Effects of <italic style="font-style: italic">Jianpi Yishen Huatan Formula</italic> （健脾益肾化痰方）-Medicated Serum on Apoptosis，Migration and the LncRNA ALAL-1/USP4/HDAC2 Pathway in Human Lung Squamous Carcinoma Cells

FANG Yijun; WANG Xuemei; XIONG Changzhou; LI Liubang; QIN Huimin; WANG Zhiguang

doi:10.13288/j.11-2166/r.2025.14.012

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Effects of Jianpi Yishen Huatan Formula （健脾益肾化痰方）-Medicated Serum on Apoptosis，Migration and the LncRNA ALAL-1/USP4/HDAC2 Pathway in Human Lung Squamous Carcinoma Cells

更新时间：2025-07-11

- Effects of Jianpi Yishen Huatan Formula （健脾益肾化痰方）-Medicated Serum on Apoptosis，Migration and the LncRNA ALAL-1/USP4/HDAC2 Pathway in Human Lung Squamous Carcinoma Cells
- Journal of Traditional Chinese Medicine Vol. 66, Issue 14, Pages: 1481-1488(2025)
- 作者机构：
  
  1.广西中医药大学研究生院，广西壮族自治区南宁市青秀区五合大道13号，530200
  2.广西中医药大学第一附属医院
- 作者简介：
- 基金信息：
- DOI：10.13288/j.11-2166/r.2025.14.012
  CLC：
- Received：03 March 2025，
  
  Revised：2025-04-14，
  
  Published：17 July 2025
- 稿件说明：
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方以峻,王雪梅,熊常州等.健脾益肾化痰方含药血清对人肺鳞癌细胞凋亡、迁移及LncRNA ALAL-1/USP4/HDAC2通路的影响[J].中医杂志,2025,66(14):1481-1488.

FANG Yijun,WANG Xuemei,XIONG Changzhou,et al.Effects of Jianpi Yishen Huatan Formula （健脾益肾化痰方）-Medicated Serum on Apoptosis，Migration and the LncRNA ALAL-1/USP4/HDAC2 Pathway in Human Lung Squamous Carcinoma Cells[J].Journal of Traditional Chinese Medicine,2025,66(14):1481-1488.
方以峻,王雪梅,熊常州等.健脾益肾化痰方含药血清对人肺鳞癌细胞凋亡、迁移及LncRNA ALAL-1/USP4/HDAC2通路的影响[J].中医杂志,2025,66(14):1481-1488. DOI： 10.13288/j.11-2166/r.2025.14.012.

FANG Yijun,WANG Xuemei,XIONG Changzhou,et al.Effects of Jianpi Yishen Huatan Formula （健脾益肾化痰方）-Medicated Serum on Apoptosis，Migration and the LncRNA ALAL-1/USP4/HDAC2 Pathway in Human Lung Squamous Carcinoma Cells[J].Journal of Traditional Chinese Medicine,2025,66(14):1481-1488. DOI： 10.13288/j.11-2166/r.2025.14.012.

摘要

目的

从LncRNA ALAL-1/USP4/HDAC2信号通路探讨健脾益肾化痰方治疗肺鳞癌的可能作用机制。

方法

40只SD大鼠随机分为对照组和高、中、低浓度中药组，每组10只。高、中、低浓度中药组大鼠分别灌胃45、30、15 g/（kg·d）健脾益肾化痰方浓缩液，对照组灌胃10 ml/（kg·d）生理盐水，每日1次，连续10天后取血制备含药血清。人肺鳞癌细胞株SK-MES-1细胞分为对照血清组及健脾益肾化痰方低、中、高含药血清组，对照血清组加入10%含生理盐水血清培养，健脾益肾化痰方低、中、高含药血清组分别加入10%低、中、高浓度中药含药血清培养。孵育48 h后采用流式细胞术检测各组细胞凋亡情况，计算总凋亡率；细胞划痕实验观察0 h和培养24 h各组细胞迁移面积，计算迁移率。另将SK-MES-1细胞分为对照血清组，健脾益肾化痰方低、中、高含药血清组，LncRNA沉默组，USP4抑制组，HDAC2抑制组，对照血清组加入10%含生理盐水血清培养，健脾益肾化痰方低、中、高含药血清组分别加入10%低、中、高浓度中药含药血清培养，LncRNA沉默组转染ALAL-1 siRNA，USP4抑制组加入35 μmol/L去泛素化酶抑制剂（PR-619），HDAC2抑制组加入60 μmol/L伏立诺他，培养24、48 h后，CCK-8法检测各组细胞存活率；培育24 h后qPCR法检测各组细胞

