The involvement of TGF-β1 /FAK/α-SMA pathway in the antifibrotic impact of rice bran oil on thioacetamide-induced liver fibrosis in rats

Abstract

The objective of the current study is to investigate the effect of rice bran oil (RBO) on hepatic fibrosis as a characteristic response to persistent liver injuries. Rats were randomly allocated into five groups: the negative control group, thioacetamide (TAA) group (thioacetamide 100 mg/kg thrice weekly for two successive weeks, ip), RBO 0.2 and 0.4 groups (RBO 0.2mL and 0.4 mL/rat/day, po) and standard group (silymarin 100 mg/kg/day, po) for two weeks after TAA injection. Blood and liver tissue samples were collected for biochemical, molecular, and histological analyses. Liver functions, oxidative stress, inflammation, liver fibrosis markers were assessed. The obtained results showed that RBO reduced TAA-induced liver fibrosis and suppressed the extracellular matrix formation. Compared to the positive control group, RBO dramatically reduced total bilirubin, AST, and ALT blood levels. Furthermore, RBO reduced MDA and increased GSH contents in the liver. Simultaneously RBO downregulated the NF-κβ signaling pathway, which in turn inhibited the expression of some inflammatory mediators, including Cox-2, IL-1β, and TNF-α. RBO attenuated liver fibrosis by suppressing the biological effects of TGF-β1, α-SMA, collagen I, hydroxyproline, CTGF, and focal adhesion kinase (FAK). RBO reduced liver fibrosis by inhibiting hepatic stellate cell activation and modulating the interplay among the TGF-β1 and FAK signal transduction. The greater dosage of 0.4 mL/kg has a more substantial impact. Hence, this investigation presents RBO as a promising antifibrotic agent in the TAA model through inhibition of TGF-β1 /FAK/α-SMA.

Fig 1. GC-MS chromatogram of RBO analysis.

Fig 2. Effect of RBO on serum levels of ALT, AST, total protein, albumin and A/G ratio in TAA-induced liver fibrosis in rats.

Control rats, treated with saline; TAA rats, treated with thioacetamide (100 mg/kg; three times per week for 2 weeks, ip); RBO rats, treated with TAA and RBO (0.2 and 0.4 mL/rat; daily for 2 weeks, po) and silymarin (100 mg/kg, po). All data are presented as Mean± SEM, (n = 6). ^a P≤0.05 was assumed to denote statistical significance compared to the negative control, ^b P≤0.05 was assumed to denote statistical significance compared to TAA group. TAA, Thioacetamide; RBO, Rice bran oil; A/G, Albumin/Globulin.

Fig 3. Effect of RBO on tissue levels of GSH and MDA in TAA-induced liver fibrosis in rats.

Control rats, treated with saline; TAA rats, treated with thioacetamide (100 mg/kg; three times per week for 2 weeks, ip); RBO rats, treated with TAA and RBO (0.2 and 0.4 mL/rat; daily for 2 weeks, po) and silymarin (100 mg/kg, po). All data are presented as Mean± SEM, (n = 6). ^a P≤0.05 was assumed to denote statistical significance compared to the negative control, ^b P≤0.05 was assumed to denote statistical significance compared to TAA group. TAA, Thioacetamide; RBO, Rice bran oil; GSH, Reduced glutathione; MDA, Malondialdehyde.

Fig 4. Effect of RBO on TGF-β1, FAK and α-SMA in TAA-induced liver fibrosis in rats.

Control rats, treated with saline; TAA rats, treated with thioacetamide (100 mg/kg; three times per week for 2 weeks, ip); RBO rats, treated with TAA, RBO (0.2 and 0.4 mL/rat; daily for 2 weeks, po) and silymarin (100 mg/kg, po). All data are presented as Mean± SEM, (n = 6). ^a P≤0.05 was assumed to denote statistical significance compared to the negative control, ^b P≤0.05 was assumed to denote statistical significance compared to TAA group. TAA, Thioacetamide; RBO, Rice bran oil; TGF-β1, Transforming growth factor beta; FAK, Focal adhesion kinase; α-SMA, Alpha-Smooth Muscle Actin.

