Lactobacillus plantarum NA136 improves the non-alcoholic fatty liver disease by modulating the AMPK/Nrf2 pathway
Zijian Zhao、Chao Wang、Li Zhang、Yujuan Zhao、Cuicui Duan、Xue Zhang、Lei Gao1、Shengyu Li
Received: 3 December 2018 /Revised: 15 February 2019 /Accepted: 15 February 2019/Publishedonline:21May2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019
Hepatic lipid metabolic disorders and oxidative stress are involved in the development of non-alcoholic fatty liver disease (NAFLD). This study is to determine the protective effects ofLactobacillus plantarumNA136 on high-fat diet and fructose (HFD/F)-induced NAFLD and to elucidate its underlying molecular mechanisms. Male C57BL/6J mice had been fed with normal diet (ND), HFD/F, or HFD/F supplemented withL. plantarumNA136 for 16 weeks. Treatment withL. plantarum NA136 significantly lowered the body weight gain and decreased the mass of fat tissues, lipids, AST, and ALT levels of HFD/F-treated mice. Our results showed thatL. plantarumNA136 activated AMPK pathway to phosphorylate ACC and to suppress the SREBP-1/FAS signaling to inhibit the de novo lipogenesis and increase the fatty acid oxidation. Furthermore,with treatment ofL. plantarumNA136, the nuclear translocation of NF-E2-related factor 2 (Nrf2) was also increased which could activate antioxidant pathway. These findings suggested thatL. plantarumNA136 improved NAFLD by regulating the fatty acid metabolism and defending against oxidative stress through AMPK and Nrf2 pathways, respectively.
Total cholesterol (TC) and triglycerides (TG) were deter-mined by commercial kits (Jiancheng Institute, Nanjing,China). Free fatty acid (FFA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), alanine aminotrans-ferase (ALT), aspartate aminotransferase (AST), superox-ide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were determined by mouse ELISA kit (Jianglai,Shanghai, China).
Effects of L. plantarumNA136 on body weight and lipid levels in serum
HFD/F-fed mice showed higher body weights than the ND group mice (Table 1). After treatment withL. plantarum NA136, the body weight was decreased by nearly 17%compared with that of HFD/F group. The results of various serum parameters of each group are shown in Table 1. The levels of serum TC, TG, and LDL significantly increased in the HFD/F group, butL. plantarumNA136 treatment could attenuate these elevations. In addition, HDL level was con-siderably decreased by HFD feeding, and this decrease was blocked byL. plantarumNA136 supplementation.
L. plantarumNA136 ameliorated fat accumulation in the liver
According to a previous study, HFD and fructose induced substantial steatosis (Ritze et al.2014). We measured hepatic steatosis by histopathological study to investigate the effect ofL. plantarumNA136 on hepatic lipid accumulation.Compared with that of the ND group livers, H&E staining of the HFD/F group livers revealed a diffuse macrovesicu-lar steatosis (Fig. 1a). More importantly, HFD/F-induced hepatic steatosis and inflammation were markedly reversed by administration ofL. plantarumNA136. Furthermore, the levels of FFA in serum and hepatic were increased signifi-cantly by HFD and fructose, and this increase was normal-ized by treatment withL. plantarumNA136 (Fig. 1b, c).These results suggested that treatment withL. plantarum NA136 could attenuate hepatic steatosis induced by HFD and fructose.
Effects of L. plantarumNA136 on liver injury
The levels of ALT and AST in the liver are routinely used as markers of liver function. The activities of ALT and AST of the HFD/F group increased significantly compared with those of the ND group, which indicated that HFD/F feeding induced a massive liver injury in the mice (Fig. 2). Nevertheless, ele-vated ALT and AST concentrations were almost normalized by treatment withL. plantarumNA136. These results sug-gested that treatment withL. plantarumNA136 could attenu-ate liver injury and improve the liver function.
Effects of L. plantarumNA136 on hepatic AMPK signaling
SREBP-1 is a major regulator for transcription of lipogenic enzymes, and it also positively regulates hepatic lipogenic proteins including FAS and ACC (Horton et al.2002;Shimano2001). We measured the hepatic levels of SREBP-1 to determine the effects ofL. plantarumNA136 on HFD/F--induced lipid synthesis and clearance. As shown in Fig. 3a and c, the expression of SREBP-1 in HFD/F+NA136 group was significantly decreased and was accompanied with the decrease of FAS expression compared to the HFD/F group.
AMPK regulates the transcription factors such as SREBP-1 to improve lipid metabolism (Long and Zierath2006). The pro-tein levels of AMPK, ACC, and phosphorylation of AMPK and ACC were examined. The phosphorylation of AMPK was obviously decreased in the HFD/F group, and when treated withL. plantarumNA136, the phosphorylation of AMPK was increased in HFD/F+NA136 groups (Fig. 3b). Phosphorylation of AMPK is known to decrease the ACC activity via phospho-rylation, which subsequently downregulated the expression of FAS (Yang et al.2014). HFD/F-induced NAFLD mice demon-strated a lower level of ACC phosphorylation compared with the ND and HFD/F+NA136 groups (Fig. 3b).
L. plantarumNA136 reduced the oxidative stress in the liver
CAT could trigger Nrf2 nuclear translocation and HO-1 gene expression to activate the antioxidative action (Chang et al.2012). The results exhibited that the level of CAT in the HFD/F group markedly decreased more than that in the ND and HFD/F+NA136 groups (Fig. 4a). As shown in Fig. 4b, the activities of SOD increased evidently in the HFD/F+NA136 group compared with those in the HFD/F group. Liver MDA contents are shown in Fig. 4c. There was a marked increased content of hepatic MDA in the HFD/F group. As expected, the treatment withL. plantarumNA136 reduced the content of MDA.
Nrf2 and its repressor Keap1 regulate numerous cellular functions (Ludtmann et al.2014). When exposed to oxida-tive stress, the Nrf2 pathway was adaptively activated to alleviate cell damage. As shown in Fig. 4d, the total Nrf2 levels displayed no obvious changes in the three groups.Furthermore, the expressions of HO-1 and the contents of nuclear Nrf2 were markedly increased and the expression of Keap1 was decreased significantly in the ND and HFD/F+NA136 groups as compared to that of the HFD/F group.
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