Abstract：
BACKGROUND: Gouty arthritis (GA), a common inflammatory condition triggered by monosodium urate crystal accumulation, often necessitates safer treatment alternatives due to the limitations of current therapies. Astilbin, a flavonoid from Smilax glabra Roxb, has demonstrated potential in traditional Chinese medicine for its anti-inflammatory properties. However, the anti-GA effect and its underlying mechanism have not been fully elucidated.
OBJECTIVE: This study aimed to investigate the therapeutic potential of astilbin in GA, focusing on its effects on neutrophil extracellular traps (NETs), as well as the potential molecular target of GA both in vitro and in vivo.
METHODS: Firstly, astilbin inhibited the citrullinated histone H3 (Cit h3) protein levels and reduced the NETs formation in neutrophils stimulated by monosodium urate (MSU). Secondly, we wondered the effect of astilbin on migration of neutrophils and dimethyl-sulfoxide (DMSO)-differentiated HL-60 (dHL-60) cells under the stimulation of MSU. Then, the effect of astilbin on suppressing NETs through purinergic P2Y6 receptor (P2Y6R) and Interlukin-8 (IL-8)/ CXC chemokine receptor 2 (CXCR2) pathway was investigated. Also, the relationship between P2Y6R and IL-8/CXCR2 was explored in dHL-60 cells under stimulation of MSU. Finally, we testified the effect of astilbin on reducing NETs in GA through suppressing P2Y6R and then down-regulating IL-8/CXCR2 pathway.
METHODS: MSU was used to induce NETs in neutrophils and dHL-60 cells. Real-time formation of NETs and migration of neutrophils were monitored by cell living imaging with or without MSU. Then, the effect of astilbin on NETs formation, P2Y6R and IL-8/CXCR2 pathway were detected by immunofluorescence (IF) and western blotting. P2Y6R knockdown dHL-60 cells were established by small interfering RNA to investigate the association between P2Y6R and IL-8/CXCR2 pathway. Also, plasmid of P2Y6R was used to overexpress P2Y6R in dHL-60 cells, which was employed to explore the role of P2Y6R in astilbin inhibiting NETs. Within the conditions of knockdown and overexpression of P2Y6R, migration and NETs formation were assessed by transmigration assay and IF staining, respectively. In vivo, MSU-induced GA mice model was established to assess the effect of astilbin on inflammation by haematoxylin-eosin and ELISA. Additionally, the effects of astilbin on neutrophils infiltration, NETs, P2Y6R and IL-8/CXCR2 pathway were analyzed by IF, ELISA, immunohistochemistry (IHC) and western blotting.
RESULTS: Under MSU stimulation, astilbin significantly suppressed the level of Cit h3 and NETs formation including the fluorescent expressions of Cit h3, neutrophils elastase, myeloperoxidase, and intra/extracellular DNA. Also, results showed that MSU caused NETs release in neutrophils as well as a trend towards recruitment of dHL-60 cells to MSU. Astilbin could markedly decrease expressions of P2Y6R and IL-8/CXCR2 pathway which were upregulated by MSU. By silencing P2Y6R, the expression of IL-8/CXCR2 pathway and migration of dHL-60 cells were inhibited, leading to the suppression of NETs. These findings indicated the upstream role of P2Y6R in the IL-8/CXCR2 pathway. Moreover, overexpression of P2Y6R was evidently inhibited by astilbin, causing a downregulation in IL-8/CXCR2 pathway, migration of dHL-60 cells and NETs formation. These results emphasized that astilbin inhibited the IL-8/CXCR2 pathway primarily through P2Y6R. In vivo, astilbin administration led to marked reductions in ankle swelling, inflammatory infiltration as well as neutrophils infiltration. Expressions of P2Y6R and IL-8/CXCR2 pathway were evidently decreased by astilbin and P2Y6R inhibitor MRS2578 either alone or in combination. Also, astilbin and MRS2578 showed notable effect on reducing MSU-induced NETs formation and IL-8/CXCR2 pathway whether used alone or in combination, parallelly demonstrating that astilbin decreased NETs formation mainly through P2Y6R.
