Xuan Zou

Stimulation of GSH synthesis to prevent oxidative stress-induced apoptosis by hydroxytyrosol in human retinal pigment epithelial cells: activation of Nrf2 and JNK-p62/SQSTM1 pathways

The Nrf2-Keap1 pathway is believed to be a critical regulator of the phase II defense system against oxidative stress. By activation of Nrf2, cytoprotective genes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO-1) and γ-glutamyl-cysteine ligase (GCL) are induced. GCL-induced glutathione (GSH) production is believed to affect redox signaling, cell proliferation and death. We here report that tert-butyl hydroperoxide (t-BHP)-induced GSH reduction led to mitochondrial membrane potential loss and apoptosis in cultured human retinal pigment epithelial cells from the ARPE-19 cell line. Hydroxytyrosol (HT), a natural phytochemical from olive leaves and oil, was found to induce phase II enzymes and GSH, thus protect t-BHP-induced mitochondrial dysfunction and apoptosis. Depletion of GSH by buthionine-[S,R]-sulfoximine enhanced t-BHP toxicity and abolished HT protection. Overexpression of Nrf2 increased GSH content and efficiently protected t-BHP-induced mitochondrial membrane potential loss. Meanwhile, HT-induced GSH enhancement and induction of Nrf2 target gene (GCLc, GCLm, HO-1, NQO-1) messenger RNA (mRNA) were inhibited by Nrf2 knockdown, suggesting that HT increases GSH through Nrf2 activation. In addition, we found that HT was able to activate the PI3/Akt and mTOR/p70S6-kinase pathways, both of which contribute to survival signaling in stressed cells. However, the effect of HT was not inhibited by the PI3K inhibitor LY294002. Rather, c-Jun N-terminal kinase (JNK) activation was found to induce p62/SQSTM1 expression, which is involved in Nrf2 activation. Our study demonstrates that Nrf2 activation induced by the JNK pathway plays an essential role in the mechanism behind HTs strengthening of the antiapoptotic actions of the endogenous antioxidant system.

Maternal Docosahexaenoic Acid Feeding Protects Against Impairment of Learning and Memory and Oxidative Stress in Prenatally Stressed Rats: Possible Role of Neuronal Mitochondria Metabolism

AIMS Docosahexaenoic acid (22:6n-3; DHA) is known to play a critical role in postnatal brain development. However, no study has been performed to investigate its preventive effect on prenatal stress-induced behavioral and molecular alterations in offspring. In the present study, rats were exposed to restraint stress on days 14-20 of pregnancy, three times a day, 2 hours each time; DHA was given at the doses of 100 and 300 mg/kg/day for two weeks. RESULTS We showed that prenatal restraint stress caused (1) learning and memory impairment, (2) BDNF mRNA level decrease, (3) oxidative damage to proteins, (4) enhanced expression of nitric oxide synthase and apoptosis, and (5) abnormalities in mitochondrial metabolism that included changes in mitochondrial complexes I-V, and enhancement of expression of proteins involved in mitochondrial fusion/fission (Mfn-1, Mfn-2, Drp-1) and autophagy (Atg3, Atg7, Beclin-1, p-Akt, and p-mTOR) in the hippocampus of offspring. INNOVATION Besides the well-known role in child brain development, we reported the novel finding of DHA in protecting prenatal stress-induced cognitive dysfunction involving the modulation of mitochondrial function and dynamics. CONCLUSION Maternal feeding of DHA significantly prevented prenatal stress-induced impairment of learning and memory and normalized the biomarkers of oxidative damage, apoptosis, and mitochondrial metabolism in the hippocampus of both male and female offspring. These results suggest that maternal feeding of DHA exerts preventive effects on prenatal stress-induced brain dysfunction and that modulation of mitochondrial metabolism may play critical role in DHA protection.

Hydroxytyrosol prevents diet-induced metabolic syndrome and attenuates mitochondrial abnormalities in obese mice.

