The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90.The online version contains supplementary material available at 10.1007/s00401-022-02491-8. SNCA) is causally linked to Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB) 1/2, where ∆δ1H and ∆δ15N are the changes in 1H and 15 N chemical shifts (in ppm) upon the addition of binding target. The binding affinities were determined based on the chemical shift perturbations and protein concentrations determined by amino acid analysis , assuming fast exchange between the free and ligand-bound forms [NMR experiments were conducted at 25 °C on a 600 MHz Varian Inova spectrometer equipped with a cryogenic triple resonance HCN probe. The nd forms .For M83+/−:TgA and M83+/− ::ΔTPR1 PFF and PBS-injected mice, motor assessments were performed at 14–16 wpi (the time-point when the majority of cohort began to significantly deteriorate and show slow movement and gait disturbance). The experimenter was blind to genotype during motor assessments, but not during data analyses.Grip force in the forelimbs was measured using Columbus Instruments Grip Strength Meter. Animals were lowered, allowing them to grip onto grip strength bar and were pulled horizontally by the tail until they let go, and placed in a large plexiglass bucket filled with bedding. The force exerted as the mouse let go of the bar was recorded in Newtons, with a total of 10 pulls per mouse being recorded. The largest value out of ten was used for analyses as described previously , 72, 98.Wire hang was performed as described elsewhere , 72. Micwww.mousebytes.ca [All behavioral tests were conducted during the light phase. Paired visual discrimination task with reversal (PVD-R) and the 5-Choice serial reaction time task (5-CSRTT) were used to evaluate reversal learning and attention in mice, respectively, as previously described , 72, 104bytes.ca ). The gebytes.ca , 72.n = 10) and non-transgenic WT controls (n = 12) at 8 months of age.The 5-CSRTT was performed to measure attention in mice as previously described , 71. A mn = 20) and littermate M83+/+ :ΔHET mice (n = 18) and WT controls (n = 20).In the acquisition phase, mice were required to choose between a rewarded  and unrewarded  stimulus displayed in a mask with two windows placed in front of the touchscreen. The location of the S+ and S- stimuli was pseudo-randomly either at the left or right window and the same stimulus arrangement was not presented more than 3 times. When the mouse touched the S+ (correct), the stimuli were removed, and the strawberry milkshake reward was delivered along with illumination of the magazine light and a tone. An incorrect response (touching the S− image) was followed by a 5 s timeout with the house light on. After an incorrect response, the mouse initiated the correction trial by entering the magazine. Correction trials preserved the left/right arrangement of the S+/S− images from the incorrect trial until a correct choice was made. The results of correction trials do not contribute to the overall trial count or correct/incorrect responses. Each session ended once the mouse completed 30 trials or reached a 60-min timeout. The mouse was required to achieve at least 80% correct responses  for 2 out of 3 consecutive days to reach acquisition criterion. Once all animals in a cohort had reached the acquisition criterion, they were put back on the task and received 2 further task sessions that served as baseline performance (B1 and B2). Baseline sessions were identical to the PVD acquisition and there were no criteria required. Following the baseline sessions, mice were tested on the reversal learning task for 30 trials per session, for 10 sessions. In the reversal phase, the S+ and S− contingencies were reversed, i.e., S+ was the marble image and the S− the fan image. Trial initiation and correction trials happened in the same fashion as during acquisition and there were no criteria required for the reversal phase. The session also ended either after completion of 30 trials or in 60 min. The measurements were compared between M83+/+ : STI1WT mice (n = 8), M83+/+ :STI1WT mice (n = 5) and M83+/+ :ΔHET mice (n = 8). These mice were perfused with 0.9% (wt/vol) phosphate-buffered saline with 0.4% ProHance , followed by 4% paraformaldehyde with 0.1% heparin. To best acquire ex vivo images, the brains were kept in-skull and the surrounding tissue  was also kept, as this reduces the likelihood of scanner artifacts, specifically caused by air bubbles [A subset of mice used for touchscreen testing was prepared for ex vivo MRI; WT control mice ( bubbles . Prior tThe MRI acquisition was performed on 7.0-T Bruker Biospec (70/30 USR) 30-cm inner bore diameter; AVANCE electronics) at the Douglas Research Centre . High-resolution ex vivo T1-weighted images  were acquired for each subject .http://bic-mni.github.io). Next, the images were stripped of their native coordinate system, left–right flipped to compensate for Bruker’s native radiological coordinate system, denoised using patch-based adaptive non-local means algorithm [All brain images were converted to NIfTI format (Neuroimaging Informatics Technology Initiative) from the native Bruker format, and then converted to the MINC  file format for processing. Image processing was performed using the MINC suite of software tools  tolgorithm  at a minn = 1) ).The images were manually inspected for artifacts (hardware and software artifacts or tissue heterogeneity and foreign bodies) both prior to and after preprocessing, and images with said artifacts were excluded (https://github.com/vfonov/mincANTS), nonlinear registration techniques are implemented to first propagate the atlas segmentations onto a subset of the subject data, known as the template library. From there, the segmentations from the template library are then propagated to each subject image yielding multiple segmentations per subject, which are then fused using majority-vote label fusion to generate a final segmentation. From these final segmentations, volumes of brain structures were computed and analyzed for group differences. A full list of the anatomical regions segmented in the modified Allen brain atlas is described in Supplementary Table 3.The preprocessed MRI images were segmented into 355 structures per hemisphere (with 53 interhemispheric regions) using the Multiple Automatically Generated Templates (MAGeT) Brain segmentation algorithm , and a dPrior to performing MRI analysis, quality control of the segmentations was performed by visual inspection of overlaying the final segmentations atop each subject image to ensure the labels matched the underlying anatomy , 60, 103https://wiki.phenogenomics.ca/display/MICePub/RMINC). The analysis was performed using linear models to examine group differences at each region using the MAGeT-Brain volume outputs, specifically to examine group differences between WT control mice, M83+/+ : STI1WT, and M83+/+ :ΔHET mice. All results were corrected for multiple comparisons using the FDR [https://doi.org/10.5281/zenodo.6620797.Statistical analyses were carried out using R software (3.5.0) and the RMINC package . For the 5-CSRTT, accuracy, omission, reward collection latency, correct touch latency, premature responses, number of trials completed, and perseverative response were recorded and analyzed using repeated-measures two-way ANOVA. For the PVD-R task, the parameters number of sessions to reach acquisition criteria , the percentage of accuracy, number of correction trials, correct touch latency (s), reward collection latency and trials completed  were recorded. Data are presented as mean ± SEM and (*) denotes p < 0.05, (**) denotes p < 0.01, (***) denotes p < 0.001. For co-immunoprecipitation experiments, individual experiments were repeated three times, with the cortices of two different animals constituting each IP condition (n = 6) different animal tissue lysates being used in each technical replicate.Data were analyzed using GraphPad Prism v8 software. Two group comparisons were analyzed using unpaired two-tailed t test. Whereas analyses comparing three groups were analyzed using one-way ANOVA with appropriate post hoc comparisons, and multivariate two group comparisons were analyzed using two-way ANOVA with appropriate post hoc comparisons when required. Normality for behavioral data was evaluated using Shapiro–Wilk test and non-normal data were analyzed using Kruskal–Wallis test (non-parametric One-Way ANOVA) or non-parametric 1H-15 N HSQC experiments on 15 N-labelled recombinant human α-synuclein in the absence and presence of STI1. Upon adding recombinant mouse STI1 protein to a solution of 15 N-labelled human α-synuclein, we observed noticeable chemical shift perturbations in the C-terminal region of α-synuclein . The apparent KD derived from the chemical shift perturbations is 176 ± 15 µM.In vitro evidence suggests that most α-synuclein in cells form complexes with chaperones, including Hsp70 and Hsp90, and interactome analyses immunoprecipitating α-synuclein indicate that STI1 may also be an interactor of α-synuclein . We testD of 79 ± 5 µM, Supp Fig. S1d, online resource). In contrast, neither TPR1 nor TPR2B induced any significant chemical shift changes on α-synuclein . We also investigated whether α-synuclein can trigger chemical shift changes in the STI1 TPR2A domain. Upon addition of an equimolar concentration of α-synuclein to 15 N-labelled STI1 TPR2A, noticeable chemical shift changes were observed for a significant number of residues, including K239, D249, T260, Y261, T263, Y296, and R331 to name a few. Remarkably, many of these amino acid residues in TPR2A that show chemical shift perturbations are adjacent to the regions interacting with Hsp90  domains that are modular protein–protein interaction sites. Therefore, we repeated the NMR experiment with each of these individual domains separately to investigate their potential interaction with α-synuclein  and electron microscopy, confirming that the sonicated fibrils were between 5 and 100 nm in diameter . M83+/− mice injected with PFFs showed robust psyn129 labelling throughout the brain in both hemispheres, whereas PBS-injected M83+/− mice had no labelling for psyn129 . PFF-injected mice became symptomatic between 12 and 16 weeks post-injection, presenting decreased mobility and grooming, as well as weight loss, at which point they were euthanized. Immunocytochemistry experiments show that ubiquitin and Hsp90 partially co-localize