CONCLUSIONS: This study revealed that astilbin suppressed NETs formation via downregulating P2Y6R and subsequently the IL-8/CXCR2 pathway, which evidently mitigated GA induced by MSU. It also highlighted the potential of astilbin as a promising natural therapeutic for GA.
OBJECTIVE: This study aimed to investigate the therapeutic potential of astilbin in GA, focusing on its effects on neutrophil extracellular traps (NETs), as well as the potential molecular target of GA both in vitro and in vivo.
METHODS: Firstly, astilbin inhibited the citrullinated histone H3 (Cit h3) protein levels and reduced the NETs formation in neutrophils stimulated by monosodium urate (MSU). Secondly, we wondered the effect of astilbin on migration of neutrophils and dimethyl-sulfoxide (DMSO)-differentiated HL-60 (dHL-60) cells under the stimulation of MSU. Then, the effect of astilbin on suppressing NETs through purinergic P2Y6 receptor (P2Y6R) and Interlukin-8 (IL-8)/ CXC chemokine receptor 2 (CXCR2) pathway was investigated. Also, the relationship between P2Y6R and IL-8/CXCR2 was explored in dHL-60 cells under stimulation of MSU. Finally, we testified the effect of astilbin on reducing NETs in GA through suppressing P2Y6R and then down-regulating IL-8/CXCR2 pathway.
METHODS: MSU was used to induce NETs in neutrophils and dHL-60 cells. Real-time formation of NETs and migration of neutrophils were monitored by cell living imaging with or without MSU. Then, the effect of astilbin on NETs formation, P2Y6R and IL-8/CXCR2 pathway were detected by immunofluorescence (IF) and western blotting. P2Y6R knockdown dHL-60 cells were established by small interfering RNA to investigate the association between P2Y6R and IL-8/CXCR2 pathway. Also, plasmid of P2Y6R was used to overexpress P2Y6R in dHL-60 cells, which was employed to explore the role of P2Y6R in astilbin inhibiting NETs. Within the conditions of knockdown and overexpression of P2Y6R, migration and NETs formation were assessed by transmigration assay and IF staining, respectively. In vivo, MSU-induced GA mice model was established to assess the effect of astilbin on inflammation by haematoxylin-eosin and ELISA. Additionally, the effects of astilbin on neutrophils infiltration, NETs, P2Y6R and IL-8/CXCR2 pathway were analyzed by IF, ELISA, immunohistochemistry (IHC) and western blotting.
RESULTS: Under MSU stimulation, astilbin significantly suppressed the level of Cit h3 and NETs formation including the fluorescent expressions of Cit h3, neutrophils elastase, myeloperoxidase, and intra/extracellular DNA. Also, results showed that MSU caused NETs release in neutrophils as well as a trend towards recruitment of dHL-60 cells to MSU. Astilbin could markedly decrease expressions of P2Y6R and IL-8/CXCR2 pathway which were upregulated by MSU. By silencing P2Y6R, the expression of IL-8/CXCR2 pathway and migration of dHL-60 cells were inhibited, leading to the suppression of NETs. These findings indicated the upstream role of P2Y6R in the IL-8/CXCR2 pathway. Moreover, overexpression of P2Y6R was evidently inhibited by astilbin, causing a downregulation in IL-8/CXCR2 pathway, migration of dHL-60 cells and NETs formation. These results emphasized that astilbin inhibited the IL-8/CXCR2 pathway primarily through P2Y6R. In vivo, astilbin administration led to marked reductions in ankle swelling, inflammatory infiltration as well as neutrophils infiltration. Expressions of P2Y6R and IL-8/CXCR2 pathway were evidently decreased by astilbin and P2Y6R inhibitor MRS2578 either alone or in combination. Also, astilbin and MRS2578 showed notable effect on reducing MSU-induced NETs formation and IL-8/CXCR2 pathway whether used alone or in combination, parallelly demonstrating that astilbin decreased NETs formation mainly through P2Y6R.
