| OBJECTIVETo investigate the effects of triptolide(TP) on the regulation of Ras-MAPKs and G protein-cAMP signal transduction pathway and elucidate the relationship between them in human fibroblast-like synoviocytes of rheumatoid arthritis (RA-HFLS). This study may offer new evidence for the molecule mechanism of therapeutic action acted by TP on rheumatoid arthritis (RA). Meanwhile, the research might provide experiment basis for the reasonable use of TP on clinic.METHODSRA-HFLS were cultured with triptolide (0.28,2.8,28,140 nM) in presence or absence of TNF-a in vitro. Cell proliferation was evaluated by MTS assay. The phosphorylation status of Ras-MAPKs-associated proteins(P38, ERK,JNK), and the expression of Gi and Gs were detected by Western blot, while the expression of Gi mRNA, Gs mRNA were detected by RT-PCR and the cAMP level was measurd by radio immunoassay (RIA) in RA-HFLS that treated with TP. CT(a selective activator of Gs), PT(a selective inhibiter of Gi), SB203580(a selective inhibiter of P38), PD98059(a selective inhibiter of ERK), SP600125(a selective inhibiter of JNK) were used to treated RA-HFLS for 1 h before induced by TNF-a, then the phosphorylation status of Ras-MAPKs-associated proteins and the expression of Gi and Gs were detected by Western blot.RESULTS1. Effect of TP on RA-HFLS proliferationCompared with blank group, The density of RA-HFLS increased, but the morphous looked similarly in TNF-a-induced group.Compared with TNF-a-induced group, after treated with TP for 24h, the density of RA-HFLS decreased, and the morphous changed in TNF-a+TP group.TP (2.8nM～140nM) could obviously decrease the RA-HFLS viability in a dose-dependent style and the inhibition ratio were 5.05%,30.83% and 43.77% (P<0.05,P<0.01).2. Phosphorylation status of RA-HFLS Ras-MAPKs signal transduction pathway induced by TNF-a for different timeTNF-a could induce phosphorylation of P38, ERK and JNK in RA-HFLS, and the peak appearanced at 15 min(P<0.05, P<0.01).The phosphorylation levels of that three proteins were 1.95 times,3.15 times and 2.78 times compared with untreated RA-HFLS.3. Effects of TP on Ras-MAPKs signal transduction pathway in RA-HFLSCompared with blank group, TNF-a could significantly increase the expression of Ras, p-P38, p-ERKand p-JNK in RA-HFLS (P<0.01).Compared with TNF-a-induced group,2.8 nM TP could significantly inhibit the expression of Ras, p-P38, p-ERK and p-JNK in RA-HFLS induced by TNF-a (P<0.05, P<0.01), when the concentration of TP reached to 140 nM, the inhibition ratio of the proteins above were 62.6%,57.5%,6.9% and 59.0%.Compared with blank group,2.8 nM TP could not significantly inhibit the expression of Ras, p-P38, p-ERK and p-JNK in RA-HFLS untreated with TNF-a (P>0.05), when its concentration reached to 140 nM from 28 nM, TP could display significantly inhibitory effect (P<0.05, P<0.01),140 nM TP could inhibit the expression of Ras, p-P38, p-ERK and p-JNK by 32.0%,32.9%,33.9% and 19.7%, but among difftent TP concentration, the inhibit effect were not significantly different.Comparing TP group with TNF-a+TP group, with regard to 2.8 nM TP, the inhibition ratio of Ras, P-P38 and p-ERK were not significantly different (P>0.05), while the inhibition ratio of p-JNK was significantly different (P<0.05), with regard to 28 nM TP, the inhibition ratio of Ras and p-P38 were not significantly different (P>0.05), while the inhibition ratio of p-ERKand p-JNK were significantly different (P<0.05, P<0.01), with regard to 140 nM TP, the inhibition ratio of the proteins above were significantly different (P<0.05, P<0.01).4. Effects of TP on the expression of Gi2, Gi3 and Gs in RA-HFLSCompared with blank group, TNF-a could increase the expression of Gi2, Gi3 and decrease the expression of Gs in RA-HFLS (P<0.01). Compared with TNF-a-induced group,2.8 nM TP could significantly inhibit the expression of Gi2 and increase the expression of Gs in RA-HFLS induced by TNF-a(P<0.05, P<0.01), when the concentration of TP reached to 140 nM, the inhibition ratio of the proteins above were 49.2%,54.1% and the enhancement radio was 90.3%, among