The Effecte Of Isoorientin-2"-O-A-L-Arabinopyranosyl On NF-κB Signaling Pathway In Hepatic Stellate Cells

Objective:Nuclear factor-KB (NF-κB) that is an essential regulator of the nuclear transcription factor with wide tissue expression involves in immune, inflammatory and cell growth. It has been shown that HSCs activation is associated with the elevation of NF-κB activity. Thus, inhibiting the activation of NF-κB pathway is a crucial measure for the treatment of hepatic fibrosis. In this study, to investigate the underlying mechanism of isoorientin-2"-O-a-L-arabinopyranosyl (IOA) on hepatic fibrosis, the effects of IOA on HSCs proliferation and apoptosis and the NF-κB signal transduction pathway were detected.Method:Hepatic stellate cells including HSC-T6, LX2 and 7702 cells were used in this study. Cells were divided into 6 groups:the normal control group, the IL-1β-stimulation group, the positive control group (PDTC group), and the low-medium-and high-dosages IOA-treated groups (IOAL, IOAM and IOAH groups). The effects of IOA on cell proliferation, clonogenicity, migration, apoptosis and cell cycle were analyzed by MTT method, clonogenicity observation, wound-healing assay, Annexin V-FITC/PI and AO-EB staining, respectively. The contens of Col-Ⅲ, TNF-a and TGF-(31 were assessed by using ELISA commercially available kits. The expressions of NF-κB p65, MMP-9 and Col-III were observed by immunohistochemical staining. The mRNA expressions of Col-I, Col-Ⅲ, NF-κB p65, IκBα, TNF-αand TGF-β1 were analyzed by RT-PCR. And the protein expressions of phospho-NF-κB p65 (p-p65), NF-κB p50, Col-I, IκBα, cytoplasm p-IκBα, NF-κB p65 and nuclear NF-κB p65 were detected by Western blot.Result:1. The effects of IOA on cell proliferation, clonogenicity, migration, apoptosis and cell cycleOur results showed that IOA significatnly inhibited hepatic stellate cell proliferation in a dose-dependent manner. Compared to the normal control group, IL-1β markedly increased the proliferation, clonogenicity and migration. IL-1β stimulation did not affect the apoptotic rate, cell cycle and morphology. Treatment with PDTC or IOA significantly reduced the proliferation, clonogenicity and migration. Furthermore, IAO treatment significantly increased the percentage of apoptotic cell, induced cell cycle arrest in G2 phase, and led to significant change of morphology. These results suggest that IOA can inhibit cell growth and migration, but promote cell apoptosis, indicating that the underlying mechanism of IOA on hepatic fibrosis is related to the inhibition of hepatic stellate cell activation.2. The effect of IOA on collagenThe results of RT-PCR showed that the mRNA expression of Col-I and Col-Ⅲ in IL-1β-stimulated group were significantly increased compared to the normal control group. ELISA assay and immunohistochemistry results revealed that IL-1β promoted the synthesis and secretion of Col-Ⅲ. Western blot showed that IL-1β significantly increased the expression of Col-I protein. Treatment with PDTC or IOA markedly decreased the mRNA and protein expressions of Col-I and Col-Ⅲ, inhibited the synthesis and secretion of Col-Ⅲ, and decreased the content of Col-I. These results indicate that IOA obviously inhibits the synthesis and deposition of collagen in hepatic stellate cells, thereby inhibiting the generation of ECM, which may be the pharmacological basis of its anti-hepatic fibrosis.3. The effects of IOA on inflammatory cytokinesCompared to the the normal control, the results of ELISA assay and RT-PCR analysis showed that IL-1β stimulation notably increased the content and mRNA expression of TNF-a and TGF-β1 in HSC-T6 and LX2 cells. Treatment with PDTC or IOA significantly decreased the content and the mRNA expression of TNF-a and TGF-β1. These results suggest that the possible mechanism of IOA in counteracting IL-1β-induced hepatotoxicity may be associated with the inhibition of inflammatory response.4. The effect of IOA on the expression of matrix metalloproteinase (MMP-9)In the HSC-T6 and LX2 cell experiments, the expression of MMP-9 in IL-1β-stimulated group was significantly higher than that of the normal control group, and the up-regμations of MMP-9 induced by IL-1β was markedly inhibited by treatment with PDTC or IOA. These data indicate that IOA inhibits the generation of ECM in hepatic stellate cells via the down-regulation of MMP-9 activity.5. The effect of IOA on NF-κB signaling pathwayThe RT-PCR results showed that PDTC or IOA significantly decreased NF-κB P65 mRNA expression, but increased IκBβ mRNA level. The Western blot analysis showed that IL-1β stimulation significantly increased the expressions of p-p65, NF-κB p50, cytoplasm p-IκBα and nuclear NF-κB p65, but decreased the expression of NF-κB p65 and IκBβ in cytoplasm. Treatment with IOA significantly reversed these abnormal changes induced by IL-1β stimulation. The immunocytochemical staining revealed that IL-1β treatment induced the activation of NF-κB, as evidenced by the increase in percentage of nuclear NF-κB p65 positive staining cells. Treatment with IOA notably decreased the nuclear NF-κB p65 positive staining cells, suggesting that IOA decreased the protein expression of nuclear NF-κB and inhibited the nuclear translocation of NF-κB, thereby inhibiting NF-κB pathway activation.Conclusion:IOA significatnly inhibited the proliferation of hepatic stellate cells, ptomoted cell apoptosis, and induced cell cycle arrest at G2 phase. Moreover, IOA reduced the synthesis and secretion of collagen, decreased the release of inflammatory cytokines, and regulated the level of matrix metalloproteinase. In addition, IOA inhibited the expresstion of IκBα and NF-κB p65 and prevented nuclear translocation of NF-κB, thereby resulting in inactivation of NF-κB. Our study indicates that IOA significantly inhibits HSCs activation and proliferation, and its underlying mechanism may be related to the inhibition of NF-κB signaling pathway.