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Ethyl acetate extract of Wedelia chinensis inhibits tert-butyl hydroperoxide-induced damage in PC12 cells and D-galactose-induced neuronal cell loss in mice.

Lin WL, Wang SM, Ho YJ, Kuo HC, Lee YJ, Tseng TH - BMC Complement Altern Med (2014)

Bottom Line: EAW decreased t-BHP-induced reactive oxygen species (ROS) accumulation, cytotoxicity and apoptosis in PC12 cells.EAW and its major constituents blocked t-BHP-induced cytochrome C release and Bcl-2 family protein ratio change.These results demonstrate that W. chinensis has neuroprotective potential through blocking oxidative stress-induced damage and that luteolin and wedelolactone contribute to the protective action.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Applied Chemistry, Chung Shan Medical University, No, 110, Section 1, Chien-Kuo N, Road, Taichung 402, Taiwan. tht@csmu.edu.tw.

ABSTRACT

Background: Wedelia chinensis is traditionally used as a hepatoprotective herb in Taiwan. The aim of this study was to evaluate the neuroprotective potential of W. chinensis.

Methods: An ethyl acetate extract of W. chinensis (EAW) was prepared and analyzed by HPLC. The neuroprotective potential of EAW was assessed by tert-butylhydroperoxide (t-BHP)-induced damage in PC12 cells and D-galactose-induced damage in mouse cortex.

Results: EAW exhibited potent radical scavenging property and highly contained luteolin and wedelolactone. EAW decreased t-BHP-induced reactive oxygen species (ROS) accumulation, cytotoxicity and apoptosis in PC12 cells. EAW and its major constituents blocked t-BHP-induced cytochrome C release and Bcl-2 family protein ratio change. EAW and its major constituents increased the endogenous antioxidant capacity evaluated by the binding activity assay of nuclear factor E2-related factor 2 (Nrf2) to antioxidant response element (ARE) and nuclear translocation of Nrf2 respectively in PC12 cells. Finally, EAW inhibited D-galactose-induced lipid peroxidation, apoptosis and neuron loss in the cerebral cortex of mice.

Conclusion: These results demonstrate that W. chinensis has neuroprotective potential through blocking oxidative stress-induced damage and that luteolin and wedelolactone contribute to the protective action.

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Related in: MedlinePlus

Effects of EAW, luteolin and wedelolactone on t-BHP-induced cytochrome C release and changes in Bcl-2 family proteins. PC12 cells were pretreated with EAW (25 and 50 μg/mL) or luteolin (L10, 10 μM; L20, 20 μM) or wedelolactone (W10, 10 μM; W20, 20 μM) for 24 h, then 100 μM of t-BHP was added for 3 h. (A) Cells were harvested after incubation, and the cytosolic protein extracts were prepared as described in the text and subjected to Western immunoblotting analysis against anti- cytochrome C. The average densitometric value of cytochrome C is shown as the mean ± SD (n = 3). # P < 0.05, compared to the solvent control (0.1% DMSO). *P < 0.05, compared to t-BHP treatment alone. (B) After treatment, the total cell lysates were prepared and subjected to Western immunoblotting analysis against against anti-Bcl-2, -Bcl-xL, -Bax and anti-actin. Actin was used as the loading control. The ratio of Bcl-2/Bax and Bcl-xL/Bax are shown as the mean ± SD (n = 3). # P < 0.05, compared to the solvent control (0.1% DMSO). *P < 0.05, compared to t-BHP treatment alone.
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Fig5: Effects of EAW, luteolin and wedelolactone on t-BHP-induced cytochrome C release and changes in Bcl-2 family proteins. PC12 cells were pretreated with EAW (25 and 50 μg/mL) or luteolin (L10, 10 μM; L20, 20 μM) or wedelolactone (W10, 10 μM; W20, 20 μM) for 24 h, then 100 μM of t-BHP was added for 3 h. (A) Cells were harvested after incubation, and the cytosolic protein extracts were prepared as described in the text and subjected to Western immunoblotting analysis against anti- cytochrome C. The average densitometric value of cytochrome C is shown as the mean ± SD (n = 3). # P < 0.05, compared to the solvent control (0.1% DMSO). *P < 0.05, compared to t-BHP treatment alone. (B) After treatment, the total cell lysates were prepared and subjected to Western immunoblotting analysis against against anti-Bcl-2, -Bcl-xL, -Bax and anti-actin. Actin was used as the loading control. The ratio of Bcl-2/Bax and Bcl-xL/Bax are shown as the mean ± SD (n = 3). # P < 0.05, compared to the solvent control (0.1% DMSO). *P < 0.05, compared to t-BHP treatment alone.

