Journal: NPJ Biofilms and Microbiomes

Article Title: Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling

doi: 10.1038/s41522-023-00415-2

Figure Lengend Snippet: Heatmaps displaying 36 highly expressed genes ( a ) and 76 lowly expressed genes ( b ) in the liver of darkness rats compared with control and DL.reuteri rats through DEG analysis using Ballgown software (|fold change | > 0.6 in the log 2 ratio value, raw P < 0.05). c Top terms from GO analysis (above) and terms from KEGG analysis (below) of the 112 differential genes in the DEG analysis. The P values of GO and KEGG analyses were determined on the DAVID website. d Visualization of the top GO and KEGG terms related with lipid metabolism and circadian rhythm in the DEG analysis. e Spearman rank correlations between module eigengenes (ME) and clinical biochemical index in the WGCNA analysis (|ρ | > 0.3, * P < 0.05). f Visualization of the top GO and KEGG terms related with lipid metabolism and circadian rhythm of purple module genes (102) in the WGCNA analysis. Circle, genes; Square, KEGG terms; Hexagon, GO terms. The bigger the square or hexagon, the more genes involved. g Crosstalk among different groups of genes identified the possible target genes taking essential roles in the lipid metabolism of L. reuteri -treated darkness rats. h mRNA abundances of Galr1 , Galr2 , Nr1d1 , Nr1d2 , Insig2 , Srebf1 , Lxra , and Rxra in rat liver detected by qPCR. β-Actin was used as a loading control for qPCR analyses. i Serum galanin concentration detected by ELISA. Statistical analysis ( h, i ) was performed with one-way ANOVA followed by Newman–Keuls multiple comparison test. n = 8 per group. Data present means ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: The nonspecific binding sites of membrane were blocked and incubated with diluted GALR1 antibody (1:500; #47567, Signalway Antibody, Maryland, USA), GALR2 antibody (1:500; #26459-1-AP, Proteintech, Wuhan, China), NR1D1 antibody (1:1000, #13418 S, Cell Signaling Technology, Massachusetts, USA; 1:1000, #14506-1-AP, Proteintech), NR1D2 antibody (1:500; #13906-1-AP, Proteintech), SREBP1 antibody (1:1000; #41878, Signalway Antibody), LXRa antibody (1:1000; #ab176323, Abcam, Cambridge, UK), RXRa antibody (1:1000; #ab125001, Abcam), INSIG2 antibody (1:500; #24766-1, Proteintech), P-AKT antibody (1:1000; #4060, Cell Signaling Technology), T-AKT antibody (1:1000; #4691, Cell Signaling Technology), P-ERK antibody (1:1000; #4370, Cell Signaling Technology) and T-ERK antibody (1:1000; #4695, Cell Signaling Technology) at 4°C for overnight.

Techniques: Software, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: NPJ Biofilms and Microbiomes

Article Title: Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling

doi: 10.1038/s41522-023-00415-2

Figure Lengend Snippet: mRNA and protein abundances of NR1D1, NR1D2, INSIG2, SREBP1, LXR, and RXR after NR1D1 knockdown or NR1D2 knockdown ( a , b ) as well as NR1D1 overexpression or NR1D2 overexpression ( c , d ) in HepG2 cells. a , c Left, representative images of western blot were shown. Right, immunoreactive bands were densitometrically quantified. b , d mRNA abundance detected by qPCR was presented. e mRNA and protein abundances of NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-ERK, T-ERK, P-AKT, and T-AKT after galanin treatment (#1179, Tocris Bioscience, Bristol, UK) at the concentration of 0, 50, 150, and 300 pg/mL for 24 h in HepG2 cells. Left, representative images of western blot were shown. Right, immunoreactive bands were densitometrically quantified (above); mRNA abundance detected by qPCR was presented (below). f mRNA and protein abundances of GALR1, GALR2, NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-ERK, T-ERK, P-AKT and T-AKT after GALR1 knockdown or GALR2 knockdown and further treatment with 300 pg/mL galanin for 24 h in HepG2 cells. g mRNA and protein abundances of GALR1, GALR2, NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-ERK, T-ERK, P-AKT, and T-AKT after GALR1 overexpression or GALR2 overexpression in HepG2 cells. h mRNA and protein abundances of NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-ERK, T-ERK, P-AKT, and T-AKT after treatment with(out) 300 pg/mL galanin, with(out) 10 μM LY294002 (#19-142, Sigma-Aldrich, St. Louis, USA) and with(out) 20 μM PD98059 (#19-143, Sigma-Aldrich). i mRNA and protein abundances of GALR1, NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-AKT, and T-AKT after GALR1 overexpression and further treatment with 10 μM LY294002 in HepG2 cells. GAPDH or β-ACTIN were used as loading controls for western blot and qPCR analyses. Representative Oil Red O staining and Nile Red staining after the induction of oleic acid and palmitic acid (OPA) and treatment with 300 pg/mL galanin for 24 h ( j ) or NR1D1 siRNA ( k ) in HepG2 cells. Left, representative images were shown. Right, intensity was quantified. Blots and images are representative. Statistical analysis was performed with unpaired Student’s t-test or one-way ANOVA followed by Newman–Keuls multiple comparison test. Data present means ± SEM from 3 to 5 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 against si-NC cells or against Vec-NC cells or against control cells; # P < 0.05, ## P < 0.01 against si-GALR2 cells or against PD98059 cells or against Vec-GALR1 cells.

