Membrane Potential Levels Research Articles

Synthesis, structure-activity relationship, and biological evaluation of quinolines for development of anticancer agents.

Tetrahydro-β-carbolines (THβCs) are a kind of natural alkaloids with multiple pharmaceutical activities. Herein, a focused compound library derived from THβCs was synthesized and their anticancer activities were studied in several cancer cell lines. Among them, three compounds showed considerable anticancer activities with low micromolar to submicromolar IC50 values. The abilities to induce apoptosis and alter mitochondrial membrane potential levels, which are comparable to those of the commercial anticancer drug adriamycin, were confirmed by one representative compound (21) on the B16/F10 cell line. Our preliminary structure-activity relationship studies indicated that alkylamines with suitable lengths are very important for potency improvement.

Synergistic Effect of Anethole and Platinum Drug Cisplatin against Oral Cancer Cell Growth and Migration by Inhibiting MAPKase, Beta-Catenin, and NF-κB Pathways.

Cisplatin is a common drug used to treat patients with oral squamous cell carcinoma. However, cisplatin-induced chemoresistance poses a major challenge to its clinical application. Our recent study has shown that anethole possesses an anti-oral cancer effect. In this study, we examined the combined effect of anethole and cisplatin on oral cancer therapy. Gingival cancer cells Ca9-22 were cultured in the presence of various concentrations of cisplatin with or without anethole. The cell viability/proliferation and cytotoxicity were evaluated, respectively, by MTT, Hoechst staining, and LDH assay, while colony formation was measured by crystal violet. Oral cancer cell migration was evaluated by the scratch method. Apoptosis, caspase activity, oxidative stress, MitoSOX, and mitochondrial membrane potential (ΔΨm) levels were evaluated by flow cytometry, and the inhibition of signaling pathways was investigated by Western blot. Our results show that anethole (3 µM) potentiates cisplatin-induced inhibition of cell proliferation and decreases the ΔΨm on Ca9-22 cells. Furthermore, drug combination was found to inhibit cell migration and enhanced cisplatin cytotoxicity. The combination of anethole and cisplatin potentiates cisplatin-induced oral cancer cell apoptosis through the activation of caspase, while we also found anethole and cisplatin to enhance the cisplatin-induced generation of reactive oxygen species (ROS) and mitochondrial stress. In addition, major cancer signaling pathways were inhibited by the combination of anethole and cisplatin such as MAPKase, beta-catenin, and NF-κB pathways. This study reports that the combination of anethole and cisplatin might provide a beneficial effect in enhancing the cisplatin cancer cell-killing effect, thus lowering the associated side effects.

Pterosin sesquiterpenoids from Pteris laeta Wall. ex Ettingsh. protect cells from glutamate excitotoxicity by modulating mitochondrial signals.

The genus Pteris (Pteridaceae) has been used as a traditional herb for a long time. In particular, Pteris laeta Wall. ex Ettingsh. has been widely used in traditional Chinese medicine to treat nervous system diseases and some pterosin sesquiterpenes from Pteris show neuroprotective activity, but their underlying molecular mechanisms remain elusive. Therefore, to investigate the neuroprotective activity and working mechanism of pterosin sesquiterpenes from P. laeta Wall. ex Ettingsh. will provide a better understanding and guidance in using P. laeta Wall. ex Ettingsh. as a traditional Chinese medicine. We aim to develop effective treatments for neurodegenerative diseases from pterosin sesquiterpenes by evaluating their neuroprotective activity and investigating their working mechanisms. Primary screening on the glutamate-induced excitotoxicity cell model was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Fluorescent-activated cell sorting (FACS) was used to analyze the activation level of glutamate receptors and mitochondria membrane potential after treatment. Transcriptomics and proteomics analysis was performed to identify possible targets of pterosin B. The key pathways were enriched by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis through the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The core targets were visualized by a protein-protein interaction network using STRING. The mRNA and protein expressions were evaluated using real-time quantitative polymerase chain reaction (Q-PCR) and western blot, respectively. Immunocytochemistry was performed to monitor mitochondrial and apoptotic proteins. Cellular reactive oxygen species (ROS) were measured by ROS assay, and Ca2+ was stained with Fluo-4 AM to quantify intracellular Ca2+ levels. We found pterosin B from Pteris laeta Wall. ex Ettingsh. showed significant neuroprotective activity against glutamate excitotoxicity, enhancing cell viability from 43.8% to 105% (p-value: <0.0001). We demonstrated that pterosin B worked on the downstream signaling pathways of glutamate excitotoxicity rather than directly blocking the activation of glutamate receptors. Pterosin B restored mitochondria membrane potentials, alleviated intracellular calcium overload from 107.4% to 95.47% (p-value: 0.0006), eliminated cellular ROS by 36.55% (p-value: 0.0143), and partially secured cells from LPS-induced inflammation by increasing cell survival from 46.75% to 58.5% (p-value: 0.0114). Notably, pterosin B enhanced the expression of nuclear factor-erythroid factor 2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) by 2.86-fold (p-value: 0.0006) and 4.24-fold (p-value: 0.0012), and down-regulated Kelch-like ECH-associated protein 1 (KEAP1) expression by 2.5-fold (p-value: 0.0107), indicating that it possibly promotes mitochondrial biogenesis and mitophagy to maintain mitochondria quality control and homeostasis, and ultimately inhibits apoptotic cell death. Our work revealed that pterosin B protected cells from glutamate excitotoxicity by targeting the downstream mitochondrial signals, making it a valuable candidate for developing potential therapeutic agents in treating neurodegenerative diseases.

