Betaine inhibits toll-like receptor 4 expression in rats with ethanol-induced liver injury

Abstract

Aim: To test whether ethanol feeding could induce Toll-like receptor 4 (TLR4) responses, assess the hepatoprotective effect of betaine and its inhibitive effect on TLR4 in animal models of alcoholic liver injury.

Methods: Forty-eight female Sprague-Dawley rats were randomly divided into four groups as control, model, low and high dose betaine groups. Except control group, all rats were fed with high fat-containing diet plus ethanol and fish oil gavages for 8 wk. Betaine was administered intragastrically after exposure of ethanol for 4 wk. The changes of liver histology were examined. The expression of TLR4 mRNA and protein was detected by RT-PCR and Western blot, respectively. The serum aminotransferase activity [alanine transarninase (ALT), aspartate aminotransferase (AST)], serum endotoxin, and liver inflammatory factors [tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-18 (IL-18)] were also assayed.

Results: Compared with control group, rats of model group developed marked liver injury, accompanied by an increase of ALT (159.41 +/- 7.74 U/L vs 59.47 +/- 2.34 U/L, P < 0.0001), AST (248.25 +/- 1.40 U/L vs 116.89 +/- 3.48 U/L, P < 0.0001), endotoxin (135.37 +/- 30.17 ng/L vs 44.15 +/- 7.54 ng/L, P < 0.0001), TNF-alpha (20.81 +/- 8.58 pg/mL vs 9.34 +/- 2.57 pg/mL, P = 0.0003), IFN-gamma (30.18 +/- 7.60 pg/mL vs 16.86 +/- 9.49 pg/mL, P = 0.0039) and IL-18 (40.99 +/- 8.25 pg/mL vs 19.73 +/- 9.31 pg/mL, P = 0.0001). At the same time, the expression of TLR4 mRNA and protein was markedly induced in the liver after chronic ethanol consumption (1.45 +/- 0.07 vs 0.44 +/- 0.04, P < 0.0001; 1.83 +/- 0.13 vs 0.56 +/- 0.08, P < 0.0001). Compared with model group, betaine feeding resulted in significant decreases of ALT (64.93 +/- 6.06 U/L vs 159.41 +/- 7.74 U/L, P < 0.0001), AST (188.73 +/- 1.11 U/L vs 248.25 +/- 1.40 U/L, P < 0.0001), endotoxin (61.80 +/- 12.56 ng/L vs 135.37 +/- 30.17 ng/L, P < 0.0001), TNF-alpha (9.79 +/- 1.32 pg/mL vs 20.81 +/- 8.58 pg/mL, P = 0.0003), IFN-gamma (18.02 +/- 5.96 pg/mL vs 30.18 +/- 7.60 pg/mL, P = 0.0008) and IL-18 (18.23 +/- 7.01 pg/mL vs 40.99 +/- 8.25 pg/mL, P < 0.0001). Betaine also improved liver steatosis. The expression levels of TLR4 mRNA or protein in liver tissues were significantly lowered (0.62 +/- 0.04 vs 1.45 +/- 0.07, P < 0.0001; and 0.65 +/- 0.06 vs 1.83 +/- 0.13, P < 0.0001). There was a statistical difference of TLR4 mRNA and protein expression between high- and low-dose betaine groups (0.62 +/- 0.04 vs 0.73 +/- 0.05, P < 0.0001, and 0.65 +/- 0.06 vs 0.81 +/- 0.09, P < 0.0001).

Conclusion: Betaine can prevent the alcohol-induced liver injury effectively and improve the liver function. The expression of TLR4 increases significantly in ethanol-fed rats and betaine administration can inhibit TLR4 expression.

Figure 1

Expression of TLR4 in rat liver tissues. A: RT-PCR assay of TLR4 mRNA; B: Western blotting assay of TLR4 protein. 1: Control; 2: High dose betaine group; 3: Low dose betaine group; 4: Model; M: DNA marker.

Figure 2

Histopathological changes of rat liver after betaine intervention. A: In control group, liver structure was normal, without obvious inflammation and hepatic steatosis; B: In model group, the structure of hepatic cord was deranged, with various degrees of diffuse hepatic steatosis and intralobular inflammation; C: In low dose betaine group, the degree of hepatic steatosis and inflammation was greatly reduced compared with model group; D: High dose betaine group, showing more significant improvement of hepatic steatosis and inflammation than the low does betaine group. Original magnification × 400.