Electromagnetic Fields Ameliorate Hepatic Lipid Accumulation and Oxidative Stress by Activating the CaMKKβ/AMPK/SREBP-1c and Nrf2 Pathways in High-Fat Diet-Fed Mice

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, and is related to disturbed lipid metabolism and redox homeostasis. However, a denitive drug treatment has not been approved for this disease. Studies have found that electromagnetic elds (EMFs) can ameliorate hepatic steatosis and oxidative stress. Nevertheless, the mechanism remains unclear. Methods: NAFLD models were established by feeding mice a high-fat diet. Simultaneously, EMF exposure is performed. The effects of the EMF on hepatic lipid deposition and oxidative stress were investigated. Additionally, the AMPK and Nrf2 pathways were analysed to conrm whether they were activated by the EMF. Results: Administration of the EMF decreased the body weight, liver weight and serum triglyceride (TG) levels and restrained the excessive hepatic lipid accumulation caused by feeding the HFD. This EMF function is achieved by boosting CaMKKβ protein expression, activating AMPK phosphorylation and suppressing mature SREBP-1c protein expression. Meanwhile, the activity of GSH-Px was enhanced following an increase in nuclear Nrf2 protein expression by EMF. However, no change was observed in the activities of SOD and CAT. Consequently, EMF reduced hepatic reactive oxygen species (ROS) and MDA levels, which means that EMF relieved liver damage by oxidative stress in HFD-fed mice. Conclusions: EMF can activate the CaMKKβ/AMPK/SREBP-1c and Nrf2 pathways to control hepatic lipid deposition and oxidative stress. This investigation indicates that EMF may be a novel therapeutic method for NAFLD.


Background
Nonalcoholic fatty liver disease (NAFLD), also called metabolic associated fatty liver disease (MAFLD), is increasing worldwide and has a prevalence of one-fourth of the general population [1]. NAFLD ranges from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH), liver brosis and cirrhosis, ultimately hepatocellular carcinoma [2]. Due to the unclear pathomechanism of NAFLD and unsatisfactory clinical drug experimental results, a speci c therapy has not been approved for NAFLD [3].
The pathological condition of NAFLD is characterized by hepatic lipid accumulation (in more than 5% of hepatocytes) without alcohol consumption or viral infection [4]. Excessive lipid deposition can directly cause the overproduction of reactive oxygen species (ROS) in the liver that cannot be cleared by antioxidant pathways [5], which disrupts redox homeostasis and leads to oxidative stress. Oxidative stress activates cellular dysfunction, cell necrosis and apoptosis in hepatocytes, which contributes remarkably to the pathogenesis of NAFLD and progresses to NASH [4,6].
AMP-activated protein kinase (AMPK) is critical for balancing cellular energy homeostasis and regulating lipid synthesis by restraining the cleavage of sterol regulatory element binding proteins (SREBPs), including the subtype of SREBP-1c [7,8]. As a major subtype of SREBPs in the liver, SREBP-1c is a master transcription factor of lipogenesis [9]. The bene t of the AMPK/SREBP-1c pathway in counteracting hepatic steatosis is widely recognized[8, 10,11].
Excessive oxidative stress is the result of an imbalance between oxidant and antioxidant functions; thus, boosting the antioxidant capacity can antagonize oxidative stress. Nuclear factor erythroid 1-related Factor 2 (Nrf2) is a key transcription factor that plays an important role in regulating antioxidant enzymes. The activation of antioxidant enzymes can protect tissues against oxidative injury [12]. Based on clinical and experimental evidence, Nrf2 has been proposed as a therapeutic target in NAFLD [13], and the administration of lipid metabolism and redox homeostasis is an effective therapeutic in NAFLD [4,6,14,15]. Our previous research demonstrated the positive effects of electromagnetic elds (EMFs) on hepatic steatosis and oxidative stress; however, the mechanism underlying the effect off EMFs on the liver has not been clari ed [16]. In this study, we investigated the bioeffects of the EMF in mice fed a highfat diet (HFD) and assessed the expression of AMPK/SREBP-1c and Nrf2 signalling pathways in the liver.

Animals and Study Design
This experimental procedure was conducted on male C57BL/6 mice (eight mice in each group, 7 weeks old and 21-23 g of initial body weight) housed in approved animal facilities (21 ± 2°C and 12 h/day light and 12 h/day dark conditions, and ad libitum access to water and commercial chow). These mice were randomly divided into three groups, and the experiments were started at the same time: (1) the control group was fed a standard chow diet, (2) the HFD group received a commercial diet rich in fatty acids (BiotechHD Co. Ltd., Beijing, China), and (3) the HFD + EMF group was fed the same diet as the HFD group and continuously exposed to an EMF 2 hours per day. Five weeks later, the mice were sacri ced to collect samples. The animal experiments were approved by the Institutional Animal Care and Use Committee at the Fourth Military Medical University and carried out according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH).

Electromagnetic Field
The electromagnetic exposure system used in this research was described in our previous articles. The EMF pulsed burst consists of a 5 ms burst width, 0.2 µs pulse rise, 0.02 ms pulse wait, 60 ms burst wait, 0.3 µs pulse rise and 0.2 µs pulse fall, and it is repeated at 15 Hz[16,17]. The peak value of the EMF was approximately 1.6 mT, as measured by a Gaussmeter (Lake Shore Cryotronics, Westerville, OH). A schematic representation of the EMF system is shown in Figure 1A

Analysis of the General Parameters
Five weeks after the intervention, the body weight was measured, and the animals were sacri ced. Following overnight fasting, blood was sampled from the eyes to obtain serum according to a previous study [18]. The liver of each mouse was collected immediately on ice after sacri ce, rinsed with 0.9% saline, dried with lter paper and weighed. Liver samples were stored at −80°C before evaluation. The procedures applied to the liver tissues were consistent with our previous report[16, 19].

