Fig 2: Proposed mechanisms for the amelioration of L. reuteri on dyslipidemia in circadian dysrhythmia-induced PCOS-like rats.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.

Fig 3: TSI environment causes mitophagy deficiency and mitochondrial dysfunction in neurons of SCN. (A) TEM images show the mitochondria in neurons of the SCN from both the control and model groups at ZT3 and ZT11 (n = 4). The red dashed square indicates the zoom region. Scale bars: 1 μm. (B) Quantification of damaged mitochondria relative to total mitochondria. (C) Mitochondrial membrane potential analysis conducted in the SCN tissues of the control and model groups at ZT3 and ZT11 (n = 4). (D) TEM images show mitophagy in neurons of the SCN from the control and model groups at ZT3 and ZT11 (n = 4). Mitophagosome-like structures are indicated by red arrowheads. The red dashed square indicates the zoom region. Scale bars: 1 μm. (E) Quantification of mitophagy numbers. (F) qPCR analysis depicts the mtDNA/nDNA ratio in the SCN of the control and model groups at ZT3 and ZT11 (n = 4). (G) Western blotting analysis showcases the protein expression of NR1D1, BMAL1, TOMM20, and LC3 II/I after cells were treated with UA (25 μM, 50 μM, 100 μM, or a vehicle) for 48 h (n = 3). (H) Quantification of NR1D1, BMAL1, TOMM20, and LC3 II/I protein expression. (I) Western blotting analysis shows the protein expression of NR1D1, BMAL1, TOMM20, and LC3 II/I after cells were treated with mdivi-1 (12.5 μM, 25 μM, 50 μM, or a vehicle) for 48 h (n = 3). (J) Quantification of NR1D1, BMAL1, TOMM20, and LC3 II/I protein expression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Data are means ± S.E.M. Two-sided Student’s t tests were used to measure the significance.

Fig 4: The WAS-induced transcriptome changes correlated with IBS phenotypes(A) Western blot analysis of control (CT) and water avoidance stressed (WAS) mouse colon IECs.(B) Typical hematoxylin-eosin (HE) staining of CT and WAS colon epithelium with/without NR1D1 agonist SR9009 intervention. Inflammatory infiltration was present in WAS mice, and SR9009 significantly prevented morphology changes.(C) Pathology scores increased in WAS mice, and SR9009 prevented this effect.(D) Stress increased FD4 (fluorescein isothiocyanate-dextran 4 kDa) permeability, and this effect was antagonized by SR9009 intervention.(E) Stress reduced the thresholds of pain responses. Data are expressed as means ± standard error, and statistical significance between groups was determined using an unpaired t test with Welch’s correction (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; N = 3, 4, 5).(F) Visceral hyperalgesia was evaluated with AWR (abdominal withdrawal reflex) scores in response to CRD (colorectal distension). AWR data were analyzed with two-way ANOVA analysis (with Tukey’s multiple comparison test); the significance between WAS/CT and WAS+SR9009/WAS is illustrated (∗, p < 0.05; ∗∗, p < 0.01; N = 4, 5).

Fig 5: Genetic and pharmacological NR1D1 inhibition rescues meiosis initiation arrest in Stra8-deficient testes.(A) Photomicrographs of hematoxylin/eosin-stained testicular cross sections from testes with indicated genotypes at 10 d.p.p.. Insets show germ cells exhibiting chromosome condensation at early meiotic prophase. Percentages of tubules containing germ cells exhibiting chromosome condensation at early meiotic prophase are shown underneath. Data represent mean ± SD. n = 2–3 mice per genotype. (B) Dual immunofluorescence staining of γ-H2AX and SYCP3 in Stra8-/-;Nr1d1+/+ and Stra8-/-;Nr1d1-/- testes at 10 d.p.p.. Note the brighter foci of SYCP3 staining in Stra8-/-;Nr1d1+/+ testes indicative of premeiotic status of their germ cells in contrast to the germ cells with nuclear distribution of SYCP3 and foci of γ-H2AX found in Stra8-/-;Nr1d1-/- testes. (C) qRT-PCR analysis of testicular Spo11, Dmc1, and Sycp3 expression normalized to β-actin from mice with indicated genotypes at 10 d.p.p.. Data are mean ± SD; n = 2–3 mice per group. *P < 0.05 (Student’s t test). (D) Photomicrographs of hematoxylin/eosin-stained testicular cross sections from wild-type and Stra8-deficient testes treated with vehicle or SR8278 (100 mg/kg) for 3 days. Testes were collected at 10 d.p.p.. Insets show germ cells exhibiting chromosome condensation at early meiotic prophase. Percentages of tubules containing germ cells exhibiting chromosome condensation at early meiotic prophase are shown underneath. Data represent mean ± SD. n = 3 mice per genotype. (E) Dual immunofluorescence staining of γ-H2AX and SYCP3 in Stra8-deficient testes treated with vehicle or SR8278. (F) qRT-PCR analysis of testicular Spo11, Dmc1, and Sycp3 expression normalized to β-actin from mice treated with vehicle or SR8278. Data are mean ± SD; n = 4–5 mice per group. *P < 0.05 (Student’s t test).