CONCLUSIONS: This study revealed that astilbin suppressed NETs formation via downregulating P2Y6R and subsequently the IL-8/CXCR2 pathway, which evidently mitigated GA induced by MSU. It also highlighted the potential of astilbin as a promising natural therapeutic for GA.
摘要：
背景：痛风性关节炎（GA），由尿酸单钠晶体积聚引发的常见炎症，由于当前疗法的局限性，通常需要更安全的治疗方法。Astilbin,一种黄酮类化合物，来自土黄Roxb,已证明其抗炎特性在中药中具有潜力。然而,抗GA效应及其潜在机制尚未完全阐明。
目的：本研究旨在探讨astilbin在GA中的治疗潜力，专注于其对中性粒细胞胞外陷阱（NET）的影响，以及GA在体外和体内的潜在分子靶标。
方法：首先，astilbin抑制瓜氨酸化组蛋白H3（Cith3）蛋白水平，并减少尿酸单钠（MSU）刺激的中性粒细胞中NETs的形成。其次,我们想知道在MSU刺激下，astilbin对中性粒细胞和二甲基亚砜（DMSO）分化的HL-60（dHL-60）细胞迁移的影响。然后，研究了astilbin通过嘌呤能P2Y6受体（P2Y6R）和Interlukin-8（IL-8）/CXC趋化因子受体2（CXCR2）途径抑制NETs的作用。此外，研究了在MSU刺激下dHL-60细胞中P2Y6R与IL-8/CXCR2的关系。最后,我们证明了astilbin通过抑制P2Y6R然后下调IL-8/CXCR2通路来减少GA中的NETs的作用。
方法：MSU用于在中性粒细胞和dHL-60细胞中诱导NETs。通过有或没有MSU的细胞活成像监测NETs的实时形成和嗜中性粒细胞的迁移。然后，astilbin对NET形成的影响，免疫荧光（IF）和蛋白质印迹法检测P2Y6R和IL-8/CXCR2通路。通过小干扰RNA建立P2Y6R敲低dHL-60细胞，以研究P2Y6R与IL-8/CXCR2通路之间的关联。此外，P2Y6R质粒用于在dHL-60细胞中过表达P2Y6R，探讨P2Y6R在astilbin抑制NETs中的作用。在P2Y6R敲低和过表达的条件下，通过迁移测定和IF染色评估迁移和NETs形成，分别。在体内,建立MSU诱导的GA小鼠模型，通过苏木精-伊红和ELISA评估astilbin对炎症的影响。此外,astilbin对中性粒细胞浸润的影响，NET,通过IF分析P2Y6R和IL-8/CXCR2通路，ELISA,免疫组织化学（IHC）和蛋白质印迹。
结果：在MSU刺激下，astilbin显著抑制Cith3的水平和NETs的形成，包括Cith3，中性粒细胞弹性蛋白酶的荧光表达，髓过氧化物酶,和细胞内/细胞外DNA。此外，结果表明，MSU引起NETs在中性粒细胞中的释放以及dHL-60细胞向MSU募集的趋势。Astilbin可以显着降低MSU上调的P2Y6R和IL-8/CXCR2通路的表达。通过沉默P2Y6R,IL-8/CXCR2通路的表达和dHL-60细胞的迁移受到抑制,导致网络压制。这些发现表明P2Y6R在IL-8/CXCR2途径中的上游作用。此外，astilbin明显抑制P2Y6R的过表达，导致IL-8/CXCR2通路下调，dHL-60细胞的迁移和NET的形成。这些结果强调了astilbin主要通过P2Y6R抑制IL-8/CXCR2途径。在体内,astilbin给药导致踝关节肿胀明显减轻，炎性浸润以及中性粒细胞浸润。astilbin和P2Y6R抑制剂MRS2578单独或联合使用可明显降低P2Y6R和IL-8/CXCR2通路的表达。此外，astilbin和MRS2578对减少MSU诱导的NETs形成和IL-8/CXCR2途径有显著作用，无论是单独使用还是联合使用，同时证明了astilbin主要通过P2Y6R减少了NETs的形成。