Article Snippet: The nonspecific binding sites of membrane were blocked and incubated with diluted GALR1 antibody (1:500; #47567, Signalway Antibody, Maryland, USA), GALR2 antibody (1:500; #26459-1-AP, Proteintech, Wuhan, China), NR1D1 antibody (1:1000, #13418 S, Cell Signaling Technology, Massachusetts, USA; 1:1000, #14506-1-AP, Proteintech), NR1D2 antibody (1:500; #13906-1-AP, Proteintech), SREBP1 antibody (1:1000; #41878, Signalway Antibody), LXRa antibody (1:1000; #ab176323, Abcam, Cambridge, UK), RXRa antibody (1:1000; #ab125001, Abcam), INSIG2 antibody (1:500; #24766-1, Proteintech), P-AKT antibody (1:1000; #4060, Cell Signaling Technology), T-AKT antibody (1:1000; #4691, Cell Signaling Technology), P-ERK antibody (1:1000; #4370, Cell Signaling Technology) and T-ERK antibody (1:1000; #4695, Cell Signaling Technology) at 4°C for overnight.

Techniques: Over Expression, Western Blot, Concentration Assay, Staining

Journal: NPJ Biofilms and Microbiomes

Article Title: Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling

doi: 10.1038/s41522-023-00415-2

Figure Lengend Snippet: a Timeline depicting the treatments of darkness, L. reuteri , and M617 in different groups of the M617-treated rat model ( n = 6 per group). b Body weight changes. c Representative Oil Red O staining of liver. Scale bar: 50 μm and 25 μm. d Left to right, serum concentrations of TG, CHOL, HDL-C, LDL-C, and NEFA detected by ELISA. e mRNA abundances of Nr1d1 and Srebf1 in rat liver. f Protein abundances of NR1D1 and SREBP1 in rat liver. Left, representative images of western blot were shown. Right, immunoreactive bands were densitometrically quantified. g Timeline depicting the treatments of darkness and M40 in different groups of the M40-treated rat model ( n = 8 per group). h Body weight changes. i Representative Oil Red O staining of liver. Scale bar: 50 μm and 25 μm. j Left to right, serum concentrations of TG, CHOL, HDL-C, LDL-C, and NEFA detected by ELISA. k mRNA abundances of Nr1d1 and Srebf1 in rat liver. l Protein abundances of NR1D1 and SREBP1 in rat liver. Left, representative images of western blot were shown. Right, immunoreactive bands were densitometrically quantified. GAPDH or β-Actin were used as loading controls for western blot and qPCR analyses. Statistical analysis was performed with one-way ANOVA followed by Newman–Keuls multiple comparison test. Data present means ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: The nonspecific binding sites of membrane were blocked and incubated with diluted GALR1 antibody (1:500; #47567, Signalway Antibody, Maryland, USA), GALR2 antibody (1:500; #26459-1-AP, Proteintech, Wuhan, China), NR1D1 antibody (1:1000, #13418 S, Cell Signaling Technology, Massachusetts, USA; 1:1000, #14506-1-AP, Proteintech), NR1D2 antibody (1:500; #13906-1-AP, Proteintech), SREBP1 antibody (1:1000; #41878, Signalway Antibody), LXRa antibody (1:1000; #ab176323, Abcam, Cambridge, UK), RXRa antibody (1:1000; #ab125001, Abcam), INSIG2 antibody (1:500; #24766-1, Proteintech), P-AKT antibody (1:1000; #4060, Cell Signaling Technology), T-AKT antibody (1:1000; #4691, Cell Signaling Technology), P-ERK antibody (1:1000; #4370, Cell Signaling Technology) and T-ERK antibody (1:1000; #4695, Cell Signaling Technology) at 4°C for overnight.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: NPJ Biofilms and Microbiomes

