Oxidative stress-induced DNA damage, the senescence-associated secretory phenotype (SASP), and impaired autophagy all are general features of senescent cells. However, the cross-talk among these events and processes is not fully understood. Here, using NIH3T3 cells exposed to hydrogen peroxide stress, we show that stress-induced DNA damage provokes the SASP largely via cytosolic chromatin fragment (CCF) formation, which activates a cascade comprising cGMP-AMP synthase (cGAS), stimulator of interferon genes protein (STING), NF-κB, and SASP, and that autolysosomal function inhibits this cascade. We found that CCFs accumulate in senescent cells with activated cGAS-STING-NF-κB signaling, promoting SASP and cellular senescence. We also present evidence that the persistent accumulation of CCFs in prematurely senescent cells is partially associated with a defect in DNA-degrading activity in autolysosomes and reduced abundance of activated DNase 2α. Intriguingly, we found that metformin- or rapamycin-induced activation of autophagy significantly lessened the size and levels of CCFs and repressed the activation of the cGAS-STING-NF-κB-SASP cascade and cellular senescence. These effects of autophagy activators indicated that autolysosomal function contributes to CCF clearance and SASP suppression, further supported by the fact that the lysosome inhibitor bafilomycin A1 blocked the role of autophagy-mediated CCF clearance and senescence repression.

Aim To explore the function and mechanisms of NLRP6 (NOD‐, LRR‐ and pyrin domain‐containing 6) in the inflammatory response of human periodontal ligament cells (HPDLCs). Methodology Tissues associated with apical periodontitis were obtained from three patients who underwent endodontic microsurgery. The expression of NLRP6 in 3 human apical periodontitis tissues and HPDLCs was examined by immunohistochemistry and immunofluorescence, respectively. The expressions of NLRP6, Phospho(p)‐ p65, p65, IκB‐α, p‐ IκB‐α, ERK, p‐ ERK, NLRP3, Pro interleukin (IL)‐1β, Pro caspase‐1 and apoptosis‐associated speck‐like protein containing a CARD (ASC) were examined by western blot. The gene expression and secretion of proinflammatory cytokines were detected using quantitative real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay. Data were analysed statistically with independent sample t‐tests. Results NLRP6 was expressed in inflammatory periapical tissues and HPDLCs. Lipopolysaccharide (LPS) from Escherichia coli induced NLRP6 in HPDLCs (P < 0.05). After silencing NLRP6, E. coli LPS‐induced activation of NF‐κB and ERK signalling was enhanced, which was also accompanied by elevated levels of IL‐6 and tumour necrosis factor‐α (TNF‐α) (P < 0.05). Moreover, knockdown of NLRP6 led to up‐regulation of NLRP3, Pro IL‐1β and Pro caspase‐1 (P < 0.05), whereas down‐regulation of ASC (P < 0.05), which may contribute to unchanged levels of IL‐1β in HPDLCs inflammation. Conclusion NLRP6 was functionally expressed in inflamed periapical tissues and HPDLCs. NLRP6 negatively regulated the production of IL‐6 and TNF‐α in HPDLCs inflammation by inhibiting NF‐κB and ERK signal pathways.

Nicotinamide adenine dinucleotide (NAD+) is indispensable for the anti-aging activity of the sirtuin (SIRT) family enzymes. AMP-activated protein kinase (AMPK) upregulates NAD+ synthesis and SIRT activity in a nicotinamide phosphoribosyltransferase (NAMPT)-dependent manner. However, the molecular mechanisms that affect AMPK-driven NAMPT expression and NAD+/SIRT activation remain unclear. In this study, we tried to identify senescence-associated microRNAs (miRNAs) that negatively regulate the cascade linking AMPK and NAMPT expression. miRNA-screening experiments showed that the expression of miR-146a increased in senescent cells but decreased following AMPK activation. Additionally, miR-146a overexpression weakened the metformin-mediated upregulation of NAMPT expression, NAD+ synthesis, SIRT activity, and senescence protection, whereas treatment with the miR-146a inhibitor reversed this effect. Importantly, these findings were observed both in vitro and in vivo. Mechanistically, miR-146a directly targeted the 3′-UTR of Nampt mRNA to reduce the expression of NAMPT. AMPK activators metformin and 5-aminoimidazole-4-carboxamide (AICAR) hindered miR-146a expression at the transcriptional level by promoting IκB kinase (IKK) phosphorylation to attenuate nuclear factor-kappaB (NF-κB) activity. These findings identified a novel cascade that negatively regulates the NAD+/SIRT pathway by suppressing miR-146a-mediated NAMPT downregulation. Furthermore, our results showed that miR-146a impedes the anti-aging effect of AMPK. This mutual inhibitory relationship between miR-146a and AMPK enriches our understanding of the molecular connections between AMPK and SIRT and provides new insight into miRNA-mediated NAD+/SIRT regulation and an intervention point for the prevention of aging and age-related diseases.

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Signal transducer and activator of transcription 3 (STAT3) is implicated in inflammation processing, but the mechanism of its regulation mostly remains limited to Janus kinase (JAK)-mediated phosphorylation. Although AMP-activated protein kinase (AMPK)-mediated STAT3 inactivation has got documented, the molecular signaling cascade connecting STAT3 inactivation and the anti-inflammatory role of AMPK is far from established. In the present study, we addressed the interplay between AMPK and STAT3, and revealed the important role of STAT3 inactivation in the anti-inflammatory function of AMPK in lipopolysaccharide-stressed macrophages and mice. Firstly, we found that pharmacological inhibition of STAT3 can improve the anti-inflammatory effect of AMPK in wild-type mice, and the expression of STAT3 in macrophage of mice is a prerequisite for the anti-inflammatory effect of AMPK. As to the molecular signaling cascade linking AMPK to STAT3, we disclosed that AMPK suppressed STAT3 not only by attenuating JAK signaling but also by activating nuclear factor erythroid-2-related factor-2 (Nrf2), a redox-regulating transcription factor, which consequently increased the expression of small heterodimer protein (SHP), thus repressing the transcriptional activity of STAT3. In summary, this study provided a unique set of evidence showing the relationship between AMPK and STAT3 signaling and explored a new mechanism of AMPK-driven STAT3 inactivation that involves Nrf2-SHP signaling cascade. These findings expand our understanding of the interplay between pro-and anti-inflammatory signaling pathways and are beneficial for the therapeutic development of sepsis treatments.