A Magnetic Field Prevents Alcoholic Liver Disease by Reducing Oxidative Stress

The radical-pair recombination change will affect the generation of free radicals, which can be regulated by static magnetic fields (SMFs) in a SMF setting dependent way. It is well known that alcohol consumption leads to significantly increased free radical levels and health risks, which lacks effective treatment method besides alcohol abstinence. Here we compared different SMF settings and found that a downward SMF of ∼0.1 T with magnetic flux of ∼4.5×10−3 Wb could effectively alleviate alcohol-induced liver damage and lipid accumulation, and improve liver function. The inflammation, reactive oxygen species (ROS) level and oxidative stress were significantly reduced. EPR (electron paramagnetic resonance) experiments also confirmed the reduced amount of free radical by SMF treatment. Moreover, the lifespan of heavy alcohol drinking mice was also significantly changed due to the SMF effects on liver cell ROS level, DNA synthesis and liver cell regeneration. Our study shows that moderate SMFs with specific parameters have great promises to be developed into a physical method to reduce alcohol-induced liver damage in the future.


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Alcoholic liver disease (ALD) caused by excessive drinking is one of the most 33 common chronic liver diseases, including steatosis, steatohepatitis, fibrosis, cirrhosis 34 and hepatocellular carcinoma. ALD with a high short-term mortality has become a  (Yu et al., 2020). Therefore, modulating ROS and oxidative stress by 76 electromagnetic field could potentially provide a non-invasive physical tool to 77 regulate physiological and pathological processes. 78 In this study, we sought to examine the effects of SMFs in alcohol-induced liver 79 damage by using different mice models and SMF settings. We found that for both 80 shorter-term lighter drinking mice and longer-term heavier drinking mice, the alcohol-81 induced liver damage can both be significantly relieved by a downward SMF of ~0.1 82 T with magnetic flux of ~4.5x10 -3 Wb. The lifespan of the heavy drinking mice was 83 significantly prolonged by this magnetic field exposure. In contrast, a different SMF 84 setting did not have such effects, and even be detrimental to heavy drinking mice.

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The downward SMF alleviates alcohol-induced mice liver damage 87 To assess the effects of SMF on alcohol-induced ALD, we used magnetic plates 88 4 with different settings. The magnet plates were composed of twelve closely connected 89 N38 Neodymium (NdFeB) magnet cubes, with either the North or South pole facing 90 up, which provided a relatively uniform magnetic flux density at a given horizontal 91 plane ( Figure 1A). We measured the magnetic field intensity at 2 cm above the 92 magnet plate, which corresponds to the position of the mice bodies when they stand.

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The average magnetic field intensity of the three groups at this horizontal level are Wb, 4.36×10 -3 Wb and 4.47×10 -3 Wb, respectively ( Figure 1A). To reduce any 96 potential placebo effects, we used un-magnetized NdFeB as sham control. The whole 97 mice cages were placed on the top of these magnetic plates ( Figure 1B). For the mice 98 that were exposed to the magnetized plates, the magnetic flux density on their bodies 99 were ~0.08-0.19 T ( Figure 1B). We fed mice with the Lieber-DeCarli alcohol liquid 100 diet (Lieber et al., 1989) containing either ethanol (EtOH-fed) or maltodextrin diet as a 101 control (pair-fed) with unlimited access ( Figure 1C). For the EtOH-fed group, we 102 gradually increased the concentration of EtOH, from 1% (week 1), 2% (week 2) to 103 3% (week 3). We started the alcohol feeding and SMF exposure simultaneously from 104 the beginning and continued for 3 weeks (SMF exposure was 12 h/d, 7 d/week) 105 ( Figure 1C).

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To get comprehensive information about the effect of SMFs on these mice, we 107 monitored their body weight, food consumption and their vital signs. No significant 108 differences in body weight or food consumption were observed among EtOH-fed 109 groups ( Figure 1D). For their vital signs, it seems that three-weeks of alcohol 110 consumption reduced both the heart rate (from 178.2 to 160.7, P < 0.05) and the 111 arterial O 2 (from 97.6% to 93.96%, P < 0.05). However, it is interesting that both the 112 SMFs can increase the mice arterial O 2 (Figure 1-figure supplement 1). 113 Next, H&E staining were used to analyze the mice tissue. No significant 114 difference was observed in spleen, lung, kidney or heart (Figure 1-figure supplement   115 2). However, all EtOH-fed mice had obvious liver damage, which appear as a large 116 number of vacuoles on the liver sections. It is interesting that although both the 117 5 upward and downward SMFs reduced the vacuoles, the downward SMF had a much 118 more obvious alleviation effect ( Figure 1E). Similarly, although both the upward and 119 downward SMFs reduced the ethanol-induced liver cell apoptosis, the downward 120 SMF had a much more significant alleviation effects as shown by the TUNEL assay 121 ( Figure 1F). Consistently, Western Blot analysis also shows that the alcohol-induced 122 cleaved-caspase 3 was reduced by SMF treatment, especially by the downward SMF.

