Fig 2: The protective effect on circulating EPCs in female mice with PM exposure was independent of estrogen. (A) The size of the uterus and ovary tissue from female mice with ovariectomy was much smaller than that from female mice with sham operation. (B) Serum level of estradiol was significantly decreased in female mice with ovariectomy to a level similar to male mice. (C,D) Pro-inflammatory cytokines IL-6 (C) and IL-1β (D) in male mice with PM exposure were significantly higher than female mice with or without ovariectomy. (E) Elevated serum levels of TNF-α induced by PM exposure were only observed in male mice, not in female mice with or without ovariectomy. (F) Flow cytometry analysis was used to detect the ROS in blood monocytes, with summary data (G) showing that a significant increase in ROS production was observed in male mice with PM exposure, but not in female mice with or without ovariectomy after PM exposure. (H) Blood cells were incubated with annexin V and PI for apoptosis analysis, with summary data (I) showing a significant increase in the apoptotic rate only in male mice with PM exposure, not in female mice with or without ovariectomy (G). (J,L) Flow cytometry analysis for circulating EPC (CD34+/CD133+) or (Sca-1+/Flk-1+), with summary data (K,M) showing a significantly decreased circulating EPC population in male mice after PM exposure, not in female mice with or without ovariectomy. MPBS: male mice with PBS treatment (n = 6); MPM: male mice with PM exposure (n = 6); Sham-FPBS: female mice with intact ovary and PBS treatment (n = 8); Sham-FPM: female mice with intact ovary and PM exposure (n = 8); Surgery-FPBS: female mice with ovariectomy and PBS treatment (n = 8); Surgery-FPM: female mice with ovariectomy and PM exposure (n = 8). (C–E) n = 5 for each group. * p < 0.05, ** p < 0.01, and *** p< 0.001.

Fig 3: PCV2 infection induces IL-10 production to promote viral replication. Wild-type mice and il10−/− mice were infected with PCV2, and samples were collected at 7, 14, and 28 d.p.i. (A) The serum IL-10 expression was measured by ELISA n = 15. (B) The PCV2 copy numbers in lungs were detected by qPCR. The data are presented as mean ± SEM of three independent experiments n = 15. (C–H) Other groups of wild-type mice and il10−/− mice were infected with PCV2. The IL-2, IL-6, IL-12, TNF-α, IFN-α, and IFN-γ production were measured by ELISA at 0, 1, 3, and 6 h.p.i., respectively. The value of the cytokines in wild-type mice is the value of PCV2-infected wild-type mice minus the value of mock-infected wild-type mice; the value of pro-inflammatory cytokines in il10−/− mice is the value of PCV2-infected il10−/− mice minus the value of mock-infected il10−/− mice. The production of cytokines at 0 h post-infection was undetectable. The data are presented as mean ± SEM of three independent experiments n = 15 mice. (B) *p < 0.05, **p < 0.01 versus same group at 7 d.p.i.; &p < 0.05, &&p < 0.01 versus same group at 14 d.p.i.; ##p < 0.01 versus wild-type mice at same infection time. (C–H) *p < 0.05, **p < 0.01 versus same group at 0 h post-infection; &p < 0.05, &&p < 0.01 versus same group at 1 h post-infection; #p < 0.05 versus wild-type mice at same infection time.

Fig 4: Effects of naringenin on the expression of inflammatory cytokine expression in ethanol-stimulated KATO III cells. Cells (0.5 × 106) were incubated with ethanol in the absence or presence of naringenin (Nar, 10 and 20 μM). (A–C) Cytokine concentrations (TNF-α, IL-6, and IL-8) in the medium were assessed by ELISA, respectively. (D) Effects on the cytotoxicity. Cell viability was determined by MTT assay. All data were shown as mean ± standard deviation (SD) (n = 3). Statistical comparison was analyzed by a one-way ANOVA followed by Tukey’s multiple comparison tests. Bars not sharing a common letter represent a statistically significant difference from each other (p < 0.05).

Fig 5: Malva parviflora hydroalcoholic extract attenuates microglia pro-inflammatory M1 phenotype in the cortex of 5XFAD mice. Total RNA was isolated from the cortex of Wt or 5XFAD mice fed with either normal diet (ND) or high-fat diet (HFD) non-treated (Vehicle) or treated with MpHE (M. parviflora) for 8 months. a The transcript levels of CD86 (marker of M1 state) were determined by RT-qPCR as described in the “Methods” section. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,16) = 42.45, p < 0.001; for the diet F(1,16) = 0.4022, p = 0.53; for the M. parviflora treatment F(1,16) = 29.79, p < 0.001; for the genotype and diet interaction F(1,16) = 0.04041, p = 0.84; for the M. parviflora treatment and diet interaction F(1,16) = 0.2594 p = 0.62; for the genotype and M. parviflora treatment interaction F(1,16) = 20.67 p < 0.001; for the genotype, M. parviflora treatment and diet interaction F(1,16) = 2.037, p = 0.17. b TNF (marker of M1 state) mRNA levels. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,16) = 25.65 p < 0.001; for the diet F(1,16) = 5.758, p = 0.03; for the M. parviflora treatment F(1,16) = 32.4, p < 0.001; for the genotype and diet interaction F(1,16) = 4.955, p = 0.04; for the M. parviflora treatment and diet interaction F(1,16) = 2.259 p = 0.15; for the genotype and M. parviflora treatment interaction F(1,16) = 26.77 p < 0.001; for the genotype, M. parviflora treatment and diet interaction F(1,16) = 2.189, p = 0.16. c Mgl1 (marker of M2 state) mRNA levels. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test, and d TREM-2 mRNA levels. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. Microglia from 8-month-old Wt or 5XFAD mice were unstimulated or stimulated with LPS (100 ng/mL) in the presence or absence of MpHE (M. parviflora; 1 mg/mL) for 24 h. Control cells were treated with PBS (Ctrl) or MpHE alone (M. parviflora). Supernatants were used to determine TNF and IL6 levels by ELISA as described in the “Methods” section. e TNF levels. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,16) = 7.878, p = 0.0127; for the LPS treatment F(1,16) = 17.74, p = 0.0007; for the M. parviflora treatment F(1,16) = 66.30, p < 0.0001; for the genotype and LPS treatment interaction F(1,16) = 6.105, p = 0.0251. f IL6 levels. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,16) = 25.76, p = 0.0001; for the LPS treatment F(1,16) = 19.86, p = 0.0004; for the M. parviflora treatment F(1,16) = 309.3, p < 0.0001; for the genotype and LPS treatment interaction F(1,16) = 20.71, p = 0.0003