Fig 2: The effect of NFATc1 on HDAC1 expression and inflammatory response and apoptosis in H/R-exposed hepatocytes. (a) The expression of HDAC1 in the liver tissues of sham-operated and HIRI mice determined by RT-qPCR (*p < 0.05). (b) The protein expression of HDAC1 in the liver tissues of sham-operated and HIRI mice determined by western blot analysis (*p < 0.05). (c) The binding relationship between NFATc1 and HDAC1 in HEK-293 T cells transduced with oe-NFATc1 verified by dual luciferase reporter gene assay (*p < 0.05). (d) The expression of HDAC1 in AML12 cells transduced with oe-NFATc1 or sh-NFATc1 determined by RT-qPCR (*p < 0.05 vs. oe-NC, #p < 0.05 vs. sh-NC). (e) The silencing efficiency of HDAC1 in AML12 cells determined by RT-qPCR (*p < 0.05vs. sh-NC). (f) RT-qPCR detection of NFATc1 and HDAC1 expression in AML12 cells transduced with oe-NFATc1 or combined with sh-HDAC1. (g) The mRNA expression of TNF-a, IL-1ß, IL-6, Bax, Bcl2, and caspase-3 in AML12 cells transduced with oe-NFATc1 or combined with sh-HDAC1 determined by RT-qPCR. (h) The protein expression of TNF-a, IL-1ß, IL-6, Bax, Bcl2. and cleaved caspase-3 in AML12 cells transduced with oe-NFATc1 or combined with sh-HDAC1 determined by western blot analysis. (i) AML12 cell apoptosis following transduction with oe-NFATc1 or combined with sh-HDAC1 detected by TUNEL staining. In (f)–(i), *p < 0.05 vs. oe-NC + sh-NC, #p < 0.05 vs. oe-NFATc1 + sh-NC. All cell experiments were repeated 3 times.

Fig 3: Trans-interleukin-6 signaling is necessary and sufficient for neuronal iron accumulation(A) Brainstem neurons from hA53T transgenic mice and substantia nigra neurons from PFF-injected mice accumulate iron measured using iCP-MS (see Figure S1 for sorting paradigm). n= 10 biological replicates per group.(B) Neuronal iron accumulation secondary to PFFs occurs via a non-cell-autonomous mechanism measured using iCP-MS. n= 10 biological replicates per group.(C) PFF-microglia conditioned media (MCM) is sufficient for neuronal iron accumulation measured using iCP-MS. n = 10 biological replicates per group.(D) 14 secreted factors are significantly elevated in PFF MCM compared with in monomer MCM measured using Procartaplex Cytokine Multiplex assay. n = 10 biological replicates per group.(E) IL-6 is necessary for PFF-MCM-mediated neuronal iron accumulation measured using iCP-MS. n = 5 biological replicates per group.(F) Trans-IL-6 signaling is sufficient for neuronal iron accumulation from ferrous, but not ferric, iron sources. Iron accumulation measured using iCP-MS. n = 5 biological replicates per group.(G) IL-6 overexpression is sufficient for neuronal iron accumulation in vivo. Iron measured in isolated neurons (see Figure S1) using iCP-MS. n = 5 biological replicates per group.(H) Seven iron transport genes are differentially expressed in neurons secondary to PFF MCM treatment in an IL-6-dependent manner. Gene expression measured using a custom Luminex Quantigene assay. n = 6 biological replicates per group.(I) IL-6 overexpression is sufficient for increased expression of Zip14, Tfrc, Dmt1, Ftl, Fth, and Hampin vivo. Gene expression measured by qPCR. n = 5 biological replicates per group.(J) Schematic showing gene expression changes in (H)–(I) (genes of interest in red) overlayed on a diagram of cellular iron transport highlighting upregulation of ferric and ferrous iron import and downregulation of iron export.(A–C, E–G, and I) Data indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by unpaired student’s two-tailed t test (A–D, G, and I) and two-way ANOVA (E and F) with Tukey’s honestly significant difference (HSD) post hoc test.

Fig 4: Interleukin-6 is necessary for a-syn-PFF-mediated neuronal CISR and behavioral deficits(A) a-syn PFF induced IL-6-dependent neuronal iron accumulation. Iron accumulation measured in sorted neurons (see Figure S1 for sorting paradigm) by iCP-MS. n = 4–6 biological replicates per group.(B) a-syn PFF induced IL-6-dependent Hmox1 mRNA upregulation in neurons measured by qPCR. n = 4–6 biological replicates per group.(C) a-syn PFF induced IL-6-dependent increase in neuronal MDA measured by ELISA. n = 4–6 biological replicates per group.(D) a-syn PFF induced IL-6-dependent CISR mRNA elevation in neurons. Gene expression determined using qPCR. n = 4–6 biological replicates per group.(E) a-syn PFF induced IL-6-dependent TH- and Nissl-positive neuronal loss. n = 4–6 biological replicates per group. Scale bar, 400 µm.(F) a-syn PFF induced IL-6-dependent decrease in neuronal TH and DAT protein levels by western analysis. n = 3 biological replicates per group.(G) a-syn PFF induced IL-6-dependent deficit in pole test performance. n = 9–13 biological replicates per group.(H–I) a-syn PFF induced IL-6-dependent deficit in grip strength. Total grip strength shown in (H). Forelimb strength only shown in (I). n = 9–13 biological replicates per group.Data indicate mean ± SEM. */#p < 0.05, **/##p < 0.01, ***/###p < 0.001, ****/####p < 0.0001, by two-way ANOVA (A–I) with Tukey’s HSD post hoc test.

Fig 5: Dihydromyricetin (DHM) rescues the osteogenic differentiation phenotypes induced by interleukin-6 (IL-6) in human valvular interstitial cells (hVICs). Western blotting(A) and immunofluorescent staining (B) were used to detect the protein levels of osteogenesis-specific genes (alkaline phosphatase, ALP, and runt-related transcription Factor 2, RUNX2) in hVICs stimulated with osteogenic induction medium (OM) and then cotreated with IL-6 or IL-6+DHM. One-way ANOVA followed by Bonferroni post hoc test. (C) Semiquantification of the fluorescence intensity of ALP and RUNX2. One-way ANOVA followed by Bonferroni post hoc test. (D) Alizarin red staining of mineralization nodules in hVICs stimulated with OM and then cotreated with IL-6 or IL-6+DHM. One-way ANOVA followed by Bonferroni post hoc test. N = 3 per group. Values are the mean ± SD. *p < 0.05 indicates a significant difference.