Mentions: Oxidative stress initiates mitochondrial apoptotic signals [22, 23]. The loss of mitochondrial cytochrome C and its release into the cytoplasm are essential events in the initiation of mitochondrial caspase signaling. It was found that t-BHP treatment increased cytosolic cytochrome C, which was decreased by pretreatment with EAW or luteolin or wedelolactone (Figure 5A). The Bcl-2 family members have been found to play important roles in regulating mitochondrial-mediated apoptosis. High ratio of Bcl-2/Bax and Bcl-XL/Bax were observed to promote cell survival by preserving the integrity of the external mitochondrial membrane, which prevents the released of cytochrome C from the mitochondria, inducing cell death. It has been reported that overexpression of Bcl-2 in neurons was found to protect from neuronal loss in mouse model of cerebral ischemia [24]. In addition, Bax inhibition prevented neuronal death in a mouse model of Parkinson’s disease [25]. The ratio of Bcl-2/Bax and Bcl-XL/Bax are important in determining sensitivity to apoptotic stimuli. Therefore, we determined the effect of EAW, luteolin and wedelolactone on t-BHP–induced changes in these proteins. The treatment of PC12 cells with 100 μM t-BHP for 3 h decreased the ratio of Bcl-2/Bax and Bcl-XL/Bax significantly, which were reversed by the pretreatment of EAW or luteolin or wedelolactone (Figure 5B). However, PC12 cells cannot totally represent the characters of primary cultured neurons, so, the further work needs to focus directly on primary neurons.Figure 5


Ethyl acetate extract of Wedelia chinensis inhibits tert-butyl hydroperoxide-induced damage in PC12 cells and D-galactose-induced neuronal cell loss in mice.

Lin WL, Wang SM, Ho YJ, Kuo HC, Lee YJ, Tseng TH - BMC Complement Altern Med (2014)

