柴芪益肝方调控巨噬细胞极化对肝癌HepG2细胞恶性生物学行为的抑制作用

doi:10.13481/j.1671-587X.20260212

Abstract

Abstract:

Objective To discuss the inhibitory effect of Chaiqi Yigan （CQ） formula on the malignant biological behaviors of hepatocellular carcinoma HepG2 cells by regulating macrophage polarization through activation of nuclear factor κB （NF-κB） signaling pathway， and to clarify the theoretical basis for the application of CQ in the treatment of hepatocellular carcinoma. Methods CQ-containing serum was prepared and diluted to concentrations of 2.5%， 5.0%， 10.0%， 20.0%， and 30.0%.Cell counting kit-8 （CCK-8） method was used to detect the activities of RAW264.7 cells and HepG2 cells in various groups after treated with CQ-containing serum； enzyme-linked immunosorbent assay （ELISA） kits were used to detect the levels of interleukin （IL）-6 and IL-10 in supernatant of RAW264.7 cells in various groups after treated with CQ-containing serum to screen the optimal concentration of CQ for treatment. A co-culture system of RAW264.7 and HepG2 cells was established， and a nude mouse model bearing HepG2 cell xenograft tumor was constructed. The co-cultured cells and tumor-bearing mice were randomly divided into control group， CQ group， NF-κB inhibitor pyrrolidine dithiocarbamate （PDTC） group， and CQ+PDTC group. CCK-8 method and Transwell chamber assay were used to detect the activities， numbers of migration cells， and numbers of invasion cells of HepG2 cells in various groups， respectively. The volumes and mass of xenograft tumors of the nude mice in various groups were measured. Flow cytometry was used to detect M1 and M2 polarization of RAW264.7 cells in co-culture system and tumor tissue of the nude mice in various groups； ELISA method was used to detect the levels of M1-type cytokines IL-6， tumor necrosis factor α （TNF-α）， and inducible nitric oxide synthase （iNOS）， and M2-type cytokines transforming growth factor β （TGF-β）， arginase 1 （Arg-1）， and IL-10 in supernatant of the RAW264.7 cells in co-culture system and serum of the nude mice in various groups； Western blotting method was used to detect the expression levels of NF-κB pathway-related proteins NF-κB inhibitor protein α （IκB-α）， phosphorylated IκB-α （p-IκB-α）， NF-κB p65， and p-NF-κB p65 in RAW264.7 cells in the co-culture system and serum of the nude mice in various groups. Results Compared with control group， the activity， number of migration cells， and number of invasion cells of HepG2 cells in CQ group were significantly decreased （P<0.05）， while those in PDTC group were significantly increased （P<0.05）； compared with CQ group， the activity， number of migration cells， and number of invasion cells of HepG2 cells in CQ+PDTC group were significantly increased （P<0.05）. Compared with control group， the volume and mass of xenograft tumor of the nude mice in CQ group were significantly decreased （P<0.05）， while those in PDTC group were significantly increased （P<0.05）； compared with CQ group， the volume and mass of xenograft tumor of the nude mice in CQ+PDTC group were significantly increased （P<0.05）. Compared with control group， the percentages of M1-type macrophages in RAW264.7 cells and tumor tissue of the nude mice in CQ group were significantly increased （P<0.05）， while the percentages of M2-type macrophages were significantly decreased （P<0.05）； the percentages of M1-type macrophages in RAW264.7 cells and tumor tissue of the nude mice in PDTC group were significantly decreased （P<0.05）， while the percentages of M2-type macrophages were significantly increased （P<0.05）. Compared with CQ group， the percentages of M1-type macrophages in RAW264.7 cells and tumor tissue of the nude mice in CQ+PDTC group were significantly decreased （P<0.05）， while the percentages of M2-type macrophages were significantly increased （P<0.05）. Compared with control group， the levels of IL-6， TNF-α， and iNOS in supernatant of the RAW264.7 cells and serum of the nude mice in CQ group were significantly increased （P<0.05）， while the levels of TGF-β， Arg-1， and IL-10 were significantly decreased （P<0.05）； the levels of IL-6， TNF-α， and iNOS in supernatant of the RAW264.7 cells and serum of the nude mice in PDTC group were significantly decreased （P<0.05）， while the levels of TGF-β， Arg-1， and IL-10 were significantly increased （P<0.05）. Compared with CQ group， the levels of IL-6， TNF-α， and iNOS in supernatant of the RAW264.7 cells and serum of the nude mice in CQ+PDTC group were significantly decreased （P<0.05）， while the levels of TGF-β， Arg-1， and IL-10 were significantly increased （P<0.05）. Compared with control group， the ratios of p-IκB-α/IκB-α and p-NF-κB p65/NF-κB p65 in RAW264.7 cells and tumor tissue of the nude mice in CQ group were significantly increased （P<0.05）； the ratios of p-IκB-α/IκB-α and p-NF-κB p65/NF-κB p65 in RAW264.7 cells and tumor tissue of the nude mice in PDTC group were significantly decreased （P<0.05）. Compared with CQ group， the ratios of p-IκB-α/IκB-α and p-NF-κB p65/NF-κB p65 in RAW264.7 cells and tumor tissue of the nude mice in CQ+PDTC group were significantly decreased （P<0.05）. Conclusion CQ can promote macrophage polarization from M2 to M1 type by activating NF-κB signaling pathway， thereby inhibiting the proliferation， migration， and invasion of hepatocellular carcinoma HepG2 cells and the growth of subcutaneous xenograft tumor in the nude mice.

