Effect of Qiling Baitouweng Tang on Proliferation and Apoptosis in Diffuse Large B-cell Lymphoma Through JAK2/STAT3 Signaling Pathway

LIU Ning; ZHAN Xinzhuo; YU Hui; CHEN Xiaoli; KONG Xiangtu; NI Haiwen

doi:10.13422/j.cnki.syfjx.20222430

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Effect of Qiling Baitouweng Tang on Proliferation and Apoptosis in Diffuse Large B-cell Lymphoma Through JAK2/STAT3 Signaling Pathway

更新时间：2023-06-02

- Effect of Qiling Baitouweng Tang on Proliferation and Apoptosis in Diffuse Large B-cell Lymphoma Through JAK2/STAT3 Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 13, Pages: 10-19(2023)
- 作者机构：
  
  1.南京中医药大学第一临床医学院，南京 210023
  2.南京中医药大学附属医院江苏省中医院，南京 210029
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20222430
  CLC： R22;R242;R2-031;R285.5
- Published：05 July 2023，
  
  Published Online：25 October 2022，
  
  Received：11 July 2022，
- 稿件说明：
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刘宁,战昕卓,于慧等.基于JAK2/STAT3信号通路探讨芪苓白头翁汤对弥漫大B细胞淋巴瘤细胞增殖和凋亡的影响[J].中国实验方剂学杂志,2023,29(13):10-19.

LIU Ning,ZHAN Xinzhuo,YU Hui,et al.Effect of Qiling Baitouweng Tang on Proliferation and Apoptosis in Diffuse Large B-cell Lymphoma Through JAK2/STAT3 Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(13):10-19.
刘宁,战昕卓,于慧等.基于JAK2/STAT3信号通路探讨芪苓白头翁汤对弥漫大B细胞淋巴瘤细胞增殖和凋亡的影响[J].中国实验方剂学杂志,2023,29(13):10-19. DOI： 10.13422/j.cnki.syfjx.20222430.

LIU Ning,ZHAN Xinzhuo,YU Hui,et al.Effect of Qiling Baitouweng Tang on Proliferation and Apoptosis in Diffuse Large B-cell Lymphoma Through JAK2/STAT3 Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(13):10-19. DOI： 10.13422/j.cnki.syfjx.20222430.

摘要

目的

探讨芪苓白头翁汤对弥漫大B细胞淋巴瘤（DLBCL）细胞增殖、凋亡、非受体型酪氨酸蛋白激酶2/信号转导及转录激活因子3（JAK2/STAT3）信号通路及炎症因子白细胞介素-10（IL-10）的影响。

方法

以人DLBCL细胞OCI-LY10、U2932细胞为研究对象，细胞增殖与活性检测法（CCK-8）检测细胞增殖情况，并计算出0、4.6、9.3、18.7、37.5、75、150 mg·L

-1

芪苓白头翁汤处理OCI-LY10、U2932细胞24 h后的半抑制浓度（IC

）分别为9.33、16.13 mg·L

-1

。后续相关实验根据芪苓白头翁汤作用于OCI-LY10、U2932细胞24 h 半抑制浓度（IC

），选用芪苓白头翁汤9.5、19、38 mg·L

-1

开展实验。用胱天蛋白酶（Caspase）-3、Caspase-8、Caspase-9酶原活化检测试剂盒检测经0、9.5、19、38 mg·L

-1

芪苓白头翁汤处理OCI-LY10、U2932细胞24 h后OCI-LY10、U2932细胞中Caspase-3、Caspase-8、Caspase-9的酶原活化情况。酶联免疫吸附测定法（ELISA）检测经0、9.5、19、38 mg·L

-1

芪苓白头翁汤处理OCI-LY10、U2932细胞24 h后OCI-LY10、U2932细胞中IL-10炎症因子表达情况。流式细胞仪检测不同浓度芪苓白头翁汤作用OCI-LY10、U2932细胞24 h后细胞凋亡率及细胞周期。蛋白免疫印迹法（Western blot）检测0、9.5、19、38 mg·L

-1

芪苓白头翁汤作用OCI-LY10、U2932细胞24 h，OCI-LY10、U2932细胞中B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、活化的聚腺苷二磷酸核糖聚合酶（cleaved PARP）、活化的Caspase-3 （cleaved Caspase-3）凋亡蛋白表达情况及JAK2、STAT3、磷酸化（p）-JAK2、p-STAT3通路蛋白表达情况，不同浓度药物作用OCI-LY10、U2932细胞24 h，OCI-LY10、U2932细胞中癌基因（c-Myc）蛋白表达情况。

