| Objective: Rheumatoid arthritis(RA) is an autoimmune disease and widely distributed throughout the body especially in hands and wrist, while its etiology and pathogenesis have not yet been found out. This disease is found around the world and in every race. The world average prevalence rate of RA is 1%-1.5% while its counterpart in China is 0.34-0.36%, and mostly happens to female, the ratio of prevalence rate between female and male is 1:3. RA is a disease that can be seen in any age while mostly occurs in the range of 30-50, also it becomes the main cause that leads the depletion of China’s young labour force. The traditional treatment medicine of RA includes nonsteroidal anti-inflammatory drugs, glucocorticoid and anti-rheumatic drugs. These medicines are mainly used to control and relieve symptoms of the disease but prolonged use of these medicines will cause serious adverse effect. According to this laboratory report, gardenias and its monomer Ge can effectively inhibit the level of proinflammatory cytokine IL-1β and TNF-α in CIA rats blood serums. Ge can inhibit the cell proliferation of CIA rat synovial cell by inducing its apoptosis. Macrophages play an important role in destruction of RA joint by secreting a variety of inflammatory mediators, matrix metalloproteinases, cytokines and growth factors. Ge is a kind of Iridoid compounds, also called gardenoside. Ge mainly exists in gardenias, eucommia and other Chinese herbs, and its main function is anti-inflammatory, anti-tumor, antithromboric, neuron protection, also it has some effects on osteoporosis treatment. In this study, PMA(phorbol myristate acetate) induced THP-1 cells to differentiate into macrophages. Stimulated by LPS(lipopolysaccharide), the macrophages culture supernatants and cause the inflammation model of synovial cell RSC-364, then add different concentration of Ge, to study the effect of the stimulation of Ge on macrophages supernatant RSC-364 cells and its signaling transduction, in order to discuss its medicinal property and mechanism further.Methods:1 Phorbol Myristate acetate(PMA) induced THP-1to differentiate into macrophages, then add lipopolysaccharide(LPS) to stimulate them to get culture supernatants.2 After that divided RSC-364 cells into a comparison(culture medium) group, a model(supernatants) group, a methotrexate(MTX 1×10-6 mol/L) group, and a geniposide-acid high(1×10-5 mol/L), a medium(1×10-6 mol/L) and a low(1×10-7 mol/L) group respectively, to conduct the MTT cell proliferation assay and to observe the cell proliferation rate of synovial cells after 24 hours, 48 hours and 72 hours.3 Then continue to cultivate different groups of RSC-364 for 72 hours, and used Hoechst33342 and AO-PI respectively to stain. The apoptotic morphological changes of RSC-364 with different concentration of Ge can be observed under the fluorescence microscope.4 Used flow cytometry to measure the total amount of DNA of RSC-364 cells with different concentration of Ge and calculated the apoptotic rate of the cells.5 The expression level of RSC-364’s supernatants of TNF-a、IL-1b、IL-6、IL-10、MMP-3 and MMP-9 is measured with ELISA.6 Radioimmunoassay is applied to measure the protein level of cyclic adenosine monophosphate( c AMP) in the comparison(culture medium) group, the model(supernatants) group, the methotrexate group(1×10-6 mol/L), and the geniposide-acid high(1×10-5 mol/L), the medium(1×10-6 mol/L) and the low(1×10-7 mol/L) group.7 Abstract and measure the total RNA of RSC-364 cells in different groups, then conduct the purity determination. Primer design, the m RNA expression of gene Gs, Gi is measured through RT-PCR.8 Abstract and measure the total protein of RSC-364 cells in different groups. The protein expression of total JNK1/2 、 ERK1/2 、 p38 and phosphorylated JNK1/2、ERK1/2、p38 are detected through Western Blot.Results:1 After 48 hours PMA induction, the THP-1 cells are completely adhered. Pseudopodium is generated on the cell surface. The growth speed of RSC-364 cells which are stimulated by the macrophage supernatant is evidently faster than the comparison group.2 After 72 hours continuous action, by comparing with comparison groups, the cell proliferation rate of model groups grew evidently(P<0.01). By comparing with model groups, the cell proliferation rate of MTX group, Ge higher dose groups, Ge medium groups and Ge lower dose group decreased evidently(P<0.01 or P<0.05). The cell proliferation rate of MTX group, Ge higher dose group and