LncRNA ALAL

1、USP4、HDAC2

mRNA表达；培育48 h后免疫印迹法检测各组细胞USP4、HDAC2蛋白水平。

结果

与对照血清组比较，健脾益肾化痰方中、高含药血清组细胞总凋亡率显著升高，健脾益肾化痰方低、中、高含药血清组细胞迁移率显著降低（

＜0.05或

＜0.01）。健脾益肾化痰方低、中、高含药血清组细胞迁移率随浓度的升

高而降低，呈现浓度依赖性（

＜0.0

或

＜0.01）。与对照血清组同时间比较，各组细胞24、48 h时存活率均显著下降（

＜0.05或

＜0.01）；与健脾益肾化痰方低含药血清组同时间比较，健脾益肾化痰方高含药血清组和LncRNA沉默组细胞在24、48 h时存活率亦降低（

＜0.05）。与对照血清组比较，健脾益肾化痰方低、中含药血清组和USP4抑制剂组

USP4、HDAC2

mRNA表达降低，健脾益肾化痰方高含药血清组、LncRNA沉默组

LncRNA ALAL

1、USP4、HDAC2

mRNA表达降低，HDAC2抑制剂组

HDAC2

mRNA表达亦降低；除HDAC2抑制剂组USP4蛋白水平外，其余各组细胞中USP4、HDAC2蛋白水平均降低（

＜0.05或

＜0.01）。

结论

健脾益肾化痰方含药血清能够抑制人肺鳞癌细胞增殖，促进凋亡，其作用机制可能与其抑制LncRNA ALAL-1/USP4/HDAC2通路相关，且以高浓度效果最好。

Abstract

Objective

To investigate the potential mechanism of the

Jianpi Yishen Huatan Formula

（健脾益肾化痰方，JPYSHF） in treating squamous cell lung cancer through the LncRNA ALAL-1/USP4/HDAC2 signaling pathway.

Methods

Forty Sprague-Dawley （SD） rats were randomly divided into a control group and high-， medium-， and low-dose JPYSHF group with 10 rats in each group. Rats in the JPYSHF groups were administered JPYSHF concentrated liquid at doses of 45， 30， and 15 g/（kg·d） via intragastric gavage， respectively， while the control group received 10 ml/（kg·d） of normal saline， once daily for 10 consecutive days before preparation of drug containing serum. Human lung squamous carcinoma SK-MES-1 cells were divided into a control group and low-， medium-， and high-dose JPYSHF-medicated serum groups. The control group was cultured with 10% saline-containing serum， while the JPYSHF groups were cultured with 10% low-， medium-， or high-dose medicated serum. After 48 hours of incubation， flow cytometry was used to detect apoptosis rates， and a cell scratch assay was performed to evaluate migration areas at 0 h and 24 h to calculate migration rate. Additional SK-MES-1 cells were divided into control serum， JPYSHF-medicated serum （low-， medium-， high-） do

se， LncRNA-silenced group （transfected with ALAL-1 siRNA）， USP4-inhibited group （treated with 35 μmol/L PR-619， a deubiquitinase inhibitor）， and HDAC2-inhibited group （treated with 60 μmol/L Vorinostat）. After 24 and 48 hours of culture， cell viability was assessed using the CCK-8 assay；

LncRNA ALAL

，

USP4

， and

HDAC2

mRNA levels were quantified by qPCR after 24 hours； USP4 and HDAC2 protein levels were measured by Western Blot after 48 hours.

Results

Compared with the control serum group， the total apoptosis rate of cells in middle- and high-JPYSHF-medicated serum group significantly increased， and the cell migration rate of cells in the low-， middle- and high-JPYSHF-medicated serum group significantly decreased （

＜0.05 or

＜0.01）. The cell migration rate of the low-， medium- and high-JPYSHF-medicated serum groups decreased with the increase of concentration in a concentration-dependent manner （

＜0.05 or

＜0.01）. Compared with the control serum group at the same time， the cell viability at 24 h and 48 h significantly decreased in all groups （

＜0.05 or

＜0.01）. Compared with the low-JPYSHF-medicated serum group at the same time， the cell viability at 24 h and 48 h also decreased in the high-JPYSHF-medicated serum group and the LncRNA silencing group （

＜0.05）. Compared with the control serum group， the expression of

USP4

and

HDAC2

mRNA reduced in the low- and medium-dose JPYSHF-medicated serum groups and the USP4 inhibitor group， and the expression of

LncRNA ALAL

，

USP4

and

HDAC2

mRNA reduced in the high-dose JPYSHF-medicated serum group and LncRNA-silencing group， and

HDAC2

mRNA expression reduced in the HDAC2 inhibitor group. USP4 and HDAC2 protein levels were reduced in cells of

all groups except for USP4 protein level in HDAC2 inhibitor group （

＜0.05 or

＜0.01）.

Conclusion

JPYSHF-medicated serum inhibits proliferation and promotes apoptosis of human lung squamous carcinoma cells， and its mechanism of action may be related to its inhibition of the LncRNA ALAL-1/USP4/HDAC2 pathway， with best effect at a high concentration.

关键词

Keywords

references

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School of Life Sciences，Zhejiang Chinese Medical University

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Effects of Jianpi Yishen Huatan Formula （健脾益肾化痰方）-Medicated Serum on Apoptosis，Migration and the LncRNA ALAL-1/USP4/HDAC2 Pathway in Human Lung Squamous Carcinoma Cells

DOI：10.13288/j.11-2166/r.2025.14.012

摘要

Abstract

关键词

Keywords

references