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. Author manuscript; available in PMC: 2018 Jun 6.
Published in final edited form as: Free Radic Biol Med. 2016 Nov 29;102:260–273. doi: 10.1016/j.freeradbiomed.2016.11.047

Fig. 4. Nitric oxide (NO) release and its role in attenuating CYP2E1 activity and resulting in decreased inflammation in progressive NAFLD.

Fig. 4

(A) Nitric oxide release measured as total nitrite (μM) in mouse serum of WT-NAFLD, WT-NAFLD+PYR, Trpv4 KO-NAFLD and Trpv4 KO-NAFLD+PYR mice. (B) Immunoblot of phosphorylated NOS3- Ser 1177 (pNOS3) and NOS3 protein in liver homogenate of both wild-type (WT) and Trpv4 KO mice fed with chow diet (LEAN, n=3), high-fat- diet fed (NAFLD, n=4) and high-fat-diet fed mice exposed with pyrazole (NAFLD+PYR, n=3). (C) Bar graphs represents protein band quantification analysis of western blot and β-actin normalized pNOS3/NOS3 ratio in respective groups of mice was plotted on y-axis. (D and E) To study the role of nitric oxide, both WT and Trpv4 KO mice groups were co-exposed with pyrazole and nitric oxide donor (NO, DETANONOate). Representative immunohistochemistry images depicting 4-HNE immunoreactivity (D) and CD68 immunoreactivity (E) in WT-NAFLD+PYR (n=3), WT-NAFLD+PYR+NO (n=3), Trpv4 KO-NAFLD+PYR (n=4) and Trpv4 KO-NAFLD+PYR+NO (n=3) liver. Images were taken at 20X magnification. (G and H) Bar graph showed morphometric analysis (% ROI) of 4-HNE (G) and CD68 (H) immunoreactivity (n=3). (F) Representative Hematoxylin and Eosin stained images of liver sections showed pathophysiology of WT-NAFLD+PYR (n=3), WT-NAFLD +PYR+NO (n=3), Trpv4 KO-NAFLD+PYR (n=4) and Trpv4 KO-NAFLD+PYR+NO (n=3). Images were taken at 20X magnification. (I and J) Immunoblot of CYP2E1 (I) and morphometric analysis (J) in WT-NAFLD (n=4), WT-NAFLD+PYR (n=3), Trpv4 KO-NAFLD (n=3), Trpv4 KO-NAFLD+PYR (n=4), WT-NAFLD+PYR+NO (n=3) and Trpv4 KO-NAFLD+PYR+NO (n=3) liver homogenate. Data points were represented with means ± SEM, unpaired t-test.