Fig 2: Inhibition of CYLD expression reversed the results that blocking of the Notch signaling pathway inhibited fibrogenesis and the release of inflammatory factors. siRNA and siRNA Scramble of CYLD were transfected into bone marrow-derived macrophages Mø-sh-NC group and Mø-sh-RBP-J group and stimulated with LPS + INF-γ for 24 h. (A): mRNA expression of CYLD in macrophages of each group was detected by qRT-PCR; (B): p65 in macrophage nuclei was detected by immunofluorescence; (C): Expression of fibrosis-related factors TGF-β1, PDGF-B, COL1, and COL3 in fibroblasts was detected by qRT-PCR; (D): Expressions of inflammatory factors TNF-α, IL-1β, and IL-6 in macrophages were detected by ELISA; Three independently repeated tests were performed, and the data were expressed as mean ± SD. One-way ANOVA was used for the analysis of variance, and Tukey's multiple comparisons test was used for post- hoc test; ** P < 0.01 (compared with Mø-sh-NC + Scramble group), and ## P < 0.01 (compared with Mø-sh-RBP-J + Scramble group).

Fig 3: Blocking Notch signaling pathway improved myocardial injury in mice. (A–D): Left ventricular end-systolic diameter (LVES), left ventricular end-diastolic diameter (LVED), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were analyzed by micro-ultrasound machine, N = 23/per group; (E): Protein expression of RBP-J was detected, N = 6; (F): Infarction area was detected by TTC method. (G): TUNEL assay of the tissue apoptosis in the mouse heart. N = 3. Three independently repeated tests were performed, and the data were expressed as mean ±SD. One-way ANOVA was used for the analysis of variance, and Tukey's multiple comparisons test was used for the post-hoc test; ** P < 0.01 (compared with the sham group), ## P < 0.01, and # P < 0.05 (compared with the sh-NC group).

Fig 4: GAS suppresses Notch-1, NICD, RBP-JK, and Hes-1 expression in OGD-activated TNC1 astrocytes. (A-D) Immunofluorescence images show that the expressions of Notch-1, NICD, RBP-JK, and Hes-1 (red) in GFAP-positive (green) astrocytes. (E) The quantitative analysis showed the expression of these proteins were increased in OGD group compared to the control, but were reduced following treatment with GAS. DAPI (blue) shows nuclei. Significant differences are expressed as: #, P<0.05 vs. CON group; *, P<0.05 vs. OGD group. Scale bars: 20 µm. The values represent the mean ± SD in triplicate. CON, control; GAS, gastrodin; GFAP, glial fibrillary acidic protein; Hes-1, transcription factor hairy and enhancer of split-1; NICD, intracellular Notch receptor domain; OGD, oxygen-glucose deprivation; RBP-JK, recombining binding protein suppressor of hairless; SD, standard deviation.

Fig 5: Blocking of the Notch signaling pathway in vitro promoted M2 macrophage polarization and CYLD expression and inhibited fibrosis-related factors expression and cellular inflammatory levels. (A): Bone marrow-derived macrophages in mice were identified by flow cytometry; (B): Heart-derived fibroblasts were identified by immunohistochemistry. Macrophages with different treatments were isolated and divided into Mø-Sham, Mø-MI, Mø-sh-NC, and Mø-sh-RBP-J groups. Macrophages and fibroblasts stimulated with LPS + INF-γ for 24 h were co-cultured by Transwell to establish an in vitro inflammatory model; (C): Macrophage polarization was detected by flow cytometry; (D): p65 in macrophage nuclei of each group was detected by immunofluorescence; (E): Expression of fibrosis-related factors TGF-β1, PDGF-B, COL1, and COL3 in fibroblasts was detected by qRT-PCR; (F): Expression of inflammatory factors TNF-α, IL-1β, and IL-6 in macrophages was detected by ELISA; (G): The expression of CYLD in macrophages was detected by qRT-PCR. Three independently repeated tests were performed, and the data were expressed as mean ± SD. One-way ANOVA was used for analysis of variance, and Tukey's multiple comparisons test was used for post-hoc test; ** P < 0.01, * P < 0.05 (compared with Mø-Sham group), and ## P < 0.01, # P < 0.05 (compared with Mø-sh-NC group).