Combination of L-Carnitine and Angiotensin-II type 1 Receptor Blocker has Beneficial Effects on Hepatic Fibrosis in a Non-Alcoholic Steatohepatitis Rat Model

Experimental Design

After one week of acclimatization, 30 rats weighing 160-175 g were randomly divided into five groups. A control group that was fed the CSAA diet for normal non-NASH baseline reference. Four other groups were provided the CDAA diet to establish diet-induced hepatic steatosis and fibrosis, which simulates human NASH. One group received no further treatment, while the second group received additional L-carnitine at 200 mg/kg/day and the third group received the ARB losartan at 30 mg/kg/day. Finally, a combination treatment group received both L-carnitine and losartan. These treatment conditions were maintained for 8 weeks, during which the rats had free access to tap water. At the end of the experimental period, the rats were anesthetized with pentobarbital and sacrificed, and the liver samples were collected. All animal procedures were performed in accordance with the Declaration of Helsinki and in compliance with the standard recommendations for the proper care and use of laboratory animals. The protocol was approved by the Animal Care and Use Committee of Nara Medical University.

Histological and Immunohistochemical Analyses

Five-micrometer-thick sections of formalin-fixed and paraffinembedded liver specimens were stained using hematoxylin and eosin and Azan stains to evaluate fibrosis. Besides, immunohistochemical staining of alpha smooth muscle actin (α-SMA; DAKO, Kyoto, Japan), which correlates with Ac-HSCs, was performed as previously described [15,16]. The stained sections were analyzed using Adobe Photoshop software ver. 6 (Adobe, Tokyo, Japan).

Quantitative RT-PCR Analysis

mRNA was extracted from frozen liver tissues using the RNeasy Mini Kit (QIAGEN, Tokyo, Japan). The total RNA (1 μg) extracted from each sample was reverse transcribed into cDNA using a high capacity RNA-to-cDNA kit (Applied Biosystems Inc., Foster City, CA, USA). As TNF-α correlates with hepatic inflammation, it leads to liver fibrosis. As direct detection of LPS is difficult, we evaluated hepatic LPS-binding protein (LBP) because this directly correlates with LPS [17]. LPS stimulates TLR4 and activates Kupffer cells, which leads to hepatic inflammation. Hepatic oxidative stress was evaluated using sterol-regulatory element-binding protein 1 (SREBP-1) mRNA. The expression of mRNA encoding liver tissuederived TNF-α, LBP, and SREBP1 was analyzed using the PowerUp SYBR Green Master Mix and Step One Sequence Detection System (Applied Biosystems Inc., Foster City, CA, USA) by qRT-PCR. The PCR conditions were as follows: 95°C for 20 s, and 40 cycles at 95°C for 3 s and 60°C for 30 s. β-Actin was used as the endogenous control. The sequence of primers used is shown in Table 1.

Measurement of Thiobarbituric Acid Reactive Substances (TBARS)

We evaluated hepatic oxidative stress using hepatic TBARS concentration. Liver homogenates were prepared with PBS using a tissue homogenizer (Power Masher II®; Nippi, Tokyo, Japan), and then protein was extracted. TBARS concentration was measured using a commercially available kit, with malondialdehyde as the standard (Cayman Chemical, MI, USA).

Statistical Analyses

All results are expressed as mean ± SD. Statistical analyses were performed using EZR ver. 1.40 (Saitama Medical Center, Jichi Medical University). All tests were two-tailed and the results with p values < 0.05 were considered statistically significant.

Results

Inhibitory Effect of L-Carnitine and ARB on Hepatic Fibrosis

The groups fed the CDAA diet showed activated liver fibrosis compared with that of the group fed the CSAA diet. In the L-carnitine group, liver fibrosis was slightly improved compared with that in the CDAA-only group (p = 0.07), and significantly improved in the ARB group (p < 0.05). The combination of L-carnitine and ARB resulted in a greater improvement than with either drug alone (Figure 1A & 1B). Immunohistochemistry of α-SMA indicated a significant decrease in α-SMA-immunopositive Ac-HSCs in groups treated with ARB (Figure 2A). The semiquantitative analysis of α-SMA immunohistochemistry showed reduced α-SMA positive staining along with the inhibition of hepatic fibrosis (Figure 2B). A significant inhibition of TNF-α mRNA expression was also observed in the ARB group compared with that in the CDAA group (Figure 3A) (p < 0.05). However, L-carnitine did not inhibit TNF-α mRNA expression compared with that in the CDAA group. The combination of L-carnitine and ARB resulted in an inhibitory effect equal to that of ARB alone.

