Fig 2: Cumulative mild stress induced increase of inflammatory cytokines and microglia activation in young adult CPMS mice. a Immunofluorescence staining for Iba1(red) was performed in the 8-week-old mice (n = 3 per group). scale bar 100 µm. b Microglia intensity was quantified in each region (Hip; 1.362 ± 0.08311, Amy; 1.377 ± 0.1037, PFC; 1.172 ± 0.05855). c Western blot analysis was performed using the cytosol isolated from hippocampus (Hip), amygdala (Amy) and prefrontal cortex (PFC). In each region, d CD86 (Hip; 2.227 ± 0.1656, Amy; 2.575 ± 0.2834, PFC; 3.479 ± 0.6652 vs CTL). e TNF-a (Hip; 3.054 ± 0.8463, Amy; 1.577 ± 0.09831, PFC; 2.311 ± 0.3439 vs CTL), f IL-6 (Hip; 1.850 ± 0.2804, Amy; 3.334 ± 0.8327, PFC; 1.923 ± 1.853 vs CTL), g IL-1ß (Hip; 3.363 ± 0.05453, Amy; 3.459 ± 0.6625, PFC; 2.442 ± 0.4368 vs CTL), h IL-18 (Hip; 3.985 ± 0.6116, Amy; 2.511 ± 0.6282, PFC; 2.511 ± 0.6107 vs CTL), and i IL-17 (Hip; 3.082 ± 0.7783, Amy; 1.861 ± 0.1675, PFC; 2.908 ± 0.8383 vs CTL) were normalized by GAPDH. All data are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTL

Fig 3: Protein expression of pro- and anti-inflammatory cytokines and CD200 in cerebellar lysates at day 62 p.i. (A,B) IL-1ß and IL-6 expression were less in Fgfr1ind-/- mice. IL-4, IL-12, IL-17, TNF-a, and IFN? expressions were not different between controls and Fgfr1ind-/- mice. (C,D) CD200 expression was reduced in Fgfr1ind-/- mice. n = 3–4/group. Data are expressed as the mean ± SEM. ns = not significant, * p < 0.05.

Fig 4: Summary picture. Cumulative mild stress during brain development (from fetus to adolescence) induced long-lasting depressive symptoms in young adulthood. Naïve T cell was differentiated to Th17 cells under the increase of differentiation factors such as IL-6 and TGF-ß with the hyperactive form of STAT3. According to the increase of Th17 cell population, the high level of IL-17 was produced and promoted the activation of microglia. As a result, the abnormal inflammatory response induced by IL-17 affects more long-lasting anxiety- and depression-like behavior in young adulthood

Fig 5: Network pharmacological analysis of active ingredients in HDW on the regulation of the IL-6/STAT3 pathway. (A) The 27 compounds-targets Network Diagram. (B) PPI of the top 30 targets filtered by degree algorithm. (C) PPI Map of the top 30 targets and IL-17, the targets are sorted in descending order in a circular layout based on the number of degrees. The size of the target reflects the number of degrees. (D) The compounds whose binding energy with STAT3 or IL-6R was lower than -7.0, with colors closer to red indicating a lower score. (E) The docking pose of STAT3 with glycyrrhetinic acid, oleanolic acid, ursolic acid, and wilforlide A (left); The docking pose of IL-6R with asiatic acid and neoandrographolide (right).