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With population aging, the incidence of aging-related Alzheimer's disease (AD) is increasing, accompanied by decreased autophagy activity. At present, Caenorhabditis elegans (C. elegans) is widely employed to evaluate autophagy and in research on aging and aging-related diseases in vivo. To discover autophagy activators from natural medicines and investigate their therapeutic potential in antiaging and anti-AD effects, multiple C. elegans models related to autophagy, aging, and AD were used. In this study, we employed the DA2123 and BC12921 strains to discover potential autophagy inducers using a self-established natural medicine library. The antiaging effect was evaluated by determining the lifespan, motor ability, pumping rate, lipofuscin accumulation of worms, and resistance ability of worms under various stresses. In addition, the anti-AD effect was examined by detecting the paralysis rate, food-sensing behavior, and amyloid-β and Tau pathology in C. elegans. Moreover, RNAi technology was used to knock down the genes related to autophagy induction. We discovered that Piper wallichii extract (PE) and the petroleum ether fraction (PPF) activated autophagy in C. elegans , as evidenced by increased GFP-tagged LGG-1 foci and decreased GFP-p62 expression. In addition, PPF extended the lifespan and enhanced the healthspan of worms by increasing body bends and pumping rates, decreasing lipofuscin accumulation, and increasing resistance to oxidative, heat, and pathogenic stress. Moreover, PPF exhibited an anti-AD effect by decreasing the paralysis rate, improving the pumping rate and slowing rate, and alleviating Aβ and Tau pathology in AD worms. However, the feeding of RNAi bacteria targeting unc-51, bec-1, lgg-1 , and vps-34 abolished the antiaging and anti-AD effects of PPF. : Piper wallichii may be a promising drug for antiaging and anti-AD. More future studies are also needed to identify autophagy inducers in Piper wallichii and clarify their molecular mechanisms. [ABSTRACT FROM AUTHOR]

Protein aggregates are considered key pathological features in neurodegenerative diseases (NDs). The induction of autophagy can effectively promote the clearance of ND-related misfolded proteins. In this study, we aimed to screen natural autophagy enhancers from traditional Chinese medicines (TCMs) presenting potent neuroprotective potential in multiple ND models. The autophagy enhancers were broadly screened in our established herbal extract library using the transgenic Caenorhabditis elegans (C. elegans) DA2123 strain. The neuroprotective effects of the identified autophagy enhancers were evaluated in multiple C. elegans ND models by measuring Aβ-, Tau-, α-synuclein-, and polyQ40-induced pathologies. In addition, PC-12 cells and 3 × Tg-AD mice were employed to further validate the neuroprotective ability of the identified autophagy enhancers, both in vitro and in vivo. Furthermore, RNAi bacteria and autophagy inhibitors were used to evaluate whether the observed effects of the identified autophagy enhancers were mediated by the autophagy-activated pathway. The ethanol extract of Folium Hibisci Mutabilis (FHME) was found to significantly increase GFP::LGG-1-positive puncta in the DA2123 worms. FHME treatment markedly inhibited Aβ, α-synuclein, and polyQ40, as well as prolonging the lifespan and improving the behaviors of C. elegans , while siRNA targeting four key autophagy genes partly abrogated the protective roles of FHME in C. elegans. Additionally, FHME decreased the expression of AD-related proteins and restored cell viability in PC-12 cells, which were canceled by cotreatment with 3-methyladenine (3-MA) or bafilomycin A1 (Baf). Moreover, FHME ameliorated AD-like cognitive impairment and pathology, as well as activating autophagy in 3 × Tg-AD mice. FHME was successfully screened from our natural product library as a potent autophagy enhancer that exhibits a neuroprotective effect in multiple ND models across species through the induction of autophagy. These findings offer a new and reliable strategy for screening autophagy inducers, as well as providing evidence that FHME may serve as a possible therapeutic agent for NDs. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Emerging evidences indicate the important roles of autophagy in anti-oxidative stress, which is closely associated with cancer, aging and neurodegeneration.Objective: In the current study, we aimed to identify autophagy inducers with potent anti-oxidative effect from traditional Chinese medicines (TCMs) in PC-12 cells and C. elegans.Methods: The autophagy inducers were extensively screened in our herbal extracts library by using the stable RFP-GFP-LC3 U87 cells. The components with autophagic induction effect in Trillium tschonoskii Maxim. (TTM) was isolated and identified by using the autophagic activity-guided column chromatography and Pre-HPLC technologies, and MS and NMR spectroscopic analysis, respectively. The anti-oxidative effect of the isolated autophagy inducers was evaluated in H2O2-induced PC-12 cells and C. elegans models by measuring the viability of PC-12 cells and C. elegans, with quantitation on the ROS level in vitro and in vivo using H2DCFDA probe.Results: The total ethanol extract of TTM was found to significantly increase the formation of GFP-LC3 puncta in stable RFP-GFP-LC3 U87 cells. One novel steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-Deoxytrillenogenin, (Deoxytrillenoside CA, DTCA) and one known steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-O-acetyl-epitrillenogenin (Epitrillenoside CA, ETCA) were isolated, identified and found to have novel autophagic effect. Both DTCA and ETCA could activate autophagy in PC-12 cells via the AMPK/mTOR/p70S6K signaling pathway in an Atg7-dependent. In addition, DTCA and ETCA could increase the cell viability and decrease the intracellular ROS level in H2O2-treated PC-12 cells and C. elegans, and the further study demonstrated that the induced autophagy contributes to their anti-oxidative effect.Conclusion: Our current findings not only provide information on the discovery of novel autophagy activators from TTM, but also confirmed the anti-oxidative effect of the components from TTM both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Hexavalent chromium (Cr(VI)) is a ubiquitous environmental pollutant that can cause reproductive toxicity. However, the exact mechanism of Cr(VI)-induced testis toxicity remains largely elusive. This study aims to explore the possible molecular mechanism of Cr(VI)-provoked testicular toxicity. Male Wistar rats were intraperitoneally injected with 0, 2, 4, or 6 mg/kg body weight/day of potassium dichromate (K 2 Cr 2 O 7), respectively, for 5 weeks. The results revealed that Cr(VI)-treated rat testis presented varying degrees of damage in a dose-dependent manner. Concretely, Cr(VI) administration suppressed Sirtuin 1/Peroxisome proliferator-activated receptor-γ coactivator-1α pathway and led to mitochondrial dynamics disorder, along with the elevation of mitochondrial division and the repression of mitochondrial fusion. Meanwhile, the downstream effector of Sirt1, nuclear factor-erythroid-2-related factor 2 (Nrf2), was downregulated, and correspondingly exacerbated oxidative stress. Mitochondrial dynamics disorder and Nrf2 inhibition collectively contribute to abnormal mitochondrial dynamics in testis, which further promotes apoptosis and autophagy, evidenced by dose-dependently increasing the protein levels and gene expressions of apoptosis-related (including Bcl-2-associated X protein, cytochrome c , and cleaved-caspase 3) and autophagy-related (Beclin-1, ATG4B, and ATG5). Collectively, our results demonstrate that Cr(VI) exposure induced testis apoptosis and autophagy by disrupting the balance of mitochondrial dynamics and the oxidation-reduction process in rats. • K 2 Cr 2 O 7 induces mitochondrial dynamics disorder in rat testis by inhibiting Sirt1/PGC-1α axis. • K 2 Cr 2 O 7 exposure aggravates inhibition of Nrf2 pathway and promotes oxidative stress. • K 2 Cr 2 O 7 exposure promotes apoptosis and autophagy, leading to testicular damage. [ABSTRACT FROM AUTHOR]

Activation of the NLRP3 inflammasome and its mediated neuroinflammation are implicated in neurodegenerative diseases, while mitophagy negatively regulates NLRP3 inflammasome activation. SHP-2, a protein-tyrosine phosphatase, is critical for NLRP3 inflammasome regulation and inflammatory responses. In this study, we investigated whether triterpenoid saponins in Radix Polygalae inhibit the NLRP3 inflammasome via mitophagy induction. First, we isolated the active fraction (polygala saponins (PSS)) and identified 17 saponins by ultra-performance liquid chromatography coupled with diode-array detection and tandem quadrupole time-of-flight mass spectrometry (UHPLC-DAD-Q/TOF-MS). In microglial BV-2 cells, PSS induced mitophagy as evidenced by increased co-localization of LC3 and mitochondria, as well as an increased number of autophagic vacuoles surrounding the