A Mediterranean diet rich in olive oil has profound influence on health outcomes including metabolic syndrome. However, the active compound and detailed mechanisms still remain unclear. Hydroxytyrosol (HT), a major polyphenolic compound in virgin olive oil, has received increased attention for its antioxidative activity and regulation of mitochondrial function. Here, we investigated whether HT is the active compound in olive oil exerting a protective effect against metabolic syndrome. In this study, we show that HT could prevent high-fat-diet (HFD)-induced obesity, hyperglycemia, hyperlipidemia, and insulin resistance in C57BL/6J mice after 17 weeks supplementation. Within liver and skeletal muscle tissues, HT could decrease HFD-induced lipid deposits through inhibition of the SREBP-1c/FAS pathway, ameliorate HFD-induced oxidative stress by enhancing antioxidant enzyme activities, normalize expression of mitochondrial complex subunits and mitochondrial fission marker Drp1, and eventually inhibit apoptosis activation. Moreover, in muscle tissue, the levels of mitochondrial carbonyl protein were decreased and mitochondrial complex activities were significantly improved by HT supplementation. In db/db mice, HT significantly decreased fasting glucose, similar to metformin. Notably, HT decreased serum lipid, at which metformin failed. Also, HT was more effective at decreasing the oxidation levels of lipids and proteins in both liver and muscle tissue. Similar to the results in the HFD model, HT decreased muscle mitochondrial carbonyl protein levels and improved mitochondrial complex activities in db/db mice. Our study links the olive oil component HT to diabetes and metabolic disease through changes that are not limited to decreases in oxidative stress, suggesting a potential pharmaceutical or clinical use of HT in metabolic syndrome treatment.

Mitochondrial Dysfunction in Obesity-Associated Nonalcoholic Fatty Liver Disease: The Protective Effects of Pomegranate with Its Active Component Punicalagin

AIMS Punicalagin (PU) is one of the major ellagitannins found in the pomegranate (Punica granatum), which is a popular fruit with several health benefits. So far, no studies have evaluated the effects of PU on nonalcoholic fatty liver disease (NAFLD). Our work aims at studying the effect of PU-enriched pomegranate extract (PE) on high fat diet (HFD)-induced NAFLD. RESULTS PE administration at a dosage of 150 mg/kg/day significantly inhibited HFD-induced hyperlipidemia and hepatic lipid deposition. As major contributors to NAFLD, increased expression of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukins 1, 4, and 6 as well as augmented oxidative stress in hepatocytes followed by nuclear factor (erythroid-derived-2)-like 2 (Nrf2) activation were normalized through PE supplementation. In addition, PE treatment reduced uncoupling protein 2 (UCP2) expression, restored ATP content, suppressed mitochondrial protein oxidation, and improved mitochondrial complex activity in the liver. In contrast, mitochondrial content was not affected despite increased peroxisomal proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and elevated expression of genes related to mitochondrial beta-oxidation after PE treatment. Finally, PU was identified as the predominant active component of PE with regard to the lowering of triglyceride and cholesterol content in HepG2 cells, and both PU- and PE-protected cells from palmitate induced mitochondrial dysfunction and insulin resistance. INNOVATION Our work presents the beneficial effects of PE on obesity-associated NAFLD and multiple risk factors. PU was proposed to be the major active component. CONCLUSIONS By promoting mitochondrial function, eliminating oxidative stress and inflammation, PU may be a useful nutrient for the treatment of NAFLD.

Zeaxanthin induces Nrf2-mediated phase II enzymes in protection of cell death

Zeaxanthin (Zea) is a major carotenoid pigment contained in human retina, and its daily supplementation associated with lower risk of age-related macular degeneration. Despite known property of Zea as an antioxidant, its underlying molecular mechanisms of action remain poorly understood. In this study, we aim to study the regulation mechanism of Zea on phase II detoxification enzymes. In normal human retinal pigment epithelium cells, Zea promoted the nuclear translocation of NF-E2-related factor 2 (Nrf2) and induced mRNA and protein expression of phase II enzymes, the induction was suppressed by specific knockdown of Nrf2. Zea also effectively protected against tert-butyl hydroperoxide-induced mitochondrial dysfunction and apoptosis. Glutathione (GSH) as the most important antioxidant was also induced by Zea through Nrf2 activation in a time- and dose-dependent manner, whereas the protective effects of Zea were decimated by inhibition of GSH synthesis. Finally, Zea activated the PI3K/Akt and MAPK/ERK pathway, whereas only PI3K/Akt activation correlated with phase II enzymes induction and Zea protection. In further in vivo analyses, Zea showed effects of inducing phase II enzymes and increased GSH content, which contributed to the reduced lipid and protein peroxidation in the retina as well as the liver, heart, and serum of the Sprague–Dawley rats. For the first time, Zea is presented as a phase II enzymes inducer instead of being an antioxidant. By activating Nrf2-mediated phase II enzymes, Zea could enhance anti-oxidative capacity and prevent cell death both in vivo and in vitro.

Maternal hydroxytyrosol administration improves neurogenesis and cognitive function in prenatally stressed offspring.

