NAFLD is currently a globally common liver disease, and it is predicted to affect up to one quarter of the population[14]. NAFLD is indicated by hepatic steatosis and associated with multiple detrimental effects and even increased mortality[21]. Therefore, the most globally pressing challenges are developing prevention and therapy strategies for NAFLD. Although the underlying mechanism is not well understood, applying an EMF can regulate lipid metabolism, insulin resistance, inflammation and redox homeostasis, which are related to the pathological mechanisms in NAFLD[16, 22–25]. Accordingly, the present research confirmed the bioeffects and explored the potential mechanism of action of EMFs on hepatic lipid deposition and oxidative stress in an HFD-induced animal model. The results showed that the EMF enhanced the expression of the CaMKKβ/AMPK/SREBP-1c and Nrf2 signalling pathways in the liver.
In modern societies, people have shifted away from healthy lifestyles towards sedentariness (lacking physical activity), and excessive energy intake (particularly food rich in fat) is considered one of the main factors that cause metabolic disorders, including NAFLD. Long-term and overconsumption of dietary fat can change lipid metabolism. In this study, we demonstrated that a HFD induced massive weight gain in the body and liver and abnormal blood TG levels. Interestingly, the EMF effectively weakened these changes. TG is mainly assembled and secreted in the endoplasmic reticulum of the liver. As an essential organ in regulating lipid metabolism, the liver is responsible for orchestrating the synthesis of fatty acids. When matured, TG is subsequently released into the blood and then redistributed to other tissues[26]. The abnormally increased serum TG level implies the disruption of lipid metabolism in the liver. To prove this hypothesis, we histologically determined the amount and size of lipid droplets in hepatocytes. The HFD treatment led to visible changes in hepatic lipid accumulation at five weeks. This result is consistent with earlier reports[27]. At the same time, we found that the EMF significantly inhibited lipid deposition. It is possible that the EMF may control hepatic lipid homeostasis to influence blood TG levels and liver weight. Additionally, the EMF regulates lipid production to diminish body weight gain.
Lipogenesis and lipid catabolism are involved in lipid metabolism; however, this study is focused on lipid synthesis. The process of lipogenesis is regulated by complex interactions with transcription factors, such as SREBP-1c[14]. SREBP-1c is expressed in the majority of tissues in mice and humans, with an especially high level in the liver[28]. A high-fat diet markedly increases SREBP-1c transcription, which increases de novo lipogenesis. Data from hepatocytes and animals showed that the transcription of mature SREBP-1c is inhibited by AMPK[21, 29]. AMPK is an essential intracellular energy regulator that has been verified to be closely linked to hepatic steatosis and insulin resistance[30]. Phosphorylation activation of AMPK reduces de novo lipogenesis and augments fatty acid oxidation in NAFLD by downstream factors covering SREBP-1c, ACC1 and FAS[21, 31]. Hence, we analysed whether the EMF functions in lipogenesis by modulating the AMPK/SREBP-1c pathway. In this investigation, we found a decrease in p-AMPK/AMPK levels and high mature SREBP-1c levels in NAFLD, which was reversed by EMF exposure. Simultaneously, we discovered that the EMF inverted the HFD-induced suppression of CaMKKβ protein expression. Both CaMKKβ and liver kinase B1 (LKB1) can phosphorylate and activate AMPK, and CaMKKβ plays a critical role in AMPK phosphorylation in LKB1-deficient cells[32, 33]. Therefore, the EMF likely activates CaMKKβ to phosphorylate and activate AMPK. Similar to previous reports, nanosecond-pulsed electric fields activated AMPK by CaMKKβ[33]. However, a limitation of this study was that we did not inquire about whether the EMF requires CaMKKβ to act on AMPK. Therefore, we can only speculate that the hepatic CaMKKβ/AMPK/SREBP-1c pathway is activated by EMF exposure in HFD-fed mice.
To fully explore the potential of EMFs as an effective method of improving fatty liver, we also focused on oxidative stress, which is vital for the development of NAFLD. This consequence is consistent with our early report that EMF attenuated oxidative stress[16]. The hepatic DHE staining and MDA levels in the present research revealed that EMFs likely eliminate the excess ROS induced by HFD feeding to prevent oxidative stress damage. Altering ROS homeostasis, especially O2− products, has been identified as a signal for enhancing the antioxidant capacity to fight against the harmful consequences of oxidative distress[34]. In addition, EMFs have previously been shown to improve antioxidant capacity in the liver in db/db mice and combined static magnetic and electric fields have been shown to scavenge overproduced superoxide by modulating the reduced glutathione (GSH)/oxidized glutathione (GSSG) redox environment[16, 23]. Similar results were observed in this study. The EMF increased the protein expression of Nrf2 and the activation of GSH-Px but not SOD or CAT. Nrf2 plays a key role in cellular resistance to oxidative stress. Although Nrf2 is present in the cytoplasm in a steady state, it will enter the nucleus to initiate antioxidant response elements when activated[35]. GSH-Px, as a downstream target of Nrf2, can metabolize lipid hydroperoxides and hydrogen peroxide into inoffensive compounds trading on GSH as a cosubstrate into GSSG[19]. A previous study reported that strengthening the activities of GSH-Px and paraoxonase (PON) enzymes provide protection against oxidative injury in rat liver[36]. In summary, the EMF activated Nrf2 to boost GSH-Px rather than SOD and CAT, which resulted in enhanced antioxidant ability and ameliorated hepatic oxidative stress induced by HFD. Furthermore, the expression of GSH-Px, GSH and GSSG proteins needs to be analysed, which is a limitation of this study.
According to the findings presented here, we found that the EMF can regulate the CaMKKβ/AMPK/SREBP-1c and Nrf2 pathways to attenuate hepatic lipid accumulation and oxidative stress. The findings of the present study suggest a promising physical therapeutic strategy for improving NAFLD.