mitochondria. Furthermore, the mechanistic study found that PSS activated the AMPK/mTOR and PINK1/parkin signaling pathways via the upregulation of SHP-2. In Aβ(1-42)-, A53T-α-synuclein-, or Q74-induced BV-2 cells, PSS significantly inhibited NLRP3 inflammasome activation, which was attenuated by bafilomycin A1 (an autophagy inhibitor) and SHP099 (an SHP-2 inhibitor). In addition, the co-localization of LC3 and ASC revealed that PSS promoted the autophagic degradation of the NLRP3 inflammasome. Moreover, PSS decreased apoptosis in conditioned medium-induced PC-12 cells. In APP/PS1 mice, PSS improved cognitive function, ameliorated Aβ pathology, and inhibited neuronal death. Collectively, the present study, for the first time, shows that PSS inhibit the NLRP3 inflammasome via SHP-2-mediated mitophagy in vitro and in vivo, which strongly suggests the therapeutic potential of PSS in various neurodegenerative diseases. [Display omitted] • Polygala saponins (PSS) activate mitophagy via SHP-2-mediated AMPK/mTOR and PINK1/Parkin signaling pathways in BV-2 cells. • PSS promote the autophagic degradation of NLRP3 inflammasome in Aβ-, α-synuclein- or mHtt-treated BV-2 cells. • PSS rescue PC-12 cells which were damaged by the over-generated proinflammatory cytokines. • PSS improve cognitive function and ameliorate the pathology in APP/PS mice via activating the SHP-2-mediated mitophagy. [ABSTRACT FROM AUTHOR]

Abstract: Tumor necrosis factor alpha (TNFα) induces necroptosis and autophagy; however, the detailed molecular mechanism is not fully understood. In this study, we found that TNFα administration caused mitochondrial dysfunction and reactive oxygen species (ROS) production, which led to necroptosis and autophagy in murine fibrosarcoma L929 cells. Notably, the RIP1 (serine–threonine kinase receptor-interacting protein 1, a main adaptor protein of necroptosis) specific inhibitor necrostatin-1 (Nec-1) recovered mitochondrial dysfunction and ROS production due to TNFα administration. Moreover, pan-caspase inhibitor z-VAD-fmk (zVAD) increased RIP1 expression and exacerbated TNFα-induced mitochondrial dysfunction and ROS production, indicating that RIP1 led to mitochondrial dysfunction and ROS production. In addition, cytochrome c release from mitochondria was accompanied with TNFα administration, and Nec-1 blocked the release of cytochrome c upon TNFα administration, while zVAD enhanced the release. These further suggested that RIP1 induced mitochondrial dysfunction accompanied with cytochrome c release. Furthermore, autophagy inhibitor 3-methyladenine (3MA) did not affect RIP1 expression as well as mitochondrial dysfunction and ROS production. Together with our previous publication that autophagy was a downstream consequence of necroptosis, we concluded that TNFα induced mitochondrial dysfunction accompanied with ROS production and cytochrome c release via RIP1, leading to necroptosis and resulting autophagic cell death. [Copyright &y& Elsevier]

Abstract Aims In myocardial ischemia-reperfusion (MI/R) injury, impaired autophagy function worsens cardiomyocyte death. AMP-activated protein kinase (AMPK) is a heterotrimeric protein that plays an important role in cardioprotection and myocardial autophagic function. AMPKα1 and α2 are localized primarily in the cytoplasm and nucleus, respectively, in cardiomyocytes, but the isoform-specific autophagy regulation of AMPK during MI/R remains unclear. Materials and methods An MI/R model was built, and the protein expression of AMPKα1/α2, p-AMPK, mTOR, p-mTOR, TFEB, p-FoxO3a, SKP2, CARM1, TBP, Atg5, LAMP2, LC3B, and p62 during ischemia and reperfusion was determined by western blotting. Recombinant adeno-associated virus (serotype 9) vectors carrying tandem fluorescent-tagged LC3 or mRFP-GFP-LC3/GFP-LC3 were used to evaluate the autophagy status. AMPKα2 knockout mice were used for in vivo studies. Key findings Both cytoplasmic AMPKα1 and nuclear α2 subunit expression decreased during the reperfusion period, which led to AMPKα1-mTOR-TFEB and AMPKα2-Skp2-CARM1-TFEB signaling inhibition, respectively. The decreased TFEB level during reperfusion suppressed autophagy. Metformin could activate both the AMPKα1- and α2- mediated pathways, thus restoring autophagy flux during reperfusion. Nevertheless, in AMPKα2 knockout mice, nuclear α2-regulated Skp2-CARM1-TFEB signaling was inhibited, while α1-related signaling was comparatively unaffected, which partially impaired metformin-enhanced autophagy. Significance Our study suggests that metformin had the dual effects of promoting both cytoplasmic AMPKα1- and nuclear AMPKα2-related signaling to improve autophagic flux and restore cardiac function during MI/R. [ABSTRACT FROM AUTHOR]