Oxaloacetate regulates mitochondrial complex II respiration in brown fat: dependence on UCP1 expression

Objective: Oxaloacetate (OAA) is a long-known potent inhibitor of succinate dehydrogenase (SDH), a phenomenon that has, however, received only limited attention. We previously found that skeletal muscle mitochondria incubated at low membrane potential (Δψ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein Δψ is intrinsically low, accumulate OAA in amounts sufficient to inhibit SDH (complex II) respiration. We proposed a mechanism wherein low Δψ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance we carried out studies of mice with inherently different levels of IBAT mitochodnrial inner membrane potential. Hypothesis: Mice with differential expression of UCP1 would manifest Δψ-dependent differences in OAA-regulated mitochondrial respiration. Methodology: We examined several parameters in complex II and complex I energized IBAT mitochondria isolated from obesity prone C57BL/6J compared to obesity resistant 129SVE mice and from UCP1 knock-out (UCP1KO) compared to wildtype (WT) mice. Data were analyzed by unpaired, 2-tailed t-test and expressed as mean ± SE. Data: Isolated complex II (succinate)-energized IBAT mitochondria from 129SVE mice versus C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower Δψ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower Δψ, exhibited much lower respiration (AUC of respiration versus time, 19963 ± 1764 vs 36015 ± 2527, p = 0.0004, n = 6), greater OAA concentrations (22.9 ± 1.8 vs 12.1 ± 3.3, p = 0.016, n = 6), much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared to the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater Δψ, ROS, and NADH, but equal or greater O2 flux compared to WT mitochondria. GDP addition, compared to no GDP, increased Δψ and complex II respiration in WT mice associated with much less OAA. Respiration on complex I substrates for mitochondria of both strains of mice and for WT and UCP1KO mice followed the more classical dynamics of greater O2 flux at lower Δψ. Summary: These findings support the above-mentioned mechanism for OAA- and Δψ-dependent complex II respiration and support its physiologically relevance. Conclusions: Oxaloacetate regulates mitochondrial complex II dynamics in a manner dependent on inner membrane potential as intrinsically determined by mouse strain or genetic manipulation. NIH award, 1 R01 DK123043-01A1, VA Merit Review Award 2 I01 BX000285-06, and the Iowa Fraternal Order of the Eagles This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Oxaloacetate regulates complex II respiration in brown fat: dependence on UCP1 expression.

We previously found that skeletal muscle mitochondria incubated at low membrane potential (ΔΨ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low, accumulate oxaloacetate (OAA) in amounts sufficient to inhibit complex II respiration. We proposed a mechanism wherein low ΔΨ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance, we carried out studies of mice with inherently different levels of IBAT mitochondrial inner membrane potential. Isolated complex II (succinate)-energized IBAT mitochondria from obesity-resistant 129SVE mice compared with obesity-prone C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower ΔΨ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower ΔΨ, exhibited much lower respiration, twofold greater OAA concentrations, much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared with the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater ΔΨ, ROS, and NADH, but equal or greater O2 flux compared with WT mitochondria. GDP addition, compared with no GDP, increased ΔΨ and complex II respiration in wild-type (WT) mice associated with much less OAA. Respiration on complex I substrates followed the more classical dynamics of greater respiration at lower ΔΨ. These findings support the abovementioned mechanism for OAA- and ΔΨ-dependent complex II respiration and support its physiological relevance.NEW & NOTEWORTHY We examined mitochondrial respiration initiated at mitochondrial complex II in mice with varying degrees of brown adipose tissue UCP1 expression. We show that, by affecting inner membrane potential, UCP1 expression determines reverse electron transport from complex II to complex I and, consequently, the NADH/NAD+ ratio. Accordingly, this regulates the level of oxaloacetate accumulation and the extent of oxaloacetate inhibition of complex II.

