High glucose suppresses autophagy through the AMPK pathway while it induces autophagy via oxidative stress in chondrocytes.
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ABSTRACT:
SUBMITTER: Wang B
PROVIDER: S-EPMC8131591 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Crassostrea hongkongensis (Hong Kong oyster) is an ecologically and economically valuable shellfish endemic to South/Southeast Asia. Due to ocean acidification and warming waters, they have become increasingly vulnerable to invading microbes including Vibrio parahaemolyticus, a significant foodborne human pathogen. In recent years, outbreaks of V. parahaemolyticus have emerged as a perennial phenomenon in parts of the world, necessitating to better understand the biology of host-pathogen interactions in this under-examined marine invertebrate. Although an immunologically relevant autophagy apparatus has been identified in Crassostrea gigas, an evolutionarily close mollusk cousin, the precise mechanistic details of C. hongkongensis autophagy during V. parahaemolyticus infection are still wanting. Here, we compellingly demonstrated that in vivo V. parahaemolyticus challenge robustly triggered autophagic signaling in C. hongkongensis hemocytes peaking at 6 h post-infection, which subsequently promoted bacterial clearance and dampened premature apoptosis. Simultaneously, a large surplus of adenosine monophosphate (AMP) and elevations in reactive oxygen species (ROS, specifically mitochondrial O2 - and cellular H2O2) formation were observed post-infection. Extrinsically applied AMP and ROS could synergistically induce AMP-activated protein kinase (AMPK) phosphorylation to stimulate downstream autophagic events. V. parahaemolyticus infection-induced autophagy was pharmacologically shown to be AMPK-dependent in vivo. Overall, our results establish autophagy as a crucial arm of host defense against Vibrio infections in mollusks, and provide new insights into the underappreciated roles of ROS and AMP as co-regulators of autophagy.
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Project description:Increasing evidence reveals that estrogen, especially 17β-estradiol (17β-E2), is associated with articular cartilage metabolism disorder and postmenopausal osteoarthritis (OA). SIRT1, AMPK, and mTOR are regarded as critical mitophagy regulators. Recent studies have shown that mitophagy displays a protective effect against OA, but the molecular mechanism is not well known. This study aimed to investigate the effect of 17β-E2 on Sirtuin-1 (SIRT1) expression and the induction of mitophagy upregulation by 17β-E2 via the SIRT1-mediated AMP-activated protein kinase (AMPK)/mammalian target of the rapamycin (mTOR) signaling pathway to protect chondrocytes. ATDC5 chondrocytes were treated with different concentrations of 17β-E2 (0 M, 1 × 10-9 M, 1 × 10-8 M, and 1 × 10-7 M) for 24 h or pretreatment with or without NAM (SIRT1 inhibitor), Compound C (AMPK inhibitor) and S1842 (mTOR inhibitor) for 30 min prior to treatment with 17β-E2 (1 × 10-7 M) for 24 in each groups. Expression of SIRT1 was evaluated by real-time PCR, Western blotting and confocal immunofluorescence staining. Then, the mitophagosomes in cells were observed under a transmission electron microscopy (TEM), and the AMPK/mTOR signaling pathway was detected by Western blotting. The mitophagy-related proteins, p-AMPK, p-mTOR, p-JNK, and p-p38 were also identified by Western blot analysis. The chondrocytes viability and proliferation were determined by MTT and 5-Bromo-2'-deoxyuridine (BrdU) assay. These experiments were independently repeated 3 times The study found that 17β-E2 increased the expression level of SIRT1, p-AMPK, and mitophagy-related proteins but decreased p-mTOR expression, and then induced mitophagy upregulation in chondrocytes. More mitochondrial autophagosomes were observed in 17β-E2-treated chondrocytes under a transmission electron microscope. Also, 17β-E2 improved cell viability and proliferation with the higher expression of SIRT1 and activation of the AMPK/mTOR signaling pathway. However, SIRT1 inhibitor nicotinamide (NAM) and AMPK inhibitor Compound C blocked the beneficial effect of 17β-E2. In summary, this study was novel in demonstrating that 17β-E2 induced mitophagy upregulation to protect chondrocytes via the SIRT1-mediated AMPK/mTOR signaling pathway.
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Project description:Hepatocellular carcinoma (HCC) is an aggressive malignancy with high rates of metastasis and relapse. Isoquercitrin (ISO), a natural flavonoid present in the Chinese bayberry and other plant species, reportedly exerts notable inhibitory effects on tumor cell proliferation, though the mechanism is unknown. In the present study, we exposed HepG2 and Huh7 human liver cancer cells to ISO and examined the roles of autophagy and apoptosis in ISO-mediated cell death. We found that ISO exposure inhibited cell viability and colony growth, activated apoptotic pathway, and triggered dysregulated autophagy by activating the AMPK/mTOR/p70S6K pathway. Autophagy inhibition using 3-methyladenine (3-MA) or Atg5-targeted siRNA decreased the Bax/Bcl-2 ratio, caspase-3 activation, and PARP cleavage and protected cells against ISO-induced apoptosis. Moreover, autophagy inhibition reversed the upregulation of AMPK phosphorylation and downregulation of mTOR and p70S6K phosphorylation elicited by ISO. By contrast, application of a broad-spectrum caspase inhibitor failed to inhibit autophagy in ISO-treated cells. These data indicate that ISO simultaneously induced apoptosis and autophagy, and abnormal induction of autophagic flux contributed to ISO-triggered caspase-3-dependent apoptosis.
