Novel 18-norspirostane steroidal saponins: Extending lifespan and mitigating neurodegeneration through promotion of mitophagy and mitochondrial biogenesis in Caenorhabditis elegans.

Pharmacological strategies to delay aging and combat age-related diseases are increasingly promising. This study explores the anti-aging and therapeutic effects of two novel 18-norspirostane steroidal saponins from Trillium tschonoskii Maxim, namely deoxytrillenoside CA (DTCA) and epitrillenoside CA (ETCA), using Caenorhabditis elegans (C. elegans). Both DTCA and ETCA significantly extended the lifespan of wild-type N2 worms and improved various age-related phenotypes, including muscle health, motility, pumping rate, and lipofuscin accumulation. Furthermore, these compounds exhibited notable alleviation of pathology associated with Parkinson's disease (PD) and Huntington's disease (HD), such as the reduction of α-synuclein and poly40 aggregates, improvement in motor deficits, and mitigation of neuronal damage. Meanwhile, DTCA and ETCA improved the lifespan and healthspan of PD- and HD-like C. elegans models. Additionally, DTCA and ETCA enhanced the resilience of C. elegans against heat and oxidative stress challenges. Mechanistic studies elucidated that DTCA and ETCA induced mitophagy and promoted mitochondrial biogenesis in C. elegans, while genetic mutations or RNAi knockdown affecting mitophagy and mitochondrial biogenesis effectively eliminated their capacity to extend lifespan and reduce pathological protein aggregates. Together, these compelling findings highlight the potential of DTCA and ETCA as promising therapeutic interventions for delaying aging and preventing age-related diseases.

Lychee seed polyphenol ameliorates DR via inhibiting inflammasome/apoptosis and angiogenesis in hRECs and db/db mice.

Blood retinal barrier (BRB) damage is an important pathogenesis of diabetic retinopathy, and alleviating BRB damage has become a key target for DR treatment. We previously found that Lycopene seed polyphenols (LSP) maintained BRB integrity by inhibiting NLRP3 inflammasome-mediated inflammation. However, it is still unknown whether LSP inhibits retinal neovascularization with abnormal capillaries and its mechanism of action. Here, we employed db/db mice and hRECs to find that LSP increases the level of glycolipid metabolism, maintains the morphology of retinal endothelial cells and inhibits acellular capillary neogenesis. Mechanistic studies revealed that LSP inhibits the NLRP3 inflammasome, reduces cell apoptosis in retinal tissue, increases tight junction protein (TJ) expression, and reduces vascular endothelial growth factor (VEGF) and Ve-Cadherin in vivo and in vitro. Collectively, this study finds that LSP inhibits inflammation and angiogenesis to improve BRB function to ameliorate DR.

Folium Hibisci Mutabilis extract, a potent autophagy enhancer, exhibits neuroprotective properties in multiple models of neurodegenerative diseases.

BACKGROUND: 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. OBJECTIVE: In this study, we aimed to screen natural autophagy enhancers from traditional Chinese medicines (TCMs) presenting potent neuroprotective potential in multiple ND models. METHODS: 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 Aß-, 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. RESULTS: 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 Aß, 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. CONCLUSION: 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.

Chlorogenic acid delays the progression of Parkinson's disease via autophagy induction in Caenorhabditis elegans.

OBJECTIVES: Parkinson's disease (PD) is the second most common neurodegenerative disease. Chlorogenic acid (CGA) is a polyphenolic substance derived from various medicinal plants. Although CGA is reported to have potential anti-PD effect, the beneficial effect and the underlying mechanism remain unclear. In this study, we aimed to further investigate the protective effect and clarify the mechanism of action of CGA in Caenorhabditis elegans (C. elegans) models of PD. METHODS: Measurements of a-synuclein aggregation, movement disorders, and lipid, ROS and malondialdehyde (MDA) contents were observed in NL5901 nematodes. Determinations of dopamine (DA) neuron degeneration, food perception, and ROS content were performed in 6-OHDA-exposed BZ555 nematodes. The autophagy activation of CGA was monitored using DA2123 and BC12921 nematodes. Meanwhile, RNAi technology was employed to knockdown the autophagy-related genes and investigate whether the anti-PD effect of CGA was associated with autophagy induction in C. elegans. RESULTS: CGA significantly reduced α-synuclein aggregation, improved motor disorders, restored lipid content, and decreased ROS and MDA contents in NL5901 nematodes. Meanwhile, CGA inhibited DA neuron-degeneration and improved food-sensing behavior in 6-OHDA-exposed BZ555 nematodes. In addition, CGA increased the number of GFP::LGG-1 foci in DA2123 nematodes and degraded p62 protein in BC12921 nematodes. Meanwhile, CGA up-regulated the expression of autophagy-related genes in NL5901 nematodes. Moreover, the anti-PD effect of CGA was closely related to autophagy induction via increasing the expression of autophagy-related genes, including unc-51, bec-1, vps-34, and lgg-1. CONCLUSIONS: The present study indicates that CGA exerts neuroprotective effect in C. elegans via autophagy induction.

