Effect of Calycosin-mediated GP130/JAK/STAT Signaling Pathway on Oxidative Injury of Astrocytes in Spinal Cord

Dehua GUO; Xiaozhou LIU; Chenglin WU; Yang XU; Guofu ZHANG

doi:10.13422/j.cnki.syfjx.20221893

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Effect of Calycosin-mediated GP130/JAK/STAT Signaling Pathway on Oxidative Injury of Astrocytes in Spinal Cord

更新时间：2022-08-11

- Effect of Calycosin-mediated GP130/JAK/STAT Signaling Pathway on Oxidative Injury of Astrocytes in Spinal Cord
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 18, Pages: 54-61(2022)
- 作者机构：
  
  1.江西中医药大学，南昌 330004
  2.江西中医药大学附属医院，南昌 330006
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20221893
  CLC： R285.5;R966;R744
- Published：20 September 2022，
  
  Published Online：23 June 2022，
  
  Received：01 March 2022，
- 稿件说明：
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郭德华,刘小舟,吴成林等.毛蕊异黄酮介导GP130/JAK/STAT信号通路对脊髓星形胶质细胞氧化损伤的影响[J].中国实验方剂学杂志,2022,28(18):54-61.

GUO Dehua,LIU Xiaozhou,WU Chenglin,et al.Effect of Calycosin-mediated GP130/JAK/STAT Signaling Pathway on Oxidative Injury of Astrocytes in Spinal Cord[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(18):54-61.
郭德华,刘小舟,吴成林等.毛蕊异黄酮介导GP130/JAK/STAT信号通路对脊髓星形胶质细胞氧化损伤的影响[J].中国实验方剂学杂志,2022,28(18):54-61. DOI： 10.13422/j.cnki.syfjx.20221893.

GUO Dehua,LIU Xiaozhou,WU Chenglin,et al.Effect of Calycosin-mediated GP130/JAK/STAT Signaling Pathway on Oxidative Injury of Astrocytes in Spinal Cord[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(18):54-61. DOI： 10.13422/j.cnki.syfjx.20221893.

摘要

目的

探究毛蕊异黄酮介导糖蛋白130（GP130）/Janus酪氨酸蛋白激酶（JAK）/信号转导及转录激活因子（STAT）信号通路对脊髓星形胶质细胞氧化损伤的影响。

方法

原代分离大鼠脊髓星形胶质细胞，并利用免疫荧光检测胶质纤维酸性蛋白（GFAP）进行鉴定。分别加入5、10、20 μmol·L

-1

毛蕊异黄酮预处理脊髓星形胶质细胞12 h，然后加入100 μmol·L

-1

过氧化氢（H

）处理24 h造成氧化损伤模型，细胞增殖与活性检测（CCK-8）法检测各组细胞增殖情况选择合适的毛蕊异黄酮处理浓度。实验分为空白组、模型组（H

100 μmol·L

-1

）、毛蕊异黄酮组（毛蕊异黄酮 20 μmol·L

-1

）、毛蕊异黄酮+LY294002组（毛蕊异黄酮 20 μmol·L

-1

+LY294002 10 μmol·L

-1

）、毛蕊异黄酮+Stattic组（毛蕊异黄酮 20 μmol·L

-1

+Stattic 3 μmol·L

-1

）。CCK-8法、免疫荧光检测各组细胞增殖情况，流式细胞术检测各组细胞凋亡和周期情况；蛋白免疫印迹法（Western blot）检测各组细胞中磷酸化（p）-JAK2、p-STAT3、p-蛋白激酶B（Akt）、GP130、白细胞介素-6（IL-6）的蛋白表达水平。

结果

与空白组比较，模型组细胞的增殖活力明显降低，细胞凋亡率明显升高（

0.05）；与模型组比较，毛蕊异黄酮（20 μmol·L

-1

）组细胞的增殖活力明显增加，细胞凋亡率明显降低（

0.05）；与毛蕊异黄酮（20 μmol·L

-1

）组比较，PI3K抑制剂LY294002和STAT3抑制剂Stattic均能明显降低细胞的增殖活力，增加细胞凋亡率（

0.05）。与空白组比较，模型组细胞中p-JAK2、p-STAT3、p-Akt、GP130、IL-6的蛋白表达水平明显增加（

0.05）；与模型组比较，各用药组p-JAK2、p-STAT3、p-Akt、GP130、IL-6的蛋白表达水平均明显降低（

0.05）。

结论

毛蕊异黄酮能够促进氧化损伤的星形胶质细胞增殖并抑制其凋亡，并能通过抑制磷脂酰肌醇3-激酶（PI3K）/Akt通路磷酸化、JAK2/STAT3通路磷酸化起作用。

Abstract

Objective

To investigate the effect of calycosin-mediated glucoprotein130/Janus kinase/signal transducer and activator of transcription factor （GP130/JAK/STAT） signaling pathway on oxidative injury of astrocytes in spinal cord.

Method

Astrocytes in rat spinal cord were isolated and identified by immunofluorescence detection of glial fibrillary acidic protein （GFAP）. The cells were respectively pre-treated with 5， 10， 20 μmol·L

-1

calycosin for 12 h， and then 100 μmol·L

-1

（24 h） was added to induce oxidative injury. Cell counting kit-8 （CCK-8） assay was employed to detect cell proliferation and select the optimal concentration of calycosin. The following experimental groups were designed： control group， model group （100 μmol·L

-1

）， calycosin group （20 μmol·L

-1

calycosin）， calycosin + LY294002 group （20 μmol·L

-1

calycosin + 10 μmol·L

-1

LY294002）， and calycosin + Stattic group （20 μmol·L

-1

calycosin + 3 μmol·L

-1

Stattic）. CCK-8 assay and immunofluorescence method were used to detect the proliferation of cells and flow cytometry was applied to detect cell apoptosis and cycle. The protein expression of phosphorylated （p）-JAK2， p-STAT3， p-protein kinase B （Akt）， GP130， and interleukin-6 （IL-6） was detected by Western blotting.

Result

Compared with the control group， the model group showed low proliferation activity and high apoptosis rate of cells （

0.05）. Compared with the model group， calycosin （20 μmol·L

-1

） group displayed high proliferation activity and low apoptosis rate of cells （

0.05）. Compared with calycosin （20 μmol·L

-1

） group， both phosphatidylinosirtol-3-kinases （PI3K） inhibitor LY294002 and STAT3 inhibitor Stattic significantly reduced the proliferation activity and increased the apoptosis rate of cells （

0.05）. The protein expression of p-JAK2， p-STAT3， p-Akt， GP130， and IL-6 in the model group was higher than that in the control group （

0.05）， and the expression of the above indicators was lower in each treatment group than in the model group

（P

0.05）.

Conclusion

Calycosin can promote the proliferation and inhibit the apoptosis of astrocytes with oxidative injury by inhibiting the phosphorylation of PI3K/Akt pathway and JAK2/STAT3 pathway.

关键词

脊髓星形胶质细胞毛蕊异黄酮氧化损伤毛蕊异黄酮介导糖蛋白130（GP130）/Janus酪氨酸蛋白激酶（JAK）/信号转导及转录激活因子（STAT）信号通路脊髓损伤补阳还五汤

Keywords

astrocytes in spinal cordcalycosinoxidative injuryglucoprotein130/Janus kinase/signal transducer and activator of transcription factor （GP130/JAK/STAT） signaling pathwayspinal cord injuryBuyang Huanwutang

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