with psyn129  in PFF-injected mice, confirming previous results that these proteins are deposited in filamentous inclusions , 121. NoTo evaluate how spreading of misfolded α-synuclein affects the chaperome, we compared STI1, Hsp90, and Hsp70 presence in detergent-soluble or insoluble fractions in PFF-injected mice and PBS-injected mice, using immunoblots. In RIPA-soluble fractions, psyn129 is increased in PFF-injected mice, and the levels of STI1, Hsp90, and Hsp70 did not change significantly when compared to PBS-injected M83 mice  in four brain regions of young and aged  men and women donors of the GTEx dataset . These results propose that that two of the regions significantly affected in synucleinopathies could be impacted by levels of STI1 and its regulation of the chaperome [The above experiments and previous work , 86, 121haperome , 108.Given the increased sequestration of Hsp70, Hsp90, and STI1 to insoluble fractions in the brains of PFF-injected M83+/− mice, and the increased levels of STI1 and other chaperone transcripts in PD Fig. a in thesWe further evaluated the effect of over-expressing STI1 on psyn129 quantitatively using immunoblotting in the ipsilateral cortex and hippocampus with RIPA-soluble lysates, to extract cytoplasmic and membrane-bound proteins. We found twofold more psyn129 in M83+/−:TgA PFF-injected mice normalized by the levels of human α-synuclein in the ipsilateral cortex when compared with mice with normal levels of STI1  WT and STI1 over-expressing mice (TgA), with no mutations in the p < 0.05, Supp. Fig. S4a); however, in the contralateral hemisphere, psyn129 pathology showed only a trend to be reduced, which did not reach statistical significance , likely due to the lower amount of pathology found in this hemisphere.Given that augmented levels of STI1 and Hsp90 favor the accumulation of the psyn129 form of α-synuclein, which normally labels misfolded protein , 105, wep = 0.052) compared to M83+/− PFF-injected mice. Additionally, we observed partial co-localization of psyn129 with ubiquitin , albeit in the hippocampus the decrease just failed statistical significance  α-synuclein. We found reduced psyn129 protein levels in the cortical fraction Fig. k, l, witMice injected with PFFs or PBS were followed for 13–16 weeks after injection, until weight loss and health decline were observed in the cohort. In general, all PFF-injected mice lost weight when compared to PBS-injected mice , accomp2 months , 126.We first tested whether STI1 and chaperones are present in insoluble fractions in M83+/+ mice cortical lysates, as observed in M83+/− PFF-injected mice. Analysis of cortical brain protein extracts from 11–12-month-old non-transgenic (WT) and M83+/+ male mice (when 50% of the mice were symptomatic ) revealeGiven that STI1 interacts with endogenous mouse α-synuclein, we further tested whether STI1 could form a complex with phosphorylated human α-synuclein in M83+/+ mouse brains. For these experiments, we used cortical tissue from control and M83+/+ male mice aged 10–11-months, which was the time-point where we observed at least 50% of the cohort to be physically declining, as reported previously . STI1 coD of 56 ± 27 µM) for STI1 TPR2A similar to that of α-synuclein . To test the possibility that STI1 may regulate phosphorylation of α-synuclein at S129, we incubated α-synuclein with PLK3 and the TPR2A domain (which reproduced the binding of STI1 to α-synuclein) or the TPR1 domain (which does not bind to α-synuclein) for 10 or 20 min, then resolved proteins by SDS-PAGE, and blotted for α-synuclein and psyn129 , which is part of a family of kinases that is thought to phosphorylate α-synuclein in vivo , 89. Com129 Fig. l. This a129 Fig. l, m. PreTo further investigate the role of STI1 for α-synuclein toxicity in vivo, we generated mice with one or two copies of the STI1 hypomorphic allele ΔTPR1  expressing two copies of the human A53T transgene (M83+/+). Immunoblotting analysis demonstrated reduced levels of psyn129 in M83+/+ animals crossed with ΔTPR1 mice compared to littermate M83+/+ mice (M83+/+:STI1WT) . HoweveTo understand whether increased psyn129 can cause high-level cognitive deficits and affect brain function, we used touchscreen tests that probe cognitive domains that have been previously shown to be affected in synucleinopathies , 118, wiex vivo T1-weighted magnetic resonance imaging (MRI) (70-micron isotropic voxel resolution acquired using a 7 T Bruker Biospec) on the brains of a subset of mice that performed the touchscreen testing. We sought to investigate brain abnormalities 1) due to over-expression of human A53T SNCA (in M83+/+ mice compared to non-transgenic mice) and 2) to examine whether the brain differences are reverted with the presence of one copy of the ΔTPR1 allele. Moreover, since volumetric differences were previously found in ΔTPR1 but not ΔHET mice [t-, p-, and q-values. Overall, these experiments suggest that pathology and toxicity of α-synuclein can be mitigated by decreasing the gene dosage of STI1.To further evaluate brain toxicity in conditions in which mice had cognitive deficits or were rescued, we performed HET mice , we focuUsing mouse models of spontaneous and seeded α-synuclein misfolding, we identified a vicious cycle in which accelerated proteostatic stress induced by α-synuclein misfolding facilitates accumulation of the chaperone machinery in insoluble species. Conversely, we found that STI1 interaction with α-synuclein contributes to its toxicity, tissue inclusions, and cognitive dysfunction Fig. . MechaniPhosphorylation of α-synuclein at S129 is a well-established and abundant pathological marker in PD and DLB , 53. α-S. Nonetheless, these results suggest that α-synuclein misfolding and its propensity to be phosphorylated at S129 and form inclusions are regulated by STI1 and the Hsp90 chaperone machinery in vivo.In both mouse models of α-synuclein misfolding (M83+/+ and M83+/− PFF injection), reduced levels of STI1 led to less psyn129, either in RIPA-soluble or insoluble forms. In contrast, increased expression of STI1 and Hsp90 augmented psyn129 and insoluble α-synuclein deposition. It is unlikely that the regulation of psyn129 by STI1 is a result from increased proteasome activity due to reduced STI1, given that normal levels of STI1 seem to be necessary for optimal proteasome function . STI1 isSTIP1 gene was found to be one of the key nodes regulating abnormal proteostatic stress [The role of STI1 in protein misfolding and aggregation in vivo is still far from being understood. In yeast, STI1 buffered proteotoxicity by rearranging amyloid-like proteins into SDS-resistant inclusions , whereasc stress .Although chaperones are thought in general to protect against protein misfolding , 32, 73,Augmented proteostatic stress in proteinopathies causes a reorganization of chaperome connectivity leading to the formation of abnormal complexes known as epichaperomes . This abOur data reveal that STI1 binding to the C-terminal region of mutated misfolded α-synuclein facilitates its phosphorylation at S129. This process is involved in the toxicity of α-synuclein, which we functionally evaluated via motor behavior deficits, high-level cognitive function, and atrophy of specific brain regions. Of note, our analysis of reversal learning using touchscreens was much more sensitive than motor function assessments in M83+/+ mice. Executive dysfunction and deficits in reversal learning and behavioral flexibility are cognitive alterations found in human synucleinopathies , 118; thSTI1 regulates multiple other co-chaperones and expression of one ΔTPR1 hypomorphic allele can also decrease the levels of co-chaperones such as cyclophilin A (CypA), which has recently been shown to favor α-synuclein misfolding , providiSupplementary Figure 1: Characterization of the interactions between STI1 and α-synuclein by NMR titration. a. 600 MHz 1H-15N HSQC spectrum of 100 µM α-synuclein  in the absence (black) and the presence (green) of 200 µM of STI1 TPR1 domain. b. 600 MHz 1H-15N HSQC spectrum of 100 µM α-synuclein  in the absence (black) and the presence (cyan) of 200 µM of STI1 TPR2B domain. c. NMR titration of unlabeled STI1 to 15N-labeled α-synuclein. The concentration of α-synuclein was kept constant at 100 µM (estimated by Lowry assay) while the concentrations of STI1 varied from 0 µM to 200 µM in increments of 25 µM (estimated by Lowry assay). The KD value was determined based on the titration data of D119, D121, and E137, the three residues that displayed the largest chemical shift changes upon the addition of STI1. Protein concentrations determined by amino acid analysis were used in fitting the KD value. d. NMR titration of unlabeled TPR2A to 15N-labelled α-synuclein. The concentration of α-synuclein was kept constant at 100 µM (estimated by Lowry assay) while the concentrations of TPR2A varied from 0 µM to 200 µM (estimated by Lowry assay) in increments of 25 µM. The KD value was determined based on the titration data of D119, D121, and E137, the three residues that displayed the largest chemical shift changes upon the addition of TPR2A. Protein concentrations determined by amino acid analysis were used in fitting the KD value. e. Overlay of 1H-15N HSQC spectra of 100 µM 15N-labelled α-synuclein (black), 100 µM 15N-labelled α-synuclein + 100 µM TPR2A (red), and 100 µM 15N-labeled α-synuclein + 100 µM TPR2A + 100 µM Hsp90 pentapeptide (green). Insets in panels c, d, and e highlight the chemical shift perturbations of several selected residues upon addition of binding ligands.Supplementary Figure 2. Characterization of WT human α-synuclein PFFs and pathology induced in M83+/- mice. a. Electron microscopy images of unsonicated and sonicated PFFs. Sonication of α-syn fibrils results in fibrils of smaller length following aggregation, leading to α-syn PFFs with a length <100 nm. b. Quantification of sonicated PFF diameter by Dynamic Light Scattering (DLS). The mean diameters for batches of PFF used in mouse injections, as quantified by DLS in three separate measurements per batch. c. Whole section tiled imaging of nuclear marker and psyn129 labelling in M83+/- PBS and PFF mice, images acquired at 20x magnification near coordinates for injection. Scale bar = 1 mm. d-f. Higher resolution imaging of colocalization analyzes in the ipsilateral cortex of M83+/- PFF injected mice. d. Colocalization of Ubiquitin with psyn129. e. Colocalization of Hsp90 with psyn129. f. Colocalization