Astaxanthin attenuates nonalcoholic steatohepatitis with downregulation of osteoprotegerin in ovariectomized mice fed choline-deficient high-fat diet

概要

【Introduction】
Postmenopausal estrogen decline increases the risk of developing nonalcoholic steatohepatitis (NASH), and it might accelerate progression to cirrhosis and hepatocellular carcinoma. This study aimed to investigate a novel therapy for postmenopausal women who are diagnosed with NASH.

【Methods】
Seven-week-old female C57BL/6J mice were divided into three experimental groups as follows: (1) sham operation (SHAM group), (2) ovariectomy (OVX group), and (3) ovariectomy + 0.02 % astaxanthin (OVX + ASTX group). These three groups of mice were fed a cholinedeficient high-fat (CDHF) diet for 8 weeks. Blood serum and liver tissues were collected to examine liver injury, histological changes, and hepatic genes associated with NASH. An in vitro study was performed with the hepatic stellate cell line LX-2.

【Results】
Astaxanthin attenuated fibrosis and steatosis in liver pathologies in OVX mice.
Hepatic collagen content was significantly increased in the OVX group compared to that in the SHAM group, while it was significantly reduced by ASTX treatment in the OVX+ASTX group compared to that in the OVX group (Fig. 1B). HE staining showed histological characteristics of the liver including lipid accumulation (Fig. 1A). Quantitative analysis showed that OVX-induced steatosis was significantly lowered by ASTX treatment in the OVX + ASTX group (Fig. 1C). Hydroxyproline assay analysis showed that the amount of hydroxyproline is significantly increased in OVX group compared with that in SHAM group and decreased in ASTX group compared with the OVX group (Fig.1D).

Astaxanthin attenuates liver injury in NASH mice.
Compared with the SHAM group, the OVX group exhibited a significant increase in inflammatory cytokines, including interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α), however, these elevations were efficiently suppressed by ASTX in the OVX+ASTX group (Fig. 2A). In addition, fibrosis markers such as α-smooth muscle actin (α-SMA) and transforming growth factor beta (TGF-β) and liver injury markers such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) showed the same tendency of expression change as inflammation markers.

Astaxanthin suppressed osteoprotegerin in liver tissues of mice.
The mRNA level of osteoprotegerin (OPG) was significantly suppressed by ASTX treatment in the OVX+ASTX group compared to that in the OVX group (Fig. 3A). The mRNA levels of OPG were decreased in the OVX+ASTX group without significant difference compared with that in the SHAM group. Consistently, western blot analysis (Fig. 3B, 3C) and immunohistochemistry results (Fig. 3D) showed that the protein levels of OPG in the liver tissues were significantly reduced in the OVX+ASTX group compared with that in the OVX group.

Astaxanthin repressed OPG production in LX-2 cells.
We performed an in vitro study to investigate the effects of ASTX on HSCs (LX-2 cells). TGF-β significantly induced OPG expression in LX-2 cells, and this elevation was suppressed by ASTX supplementation (Fig 4). In addition, ASTX decreased OPG expression in LX-2 cells without TGF-β stimulation.

【Discussion】
In our study, we showed that ASTX effectively improves NASH in CDHF-diet-fed OVX mice, with attenuated steatosis, inflammation, and fibrogenesis. Further investigation showed that this effect might have occurred through downregulation of OPG mediated by TGF-β suppression, resulting in recovery of liver injury, as indicated by decreased AST and ALT levels. These results indicate that ASTX could serve as a treatment for postmenopausal women who have lost active estrogen production.

The CDHF diet mouse model is widely used in investigations of NAFLD progression. Because choline is necessary for lipid transport and the production of very low-density lipoprotein choline deficiency triggers NAFLD by inhibiting hepatic triglyceride export and inducing fatty liver. Our previous study revealed that hepatic lipid accumulation in CDHFfed mice was significantly increased compared with that in choline-sufficient high fat dietfed mice. Because estrogen is contributing to choline synthesis through inducing the expression of PEMT (phosphatidylethanolamine-N-methyltransferase), the hepatic enzyme enabling endogenous biosynthesis of choline, postmenopausal women with the declined estrogen level have decreased choline synthesis. OVX is performed to induce estrogen loss. The combination of CDHF-diet with OVX operation was supposed to further enhance choline deficiency to mimic human postmenopausal conditions.

Activated HSCs are the key cells responsible for collagen production and hepatic fibrogenesis. Estrogen has been shown to prevent HSC activation in vitro and regression of fibrogenesis in vivo. In this study, our results showed that ASTX treatment reduced OVXinduced collagen deposition and markers of HSC activation, such as α-SMA and TGF-β, which suggested that ASTX treatment could exert protective effects against estrogen loss inducedHSC activation.

OPG is controlled by the opposing effects between active estrogen production in the premenopausal stage and increasing oxidative stress caused by estrogen loss in the postmenopausal stage. Functionally, OPG is an important player responsible for the major characteristics of NASH and not only fibrosis caused by activated HSCs but also the process of steatosis. In our study, OPG expression was significantly lower in the OVX+ASTX group than in the OVX group. Besides, we found that OPG was abundant in the portal vein area where the myofibroblast cells locate, among what 80% are contributed by activated pattern of HSCs. Another important outcome of our study – first assessed whether the previously reported existence of OPG in HSCs was a result of secretion by HSCs. We indeed found that TGF-β treatment increased OPG expression in LX-2 cells. ASTX could downregulate OPG expression with and without TGF-β stimulation. With the evidence that OPG stimulates the expression genes associated with fibrogenesis through TGF-β in vivo and that the inhibition of TGF-β suppresses the production of OPG, we speculate that the downregulation of OPG induced by ASTX might be a consequence of TGF-β suppression, resulting in the attenuated OVX-CDHFinduced NASH pathophysiology. Additionally, high expression of OPG has been found to be involved in arterial stiffness and insulin resistance in postmenopausal women. Thus, we propose that low levels of OPG might be beneficial for postmenopausal women.

【Conclusion】
ASTX may slow the progression of liver steatosis and fibrosis associated with CDHF-dietinduced NASH in ovariectomized female mice, and this preventive effect is attributed to the downregulation of OPG production by suppressed TGF-β in HSCs. Through our study, we present the novel view that ASTX may be used for the treatment of postmenopausal women.