Histological Analysis
Frozen liver slices were used to detect reactive oxygen species (ROS) activation and lipid deposition. Fresh liver samples were stored at -80°C and sliced into 10 µm thick tissue sections. The hepatic cryosections were incubated with a 10 µM dihydroethidium (DHE) uorescence probe (S0063, Beyotime, Beyotime Biotechnology., Shanghai, China) for 1 h in the dark. After rinsing with PBS, Hoechst 33258 (C0003, Beyotime, Beyotime Biotechnology., Shanghai, China) was used to stain the cell nucleus. A modi ed Oil Red O staining kit (C0158S, Beyotime, Beyotime Biotechnology., Shanghai, China) was applied to examine lipid accumulation in the hepatocytes. The liver slice was stained with oil-saturated O liquid for 10 minutes in the dark and then washed in distilled water. Next, haematoxylin and eosin (HE) was applied to stain the nucleus and cytoplasm. We used Image-Pro Plus 6.0 software to analyse the histology results.

Statistical Analysis
All data are expressed as the mean ± standard deviation (SD). One-way ANOVA with LSD t-test was used to evaluate the signi cant intergroup differences. Statistical analysis of data was managed with SPSS 16.0 at a signi cance level of P < 0.05.

EMF attenuates HFD-induced elevation of body weight, liver weight, serum TG and hepatic lipid deposition
The mouse model was induced by a HFD for ve weeks. To assess the positive effects of the EMF, we evaluated the body and liver weights. Both the body and liver weights in the EMF group were signi cantly lower than those in the HFD group (P < 0.05, Figure 1B and 1C). The serum TG level was reduced in the EMF group compared with the HFD group (P < 0.05, Figure 1D). Moreover, hepatic lipid accumulation had the same result (P < 0.05, Figure 1E and 1F). These ndings imply that the EMF could improve hepatic lipid metabolism.

EMF reduces HFD-induced lipid accumulation by activating the CaMKKβ/AMPK signalling pathway in the liver
To determine the potential mechanism by which EMF improves hepatic lipid metabolism, we investigated the protein expression of the CaMKKβ/AMPK/SREBP-1c signalling pathway. Figure 2 con rms the signi cant suppression of CaMKKβ and AMPK activation by HFD (P < 0.05). In addition, HFD induced an increase in mature SREBP-1c compared with the control group (P < 0.05). Nevertheless, the EMF inhibited the damage of HFD according to the western blot results, based on the alterations of target band intensity in the HFD group when compared with the EMF group (P < 0.05). The western blot results demonstrated that the EMF activates the CaMKKβ/AMPK/SREBP-1c signalling pathway to improve lipid deposition.

EMF alleviates HFD-induced oxidative stress in liver
A HFD can induce oxidative stress in the liver. To clarify the effects of the EMF on hepatic redox homeostasis, we measured oxidative stress indices. Figure 3A shows that DHE uorescence intensity, an indicator of ROS, was higher in the HFD group than in the EMF group. The mean IOD data in Figure 3B also support this nding (P < 0.05). The level of hepatic MDA as a redox status marker was measured.
The hepatic MDA content increased signi cantly due to HFD administration compared to the control group (P < 0.05). However, the EMF downregulated the MDA level, and a signi cant effect was seen in the EMF group compared with the HFD group (P < 0.05, Figure 3C). These results hint that the EMF rebalances redox homeostasis.

EMF improves hepatic antioxidant via the Nrf2 pathway
To verify whether the EMF regulated liver redox through antioxidant enzyme activation, we determined the hepatic nuclear Nrf2 protein expression and downstream antioxidant enzyme levels. Figure 4 shows that the nuclear Nrf2 protein expression levels were lower in the HFD group than the control group but were remarkably upregulated following EMF exposure (P < 0.05, Figure 4A). Compared to the HFD-fed animals, the hepatic SOD and CAT levels were not changed after EMF exposure ( Figure 4B and 4C). However, the GSH-Px level was notably increased by the EMF (P < 0.05, Figure 4D). These results verify that applying an EMF ameliorates hepatic oxidative stress in HFD-induced mice by strengthening antioxidants through Nrf2 activation.

Discussion
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, in ammation and redox homeostasis, which are related to the pathological mechanisms in NAFLD[16, [22][23][24][25]. Accordingly, the present research con rmed 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 ve weeks. This result is consistent with earlier reports [27]. At the same time, we found that the EMF signi cantly inhibited lipid deposition. It is possible that the EMF may control hepatic lipid homeostasis to in uence 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 veri ed 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-de cient cells [32,33]. Therefore, the EMF likely activates CaMKKβ to phosphorylate and activate AMPK. Similar to previous reports, nanosecond-pulsed electric elds 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 O 2 − products, has been identi ed as a signal for enhancing the antioxidant capacity to ght 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 elds 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 ndings 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 ndings of the present study suggest a promising physical therapeutic strategy for improving NAFLD.

Conclusions
In conclusion, the use of an EMF as a noninvasive physical intervention method had very desirable bioeffects on lipogenesis and the redox rebalance in fatty liver. This study provides new insights into treating NFALD by a single therapy or a combination of therapy and medication. Further research is expected to overcome the current dilemma of the lack of drugs approved for NAFLD.   Representative western blotting of hepatic CaMKKβ/AMPK/SREBP-1c signalling pathway. Values are all expressed as mean ± SD, n = 3. * P 0.05, compared with control. # P 0.05, compared with HFD.