Kim, Kkot Byeol
(Research Institute, Seoul Medical Center)
,
Lee, Seonah
(Research Institute, Seoul Medical Center)
,
Kim, Jung Hee
(Research Institute, Seoul Medical Center)
BACKGROUND/OBJECTIVES: Oxidative stress causes cell damage and death, which contribute to the pathogenesis of neurodegenerative diseases. Urolithin A (UA), a gut microbial-derived metabolite of ellagitannins and ellagic acid, has high bioavailability and various health benefits such as antioxidant a...
BACKGROUND/OBJECTIVES: Oxidative stress causes cell damage and death, which contribute to the pathogenesis of neurodegenerative diseases. Urolithin A (UA), a gut microbial-derived metabolite of ellagitannins and ellagic acid, has high bioavailability and various health benefits such as antioxidant and anti-inflammatory effects. However, it is unknown whether it has protective effects against oxidative stress-induced cell death. We investigated whether UA ameliorates H2O2-induced neuronal cell death. MATERIALS/METHODS: We induced oxidative damage with 300 μM H2O2 after UA pretreatment at concentrations of 1.25, 2.5, and 5 μM in SK-N-MC cells. Cytotoxicity and cell viability were determined using the CCK-8 assay. The formation of reactive oxygen species (ROS) was measured using a 2,7-dichlorofluorescein diacetate assay. Hoechst 33342 staining was used to characterize morphological changes in apoptotic cells. The expressions of apoptosis proteins were measured using Western blotting. RESULTS: UA significantly increased cell viability and decreased intracellular ROS production in a dose-dependent manner in SK-N-MC cells. It also decreased the Bax/Bcl-2 ratio and the expressions of cytochrome c, cleaved caspase-9, cleaved caspase-3, and cleaved PARP. In addition, it suppressed the phosphorylation of the p38 mitogen-activated protein kinase (MAPK) pathway. CONCLUSIONS: UA attenuates oxidative stress-induced apoptosis via inhibiting the mitochondrial-related apoptosis pathway and modulating the p38 MAPK pathway, suggesting that it may be an effective neuroprotective agent.
BACKGROUND/OBJECTIVES: Oxidative stress causes cell damage and death, which contribute to the pathogenesis of neurodegenerative diseases. Urolithin A (UA), a gut microbial-derived metabolite of ellagitannins and ellagic acid, has high bioavailability and various health benefits such as antioxidant and anti-inflammatory effects. However, it is unknown whether it has protective effects against oxidative stress-induced cell death. We investigated whether UA ameliorates H2O2-induced neuronal cell death. MATERIALS/METHODS: We induced oxidative damage with 300 μM H2O2 after UA pretreatment at concentrations of 1.25, 2.5, and 5 μM in SK-N-MC cells. Cytotoxicity and cell viability were determined using the CCK-8 assay. The formation of reactive oxygen species (ROS) was measured using a 2,7-dichlorofluorescein diacetate assay. Hoechst 33342 staining was used to characterize morphological changes in apoptotic cells. The expressions of apoptosis proteins were measured using Western blotting. RESULTS: UA significantly increased cell viability and decreased intracellular ROS production in a dose-dependent manner in SK-N-MC cells. It also decreased the Bax/Bcl-2 ratio and the expressions of cytochrome c, cleaved caspase-9, cleaved caspase-3, and cleaved PARP. In addition, it suppressed the phosphorylation of the p38 mitogen-activated protein kinase (MAPK) pathway. CONCLUSIONS: UA attenuates oxidative stress-induced apoptosis via inhibiting the mitochondrial-related apoptosis pathway and modulating the p38 MAPK pathway, suggesting that it may be an effective neuroprotective agent.
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가설 설정
Therefore, metabolites derived from gut microbes are expected to be effective protective agents for the prevention and treatment of brain-related diseases. In this study, we hypothesized that UA, a representative polyphenol metabolite, may have neuroprotective effects by inhibiting cell death induced by oxidative stress.
In this study, we investigated the protective effects of UA against H2O2-induced oxidative stress in human neuroblastoma SK-N-MC cells. We also examined possible mechanisms associated with the action of UA.
대상 데이터
Cell counting Kit-8 (CCK-8) assay reagents were purchased from Dojindo Molecular Technologies (Gaithersburg, MD, USA), and the intracellular ROS assay kit was purchased from Cell Biolabs (San Diego, CA, USA). Anti-Bax (#2772), anti-total p38 (#9212), anti-caspase-9 (#9502), anti-caspase-3 (#9665), anti-PARP (poly (ADP-ribose) polymerase, #9532), anti-mouse IgG horseradish peroxidase [HRP]-conjugated antibody (#7076), anti-rabbit IgG HRP-conjugated antibody (#7074), and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (#5633) were purchased from Cell Signaling Technology (Danvers, MA, USA).
Eagle’s minimum essential medium (EMEM), trypsin-EDTA, antibiotics, Dulbecco’s phosphate-buffered saline (PBS), and Hank’s balanced salt solution (HBSS) were purchased from WelGENE (Daegu, Republic of Korea). Fetal bovine serum (FBS), urolithin A (UA), and general reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell counting Kit-8 (CCK-8) assay reagents were purchased from Dojindo Molecular Technologies (Gaithersburg, MD, USA), and the intracellular ROS assay kit was purchased from Cell Biolabs (San Diego, CA, USA).
The SK-N-MC human neuroblastoma cell line was purchased from American Type Culture Collection (Rockville, MD, USA). Eagle’s minimum essential medium (EMEM), trypsin-EDTA, antibiotics, Dulbecco’s phosphate-buffered saline (PBS), and Hank’s balanced salt solution (HBSS) were purchased from WelGENE (Daegu, Republic of Korea).
데이터처리
A one-way ANOVA followed by Duncan’s multiple range test was used to determine differences among the treatment groups.
Statistical analyses included t-tests and one-way analysis of variance (ANOVA) using SPSS statistical software for Windows, version 20.0 (SPSS, Chicago, IL, USA).
이론/모형
Protective effects of urolithin A (UA) on H2O2-induced cell death and cytotoxicity in SK-N-MC cells. Cell viability was determined by CCK-8 assay. (A) The molecular of UA.
Human neuroblastoma SK-N-MC cells have been widely used as an in vitro model to study the pathogenesis of NDs such as AD because of their high stability and homogeneity [22,23]. The cell viabilities were determined using the CCK-8 assay. Cell viability was not significantly affected at UA concentrations up to 10 μM (Fig.
The cell viability was evaluated using a CCK-8 assay [19]. CCK-8 is reduced by dehydrogenases in cells to give a yellowcolored product (formazan).
(B) The cells were pretreated with SB206580 (p38 MAPK inhibitor) for 1 h, treated 5 µM UA for 6 h, and then treated with 300 µM H2O2 for 18 h. The cell viability was measured using the CCK-8 assay. The results are expressed as mean ± SD.
성능/효과
In conclusion, UA inhibited apoptotic cell death involving oxidative stress by decreasing intracellular ROS production, inhibiting the mitochondrial-related apoptosis pathway, and modulating the p-38 MAPK pathway. Taken together, our results indicate that UA protects against H2O2-induced growth inhibition and apoptosis induction in SK-N-MC cells.
후속연구
Thus, we suggest that it could be a useful neuroprotective agent against oxidative stress-related brain diseases. Future studies should examine the metabolic processes of UA and its protective effects on brain tissue in animal models of AD and PD.
참고문헌 (52)
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