Rho-kinase inhibition ameliorates non-alcoholic fatty liver disease in type 2 diabetic rats

Abstract

Non-alcoholic fatty liver disease (NAFLD) is linked to type 2 diabetes mellitus (T2DM), obesity, and insulin resistance. The Rho/ROCK pathway had been involved in the pathophysiology of diabetic complications. This study was designed to assess the possible protective impacts of the Rho/Rho-associated coiled-coil containing protein kinase (Rho/ROCK) inhibitor fasudil against NAFLD in T2DM rats trying to elucidate the underlying mechanisms. Animals were assigned into control rats, non-treated diabetic rats with NAFLD, and diabetic rats with NAFLD that received fasudil treatment (10 mg/kg per day) for 6 weeks. The anthropometric measures and biochemical analyses were performed to assess metabolic and liver function changes. The inflammatory and oxidative stress markers and the histopathology of rat liver tissues were also investigated. Groups with T2DM showed increased body weight, serum glucose, and insulin resistance. They exhibited disturbed lipid profile, enhancement of inflammatory cytokines, and deterioration of liver function. Fasudil administration reduced body weight, insulin resistance, and raised liver enzymes. It improved the disturbed lipid profile and attenuated liver inflammation. Moreover, it slowed down the progression of high fat diet (HFD)-induced liver injury and reduced the caspase-3 expression. The present study demonstrated beneficial amelioration effect of fasudil on NAFLD in T2DM. The mechanisms underlying these impacts are improving dyslipidemia, attenuating oxidative stress, downregulated inflammation, improving mitochondrial architecture, and inhibiting apoptosis.

Fig. 1

representative photomicrograph of H&E stain of liver tissue of normal control group (A): showing central vein (cv) surrounded with normal hepatocytes (arrow head) arranged in cords and separated by blood sinusoids (s); HFD+T2DM group (B): liver tissue showing dilated central veins (CV), NASH with marked micro (wavy arrow) and macro (bifid arrow) -steatosis in hepatocytes with ballooning degeneration (short arrow) along with inflammatory cellular infiltration (if); HFD+T2DM+Fasudil group (C): showing mildly dilated central veins (cv) and partially restoring the normal architecture of the liver where most hepatocytes show normal vesicular nuclei (arrow head), but still showing mild fatty changes in the form of macrosteatosis in hepatocytes (bifid arrow) and hydrobic degeneration (short arrow) (H&E ×400).

Fig. 2

Representative image of Sirius red stained liver tissue collected from all rats’ groups; (A) Control (B) HFD+T2DM group (C) HFD+T2DM+Fasudil group. Arrows in (A) point to the fine collagen deposition in the portal area (P) and surrounding the central vein (V), arrows and arrow head in (B) point to the heavy collagen deposition encircling the portal region (P) and extending in the septa. Whereas, arrows in (C) point to the fine collagen deposition around both portal area (P) and central vein (V). Magnification, ×200. (D) Represents a quantitative analysis of liver fibrosis determined by % collagen deposition calculation from Sirius red stain. Data are displayed as mean ± SD. *** P<0.001 vs. control group and ^### P<0.001 vs. HFD+T2DM group.

Fig. 3

Representative image of immunohistochemical staining of liver sections with anti-caspase-3 antibody from various studied groups (A) Control (B) HFD+T2DM group (C) HFD+T2DM+Fasudil group. Arrowhead points to the brown coloration of the immuno-positive cells. (D) Histogram shows the % area of immuno-positive cells from the various experimental groups. HFD+T2DM group showed significant increase in caspase-3 immunostaining compared to other groups. HFD+T2DM+Fasudil group revealed weakly positive immunostaining.

Fig. 4

TEM representative of the liver tissue of normal control rats. (A) Revealed normal hepatocytes as well as normal sinusoids with no abnormal features. The hepatocytes showed normal nucleoplasm with round nuclei surrounded by obvious nuclear envelop with fine granular chromatin. The cytoplasm showed mitochondria (M), rough endoplasmic reticulum (RER). (B) Revealed normal hepatocytes with normal nucleoplasm with round nuclei and nuclear envelop. The cytoplasm showed mitochondria (M), glycogen inclusions (GL).

Fig. 5

TEM examination of the liver tissue of HFD+T2DM group. (A) Showed abnormal hepatocytes with wide sinusoids, marked fat droplets infiltration (L) with glycogen inclusions depletion. (B) Abnormal hepatocytes with wide sinusoids, marked fat droplets infiltration (L). (C) Abnormal hepatocytes with wide sinusoids swollen mitochondria (M), marked fat droplets infiltration (L) with infrequent glycogen inclusions. (D) The abnormal hepatocytes with abnormal nuclear chromatin distribution (N), significant fat droplets infiltration (L).

Fig. 6

TEM examination of the liver tissue of HFD+T2DM+Fasudil group, revealed improved hepatocytes appearance. The hepatocytes showed normal nucleoplasm with round nuclei surrounded by obvious nuclear envelop with fine granular chromatin. The cytoplasm showed mitochondria (M), rough endoplasmic reticulum (RER), reappearance of glycogen inclusions (GL), and Mallory body (MB).

Fig. 7

A summarized graph of the anti-NAFLD mechanistic activity of fasudil.