Enhancing the clearance of proteins associated with Alzheimer's disease (AD) emerges as a promising approach for AD therapeutics. This study explores the potential of Radix Stellariae , a traditional Chinese medicine, in treating AD. Utilizing transgenic C. elegans models of AD, we demonstrated that a 75% ethanol extract of Radix Stellariae (RSE) (at 50 µg/mL) effectively diminishes Aβ and Tau protein expression, and alleviates their induced impairments including paralysis, behavioral dysfunction, neurotoxicity, and ROS accumulation. Additionally, RSE enhances the stress resistance of C. elegans. Further investigations revealed that RSE promotes autophagy, a critical cellular process for protein degradation, in these models. We found that inhibiting autophagy-related genes negated the neuroprotective effects of RSE, suggesting a central role for autophagy in the actions of RSE. In PC-12 cells, we observed that RSE not only inhibited Aβ fibril formation but also promoted the degradation of AD-related proteins and reduced their cytotoxicity. Mechanistically, RSE was found to induce autophagy via modulating PI3K/AKT/mTOR and AMPK/mTOR signaling pathways. Importantly, inhibiting autophagy counteracted the beneficial effects of RSE on the clearance of AD-associated proteins. Moreover, we identified Dichotomine B, a β-carboline alkaloid, as a key active constituent of RSE in mitigating AD pathology in C. elegans at concentrations ranging from 50 to 1000 µM. Collectively, our study presents novel discoveries that RSE alleviates AD pathology and toxicity primarily by inducing autophagy, both in vivo and in vitro. These findings open up new avenues for exploring the therapeutic potential of RSE and its active component, Dichotomine B, in treating neurodegenerative diseases like AD. [Display omitted] • RSE ameliorates AD-related pathology in both C. elegans and cellular models of AD. • RSE induces autophagy via the PI3K/AKT/mTOR and AMPK/mTOR signaling pathways. • RSE protects against AD by activating autophagy pathway in vivo and in vitro. [ABSTRACT FROM AUTHOR]

To validate the mechanism by which miR-21-5p mediates autophagy in drug-resistant cells in hepatocellular carcinoma (HCC), aggravating sorafenib resistance and progression of HCC. HCC cells were treated with sorafenib to establish sorafenib-resistant cells, and nude mice were subcutaneously injected with hepatoma cells to establish animal models. RT–qPCR was used to determine the level of miR-21-5p, and Western blotting was used to determine the level of related proteins. Cell apoptosis, cell migration, the level of LC3 were accessed. Immunohistochemical staining was used for detection of Ki-67 and LC3. A dual-luciferase reporter assay certified that miR-21-5p targets USP42, and a co-immunoprecipitation assay validated the mutual effect between USP24 and SIRT7. miR-21-5p and USP42 were highly expressed in HCC tissue and cells. Inhibition of miR-21-5p or knockdown of USP42 inhibited cell proliferation and cell migration, upregulated the level of E-cadherin, and downregulated the level of vimentin, fibronectin and N-cadherin. Overexpression of miR-21-5p reversed the knockdown of USP42. Inhibition of miR-21-5p downregulated the ubiquitination level of SIRT7, downregulated the levels of LC3II/I ratio and Beclin1, and upregulated the expression of p62. The tumor size in the miR-21-5p inhibitor group was smaller, and Ki-67 and LC3 in tumor tissue were reduced, while the overexpression of USP42 reversed the effect of the miR-21-5p inhibitor. miR-21-5p promotes deterioration and sorafenib resistance in hepatocellular carcinoma by upregulating autophagy levels. Knockdown of miR-21-5p inhibits the development of sorafenib-resistant tumors by USP24-mediated SIRT7 ubiquitination. Schematic model of the mechanism underlying miR-21-5p on sorafenib resistance in HCC. miR-21-5p promotes the deterioration and Sorafenib resistance in hepatocellular carcinoma by upregulating SIRT7-mediating autophagy levels through deubiquitinating enzyme USP24. [Display omitted] [ABSTRACT FROM AUTHOR]