Ge medium groups decreased evidently on 72 hours(P<0.01).3 Most RSC-364 cells in the comparison groups and the model showed homogeneous green fluorescence after stained by AO-PI. The large amount of apoptosis in Ge higher dose group(1×10-5 mol/L), Ge medium dose group(1×10-6 mol/L), Ge lower dose group(1×10-7 mol/L) and positive drug group(MTX 1×10-6 mol/L) showed in intense green fluorescent, membrane folds, caryon pyknosis or rhexis, especially for cells in positive drug group and Ge high dose group. After stained by Hoechst33342, most synovial cell in comparison group and model group showed in homogeneous light blue fluorescence. With the increase of Ge concentration, the chromosome DNA broke and got together, apoptotic bodies occurred, cells showed in high bright blue fluorescence, especially in positive drug group and Ge high dose group.4 The apoptosis rate of control group and model group is 3.8% and 4.3% respectively but for the higher dose group, Ge medium dose group(1×10-6 mol/L),Ge lower dose group(1×10-7 mol/L) and positive drug group(MTX 1×10-6 mol/L) the rate is 31.3%, 26.5%, 9.3% and 35.6% respectively. Therefore, by comparing with comparison group and model group, the apoptosis rate of RSC-364 in the other groups show a positive trend and a dose dependent trend.5 By comparing with the comparison group, the concentration of IL-1b,TNF-a、IL-6、MMP-3 and MMP-9 increased evidently in model group(P<0.01). By comparing with the model group, the concentration of IL-1b,TNF-a、IL-6、MMP-3and MMP-9 showed a decrease trend(P<0.01 or P<0.05). By comparing with the comparison group, the concentration of IL-10 decreased evidently in model group(P<0.01). By comparison with the model group, the expression level of IL-10 showed a positive and dose dependent relationship the concentration of Ge, especially in Ge high dose group(1×10-5 mol/L).6 By comparing with the comparison group, the c AMP level in model group decreased evidently(P<0.01). By comparing with the model group, the concentration level of c AMP showed a positive and dose dependent relationship the concentration of Ge(P<0.01 or P<0.05), especially in Ge high dose group(1×10-5 mol/L)(P<0.01). By comparing with the model group, the c AMP level of MTX group increased evidently(P<0.01).7 By comparing with the comparison group, the Gi m RNA gene expression in model group increased evidently(P<0.01). By comparing with the model group, Ge higher, medium dose group and positive drug group significantly inhibited the Gi m RNA gene expression of synovial cell(P<0.01). By comparing with the comparison group, the Gs m RNA gene expression in model group decreased evidently(P<0.01). By comparing with the model group, Ge higher, medium dose group and positive drug group increase the gene expression level of Gs m RNA(P<0.01 or P<0.05), but Ge lower dose group is ineffective(P<0.01).8 By comparing with the comparison group, the protein expression level of p-p38、p-JNK1/2、p-ERK1/2 in model group is enhanced evidently(P<0.01). By comparing with the model group, Ge higher, medium, lower dose group can effectively inhibit the protein expression level of p-p38 、p-JNK1/2、p-ERK1/2(P<0.01 or P<0.05). By comparing with the model group, the protein expression level of p-p38、p-JNK1/2、p-ERK1/2 in positive drug group is enhanced evidently(P<0.01). There is no evidence to show that Ge higher, medium, lower dose group and positive drug group has an significant effect on the protein expression level of p38、JNK1/2、ERK1/2.Conclusion:1 Induce the monocyte THP-1 to macrophages in Vitro, and use LPS to stimulate it to produce inflammatory cytokines such as IL-1b,TNF-a that can make RSC-364 cells over-propagate2 Ge can effective decrease the cell proliferation of RSC-364 cells under Inflammatory milieu while has an impact on the production of Pro- inflammatory cytokines and relevant protein.3 Ge can help c AMP to grow and decrease the cell proliferation of RSC-364 cells by increasing the expression level of Gs gene’s m RNA and decrease the m RNA expression of Gi gene. Notice: Ge may has the anti-inflammatory function by adjust G protein coupled signal transduction pathways.4 Ge can effectively inhibit the protein expression level of p-p38, p-JNK1/2, p-ERK1/2 in RSC-364 cells which are induced by inflammatory cytokines. Notice: Ge may inhibit MAPKs signal transduction activating to further inhibit the proliferation and secretion of synovial cell. |
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