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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 inhibits NLRP3 inflammasome activation. RAW264.7 cells were pretreated with HSYA at indicated doses (25, 50, and 100 μM) for 3 h and then treated with 1 μg/mL LPS for 8 h. The GAPDH, NLRP3, ASC, procaspase-1, and caspase-1 proteins expressions were analyzed by Western Blotting. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, $$P < 0.01, ##P < 0.01, △P < 0.05, and △△P < 0.01 indicate significant differences compared with the control group of indicated proteins, respectively.
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fig7: HSYA inhibits NLRP3 inflammasome activation. RAW264.7 cells were pretreated with HSYA at indicated doses (25, 50, and 100 μM) for 3 h and then treated with 1 μg/mL LPS for 8 h. The GAPDH, NLRP3, ASC, procaspase-1, and caspase-1 proteins expressions were analyzed by Western Blotting. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, $$P < 0.01, ##P < 0.01, △P < 0.05, and △△P < 0.01 indicate significant differences compared with the control group of indicated proteins, respectively.

Mentions: Since latest research demonstrated that activation of NLRP3 inflammasome requires the ROS generated by XO, we further detected the effect of HSYA on activation of NLRP3 inflammasome. As shown in Figure 7, 1 μg/mL LPS challenge significantly enhanced the expression of NLRP3, ASC, and procaspase-1 proteins (P < 0.01), indicating that NLRP3 inflammasome was activated by LPS in macrophages. HSYA treatment from 50 to 100 μM notably inhibited the expression of NLRP3 (P < 0.05); however, no significant regulatory effects were observed on expression of ASC and procaspase-1 (P > 0.05). NLRP3 is essential to activate caspase-1 by cleaving procaspase-1 into mature form. Hence, we detected the effect of HSYA on cleaved caspase-1 expression. Result showed that HSYA treatment from 50–100 μM exhibited remarked inhibitory effect on the mature caspase-1 expression in macrophages (P < 0.05).


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 inhibits NLRP3 inflammasome activation. RAW264.7 cells were pretreated with HSYA at indicated doses (25, 50, and 100 μM) for 3 h and then treated with 1 μg/mL LPS for 8 h. The GAPDH, NLRP3, ASC, procaspase-1, and caspase-1 proteins expressions were analyzed by Western Blotting. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, $$P < 0.01, ##P < 0.01, △P < 0.05, and △△P < 0.01 indicate significant differences compared with the control group of indicated proteins, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940575&req=5

fig7: HSYA inhibits NLRP3 inflammasome activation. RAW264.7 cells were pretreated with HSYA at indicated doses (25, 50, and 100 μM) for 3 h and then treated with 1 μg/mL LPS for 8 h. The GAPDH, NLRP3, ASC, procaspase-1, and caspase-1 proteins expressions were analyzed by Western Blotting. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, $$P < 0.01, ##P < 0.01, △P < 0.05, and △△P < 0.01 indicate significant differences compared with the control group of indicated proteins, respectively.
Mentions: Since latest research demonstrated that activation of NLRP3 inflammasome requires the ROS generated by XO, we further detected the effect of HSYA on activation of NLRP3 inflammasome. As shown in Figure 7, 1 μg/mL LPS challenge significantly enhanced the expression of NLRP3, ASC, and procaspase-1 proteins (P < 0.01), indicating that NLRP3 inflammasome was activated by LPS in macrophages. HSYA treatment from 50 to 100 μM notably inhibited the expression of NLRP3 (P < 0.05); however, no significant regulatory effects were observed on expression of ASC and procaspase-1 (P > 0.05). NLRP3 is essential to activate caspase-1 by cleaving procaspase-1 into mature form. Hence, we detected the effect of HSYA on cleaved caspase-1 expression. Result showed that HSYA treatment from 50–100 μM exhibited remarked inhibitory effect on the mature caspase-1 expression in macrophages (P < 0.05).

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