Journal: Pharmaceutical Biology

Article Title: Indirubin regulates M1/M2 polarization and inhibits ferroptosis in dextran sulfate sodium induced colitis and in cultured THP-1 cells

doi: 10.1080/13880209.2025.2568215

Figure Lengend Snippet: IDR inhibited ferroptosis in THP-1 mocytes and in THP-1 derived macrophages with M1- or M2- polarized status. (a–d) IDR inhibited RSL3 induced cell loss in THP-1 derived macrophages, including M0 (a), M1 (b), and M2 (c) polarized macrophages, as well as in THP-1 monocytes (d). The viable cell counts were determined with CCK-8 assay. Data were presented as Mean ± S.D. (n = 4). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the RSL3 group. (e) The representative plots of IDR inhibiting RSL3 induced cell death determined by PI staining with flow-cytometry analysis. The proportion of PI-positive cells (f) and the preserved cell counts normalized to count beads (g) were also shown in the histogram plots. Data were presented as Mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01. (h) Reactive oxygen species (ROS) levels were determined with DHCF-DA. Data were presented as Mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01. (i) Malondialdehyde (MDA) levels were determined. Data were presented as Mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01. (j) Ferrous iron (Fe 2+ ) levels were quantified using a fluorometric assay. Data were normalized to cell counts and presented as Mean ± S.D. (n = 3). # p < 0.05 compared to the NC group. *** p < 0.05 compared to the DSS group. (k) The effects of IDR treatments on the mRNA expression levels of ferroptosis-associated genes, including Gpx4, Acsl4, Nrf2, and Slc7a11. Data were normalized to the level of reference gene Gapdh and presented as Mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 compared to the RSL3 group. (l) The representative images of Western blotting analysis of ferroptosis-associated proteins, including 4-HNE, GPX4, NRF2, FTL, and GAPDH. (m) Column plots of 4-HNE, GPX4, NRF2 and FTL abundance after normalizing to GAPDH. Mean ± S.D. (n = 3). * p < 0.05 compared to the RSL3 group.

Article Snippet: A Cell Ferrous Iron (Fe2+) Assay Kit (Fluorometric) (HY-K0322, MedChemExpress LLC, China) was applied to quantify iron concentration in THP-1 cells according to the manufacturer’s manual.

Techniques: Derivative Assay, CCK-8 Assay, Staining, Flow Cytometry, Expressing, Western Blot

Journal: Pharmaceutical Biology

Article Title: Indirubin regulates M1/M2 polarization and inhibits ferroptosis in dextran sulfate sodium induced colitis and in cultured THP-1 cells

doi: 10.1080/13880209.2025.2568215

Figure Lengend Snippet: IDR augmented M2-polarization and inhibited ferroptosis in peritoneal macrophages. (a) The effects of IDR treatments on the surface staining of Arg-1 in M2-polarized mouse peritoneal macrophages were determined by flow-cytometry analysis. (b) Histogram plot of the proportion and MFI (mean fluorescence intensity) of Arg-1 positive cells. Data were presented a Mean ± S.D. (n = 3). ## p < 0.01 compared to the NC group. * p < 0.05 and ** p < 0.01 compared to the M2 group. (c) The viable cell counts were determined with CCK-8 assay. Data were presented as Mean ± S.D. (n = 5). ## p < 0.01 compared to the NC group. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the M2 group. (d) The representative images of cellular ferrous iron fluorescence. (e) Histogram plot of the MFI of ferrous iron. Data were presented as Mean ± S.D. (n = 5). ### p < 0.01 compared to the NC group. ** p < 0.01 and *** p < 0.001 compared to the RSL3 group.

Article Snippet: A Cell Ferrous Iron (Fe2+) Assay Kit (Fluorometric) (HY-K0322, MedChemExpress LLC, China) was applied to quantify iron concentration in THP-1 cells according to the manufacturer’s manual.

Techniques: Staining, Flow Cytometry, Fluorescence, CCK-8 Assay

Journal: Viruses

Article Title: Infectious Spleen and Kidney Necrosis Virus Triggers Ferroptosis in CPB Cells to Enhance Virus Replication.

doi: 10.3390/v17050713

Figure Lengend Snippet: Figure 3. ISKNV infection induces ferroptosis in CPB cells. (A) Transmission electron microscopy of CPB cells treated with DMSO (72 h), erastin (10 µmol/L, 72 h), and ISKNV (100 MOI, 24 h, 48 h, 72 h). Scale bars = 1 µm. (B) Analysis of Fe2+ levels in CPB cells after treatment with ISKNV (100 MOI) using the fluorescent probe FerroOrange by laser scanning confocal microscopy. Scale bars = 20 µm. (C) Quantitative analysis of the mean fluorescence intensity of (B) using Image J. (D) Analysis of intracellular ROS levels using DCFH-DA staining, and laser scanning confocal microscopy of CPB cells treated with ISKNV (100 MOI) for 24–72 h. Scale bars = 10 µm. (E) Quantitative analysis of the mean fluorescence intensity of (D) using Image J. (F–H) Detection of Fe2+, ROS, and MDA levels in cell lysates treated with ISKNV (100 MOI) for 24–72 h by microplate reader. * p < 0.05, ** p < 0.01, and *** p < 0.001, with p > 0.05 considered not significant (ns).

