ERK, and ERK activation may partially involve the increased expression of p27 and Bax in apoptotic macrophages. Therefore, induction of macrophage apoptcsis through ERK activation may be one of mechanisms by which SN exhibits its immunosuppressive function.In conclusion, SN can induce the apoptosis of macrophages through ERK activation, and ERK activation is partially involved in the increased expression of p27 and Bax in SN-induced apoptotic macrophages. Our study outlines n...