of STI1 with psyn129. g & h. Labeling for psyn129 and Amytracker in the piriform pyramidal layer of M83+/- PBS and M83+/- PFF injected mice. psyn129 partially colocalizes with misfolded and potentially aggregated proteins. Arrows indicate regions with high co-labelling between psyn129 and Amytracker, whereas arrowheads indicate cells with predominant Amytracker labeling, but little psyn129. Images acquired at 63X magnification and post-processed 2X zoom factor (zoom factor only applied to d-f). Scale bar = 25 µm. g. As expected, little to no staining is observed in M83+/- PBS injected mice. h. pathology labelling is abundant in M83+/- PFF injected mice.Supplementary Figure 3. a. Representative immunoblots of panHsp90, Hsp70, STI1, pSer129 α-synuclein (psyn129), human α-synuclein, and total (mouse and human) α-synuclein in RIPA lysates from cortex of WT and M83+/- mice (no inoculations with PFFs) b. Densitometric quantification of Hsp90 (t(7)= 2.605, p=0.0352). c. Densitometric quantification of constitutive Hsp70 (t(7) = 2.869, p=0.0240). d. Densitometric quantification of STI1 (t(7) = 3.282, p=0.0134). e. Representative immunoblots from 2% SDS and 4 M Urea extraction in cortical lysates for panHsp90, Hsp70, STI1, psyn129, human α-synuclein, and total (mouse and human) α-synuclein. f. Densitometric quantification of Hsp90 (t(7) = 0.563, p=0.591), g. Densitometric quantification of Hsp70 (t(7) = 0.361, p = 0.729), h. Densitometric quantification of STI1 (t(7) = 1.072, p=0.320). i. Representative immunolabelling for psyn129 (red), and nuclear marker (blue) in the ipsilateral ACC j. Representative immunolabelling for psyn129 (red), and nuclear marker (blue) in the dentate gyrus of M83+/- and M83+/-:TgA PFF injected mice. 40X images, 2X zoom, with scale bars = 25 µm. k. Immunoblots from C57BL/6 non-M83 transgenic WT and TgA mouse cortical lysates. l. Densitometric quantification of total mouse α-synuclein normalized to actin loading control (t(8) = 0.6609, p=0.5272). Data represented as mean ± SEM and analyzed using unpaired t-test.Supplementary Figure 4. Quantification of the percent area immunolabeled with psyn129 in the a. ipsilateral hemisphere (t(9) = 3.006, p=0.015) and b. contralateral hemisphere (t(9) = 1.769, p=0.1107). Imaged at 20X magnification.  in the ipsilateral caudate putamen (region of injection) with nuclear marker (blue), GFAP , psyn129 (red) and d. quantification of percent area with psyn129 immunoreactivity in the ipsilateral caudate putamen . N=5/genotype. e. Representative microscopy images of the ipsilateral ACC of M83+/- (N=6) and M83+/-::ΔTPR1 PFF-injected mice (N=5). f. quantification of percent area with psyn129 immunoreactivity in the ipsilateral ACC (t(8) = 2.616, p=0.0308). g. Representative images in the ventrolateral cortical area, the piriform pyramidal layers  within M83+/- (N=6) and M83+/-::ΔTPR1 PFF-injected mice (N=5). h. Quantification of percent area with psyn129 immunoreactivity in the ipsilateral PPLs (t(8) = 2.911, p=0.0196). i. Representative images of the ipsilateral hippocampus (image set within the dentate gyrus) and j. respective quantification of percent area with psyn129 immunoreactivity in the whole ipsilateral hippocampus (t(8)= 2.288, p=0.0514). N=5/genotype. Scale bars are 50 µm and images taken at 40x with post processing at 2X zoom. Images used for quantification were tiling images of the whole section, taken at 20X. Individual circles (M83+/- PFF) and filled triangles (M83+/-:ΔTPR1 PFF) represent individual animals. Data represented as mean ± SEM. Data were analyzed with unpaired t-test and assessed for normality using Shapiro-Wilk test. If data were non-normal, Mann Whitney non-parametric test was used. *p<0.05, **p<0.01.               Supplementary Figure 5. Qualitative representative pattern of staining of human pSer129 (MJF-R13) antibody in M83+/- PFF injected mutant STI1 mice. M83+/-, M83+/-:TgA and M83+/-::ΔTPR1 PFF mice immunolabelling for human pSer129 (red) in the a. Anterior Cingulate Cortex (ACC) and b. piriform pyramidal layers (PPLs). Both are regions with abundant pathology. Images were acquired at 40X magnification. Scale bars are 50 µm.Supplementary Figure 6. Ubiquitin, Hsp90, and STI1 cytoplasmic staining co-labels with psyn129 aggregates in M83+/- PFF injected mice. Representative images of inclusions in M83+/- and M83+/-:ΔTPR1-PFF injected mice. a. partial colocalization between psyn129 and ubiquitin in ipsilateral piriform pyramidal layer (PPL) b. partial colocalization between psyn129 and ubiquitin in the ipsilateral Dentate gyrus. Likewise, Hsp90 and psyn129 partial colocalization in c. PPL. d. partial colocalization between psyn129 and Hsp90 in the Dentate gyrus. e. partial colocalization between psyn129 and STI1 in the PPL. f. partial colocalization between psyn129 and STI1 in the Dentate gyrus. Arrows illustrate areas where co-localization was observed. Images were taken at 63X magnification and post processed to 2X zoom. Scale bars are 25 µm for all images. g. Representative immunoblot and h. Densitometric quantification of mouse α-synuclein protein levels in WT and ΔTPR1 mouse tissues with 50% less STI1 (ΔHET) or 80% less STI1 (ΔTPR1) (N=4 mice/genotype). Data are mean ± SEM and analyzed using One-way ANOVA with no significant different between genotypes  = 0.494, p=0.63).Supplementary Figure 7. NMR titration of unlabeled TPR2A to 15N-labelled psyn129. a. The concentration of psyn129 was kept constant at 100 µM (estimated by Lowry assay), while the concentrations of TPR2A varied from 0 µM to 200 µM (estimated by Lowry assay) in increments of 25 µM. The KD value was determined based on the titration data of D119, D121, and E137, the three residues that displayed the largest chemical shift changes upon the addition of TPR2A. Protein concentrations determined by amino acid analysis were used to in fitting the KD value. Insets in the figure highlight the chemical shift perturbations of several selected residues upon addition of TPR2A. The dashed square indicates the location of the S129 peak of non-phosphorylated α-synuclein obtained in the other titrations. The lack of signal in that position indicates that α-synuclein was almost completely phosphorylated. Supplementary Figure 8. Five-choice serial reaction time task (5-CSRTT) used to measure attention in M83 mice and their non-transgenic controls at 8 months old. a. Accuracy during probe trial sessions. Repeated measures two-way ANOVA  = 24.62, p < 0.0001; Genotype: F = 3.263, p = 0.0859; Interaction: F = 1.628, p = 0.1930). b. Rate of omission. Repeated measures two-way ANOVA  = 24.23, p < 0.0001; Genotype: F =0.2109, p = 0.6510; Interaction: F = 0.8955, p = 0.4491). c. Number of Premature responses. Repeated measures two-way ANOVA  = 3.310, p = 0.0318; Genotype: F = 0.6922, p = 0.4152; Interaction: F = 0.4390, p = 0.7260). d. Correct touch latency (s). Repeated measures two-way ANOVA  = 9.142, p < 0.0001; Genotype: F = 5.540e-009, p = 0.9999; Interaction: F = 0.6604, p = 0.5791). e. Reward collection latency (s). Repeated measures two-way ANOVA  = 1.609, p = 0.2046; Genotype: F = 0.1751, p = 0.6801; Interaction: F = 2.969, p = 0.0393). f. Number of Perseverative responses. Repeated measures two-way ANOVA  = 1.725, p = 0.2037; Genotype: F = 0.01583, p = 0.9011; Interaction: F = 0.5263, p = 0.6660). g. Number of trials completed. Repeated measures two-way ANOVA  = 0.5924, p = 0.2037; Genotype: F = 0.001581, p = 0.9684; Interaction: F = 1.235, p = 0.3025). Attention was measured, in M83+/+:STI1WT mice (n=10) and their non-transgenic WT controls (n=12) at 8 months old, using the 5-choice Serial Reaction Time Testing (5-CSRTT) performance probe trials with 1.5-, 1.0-, 0.8- and 0.6-second stimulus durations. Results are expressed as mean ± SEM. Asterisks indicate statistical differences between groups: *p<0.05. Individual unfilled black circles (WT), dark blue circles (M83+/+:STI1WT), half-filled pink triangles (M83+/+:ΔHET) represent individual animals. Supplementary Figure 9. Additional parameters for cognitive behavior measured in the PVD-R task. a. Timeline for the testing mice on the PVD-R test. b. Correct touch latency (s). Repeated measures two-way ANOVA  = 30.14, p < 0.0001; Genotype: F = 10.46, p = 0.0001; Interaction: F = 5.527, p < 0.0001, Adjusted for multiple comparisons with Tukey's multiple comparisons test: WT vs. M83+/+: STI1WT p= 0.5341, WT vs. M83+/+:ΔHET p < 0.0001, M83+/+:STI1WT vs. M83+/+:ΔHET p < 0.0001). c. Reward collection latency (s). Repeated measures two-way ANOVA  = 2.737, p= 0.0071; Genotype: F = 12.62, p 0.0001; Interaction: F=1.982, p=0.0050, adjusted for multiple comparisons with Tukey's multiple comparisons test: WT vs. M83+/+:STI1WT p < 0.0001, WT vs. M83+/+:ΔHET p < 0.0001, M83+/+:STI1WT vs. M83+/+:ΔHET p < 0.0001). d. Number of trials completed in the PVD reversal learning (PVD-R) task. Repeated measures two-way ANOVA  = 7.461, p < 0.0001; Genotype: F = 3.001, p=0.0579; Interaction: F = 1.749, p = 0.0188, adjusted for multiple comparisons with Tukey's multiple comparisons test: WT vs. M83+/+:STI1WT p = 0.5299, WT vs. M83+/+:ΔHET p = 0.3415, M83+/+:STI1WT vs. M83+/+:ΔHET p = 0.0407).). WT control mice (n=20), M83+/+: STI1WT mice (n=20) and M83+/+:ΔHET mice (n=18). Marble (S-) and fan (S+) stimuli were used during the PVD-R acquisition phase. Parameters were measured across baseline days 1 and 2  and reversal days 1 to 10 (R1-R10). Results are expressed as mean ± SEM. Asterisks indicate statistical differences between groups: *p<0.05, **p<0.001, ***p<0.0001. Individual unfilled black circles (WT), dark blue circles (M83+/+:STI1WT), half-filled pink triangles (M83+/+:ΔHET) represent individual animals.Supplementary Table 1: List of anonymized control and PD samples analyzed using RNAseq per age group and brain region, Lewy body pathology severity and length of disease. Supplementary Table 2: Healthy control brain tissue information from the GTEX database used for RNA seq analyses.Supplementary Table 3: A full list of the anatomical regions segmented in the modified Allen brain atlas.Supplementary Table 4: Correlations, p value and FDR information for Transcriptomic Analysis.Supplementary Table 5: Volumetric statistical results for each structure in the modified Allen Brain Atlas in terms of standardised betas, t-, p- and q- values for each of the comparisons performed . Supplementary file 1 (PDF 35970 KB)Below is the link to the electronic supplementary material.


Rheumatoid arthritis (RA) is a chronic and refractory autoimmune disease. Deficiency pattern (DP) and excess pattern (EP), as crucial types of Chinese medicine pattern diagnoses published by International Classification of Diseases 11th Revision (ICD-11), could provide new strategies for RA diagnosis. However, the biological basis of DP and EP of RA is not explicit.19 female RA DP patients, 41 female RA EP patients and 30 female healthy participants were included in the study. The serums of participants were collected and analyzed by metabolomics based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry to profile metabolic characteristics of RA DP and EP. Furthermore, bioinformatics analysis results were obtained by using Ingenuity Pathway Analysis (IPA) and statistical analysis was performed by SAS version 9.4 for further identification of potential biomarkers.α signaling.Serum metabolic profiling revealed 25 and 24 differential metabolites in RA DP and EP respectively, and 19 metabolites were common to RA DP and EP. Compared with DP group, L-Homocysteic acid, LysoPE(P-16:0/0:0), N(omega)-Hydroxyarginine and LysoPC(16:0/0:0) decreased (P < 0.05), and Pyruvic acid, D-Ribose, Gamma-Glutamylserine, PE(22:0/24:1(15Z)), Inosinic acid increased (P < 0.05) in EP group. Menawhile, S-Nitrosoglutathione, 5-Thymidylic acid, SN38 glucuronide, PE(22:0/24:0), PC(24:0/24:1(15Z)) and Bisdiphosphoinositol tetrakisphosphate increased significantly in DP group compared to EP group (P < 0.05). For the unique metabolites, bioinformatics analysis results showed that 5-Methoxytryptamine involved in Melatonin Degradation II and Superpathway of Melatonin Degradation is the key metabolite to RA DP. Meanwhile, GABA is the key metabolite in EP group, which involved in Glutamate Dependent Acid Resistance, GABA Receptor Signaling, Glutamate Degradation III (via 4-aminobutyrate) and 4-aminobutyrate Degradation I. Bioinformatics analysis between unique metabolites of RA DP and EP groups with human target genes for RA showed that 5-methoxytryptamine and LysoPC(18:1(9Z)/0:0), the unique metabolites of RA DP, might participate in colorectal cancer metastasis signaling, tumor microenvironment pathway, apoptosis signaling, MYC mediated apoptosis signaling, erythropoietin signaling pathway and LXR/RXR activation. Simultaneously, GABA, LysoPA(18:1(9Z)/0:0) and L-Targinine, the unique metabolites of RA EP, might participate in neuroinflammation signaling pathway, osteoarthritis pathway, glucocorticoid receptor signaling, ILK signaling, IL-17 signaling and HIF1The study indicates that serum metabolomics preliminarily revealed the biological basis of RA DP and EP. 5-methoxytryptamine, LysoPC(18:1(9Z)/0:0) and GABA, LysoPA(18:1(9Z)/0:0), L-Targinine might be the predictors to distinguish the DP and EP of RA respectively. These interesting results provide thoughts for further study of traditional medicine patterns of ICD-11. It also contributes to provide strategy for personalized precision treatment of RA and further validation is needed. Rheumatoid arthritis (RA) is a heterogeneous and refractory disease, which primarily involves the joints and cause disability . The etiDeficiency pattern (DP) and excess pattern (EP) are two crucial types of CM pattern diagnoses, which belongs to the principle-based patterns in the category of traditional medicine patterns published by International Classification of Diseases 11th Revision (ICD-11) –7. PatteMetabolomics, the analysis of concentration profiles of low molecular weight metabolites present in biological fluids, has immense potential as a novel diagnostic tool in complex disorders such as RA, systemic lupus erythematosus , 12. SimPrevious studies have studied the biological basis of cold pattern and heat pattern of RA by using plasma metabolomics , 16. MeaThe participants including female RA DP patients, female RA EP patients and healthy females were recruited from November 2018 to June 2019 in The First Affiliated Hospital of Henan University of CM. The sample size was based on previous studies , 19, 27.Each blood sample collected in a fasting condition was left standing at room temperature for 30 min. Then blood samples were centrifuged for 20 min at 3000 g at 4 °C to obtain the serum and transferred into clean tubes. All serum samples were stored at − 80 °C until LC-MS analysis.Chromatographic separation was performed on a 1.8 μm, 2.1 mm × 100 mm ACQUITY UPLC®HSS T3 column  using an ACQUITY Ultra Performance LC system  equipped with a binary solvent delivery system, an auto-sampler, and high temperature column oven. The column was maintained at 30 °C. The flow rate was set at 0.4 ml/min. The sample injection volume was 1.2 µl. Solvent A was water mixed with 0.1% formic acid, and solvent B was acetonitrile mixed with 0.1% formic acid. The gradient elution programmes were: 0–0.5 min 5% B, 0.5–7 min 100% B, 7–8.6 min 100% B, 8.6–11.5 min 100% B, 11.5–11.6 min 5% B, 11.6–16 min 5% B.Mass spectrometry detection was acquired on a quadrupole time-of-flight mass spectrometer  equipped with an electrospray ion source. The positive ion mode detection: The desolvation gas was set at 600 L/h at a temperature of 350 °C. The cone gas was set at 37 L/h. The source temperature was set at 100 °C. The capillary and cone voltage were optimized at 1.5 kV and 40 V, respectively. Full scan spectra were performed between 50 and 1000 m/z with a 0.2 s scan time and a 0.1 s interscan delay. The negative ion mode detection: The desolvation gas was set at 800 L/h at a temperature of 500 °C. The cone gas was set at 50 L/h. The source temperature was set at 120 °C. The capillary and cone voltage were optimized at 2 kV and 40 V, respectively. Full scan spectra were performed between 50 and 1200 m/z with a 0.2 s scan time and a 0.1 s interscan delay. t-test and fold-change were also applied to these ions. Ions with P-values < 0.05, fold-changes > 2.0 (or < 0.5) and relative standard deviation (RSD) < 0.3 were finally regarded as differentiated metabolite ions. Then they were structurally identified and interpreted based on searches of their accurate masses in HMDB (www.hmdb.ca) database. Lastly, the isotopic distribution, retention time and fragments of commercial standards were further confirmed for the metabolites of interest.The raw serum LC-MS data was pre-processed using Waters Progenesis QI 2.0 software . Progenesis QI can provide an accurate measurement of the compounds in the bio-samples and facilitate flexible comparisons across runs. It included steps of importing data, reviewing alignment, experiment design setup, picking peaks, identifying and reviewing compounds and performing compound statistical analysis. Then, the data were exported into SIMCA-P software (v13.0)  for multivariable statistical analysis (MVA). The MVA of principal component (PCA), partial least squares discriminant (PLS-DA) and orthogonal partial least square-discriminant (OPLS-DA) models were both applied to observe the classifications for different groups in score plots. Next, to find significant metabolites contributing to the classifications, ions with variable importance in the projection (VIP) > 1 were picked out from the OPLS-DA loading plots. Then univariate analysis ofhttp://www.ingenuity.com) for the candidate metabolites and proteins to gain further insights into the typical metabolic alterations.The analysis of canonical pathways, bio-functions and the networks were conducted by using the Ingenuity Pathway Analysis system , but there was statistical difference between DP and EP (P = 0.003). There were no significant differences in BMI, WBC, RBC, HGB, PLT, ALB, between the three groups (P > 0.05). In terms of duration of RA, ESR, CRP, RF-IgG, RF-IgA, RF-IgM, smoked, hypertension, diabetes and coronary heart disease, there were no statistically significant differences between DP and EP (P > 0.05).A total of 90 participants including 19 female RA DP patients, 41 female RA EP patients , R2Y = 0.862 (cum), Q2 = 0.739(cum). And in PLS-DA negative ion mode: R2X = 0.323 (cum), R2Y = 0.808 (cum), Q2 = 0.772 (cum).PCA was used to determine the presence of inherent similarities in spectral profiles and the corresponding PLS-DA analysis was used to identify discriminating metabolites and differentiate the three groups. Although there was some overlap between DP and EP group, the PCA Fig. A, B and 2X = 0.689 (cum), R2Y = 0.992 (cum), Q2 = 0.972 (cum). And in OPLS-DA negative ion mode: R2X = 0.446 (cum), R2Y = 0.99 (cum), Q2 = 0.972 (cum). By metabolite identification, 25 differential metabolites including 12 in positive ion mode and 13 in negative ion mode were obtained between DP and healthy groups (Table P < 0.01).PCA Fig. A, B and 2X = 0.667 (cum), R2Y = 0.985 (cum), Q2 = 0.968 (cum). And in OPLS-DA negative ion mode: R2X = 0.496 (cum), R2Y = 0.983 (cum), Q2 = 0.965 (cum). By metabolite identification, 24 differential metabolites including 11 in positive ion mode and 13 in negative ion mode were obtained between DP and healthy groups (Table P < 0.01).PCA Fig. A, B and 19 metabolites were common to DP and EP groups, including 8 in positive ion mode: L-Homocysteic acid, S-Nitrosoglutathione, LysoPE(P-16:0/0:0), LysoPC(16:0/0:0), LysoPC(P-18:0/0:0), Bisdiphosphoinositol tetrakisphosphate, PE(22:0/24:0), PC(24:0/24:1(15Z)), and 11 in negative ion mode: Pyruvic acid, D-Ribose, N(omega)-Hydroxyarginine, Citric acid, Gamma-Glutamylserine, 5-Thymidylic acid, Inosinic acid, SN38 glucuronide, PC(O-22:1(13Z)/22:3), PE(22:0/24:1(15Z)), Octanoyl-CoA.P < 0.05), and Pyruvic acid  in EP group. Menawhile, S-Nitrosoglutathione . However, there was no statistical difference in Citric acid .Compared with DP group, L-Homocysteic acid Fig. A, LysoPEcid Fig. A, Octanocid Fig. B, LysoPCcid Fig. C and PC/0:0), LysoPC(20:1(11Z)/0:0), Glucosylceramide (d18:1/12:0), PE /22:6 ), and 5 unique metabolites in EP group: Gamma-Aminobutyric acid (GABA), LysoPA(18:1(9Z)/0:0), DG/22:6  /0:0), L-Targinine, PC(24:0/24:0). IPA core function analysis results show that Melatonin Degradation II, Tyrosine Degradation I, Superpathway of Melatonin Degradation are biological pathways closely related to DP group  The sample size of this study was small, but representative RA DP and EP samples were still collected based on the rigorous diagnostic criteria, inclusion criteria and exclusion criteria, and meaningful results had been obtained; (2) The participants included in the study were all from Henan Province, which might reduce the influence of environmental factors on traditional medicine patterns of RA to some extent. However, the results could also reflect the traditional medicine patterns of RA in central China. Future studies under different regions and environments are still needed; (3) Since there is no animal and cell models of DP and EP, the study did not verify the findings based on cell biology experiments. However, metabolomics and bioinformatics analysis found some interesting results, which can provide a scientific basis for the biological basis of RA DP and EP and thoughts for further study of traditional medicine patterns of ICD-11; (4) DP and EP are the basic steps of the principle-based patterns for the diagnosis of RA. Subgroups can still be classified under each CM pattern diagnosis. For example, liver and kidney deficiency syndrome is one of the important subgroups of DP, while cold and dampness syndrome is one of the important subgroups of EP, which needs to be further studied.In conclusion, serum metabolomics preliminarily revealed the biological basis of RA DP and EP. 5-methoxytryptamine, LysoPC(18:1(9Z)/0:0) and GABA, LysoPA(18:1(9Z)/0:0), L-Targinine might be the predictors to distinguish the DP and EP of RA respectively, which provides a potential basis for the understanding of CM pattern diagnoses. These interesting results provide thoughts for further study of traditional medicine patterns of ICD-11. It also contributes to provide strategy for personalized precision treatment of RA and further validation is needed.