Analyzing mechanisms of action of antimicrobial peptides on bacterial membranes requires multiple complimentary assays and different bacterial strains

Antimicrobial peptides (AMPs) commonly target bacterial membranes and show broad-spectrum activity against microorganisms. In this research we used three AMPs (nisin, epilancin 15×, [R4L10]-teixobactin) and tested their membrane effects towards three strains (Staphylococcus simulans, Micrococcus flavus, Bacillus megaterium) in relation with their antibacterial activity. We describe fluorescence and luminescence-based assays to measure effects on membrane potential, intracellular pH, membrane permeabilization and intracellular ATP levels. The results show that our control peptide, nisin, performed mostly as expected in view of its targeted pore-forming activity, with fast killing kinetics that coincided with severe membrane permeabilization in all three strains. However, the mechanisms of action of both Epilancin 15× as well as [R4L10]-teixobactin appeared to depend strongly on the bacterium tested. In certain specific combinations of assay, peptide and bacterium, deviations from the general picture were observed. This was even the case for nisin, indicating the importance of using multiple assays and bacteria for mode of action studies to be able to draw proper conclusions on the mode of action of AMPs.

Protective Effects of a Red Grape Juice Extract against Bisphenol A-Induced Toxicity in Human Umbilical Vein Endothelial Cells.

Human exposure to bisphenol A (BPA) occurs through the ingestion of contaminated food and water, thus leading to endothelial dysfunction, the first signal of atherosclerosis. Vitis vinifera L. (grape) juice is well known for its health-promoting properties, due to its numerous bioactive compounds among which are polyphenols. The aim of this study was to evaluate the protective effect of a red grape juice extract (RGJe) against the endothelial damage induced by BPA in human umbilical vein endothelial cells (HUVECs) as an in vitro model of endothelial dysfunction. Our results showed that RGJe treatment counteracted BPA-induced cell death and apoptosis in HUVECs, blocking caspase 3 and modulating p53, Bax, and Bcl-2. Moreover, RGJe demonstrated antioxidant properties in abiotic tests and in vitro, where it reduced BPA-induced reactive oxygen species as well as restored mitochondrial membrane potential, DNA integrity, and nitric oxide levels. Furthermore, RGJe reduced the increase of chemokines (IL-8, IL-1β, and MCP-1) and adhesion molecules (VCAM-1, ICAM-1, and E-selectin), caused by BPA exposure, involved in the primary phase of atheromatous plaque formation. Overall, our results suggest that RGJe prevents BPA-induced vascular damage modulating specific intracellular mechanisms, along with protecting cells, owing to its antioxidant capability.

Glycolysis mediates neuron specific histone acetylation in valproic acid-induced human excitatory neuron differentiation.

Pregnancy exposure of valproic acid (VPA) is widely adopted as a model of environmental factor induced autism spectrum disorder (ASD). Increase of excitatory/inhibitory synaptic transmission ratio has been proposed as the mechanism of VPA induced ASD. How this happened, particularly at the level of excitatory neuron differentiation in human neural progenitor cells (NPCs) remains largely unclear. Here, we report that VPA exposure remarkably inhibited human NPC proliferation and induced excitatory neuronal differentiation without affecting inhibitory neurons. Following VPA treatment, mitochondrial dysfunction was observed before neuronal differentiation, as showed by ultrastructural changes, respiratory complex activity, mitochondrial membrane potential and oxidation levels. Meanwhile, extracellular acidification assay revealed an elevation of glycolysis by VPA stimulation. Interestingly, inhibiting glycolysis by 2-deoxy-d-glucose-6-phosphate (2-DG) efficiently blocked the excitatory neuronal differentiation of human NPCs induced by VPA. Furthermore, 2-DG treatment significantly compromised the VPA-induced expression of H3ac and H3K9ac, and the VPA-induced binding of H3K9ac on the promoter of Ngn2 and Mash1, two key transcription factors of excitatory neuron fate determination. These data, for the first time, demonstrated that VPA biased excitatory neuron differentiation by glycolysis-mediated histone acetylation of neuron specific transcription factors.

