BACKGROUND/OBJECTIVES: The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS: Methanolic extract from black rice was evaluated for the protective effect on TBHP-induc...
BACKGROUND/OBJECTIVES: The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS: Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined. RESULTS: TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors. CONCLUSIONS: These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress.
BACKGROUND/OBJECTIVES: The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS: Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined. RESULTS: TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors. CONCLUSIONS: These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress.
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문제 정의
This study demonstrates that BRE (black rice extract) has the ability to protect HepG2 cells from oxidative stress-induced cell death by modulating ERK1/2 and Akt activation. We examined the effect of BRE on the response of cultured human HepG2 cells to oxidative stress induced by TBHP.
가설 설정
Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. Based on the results from ERKs and Akt inhibitor experiments, we hypothesized that that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE. While, as discussed above in regard to the complexity of composition of black rice extracts, further studies are needed to clearly elucidate which particular compounds are responsible for the protective effect.
제안 방법
Cells were treated for 0, 3, 6 and 12 h with different concentration of TBHP (50, 100, 200, 500, 1000 μM), and cell viability was determined by MTT assay.
Finally, to investigate the signaling pathway involved in the protective effect of BRE, we explored the Akt and ERK pathways using the appropriate inhibitors, LY294002 and PD98059, respectively. The results showed that ERKs and Akt inhibitors blunted the protective effect of BRE on TBHP-induced cell death and ROS generation.
However, limited studies are available concerning the cytoprotective effects and its possible mechanism of black rice extract against oxidative stress. In this study, we evaluated the protective effect of black rice extract on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells and also defined several biomarkers that modulate cell survival and death.
The first goal of our study was to determine the oxidative conditions leading to cell death in HepG2. Cells were treated for 0, 3, 6 and 12 h with different concentration of TBHP (50, 100, 200, 500, 1000 μM), and cell viability was determined by MTT assay.
To determine the protective effect of BRE against oxidative stress, cells were pretreated with BRE for 12 h and then exposed to 500 μM TBHP for 6 h and then cell death and apoptosis were evaluated.
대상 데이터
, Winooski, VT, USA). All experiments were performed in triplicate.
Blakc rice (Oryza sativa cv. Heugjinjubyeo) was purchased from local market (Cheongju, Korea). Extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor (PD98059), Akt inhibitor (LY294002), Caspase-3 assay kit, 2´,7´-dichlorofluorescin diacetate (DCFH-DA), tert-butyl hydroperoxide (TBHP), and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-terazolium bromide (MTT) were purchased from Sigma-Aldrich (St.
Extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor (PD98059), Akt inhibitor (LY294002), Caspase-3 assay kit, 2´,7´-dichlorofluorescin diacetate (DCFH-DA), tert-butyl hydroperoxide (TBHP), and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-terazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
데이터처리
Significant differences among treatment means were determined using a one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test using SAS version 8.1 (SAS Institute, Cary, NC, USA).
성능/효과
In conclusion, our results show that pretreatment of HepG2 cells with BRE strongly protects the cells against TBHP-induced oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERK1/2 deactivation and the prolonged JNK activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins.
Finally, to investigate the signaling pathway involved in the protective effect of BRE, we explored the Akt and ERK pathways using the appropriate inhibitors, LY294002 and PD98059, respectively. The results showed that ERKs and Akt inhibitors blunted the protective effect of BRE on TBHP-induced cell death and ROS generation. Taken together, our results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress.
후속연구
Based on the results from ERKs and Akt inhibitor experiments, we hypothesized that that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE. While, as discussed above in regard to the complexity of composition of black rice extracts, further studies are needed to clearly elucidate which particular compounds are responsible for the protective effect. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress.
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