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The alcohol induced BCL2 reduction was also reversed by SMF treatment, especially 124 by the downward SMF ( Figure 1G).

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These results indicate that EtOH-induced oxidative stress can be reduced by the 193 downward SMF treatment.

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The downward SMF protects hepatocyte through suppressing oxidative stress 195 To investigate the effects of SMFs on the oxidative stress in hepatocyte, we 196 exposed HL7702 cells to NdFeB magnets that can be placed in the regular cell 197 incubator and their magnetic field flux density is similar to the mice SMF exposure 198 system ( Figure 4A). For cells on the magnet, the SMFs are in the range of 0.1~0.4 T. 199 We first confirmed that the ROS levels of the hepatocyte HL7702 cells were  Figure 4E).

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In order to analyze the effect of SMF on EtOH-induced cellular ROS elevation, 209 12 we treated HL7702 hepatocytes that were exposed to SMFs with 340 mM ethanol for 210 24 h. The downward SMF significantly reduced EtOH-induced cellular ROS while 211 the upward SMFs were less effective ( Figure 4F). Since NRF2 is an oxidative stress 212 marker, which may active antioxidant enzyme to reduce intracellular ROS caused by      The black arrows indicate the spectrum of hydroxyl radical (OH·).   Ki67 and Top2A in EtOH-fed mice. We speculated that the hepatocytes regeneration 390 was inhibited by the upward SMF in EtOH-fed mice. Therefore, the EtOH-fed mice 391 had a low survival rate when exposed to the upward SMF.  Lastly, the SMF direction dependent effects exist, but the mechanism is still 423 elusive. We found that the downward SMF had significantly beneficial effects, 424 including reduced liver cell apoptosis and lipid accumulation, serum ALT, AST and 425 TG, as well as increased life span. However, the upward SMFs did not affect the light 426 drinking mice, but surprisingly shortened the lifespan of heavy drinking mice, which 427 is likely due to the impaired liver regeneration of the heavy drinking mice. This shares 428 the similar mechanism with the tumor cell proliferation inhibition effects by SMFs.

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Moreover, we have also previously reported that the blood sugar regulation effects are 430 also SMF direction dependent. Although the Lorenz forces exerted on negatively 431 charged moving DNA can explain some of the phenotypes, we believe there must be 432 other unraveled mechanisms underlying the SMF direction dependent bioeffects.

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In summary, our study revealed that a moderate SMF with a downward direction 434 could alleviate ALD through reduced ROS, oxidative stress, and inflammation. 435 Unexpectedly, the upward SMF setting in our study may be a taboo for heavy 436 drinkers, which might be a potential precaution to some open MRI operations, which 437 usually have upright magnetic field of ~0.5 T. Whether other magnetic settings, for 438 example, different magnetic field flux density and distribution, will provide improved 439 beneficial effects or more detrimental effects, still need further investigations.   The formulas of diets were formulated by Research Diets. The mice were exposed to 457 sham, upward and downward SMFs for 3 weeks (chronic model) or 6 weeks (acute   Human hepatocyte HL7702 cells (ATCC) were exposed to alcohol and magnetic 526 fields before they were collected in 1.5 mL centrifuge tube by 0.25% trypsin 527 digestion. Cell number were counted by flow cytometry. Additionally, cultured cells 528 were exposed to alcohol and SMFs for 24 h. Annexin V-FITC apoptosis detection kit 529 (BD Biosciences, 0076884, USA) was used to analyze hepatocyte apoptosis according 530 to manufacturer's protocol. Cell apoptosis was detected by flow cytometry.

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Western blot analysis 532 The protein isolations were collected with protease inhibitors from liver tissues and 533 cultured cells, and western blotting were performed as previously described. The

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Mice exposed to the short-term alcoholic diet were executed to collect liver tissues.

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And then liver tissues were polished on ice and were used to extract RNA by a 541 RNAeasy TM animal RNA isolation kit with spin column (R0027, Beyotime).

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Novoscript R plus all-in-one 1 st strand cDNA synthesis supermix (E047-01A, 543 Novoprotein) was used to RNA reverse transcription, and Novostart R SYBR qPCR 544 supermix plus (E096-01A, Novoprotein) were used to amplify target gene under the 545 action of specific primers. The detailed primers sequences were shown in table S1.