Article Snippet: The Fe2+ content of the cells was detected by laser scanning confocal microscopy and a microplate reader using the fluorescent probe FerroOrange (Elabscience, E-BC-F101, Wuhan, China).

Techniques: Infection, Transmission Assay, Electron Microscopy, Confocal Microscopy, Fluorescence, Staining

Journal: Frontiers in Physiology

Article Title: Adolescent Development of Biological Rhythms in Female Rats: Estradiol Dependence and Effects of Combined Contraceptives

doi: 10.3389/fphys.2021.752363

Figure Lengend Snippet: High frequency fecal estradiol (fE2) enables monitoring of estrous cycle emergence, sex steroid manipulation, and ovarian status during adolescence. Group mean (+SEM) fE2 concentrations of Intact (light blue), Intact+short-term pubertal contraceptives (Intact+C; dark blue), Ovariectomized (OVX; gray), and OVX+E2 (orange) groups did not significantly differ prior to puberty (p24–p30; A ). Fecal estradiol in Intact and Intact+C groups increased over that of OVX animals beginning at the first cycle following vaginal opening or relative to the time of silastic implant in OVX+E2 animals (p30–p37; B ) and remained significantly elevated thereafter at mid puberty (p43–p49; C ) and during late puberty (p55–p61; D ). Dark horizontal bar in D indicates Intact+C data were gathered during contraceptive administration. Color of letters at the top of B , C , and D indicate experimental group; letters indicate statistical differences, with groups not sharing the same letter being significantly different ( p <0.03).

Article Snippet: A commercially available fE2 ELISA kit was used to quantify E2 in fecal samples (Arbor Assays, Ann Arbor, MI, United States).

Techniques:

Journal: Frontiers in Physiology

Article Title: Adolescent Development of Biological Rhythms in Female Rats: Estradiol Dependence and Effects of Combined Contraceptives

doi: 10.3389/fphys.2021.752363

Figure Lengend Snippet: Level and rate of early adolescent rise in core body temperature (CBT) circadian power are tied to fecal estradiol (fE2). Circadian, but not ultradian power rises across early adolescence (A-D) . Linear plots of group mean (±SD) of CBT circadian (solid) and ultradian (dashed) power in Intact (light blue, A ), Intact+C (dark blue, B ), OVX (gray, C ), and OVX+E2 (orange, D ) animals. * indicates significant trend over time for the bracketed time region ( p <0.05). Phase of adolescent time periods (pre to mid, mid to late, and late to adult) are indicated by black dividers in the colored x-axis at p42 and p58. Although, CBT circadian power rises over early adolescence in all groups, estradiol increases the rate of this rise (L) . Violin plots (E–G) of circadian power in each group analyzed by segment of life: pre to mid adolescence from p26 to p41 (E) , mid to late adolescence from p42 to p57 (F) , and late adolescence through early adulthood from p58 to p73 (G) illustrate that circadian rhythm (CR) power is highest in Intact animals, with a reduction following hormonal contraceptive administration and OVX, and a partial rescue in OVX+E2 animals. Black lines indicate mean and red lines indicate median of each plot. Scatters of fE2 level by CR power indicate that hormonal manipulation reduces or eliminates the correlation between CR power and fE2 concentrations (H-K) . Note that each individual within a group is plotted in a unique color. CR power and fE2 are significantly correlated in Intact (light blue, H ), Intact+C animals prior to contraceptive administration (dark blue, I ), with inset depicting abolished correlation during and after contraceptive administration. CR power and fE2 are weakly correlated in OVX+E2 (orange, K ) but not OVX animals (J) . * indicates significant positive correlation between fE2 and CR power. AIC indicates relative performance of the mixed effects model. Color of letters at the top in E–G and L indicate experimental group; letters indicate statistical differences, with groups not sharing the same letter being significantly different ( p <0.03).

Article Snippet: A commercially available fE2 ELISA kit was used to quantify E2 in fecal samples (Arbor Assays, Ann Arbor, MI, United States).

Techniques:

Journal: Frontiers in Physiology

Article Title: Adolescent Development of Biological Rhythms in Female Rats: Estradiol Dependence and Effects of Combined Contraceptives

doi: 10.3389/fphys.2021.752363

Figure Lengend Snippet: Contraceptive administration in adolescence persistently perturbs 4-day temperature rhythms. Normalized CBT mean (±SD) minus ultradian rhythm (UR) power relative to within-individual first day of fE2 rise in Intact (A) and Intact+C (B) rats (see: Materials and Methods and ). Dark bars along the x-axis for Intact+C animals indicate average time of contraceptive administration relative to fE2 rise. * indicates regions of time over which every 4th day’s CBT values are significantly elevated compared to other days of cycle ( p <0.003). Twenty-four hour smoothed average plots of normalized linear CBT in Intact (C) and Intact+C (D) individuals from the time of contraceptive administration illustrate a reduction in regularity of 4-day oscillations. Insets show FFT centered at 4–5days. * indicates significantly higher AUCs in the 4–5day range for Intact (panel C ) compared to Intact+C rats (panel D ). Individual animals (E–T) comprising Intact (light blue) and Intact+C (dark blue) groups prior to and following hormonal contraceptive administration. Dark bars along horizontal axes indicate time of contraceptive administration; administration days differ based on an individual’s day of fE2 rise.

Article Snippet: A commercially available fE2 ELISA kit was used to quantify E2 in fecal samples (Arbor Assays, Ann Arbor, MI, United States).

Techniques: