羌活通过调节P2X3抑制PKC诱导的炎症反应治疗

doi:10.5246/jcps.2022.03.014

中国药学(英文版) ›› 2022, Vol. 31 ›› Issue (3): 163-175.DOI: 10.5246/jcps.2022.03.014

• 【研究论文】 • 下一篇

羌活通过调节P2X3抑制PKC诱导的炎症反应治疗"上肢痹痛"

王颖峥¹^,^#, 杨策¹^,²^,^#, 朱翠萍¹, 赵茜茜¹, 卢雪花³, 苏晓宇¹^,^*(), 王英豪¹^,^*()

1. 福建中医药大学药学院, 福建福州 350122
2. 重庆三峡医药高等专科学校, 重庆 404120
3. 福建省医学科学研究院, 福建福州 350001

收稿日期:2021-11-14 修回日期:2021-12-08 接受日期:2022-01-12 出版日期:2022-03-31 发布日期:2022-03-31
通讯作者: 苏晓宇, 王英豪
作者简介:
+ Tel./Fax: +86-591-22861136, E-mail: wyhtcm@163.com
+ E-mail: 30109053@qq.com
基金资助:
The Department of Science & Technology of Fujian Province (Grant No. 2017J01539 and 2020J01729), the Education Department Of Fujian Province (Grant No. JAT190239).

Upper limb arthromyodynia can be treated with Notopterygium by down-regulating P2X3 to mediate PKC-induced inflammatory response

Yingzheng Wang¹^,^#, Ce Yang¹^,²^,^#, Cuiping Zhu¹, Qianqian Zhao¹, Xuehua Lu³, Xiaoyu Su¹^,^*(), Yinghao Wang¹^,^*()

1 College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
2 Medical School, Chongqing Three Gorges Medical College, Chongqing 404120, China
3 Fujian Academy of Medical Sciences, Fuzhou 350001, China

Received:2021-11-14 Revised:2021-12-08 Accepted:2022-01-12 Online:2022-03-31 Published:2022-03-31
Contact: Xiaoyu Su, Yinghao Wang
About author:
# These authors contributed equally to this work.

摘要/Abstract

摘要：

类风湿性关节炎(RA)是世界上最难以治愈的疾病之一。中药羌活(NE)常用于治疗上肢疼痛。NE可显著降低上肢关节炎大鼠炎性疼痛靶点P2X3受体的表达。为了验证NE治疗"上肢痹痛"的机制与P2X3受体介导的PKC炎症反应途径有关, 本研究采用超高效液相色谱法测定NE乙酸乙酯部位的药效物质, 将SD大鼠随机分为空白组、模型组、羌活组和阳性药组, 21天后取关节组织。体外培养的RA-FLS细胞, 分为模型组、羌活组和阳性药组。Western blot检测P2X3和PKC炎症途径指标的表达。P2X3抑制剂(A-317491)作用于RA-FLS细胞, 并设模型组和阳性药组, 检测PKC蛋白的表达。实验发现, NE降低大鼠和RA-FLS细胞中P2X3、Rab7、PKC、NF-κB的蛋白表达。NE和P2X3受体拮抗剂对RA-FLS细胞PKC通路的关键蛋白表达的降低程度相似, 且作用没有叠加性。结果表明, NE能够有效改善RA大鼠"上肢痹痛", 其机制与P2X3/Rab7/PKC/NF-κB通路密切相关。

关键词: 羌活, P2X3受体, PKC, 炎症反应通路

Abstract:

Rheumatoid arthritis (RA) is one of the most common refractory diseases in the world, and traditional Chinese medicine Notopterygium (NE) has been used in the treatment of upper limb pain for a long time. NE can significantly reduce the expression of inflammatory pain target P2X3 receptor in rats with upper-limb arthritis. To verify the relationship between the mechanism of NE for "upper limb paralysis" and the P2X3 receptor-mediated PKC inflammatory response pathway, UPLC was taken to measure the exact medicinal substance of ethyl acetate from NE. Sprague Dawley rats were randomly divided into a blank group, a model group, a live-action group, and a positive group. The joint cavity was removed after 21 d. Moreover, a model group, a live group, and a positive group were also set up with RA-FLS cells in our in vitro study. The expressions of P2X3 and PKC inflammation pathway indicators were detected by Western blotting analysis. A P2X3 inhibitor (A-317491) acted on RA-FLS cells, and a model group and a positive group were set. Then the protein expression of PKC was detected. NE reduced the expressions of P2X3, Rab7, PKC, and NF-κB at the protein level in both systems. NE and P2X3 receptor antagonists reduced the expressions of key proteins in the PKC pathway in RA-FLS cells to similar extents, and their effects were not additive. NE could effectively improve the "forelimb pain" of RA rats, with a mechanism closely related to the P2X3/Rab7/PKC/NF-κB pathway.

