Reduction of Hepatic Steatosis, Oxidative Stress, Inflammation, Ballooning and Insulin Resistance After Therapy with Safranal in NAFLD Animal Model: A New Approach

Abstract

Introduction: Non-alcoholic fatty liver disease (NAFLD) is intimately linked to hepatic steatosis, inflammation, insulin resistance (IR), oxidative stress (OS), and ballooning. A high fat diet (HFD) is considered a major etiological factor that primarily covers the numerous features of NAFLD.

Methods: The present study aimed to evaluate the protective effect of safranal on hepatic steatosis, OS, liver index, IR index, liver function enzymes, plasma lipids, TNF-α, malondialdehyde (MDA), advanced oxidation protein products (AOPPs) and nitrite (NO₂ ^-) levels in a NAFLD rat model fed with a HFD for 12 weeks. The ELISA kits were used to measure TNF-α and insulin in serum and plasma, respectively.

Results: HFD significantly induced hepatic steatosis, OS, IR, liver, and oxidative enzyme elevation and inflammation in experimental animals. Rats treated with safranal in ascending order of doses 250 and 500 mg/kg orally for 4-weeks showed a reduction in hepatic lipid's accumulation, liver index, hepatic enzymes, collagen, hepatic oxidonitrative stress markers (like AOPP, MDA and NO₂ ^-), and raised the levels of catalase (CAT) and superoxide dismutase (SOD) enzymes. Glutathione system components, namely glutathione (GSH), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) levels were also restored in the safranal-treated groups. The reduction in serum TNF-α and IR provided further support to the anti-NAFLD effect of safranal. Moreover, the histopathological images indicated reverse of NAFLD activity score (NAS) through mild fatty degeneration, ballooning and inflammation in hepatocytes of treated groups.

Conclusion: Findings of blood and tissue analysis concluded that safranal can be a good choice in the management and cure of NAFLD.

Keywords: glutathione; inflammation; insulin resistance; non-alcoholic fatty liver disease; safranal.

Graphical abstract

Figure 1

Effect of safranal on body weight of rats over 12-weeks (A) and % weight gain during treatment period (B). Results are expressed as Mean ± SEM (n=6). Where ^ap<0.001: statistically significant as compared to disease control (DC) group by using two-way and one-way method of ANOVA following the Bonferroni multiple comparisons and Dunnett’s tests, respectively.

Figure 2

Effects of safranal on food (A) water (B) and energy intake (C). Results are expressed as mean ± SEM (n=6). Results are expressed as Mean ± SEM (n=6). Where ^ap<0.001: statistically significant as compared to disease control (DC) group by using two-way and one-way method of ANOVA following the Bonferroni multiple comparisons and Dunnett’s tests, respectively.

Figure 3

Effect of safranal on random blood glucose levels (A), oral glucose tolerance test (B) and AUC (C) in NAFLD rat model. Results are expressed as Mean ± SEM (n=6). Where ^ap<0.001, ^bp<0.01 and ^cp<0.05: statistically significant as compared to disease control (DC) group by using two-way and one-way method of ANOVA following the Bonferroni multiple comparisons and Dunnett’s tests, respectively.

Figure 4

Effect of safranal on hepatic oxidative stress markers namely MDA (A), AOPP (B), nitrites (C), and glutathione system GSH (D), GPx (E) and GST (F) levels in HFD-induced NAFLD rat model. Results are expressed as mean± SEM (n=6) and statistically significant as compared to disease control (DC) group by using one-way method of ANOVA following the Dunnett’s test. Where ^ap<0.001, ^bp<0.01 and ^cp<0.05.

Figure 5

Effect of safranal on hepatic antioxidant enzymes namely CAT (A) and SOD (B) levels in NAFLD rat model. Results are expressed as mean± SEM (n=6) and statistically significant as compared to disease control (DC) group by using one-way method of ANOVA following the Dunnett’s test. Where ^ap<0.001, ^bp<0.01 and ^cp<0.05.

Figure 6

Effects of safranal on serum tumor necrosis factor-α (TNF-α) (A), and hepatic collagen levels (B). Results are expressed as mean± SEM (n=6) and statistically significant as compared to disease control (DC) group by using one-way method of ANOVA following the Dunnett’s test. Where ^ap<0.001 and ^bp<0.01.

Figure 7

Histology images of rat’s liver with macrovesicular and microvesicular steatosis (magnification, 100x), where black arrows shows the fat droplets (A) liver of normal control group presenting normal hepatocytes; (B) liver of rat fed with HFD showing severe steatosis and fatty degeneration of hepatocytes; (C) liver of rat treated with standard botanical mixture along with HFD, showing moderate fatty degeneration of hepatocytes; (D) liver sections of rats fed with HFD and treated with safranal 250 mg/kg/day showing moderate fatty degeneration of hepatocytes; (E) liver sections of rats fed with HFD and treated with safranal 500 mg/kg/day showing mild fatty deposition.

Figure 8

Effect of Safranal on the histopathology of HFD fed rat’s liver stained with hematoxylin-eosin. Images of representative sections of each group are shown lobular inflammation and steatosis around central vein. Where (arrow a) indicates oval cells hyperplasia, (arrow b) inflammatory cells, and (arrow c) steatosis. (A) Normal control group showing normal hepatocytes and central vein (magnification, 100x); (B) disease control group showing steatosis and severe inflammation around central vein with mixture of oval cells hyperplasia (400x); (C) group treated with standard botanical mixture showing moderate steatosis and inflammation (400x); (D) group treated with safranal-250 mg/kg dose showing moderate steatosis, inflammation and oval cells (400x); (E) group treated with safranal-500 mg/kg showing mild fatty infiltration, inflammation and oval cells (400x).

Figure 9

Effects of Safranal on scoring of rat livers for steatosis, ballooning, and lobular inflammation. Results are expressed as mean ± SEM (n=6) and statistically significant as compared to disease control (DC) group by using one-way method of ANOVA following the Dunnett’s test. Where ^ap<0.001, ^bp<0.01 and ^cp<0.001.