Study On The Anti-inflammatory Mechanism Of Extract From Ligukaria Fischeri Alcoholized Ethyl Acetate Through The Jnk Pathway

ObjectiveLigularia fischeri Turcz is a perennial herb belongs to genus Ligularia, Compositae, whose root and rhizome included in Jilin province herbs standard, distributing in Yanbian counties. As the research goes deeper, Ligularia fischeri has anti-inflammatory effects have been gradually clarified, which out of the aerial parts have anti-inflammatory activity strongly. Studies have shown that Ligularia fischeri ethanol extracts could inhibit a variety of acute and chronic inflammatory and autoimmune. In this study, we explore the anti-inflammatory mechanism of extract from Ligularia fischeri alcoholized ethyl acetate works on the JNK (c-Jun N-terminal Kinase) pathway, aims at prove its anti-inflammatory effect and then determine whether it is accomplished through the JNK pathway.Method1. MTT method was assay to test the toxic concentration of extract from Ligularia fischeri alcoholized ethyl acetate (concentration range from0to640mg·L-1) made effect on RAW264.7cells for24hours, which density was5x104cells· mL-1.2. Determine the safe and effective concentration of JNK inhibitor SP600125(final concentration was25,12.5,6.25μ mol· L-1) act on cells for24hours, which density was5x104cells· mL-1, measure the viability of the cells.3. Measure the viability which extract from Ligularia fischeri alcoholized ethyl acetate act on inhibited lipopolysaccharid(LPS)-induced on cells for12,24,36,48hours, and then calculated the concentration of drug inhibiting on cell proliferation.4. Use the extract from Ligularia fischeri alcoholized ethyl acetate act on LPS-induced cells for24hours, and then Western-Blot method was used to observe the expression of P-JNK, JNK and Cyclo-oxgenase2protein. Results1. According to the test, the maximum non-toxic of extract from Ligularia fischeri alcoholized ethyl acetate is5.23mg· L-1.270.4mg· L-1is median toxic concentration.2.The concentration of SP600125inhibit the RAW264.7cells growth is25μ mol· L-1, having statistical difference compared with the control group(P<0.05), consider that concentration of SP600125has cytotoxic. However,12.5μ mol· L-1act on cells seems no effected, compare with the control group have no statistical difference, which concentration act on LPS-induced cells could inhibit cell activity, and have statistical difference compared with the control group(P<0.05).3. The concentration of extract from Ligularia fischeri alcoholized ethyl acetate inhibited LPS-induced RAW264.7cell viability is5mg·L-1(P<0.05). However, the concentration of2.5mg·L-1and lmg·L-1effect on inflammatory cell model did not inhibit the proliferation of cells.4. Extract from Ligularia fischeri alcoholized ethyl acetate act on LPS-induced RAW264.7cells, measured the relative gray values of P-JNK, JNK, COX-2were:0.12±0.034,0.48±0.027,0.18±0.037,and the relative gray values which no drug effects of LPS-induced RAW264.7cells were:0.68±0.046,0.83±0.044,0.58±0.039, compare two sets of data have obvious differences (P<0.01). Extract from Ligularia fischeri alcoholized ethyl acetate inhibited proliferation of RAW264.7cells which induced by LPS, cut down the expression of P-JNK, JNK, COX-2and other inflammatory proteins. Conclusion1. Extract from Ligularia fischeri alcoholized ethyl acctate has anti-inflammatory effects.2. Extract from Ligularia fischeri alcoholized ethyl acetate can inhibit LPS-induced RAW264.7cells proliferation by its anti-inflammatory effects.3. JNK is one of the pathway that the extract from Ligularia fischeri alcoholized ethyl acetate bring into play its anti-inflammatory mechanism...