TXL Inhibits RAW264.7Macrophage Proliferation And Migration Induced By TNF-α

Objective:TNF (Tumor necrosis factor) is a pleiotropic cytokine which isproduced by monocytes, macrophages, T cells, natural killer (NK) cells andother immune cells. TNF-α is a important mediator of immune andinflammatory responses, which involves in modulating endothelial cellfunction, inducing other cytokines, activating anti-viral activity of the cells,stimulating bone resorption, promoting angiogenesis, accelerating fibroblastmitogenesis and so on. Under normal circumstances, it plays important role inanti-tumor, anti-infection and tissue repair which is favorable for thebody. Under pathological and abnormal physiological states, it will acceleratethe inflammatory cascade, which causes the body heat, shock, cachexia andtissue damage. Thus, inhibition of TNF-α-mediated inflammatory response isan important part of many disease treatments.Tongxinluo (TXL) is composed of Ginseng, leeches, cicada, centipede,scorpion gold, Eupolyphaga, peony, sandalwood, Semen (fried), frankincense(system), dalbergia and borneol. Studies have shown that TXL can repairendothelial function, regulate blood lipids, stabilize plaque, anti-inflammatoryand anti-oxidation action. Clinical studies have shown that TXL can reduceC-reactive protein levels and exert the inflammatory cytokine production.Some studies have found that TXL can inhibit inflammatory responsesmediated by IL-1β in the coronary vessel wall, delay intimal proliferation andreduce the stenosis. TXL may act by inhibiting the expression of cytokines andadhesion molecules, thereby inhibiting IL-1β-induced inflammation andintimal proliferation. In this study, we aimed to investigate the effect of TNF-αon RAW264.7macrophage proliferation and migration，and then examinewhether TXL could inhibit TNF-α-induced inflammatory response. Methods: The expression of cyclin D1, cyclin E1and mmp-2wasdetected by Western blotting and real-time PCR analysis. Wound healing assaywas used to detect RAW264.7macrophage migration. Animal experiments:experimental animals were divided into two groups, vehicle group and TXLgavage group. Gavaging for7days, TNF-α was injected by tail vein to induceinflammatory response. At24hours after TNF-α injection, blood andC-reactive protein were examined, and cyclin D1, cyclin E1and mmp-2mRNA expression levels were detected by RT-PCR in mouse bone marrowmacrophages.Results:1TNF-α induced the expression of cyclin D1and cyclin E1inRAW264.7macrophagesWestern blot analysis showed that after RAW264.7macrophages wereincubated with TNF-α for24h at concentrations of0,1,10,100ng/ml, theexpression of cyclin D1and cyclin E1, compared with the control, markedlyincreased.RAW264.7macrophages were stimulated with10ng/ml TNF-α for0,6,12,24h. Western blot analysis showed that TNF-α promoted the expressionof cyclin D1and cyclin E1in time-dependent manner.Real-time PCR analysis was consistent with the Western blot results.These results indicated that TNF-α promoted RAW264.7macrophageproliferation by inducing the expression of cyclin D1and cyclin E1.2TXL inhibited RAW264.7macrophage proliferation induced by TNF-αRAW264.7macrophages were pre-incubated with TXL(100ug/ml) for24h, and then treated with TNF-α for24h, Western blot analysis showed thatin TXL pretreatment group, the expression of cyclin D1and cyclin E1wasdownregulated. Real-time PCR results were consistent with Western blotresults. These results indicate that TXL inhibited RAW264.7macrophageproliferation induced by TNF-α via suppressing the expression of cyclin D1and cyclin E1.3TNF-α induced the expression of mmp-2in RAW264.7macrophages RAW264.7macrophages were treated with TNF-α (0,1,10,100ng/ml)for24h. Western blot analysis showed that with increasing concentrations ofTNF-α, the expression level of migration-related gene mmp-2had adose-dependent increase.When RAW264.7macrophages were stimulated with10ng/ml TNF-α for0,6,12,24h, mmp-2expression was upregulated intime-dependent manner. Real-time PCR analysis was consistent with theWestern blot analysis. These results indicated that TNF-α promotedRAW264.7macrophage migration by upregulating the expression of mmp-2.4TXL inhibited RAW264.7macrophage migration induced by TNF-αRAW264.7macrophages were pre-incubated with TXL(100ug/ml) for24h, and then treated with TNF-α for24h, the expression level of mmp-2wasobserved by Western blot analysis. After treatment with TXL, the expressionof mmp-2was downregulated. Real-time PCR results were consistent withWestern blot results.Wounding cell migration assay showed that RAW264.7macrophageswere pre-incubated with TXL for24h, then stimulated with10ng/ml TNF-αfor24h, TXL could inhibit TNF-α-induced RAW264.7macrophagemigration.The above results revealed that TXL inhibited RAW264.7macrophagemigration induced by TNF-α by downregulating the expression of mmp-2.5TXL inhibited TNF-α-induced inflammation in mice.Mice were divided into two groups: control group and experimentalgroup (gavage with TXL).7days after treatment with TXL, TNF-α (0.1mg/kg)was injected by tail vein of mice. The results showed that the bodytemperature, C-reactive protein (CRP), white blood cells and neutrophils weresignificantly increased in the control group. Compared with control group, thebody temperature, CRP, white blood cells and neutrophils were decreased inexperimental group. Real-time PCR analysis showed that the expression levelsof cyclin D1, cyclin E1and mmp-2were decreased in bone marrowmacrophages of experimental group. These results suggested that TXLinhibited inflammatory response induced by TNF-α by downregulating the expression of cyclin D1, cyclin E1and mmp-2in mice.Conclusion:1TNF-α induced the expression of cyclin D1and cyclin E1inRAW264.7macrophages.2TXL inhibited RAW264.7macrophage proliferation induced by TNF-αthrough suppressing the expression of cyclin D1and cyclin E1.3TNF-α induced the expression of mmp-2in RAW264.7macrophages.4TXL inhibited RAW264.7macrophage migration induced by TNF-αthrough suppressing mmp-2expression.5TXL inhibited TNF-α-induced inflammation in mice.