Citri Reticulatae Semen Extract Promotes Healthy Aging and Neuroprotection via Autophagy Induction in Caenorhabditis elegans.

Nutrition intervention has emerged as a potential strategy to delay aging and promote healthy longevity. Citri Reticulatae Semen (CRS) has diverse beneficial effects and has been used for thousands of years to treat pain. However, the health benefits of CRS in prolonging health span and improving aging-related diseases and the exact mechanisms remain poorly characterized. In this study, Caenorhabditis elegans (C. elegans) was used as a model organism to study the antiaging and health span promoting activities of 75% ethanol extract of CRS (CRSE). The results showed that treatment with CRSE at 1 000 µg/mL significantly extended the life span of worms by 18.93% without detriment to health span and fitness, as evidenced by the delayed aging-related phenotypes and increased body length and width, and reproductive output. In addition, CRSE treatment enhanced the ability of resistance to heat, oxidative, and pathogenic bacterial stress. Consistently, heat shock proteins and antioxidant enzyme-related and pathogenesis-related genes were up-regulated by CRSE treatment. Furthermore, CRSE supplementation also improved α-synuclein, 6-OHDA, and polyQ40-induced pathologies in transgenic C. elegans models of Parkinson's disease and Huntington's disease. The mechanistic study demonstrated that CRSE induced autophagy in worms, while the RNAi knockdown of 4 key autophagy-related genes, including lgg-1, bec-1, vps-34, and unc-51, remarkably abrogated the beneficial effects of CRSE on the extending of life span and health span and neuroprotection, demonstrating that CRSE exerts beneficial effects via autophagy induction in worms. Together, our current findings provide new insights into the practical application of CRS for the prevention of aging and aging-related diseases.

Ferulic Acid Exerts Neuroprotective Effects via Autophagy Induction in C. elegans and Cellular Models of Parkinson's Disease.

Parkinson's disease (PD) is a complex neurological disorder characterized by motor and nonmotor features. Although some drugs have been developed for the therapy of PD in a clinical setting, they only alleviate the clinical symptoms and have yet to show a cure. In this study, by employing the C. elegans model of PD, we found that ferulic acid (FA) significantly inhibited α-synuclein accumulation and improved dyskinesia in NL5901 worms. Meanwhile, FA remarkably decreased the degeneration of dopaminergic (DA) neurons, improved the food-sensing behavior, and reduced the level of reactive oxygen species (ROS) in 6-OHDA-induced BZ555 worms. The mechanistic study discovered that FA could activate autophagy in C. elegans, while the knockdown of 3 key autophagy-related genes significantly revoked the neuroprotective effects of FA in α-synuclein- and 6-OHDA-induced C. elegans models of PD, demonstrating that FA exerts an anti-PD effect via autophagy induction in C. elegans. Furthermore, we found that FA could reduce 6-OHDA- or H2O2-induced cell death and apoptosis in PC-12 cells. Moreover, FA was able to induce autophagy in stable GFP-RFP-LC3 U87 cells and PC-12 cells, while bafilomycin A1 (Baf, an autophagy inhibitor) partly eliminated the protective effects of FA against 6-OHDA- and H2O2-induced cell death and ROS production in PC-12 cells, further confirming that FA exerts an anti-PD effect via autophagy induction in vitro. Collectively, our study provides novel insights for FA as a potent autophagy enhancer to effectively prevent neurodegenerative diseases such as PD in the future.

Longitudinal Dynamics of the Neutralizing Antibody Response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a global pandemic with no licensed vaccine or specific antiviral agents for therapy. Little is known about the longitudinal dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) in patients with COVID-19. METHODS: Blood samples (n = 173) were collected from 30 patients with COVID-19 over a 3-month period after symptom onset and analyzed for SARS-CoV-2-specific NAbs using the lentiviral pseudotype assay, coincident with the levels of IgG and proinflammatory cytokines. RESULTS: SARS-CoV-2-specific NAb titers were low for the first 7-10 days after symptom onset and increased after 2-3 weeks. The median peak time for NAbs was 33 days (interquartile range [IQR], 24-59 days) after symptom onset. NAb titers in 93.3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34.8% (IQR, 19.6-42.4%). NAb titers increased over time in parallel with the rise in immunoglobulin G (IgG) antibody levels, correlating well at week 3 (r = 0.41, P < .05). The NAb titers also demonstrated a significant positive correlation with levels of plasma proinflammatory cytokines, including stem cell factor (SCF), TNF-related apoptosis-inducing ligand (TRAIL), and macrophage colony-stimulating factor (M-CSF). CONCLUSIONS: These data provide useful information regarding dynamic changes in NAbs in patients with COVID-19 during the acute and convalescent phases.