结论：这项研究表明，astilbin通过下调P2Y6R和随后的IL-8/CXCR2途径抑制NETs的形成，明显减轻了MSU诱导的GA。它还强调了astilbin作为GA的有前途的天然治疗剂的潜力。
目的：本研究旨在探讨astilbin在GA中的治疗潜力，专注于其对中性粒细胞胞外陷阱（NET）的影响，以及GA在体外和体内的潜在分子靶标。
方法：首先，astilbin抑制瓜氨酸化组蛋白H3（Cith3）蛋白水平，并减少尿酸单钠（MSU）刺激的中性粒细胞中NETs的形成。其次,我们想知道在MSU刺激下，astilbin对中性粒细胞和二甲基亚砜（DMSO）分化的HL-60（dHL-60）细胞迁移的影响。然后，研究了astilbin通过嘌呤能P2Y6受体（P2Y6R）和Interlukin-8（IL-8）/CXC趋化因子受体2（CXCR2）途径抑制NETs的作用。此外，研究了在MSU刺激下dHL-60细胞中P2Y6R与IL-8/CXCR2的关系。最后,我们证明了astilbin通过抑制P2Y6R然后下调IL-8/CXCR2通路来减少GA中的NETs的作用。
方法：MSU用于在中性粒细胞和dHL-60细胞中诱导NETs。通过有或没有MSU的细胞活成像监测NETs的实时形成和嗜中性粒细胞的迁移。然后，astilbin对NET形成的影响，免疫荧光（IF）和蛋白质印迹法检测P2Y6R和IL-8/CXCR2通路。通过小干扰RNA建立P2Y6R敲低dHL-60细胞，以研究P2Y6R与IL-8/CXCR2通路之间的关联。此外，P2Y6R质粒用于在dHL-60细胞中过表达P2Y6R，探讨P2Y6R在astilbin抑制NETs中的作用。在P2Y6R敲低和过表达的条件下，通过迁移测定和IF染色评估迁移和NETs形成，分别。在体内,建立MSU诱导的GA小鼠模型，通过苏木精-伊红和ELISA评估astilbin对炎症的影响。此外,astilbin对中性粒细胞浸润的影响，NET,通过IF分析P2Y6R和IL-8/CXCR2通路，ELISA,免疫组织化学（IHC）和蛋白质印迹。
结果：在MSU刺激下，astilbin显著抑制Cith3的水平和NETs的形成，包括Cith3，中性粒细胞弹性蛋白酶的荧光表达，髓过氧化物酶,和细胞内/细胞外DNA。此外，结果表明，MSU引起NETs在中性粒细胞中的释放以及dHL-60细胞向MSU募集的趋势。Astilbin可以显着降低MSU上调的P2Y6R和IL-8/CXCR2通路的表达。通过沉默P2Y6R,IL-8/CXCR2通路的表达和dHL-60细胞的迁移受到抑制,导致网络压制。这些发现表明P2Y6R在IL-8/CXCR2途径中的上游作用。此外，astilbin明显抑制P2Y6R的过表达，导致IL-8/CXCR2通路下调，dHL-60细胞的迁移和NET的形成。这些结果强调了astilbin主要通过P2Y6R抑制IL-8/CXCR2途径。在体内,astilbin给药导致踝关节肿胀明显减轻，炎性浸润以及中性粒细胞浸润。astilbin和P2Y6R抑制剂MRS2578单独或联合使用可明显降低P2Y6R和IL-8/CXCR2通路的表达。此外，astilbin和MRS2578对减少MSU诱导的NETs形成和IL-8/CXCR2途径有显著作用，无论是单独使用还是联合使用，同时证明了astilbin主要通过P2Y6R减少了NETs的形成。
结论：这项研究表明，astilbin通过下调P2Y6R和随后的IL-8/CXCR2途径抑制NETs的形成，明显减轻了MSU诱导的GA。它还强调了astilbin作为GA的有前途的天然治疗剂的潜力。