Prenatal stress is known to induce emotional and cognitive dysfunction in the offspring of both humans and experimental animals. Hydroxytyrosol (HT), a major polyphenol in olive oil with reported ability modulating oxidative stress and mitochondrial function, was performed to investigate its preventive effect on prenatal stress-induced behavioral and molecular alterations in offspring. Rats were exposed to restraint stress on days 14-20 of pregnancy. HT was given at doses of 10 and 50 mg/kg/day. The spontaneous alternation performance and Morris water maze confirmed the impaired learning capacity and memory performance induced by prenatal stress in both male and female offspring, and these effects were markedly restored in the HT supplement groups. Through tissue analysis of the hippocampi of male offspring, we found that the stress-induced downregulation of neural proteins, including BDNF, GAP43, synaptophysin, NMDAR1, NMDANR2A and NMDANR2B, was prevented by HT. Prenatal stress-induced low expression of glucocorticoid receptor was also increased by HT, although basal fetal serum corticosterone levels were not different among the four groups. Oxidative stress and mitochondrial dysfunction in prenatally stressed rats were confirmed with changes in protein oxidation, SOD activity, the expression of mitochondrial complexes and mitochondrial DNA copy number. Meanwhile, HT significantly increased transcription factors FOXO1 and FOXO3, as well as phase II enzyme-related proteins, including Nrf2 and HO-1, which may contribute to the decreased oxidative stress and increased mitochondrial function shown with HT supplementation. Taken together, these findings suggest that HT is an efficient maternal nutrient protecting neurogenesis and cognitive function in prenatally stressed offspring.

Evidence for association of mitochondrial metabolism alteration with lipid accumulation in aging rats

Adipogenesis and lipid accumulation during aging have a great impact on the aging process and the pathogenesis of chronic, age-related diseases. However, little is known about the age-related molecular changes in lipid accumulation and the mechanisms underlying them. Here, using 5-month- and 25-month-old rats (young and old, respectively), we found that epididymal fat is the only tissue to accumulate during aging. By testing tissues rich with mitochondria in old and young animals, we found that the old animals had elevated levels of triglycerides in their muscle, heart and liver tissues but not in their kidneys, while, the mRNA level of fatty acid synthase remained unchanged among the four tissues. Regarding lipid catabolism, we determined that the activities of mitochondrial ETC. complexes changed in aged rats (muscle: decreased complex I and V activities; heart: decreased complex I activity; liver: increased complex I and III activities; kidney: decreased complex I and increased complex II activities), while changes in mitochondrial content were not observed in the muscle, heart nor in the liver tissue except increased complex IV and V subunits in aged kidneys. Furthermore, decreased mitochondrial fusion marker Mfn2 and decreased PGC-1α level were observed in the aged muscle, heart and liver but remained unchanged in the kidneys. Down-regulation of Mfn2 with siRNA in 293T cells induced significant mitochondrial dysfunction including decreased oxygen consumption, decreased ATP production, and increased ROS production, followed by increased triglyceride content suggesting a contributing role of decreased mitochondrial fusion to lipid deposit. Meanwhile, judging from autophagy marker p62/SQSTM1 and LC3-II, autophagy was suppressed in the aged muscle, heart and liver but remained unchanged in the kidneys. Taken together, these data suggest that reduction in PGC-1α expression and disruption of mitochondrial dynamics and autophagy might contribute to lipid accumulation during aging.

AMPK activation prevents prenatal stress-induced cognitive impairment: Modulation of mitochondrial content and oxidative stress

Prenatal stress induces cognitive functional impairment in offspring, an eventuality in which mitochondrial dysfunction and oxidative stress are believed to be closely involved. In this study, the involvement of the AMP-activated protein kinase (AMPK) pathway was investigated. A well-known activator, resveratrol (Res), was used to induce AMPK activation in SH-SY-5Y cells. Significant mitochondrial biogenesis and phase II enzyme activation, accompanied by decreased protein oxidation and GSSG content, were observed after Res treatment, and inhibition of AMPK with Compound c abolished the induction effects of Res. Further study utilizing a prenatal restraint stress (PRS) animal model indicated that maternal supplementation of Res may activate AMPK in the hippocampi of both male and female offspring, and that PRS-induced mitochondrial loss in the offspring hippocampus was inhibited by Res maternal supplementation. In addition, Res activated Nrf2-mediated phase II enzymes and reduced PRS-induced oxidative damage in both male and female offspring. Moreover, PRS markedly decreased mRNA levels of various neuron markers, as well as resultant offspring cognitive function, based on spontaneous alternation performance and Morris water maze tests, the results of which were significantly improved by maternal Res supplementation. Our results provide evidence indicating that AMPK may modulate mitochondrial content and phase II enzymes in neuronal cells, a process which may play an essential role in preventing PRS-induced cognitive impairment. Through the coupling of mitochondrial biogenesis and the Nrf2 pathway, AMPK may modulate oxidative stress and be a promising target against neurological disorders.