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Project description:Salvia chinensia Benth (Shijianchuan in Chinese, SJC) has been used as a traditional anti-cancer herb. SJC showed good anti-esophageal cancer efficacy based on our clinical application. However, the current research on SJC is minimal, and its anti-cancer effect lacks scientific certification. This study aims to clarify the inhibitory effect of SJC on esophageal cancer and explore its underlying mechanism. Q-Orbitrap high-resolution LC/MS was used to identify the primary chemical constituents in SJC. Cell proliferation and colony formation assays showed that SJC could effectively inhibit the growth of esophageal tumor cells in vitro. To clarify its mechanism of action, proteomic and bioinformatic analyses were carried out by combining tandem mass labeling and two-dimensional liquid chromatography-mass spectrometry (LC-MS). Data are available via ProteomeXchange with identifier PXD035823. The results indicated that SJC could activate AMPK signaling pathway and effectively promote autophagy in esophageal cancer cells. Therefore, we further used western blotting to confirm that SJC activated autophagy in esophageal cancer cells through the AMPK/ULK1 signaling pathway. The results showed that P-AMPK and P-ULK1 were significantly up-regulated after the treatment with SJC. The ratio of autophagosomes marker proteins LC3II/I was significantly increased. In addition, the expression of the autophagy substrate protein P62 decreased with the degradation of autophagosomes. Using lentiviral transfection of fluorescent label SensGFP-StubRFP-LC3 protein and revalidation of LC3 expression before and after administration by laser confocal microscopy. Compared with the control group, the fluorescence expression of the SJC group was significantly enhanced, indicating that it promoted autophagy in esophageal cancer cells. Cell morphology and the formation of autophagosomes were observed by transmission electron microscopy. Our study shows that the tumor suppressor effect of SJC is related to promoting autophagy in esophageal tumor cells via the AMPK/ULK1 signaling pathway.
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Project description:The c-Jun N-terminal protein kinase (JNK) plays an important role in the regulation of cell apoptosis. Forkhead box O (FoxO) transcription factors are involved in diverse biological processes, including cellular metabolism, cell apoptosis, and cell cycle. However, the JNK/FoxO1 pathway involved in the process of apoptosis induced by oxidative stress remains to be elucidated. Here, we demonstrated that the JNK activity significantly increased in response to oxidative stress in mouse follicular granulosa cells (MGCs). SP600125, a selective JNK inhibitor, attenuated the oxidative stress-induced MGCs apoptosis. Oxidative stress enhanced the FoxO1 nuclear translocation by activating the JNK activity. Moreover, JNK mediated the dissociation of FoxO1 from 14-3-3 proteins in MGCs after the treatment with H2O2. Finally, oxidative stress up-regulated the expression of FoxO1 via JNK mediation of FoxO1 self-regulation in MGCs. Taken together, our findings suggest that JNK/FoxO1 is involved in the regulation of oxidative stress-induced cell apoptosis in MGCs.
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Project description:Salvia chinensia Benth. (Shijianchuan in Chinese, SJC) has been used as a traditional anti-cancer herb. SJC showed good anti-esophageal cancer efficacy based on our clinical application. However, the current research on SJC is minimal, and its anti-cancer effect lacks scientific certification. This study aims to clarify the inhibitory effect of SJC on esophageal cancer and explore its underlying mechanism. Q-Orbitrap high-resolution LC/MS was used to identify the primary chemical constituents in SJC. Cell proliferation and colony formation assays showed that SJC could effectively inhibit the growth of esophageal tumor cells in vitro. To clarify its mechanism of action, proteomic and bioinformatic analyses were carried out by combining tandem mass labeling and two-dimensional liquid chromatography-mass spectrometry (LC-MS). The results indicated that SJC could activate AMPK signaling pathway and effectively promote autophagy in esophageal cancer cells. Therefore, we further used WB to confirm that SJC activated autophagy in esophageal cancer cells through the AMPK / ULK1 signaling pathway. The results showed that P-AMPK and P-ULK1 were significantly up-regulated after the treatment with SJC. The ratio of autophagosomes marker proteins LC3II/I was significantly increased. In addition, the expression of the autophagy substrate protein P62 decreased with the degradation of autophagosomes. Using lentiviral transfection of fluorescent label SensGFP-StubRFP-LC3 protein and revalidation of LC3 expression before and after administration by laser confocal microscopy. Compared with the control group, the fluorescence expression of the SJC group was significantly enhanced, indicating that it promoted autophagy in esophageal cancer cells. Cell morphology and the formation of autophagosomes were observed by transmission electron microscopy. Our study shows that the tumor suppressor effect of SJC is related to promoting autophagy in esophageal tumor cells via the AMPK/ULK1 signaling pathway.
2022-10-14 | PXD035823 | Pride