| Objective:To investigate the mechanism of New Bitongling (NBTL) on HMGB1/ NF-κB signal pathway for rheumatoid arthritis (RA).Methods:Theoretical study:Point out the reliability of NBTL for treating RA and the possibility of relationship existed between its mechanism and HMGB1/NF-κB signal pathway via analyzing the latest research development on the etiology and pathogenesis, the review of comprehending and treating of Traditional Chinese medicine, and the efficacy evaluation of current clinical drug for RA.Experimental study:Apply relevant process to make extractum of NBTLTake SD rats to carry out the experiment.Randomly divid them into the normal group, the model group, the high-, moderate-, low-dose NBTL groups (3.2,1.6,0.8 g·kg-1·d-1) and the tripterygium wilfordii Hook F group (0.008 g·kg·d-1). Rats were used to be built the animal models of collagen-induced arthritis except the ones in the normal group. Rats of different groups were given corresponding drugs or distilled water at the day of secondary immune and continuously dosed for 21 days by ig. To observe the general situation of rats, measure the volume of left ankle joints and estimate the arthritis index (AI). Rats were executed to observe and evaluate the pathological tissues of joints. Detect the content of IL-1β, IL-6, TNF-α and HMGB1 in rats peripheral blood by the method of enzyme-linked immunosorbnent assay(ELISA) as well as the espression of IL-1β mRNA, IL-6 mRNA, TNF-αmRNA and HMGB1 mRNA in the synovial tissues by quantitative real time PCR(RT-qPCR).Incubate HMGB1 and LPS into HMGB1-LPS complex. The extractum of NBTL was diluted into low-, moderate- and high-concentration according to 2×10-6,10-5 and 2×10-5 times. After regular cultivation and generation passage, RSC-364 cells were randomly divided into the low-, moderate-, high-concentration NBTL groups (0.004,0.020,0.100 g·L-1), the control group and the model group. ALL of the NBTL groups were pretreated with corresponding concentration of the extractum for 24 h. Then make intervention of HMGB1-LPS complex to the NBTL groups and the model group. All groups were examined the content of IL-1B, IL-6 and TNF-α in cell culture supernatant by the method of ELISA as well as the expression of IL-1 mRNA, IL-6 mRNA and TNF-α mRNA in cells by RT-qPCR. And western blot was applied to examine the expression of NF-κB p65 and IκBα protein.Results:Theoretical study suggested that NBTL could be effective on hanshibizu syndrome of active rheumatoid arthritis.In vivo experimental study, CIA rats of the model group showed progressing joints swelling and even along with movement disturbance. Compared with the model group at corresponding period, all of the NBTL groups and the TwHF group had improvement on rats’activity, joints swelling and movement disturbance, especially for the high-dose NBTL group and the TwHF group.According to the results of evaluating the arthritis index, compared with the model group, all of the NBTL groups and the TwHF group were scored lower (P＜0.01) at 21d. According to the degree of joints swelling, the joints swelling of rats in the model group were progressing as the duration of immunity. Compared with the model group in the same time scale, the high-dose NBTL group showed lower joints swelling at 14 d (P＜0.05), as well as the high-dose NBTL and TwHF group showed lower joints swelling obviously at 21 d (P＜0.01). All of the NBTL groups and the TwHF group were alleviated on joint pathological section. According to the pathological score, the high-and middle-dose NBTL groups as well as the TwHF group were alleviated obviously (P＜0.01 or P＜0.05). Compared with the model group, the content of IL-1β, IL-6 and HMGB1 in rats peripheral blood were lower in all of the NBTL groups as well as in the TwHF group (P<0.01 or P＜0.05) and the content of TNF-α were lower in the high-and middle-dose NBTL groups (P＜0.01 or P＜0.05). While comparing with the model group, the NBTL groups and the TwHF group showed lower expression of IL-1β mRNA, IL-6mRNA and HMGB1 mRNA in the synovial tissues (P<0.01 or P<0.05), and the high-and middle-dose NBTL groups as well as the TwHF group showed decreased expression of TNF-a mRNA (P<0.01)In vitro experimental study, according to the results detected by ELISA, the content of IL-1β, IL-6 and TNF-a in cell culture supernatant were all obviously higher in the model group and all of the NBTL groups than that in the control group (P<0.01). Compared with the model group, the moderate-and high-concentration NBTL groups showed lower content of IL-1β and IL-6 (P<0.01), and the low, moderate-and high-concentration NBTL groups all showed lower content of TNF-a obviously (P＜0.01 or P＜0.05).According to the results detected by RT-qPCR, the expression of IL-1β mRNA, IL-6 mRNA and TNF-a mRNA in cells of the model group (P＜0.01) and the NBTL groups (P＜0.01 or P＜005) were all higher than that in the control group. While comparing with the model group, all of the NBTL groups showed lower expression of IL-1β mRNA and IL-6 mRNA (P<0.01 or P＜0.05), and the high-concentration NBTL group showed lower expression of TNF-α mRNA (P＜0.01).According to the results detected by Western Blot, compared with the control group, the model group and all of the NBTL groups showed higher expression of NF-κB p65 protein obviously (P＜0.01). Compared with the model group, the moderate-and high-concentration NBTL groups showed lower expression of NF-κB p65 protein (P＜0.01).Compared with the control group, the model group and the low-concentration NBTL group showed lower expression of IκBα protein in cells (P<0.01 or P＜0.05). While comparing with the model group, all of the NBTL groups showed higher expression of IκBα protein (P<0.01).Conclusion:Based on the theory of TCM, treating RA by NBTL, which composition was confirmed by screening out from optimized prescription, is safe and reliable. Experimental study confirmed that NBTL had well immunosuppressive action, as it could relieve joints swelling of CIA rats, restrain the arthropathy of the joints, and also suppress the inflammatory response of RSC-364 cells under the inducing of HMGB1-LPS complex. The immunosuppressive action of NBTL is related with HMGBI/NF-κB signal pathway, as it could inhibit HMGB1, the downstream cytokines of NF-κB signal pathway, such as IL-1β, IL-6 and TNF-α, and the activation of NF-κB. Therefore, it can further impact the inflammatory cascade reaction of HMGB1/NF-κB signal pathway, and effectively regulate the inflammatory caused by RA. |
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