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Hydroxysafflor Yellow A Inhibits LPS-Induced NLRP3 Inflammasome Activation via Binding to Xanthine Oxidase in Mouse RAW264.7 Macrophages.

Xu X, Guo Y, Zhao J, Wang N, Ding J, Liu Q - Mediators Inflamm. (2016)

Bottom Line: In this study, we predicted the potential interaction between HSYA and xanthine oxidase (XO) via PharmMapper inverse docking and confirmed the binding inhibition via inhibitory test (IC50 = 40.04 μM).Computation docking illustrated that, in this HSYA-XO complex, HSYA was surrounded by Leu 648, Leu 712, His 875, Leu 873, Ser 876, Glu 879, Phe 649, and Asn 650 with a binding energy of -5.77 kcal/M and formed hydrogen bonds with the hydroxyl groups of HSYA at Glu 879, Asn 650, and His 875.Moreover, we proved that HSYA markedly inhibited LPS-induced cleaved caspase-1 activation via suppressing the sensitization of NLRP3 inflammasome and prevented the mature IL-1β formation from pro-IL-1β form.

View Article: PubMed Central - PubMed

Affiliation: Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing 100010, China; Beijing Institute of Traditional Chinese Medicine, Beijing 100010, China; Beijing Key Laboratory of Basic Research with Traditional Chinese Medicine on Infectious Diseases, Beijing 100010, China.

ABSTRACT
Hydroxysafflor yellow A (HSYA) is an effective therapeutic agent for inflammatory diseases and autoimmune disorders; however, its regulatory effect on NLRP3 inflammasome activation in macrophages has not been investigated. In this study, we predicted the potential interaction between HSYA and xanthine oxidase (XO) via PharmMapper inverse docking and confirmed the binding inhibition via inhibitory test (IC50 = 40.04 μM). Computation docking illustrated that, in this HSYA-XO complex, HSYA was surrounded by Leu 648, Leu 712, His 875, Leu 873, Ser 876, Glu 879, Phe 649, and Asn 650 with a binding energy of -5.77 kcal/M and formed hydrogen bonds with the hydroxyl groups of HSYA at Glu 879, Asn 650, and His 875. We then found that HSYA significantly decreased the activity of XO in RAW264.7 macrophages and suppressed LPS-induced ROS generation. Moreover, we proved that HSYA markedly inhibited LPS-induced cleaved caspase-1 activation via suppressing the sensitization of NLRP3 inflammasome and prevented the mature IL-1β formation from pro-IL-1β form. These findings suggest that XO may be a potential target of HSYA via direct binding inhibition and the combination of HSYA-XO suppresses LPS-induced ROS generation, contributing to the depression of NLRP3 inflammasome and inhibition of IL-1β secretion in macrophages.

No MeSH data available.


Related in: MedlinePlus

HSYA possesses no regulatory effect on XO expression. (a) RAW264.7 macrophages were pretreated by HSYA (25, 50, and 100 μM) for 3 h and then challenged by 1 μg/mL LPS for another 8 h. The XO protein expression was analyzed by Western Blot. (b) RAW264.7 macrophages were pretreated by HSYA (25, 50, and 100 μM) for 3 h and then challenged by 1 μg/mL LPS for another 6 h. The XO mRNA expression was analyzed by RT-PCR. Data represent the mean ± SEM of three independent experiments. ∗∗P < 0.01 indicates significant difference compared with the control group.
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fig4: HSYA possesses no regulatory effect on XO expression. (a) RAW264.7 macrophages were pretreated by HSYA (25, 50, and 100 μM) for 3 h and then challenged by 1 μg/mL LPS for another 8 h. The XO protein expression was analyzed by Western Blot. (b) RAW264.7 macrophages were pretreated by HSYA (25, 50, and 100 μM) for 3 h and then challenged by 1 μg/mL LPS for another 6 h. The XO mRNA expression was analyzed by RT-PCR. Data represent the mean ± SEM of three independent experiments. ∗∗P < 0.01 indicates significant difference compared with the control group.

Mentions: To confirm if there is a direct interaction between HSYA and XO, we detected the inhibitory effect of HSYA on XO activity both out of cells and within cells. Febuxostat was used as positive control. Because HSYA liquor appears yellow, we employed fluorescence detection, other than OD detection, to reduce error. Results showed that HSYA exhibited remarkable inhibitory effect on XO activity with IC50 = 40.04 μM out of cells (Figure 3(a)). The IC50 of positive control Febuxostat was 3.24 μM (data not shown). In addition, as shown in Figure 3(b), 50–100 μM HSYA treatment started to exhibit suppression on the activity of XO in LPS-stimulated macrophages (P < 0.05). However, results demonstrated that XO treatment had no noticeable action on the protein and mRNA expression of XO in macrophages (P > 0.05) (Figure 4).


