Fig 1: H2S-induced increase in G6PD activity is critical for regulating hypertrophic responsesa Schematic representation for experimental set in panel b and c. H9c2 cells (with or without 30 min NaHS pre-treatment) were stimulated with ISO (50 µM), in the presence or absence of inhibitors; 6-AN (250 µM, 16 h) or DHEA (25 µM, 16 h). b, c Bar graphs (Mean ± S.E.) representing G6PD activity in mU/ml b or percentage change over control c from various groups (as indicated in figure). d–f Bar graph (Mean ± S.E.) depicting ANP or BNP levels (in pg/ml), as estimated from culture supernatants after 24 d or 48 h e, f of ISO treatment, with or without NaHS, in the presence or absence of G6PD inhibitors, 6-AN or DHEA. *p < 0.05, **p < 0.01 compared to control groups
Fig 2: Representative figures showing the effect of nanocurcumin formulation (NCF) supplementation on cHH-induced RVH in rats. Chronic HH-mediated RVH was evident in animals by increases in heart size, collagen accumulation (arrows), Fulton’s index and histopathology staining (× 40) (a, b). Tissue expression levels of skeletal a-actin and atrial natriuretic factor (ANF) were increased due to hypertrophic growth (b). Circulating levels of ANF and brain natriuretic peptide (BNP) further confirmed cHH-induced RVH in rats (c). Phosphorylation and activation of Akt-Gsk signaling promoted hypertrophic growth in rats under hypoxic conditions (d, e). Changes in gene expression levels of markers of myocardial matrix remodeling (Col1a1 and Col3a1, along with MMP2 and 9) (f, g) in various experimental groups. Data are expressed as the means±s.d. NCF supplementation modulated chronic hypobaric hypoxia (cHH)-induced right ventricular hypertrophy (RVH) better than nanocurcumin (NC) and pyrroloquinoline quinone (PQQ) treatments. Values were considered to be statistically significant at **P?0.01 vs NVC, #P?0.05 vs HVC and #P?0.01 vs HVC. Non-significant changes are depicted as NS. Scale bar, 10 µm.
Fig 3: Both the therapeutic and preventive chronic infusion of ANP lower mean arterial pressure in male SSWT rats.(A) The experimental protocol for the “preventive” infusion of ANP to the SSWT rats; yellow arrows denote metabolic cage urine collections. (B) The mean arterial pressure (MAP) following i.v. infusion of 100 ng/kg/day ANP (n = 8) or vehicle (saline, n = 9) performed together with the 21-day HS diet challenge; P < 0.01 in HS diet–fed groups (vehicle versus infusion) starting day 1 of the infusion. (C) The experimental protocol for the “therapeutic” infusion of ANP to the SSWT rats; yellow arrows denote metabolic cage urine collections. (D) I.v. infusion of 100 ng/kg/day ANP (n = 7) or vehicle (saline, n = 5) was started on day 14 of the 21-day HS diet challenge. P < 0.01 in HS diet–fed groups (vehicle versus infusion) starting day 1 of the infusion. (E and F) Graphs summarize 2 kidneys/body weight and heart/body weight ratios obtained at the end of the protocols. n = 14, 8, and 7 individual rats. Groups are the following: vehicle, rats infused with ANP for the last 7 days of the HS challenge (14–21 days), and throughout the HS challenge (0–21 days). (G) Plasma levels of ANP in the SSWT rats fed a NS and a HS diet, and administered with 100 ng/kg/day ANP for 21 days during a HS challenge (HS and ANP, 0–21 days). Data were analyzed with 1-way ANOVA followed by a Holm-Sidak post hoc test; significant P values are shown on the graphs. Male animals were used.
Fig 4: ANP deficiency in male rats exacerbates glomerular damage but does not aggravate microalbuminuria and renal protein cast formation induced by a HS diet.(A) Representative cortical images from Masson trichrome–stained kidneys taken at 10× (upper row), and expended regions of interest (bottom row). Blind scores are shown on the right; each point on the graph is an average of 100 scored glomeruli from 1 rat. n = 6, 10, 5, and 11 independent tissues scored for the SSWT rats on NS and HS, and SSNPPA–/– rats on NS and HS. (B) Representative images from Masson trichrome–stained renal tissues showing protein casts formation. Shown are images taken at 20×; graph on the right summarizes the percentage of the protein cast area to whole kidney section area; n = 6, 6, 5, and 11 independent tissue scans analyzed for the SSWT rats on NS and HS, and SSNPPA–/– rats on NS and HS. (C) Urinary microalbumin excretion (normalized to urine flow). n = 8, 7, 7, and 8 urine samples from independent animals were analyzed for the SSWT rats on NS and HS, and SSNPPA–/– rats on NS and HS. (D) Representative staining for megalin in the renal cortex (20×). (E) Western blot analysis of megalin expression in the renal cortex of the SSWT rats on NS and HS, and SSNPPA–/– rats on NS and HS; each lane represents an independent renal tissue sample obtained from a different rat. n = 5 per group. (F) Intensity profiles of megalin staining assessed in the cortical proximal tubules of the SSWT rats and SSNPPA–/– rats on NS and HS. n = 3 animals per group; at least 8 random images at 40× were analyzed per animal, with n = 8–10 individual tubules measured per image (total n = 172, 238, 217, 229 in SSWT rats and SSNPPA–/– rats on NS and HS). (G) Plasma albumin levels obtained from SSWT rats on NS and HS, and SSNPPA–/– rats on NS and HS; n = 7, 7, 8, 8. Data were analyzed with 2-way ANOVA or repeated measures ANOVA; if significant, P values are shown on the graphs. Male animals were used. Scale bars: 50 µm.
Fig 5: The effect of nanocurcumin formulation (NCF) on hypoxia-induced hypertrophy in human ventricular cardiomyocytes (HVCM) cells. Increments in cell size (× 40) (a, b) along with atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) levels (c) depicted hypertrophic growth. Increments in FITC-leucine and FITC-phenylalanine uptake further confirmed hypoxia-induced hypertrophy (d). NCF supplementation effectively modulated hypoxia-induced hypertrophy in vitro. Data are expressed as the means±s.d. Values were considered to be statistically significant at *P?0.05 vs NVC, **P?0.01 vs NVC, #P?0.05 vs HVC and ##P?0.01 vs HVC. Non-significant changes are depicted as NS. Scale bar, 20 µm.
Supplier Page from Abcam for Rat ANP ELISA Kit (NPPA)