结果

芪苓白头翁汤作用于OCI-LY10、U2932细胞24 h，与空白组比较，芪苓白头翁汤各组细胞增殖均受到明显抑制（

0.05，

0.01）；9.5、19、38 mg·L

-1

芪苓白头翁汤组Caspase-3、Caspase-8、Caspase-9酶原显著活化（

0.01），细胞凋亡增加（

0.05，

0.01），细胞于DNA合成前期（G

）阻滞增加（

0.05，

0.01）；9.5、19、38 mg·L

-1

芪苓白头翁汤组OCI-LY10、U2932细胞中Bcl-2、p-JAK2、p-STAT3蛋白表达显著降低（

0.01），Bax、cleaved PARP、cleaved Caspase-3蛋白表达显著增加（

0.01），JAK2、STAT3蛋白表达未见明显改变。与空白组比较，19 mg·L

-1

芪苓白头翁汤组、19 mg·L

-1

芪苓白头翁汤+10 μg·L

-1

IL-10组c-Myc、p-JAK2、p-STAT3蛋白表达水平下降（

0.05，

0.01），10 μg·L

-1

IL-10组c-Myc、p-JAK2、p-STAT3蛋白表达明显增加（

0.05，

0.01），通路蛋白JAK2、STAT3蛋白表达未见明显改变。

结论

芪苓白头翁汤可以抑制人DLBCL细胞OCI-LY10、U2932细胞的增殖并促进其凋亡，其作用机制可能与芪苓白头翁汤调控JAK2/STAT3信号通路有关。

Abstract

Objective

To investigate the effect of Qiling Baitouweng Tang （QLBTWT） on proliferation and apoptosis， Janus kinase 2 （JAK2）/signal transducer and activator of transcription 3 （STAT3） signaling pathway and interleukin-10 （IL-10） in diffuse large B-cell lymphoma （DLBCL）.

Method

With human DLBCL cells OCI-LY10 and U2932 as research objects， cell proliferation was detected by cell counting kit-8 （CCK-8） assay. After treatment with 0， 4.6， 9.3， 18.7， 37.5， 75， 150 mg·L

-1

QLBTWT for 24 h， the half-inhibitory concentration （IC

） of OCL-LY10 and U2932 cells was calculated to be 9.33， 16.13 mg·L

-1

， respectively， based on which， 9.5， 19， 38 mg·L

-1

QLBTWT were selected for subsequent experiments. After 0， 9.5， 19， 38 mg·L

-1

QLBTWT treatment for 24 h， the zymogen activities of Caspase-3， Caspase-8 and Caspase-9 in OCI-LY10 and U2932 cells were detected using corresponding activity assay kits （colorimetric）， and the IL-10 expression was detected by enzyme-linked immuno sorbent assay （ELISA）. The apoptosis rate and cell cycle of OCI-LY10 and U2932 cells treated with different concentrations of QLBTWT for 24 h were detected by flow cytometry. The expressions of apoptosis-related proteins ［B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved poly adenosine diphosphate ribose polymerase （cleaved PARP）， cleaved Caspase-3］， JAK2， STAT3， phospho-JAK2 （p-JAK2）， phospho-STAT3 （p-STAT3） pathway proteins， and c-Myc protein in OCL-LY10 and U2932 cells after 24 h treatment with 0， 9.5， 19， 38 mg·L

-1

QLBTWT were all tested by Western blot.

Result

After QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h， cell proliferation was inhibited in each QLBTWT group compared with that in the control group （

0.05，

0.01）. The zymogens of Caspase-3， Caspase-8 and Caspase-9 were activated （

0.01）， and there was an increase in cell apoptosis （

0.05，

0.01） and cell cycle arrest at Gap phase1 （G

） phase in 9.5， 19 and 38 mg·L

-1

QLBTWT group （

0.05，

0.01）. After 9.5， 19 and 38 mg·L

-1

QLBTWT treatment on OCI-LY10 and U2932 cells for 24 h， the expressions of Bcl-2， p-JAK2 and p-STAT3 proteins were decreased （

0.01）， and the expressions of Bax， cleaved PARP and cleaved Caspase-3 proteins were increased （

0.01）， but no significant change was observed in the expressions of JAK2 and STAT3 proteins. Compared with the conditions in the control group， the expressions of c-Myc， p-JAK2， and p-STAT3 proteins were down-regulated in 19 mg·L

-1

QLBTWT group and 19 mg·L

-1

QLBTWT+10 μg·L

-1

IL-10 group （

0.05，

0.01）， and up-regulated in 10 μg·L

-1

IL-10 group （

0.05，

0.01）， while there was no difference in JAK2/STAT3 proteins.

Conclusion

QLBTWT can inhibit proliferation and induce apoptosis of human DLBCL cells OCI-LY10 and U2932， and the potential mechanism may be related to the regulation of JAK2/STAT3 signaling pathway.

关键词

芪苓白头翁汤弥漫大B细胞淋巴瘤OCI-LY10细胞U2932细胞增殖细胞周期凋亡非受体型酪氨酸蛋白激酶2/信号转导及转录激活因子3（JAK2/STAT3）白细胞介素-10（IL-10）

Keywords

Qiling Baitouweng Tangdiffuse large B-cell lymphoma （DLBCL）OCI-LY10 cellU2932 cellproliferationcell cycleapoptosisjanus kinase 2/signal transducer and activator of transcription 3 （JAK2/STAT3）interleukin-10 （IL-10）

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