图1 UHPLC-HRMS总离子流图

Fig.1 UHPLC-HRMS total ion chromatogram (TIC) of Xiaoyao San (XYS). A: Positive ion mode (POS). B: Negative ion mode (NEG).

图2 XYS干预能够延长PD线虫寿命并改善其活动能力

Fig.2 XYS extends lifespan and improves locomotor activity of C. elegans PD models. A: Survival curves of C. elegans exposed to different concentrations of XYS. B: Schematic diagram of the experimental design for combined 6-OHDA and XYS treatment. C: Survival curves of 6-OHDA-induced PD model worms with or without XYS treatment. D: Quantification of the basal slowing response. E: Quantification of body bends within 20 s on days 5 and 10 post-treatment. Data represent Mean±SD from at least 3 independent biological replicates (n=50 for lifespan assays and n=30 for other assays). #P<0.05, ##P<0.01 vs control group; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs model group.

图3 XYS干预能够延长PD转基因模型NL5901线虫寿命并改善其体内的α-syn聚集

Fig.3 XYS extends lifespan and reduces α-syn aggregation in NL5901 worms. A: Survival curves of NL5901 worms with or without XYS treatment. B: Quantification of body bends within 20 s on days 5 and 10 post-treatment. C: Representative images of α‑syn::YFP fluorescence in body wall muscle cells and quantification of fluorescence intensity (Scale bar=100 μm). Data represent Mean±SD from at least 3 independent biological replicates (n=50 for lifespan assays and n=30 for other assays). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs control group.

图4 XYS干预显著改善6-OHDA诱导的PD线虫模型多巴胺能神经元变性

Fig.4 XYS protects against 6-OHDA-induced dopaminergic neurodegeneration in C. elegans. A: Representative GFP images of dopaminergic neurons with or without XYS treatment and quantification of fluorescence intensity (Scale bar=100 μm). B: Relative mRNA expression levels of dat-1, cat-2, and ced-9. Data represent Mean±SD from at least three independent biological replicates (n=30 for fluorescence analysis and n=3 for qPCR analysis). ##P<0.01, ###P<0.001, ####P<0.0001 vs control group; **P<0.01, ***P<0.001, ****P<0.0001 vs model group.

图5 XYS干预能够显著改善线虫体内抗氧化效应

Fig.5 XYS significantly enhances antioxidant capacity in C. elegans. A: Representative fluorescence images of sod-3::GFP worms and quantification of sod-3::GFP fluorescence intensity (Scale bar=100 μm). B: Relative mRNA expression levels of sod-3 and gst-4. Data represent Mean±SD from at least 3 independent biological replicates (n=30 for fluorescence analysis and n=3 for qPCR analysis). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs control group.

图6 XYS干预能够显著降低不同PD线虫模型体内氧化应激水平

Fig.6 XYS significantly reduces oxidative stress levels in different C. elegans models of PD. A: Representative DHE staining images of NL5901 worms with or without XYS treatment and quantification of DHE fluorescence intensity. B: Representative DHE staining images of 6-OHDA-treated N2 worms with or without XYS treatment and quantification of DHE fluorescence intensity. Scale bar=100 μm. Data represent Mean±SD from at least 3 independent biological replicates (n=30). ####P<0.0001 vs control group; *P<0.05, **P<0.01 vs model group.

图7 XYS通过上调SKN-1改善PD线虫模型抗氧化效应

Fig.7 XYS improves antioxidant responses in PD C. elegans models through upregulation of SKN-1. A: Representative fluorescence images of LD1 worms. B: Quantification of fluorescence intensity in LD1 worms. C: Quantification of fluorescence intensity in CF1553 worms. D: Representative fluorescence images of CF1553 worms. E: Representative fluorescence images of skn-1 RNAi-treated worms and quantification of fluorescence intensity. Scale bar=100 μm. Data represent Mean±SD from at least 3 independent biological replicates (n=30). ##P<0.01, ###P<0.001 vs control group; #P<0.05 vs skn-1 RNAi group; *P<0.05, **P<0.01 vs model group.

图8 XYS干预能够改善PD线虫模型脂质代谢异常

Fig.8 XYS ameliorates lipid metabolic dysfunction in PD C. elegans models. A: Representative fluorescence images of LIU104 worms and quantification of fluorescence intensity. B: Representative fluorescence images of DMS303 worms and quantification of fluorescence intensity. C: Representative Nile Red staining images of NL5901 worms and quantification of fluorescence intensity. Scale bar=100 μm. Data represent Mean±SD from at least 3 independent biological replicates (n=30). #P<0.05, ##P<0.01 vs control group; *P<0.05, **P<0.01 vs model group.

图9 XYS通过skn-1调节α-syn蓄积及脂质积累

Fig.9 XYS regulates α-synuclein accumulation and lipid deposition via skn-1. A: Representative fluorescence images of skn-1 RNAi worms and quantification of fluorescence intensity. B: Representative Nile Red staining images of skn-1 RNAi worms and quantification of lipid accumulation based on Nile Red staining. Scale bar=100 μm. Data represent Mean±SD from at least 3 independent biological replicates (n=30). ##P<0.01, ###P<0.001 vs control group.