Fig 2: Pathological and chemical changes in ovaries after nitrite exposure. A–E; Pathological alterations in control and treatment groups (H&E staining). A–B: Low magnification of control and treatment ovaries. The control ovary is plump, with some developing follicles (A, arrow). Conversely, the treatment group ovary shows notable atrophy and congested blood vessels (B, arrow). C–E: High magnification of control and treatment ovaries. Various follicles, including secondary follicles (arrow) and some antral follicles (*), can be observed in the control ovary (C). Corpora lutea with loose connective tissue and vessels inside can be observed in the control group tissues (D, *). After nitrite exposure, mature follicles are decreased, but atretic follicles with morphological deformation (arrow), such as oocyte autolysis, zona pellucida collapse, follicular wall collapse, are markedly increased (E). F–G: Nitrite exposure and cell apoptosis in granulosa follicular cells (TUNEL staining and DAPI counterstaining). In the control group, DAPI counterstaining (blue) shows numerous granulosa cells (blue) surrounding oocytes in the follicles, with few apoptotic cells (green, arrow) in granulosa cells (F). After high-dose nitrite exposure, numerous apoptotic cells (arrow) can be observed in granulosa cells (G). Interestingly, the matrix in the follicular cavity appears to be TUNEL-positive. H–J: Nitrite exposure and iNOS expression in granulosa cells (iNOS immunofluorescent labeling and DAPI counterstaining). In the control group (H), few iNOS positive cells with weak fluorescence (arrow) can be seen in granulosa cells (blue). The matrix in the follicular cavity exhibits iNOS-positivity. After nitrite exposure, numerous iNOS positive cells (arrow) can be observed in follicular granulosa cells (J). It should be noted that although there the iNOS positive matrix in the follicular cavity of the control group is dense, the iNOS positive granulosa cells are few. In contrast, the iNOS positive granulosa cells are enriched in treatment groups (arrow, with DAPI and iNOS double labeling). The control and high-dose exposure groups are indicated as Cont. and Treat. in the upper-left corner, respectively. Scale bar: A-B, 20 µm; C & E, 50 µm; D, 100 µm; F-J, 200 µm. H&E, hematoxylin-eosin; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase.

Fig 3: Activated NF-?B-mediated upregulation of oxidative and inflammatory factors in ovaries of aging rats. (A) Western blots and quantification of (B) NF-?B p65, (C) NF-?B p50, (D) A20, (E) I-?B alpha, and (F) pI-?B alpha, in ovaries of naturally aging SD rats. Data are presented as mean ± SD (n = 6). (G,H) NF-?B binding activity in ovaries of naturally aging SD rats. Data presented as mean ± SD (n = 6). Lane 1, blank control; Lane 2, 3 M (3-month-old); Lane 3, 9 M; Lane 4, 12 M; Lane 5, 16 M; Lane 6, 22 M; Lane 7, competitive control: 9 M and 200-fold molar excess of unlabeled DNA. (I) Western blots and quantification of (J) iNOS, (K) TNF a, and (L) TNFR1 in ovaries of aging rats. Data are presented as mean ± SD (n = 6). The differences between various age groups were compared using one-way ANOVA followed by Tukey’s multiple comparison test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with 3-month-old rats. #, p < 0.05; ##, p < 0.01; ###, p < 0.001; ####, p < 0.0001 compared with 9-month-old rats. @, p < 0.05; compared with 22-month-old rats.

Fig 4: The H2S donor downregulated the levels of microglial pro-inflammatory profile in vivo. (A,B) Representative images of iNOS (red) with Iba1 (green) immunostaining or (C,D) Arg1 (red) with Iba1 (green) immunostaining in the CA1 and CA3 regions of the hippocampus in control and the H2S donor-treated SE mice. Scale bar 25 µm in the merged panel and 5 µm in the magnified panel. Magnified images are expansions of boxed areas in corresponding panels in the left of each magnified image. (E) Quantitative analysis of Iba1+cells in CA1 and CA3 regions of the hippocampus. (F) Quantitative data are shown the ratio of iNOS+: Iba1+to total Iba1+cells. n = 6 per group. ***p < 0.001 vs SE. Two-way ANOVA with Bonferroni post hoc tests. (G) Quantitative data are shown as the ratio of Arg1+: Iba1+to total Iba1+cells. n = 6 per group. ***p < 0.001 vs SE. Student’s t test. (H–K) Western blot assay shows that expression levels of microglial pro-inflammatory markers (COX2 and TNF-a) are increased in the hippocampus of SE mice. However, the H2S donor decreased COX2 and TNF-a expression and increased Arg-1and IL-10 expression in SE mice. The results are expressed as the mean±SEM. n = 3 per group.*p < 0.05, **p < 0.01 vs Control, #p < 0.05, ##p < 0.01 vs SE. One-way ANOVA with Bonferroni post hoc tests.

Fig 5: AS-DpS-AS is more effective against DNBS-induced rat colitis than SSZ. Three days after colitis induction by DNBS, SSZ (50 mg/kg) and AS-DpS-AS (L: 17 mg/kg, equivalent to 25 mg/kg of SSZ, H: 34 mg/kg, equivalent to 50 mg/kg of SSZ) were administered orally to rats once per day, and the rats were sacrificed after 6 days of treatment. (A) Left panel: Serosal and luminal sides of the distal colon of rats were photographed. Representative images are shown. Right panel: Colon damage score (CDS) was determined for each group, as described in the Section 2. * a < 0.05 vs. DNBS control, # a < 0.05. (B) Myeloperoxidase (MPO) activity was measured in the inflamed distal colon (4 cm). * p < 0.05 vs. control, # p < 0.05. (C) CINC-3 levels in the inflamed colon were measured using an ELISA kit. (D) iNOS and COX-2 levels were measured in the inflamed distal colon using Western blotting. * p < 0.05 vs. control, # p < 0.05. The data in (A–C) represent mean ± SD (n = 5). NM: not measurable.