Key words: Notopterygium, P2X3 receptor, Protein kinase c, Inflammatory response pathway

Supporting:

王颖峥, 杨策, 朱翠萍, 赵茜茜, 卢雪花, 苏晓宇, 王英豪. 羌活通过调节P2X3抑制PKC诱导的炎症反应治疗"上肢痹痛"[J]. 中国药学(英文版), 2022, 31(3): 163-175.

Yingzheng Wang, Ce Yang, Cuiping Zhu, Qianqian Zhao, Xuehua Lu, Xiaoyu Su, Yinghao Wang. Upper limb arthromyodynia can be treated with Notopterygium by down-regulating P2X3 to mediate PKC-induced inflammatory response[J]. Journal of Chinese Pharmaceutical Sciences, 2022, 31(3): 163-175.

图/表 9

Figure 1. UPLC chromatogram of NE active site. Peaks 1–7 were the main chromatographic peaks of the active part. Peak 7 was determined to be isoimperatorin at 8.89 min, which was selected as the internal reference peak. Peaks 1 and 6 were ferulic acid and NE, respectively.

Figure 2. UPLC fingerprint of NE active site. S1–10 were the chromatographic peaks of 10 different batches of test products. R was for reference chromatography.

Figure 3. Toe volume maps for different groups of treatments. Representative photographs of the (control) control group, (AA) model group, (AA + PNE) positive group, and (AA + NE) NE group. AA, adjuvant arthritis; PNE, prednisone; NE, herbal NE extract.

Figure 4. Effects of NE and PNE on pain threshold, IL-6, TNF-α, and PGE2 in AA rats. NE and PNE prevented the decreased pain threshold (a) and reduced the increased inflammatory factor levels of IL-6 (b), TNF-α (c), and PGE2 (d) in AA rats. Inflammatory factor levels were measured by ELISA. ##P < 0.01 and ###P < 0.001 compared with the normal group, *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the AA group.

Figure 5. Effects of NE and PNE on CXCL1 in AA rats. NE and PNE reduced the increased protein level of CXCL1 in AA rats. Representative Western blots probed with the antibody against CXCL1 and re-probed with an anti-GAPDH antibody. The values of CXCL1 protein, normalized to GAPDH levels, were shown beneath their respective Western blots. ###P < 0.001 compared with the normal group, **P < 0.01 compared with the AA group.

Figure 6. Effects of NE and PNE on P2X3, Rab7, PKC, and NF-κB in AA rats. NE and PNE reduced the increased protein levels of P2X3 (a), Rab7 (b), PKC (c), and NF-κB (d) proteins in AA rats. Representative Western blots probed with the antibody against P2X3, Rab7, PKC, NF-κB. P2X3, Rab7, and PKC were re-probed with anti-GAPDH antibody, and NF-κB was re-probed with anti-H3 antibody. The values of protein, normalized to GAPDH levels, were shown beneath their respective Western blots. #P < 0.05 and ##P < 0.001 compared with the normal group, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the AA group.

Figure 7. Effects of NE and PNE on CXCL1 in RA-FLS cells. NE and PNE reduced the increased protein level of CXCL1 in RA-FLS cells. Representative Western blots probed with the antibody against CXCL1 and re-probed with an anti-β-actin antibody. The values of CXCL1 protein, normalized to β-actin levels, were shown beneath their respective Western blots. **P < 0.01 compared with the control group.

Figure 8. Effects of NE and PNE on P2X3, Rab7, PKC, and NF-κB in RA-FLS Cells. NE and PNE reduced the increased protein levels of P2X3 (a), Rab7 (b), and PKC (c), NF-κB (d) in RA-FLS cells. Representative Western blots probed with the antibody against P2X3, Rab7, PKC, NF-κB. P2X3, Rab7, and PKC were re-probed with anti-β-actin antibody, and NF-κB was re-probed with anti-H3 antibody. The values of protein, normalized to β-actin levels, were shown beneath their respective Western blots. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group.

Figure 9. Effects of NE and A-317491 on PKC in RA-FLS cells. NE and NE + A-317491 reduced the increased level of PKC protein in RA-FLS cells. Representative Western blots probed with the antibody against PKC and re-probed with an anti-β-actin antibody. The values of protein, normalized to β-actin levels, were shown beneath their respective Western blots. *P < 0.05 and **P < 0.01 compared with the control group.

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羌活通过调节P2X3抑制PKC诱导的炎症反应治疗"上肢痹痛"

Upper limb arthromyodynia can be treated with Notopterygium by down-regulating P2X3 to mediate PKC-induced inflammatory response

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