Article Title: Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling

doi: 10.1038/s41522-023-00415-2

Figure Lengend Snippet: a Multiomics correlation networks of all variables for the faecal microbiome (green), faecal metabolome (purple) and serum metabolome (yellow) within control rats (left), darkness rats (middle), and DL.reuteri rats (right). Vertices indicate omics variables, and lines indicate a significant Spearman’s rank correlation coefficient at |ρ | > 0.8 and P < 0.05. Red connections indicate positive correlation, and blue connections show negative correlations. b Procrustes analyses of faecal microbiome versus faecal metabolome (above) and of faecal metabolome versus serum metabolome (below). c Spearman correlation network between target faecal metabolites (circle) and target serum metabolites (square) ( P < 0.05). The color of each metabolite was determined by their correlations with target genera ( P < 0.05): Pink, Lactobacillus ; Blue, Clostridium sensu stricto 1 ; Green, Ruminococcaceae UCG-010 ; Yellow, Family XIII AD3011 group . d Spearman rank correlations between target serum metabolites and mRNA expression of Galr1 , Nr1d1 , and Srebf1 in rat liver (* P < 0.05, ** P < 0.01, *** P < 0.001). e Timeline depicting the treatments of darkness, L. reuteri , and capric acid in different groups of the capric acid-treated rat model ( n = 6 per group). f Body weight changes. g Left to right, serum concentrations of TG, CHOL, HDL-C, LDL-C, and NEFA detected by ELISA. h Representative Oil Red O staining of liver. Scale bar: 50 μm and 25 μm. i mRNA abundances of Galr1 , Nr1d1, and Srebf1 in rat liver. β-Actin was used as a loading control for qPCR analyses. Statistical analysis ( g , i ) was performed with one-way ANOVA followed by Newman–Keuls multiple comparison test. Data present means ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: The nonspecific binding sites of membrane were blocked and incubated with diluted GALR1 antibody (1:500; #47567, Signalway Antibody, Maryland, USA), GALR2 antibody (1:500; #26459-1-AP, Proteintech, Wuhan, China), NR1D1 antibody (1:1000, #13418 S, Cell Signaling Technology, Massachusetts, USA; 1:1000, #14506-1-AP, Proteintech), NR1D2 antibody (1:500; #13906-1-AP, Proteintech), SREBP1 antibody (1:1000; #41878, Signalway Antibody), LXRa antibody (1:1000; #ab176323, Abcam, Cambridge, UK), RXRa antibody (1:1000; #ab125001, Abcam), INSIG2 antibody (1:500; #24766-1, Proteintech), P-AKT antibody (1:1000; #4060, Cell Signaling Technology), T-AKT antibody (1:1000; #4691, Cell Signaling Technology), P-ERK antibody (1:1000; #4370, Cell Signaling Technology) and T-ERK antibody (1:1000; #4695, Cell Signaling Technology) at 4°C for overnight.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: NPJ Biofilms and Microbiomes

Article Title: Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling

doi: 10.1038/s41522-023-00415-2

Figure Lengend Snippet: Left, circadian dysrhythmia due to constant darkness resulted in dyslipidemia and reproductive hallmarks of PCOS in rats. Elevated galanin-GALR1 induced by darkness exposure functioned as an upstream factor of PI3K/AKT pathway and further suppressed NR1D1-induced SREBF1 transcription and translation, thus inducing hepatic lipid accumulation in PCOS-like rats. Right, L. reuteri supplementation ameliorated dyslipidemia and reproductive hallmarks in circadian dysrhythmia-induced PCOS-like rats. L. reuteri restructured microbiome-metabolome network in darkness rats ameliorating the abundance of Lactobacillus , Clostridium sensu stricto 1 , Ruminococcaceae UCG-010 , and Family XIII AD3011 group , followed by varied serum levels of cortisol, cis-9-palmitoleic acid, 13-methylmyristic acid, capric acid, and dUMP. Notably, capric acid mediated the inhibition of L. reuteri on hepatic GALR1-PI3K/AKT-NR1D1-SREBP1 pathway, which eventually alleviated dyslipidemia.

Article Snippet: The nonspecific binding sites of membrane were blocked and incubated with diluted GALR1 antibody (1:500; #47567, Signalway Antibody, Maryland, USA), GALR2 antibody (1:500; #26459-1-AP, Proteintech, Wuhan, China), NR1D1 antibody (1:1000, #13418 S, Cell Signaling Technology, Massachusetts, USA; 1:1000, #14506-1-AP, Proteintech), NR1D2 antibody (1:500; #13906-1-AP, Proteintech), SREBP1 antibody (1:1000; #41878, Signalway Antibody), LXRa antibody (1:1000; #ab176323, Abcam, Cambridge, UK), RXRa antibody (1:1000; #ab125001, Abcam), INSIG2 antibody (1:500; #24766-1, Proteintech), P-AKT antibody (1:1000; #4060, Cell Signaling Technology), T-AKT antibody (1:1000; #4691, Cell Signaling Technology), P-ERK antibody (1:1000; #4370, Cell Signaling Technology) and T-ERK antibody (1:1000; #4695, Cell Signaling Technology) at 4°C for overnight.

Techniques: Inhibition