Effect of L-Carnitine and ARB on LPS Signaling

Hepatic LBP mRNA expression increased in the CDAA group compared with that in the CSAA group (Figure 3B). In the ARB and combination treatment groups, hepatic LBP mRNA expression was significantly lower than that in the CDAA group (p < 0.05). Hepatic LBP mRNA expression in the L-carnitine group was slightly lower, but not significantly different.

Inhibitory Effect of L-Carnitine and ARB on Oxidative Stress

Liver SREBP1 mRNA expression increased significantly over the control after 8 weeks of CDAA feeding (p < 0.01). In the L-carnitine group, SREBP1 mRNA expression was lower than that in the CDAA group (p < 0.05; Figure 4A), but no significant effect was observed in the ARB group. Under treatment with both L-carnitine and ARB, SREBP1 mRNA expression was lower than that in the CDAA group, but not significantly different from that with L-carnitine alone. Similarly, the increase in hepatic TBARS level in mice fed the CDAA diet, was decreased by L-carnitine treatment (p < 0.05; Figure 4B) but did not change under ARB treatment. The effect of combination of L-carnitine and ARB was similar to that of L-carnitine treatment. These data suggest that the inhibitory effect of the combination treatment on hepatic oxidative stress is associated with L-carnitine rather than ARB.

Discussion

Treatment with L-carnitine and ARB ameliorated liver fibrosis and suppressed Ac-HSCs and oxidative stress in the rat NASH model. Previously, we reported that a CDAA diet caused liver fibrosis by increasing TNF-α [18]. In the present study, the combination treatment with L-carnitine and ARB resulted in a synergistic antifibrotic effect greater than that resulting from either drug alone. L-Carnitine improved hepatic oxidative stress, whereas ARB suppressed the activation of HSCs. NASH has various causative factors, including inflammation and oxidative stress. Activation of HSCs leads to liver inflammation and ultimately fibrosis, which is governed by a complex network of autocrine/paracrine fibrogenic signals promoted by the activation of HSCs and characterized by the expression of α-SMA. The interaction between AT-II and Ac- HSC plays an important role in liver fibrogenesis. We previously reported that ARB directly inhibits Ac-HSC activation and that AT-II is important for the upregulation of TLR4 expression via stimulation of AT1R in Ac-HSCs [14]. Our results showed that ARB treatment of rats with NASH reduces α-SMA-positivity and TNF-α, which leads to an improvement in hepatic inflammation and fibrogenesis.
L-Carnitine is a vitamin-like dietary compound, synthesized from the essential amino acids lysine and methionine [19]. The liver is the main tissue for carnitine synthesis [6]. L-Carnitine is important for the transport of long chain fatty acids into the mitochondrial matrix via specialized acyltransferases [19]. Thus, L-carnitine inhibits oxidative stress [20,21] and stimulates the β-oxidation of fatty acids. Under L-carnitine deficiency, mitochondrial fatty acid oxidation is impaired, and lipids accumulate in hepatocyte cytoplasm, leading to the impairment of hepatic functions [22]. Our results showed that L-carnitine treatment reduces hepatic SREBP and TBARS in a rat model of NASH, leading to an improvement in hepatic oxidative stress. In contrast, hepatic TNF-α, LBP, and α-SMA were unchanged by L-carnitine treatment. Several studies have indicated the effectiveness of L-carnitine for liver oxidative stress and inflammation; however, its effectiveness for liver fibrosis is not known [23,24].
In this study, we examined the inhibitory effect of the combination of L-carnitine and ARB on liver fibrosis. As previously reported, ARB improved only liver fibrosis. Furthermore, L-carnitine slightly improved liver fibrosis without a significant difference. However, the combination of L-carnitine and ARB resulted in a significant improvement in liver fibrosis compared with that of ARB alone. Overall, the combination of L-carnitine and ARB showed a synergistic effect on liver fibrosis. This study was notably limited using only one model animal. It is also difficult to detect the signal transduction of L-carnitine for liver fibrogenesis using in vitro study, as it does not directly affect hepatic parenchymal cells. In conclusion, we found that simultaneous administration of L-carnitine and ARB exerted a potent and synergistic inhibitory effect on hepatic fibrosis compared with either agent alone by suppressing oxidative stress and Ac-HSC proliferation, respectively. This combination therapy could be useful for inhibiting NASH progression in clinical applications.

Acknowledgment

We would like to thank Editage (www.editage.com) for English language editing.

Conflicts of Interest

The authors declare no conflict of interest.

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Combination of L-Carnitine and Angiotensin-II type 1 Receptor Blocker has Beneficial Effects on Hepatic Fibrosis in a Non-Alcoholic Steatohepatitis Rat Model

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