• We discussed the key role of microglia and sex differences in NLRP3 inflammasome-mediated neuroinflammation. • We discussed the correlation of the over-activated NLRP3 inflammasome with the pathology of neurodegenerative diseases. • We discussed microglial autophagy and summarized the modulators in NLRP3 inflammasome-mediated neurodegenerative diseases. • The combined use of microglial autophagy inducers with the inhibitors of NLRP3 inflammasome requires further validations. Neuroinflammation is considered as a detrimental factor in neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), etc. Nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3), the most well-studied inflammasome, is abundantly expressed in microglia and has gained considerable attention. Misfolded proteins are characterized as the common hallmarks of neurodegenerative diseases due to not only their induced neuronal toxicity but also their effects in over-activating microglia and the NLRP3 inflammasome. The activated NLRP3 inflammasome aggravates the pathology and accelerates the progression of neurodegenerative diseases. Emerging evidence indicates that microglial autophagy plays an important role in the maintenance of brain homeostasis and the negative regulation of NLRP3 inflammasome-mediated neuroinflammation. The excessive activation of NLRP3 inflammasome impairs microglial autophagy and further aggravates the pathogenesis of neurodegenerative diseases. In this review article, we summarize and discuss the NLRP3 inflammasome and its specific inhibitors in microglia. The crucial role of microglial autophagy and its inducers in the removal of misfolded proteins, the clearance of damaged mitochondria and reactive oxygen species (ROS), and the degradation of the NLRP3 inflammasome or its components in neurodegenerative diseases are summarized. Understanding the underlying mechanisms behind the sex differences in NLRP3 inflammasome-mediated neurodegenerative diseases will help researchers to develop more targeted therapies and increase our diagnostic and prognostic abilities. In addition, the superiority of the combined use of microglial autophagy inducers with the specific inhibitors of the NLRP3 inflammasome in the inhibition of NLRP3 inflammasome-mediated neuroinflammation requires further preclinical and clinical validations in the future. [ABSTRACT FROM AUTHOR]

• LSP, a bioactive fraction enriching polyphenol from lychee seed, activates autophagy via the LRP1/AMPK-regulated autophagy in BV-2 cells. • LRP1 negatively regulates the NLRP3 inflammasome in Aβ(1-42)-induced BV-2 cells. • LSP inhibits the NLRP3 inflammasome-mediated neuroinflammation in Aβ(1-42)-induced BV-2 cells via the LRP1/AMPK signaling pathway. • LSP improves the cognitive function and inhibits the NLRP3 inflammasome in APP/PS1 mice. Emerging evidence indicates that the enhancement of microglial autophagy inhibits the NLRP3 inflammasome mediated neuroinflammation in Alzheimer's disease (AD). Meanwhile, low density lipoprotein receptor-related protein 1 (LRP1) highly expressed in microglia is able to negatively regulate neuroinflammation and positively regulate autophagy. In addition, we have previously reported that an active lychee seed fraction enriching polyphenol (LSP) exhibits anti-neuroinflammation in Aβ-induced BV-2 cells. However, its molecular mechanism of action is still unclear. In this study, we aim to investigate whether LSP inhibits the NLRP3 inflammasome mediated neuroinflammation and clarify its molecular mechanism in Aβ-induced BV-2 cells and APP/PS1 mice. The results showed that LSP dose- and time-dependently activated autophagy by increasing the expression of Beclin 1 and LC3II in BV-2 cells, which was regulated by the upregulation of LRP1 and its mediated AMPK signaling pathway. In addition, both the Western blotting and fluorescence microscopic results demonstrated that LSP could significantly suppress the activation of NLRP3 inflammasome by inhibiting the expression of NLRP3, ASC, the cleavage of caspase-1, and the release of IL-1β in Aβ(1-42)-induced BV-2 cells. In addition, the siRNA LRP1 successfully abolished the effect of LSP on the activation of AMPK and its mediated autophagy, as well as the inhibition of NLRP3 inflammasome. Furthermore, LSP rescued PC-12 cells which were induced by the conditioned medium from Aβ(1-42)-treated BV-2 cells. Moreover, LSP improved the cognitive function and inhibited the NLRP3 inflammasome in APP/PS1 mice. Taken together, LSP inhibited the NLRP3 inflammasome-mediated neuroinflammation in the in vitro and in vivo models of AD, which was closely associated with the LRP1/AMPK-mediated autophagy. Thus, the findings from this study further provide evidences for LSP serving as a potential drug for the treatment of AD in the future. [ABSTRACT FROM AUTHOR]