Salmonella vaccine for broilers. Chitosan nanoparticle (CNP) vaccines can be used to deliver Salmonella antigens orally. We investigated the efficacy of a killed Salmonella CNP vaccine on broilers. CNP vaccine was synthesized using Salmonella enterica serovar Enteritidis (S. Enteritidis) outer membrane and flagella proteins. CNP was stable at acidic conditions by releasing 14% of proteins at pH 5.5. At 17 h post-incubation, the cumulative protein release for CNP was 75% at pH 7.4. Two hundred microliters of PBS with chicken red blood cells incubated with 20 μg/ml CNP released 0% hemoglobin. Three hundred chicks were allocated into 1) Control, 2) Challenge, 3) Vaccine + Challenge. At d1 of age, chicks were spray-vaccinated with PBS or 40 mg CNP. At d7 of age, chicks were orally-vaccinated with PBS or 20 μg CNP/bird. At d14 of age, birds were orally-challenged with PBS or 1 × 107 CFU/bird of S. Enteritidis. The CNP-vaccinated birds had higher antigen-specific IgY/IgA and lymphocyte-proliferation against flagellin (p < 0.05). At 14 days post-infection, CNP-vaccinated birds reversed the loss in gut permeability by 13% (p < 0.05). At 21 days post-infection, the CNP-vaccinated birds decreased S. Enteritidis in the ceca and spleen by 2 Log10 CFU/g, and in the small intestine by 0.6 Log10 CFU/g (p < 0.05). We conclude that the CNP vaccine is a viable alternative to conventional Salmonella poultry vaccines.There is a critical need for an oral-killed  Salmonella is a zoonotic pathogen that is currently responsible for approximately four billion dollars in total costs of foodborne illness in the United States of America (United States)  . Poultryne cases . Salmones of age . One of For chickens, spray vaccination is a standard method for delivering respiratory vaccines, such as Newcastle Disease or Infectious Bronchitis . Spray vSalmonella. The oral route provides a more tailored and effective defense against Salmonella because systemic and mucosal immune responses are highly segregated  along the gastrointestinal tract (GIT)  vaccines against Salmonella are studied as alternative vaccine candidates.Selecting the most efficient delivery route for vaccines is vital because pathogens have different natures that allow them to thrive against the host immune defenses. For example, the oral vaccination route is key to developing mucosal immune responses against enteric pathogens, such as gregated . Howeverct (GIT) . The sIg the GIT . In addiirulence  and compSalmonella enterica serovar Enteritidis  in the ceca  quantifying serum, cloacal, and bile anti-Salmonella OMPs IgY and IgA antibodies, (2) quantifying S. Enteritidis loads in the gizzard, pancreas, small intestine, spleen, liver, ceca, heart, and blood, (3) quantifying the antigen-recall response, (4) quantifying key cytokines and Toll-like receptors (TLRs) mRNA amounts, (5) quantifying fluorescein isothiocyanate dextran (FITC-d) levels in the serum, and (6) monitoring the body-weight-gain (BWG) and feed-conversion-ratio (FCR) of immunized and challenged broilers.Previous studies have explored the potential for mass vaccination delivery of the CNP vaccine through oral gavage, water, feed, n scheme . TherefoAll animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Georgia (IACUC # A2021 04-014-Y1-A1). Experimental procedures were performed following the pertinent guidelines concerning animal handling, care, and welfare.S. Enteritidis OMPs was used for the preparation of the CNP vaccine. The isolation of S. Enteritidis OMPs was done as described previously  and centrifuged three times at 4,800 ×g for 40 min. The cell pellet was collected, washed three times using 10 mM TRIS Base buffer (pH 7.5), and heat-killed at 75 C for 20 min. The cell pellet was subsequently treated with 2% Triton X-100 in 10 mM Tris HCl buffer (pH 7.5) and disrupted using a homogenizer  for two rounds of 3 min each, with a cooling period on ice of 1 min in between each round. Afterward, the cell suspension was centrifuged at 4,800 ×g for 30 min and the supernatant was collected and centrifuged at 100,000 ×g for 3 h. The protein concentration was estimated using a Pierce™ BCA Protein Assay Kit , as per the manufacturer’s instructions. The final product was freeze-dried with 5% sucrose and stored until further use.A crude protein extract of eviously . In brieS. Enteritidis flagellin was used for the preparation of the CNP vaccine. The flagellin proteins were isolated from S. Enteritidis, as described previously  for 48 h at 37°C, without shaking. The grown culture was resuspended with 1 × PBS (pH 7.4) and centrifuged three times at 4,000 ×g for 40 min. The suspension was subsequently treated with 3M Potassium Thiocyanate in 1 × PBS (pH 7.4) for 2 h at 25°C, with magnetic stirring. The treated cell suspension was centrifuged at 35,000 ×g for 30 min. The supernatant was dialyzed against 1 × PBS (pH 7.4), followed by overnight dialysis in Milli-Q water. The protein concentration was estimated using a Pierce™ BCA Protein Assay Kit, as per the manufacturer’s instructions. The end product was freeze-dried using 5% sucrose and stored until further use.A crude protein extract of eviously . A pure The loaded CNP was synthesized using the ionic gelation method, as described previously . First, The Gel-pac solution used for this study was kindly provided by Animal Science Products, Inc. and was prepared as per the manufacturer’s instructions . In brief, one hundred grams of Gel-pac were dissolved into 4 L of cold water. The solution was thoroughly homogenized using a high-speed handheld emulsifier until a uniform distribution of the green coloring in the gel spray solution was obtained. For the vaccination, a total of 40 mg of loaded CNP vaccine diluted in PBS (pH 7.4) were added to the Gel-pac solution for a total volume of 500 ml stock solution. The vaccine solution was delivered in a concentration/volume of 2 mg CNP/25 ml Gel-Pac solution per box of 100 hatched chicks, to account for the delivery of 20 μg/bird. For the mock Gel-pac vaccine solution PBS (pH 7.4) was added to the Gel-pac stock solution.After centrifuging the loaded CNP, the entrapment efficiency for the synthesized CNP was estimated by quantifying the amount of proteins that were left in the supernatant, as described previously . In brieThe cumulative protein release of the CNP was measured by using a cumulative protein release assay, as described previously . In brieA pH stability assay was used to measure the stability of the CNP at different acidic and alkaline pH, as described previously . In brieA hemolysis assay was done to study the biocompatibility of the CNP with chicken red blood cells (cRBCs) . In briead libitum feed and water during the experimental period. Broiler birds were monitored twice a day for (1) dehydration, (2) refusal to eat food, (3) loss of body weight, (4) diarrhea, (5) bloody feces, and (6) lethargy during the experimental period. Broiler birds were euthanized with CO2, as per the IACUC standards.Broiler birds  had access to ® II, K Supply Co. Inc.). Non-vaccine groups were given PBS as a mock vaccination. The vaccine group was given the CNP vaccine. At d7 of age, birds in the control and the challenge groups were given a mock booster vaccination of 0.5 ml 1 × PBS/bird, by oral gavage; while birds in the vaccine + challenge group were given a booster vaccination of 20 μg CNP/bird, by oral gavage. After vaccination, each pen was assigned 16 replicates as birds per pen. At d14 of age, birds in the control group were given a mock challenge of 0.5 ml 1 × PBS/bird by oral gavage, and birds in the challenge and the vaccine + challenge groups were orally challenged with 1 × 107 CFU/bird of nalidixic acid-resistant S. Enteritidis. For this study, the experimental unit was the pen (n = 6 pen/treatment). A summary of the experimental treatment groups is provided in At d1 of age, three hundred chicks were randomly allocated into three treatment groups: 1) Control, 2) Challenge, and 3) Vaccine + Challenge. At d1 of age, all treatments were delivered using a spray cabinet  for 6 h. Subsequently, 10 μL of the enriched supernatant were inoculated to Modified Semi-Solid Rappaport-Vassiliadis (MSRV) Agar . Samples were subsequently incubated at 41°C for 24 h. Detection of Salmonella was negative on the day of hatch.In this study, the experimental unit was the pen. Treatments consisted of S. Enteritidis was selected for nalidixic acid resistance on Xylose Lactose Tergitol™ 4 (XLT4)  agar at 500  mg/L. The nalidixic acid-resistant colonies were grown at 37°C for 24 h on Tryptic Soy Broth  containing 500 mg/L nalidixic acid and further used for the experimental challenge.For the experimental challenge, a pure culture of wild-type 7 CFU/bird of nalidixic acid-resistant S. Enteritidis. At 12 h post-challenge, cecal tonsils were collected to analyze induced nitric oxide synthase (iNOS), IFN-γ, TNF-α, IL-10, IL-6, IL-17, TGF-β, K 60, and TLR 4 mRNA levels by RT-PCR. Bodyweight and feed consumption were recorded weekly. The BWG and FCR were calculated. Blood, bile, and cloacal swabs were collected before the experimental challenge at d14 and d35 of age. The serum, bile, and cloacal swab samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for anti-OMPs IgY and IgA antibodies, respectively. At d12 of age, primary splenocytes were isolated and stimulated with either S. Enteritidis OMPs, S. Enteritidis flagellin, S. Enteritidis heat-killed antigen (HKA), S. Typhimurium HKA, S. Kentucky HKA, S. Infantis HKS, S. Heidelberg HKA, S. Hadar HKA, S. Litchfield HKA, or S. Newport HKA, to determine the recall response. At 14 days post-infection (dpi), one bird per pen was given an oral gavage of 2.2 mg FITC-d/bird, and serum was collected after 2 h for a gut permeability assay. At 21 dpi, the birds’ gizzard, pancreas, small intestine, spleen, liver, ceca, heart, and blood were collected for S. Enteritidis quantification by plating. At 21 dpi, cecal tonsils were collected to analyze IL-1β, TNF-α, IL-6, TGF-β, and IL-10 mRNA levels by RT-PCR, and jejunum samples were collected to analyze Claudin-1 and Zona Occludens-1 mRNA levels by RT-PCR. Spleen samples for the ex-vivo splenocyte recall assay were collected from two birds per pen, otherwise, samples were collected from one bird per pen (n = 6) at each time point.At d1 of age, one hundred birds per group were gel-spray vaccinated with either mock PBS or CNP vaccine. At 12 h post-vaccination, cecal tonsils were collected to analyze IL-1β, TNF-α, IFN-γ, IL-6, and TLR 5 mRNA expression by RT-PCR. At d7 of age, birds were treated as follows: (1) the control group and the challenge group were boosted with 1 × PBS (7.4 pH), and (2) the vaccine + challenge group was boosted with CNP vaccine. At d14 of age, birds were treated as follows: (1) the control group was given a mock challenge of 0.5 ml 1 × PBS/bird by oral gavage, and (2) the challenge group and the vaccine + challenge group were orally challenged with 1 × 10°C. The unbound antigens were removed by washing the plates three times with PBS-T. A two-fold serial dilution for a total of 100 μL per well was carried out for each sample. Serum and bile samples were diluted with 2.5% non-fat dry milk and cloacal swabs samples were diluted in 1 × PBS (pH 7.4). Negative serum was used as a control for serum samples, negative bile was used as a control for bile samples, and sterile 1 × PBS (pH 7.4) was used as a control for cloacal swabs samples. The samples were incubated for 2 h at 37°C and subsequently washed three times with PBS-T. Fifty microliters of the HRP-conjugated goat anti-chicken IgG  were added at 1: 10,000 in 2.5% non-fat dry milk powder in PBS-T or 50 μL of the HRP-conjugated goat anti-chicken IgA  were added at 1: 3,000 in 2.5% skim milk powder in PBS-T. The secondary antibodies were incubated for 2 h at 37°C. Plates were subsequently washed three times with PBS-T, and 50 μL per well of TMB peroxidase substrate  were added. After 5 min, the reaction was stopped by adding 50 μL per well of 2M Sulfuric Acid . The Optical Density (OD) was measured at 450 nm using a spectrophotometer and the corrected OD was calculated by subtracting the treatment OD from the blank OD. Results were reported as geometric mean titers (GMT). The cut-off values were determined by the mean (x) and standard deviation (SD) of the negative sera for serum samples, negative bile for bile samples, and PBS controls for cloacal swabs samples. The cut-off value was taken as x + 3SD, as described previously  at d12 of age. The recall-response was determined using an ex-vivo recall assay, as previously described  analysis was done to visualize the S. Enterica serovars heat-killed whole-antigenic crude extract . All samples were stored in stomacher bags and placed on ice. At the laboratory, samples were diluted with 1× (wt/vol) 1 × PBS (pH 7.4), mashed with a rubber mallet, and then stomached for 2 min. A volume of 100 μL of ceca was serially diluted into 900 μL of 1 × PBS (pH 7.4) and from every dilution, a volume of 10 μL was plated in duplicates on XLT4 agar plates. Plates were then incubated for 24 h at 41°C for the confirmation of black colonies. When no growth was observed, to corroborate true negative samples, the samples were further enriched in Tetrathionate Broth for 6 h followed by the inoculation of 10 μL of the enriched solution to XLT4 agar or MSRV Agar. The inoculated media were then incubated for 24 h at 41°C for the confirmation or the absence of black colonies on XLT-4 agar plates or the confirmation of positive or negative samples on MSRV selective motility-enrichment media. Upon double confirmation of the absence of growth, the samples were considered to be negative for Salmonella colonization. Data were recorded as CFU/g of organ and then transformed to Log 10 CFU/g of organ for statistical analysis. Further, the prevalence of S. Enteritidis in colonized organs was also calculated. In addition, the organ weight was further used to observe the CNP vaccine effect on the relative weight of different organs by calculating the organ index as Weight Index (%) = (organ weight (g))/(live weight (g)) × (100).Gizzard, pancreas, small intestine, spleen, liver, ceca, heart, and blood samples were analyzed for 2 inhalation and blood samples were collected. As a control, serum was taken from one broiler chicken of each treatment group that was not given FITC-d. The blood samples were collected and centrifuged at 3,000 rpm for 12 min at 4°C. The serum was then collected and diluted at 1:5. The OD was measured at 485 nm using a spectrophotometer.To determine the serum FITC-d levels at 14 dpi, one bird per pen was given FITC-d  by oral gavage at a dose of 2.2 mg FITC-d per bird . After 2n = 6) at 21 dpi. For gene expression analysis samples were analyzed in duplicates. The TRIzol reagent  was used for the total RNA extraction, as per the manufacturer’s instructions. The extracted RNA was dissolved in Tris-EDTA buffer (pH 7.5) and the cDNA synthesis was executed using 2 µg of total RNA template in a 20 µL reaction volume, as described previously (−ΔΔCt method). The primers sequences used for RT-PCR analysis are described in Cecal tonsils were collected to monitor the mRNA expression of key cytokines of the gut-associated lymphoid tissue. Jejunum samples were also collected to monitor the expression of key cytokines of tight-junction proteins in the gut. For this study, cecal tonsil and jejunum samples were collected from one bird per pen , followed by a Tukey’s post-hoc test. Otherwise, the statistical differences were determined using a Kruskal-Wallis test and followed by Dunn’s post-hoc test. Statistical analysis was performed using JMP Pro 14  and results were statistically significant at p < 0.05.The experimental unit was the pen, where The entrapment efficiency for the total protein content of the CNP vaccine was 87% (data not shown).At 6 h post-incubation the CNP released 3, 9, 10, 14, 31 and 26% of proteins from 3.5 to 7.5 pH, respectively .At 2 h post-incubation the CNP had released 11% of its protein cargo, at 3 h post-incubation the CNP had released 14% of its protein cargo, at 10 h post-incubation the CNP had released 21% of its protein cargo, and at 17 h post-incubation the CNP had released 75% of its protein cargo .The cRBC incubated with 20 μg/ml, 50 μg/ml, and 100 μg/ml of CNP had 0.07, 0.09 and 0.9% hemolysis respectively .p > 0.05) between treatments in the BWG or FCR of birds at all the time points, compared to control; hence, results were reported as cumulative BWG  (S. Enteritidis OMP IgY titers from serum samples between any of the treatment groups when compared to the control group (p = 0.05)      . At d35  = 0.05) . At d14  < 0.05) . At d35  < 0.05) . At d14  = 0.05) . At d35  < 0.05) .S. Enteritidis Flagellin IgY titers from serum samples between any of the treatment groups when compared to control (p > 0.05)      . At d14  < 0.05) . At d35  < 0.05)  7. At d1 > 0.05) . At d35  < 0.05) .p < 0.05) T-lymphocyte proliferation when they were stimulated with 20 μg/ml Flagellin, compared to control  T-lymphocyte proliferation when they were stimulated with 20 μg/mL S. Enteritidis HKA, 20 μg/mL S. Typhimurium HKA, and 20 μg/mL S. Litchfield HKA, compared to control . There was no S. Enteritidis detection in gizzard, pancreas, liver, heart, and blood when compared to control (p > 0.05).At 21 dpi the CNP-vaccinated birds had a 2 Logthe ceca , spleen the ceca , and smathe ceca , respectS. Enteritidis in the ceca of the immunized birds was 33% for Salmonella negative birds and 67% for Salmonella positive birds   .p < 0.05)  .p < 0.05) (p < 0.05) (p < 0.05) (p > 0.05)  . At 12 h < 0.05) . At 12 h < 0.05) . At 12 h > 0.05) .p < 0.05) (p < 0.05) (p < 0.05) (p > 0.05)  . At 12 h < 0.05) . At 12 h < 0.05) . At 12 h > 0.05) .p > 0.05)  .p > 0.05)  .Salmonella vaccine antigens. Previous studies with broiler and layer birds have investigated the efficacy of the oral Salmonella CNP vaccine when delivered by oral gavage, water, feed, in-ovo, and in a combined live followed by killed vaccination scheme  unlike commercial breeders, broilers have a shorter life span, (2) Salmonella vaccines require two doses that are three to 4 weeks apart, and (3) Salmonella vaccines have a withdrawal period of 21 days before processing  in cell proliferation of birds in the control and the challenge groups at d12 of age can be due to individual variations. Variations among individuals of the same species can result in differences in the immune responses  in the jejunum samples of the challenge group. Previous findings demonstrate that S. Typhimurium is known to increase intestinal permeability of the chicken gut due to its ability to induce tight junction protein damage, as seen by the decreased expression of a few key markers, such as Claudin-1 and Zona Occludens-1  cells . There i species , includi species . It has nfection . Howeverlagellin  and thatoduction . Moreoveoduction  in whichoduction  and resuresponse . Future Salmonella delivered by gel-spray to broilers. The CNP vaccine (1) can overcome the hurdles of conventional vaccines; (2) had no negative effects on production performance of broilers; (3) elicited antigen-specific systemic and mucosal immune responses; (4) elicited antigen-specific recall response against S. Enteritidis Flagellin, S. Enteritidis HKA, S. Typhimurium HKA, and S. Litchfield HKA; (5) reduced the Salmonella load in the ceca, spleen and small intestine of broilers at d35 of age; (6) decreased the loss in gut permeability caused by S. Enteritidis; and (7) increased IL-6, IL- 17, and TNF-α mRNA in cecal tonsils. Future studies will examine the CNP vaccine potential to convey cross-protection against heterologous Salmonella serovars, the efficacy of the CNP vaccine in reducing S. Enteritidis loads in ready-to-eat carcasses, and the relationship between the CNP vaccine and the Th17/IL-17 immune response to explore the role of Th17 cells and IL-17 cytokines on Salmonella infections in broilers. We conclude that the CNP vaccine is a viable alternative to conventional Salmonella vaccines for poultry.This study analyzed the efficacy of a CNP vaccine against


Polygonatum sibiricum (Asparagaceae) is often used as an herbal drug in the traditional medicine of Southeast Asia. Its rhizome, called “Huang Jing”, is used in traditional Chinese medicine as an immune system stimulant, hypolipidemic agent, anti-aging agent, anti-fatigue agent, and cardiovascular protectant. We investigated the antioxidant, anti-acetylcholinesterase (AChE), anti-inflammatory, and anti-α-glucosidase effects of various solvent extracts and major bioactive components of Polygonatum sibiricum (PS) and processed Polygonatum sibiricum (PPS). Dichloromethane extract of PS showed stronger antioxidant effects by DPPH, ABTS, and FRAP assays, and EtOAc extract displayed relatively high antioxidant activity by a superoxide radical scavenging test. Moreover, acetone, EtOAc, and dichloromethane extracts displayed a significant anti-α-glucosidase effect. EtOH and CH2Cl2 extracts showed effective AChE inhibitory activity. In addition, dichloromethane extract showed the best inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) accumulation in RAW264.7 macrophages. HPLC analysis was used to investigate and compare the content of major active components of various solvent extracts of PS and PPS. Rutin showed the most effective scavenging of DPPH and ABTS free radicals, while scopoletin and isoquercetin displayed the strongest anti-α-glucosidase and anti-AChE effect, respectively. Rutin showed the best inhibition against LPS-induced NO production and also inhibited inducible nitric oxide synthase (iNOS) expression in Western blot. The molecular docking of AChE and iNOS revealed that active components could have a better antagonistic effect than positive controls (common inhibitors). This study shows that the active extracts and components of Polygonatum sibiricum have the potential to be further developed as a natural anti-AChE, anti-α-glucosidase, antioxidant and anti-inflammatory agent. Polygonumsibiricum is known as “Huang Jing” in Taiwan and is listed in the Chinese Pharmacopoeia (2015 edition). According to the application, the raw medicinal herbs normally must be processed before use in clinical prescriptions. P. sibiricum can be classified as fresh PS, dried PS (PS) and processed PS (PPS) according to different processing methods. It is well known that nine cycles of steaming and sun-drying are a traditional processing method for processed PS [The dried rhizome of opoeia 205 editionessed PS , alkaloiessed PS , steroidessed PS , polysacessed PS  and theiessed PS ,8, anti-essed PS ,10, antiessed PS ,12, immuessed PS ,14, neuropoeia 20 edition.essed PS , anticanessed PS , and sleessed PS  activitiPolygonatum sibiricum and is one of the representative products of the caramelization reaction and Maillard reaction during PS