[논문]산화적 스트레스에 의한 간세포의 DNA 손상 및 apoptosis 유도에 대한 노근 추출물의 보호 효과

이희영; 홍상훈; 박상은

doi:10.22246/jikm.2021.42.6.1269

[국내논문] 산화적 스트레스에 의한 간세포의 DNA 손상 및 apoptosis 유도에 대한 노근 추출물의 보호 효과
Protective Effect of Phragmitis Rhizoma against Oxidative Stress-induced DNA Damage and Apoptosis in Chang Liver Cells 원문보기

대한한방내과학회지 = The journal of internal Korean medicine, v.42 no.6, 2021년, pp.1269 - 1284

이희영 (미소로 한의원) , 홍상훈 (동의대학교 한방내과학교실) , 박상은 (동의대학교 한방내과학교실)

Abstract ▼ AI-Helper

Objectives: Phragmitis Rhizoma is the fresh or dried rhizome of Phragmites communis Trin., which has been prescribed in traditional Korean medicine to relieve fever and vomiting and to nourish the body fluids. Recently, the protective effect of Phragmitis Rhizoma extract or its components on myelotoxicity and inflammatory responses have been reported, but no study has yet been conducted on oxidative stress. Methods: The present study investigated whether an ethanol extract of Phragmitis Rhizoma (PR) could protect against cellular damage induced by oxidative stress in Chang liver cells. Results: Pretreatment with PR significantly suppressed the hydrogen peroxide (H2O2)-induced reduction of Chang cell viability and generation of reactive oxygen species (ROS), thereby deferring apoptosis. PR also markedly inhibited H2O2-induced comet tail formation and phospho-γH2AX expression, suggesting that PR protected against oxidative stress-mediated DNA damage. PR also effectively prevented the inhibition of ATP synthesis in H2O2-treated Chang cells by inhibiting the loss of mitochondrial membrane potential, indicating that PR maintains energy metabolism through preservation of mitochondrial function while eliminating ROS generated by H2O2. Immunoblotting results indicated that PR attenuated the H2O2-induced downregulation of Bcl-2 and upregulation of Bax expression. Conclusions: PR protects against oxidative injury in Chang liver cells by regulating energy homeostasis via ROS generation blockade, which is at least partly mediated through inactivation of the mitochondria-mediated apoptosis pathway.

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표/그림 (7)

그림 Fig. 1. Effects of ethanol extract of Phragmitis Rhizoma (PR) and H₂O₂ on the cell viability in Chang liver cells. Cells were treated with various concentrations of PR (A) and H₂O₂ (B) for 24 hours. Cell viability was assessed by an MTT reduction assay. The results are the mean±SD obtained from three independent experiments (*p<0.05 compared with the control group).
그림 Fig. 2. Inhibition of H₂O₂-induced cytotoxicity by PR in Chang liver cells. Cells were treated with the indicated concentrations of PR for 24 hours, or pretreated with or without PR for 1 hour, and then stimulated with 1 mM H₂O₂ for 24 hours. (A) Cell viability was assessed by an MTT reduction assay. The results are the mean±SD obtained from three independent experiments (*p<0.05 compared with the control group, ^#p<0.05 compared with the H₂O₂-treated group). (B) To investigate morphological changes of Chang cells, cells cultured under the same
그림 Fig. 3. Suppression of H₂O₂-induced apoptosis by PR in Chang liver cells. Cells were treated with the indicated concentrations of PR for 1 hour, and then stimulated with or without 1 mM H₂O₂ for 24 hours. (A) The cells were fixed and stained with DAPI solution. The stained nuclei were observed by a fluorescence microscope (original magnification, ×400). Representative photographs are shown. (B) The cells were collected and stained with PI solution, and the percentages of apoptotic cells were then analyzed by a flow cytometer. The results are the mean±SD obtained from three independent experiments (*p<0.05 compared with the control group, ^#p<0.05 compared with the H₂O₂-treated group).
그림 Fig. 4. Attenuation of H₂O₂-induced ROS generation by PR in Chang liver cells. Cells were pretreated with the indicated concentration of PR for 1 hour, and then stimulated with or without 1 mM H₂O₂ for 1 hour. (A and B) The cells were incubated with DCF-DA, and DCF fluorescence was measured by a flow cytometer. The values represent the mean±SD obtained from three independent experiments (*p<0.05 compared with the control group, ^#p<0.05 compared with the H₂O₂-treated group). (C) After staining with DCF-DA, images were obtained by a fluorescence microscope (original magnification,×200). These images are representative of at least three independent experiments.
그림 Fig. 5. Protection of H₂O₂-induced DNA damage by PR in Chang liver cells. Cells were pretreated with the indicated concentrations of PR for 1 hour, and then stimulated with or without 1 mM H₂O₂ for 24 hours. (A) Equal amounts of cell lysates were separated on SDS-polyacrylamidegels, and transferred to PVDF membranes. The membranes were probed with specific antibodies against γH2AX and p-γH2AX, and the proteins were visualized using an ECL detection system. Actin was used as an internal control. (B) Bands were quantified using ImageJ and normalized to actin, and the ratio was determined. Data are expressed as mean±SD. All experiments were repeated three times (*p<0.05 compared with the control group, ^#p<0.05 compared with the H₂O₂-treated group). (C) To detect cellular DNA damage, the comet assay was performed, and representative photographs of the comets were taken by a fluorescence microscope (original magnification, ×200). These images are representative of at least three independent experiments.
그림 Fig. 6. Prevention of H₂O₂-induced mitochondrial dysfunction by PR in Chang liver cells. Cells were pretreated with the indicated concentrations of PR for 1 hour, and then stimulated with or without 1 mM H₂O₂ for 24 hours. (A) The cells were collected and incubated with JC-1 solution for 20 min at 37 ℃ in the dark. The values of MMP were evaluated by a flow cytometer. (B) To monitor the ATP production using a luminometer, a commercially available kit was used. The values represent the mean± SD obtained from three independent experiments (*p<0.05 compared with the control group, ^#p<0.05 compared with the H₂O₂-treated group).
그림 Fig. 7. Effect of PR on the expression of Bcl-2 and Bax in H₂O₂-treated Chang liver cells. (A) After treatment with the indicated concentrations of PR in the presence or absence of H₂O₂ for 24 hours, the cellular proteins were prepared, and Bcl-2 and Bax protein levels were assayed by Western blot analysis using an ECL detection system. Actin was used as an internal control. (B), (C) Bands were quantified using ImageJ and normalized to actin, and the ratio of Bcl-2 and Bax to actin was determined. Each point represents the mean±SD of three independent experiments (*p<0.05 compared with the control group, ^#p<0.05 compared with the tacrolimustreated group).

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