Linggui Zhugan Decoction activates the SIRT1-AMPK-PGC1α signaling pathway to improve mitochondrial and oxidative damage in rats with chronic heart failure caused by myocardial infarction.

Objective: To investigate the effects of Linggui Zhugan Decoction on mitochondrial and oxidative damage in rats with chronic heart failure after myocardial infarction and the related mechanisms. Methods: Chronic heart failure after myocardial infarction was established by coronary artery ligation. Heart failure rats were randomly divided into three groups: Model group (n = 11), Linggui Zhugan Decoction group (n = 12), and captopril group (n = 11). Rats whose coronary arteries were only threaded and not ligated were sham group (n = 11). Cardiac function, superoxide dismutase (SOD), malondialdehyde (MDA) contents, soluble growth-stimulating expression factor (ST2), and N-terminal B-type brain natriuretic peptide precursor (NTproBNP) levels were analyzed after treatment. Moreover, the level of mitochondrial membrane potential was detected by JC-1 staining, the ultrastructural of myocardial mitochondria were observed by transmission electron microscopy. The related signal pathway of silent information regulator factor 2-related enzyme 1 (SIRT1), adenylate activated protein kinase (AMPK), phosphorylated adenylate activated protein kinase (p-AMPK), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is an important pathway to regulate mitochondrial energy metabolism, and to initiate mitochondrial biogenesis. The expression level was detected by Western blot and reverse transcription to explore the mechanism of the decoction. Results: Compared with the model rats, Linggui Zhugan Decoction significantly improved cardiac function (p < 0.05), reduced MDA production (p < 0.01), increased SOD activity (p < 0.05), reduced ST-2(p < 0.01), and NT-proBNP(p < 0.05) levels, increased mitochondrial membrane potential, and improved mitochondria function. In addition, Linggui Zhugan Decoction upregulated the expression of SIRT1, p-AMPK, PGC-1α protein, and mRNA in cardiac myocytes. Conclusion: Linggui Zhugan Decoction can improve the cardiac function of heart failure rats by enhancing myocardial antioxidant capacity and protecting the mitochondrial function, the mechanism is related to activating SIRT1/AMPK/PGC-1α signaling pathway.

Abstract 4801: Laurine acid enhances GEM-chemosensitivity targeting hypoxia-induced mitochondrial disfunction and stemness in pancreatic ductal adenocarcinoma

Abstract Hypoxia is a feature of the pancreatic ductal adenocarcinoma (PDA). Although gemcitabine (GEM) is widely used in chemotherapy for PDA, drug resistance restricts its clinical effectiveness. We have found that hypoxia contributes to chemoresistance, however the mechanism is still unclear. The aim of this study is to clarify the mechanism and to develop a new therapeutic strategy. First, we examined the effect of hypoxia to GEM sensitivity, stemness and energy metabolism in three human pancreatic ductal carcinoma cell lines. The inhibition rate by GEM was lower under CoCl2-induced chemical hypoxia than normoxia. Reactive oxygen species (ROS) after GEM was increased under normoxia but not under hypoxia. Flux analysis showed that both oxidative phosphorylation (OXPHOS) and glycolysis were suppressed under hypoxia, indicating a quiescent state. Next, we investigated the relationship between hypoxia and stemness. Hypoxia increased stem cell markers mRNA (C-Myc, Oct-3/4, CD24, CD44, CD133) expression and tumorsphere formation efficiency (TFE). In addition, hypoxia and GEM administration reduced sphere size but increased the number of spheres and TFE. This indicated that hypoxia and GEM suppressed proliferation, however promoted stemness. Second, to examine the mechanism of GEM resistance, we established two GEM-resistant cell lines from human PDA cells by continuous treatment with GEM and CoCl2-induced chemical hypoxia. One resistant cell line possessed reduced OXPHOS and decreased mitochondrial ROS levels, while the other resistant cell line possessed increased stemness. In both cell lines, ethidium bromide-stained mitochondrial DNA levels decreased, suggesting mitochondrial DNA damage. In addition, tetramethylrhodamine-stained mitochondrial membrane potential levels decreased, suggesting mitochondrial impairment. This indicated that long hypoxia and treatment with GEM induced mitochondrial impairment and acquisition of stemness, causing chemoresistance. We further investigated the effect of lauric acid (LAA), a medium-chain fatty acid, on GEM resistance. We have previously reported that LAA causes reprogramming of energy metabolism and induces cell death in cancer cells. Treatment with LAA restored GEM sensitivity in both GEM-resistant cell lines. Flux analysis showed that both OXPHOS and glycolysis were promoted, indicating displacement from quiescent state by LAA. These results suggest that decreased energy production, decreased mitochondrial ROS levels, and increased stemness associated with mitochondrial damage caused by GEM lead to GEM resistance, and that hypoxia may promote this process. Furthermore, forced activation of OXPHOS by LAA could be a tool to overcome GEM resistance. Citation Format: Tadataka Takagi, Hiroki Kuniyasu, Masayuki Sho, Rina Tani, Shiori Mori, Shingo Kishi, Yukiko Nishiguchi, Yudai Hojo. Laurine acid enhances GEM-chemosensitivity targeting hypoxia-induced mitochondrial disfunction and stemness in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4801.

