| ObjectiveBy observing the effect of Miao nationality's herb Jinwujiangufang on the cell inhibition rate, apoptosis rate, nuclear factor kappa B activator 1 (ACT1), tumor necrosis factor receptor-associated factor 6 (TRAF6), expression of relevant inflammatory cytokines as TNF-?, IL-l?, IL-1?, IL-6. IL-17 in IL-17 signal pathway of rheumatoid arthritis human fibroblast-likes synoviocytes (RA-HFLS), to investigate the action mechanism of Jinwujiangufang on rheumatoid arthritis (RA)Methods1. Preparation of drug serum 36 healthy New Zealand white rabbit were randomly divided into 6 groups which were blank control group(n=6), Jinwujiangufang high(n=6), middle(n=6) and low(n=6) different doses group, Prednisone group(n=6), and Tripterygium Wilfordii (GTW) group(n=6). Except the blank control group, the rest was dosed with corresponding drug suspension by gavage. After 3 days of dosing, the blood was sampled in aseptic condition to separate serum. The Freeze-dried powder of the serum was formed by vacuum drying and stored at 4?.2. RA-HFLS was disposed by different drug serum concentration of Jinwujiangufang. Using MTT to detect the inhibition rate of RA-HFLS in different concentration of Jinwujiangufang. To detect apoptosis rate of RA-HFLS, we adopt Flow Cytometry. Employing ELISA to detect the expression of TNF-?, IL-1?, IL-1?, IL-6, and IL-17 in cell culture supernatant. PCR was used to detect the expression of ACT1 and TRA6mRNA.Results1. MTT result:The RA-HFLS of blank control group proliferated continually At the time point of 48h, compared with the blank control group, with the increasing concentration of Jinwujiangufang.OD value gradually decreased and the inhibition rate became higher and higher.2. Flow Cytometry result:Compared with blank control group and rabbit serum control group, apoptosis rate of Jinwujiangufang high, middle and low dose group and positive control group was increased; and the apoptosis rate among different doses group have statistical significance (P<0.01). In a certain scope, apoptosis of RA-HFLS was induced by Jinwujiangufang and showed a certain dose-dependency. With the increase of concentration, the induced ability enhanced. Compared with GTW control group, different doses of Jinwujiangufang high, middle and low group showed a stronger effect on promoting apoptosis (P<0.01).Compared with Prednisone group, the ability of promoting apoptosis in Jinwujiangufang middle dose group had no difference(P>0.5), but the ability of promoting apoptosis in Jinwujiangufang high dose group was stronger(P<0.01).3. ELISA result:At the time point of 48h, compared with blank control group and rabbit serum control group, the expression level of IL-17, IL-1?, IL-1?, TNF-? and IL-6 in cell culture supernatant in each group decreased with significant difference (P<0.01). All the groups of Jinwujiangufang in different concentration can significantly reduce the protein expression level of IL-17, IL-1?, IL-1?, TNF-? and IL-6(P<0.01).Compared with GTW control group, in Jinwujiangufang middle dose group, the expression level of IL-17, IL-l?, IL-1? was lower(P<0.01), but there was no obvious difference in the expression of TNF-? and IL-6(P>0.5). The effect of lower expression of IL-17, IL-1?,IL-1?, TNF-? and IL-6 within Jinwujiangufang high dose group was more obvious(P<0.01). Compared with Prednisone control group, the Jinwujiangufang high dose group had statistical significance(P<0.01), and the effect was better than Prednisone control group. Between Jinwujiangufang middle dose group and Prednisone control group, there was no difference in reducing the expression level of IL-17, IL-1?, IL-1? and IL-6 (P>0.5).4. RT-PCR result:At the time point of 48h, compared with the blank control group and rabbit serum control group, the expression of TRAF6 and ACT1 were all downregulation. In generally, there was obvious inhibition among Jinwujiangufang groups, GTW control group and Prednisone control group(P<0.01). Compared with GTW control group, Jinwujiangufang middle and high dose group had significant effect(.P<0.01). Compared with the Prednisone control group, Jinwujiangufang high dose group had significant effect(P<0.01), but Jinwujiangufang middle dose group didn't have(P>0.5).Conclusions1. Jinwujiangufang inhibit the proliferation of RA-HFLS, and promote the apoptosis of RA-HFLS. In a certain effective range, it shows dose-dependent.2. Jinwujiangufang can downregulate the expression of TNF-?, IL-1?, IL-1?, IL-6, and IL-17 in cultural supernatant of RA-HFLS and reduce the expression of ACT1 and TRAF6mRNA.3. It reveals that Jinwujiangufang has a therapeutic effect on RA by reducing the expression of its only adaptor protein ACT1 and its key substrate TRAF6 in IL-17 signal pathway, through inhibiting its activation, downregulating inflammatory cytokines, inhibiting the proliferation and promoting cell apoptosis in RA-HFLS. This would be the action mechanism of Jinwujiangufang on rheumatoid arthritis (RA). |
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