Hydroxysafflor Yellow A Inhibits LPS-Induced NLRP3 Inflammasome Activation via Binding to Xanthine Oxidase in Mouse RAW264.7 Macrophages.

Xu X, Guo Y, Zhao J, Wang N, Ding J, Liu Q - Mediators Inflamm. (2016)

HSYA possesses no regulatory effect on XO expression. (a) RAW264.7 macrophages were pretreated by HSYA (25, 50, and 100 μM) for 3 h and then challenged by 1 μg/mL LPS for another 8 h. The XO protein expression was analyzed by Western Blot. (b) RAW264.7 macrophages were pretreated by HSYA (25, 50, and 100 μM) for 3 h and then challenged by 1 μg/mL LPS for another 6 h. The XO mRNA expression was analyzed by RT-PCR. Data represent the mean ± SEM of three independent experiments. ∗∗P < 0.01 indicates significant difference compared with the control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: HSYA possesses no regulatory effect on XO expression. (a) RAW264.7 macrophages were pretreated by HSYA (25, 50, and 100 μM) for 3 h and then challenged by 1 μg/mL LPS for another 8 h. The XO protein expression was analyzed by Western Blot. (b) RAW264.7 macrophages were pretreated by HSYA (25, 50, and 100 μM) for 3 h and then challenged by 1 μg/mL LPS for another 6 h. The XO mRNA expression was analyzed by RT-PCR. Data represent the mean ± SEM of three independent experiments. ∗∗P < 0.01 indicates significant difference compared with the control group.
Mentions: To confirm if there is a direct interaction between HSYA and XO, we detected the inhibitory effect of HSYA on XO activity both out of cells and within cells. Febuxostat was used as positive control. Because HSYA liquor appears yellow, we employed fluorescence detection, other than OD detection, to reduce error. Results showed that HSYA exhibited remarkable inhibitory effect on XO activity with IC50 = 40.04 μM out of cells (Figure 3(a)). The IC50 of positive control Febuxostat was 3.24 μM (data not shown). In addition, as shown in Figure 3(b), 50–100 μM HSYA treatment started to exhibit suppression on the activity of XO in LPS-stimulated macrophages (P < 0.05). However, results demonstrated that XO treatment had no noticeable action on the protein and mRNA expression of XO in macrophages (P > 0.05) (Figure 4).

Bottom Line: In this study, we predicted the potential interaction between HSYA and xanthine oxidase (XO) via PharmMapper inverse docking and confirmed the binding inhibition via inhibitory test (IC50 = 40.04 μM).Computation docking illustrated that, in this HSYA-XO complex, HSYA was surrounded by Leu 648, Leu 712, His 875, Leu 873, Ser 876, Glu 879, Phe 649, and Asn 650 with a binding energy of -5.77 kcal/M and formed hydrogen bonds with the hydroxyl groups of HSYA at Glu 879, Asn 650, and His 875.Moreover, we proved that HSYA markedly inhibited LPS-induced cleaved caspase-1 activation via suppressing the sensitization of NLRP3 inflammasome and prevented the mature IL-1β formation from pro-IL-1β form.

View Article: PubMed Central - PubMed

Affiliation: Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing 100010, China; Beijing Institute of Traditional Chinese Medicine, Beijing 100010, China; Beijing Key Laboratory of Basic Research with Traditional Chinese Medicine on Infectious Diseases, Beijing 100010, China.

ABSTRACT
Hydroxysafflor yellow A (HSYA) is an effective therapeutic agent for inflammatory diseases and autoimmune disorders; however, its regulatory effect on NLRP3 inflammasome activation in macrophages has not been investigated. In this study, we predicted the potential interaction between HSYA and xanthine oxidase (XO) via PharmMapper inverse docking and confirmed the binding inhibition via inhibitory test (IC50 = 40.04 μM). Computation docking illustrated that, in this HSYA-XO complex, HSYA was surrounded by Leu 648, Leu 712, His 875, Leu 873, Ser 876, Glu 879, Phe 649, and Asn 650 with a binding energy of -5.77 kcal/M and formed hydrogen bonds with the hydroxyl groups of HSYA at Glu 879, Asn 650, and His 875. We then found that HSYA significantly decreased the activity of XO in RAW264.7 macrophages and suppressed LPS-induced ROS generation. Moreover, we proved that HSYA markedly inhibited LPS-induced cleaved caspase-1 activation via suppressing the sensitization of NLRP3 inflammasome and prevented the mature IL-1β formation from pro-IL-1β form. These findings suggest that XO may be a potential target of HSYA via direct binding inhibition and the combination of HSYA-XO suppresses LPS-induced ROS generation, contributing to the depression of NLRP3 inflammasome and inhibition of IL-1β secretion in macrophages.

No MeSH data available.


Related in: MedlinePlus