Jie Geng Tang reverses cisplatin resistance through the Nrf2 pathway in lung cancer

Jie Geng Tang (JGT) is an ancient traditional Chinese herbal decoction that exhibits various pharmacological activities, however, is poorly understood in the sensitivity of lung cancer to chemotherapy. Here, we explored the effect of JGT on sensitizing cisplatin (DDP)-resistant A549 cells (A549/DDP). Cell viability was assessed using cell counting kit-8 assay. Flow cytometry was applied to detected cell apoptosis, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels. Western blotting and qRT-PCR were performed to determine protein and mRNA levels. The results demonstrated that DDP co-treatment with JGT significantly increased the cytotoxicity of A549/DDP cells and exhibited efficacy in suppressing the migration and proliferation. The rate of apoptosis was increased by co-treatment with DDP and JGT, along with a higher rate of Bax/Bcl-2, and increased loss of MMP. Furthermore, the combination promoted ROS accumulation and increased γ-H2AX levels. Moreover, Nrf2 levels were suppressed in a dose- and time-dependent manner, Nrf2 stability was reduced following treatment with JGT. Notably, the combination induced inhibition of the Nrf2/ARE pathway at the mRNA and protein levels. Collectively, these results indicate that co-treatment with JGT and DDP can be considered a combinational approach to treating DDP resistance.

BaoShenTongLuo formula protects against podocyte injury by regulating AMPK-mediated mitochondrial biogenesis in diabetic kidney disease

BackgroundMitochondrial dysfunction is considered to be an important contributor in podocyte injury under diabetic conditions. The BaoShenTongLuo (BSTL) formula has been shown to reduce podocyte damage and postpone the progression of diabetic kidney disease (DKD). The potential mechanisms underlying the effects of BSTL, however, have yet to be elucidated. In this study, we aimed to investigate whether the effects of BSTL are related to the regulation of mitochondrial biogenesis via the adenosine monophosphate-activated protein kinase (AMPK) pathway.MethodsHigh-Performance Liquid Chromatography Electrospray Ionization Mass Spectrometer (HPLC–ESI–MS) analysis was performed to investigate the characteristics of pure compounds in BSTL. db/db mice and mouse podocyte clone-5 (MPC5) cells were exposed to high glucose (HG) to induce DKD and podocyte damage. Body weight, random blood glucose, urinary albumin/creatinine ratio (UACR), indicators of renal function and renal histological lesions were measured. Markers of podocyte injury, mitochondrial morphology, mitochondrial deoxyribonucleic acid (mtDNA) content, mitochondrial respiratory chain complexes activities, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) levels were assessed. Protein expressions of AMPK, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), transcription factor A (TFAM), mitochondrial fusion protein 2 (MFN2) and dynamin-related protein 1 (DRP1) were also detected. MPC5 cells were transfected with AMPKα small interfering RNA (AMPKα siRNA) to determine the underlying mechanisms of BSTL improvement of mitochondrial function under diabetic conditions.ResultsIn vivo, treatment with BSTL reduced the UACR levels, reversed the histopathological changes in renal tissues, and alleviated the podocyte injury observed in db/db mice. After BSTL treatment, the decreased mtDNA content and mitochondrial respiratory chain complex I, III, and IV activities were significantly improved, and these effects were accompanied by maintenance of the protein expression of p-AMPKαT172, PGC-1α, TFAM and MFN2. The in vitro experiments also showed that BSTL reduced podocyte apoptosis, suppressed excessive cellular ROS production, and reversed the decreased in MMP that were observed under HG conditions. More importantly, the effects of BSTL in enhancing mitochondrial biogenesis and reducing podocyte apoptosis were inhibited in AMPKα siRNA-treated podocytes.ConclusionBSTL plays a crucial role in protecting against podocyte injury by regulating the AMPK-mediated mitochondrial biogenesis in DKD.