Key words: Chaiqi Yigan formula, Nuclear factor κB, Macrophage polarization, Hepatocellular carcinoma, Tumor progression

CLC Number:

R273

Xiqian ZHANG,Zhibo DANG,Yunan DU,Pei WU,Hang XIE,Gaofeng TAN. Inhibitory effect of Chaiqi Yigan formula on malignant biological behaviors of liver cancer HepG2 cells by regulating macrophage polarization[J].Journal of Jilin University(Medicine Edition), 2026, 52(2): 398-409.

Figures/Tables 12

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References 25

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Group	IL-6	IL-10
Blank	79.04±13.35	23.01±3.92
Co-culture	96.15±12.10	82.75±5.13^*
2.5% CQ-containing serum	220.28±13.64^*△	63.52±2.87^△
5.0% CQ-containing serum	345.76±14.15^*△	45.02±3.24^△
10.0% CQ-containing serum	402.85±15.86^*△	27.93±2.90^△
20.0% CQ-containing serum	420.23±17.12^*△	25.80±3.45^△
30.0% CQ-containing serum	435.71±16.53^*△	24.14±4.16^△

Group	Cell activity (η/%)	Number of migration cells	Number of invasion cells
Control	100.00±0.00	232.50±19.23	329.00±21.36
CQ	48.90±9.62^*	119.00±14.15^*	132.50±16.22^*
PDTC	205.45±13.25^*	343.50±22.46^*	471.00±27.40^*
CQ+PDTC	92.70±10.84^△	215.00±20.14^△	314.50±23.05^△

Group	M1 type macrophage		M2 type macrophage
Group	RAW264.7	Tumor tissue	RAW264.7	Tumor tissue
Control	18.05±3.30	20.84±2.62	52.43±4.73	45.31±4.12
CQ	52.32±4.25^*	63.75±4.80^*	20.36±3.15^*	16.70±2.90^*
PDTC	1.84±0.60^*	2.56±0.79^*	81.90±5.64^*	71.89±4.53^*
CQ+PDTC	20.13±2.72^△	22.13±2.45^△	49.86±3.92^△	43.25±3.64^△

Group	IL-6	TNF-α	iNOS	TGF-β	Arg-1	IL-10
Control	93.54±9.37	42.71±5.02	11.74±1.15	31.15±3.76	126.21±9.16	82.75±6.13
CQ	234.76±15.72^*	184.69±9.20^*	29.83±2.04^*	14.49±2.53^*	49.86±7.95^*	29.10±4.51^*
PDTC	40.13±7.51^*	13.45±3.62^*	3.67±0.92^*	47.62±4.14^*	198.74±8.78^*	131.82±8.36^*
CQ+PDTC	101.08±11.13^△	47.93±4.84^△	12.95±1.48^△	29.05±3.12^△	118.54±8.20^△	76.91±6.42^△

Group	IL-6	TNF-α	iNOS	TGF-β	Arg-1	IL-10
Control	60.92±6.73	29.62±4.14	5.98±0.37	15.56±2.63	71.64±7.13	59.12±5.20
CQ	156.45±13.62^*	130.51±6.97^*	19.32±0.64^*	7.47±1.82^*	23.60±6.04^*	18.63±3.57^*
PDTC	27.63±5.21^*	8.75±2.70^*	0.83±0.25^*	24.06±3.15^*	117.95±8.20^*	105.82±7.69^*
CQ+PDTC	67.10±7.24^△	33.56±3.92^△	5.13±0.46^△	13.91±2.81^△	66.17±7.92^△	54.96±4.84^△

Inhibitory effect of Chaiqi Yigan formula on malignant biological behaviors of liver cancer HepG2 cells by regulating macrophage polarization

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