Effects of EAW, luteolin and wedelolactone on t-BHP-induced cytochrome C release and changes in Bcl-2 family proteins. PC12 cells were pretreated with EAW (25 and 50 μg/mL) or luteolin (L10, 10 μM; L20, 20 μM) or wedelolactone (W10, 10 μM; W20, 20 μM) for 24 h, then 100 μM of t-BHP was added for 3 h. (A) Cells were harvested after incubation, and the cytosolic protein extracts were prepared as described in the text and subjected to Western immunoblotting analysis against anti- cytochrome C. The average densitometric value of cytochrome C is shown as the mean ± SD (n = 3). # P < 0.05, compared to the solvent control (0.1% DMSO). *P < 0.05, compared to t-BHP treatment alone. (B) After treatment, the total cell lysates were prepared and subjected to Western immunoblotting analysis against against anti-Bcl-2, -Bcl-xL, -Bax and anti-actin. Actin was used as the loading control. The ratio of Bcl-2/Bax and Bcl-xL/Bax are shown as the mean ± SD (n = 3). # P < 0.05, compared to the solvent control (0.1% DMSO). *P < 0.05, compared to t-BHP treatment alone.
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Fig5: Effects of EAW, luteolin and wedelolactone on t-BHP-induced cytochrome C release and changes in Bcl-2 family proteins. PC12 cells were pretreated with EAW (25 and 50 μg/mL) or luteolin (L10, 10 μM; L20, 20 μM) or wedelolactone (W10, 10 μM; W20, 20 μM) for 24 h, then 100 μM of t-BHP was added for 3 h. (A) Cells were harvested after incubation, and the cytosolic protein extracts were prepared as described in the text and subjected to Western immunoblotting analysis against anti- cytochrome C. The average densitometric value of cytochrome C is shown as the mean ± SD (n = 3). # P < 0.05, compared to the solvent control (0.1% DMSO). *P < 0.05, compared to t-BHP treatment alone. (B) After treatment, the total cell lysates were prepared and subjected to Western immunoblotting analysis against against anti-Bcl-2, -Bcl-xL, -Bax and anti-actin. Actin was used as the loading control. The ratio of Bcl-2/Bax and Bcl-xL/Bax are shown as the mean ± SD (n = 3). # P < 0.05, compared to the solvent control (0.1% DMSO). *P < 0.05, compared to t-BHP treatment alone.
Mentions: Oxidative stress initiates mitochondrial apoptotic signals [22, 23]. The loss of mitochondrial cytochrome C and its release into the cytoplasm are essential events in the initiation of mitochondrial caspase signaling. It was found that t-BHP treatment increased cytosolic cytochrome C, which was decreased by pretreatment with EAW or luteolin or wedelolactone (Figure 5A). The Bcl-2 family members have been found to play important roles in regulating mitochondrial-mediated apoptosis. High ratio of Bcl-2/Bax and Bcl-XL/Bax were observed to promote cell survival by preserving the integrity of the external mitochondrial membrane, which prevents the released of cytochrome C from the mitochondria, inducing cell death. It has been reported that overexpression of Bcl-2 in neurons was found to protect from neuronal loss in mouse model of cerebral ischemia [24]. In addition, Bax inhibition prevented neuronal death in a mouse model of Parkinson’s disease [25]. The ratio of Bcl-2/Bax and Bcl-XL/Bax are important in determining sensitivity to apoptotic stimuli. Therefore, we determined the effect of EAW, luteolin and wedelolactone on t-BHP–induced changes in these proteins. The treatment of PC12 cells with 100 μM t-BHP for 3 h decreased the ratio of Bcl-2/Bax and Bcl-XL/Bax significantly, which were reversed by the pretreatment of EAW or luteolin or wedelolactone (Figure 5B). However, PC12 cells cannot totally represent the characters of primary cultured neurons, so, the further work needs to focus directly on primary neurons.Figure 5

Bottom Line: EAW decreased t-BHP-induced reactive oxygen species (ROS) accumulation, cytotoxicity and apoptosis in PC12 cells.EAW and its major constituents blocked t-BHP-induced cytochrome C release and Bcl-2 family protein ratio change.These results demonstrate that W. chinensis has neuroprotective potential through blocking oxidative stress-induced damage and that luteolin and wedelolactone contribute to the protective action.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Applied Chemistry, Chung Shan Medical University, No, 110, Section 1, Chien-Kuo N, Road, Taichung 402, Taiwan. tht@csmu.edu.tw.

ABSTRACT

Background: Wedelia chinensis is traditionally used as a hepatoprotective herb in Taiwan. The aim of this study was to evaluate the neuroprotective potential of W. chinensis.

Methods: An ethyl acetate extract of W. chinensis (EAW) was prepared and analyzed by HPLC. The neuroprotective potential of EAW was assessed by tert-butylhydroperoxide (t-BHP)-induced damage in PC12 cells and D-galactose-induced damage in mouse cortex.

Results: EAW exhibited potent radical scavenging property and highly contained luteolin and wedelolactone. EAW decreased t-BHP-induced reactive oxygen species (ROS) accumulation, cytotoxicity and apoptosis in PC12 cells. EAW and its major constituents blocked t-BHP-induced cytochrome C release and Bcl-2 family protein ratio change. EAW and its major constituents increased the endogenous antioxidant capacity evaluated by the binding activity assay of nuclear factor E2-related factor 2 (Nrf2) to antioxidant response element (ARE) and nuclear translocation of Nrf2 respectively in PC12 cells. Finally, EAW inhibited D-galactose-induced lipid peroxidation, apoptosis and neuron loss in the cerebral cortex of mice.

Conclusion: These results demonstrate that W. chinensis has neuroprotective potential through blocking oxidative stress-induced damage and that luteolin and wedelolactone contribute to the protective action.

Show MeSH
Related in: MedlinePlus