Fig 2: (A): Schematic representation of the experimental design, showing imiquimod treatment and cinnamon administration for the different groups: sham, sham treated with cinnamon (Sham Cinna), imiquimod-induced lupus (Lupus), imiquimod-induced lupus with cinnamon treatment (Lupus Cinna), and imiquimod-induced lupus with cinnamon administration starting 2 weeks before induction and continued after induction (Cinna Lupus Cinna). (B) Hippocampal immunofluorescence (scale bars 15 μm, magnification ×100) with quantification of IgG accumulation in the different groups. (C,D) Antibody array (C) with heatmap (D) of oxidative stress markers, TLR7 and MYD88, for the different groups. (E–K) Quantification of p-NRF/NRF, p-FOXO3/FOXO3, TLR7/β-actin, MYD88/β-actin, p-NOS3/NOS3, SOD1/β-actin, and SOD2/β-actin. (L,M) Western blot and quantification of p-FOXO3/FOXO3. (N,O) Western blot and quantification of p-NRF/NRF. a.u.: arbitrary units. * p < 0.05 and ** p < 0.01.

Fig 3: (A) Schematic representation of in vitro treatment of cultured hippocampal cells with imiquimod, imiquimod with cinnamon, or the plasma of different mice groups. (B–J) Antibody arrays, heatmap, and quantification of TLR7, MYD88, and oxidative stress markers in hippocampal cells treated with the plasma of different mice groups. (K–S) Antibody arrays, heatmap, and histograms of TLR7, MYD88, and oxidative stress markers in cultured hippocampal cells treated with imiquimod, cinnamon with imiquimod, or sham. a.u.: arbitrary units. * p < 0.05, ** p < 0.01.

Fig 4: Western blot of TLR pathway and platelet-related markers of the submandibular gland tissue of NOD mice. Submandibular gland was obtained at the age of 13 weeks, after intervention of CA-4948/Resiquimod/saline for 5 weeks. C: control group treated with saline; I: inhibitor group (treated with CA-4948); A: activator group (treated with Resiquimod). Compared with saline control group, Resiquimod induced significantly increased expression of the TLR7 signaling pathway molecules, interleukin (IL)-1β, IL-8, and Megakaryocyte Colony Stimulating Factor (MK-CSF), and decreased expression of TPO in the submandibular glands. While CA-4948 group showed opposite expression trends of these molecules (*p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant).

Fig 5: Detection of TLR7/8 on bovine X- and Y-sorted sperm(A) Immunofluorescence of TLR7 (green), TLR8 (green), acetyl α-tubulin (green) and DAPI (blue) on frozen-thawed un-sorted, X- and Y-sorted bovine sperm. (i–ii) Both TLR7 and TLR8 were stained at the acrosomal regions of the bovine sperm heads (red arrows), and tails (yellow arrows). Scale bar, 21 μm.(B, C, and E) (B) The percentages of positively stained bovine sperm for TLR7 and TLR8. Western blot analyses of TLR7 (C) and TLR8 (E) on bovine un-sorted (U), X-sorted (X) and Y-sorted (Y) sperm. TLR7 and TLR8 were resolved and blotted using non-reduced and reduced samples, respectively.(D and F) Quantification of the TLR7 (D) and TLR8 (F) bands from western blots. Data were analyzed using one-way ANOVA, ns = not significant (p > 0.05).(G) Flow cytometry analysis of bovine sperm stained for TLR7/8 and DAPI. In controls (left panels) the sperm were probed with the secondary antibody only, which provided the cutoff (vertical line) for stained and unstained sperm. The cells were detected by the FITC-A (x axis) and DAPI (y axis) channels. Each dot represents one sperm. The middle and right panels represent sperm stained for TLR7 and TLR8, respectively. Nearly all sperm were stained by both antibodies.(H) The percentages of positively stained bovine sperm as detected by flow cytometry. Data were analyzed using one-way ANOVA. All values are mean ± SD of at least three replicates, ns = not significant (p > 0.05).