Fig 5

H&E stained liver sections; (a) Liver of control rat shows normal histological structure of central vein (arrow) and hepatic parenchymal cells (HCs). (b-d) liver of TAA-administrated rat showing; (b) thickening of the portal triad (arrow) with fibrous proliferation, inflammatory cells infiltration and proliferated bile duct epithelium, (c) extended fibrous septa (dotted arrow) with marked parenchymal pseudolobulation (PL), (d) vacuolar to ballooning degeneration of the hepatic cells (arrow) and marked apoptosis within pseudo-lobules. (f) (e–h) livers of RBO treated group showing marked retraction of fibrous proliferation without any evidence of pseudolobulation, only at low dose treatment (0.2 mL/rat), mild limited fibroplasia in the portal area (arrow) with scattered necrotic hepatocytes (dotted arrow) and scarce ones at the high dose (0.4 mL/rat) treatment. (I and j) Silymarin treated group showing moderate retraction of parenchymal fibrous with its limitation to the portal areas (arrow). (k) The scoring of the fibrosis extension in TAA and various treated groups using Metavir scoring scale. All data are presented as Mean± SEM, (n = 6). a P≤0.05 was assumed to denote statistical significance compared to the negative control, b P≤0.05 was assumed to denote statistical significance compared to TAA group. C: control; TAA: Thioacetamide; RBO: Rice bran oil.

Fig 6. MTC stained liver sections.

Control group showing normal limited amount of fibrous tissue in the portal areas (dotted arrow), the highest significant increase in MTC stained areas of fibrous tissue in the bridging fibrosis (arrow) in TAA group. While, RBO administration gave rise to a dose dependent significant decrease in MTC-stained areas with its limitation to portal triads (arrow) compared to TAA group and silymarin administrated group. The quantitative image analysis of the MTC stained areas presented as area percentage. All data are presented as Mean± SEM, (n = 6). ^a P≤0.05 was assumed to denote statistical significance compared to the negative control, ^b P≤0.05 was assumed to denote statistical significance compared to TAA group. C: control; TAA: Thioacetamide; RBO: Rice bran oil.

Fig 7. Immunohistochemical analysis of p-Akt expression in various experimental groups showing negative expression in control group, marked increased expression along the extended fibrous bands (upper insert) in TAA group, significant dose-related decreased expression following the decreased fibroplasia in RBO treated groups, and limited expression in SIL treated group.

The quantitative image analysis of the area percent of the positive brown colour presented as intensity scores. All data are presented as Mean± SEM, (n = 6). ^a P≤0.05 was assumed to denote statistical significance compared to the negative control, ^b P≤0.05 was assumed to denote statistical significance compared to TAA group. C: control; TAA: Thioacetamide; RBO: Rice bran oil.

Fig 8. Immunohistochemical analysis of PDGF-BB expression in various experimental groups; showing negative expression in control group, significant increased along the fibrous septa and along the sinusoidal areas (upper insert) in TAA administrated group, significant dose related decreased expression in RBO which showing scattered expression in the sinusoidal areas, and mild expression along the incomplete septa as well as in sinusoidal areas in silymarin treated group.

(C) Quantitative image analysis of the area percent of the positive brown colour presented as intensity scores. All data are presented as Mean± SEM, (n = 6). ^a P≤0.05 was assumed to denote statistical significance compared to the negative control, ^b P≤0.05 was assumed to denote statistical significance compared to TAA group. C: control; TAA: Thioacetamide; RBO: Rice bran oil.

Fig 9. Effect of RBO on NF-κβ and COX-2 in TAA-induced liver fibrosis in rats.

Control rats, treated with saline; TAA rats, treated with thioacetamide (100 mg/kg; three times per week for 2 weeks, ip); RBO rats, treated with TAA, RBO (0.2 and 0.4 mL/rat; daily for 2 weeks, po) and silymarin (100 mg/kg, po). All data are presented as Mean±SEM, (n = 6). ^a P≤0.05 was assumed to denote statistical significance compared to the negative control, ^b P≤0.05 was assumed to denote statistical significance compared to TAA group. TAA, Thioacetamide; RBO, Rice bran oil; NF- κβ, Nuclear factor kappa B; COX-2, Cyclooxygenase-2.