difftent TP concentration, except the inhibitory effect of middle dose and high dose on Gi2 and the elevation effect on Gs, the impact effect were significantly different.Compared with blank group,2.8 nM TP could significantly inhibit the expression of Gi2, Gi3 (P<0.05, P<0.01), but its effect on Gs was not statistic difference in RA-HFLS untreated with TNF-α, when its concentration reached to 140 nM from 28 nM, its impact increased (P<0.05, P<0.01),140 nM TP could inhibit the expression of Gi2, Gi3 by 44.6%,30.4% and increase the expression of Gs by 17.2%, but among difftent TP concentration, only the difference between middle dose and high dose was significantly different.Comparing TP group with TNF-a+TP group, with regard to 2.8 nM TP, the inhibition ratio of Gi2, Gi3 were not significantly different (P>0.05), while the enhancement ratio of Gs was significantly different (P<0.05), with regard to 28 nM TP, the inhibition ratio of Gi2, Gi3 were not significantly different (P>0.05), while the enhancement ratio of Gs was significantly different (P<0.01), with regard to 140 nM TP, the inhibition ratio of Gi2 was not significantly different (P>0.05), while the inhibition ratio of Gi3 and the enhancement ratio of Gs were significantly different (P<0.01).5. Effects of TP on the expression of Gi mRNA and Gs mRNATNF-a could increase the expression of Gi mRNA and decrease the expression of Gs mRNA, in RA-HFLS.Compared with TNF-a-induced group,2.8 nM TP could significantly inhibit the expression of Gi mRNA and increase the expression of Gs mRNA in RA-HFLS induced by TNF-a (P<0.05, P<0.01), when the concentration of TP reached to 140 nM, the inhibition ratio of the mRNA above were 98.1% and the enhancement radio was 233.3%, among difftent TP concentration, except the inhibitory effect of low dose and middle dose on Gi mRNA and the elevation effect on Gs mRNA, the impact effect on RA-HFLS were significantly different.Compared with blank group,140 nM TP could significant inhibit the expression of Gi mRNA and increase the expression of Gs mRNA in RA-HFLS untreated with TNF-a.Comparing TP group with TNF-a+TP group, with regard to 140 nM TP, the inhibition ratio of Gi mRNA was not significantly different (P>0.05), while the enhancement ratio of Gs mRNA was significantly different (P<0.01).6. Effects of TP on the expression of cAMP in RA-HFLSCompared with blank group, TNF-a could inhibit the expression of cAMP, the inhibition ratio was 12.5% in RA-HFLS.Compared with TNF-a-induced group, TP could enhance the expression of cAMP in dose-dependent style and the enhancement radio were 10.4%,18.8%and 33.3%.Compared with blank group,140 nM TP could also significantly elevate the expression of cAMP in RA-HFLS untreated with TNF-a (P<0.05, P<0.01).7. The relationship between Ras-MAPKs and G protein-cAMP signal transduction pathway in RA-HFLSPT (1μg/mL) and CT (10μg/mL) could inhibit the enhancement of p-P38, p-ERK and p-JNK in RA-HFLS induced by TNF-α, SB203580 (10μM), PD98059 (50μM) and SB600125 (25μM)could inhibit the enhancement of Gi2, Gi3 and degression of Gs in RA-HFLS induced by TNF-a (P<0.05, P<0.01).CONCLUSION1. TNF-αcould significantly induced the proliferation of RA-HFLS, the abnormal activation of Ras-MAPKs and the abnormal inhibiton of G protein-cAMP signal transduction pathway.2. TP could significantly inhibit the proliferation of RA-HFLS.3. TP could inhibit the activation of Ras-MAPKs signal transduction pathway by inhibiton tne expression of Ras, p-P38, p-ERK and p-JNK in RA-HFLS.4. TP could adjust the imbalance of G protein-cAMP signal transduction pathway by regaining the balance between Gi and Gs and increasing the level of cAMP in RA-HFLS.5. The cross talk existed between Ras-MAPKs and G protein-cAMP signal transduction pathway in RA-HFLS.6. TP could directly inhibit the activation of Ras-MAPKs and adjust the imbalance of G protein-cAMP signal transduction pathway, meanwhile, inhibit one pathway could affect another one indirectly, and this might be the molecule mechanism of therapeutic action acted by Tripterygium wilfordii on RA. |
PDF Full Text Request