Regulative effect of tripterine on oxidative stress and senescence in hepatocellular carcinoma cells

doi:10.11958/20260496

Abstract

Abstract:

Objective To investigate the regulatory effect of tripterine on hepatocellular carcinoma (HCC) cells under oxidative stress and senescence conditions. Methods The Huh-7 human HCC cell line was routinely cultured in vitro. The cells were treated with different concentrations (0, 0.25, 0.5, 1, 2 and 4 μmol/L) of tripterine. The highest drug concentration that did not affect cell viability was determined by CCK-8 cell viability assay. The experiment was divided into four groups: the control group, the H₂O₂ group, the H₂O₂ + DMSO group and the H₂O₂ + tripterine group. The degree of cell senescence was assessed by β-galactosidase staining. The intracellular reactive oxygen species (ROS) level was detected by the DCFH-DA fluorescence probe method. The intracellular ATP level was detected by the luciferase catalysis method. The superoxide level in mitochondria was detected by the MitoSO Red fluorescence probe method. The mitochondrial membrane potential level was assessed by the JC-1 fluorescence probe method. Results The highest drug concentration of tripterine that did not affect the viability of the HCC cell line Huh-7 was 0.5 μmol/L, and this optimal drug concentration was used for the subsequent experiments. Compared with the control group, the cell senescence was enhanced in the H₂O₂ group, intracellular reactive oxygen species (ROS) level was increased, ATP level decreased, mitochondrial superoxide level increased and mitochondrial membrane potential level decreased (all P<0.05). Compared with the H₂O₂ + DMSO group, the H₂O₂ + tripterine group showed alleviated cell senescence, decreased intracellular ROS level, increased ATP level, decreased mitochondrial superoxide level and elevated mitochondrial membrane potential level (all P<0.05). Conclusion Tripterine can alleviate oxidative stress and subsequent senescence and mitigate mitochondrial damage in HCC cells.

Key words: celastrol, carcinoma, hepatocellular, cellular senescence, oxidative stress, reactive oxygen species, mitochondrial dysfunction

CLC Number:

R285.5

Figures/Tables 7

Fig.1 Confirmation of the optimal concentration of tripterine

Fig.2 Detection of cell senescence in each group (β-galactosidase staining，×200)

Tab.1 Comparison of senescence degree and intracellular ATP level between the four groups （n=3，$\stackrel{-}{x}\pm s$）

组别	衰老程度	ATP（μmol/L）
对照组	3.72±1.31	2.94±0.10
H₂O₂组	13.19±4.25^a	2.46±0.06^a
H₂O₂+DMSO组	16.15±2.45	2.76±0.00
H₂O₂+雷公藤红素组	8.25±2.40^b	3.39±0.01^b
F	11.430^＊＊	131.300^＊＊

Tab.2 Comparison of intracellular ROS level, superoxide level in mitochondria and MMP level between the four groups （n=4，$\stackrel{-}{x}\pm s$）

组别	ROS	线粒体超氧化物	MMP（R/G值）
对照组	1.00±0.03	96.25±16.84	1.02±0.09
H₂O₂组	1.33±0.09^a	127.50±8.50^a	0.83±0.07^a
H₂O₂+DMSO组	1.07±0.05	124.00±14.57	0.77±0.07
H₂O₂+雷公藤红素组	0.77±0.05^b	89.00±9.29^b	0.95±0.09^b
F	63.100^＊＊	9.207^＊＊	8.111^＊＊

Fig.3 Detection of intracellular ROS level in each group (fluorescence detection, ×200)

Fig.4 Detection of superoxide level in mitochondria in each group (fluorescence detection, ×200)

Fig.5 Detection of MMP level in each group (fluorescence detection, ×200)

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