背景:蛋白质停滞是一种关键的衰老标志,负责通过自噬-溶酶体途径(ALP)和泛素-蛋白酶体系统(UPS)改善蛋白酶体降解来去除受损或错误折叠的蛋白质及其聚集体。关于热杀灭益生菌及其结构成分对衰老标志和先天免疫反应的影响的研究很少,然而,增强这些影响可能会延缓与年龄有关的疾病。
结果:这项研究引入了一种新型的热灭活短小左旋芽孢杆菌菌株MKAK9(HKMKAK9),连同它的胞外多糖(EPS),通过改善野生型秀丽隐杆线虫的蛋白稳定和免疫反应,证明了它们延长寿命的能力。我们通过涉及mRNA和小RNA测序的综合方法阐明了潜在的机制,蛋白质组学分析,对功能丧失突变体的寿命测定,和定量RT-PCR。机械上,HKMKAK9及其EPS以DAF-16依赖性方式导致胰岛素样信号通路下调,通过激活ALP途径增强蛋白质的泛素化和随后的蛋白酶体降解,部分由mir-243microRNA介导。重要的是,自噬体吞噬泛素化蛋白,自噬受体sqst-3的表达增加,随后与溶酶体融合,由溶酶体相关膜蛋白(LAMP)lmp-1水平升高促进,表明自溶酶体的形成用于降解所选货物。此外,HKMKAK9及其EPS激活p38MAPK通路及其下游SKN-1转录因子,已知它们调节与先天免疫反应有关的基因(thn-1,ilys-1,cnc-2,spp-9,spp-21,clec-47和clec-266)和抗氧化(sod-3和gst-44),从而减少活性氧(ROS)在细胞和线粒体水平的积累。值得注意的是,SOD-3作为DAF-16和SKN-1转录因子的转录靶标出现。
结论:我们的研究通过证明热灭活益生菌及其特定的细胞成分,为未来的研究树立了基准。EPS,可以下调胰岛素信号通路,可能改善自噬-溶酶体途径(ALP),以降解泛素化蛋白并促进生物体寿命。此外,我们发现mir-243microRNA的表达增加调节胰岛素样信号及其下游ALP通路.我们的发现还表明,后生物治疗可能会增强抗氧化和先天免疫反应,为干预与衰老相关的疾病提供了一个有希望的途径。
BACKGROUND: Proteostasis is a critical aging hallmark responsible for removing damaged or misfolded proteins and their aggregates by improving proteasomal degradation through the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS). Research on the impact of heat-killed probiotic bacteria and their structural components on aging hallmarks and innate immune responses is scarce, yet enhancing these effects could potentially delay age-related diseases.
RESULTS: This study introduces a novel heat-killed Levilactobacillus brevis strain MKAK9 (HK MKAK9), along with its exopolysaccharide (EPS), demonstrating their ability to extend longevity by improving proteostasis and immune responses in wild-type Caenorhabditis elegans. We elucidate the underlying mechanisms through a comprehensive approach involving mRNA- and small RNA sequencing, proteomic analysis, lifespan assays on loss-of-function mutants, and quantitative RT-PCR. Mechanistically, HK MKAK9 and its EPS resulted in downregulation of the insulin-like signaling pathway in a DAF-16-dependent manner, enhancing protein ubiquitination and subsequent proteasomal degradation through activation of the ALP pathway, which is partially mediated by microRNA mir-243. Importantly, autophagosomes engulf ubiquitinylated proteins, as evidenced by increased expression of the autophagy receptor sqst-3, and subsequently fuse with lysosomes, facilitated by increased levels of the lysosome-associated membrane protein (LAMP) lmp-1, suggesting the formation of autolysosomes for degradation of the selected cargo. Moreover, HK MKAK9 and its EPS activated the p38 MAPK pathway and its downstream SKN-1 transcription factor, which are known to regulate genes involved in innate immune response (thn-1, ilys-1, cnc-2, spp-9, spp-21, clec-47, and clec-266) and antioxidation (sod-3 and gst-44), thereby reducing the accumulation of reactive oxygen species (ROS) at both cellular and mitochondrial levels. Notably, SOD-3 emerged as a transcriptional target of both DAF-16 and SKN-1 transcription factors.
CONCLUSIONS: Our research sets a benchmark for future investigations by demonstrating that heat-killed probiotic and its specific cellular component, EPS, can downregulate the insulin-signaling pathway, potentially improving the autophagy-lysosome pathway (ALP) for degrading ubiquitinylated proteins and promoting organismal longevity. Additionally, we discovered that increased expression of microRNA mir-243 regulates insulin-like signaling and its downstream ALP pathway. Our findings also indicate that postbiotic treatment may bolster antioxidative and innate immune responses, offering a promising avenue for interventions in aging-related diseases.