Yang-Gan-Jiang-Mei formula alleviates non-alcoholic steatohepatitis by inhibiting NLRP3 inflammasome through mitophagy

ABSTRACT As an effective formula of traditional Chinese medicine, Yang-Gan-Jiang-Mei (YGJM) formula exhibited a unique advantage in ameliorating liver injury and hepatic steatosis of non-alcoholic steatohepatitis (NASH). Nevertheless, the related pharmacological mechanism needs to be elucidated. This study aimed to explore the molecular mechanism of YGJM formula on mitophagy mediated by PINK1/parkin signaling pathway and NOD-like receptor protein 3 (NLRP3) inflammasome in NASH. High-fat-diet rats and HepG2 cells induced by free fatty acid were used as NASH models in vivo and in vitro. Liver pathology and serum indicator embodying liver function (aspartate transferase, alanine transferase, triglyceride, and total cholesterol) were applied to evaluate the extent of hepatic damage and lipid accumulation. Besides, transmission electron microscopy, JC-1 and 2‘,7’-dichlorofluorescein diacetate were utilized to observe hepatic mitochondrial morphology, as well as cellular mitochondrial membrane potential and reactive oxygen species level. Additionally, expression of PINK1/parkin-mediated mitophagy and NLRP3 inflammasome was detected to elucidate the underlying mechanism of YGJM formula by immunohistochemistry, immunofluorescence, RT-PCR (reverse transcription-polymerase chain reaction) and Western blot. The manifestations of pathology and biochemical detection confirmed the efficacy of YGJM formula in relieving hepatic damage and lipid deposition. Simultaneously, YGJM formula could obviously improve mitochondrial function. In addition, YGJM formula exhibited the promotion of PINK1/parkin-mediated mitophagy, which could perturb NLRP3 inflammasome activation, and as a result, the hepatocyte inflammation was also suppressed both in vitro and in vivo. Our preliminary results indicate that YGJM formula can ameliorate NASH mechanistically by interfering with PINK1/parkin-mediated mitophagy and NLRP3 inflammasome to exert anti-inflammation ability and promote mitochondrial function restoration.

MFN2 deficiency affects calcium homeostasis in lung adenocarcinoma cells via downregulation of UCP4.

Mitofusin-2 (MFN2) is a transmembrane GTPase that regulates mitochondrial fusion and thereby modulates mitochondrial function. However, the role of MFN2 in lung adenocarcinoma remains controversial. Here, we investigated the effect of MFN2 regulation on mitochondria in lung adenocarcinoma. We found that MFN2 deficiency resulted in decreased UCP4 expression and mitochondrial dysfunction in A549 and H1975 cells. UCP4 overexpression restored ATP and intracellular calcium concentration, but not mtDNA copy number, mitochondrial membrane potential or reactive oxygen species level. Furthermore, mass spectrometry analysis identified 460 overlapping proteins after independent overexpression of MFN2 and UCP4; these proteins were significantly enriched in the cytoskeleton, energy production, and calponin homology (CH) domains. Moreover, the calcium signaling pathway was confirmed to be enriched in KEGG pathway analysis. We also found by protein-protein interaction network analysis that PINK1 may be a key regulator of MFN2- and UCP4-mediated calcium homeostasis. Furthermore, PINK1 increased MFN2/UCP4-mediated intracellular Ca2+ concentration in A549 and H1975 cells. Finally, we demonstrated that low expression levels of MFN2 and UCP4 in lung adenocarcinoma are associated with poor clinical prognosis. In conclusion, our data suggest not only a potential role of MFN2 and UCP4 in co-regulating calcium homeostasis in lung adenocarcinoma but also their potential use as therapeutic targets in lung cancer.