Bitter Gourd Inhibits the Development of Obesity-Associated Fatty Liver in C57BL/6 Mice Fed a High-Fat Diet

Bitter gourd (BG) is a popular fruit in Asia with numerous well-known medicinal uses, including as an antidiabetic. In the current study, we aimed to explore the effects of BG on mitochondrial function during the development of obesity-associated fatty liver. C57BL/6 mice were divided into 4 experimental groups: mice fed a normal diet (control; included for reference only), mice fed a high-fat diet (HFD), and mice fed an HFD supplemented with freeze-dried BG powder through daily gavage at doses of 0.5 (HFD+0.5BG) and 5 (HFD+5BG) g/kg, respectively. After 16 wk, mice in the HFD+5BG group showed less body and tissue weight gain and less hyperglycemia and hyperlipidemia compared with those in the HFD group (P < 0.05). In both HFD+0.5BG and HFD+5BG groups, serum interleukin-6 concentration was lower than that in the HFD group (P < 0.02). The serum C-reactive protein concentration was lower in the HFD+5BG group compared with the HFD group (P < 0.04). An analysis of liver tissue revealed lower liver triglyceride and cholesterol concentrations in both HFD+0.5BG and HFD+5BG groups than in the HFD group (P < 0.01). The HFD+5BG group had less activation of the sterol regulatory element binding protein/fatty acid synthase (SREBP-1/FAS) pathway, greater superoxide dismutase activity, and less total protein and mitochondrial protein oxidation than did the HFD group (P < 0.05). Mitochondrial complex I, II, III, and V activity was greater in the HFD+0.5BG group than in the HFD group (P < 0.03). The HFD+5BG group only had greater complex V activity compared with the HFD group (P < 0.05). Mitochondrial dynamics regulators, including dynamin related protein 1 (DRP1) and mitofusin 1 (MFN1), as well as proapoptotic protein expression levels were restored by BG treatment (P < 0.02). Taken together, our results suggest that BG prevents inflammation and oxidative stress, modulates mitochondrial activity, suppresses apoptosis activation, and inhibits lipid accumulation during the development of fatty liver.

4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic Acid (C75), an Inhibitor of Fatty-acid Synthase, Suppresses the Mitochondrial Fatty Acid Synthesis Pathway and Impairs Mitochondrial Function

Background: C75 is a fatty-acid synthase inhibitor and potential anticancer drug. Results: C75 treatment leads to mitochondrial dysfunction that is rescued by overexpression of β-ketoacyl-acyl carrier protein synthase or lipoic acid. Conclusion: The effect of C75 on mitochondria is caused by inhibition of β-ketoacyl-acyl carrier protein synthase. Significance: The mitochondrial fatty acid synthesis pathway plays an important role in mitochondrial function. 4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75) is a synthetic fatty-acid synthase (FASN) inhibitor with potential therapeutic effects in several cancer models. Human mitochondrial β-ketoacyl-acyl carrier protein synthase (HsmtKAS) is a key enzyme in the newly discovered mitochondrial fatty acid synthesis pathway that can produce the substrate for lipoic acid (LA) synthesis. HsmtKAS shares conserved catalytic domains with FASN, which are responsible for binding to C75. In our study, we explored the possible effect of C75 on HsmtKAS and mitochondrial function. C75 treatment decreased LA content, impaired mitochondrial function, increased reactive oxygen species content, and reduced cell viability. HsmtKAS but not FASN knockdown had an effect that was similar to C75 treatment. In addition, an LA supplement efficiently inhibited C75-induced mitochondrial dysfunction and oxidative stress. Overexpression of HsmtKAS showed cellular protection against low dose C75 addition, whereas there was no protective effect upon high dose C75 addition. In summary, the mitochondrial fatty acid synthesis pathway has a vital role in mitochondrial function. Besides FASN, C75 might also inhibit HsmtKAS, thereby reducing LA production, impairing mitochondrial function, and potentially having toxic effects. LA supplements sufficiently ameliorated the toxicity of C75, showing that a combination of C75 and LA may be a reliable cancer treatment.