processing. Long-term steaming converts reducing sugar into 5-HMF, which can be used as the main basis to distinguish PS from PPS, and the number of 5-HMFs of processed PS are several times higher than those of PS [5-Hydroxymethylfurfural (5-HMF) is the most important component that changes before and after processing se of PS . The numse of PS .Carbohydrates are an important part of our daily diet. Most carbohydrates are digested by salivary and pancreatic amylases and further broken down into monosaccharides by enzymes (mainly for α-glucosidase) in the brush border membrane of enterocytes . α-GlucoOxidative stress plays a crucial role in Alzheimer’s disease (AD) and other related neurological diseases, as well as diabetes. Intracellular oxidative stress is caused by the production of reactive oxygen species and reactive nitrogen species and the poor antioxidant capacity of cells. Therefore, excess reactive oxygen species and reactive nitrogen species can lead to protein and nucleic acid damage, which has been shown to be directly related to diabetes and AD. Oxidative stress increases the production of advanced glycation end products through sugar oxidation and lipid peroxidation. Advanced glycation end products and lipid peroxidation products are abundant in diabetes and AD and serve as markers for both diseases . EndogenNitric oxide (NO) is a free radical involved in the regulation of many physiological processes, such as vascular relaxation, neurotransmission, platelet aggregation, and immune regulation. NO is produced by nitric oxide synthase (NOS) from molecular oxygen and l-arginine, yielding l-citrulline as a by-product. When cytotoxins produced by inflammatory substances are present in the environment, the iNOS gene is stimulated, and its expression is elevated, thereby enabling iNOS to promote the production of nitric oxide .Flavonoids are considered health-promoting substances, some of which have anti-inflammatory and antioxidant properties. Flavonoids are abundantly present in vegetables, fruits, red wine and tea, and the flavonoid mostly consumed is quercetin. Quercetin has a variety of biological effects, such as hypoglycemic and anti-inflammatory activities ,27.P. sibiricum . Disodium hydrogenphosphate, sodium carbonate, potassium peroxodisulfate, and potassium dihydrogenphosphate were obtained from SHOWA Chemical Co. .Folin–Ciocalteau’s reagent, bovine serum albumin, ABTS, acarbose, sodium dodecyl sulfate, EDTA, sodium bicarbonate, α-glucosidase, iNOS, TNF-α, IL-6, acetylcholinesterase, lipopolysaccharide, acetylcholine iodide, DTNB, TPTZ, chlorogenic acid, DMSO, and Trolox were purchased from Sigma-Aldrich . Glycine was purchased from J.T.Baker . DPPH, phenazine methosulphate, and nitroblue tetrazolium were supplied from Tokyo Chemical Industry Co., Ltd. . Thiazolyl blue tetrazolium bromide was purchased from MedChemExpress . Butyl hydroxytoluene, nicotinamide adenine dinucleotide, sodium acetate, and potassium acetate were supplied from Acros Organics . Ammonium persulfate and TEMED were purchased from Bio-Rad . Tween20 was purchased from Merck . Acetic acid was supplied from Macron Fine Chemicals . Iron (III) chloride, aluminum chloride, as well as P. sibiricum were purchased from Nantou, Taiwan, in January 2022, and the processed rhizomes of P. sibiricum were supplied from Changsheng Chinese medicine shop, Taipei city, Taiwan, in August 2021 and identified by Prof. J.-J. Chen. The voucher specimens were stored in the Department of Pharmacy, National Yang Ming Chiao Tung University, Taipei, Taiwan. First, the rhizomes of P. sibiricum (15 g) were soaked in 150 mL of different solvents  and shaken on an orbital shaker for 24 h at room temperature. Subsequently, the extracts were filtered through filter paper (Whatman No. 1) and concentrated using a rotary evaporator at 38 °C. Finally, all extracts were stored at −20 °C until further experiments.The rhizomes of Polygonatum sibiricum were extracted 3 times for 3 days with MeOH (5.0 L). The MeOH extract was concentrated under reduced pressure at 37 °C to obtain MeOH extract (120 g). The MeOH extract  was purified by column chromatography (CC)  to afford 12 fractions: A1–A12. Fraction A2 (6.8 g) was subjected to CC ; H2O/MeOH 19:1–1:1, 900 mL fractions) to give 11 subfractions: A2-1–A2-11. Fraction A2-3 (335 mg) was purified by HPLC  to obtain rutin (31.3 mg) (tR 2.8 min).The dried rhizomes (1.0 kg) of 2O/MeOH (9:1–3:7) to give 8 fractions . Fraction A3-6 (347 mg) was purified by HPLC  to afford hyperoside (41.6 mg) (tR 9.0 min) and isoquercetin (25.4 mg) (tR 9.5 min). Fraction A5 (5.3 g) was chromatographed on C18 silica gel  and eluted with H2O/MeOH (7:3–2:8) to give 10 fractions . Fraction A5-6 (326 mg) was purified by HPLC  to afford scopoletin (26.2 mg) (tR 12.8 min). Fraction A8 (6.8 g) was separated by column chromatography over Sephadex LH-20 and eluted with 100% MeOH to yield 8 fractions (A8-1–A8-8). Fraction A8-3 (530 mg) was purified by HPLC  to afford 5-HMF (32.3 mg) (tR 3.2 min). The structures of rutin, hyperoside, isoquercetin, scopoletin and 5-HMF were identified by nuclear magnetic resonance (NMR) spectra acquired using a Bruker Avance 600 MHz spectrometer  (Fraction A3 (5.6 g) was chromatographed on C18 silica gel , eluting with HGermany) .v/v) (solvent A) and acetonitrile (solvent B) as follows: 0–8 min, isocratic elution with 95% A; 8–15 min, isocratic elution with 8% B; 15–35 min, isocratic elution with 10% B; 35–45 min, isocratic elution with 12% B; 45–55 min, isocratic elution with 15% B; 55–65 min, isocratic elution with 25% B; 65–75 min, isocratic elution with 45% B; 75–95 min, isocratic elution with 55% B; 95–105 min, isocratic elution with 65% B; 105–130 min, gradient elution from 65 to 100% B; 130–140 min, back to initial 95% A. The flow rate was 1.0 mL/min, and the injection volume was 500 μL at room temperature. Peaks were detected at 280 nm. Different compounds were identified by retention time.The reversed-phase HPLC assay for the measurement of the seven components was performed as previously described, with minor modifications ,29. ReveThe TPC of different solvent extracts was measured using a previously reported method .The TFC of different solvent extracts was determined using a previously reported method .This assay was determined using the procedure previously reported .This assay was determined using the procedure previously reported .This assay was determined using the method previously reported .The FRAP assay was determined using the method previously reported .This assay was performed according to the previous method .The AChE inhibitory assay was performed according to the previous method, with slight modifications . First, 2 [Murine RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin at 37 °C, 5% COThe NO inhibitory assay was performed based on the reference method .The MTT assay was carried out according to the previous method .The Western blot analysis was performed using the reference method with slight modifications . RAW264.The in silico evaluation was performed with AutoDock Vina (ADT ver. 4.0.1) software . CrystalStatistical analysis was carried out using the t-test, and all assays were performed at least three times and expressed as mean ± standard error of the mean (SEM). Less than 0.05 was considered statisti-cally significant.Polygonatum sibiricum (PS) and processed Polygonatum sibiricum (PPS). 2Cl2, EtOAc, acetone, MeOH, EtOH, and water extracts from PS and PPS. The yields of various solvent extracts were ranged from 13.49 ± 1.53% (methanol extract) to 0.35 ± 0.12% (dichloromethane extract) of PS, and 15.90 ± 2.64% (water extract) to 1.25 ± 0.03% (dichloromethane extract) of PPS. The dichloromethane extract of PS and ethyl acetate extract of PPS showed the highest TPC with 77.50 ± 7.34 and 60.47 ± 1.91 mg/g, respectively. The highest TFC (86.02 ± 1.54 and 98.30 ± 0.47 mg/g) were found in the ethyl acetate extracts of PS and PPS, respectively, among all solvent extracts  and acetone extracts (SC50 = 278.31 ± 3.26 μg/mL) of PS showed relatively strong antioxidant effects by DPPH radical scavenging assay among all extracts.The DPPH radical scavenging ability of different solvent extracts is displayed in 50 = 229.37 ± 5.59 μg/mL) of PS showed the greatest ABTS radical scavenging effect, followed by dichloromethane extract (SC50 = 240.49 ± 5.68 μg/mL), ethanol extract (SC50 = 245.48 ± 3.59 μg/mL), and methanol extract (SC50 = 364.48 ± 3.21 μg/mL).The ABTS radical scavenging ability of different solvent extracts is displayed in 50 > 400 g/mL), except for the EtOAc extract (SC50 = 190.23 ± 1.09 μg/mL) and water extract (SC50 = 294.54 ± 7.28 μg/mL) of PS.The results are shown in The FRAP activities of all extracts are shown in P. sibiricum had the strongest anti-α-glucosidase activity (IC50 = 22.34 ± 1.66 μg/mL), followed by acetone extract (IC50 = 26.13 ± 2.48 μg/mL) and dichloromethane extract (IC50 = 34.29 ± 6.26 μg/mL).As presented in 50 = 379.07 ± 4.23 μg/mL). These results suggest that the low-polarity solvent extracts of PS had a higher α-glucosidase inhibitory effect  of PS and the ethyl acetate extract (IC50 = 32.60 ± 5.27 μg/mL) and dichloromethane extract (IC50 = 56.27 ± 7.11 μg/mL) of PPS showed the most effective AChE-inhibitory effect among all extracts.Alzheimer’s disease (AD) is a progressive, neurodegenerative disease characterized by a decline in cognitive and memory functions. Acetylcholinesterase (AChE) inhibitors block the AChE enzyme, thus revitalizing cholinergic action to improve memory and cognitive function. The AChE inhibitory effect of each extract is presented in 50 = 18.84 ± 1.80 μg/mL) of PS and dichloromethane extract (IC50 = 27.48 ± 6.99 μg/mL) of PPS show the greatest NO inhibitory effect among all solvent extracts.The NO inhibitory assay of each extract is displayed in The MTT assay is mainly used to detect the cytotoxicity of tested samples. From the MTT assay , the celPolygonatum sibiricum by reversed-phase HPLC analysis and the 1H-NMR spectra of bioactive compounds. P. sibiricum in reversed phase HPLC. The amounts of the five bioactive compounds in different solvent extracts are illustrated in 1), scopoletin (2), isoquercetin (3), hyperoside (4), and rutin (5) (utin (5) .1–5) are displayed in 5) (SC50 = 5.60 ± 0.34 μM) showed the strongest DPPH radical scavenging effect, followed by hyperoside (4) (SC50 = 12.46 ± 4.02 μM), and isoquercetin (3) (SC50 = 12.64 ± 3.21 μM). Rutin (5) (SC50 = 15.43 ± 0.25 μM) also showed the most potent ABTS scavenging effect, followed by isoquercetin (3) (SC50 = 22.73 ± 1.17 μM) and hyperoside (4) (SC50 = 29.26 ± 0.51 μM). Compounds 3, 4 and 5 displayed higher superoxide radical scavenging effects than 1 and 2. In addition, compounds 3 and 4 showed a more effective antioxidant effect than BHT by FRAP assay (Antioxidant assay results of isolated components (AP assay .2) (IC50 = 23.63 ± 7.22 μM) showed the most effective anti-α-glucosidase effect, followed by isoquercetin (3) (IC50 = 159.73 ± 3.12 μM), hyperoside (4) (IC50 = 208.14 ± 5.70 μM), and rutin (5) (IC50 = 331.15 ± 3.81 μM). Compounds 2–5 showed more potent anti-α-glucosidase effect than the positive control, acarbose (IC50 = 550.15 ± 7.65 μM) (As displayed in 7.65 μM) .3) (IC50 = 23.13 ± 3.15 μM) exhibited the strongest anti-AChE effect, followed by scopoletin (IC50 = 32.35 ± 2.05 μM), rutin (5) (IC50 = 33.09 ± 5.43 μM), 5-HMF (IC50 = 81.46 ± 11.05 μM), and hyperoside (4) (IC50 = 121.10 ± 10.70 μM). Compounds 2, 3 and 5 showed more effective anti-AChE activities than the positive control, chlorogenic acid (68.23 ± 2.90 μM).As shown in 5) (IC50 = 9.89 ± 1.36 μM) showed the strongest nitric oxide inhibitory effect, followed by isoquercetin (3) (IC50 = 17.03 ± 1.28 μM), hyperoside (4) (IC50 = 18.87 ± 1.68 μM), 5-HMF (1) (IC50 = 34.90 ± 8.80 μM) and scopoletin (2) (IC50 = 36.26 ± 4.65 μM). Compounds 3 and 5 exhibited more effective anti-NO activities than the positive control, quercetin (18.26 ± 0.54 μM).As showed in As shown in 5), isoquercetin (3), and hyperoside (4), with their better inhibitory activity against NO production, were selected for further analysis of their inhibitory effect on iNOS. As shown in 3, 4, and 5 significantly inhibited iNOS production in a concentration-dependent manner. Therefore, the reason for their inhibition of NO production can be verified.Rutin  modeling program. The crystal structure (PDB: 1C2B) of acetylcholinesterase from is study .3) , scopoletin (2) , and rutin (5)  were lower than those of chlorogenic acid , suggesting that these compounds can have better docking ability with the crystal structure of PDB: 1C2B.The molecular docking simulations were performed along with the binding affinity calculations for PDB: 1C2B (acetylcholinesterase) and active compounds. The interactions between active compounds and PDB: 1C2B were displayed in the best-docked poses for the calculations. These results demonstrate the high accuracy of the existing simulation system, thereby supporting further calculations. The lowest binding energy for each ligand was considered the optimal conformation, and the binding affinities are shown in 3) with the active sites of E. electricus acetylcholinesterase (AChE) are shown in Interactions of isoquercetin (2) and E. electricus acetylcholinesterase is shown in Molecular docking of scoloptein (5) with the active sites of E. electricus AChE are shown in 5) binds to TYR 124 and GLN 71 via conventional hydrogen bonding and to TRP 286, PRO 88, and SER 125 via carbon-hydrogen bonding. Unfavorable donor-donor and unfavorable acceptor-acceptor interactions were found on PHE 295, TYR 341, and TYR 337. There are other interactions with rutin, such as π-π T-shaped, alkyl, and π-π stacked interactions to make a stable complex of rutin and AChE.Interactions of rutin , isoquercetin (3), and hyperoside (4), all have anti-inflammatory potential. Therefore, these three compounds were used in a molecular docking model to see their binding abilities to iNOS. The 3D structure (PDB: 1M9T) of iNOS used as a docking model is from Mus musculus, and the active site consists of four pockets, where the substrate-binding S pocket contains heme [According to the results of the NO production inhibition test  and the ins heme .5) with the active sites of Mus musculus iNOS are shown in Interactions of rutin (3), which mainly forms conventional hydrogen bonds with TRP 366 and even with GLU 371, ASP 376 and ARG 382, and also forms π-alkyl interactions with PRO 344 and VAL 346. Carbon hydrogen bonds are observed between isoquercetin and GLN 371 and GLN 257. Most importantly, isoquercetin forms π-π-carbon and π-π stacked interactions with HEM 901 to remain parallel to HEM 901 and increase the affinity of isoquercetin and iNOS. (The next compound is isoquercetin (nd iNOS. .4), which mainly forms conventional hydrogen bonds with TRP 366 as well as with GLU 371, ASP 376, GLN 257, SER 256 and ARG 382. Hyperoside also forms π-alkyl interactions with PRO 344 and VAL 346. In addition, hyperoside forms π-cation, π-π stacked, and conventional hydrogen bond interactions with HEM 901 to increase their affinity (The last is hyperoside (affinity .5) was significantly higher than compounds 1–4 and the positive control quercetin (5) has the greatest binding affinity with iNOS, which is consistent with the strongest anti-NO activity of rutin (5) presented in According to the binding energy data presented in uercetin . This inPolygonum sibiricum were extracted using solvents of different polarities, and the biological activities of the extracts and isolated compounds were evaluated. The results show that by using solvents of different polarities, different extracts and components with different contents can be obtained and show different biological activities.Various approaches have been devoted to extracting natural products  to replace modern medicines. For many years, the most common extraction methods were boiling or decoction, both of which were considered cost-effective and easy to perform. Researching and finding natural ingredients with health benefits from medicinal plants is a hot trend these days. In recent studies, organic solvents have been used to obtain extracts of natural products, including various metabolites, depending on the polarity and properties of these specific compounds . Other e3+) complexes to ferrous (Fe2+) complexes [The ABTS and DPPH assays are mainly used to evaluate the antioxidant effects of natural compounds, which are usually related to the proton radical scavenging or hydrogen donating capacity of the compounds . The supomplexes . In our 2Cl2, EtOAc, acetone, EtOH, MeOH, and water) of PS and PPS was demonstrated for the first time in this study. This may give a direction for the choice of suitable solvents for TFC, TPC, and antioxidant extraction applications. According to the antioxidant assay result, rutin (5) (SC50 = 5.60 ± 0.34 μM) showed the strongest DPPH radical scavenging effect. Compounds 3, 4 and 5 displayed higher superoxide radical scavenging effects than 1 and 2. In addition, compounds 3 and 4 showed a more effective antioxidant effect than BHT by FRAP assay.A comparative assessment of the TPC, TFC, and antioxidant effects  of various solvent extracts (CH2) (IC50 = 23.63 ± 7.22 μM), isoquercetin (3) (IC50 = 159.73 ± 3.12 μM) and hyperoside (4) (IC50 = 208.14 ± 5.70 μM) showed more potent anti-α-glucosidase activity than acarbose (IC50 = 550.15 ± 7.65 μM). Compound 2 is about 10 times more potent than acarbose against α-glucosidase.Among all antidiabetic drugs used to treat type 2 diabetes, α-glucosidase inhibitors are considered to be the most effective drugs for reducing postprandial hyperglycemia. Currently, acarbose, voglibose, and miglitol are effective enzyme inhibitors for the treatment of postprandial hyperglycemia, but these inhibitors have side effects on the gastrointestinal tract; therefore, they are not suitable for long-term use . Scopole3) has attracted the attention of many researchers because of its potential to improve brain memory function through different mechanisms, so it is believed to have a certain role in delaying the progression of AD memory loss [3) (IC50 = 23.13 ± 3.15 μM), scopoletin (2) (IC50 = 32.35 ± 2.05 μM) and rutin (5) (IC50 = 33.09 ± 5.43 μM) also show potent anti-AChE effects and deserve further investigation.Alzheimer’s disease (AD) is a progressive degenerative brain disease in which cognitive and memory functions deteriorate. It is primarily treated by AChE inhibitors that increase the cholinergic effects of the brain. In contrast, chlorogenic acid, a phenolic acid derived from food (fruits and vegetables), has been shown to have neuroprotective properties associated with Alzheimer’s disease ,49. Fromory loss . In our 3 exhibited the most significant inhibitory effect among all isolated compounds; therefore, the interaction between AChE and 3 was assessed by molecular docking. Compound 3 showed the best binding energy  compared to chlorogenic acid . This revealed that 3 could dock into the pocket of the crystal structure of AChE from E. electricus more efficiently than chlorogenic acid.Based on the results of the anti-AChE assay, compound 3 (IC50 = 17.03 ± 1.28 μM) and 5 (IC50 = 9.89 ± 1.36 μM) exhibited more effective anti-NO activities than the positive control, quercetin (IC50 = 18.26 ± 0.54 μM). Compounds 3, 4, and 5 also significantly inhibited the production of iNOS, TNF-α and IL-6 in a concentration-dependent manner.The inflammatory response is a defense mechanism of the body. A general inflammatory response leads to tissue protection and regeneration after injury and, in this way, responds to injury and infection. Under inflammatory conditions, activated macrophages can exhibit detrimental effects involving the overproduction of inflammatory cytokines such as NO, TNF-α and IL-6. Therefore, inhibiting the aberrant activation of macrophages may have therapeutic potential in the treatment of inflammation-related degenerative diseases. In our study, compounds 5 showed the strongest inhibitory effect among all isolated compounds. Therefore, the interaction between iNOS and compound 5 was assessed by molecular docking. Compound 5 showed the best binding energy  compared to quercetin . This revealed that 5 could dock into the pocket of the crystal structure of iNOS from M. musculus more efficiently than quercetin. In addition, our study performed molecular docking for the first time to calculate the binding energy of 5 with M. musculus iNOS.Based on Western blot analysis of iNOS expression, compound P. sibiricum (PS) and processed P. sibiricum (PPS) were investigated with anti-AChE, anti-α-glucosidase, anti-inflammatory and antioxidant assays. In this study, the CH2Cl2 extract of PS revealed the strongest antioxidant activity among all solvent extracts by ABTS, DPPH and FRAP assays. The EtOAc extract (IC50 = 22.34 ± 1.66 μg/mL) of PS displayed the highest anti-α-glucosidase effect compared with all other solvent extracts. The CH2Cl2 extract (IC50 = 18.84 ± 1.80 μg/mL) of PS showed the strongest anti-NO effect. The EtOAc extract (IC50 = 32.60 ± 5.27 μg/mL) of PPS revealed the most potent anti-AChE activity. Five isolated components from P. sibiricum were quantified by HPLC and identified as 5-hydroxymethylfurfural (1), scopoletin (2), isoquercetin (3), hyperoside (4), and rutin (5). Furthermore, a comparative assessment for the quantification of these major active compounds (1–5) of various solvent extracts  from P. sibiricum (PS) and processed P. sibiricum (PPS) by HPLC analyses was first carried out in this research.Different solvent extracts of 5 exhibited the most potent antioxidant effects as measured by DPPH and ABTS assays. In the FRAP experiment, 3 showed the most potent antioxidant effect (3898.88 ± 23.23 mM TE/g). Compounds 2–4 had a stronger anti-α-glucosidase effect than acarbose, and compounds 2, 3, and 5 showed better anti-AChE activity than chlorogenic acid. In addition, compounds 3, 4, and 5 obviously inhibited iNOS and NO production in a concentration-dependent manner. Compounds 3, 4, and 5 also significantly inhibited the production of TNF-α and IL-6 in a concentration-dependent manner. This verifies that these compounds possess potent anti-inflammatory activity. Further molecular docking computing results supported that the binding affinity of rutin (5) was significantly higher than compounds 1–4 and the positive control quercetin. This revealed that 5 has the strongest binding affinity with iNOS, which is consistent with the highest anti-NO activity of 5.Bioactivity assays indicated that 3–5) can be applied as herbal antioxidants against oxidative damage, and scopoletin (2) may be used as a natural anti-α-glucosidase agent. Furthermore, rutin (5) and isoquercetin (3) also can be used as natural anti-inflammatory and anti-AChE agents.The above active extracts and active compounds (especially