Galuteolin inhibits cell proliferation of RA-FLS cells.
As shown in Figure 1A, we found that TNF-α (50 ng/mL) induced an obvious increase in the cell viability of RA-FLS cells (p<0.001 at 24 and 48 h). Under the condition of TNF-α treatment, the proliferation of RA-FLS cells was inhibited remarkably by pretreatment of Galu in a concentration dependent manner (Fig. 1A). These results showed that Galu (20-200 μg/mL) could counteract the auxo-action of TNF-α on the viability of RA-FLS. Therefore, we chose the maximum dose (200 μM) of Galu for the follow-up experiments.
Galuteolin promotes cell apoptosis of RA-FLS cells.
To verify the effect of Galu on the apoptosis of RA-FLS, TUNEL staining was performed. We found that the ratio of apoptosis (Brown represents apoptotic cells) in the Galu-treated alone group was significantly higher than that in the control group, while TNF-α treatment reduced cell apoptosis obviously (Figure 1B). Interestingly, pretreatment of Galu could counteract the inhibitory role of TNF-α on the apoptosis of RA-FLS cells and induced cell apoptosis (Figure 1B). These results were further confirmed by detecting the expression of apoptosis-related proteins. As shown in Figure 1C, Galu could upregulate Bax and cleaved-caspase3 expressions in the RA-FLS cells with TNF-α stimulation or not, but suppressed the expression of anti-apoptosis protein Bcl-2. Caspase3 expression levels did not change. These findings demonstrated that galuteolin may have an anti-apoptosis effect in rheumatoid arthritis.
Galuteolin inhibits inflammation of RA-FLS cells
Overproductions of pro-inflammatory cytokines have played a key role in the pathophysiology of rheumatoid arthritis. In the present study, we observed that TNF-α could significantly elevate the levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8 and MMP-1. Moreover, Galu suppressed pro-inflammatory cytokines production in the RA-FLS cells induced with or without TNF-α (Figure 2A and 2B). Both RT-qPCR and ELISA experiments show this trend, which showed that galuteolin may have a good anti-inflammatory effect for TNF-α-induced inflammation in RA-FLS cells.
Galuteolin elevated TNF-α-induced inhibition of HO-1 expression in RA-FLS cells
In order to confirm the interaction between Galu and HO-1 and their roles in RA, we examined the expression level of HO-1 in RA-FLS cells after TNF-α stimulation or not. As seen in Figure 3A, Galu notably upregulated the mRNA expression of HO-1 and TNF-α stimulation suppressed HO-1 expression in in RA-FLS cells. Interestingly, the pre-treatment of TNF-α-induced RA-FLS cells with Galu could elevate HO-1 expression when compared with TNF-α group. These findings confirmed that galuteolin could aggrandize the expression of HO-1 in RA.
Silencing of HO-1 diminishes the pro-apoptotic effect of galuteolin in RA-FLS cells
To verify whether HO-1 is involved in the pro-apoptotic role of Galu in RA-FLS cells and the relationship between them, the loss-function experiments have proceeded. We first transfected RA-FLS cells with two different small interfering RNAs (si-HO-1-1 and si-HO-1-2) to knocked down HO-1. As shown in Figure 3B, there was no significant difference between negative plasmid si-NC group when compared with control group. However, HO-1 was reduced in si-HO-1-1 and si-HO-1-2 transfected RA-FLS cells when compared with si-NC group. The plasmid of si-HO-1-2 (si-HO-1) with relatively higher interference efficiency was selected for subsequent experiments.
In addition, knockdown of HO-1 sharply increased cell proliferation, and the inhibitory effect of Galu on cell viability was largely overturned by HO-1 knock down (Figure 3C). Besides, TUNEL staining showed that silence of HO-1 could reduce the apoptosis ratio of in Galu-induced cells, and could weaken the pro-apoptotic effect of Galu in RA-FLS cells after TNF-α stimulation (Figure 3D). In the Figure 3E, using Galu pre-treatment RA-FLS cell group as the control, we found that knock down of HO-1 remarkably reduced cell apoptosis through down-regulating Bax and cleaved-caspase-3 expression, and upregulating Bcl-2 expression in the Galu pre-treatment cells. Moreover, after pre-treated with Galu of TNF-α-stimulated RA-FLS cells, silence of HO-1 could significantly inhibit apoptosis by down-regulating Bax and cleaved-caspase-3 expression and increasing Bcl-2 expression, with Caspase-3 unaffected. These findings indicated that HO-1 might be involved in the pro-apoptotic process of galuteolin and as a downstream target of galuteolin in inducing RA-FLS cell apoptosis in response to TNF-α or not.
Silencing of HO-1 diminishes the anti-inflammatory effect of galuteolin in RA-FLS cells
As shown in Figure 4A, silence of HO-1 sharply increased IL-1β, IL-6, IL-8 and MMP-1 mRNA levels, and the anti-inflammatory effect of Galu was largely overturned by HO-1 knock down. Similarly, when the expression of HO-1 was suppressed, the effect of Galu to suppress the release of pro-inflammatory cytokines was restored and distinctly enhanced in TNF-α-stimulated cells. In addition, ELISA showed that the levels of IL-1β, IL-6, IL-8 and MMP-1 were upregulated obviously by si-HO-1 plasmid after Galu pretreatment in RA-FLS cells stimulated with or without TNF-α (Figure 4B). These findings demonstrated that HO-1 might be a downstream target of galuteolin in suppressing inflammation of RA-FLS cells.
Galuteolin inhibits the Iκκβ/NF-κB signaling pathway via activating the HO-1 expression in RA-FLS cells
To explore the Galu targets the downstream signaling pathway behind HO-1, we preliminarily measured the expression level of the Iκκβ/NF-κB signaling pathway. As seen in Figure 5, we found that Galu could suppress Iκκβ/NF-κB signaling pathway through downregulating Iκκβ, p-NF-κB p65 and p-IκB expression, which were upregulated by si-HO-1 plasmid in the RA-FLS cells. Interestingly, TNF-α led to a sharp increase in the Iκκβ, p-NF-κB p65 and p-IκB expression. Similarly, in the TNF-α-stimulated cells, pre-treatment of Galu could suppress the Iκκβ/NF-κB pathway, which was activated by si-HO-1 transfection.
These findings demonstrated that galuteolin might suppresses proliferation and inflammation in TNF-α-induced RA-FLS cells by activating HO-1 to regulate IKKβ/NF-κB pathway.