A Peptide-Based Magnetic Chemiluminescence Enzyme Immunoassay for Serological Diagnosis of Coronavirus Disease 2019.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel ß-coronavirus, causes severe pneumonia and has spread throughout the globe rapidly. The disease associated with SARS-CoV-2 infection is named coronavirus disease 2019 (COVID-19). To date, real-time reverse-transcription polymerase chain reaction (RT-PCR) is the only test able to confirm this infection. However, the accuracy of RT-PCR depends on several factors; variations in these factors might significantly lower the sensitivity of detection. METHODS: In this study, we developed a peptide-based luminescent immunoassay that detected immunoglobulin (Ig)G and IgM. The assay cutoff value was determined by evaluating the sera from healthy and infected patients for pathogens other than SARS-CoV-2. RESULTS: To evaluate assay performance, we detected IgG and IgM in the sera from confirmed patients. The positive rate of IgG and IgM was 71.4% and 57.2%, respectively. CONCLUSIONS: Therefore, combining our immunoassay with real-time RT-PCR might enhance the diagnostic accuracy of COVID-19.

Hepatitis C virus core protein-induced miR-93-5p up-regulation inhibits interferon signaling pathway by targeting IFNAR1.

AIM: To investigate the mechanism by which hepatitis C virus (HCV) core protein-induced miR-93-5p up-regulation regulates the interferon (IFN) signaling pathway. METHODS: HCV-1b core protein was exogenously expressed in Huh7 cells using pcDNA3.1 (+) vector. The expression of miR-93-5p and interferon receptor 1 (IFNAR1) was measured using quantitative reverse transcription-polymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of miR-93-5p and IFNAR1 were performed using miR-93-5p agomir and antagomir, and pcDNA3.1-IFNAR1 and IFNAR1 siRNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of miR-93-5p. Cellular experiments were also conducted. RESULTS: Serum miR-93-5p level was increased in patients with HCV-1b infection and decreased to normal level after HCV-1b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum miR-93-5p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1b core protein increased miR-93-5p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of miR-93-5p, and IFNAR1 restore could rescue miR-93-5p-reduced STAT1 phosphorylation, suggesting that the miR-93-5p-IFNAR1 axis regulates the IFN signaling pathway. CONCLUSION: HCV-1b core protein-induced miR-93-5p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the miR-93-5p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1b infection.

[Construction of a full-genome HCV replicon with self-cleaving double ribozyme sequences and characterization in vitro and in vivo].

OBJECTIVE: To construct a full-genome hepatitis C virus (HCV) replicon that will allow for direct initiation of replication and generation of infectious viral particles in an in vitro and in vivo cell system. METHODS: Self-cleaving ribozyme sequences were added to each side of the HCV cDNA clone JFH1 and the replication-deficient clone JFH1/GND, then inserted into the pcDNA3.1 vector downstream of the CMV promoter. The resultant recombinant plasmids, pcDNA3.1-RZ-JFH1 and pcDNA3.1-RZ-JFH1/GND, were tested for activity in vitro and in vivo by transiently transfecting into Huh7.5 cells (5 mug/100 mm culture dish) and injecting by high-pressure tail vein injection into Kunming mice (10 - 30 mug/mouse). Quantitative reverse transcription-PCR, immunofluorescence, immunohistochemistry, and serological testing were performed to determine the replication ability and assess the properties of the recombinant plasmids in the two systems. RESULTS: HCV RNA (1 - 3 * 10(6) copies/ml) was detected in the supernatant of transfected Huh7.5 cells up to 16 weeks after transfection. In addition, the viral particles from the supernatant were able to infect nave Huh7.5 cells. However, only transient viremia was achieved upon tail vein injection of the plasmid, and no HCV antigen-positive cells were detected by immunohistochemistry nor HCV-specific antibodies by serological testing. CONCLUSION: The constructed HCV replicon was capable of stable expression in cultured cells and of efficiently generating infectious viral particles in the in vitro system over a long period. However, the HCV replicon did not show infective characteristics in an in vivo mouse system. The full-length HCV replicon may represent a useful tool for in vitro study of HCV pathological mechanisms, possibly including anti-HCV drug screening.