Pterostilbene ameliorates oxidative damage and ferroptosis in human ovarian granulosa cells by regulating the Nrf2/HO-1 pathway

The survival of ovarian granulosa cells is of great significance to the physiological maintenance of the ovary. Oxidative damage to the ovarian granulosa cells can lead to various diseases related to ovarian dysfunction. Pterostilbene exerts many pharmacological effects, such as anti-inflammatory and cardiovascular protective effects. Moreover, pterostilbene was shown to have antioxidant properties. This study aimed to investigate the effect and underlying mechanism of pterostilbene on oxidative damage in ovarian granulosa cells. Ovarian granulosa cell (OGC) lines COV434 and KGN were exposed to H2O2 to establish an oxidative damage model. After treatment with different concentrations of H2O2 or pterostilbene, the cell viability, mitochondrial membrane potential, oxidative stress, and iron levels were detected, and the expression of ferroptosis-related and Nrf2/HO-1 signaling pathway-related proteins were evaluated. Pterostilbene treatment could effectively improve cell viability, reduce oxidative stress, and inhibit ferroptosis stimulated by H2O2. More importantly, pterostilbene could up-regulate Nrf2 transcription by stimulating histone acetylation, and inhibition of Nrf2 signaling could reverse the therapeutic effect of pterostilbene. In conclusion, this research shows that pterostilbene protects human OGCs from oxidative stress and ferroptosis through the Nrf2/HO-1 pathway.

The implication of mitochondrial DNA mutation and dysfunction in periodontal diseases.

Periodontitis is a chronic oral inflammatory disease that is caused by dental plaque pathogens. Periodontal disease development and evolution are based on the host immune system, humoral and cellular immunity, the integrity of the tissues, and certain endocrine and nutritional factors. Mitochondria are significantly involved in periodontal infections and inflammation, which play a role in the inflammatory response in a variety of ways. In general, oxidative stress causes a stressful environment that subsequently leads to tissue damage and chronic inflammation. Several mutations and alterations in mitochondrial DNA lead the disease to an aggressive condition, by causing dysregulated mitochondrial function. Such mutations are significantly associated with various diseases. Numerous studies indicate chronic periodontitis patients have a decreased level of mitochondrial membrane potential, as well as adenosine triphosphate, and an increased level of reactive oxygen species production, which causes cell death in the periodontium and affects tissue growth. Further studies into the association between mitochondria and periodontitis might be helpful for the treatment and prevention of the diseases.

In Vitro Inhibition of Growth and Induction of Apoptosis by Mosinone-A in HEp-2 Oral Cancer Cells

The present study evaluated the influence of a Mosinone-A on the induction of apoptosis in HEp-2 human pharyngeal carcinoma cell lines as evidenced by cytotoxicity morphological changes, inhibition of cell proliferation in a time and dose dependent manner. Oral cancer is one of the leading causes of cancer worldwide. Many chemotherapeutics from plants have been tested in cancer, such as Mosinone-A, isolated from the bark of Annona squamous, acetogenin. further we analyzed anticancer activity by MTT assay, the generation of reactive oxygen species (ROS), the level of mitochondrial membrane potential and apoptotic morphological changes were analyzed by AO/EtBr. The results of an MTT assay demonstrated that Mosinone-A inhibited the growth of HEp2 cells in a dose-dependent manner. In addition, the induction of apoptosis was detected, as determined by morphological observation. These results indicated that Mosinone-A may induce apoptosis of HEp2 cells through the mitochondrial pathway.

Interactions Among Sucrose and Concentrations of Serum Fetal Bovine on the Cryopreservation of Somatic Cells Derived from Red-Rumped Agoutis

BACKGROUND: The synergistic action among the different extracellular cryoprotectants could improve somatic cell quality after thawing and provide bases for the formation of biobanks for redrumped agoutis. OBJECTIVE: This study evaluated the interactions among sucrose (SUC) and concentrations of serum fetal bovine (FBS) on the cryopreservation of somatic cells derived from redrumped agoutis. MATERIALS AND METHODS: Cells were cryopreserved with 10% dimethyl sulfoxide and different concentrations of FBS (10%, 40%, and 90%) with or without 0.2 M SUC, totaling six comparison groups. Non-cryopreserved cells were used as a control. Cells were evaluated for viability, metabolic activity, proliferative activity, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and apoptosis levels. RESULTS: No difference was observed among cryopreserved with DMSO containing (10FBS, 10FBS-SUC, 40FBS, 40FBS-SUC, 90FBS, 90FBSSUC) and non-cryopreserved groups for viability, metabolic activity, proliferative activity, and ROS levels. Interestingly, only cells cryopreserved with 90% FBS and SUC maintained the ΔΨm like the control. This indicates that at high concentrations of FBS, SUC contributes to the maintenance of this parameter in cryopreserved cells. Moreover, at concentrations of 10% and 40% of FBS, SUC contributed to the maintenance of viability evaluated by the levels of apoptosis evaluated after thawing. In summary, we verified that 90% FBS and 0.2 M SUC promote greater ability of cells after thawing. Additionally, SUC positively acts in cryopreservation solutions containing 10% and 40% FBS. CONCLUSION: This information is essential to an understanding of the mechanisms involved in the interactions of extracellular cryoprotectants in somatic cell cryopreservation solutions of red-rumped agoutis.

Inhibition of Polo-like kinase 1 (PLK1) triggers cell apoptosis via ROS-caused mitochondrial dysfunction in colorectal carcinoma.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers. Polo-like kinase 1 (PLK1), a member of the serine/threonine kinase PLK family, is the most investigated and essential in the regulation of cell cycle progression, including chromosome segregation, centrosome maturation and cytokinesis. However, the nonmitotic role of PLK1 in CRC is poorly understood. In this study, we explored the tumorigenic effects of PLK1 and its potential as a therapeutic target in CRC. GEPIA database and immunohistochemistry analysis were performed to evaluate the abnormal expression of PLK1 in CRC patients. MTT assay, colony formation and transwell assay were performed to assess cell viability, colony formation ability and migration ability after inhibiting PLK1 by RNAi or the small molecule inhibitor BI6727. Cell apoptosis, mitochondrial membrane potential (MMP) and ROS levels were evaluated by flow cytometry. Bioluminescence imaging was performed to evaluate the impact of PLK1 on CRC cell survival in a preclinical model. Finally, xenograft tumor model was established to study the effect of PLK1 inhibition on tumor growth. First, immunohistochemistry analysis revealed the significant accumulation of PLK1 in patient-derived CRC tissues compared with adjacent healthy tissues. Furthermore, PLK1 inhibition genetically or pharmacologically significantly reduced cell viability, migration and colony formation, and triggered apoptosis of CRC cells. Additionally, we found that PLK1 inhibition elevated cellular reactive oxygen species (ROS) accumulation and decreased the Bcl2/Bax ratio, which led to mitochondrial dysfunction and the release of Cytochrome c, a key process in initiating cell apoptosis. These data provide new insights into the pathogenesis of CRC and support the potential value of PLK1 as an appealing target for CRC treatment. Overall, the underlying mechanism of inhibiting PLK1-induced apoptosis indicates that the PLK1 inhibitor BI6727 may be a novel potential therapeutic strategy in the treatment of CRC.

PM2.5 induces cardiac malformations via PI3K/akt2/mTORC1 signaling pathway in zebrafish larvae

Growing evidence indicates that maternal fine particulate matter (PM2.5) exposure is linked with congenital heart diseases in the offspring. To explore the underlying molecular mechanisms, we tested the effects of a number of pharmaceutical inhibitors, and found that suppressing the PI3K/akt signaling pathway had a protective effect against cardiac defects in zebrafish larvae exposed to extractable organic matter (EOM) from PM2.5. Using genetic knockdown and a specific akt2 pharmacological inhibitor, CCT128930, we demonstrated that akt2 activation is essential to EOM-induced heart malformations. Next, we found that the EOM-induced akt2 overactivation enhances intracellular reactive oxygen species (ROS)/mitochondrial ROS production, decreases mitochondrial membrane potential levels, and elicits intrinsic apoptosis in the heart of zebrafish embryos. In addition, EOM-induced akt2 activation decreased active β-catenin levels and inhibited the expression of Wnt target genes axin2 and nkx2.5. We further demonstrated that mTORC1 phosphorylation mediates the adverse effects of akt2 on intrinsic apoptosis and canonical Wnt signaling in the heart of zebrafish larvae exposed to EOM. Moreover, EOM-induced akt2 activation is mediated via aryl hydrocarbon receptor (AHR)/ROS-induced PTEN inhibition. In conclusion, our results indicate that PM2.5 activates PI3K/akt2/mTORC1 signaling via AHR/ROS-induced PTEN suppression, which leads to mitochondrial-mediated intrinsic apoptosis and Wnt signaling suppression, resulting in cardiac defects in zebrafish larvae.