Fermented Soybean Lipid And Its Effects On Regulating Lipid Metabolism Against Microwave-Induced Cognitive Impairment

BackgroundMicrowave techniques contribute to promoting the progress of humans and society and simultaneously have become a source of omnipresent electromagnetic pollution.Microwave radiation is harmful to special people who work or live around the microwave environment and lead them to suffer from electromagnetic hypersensitivity,involving headache,nausea,chest pain,sleep disturbance,and cognitive impairment.It is urgent to develop safe and effective anti-microwave agents.The antioxidant is the main strategy in the development of anti-microwave agents due to the biological effects of microwave radiation injury,and natural products with potent antioxidant activity are promising.However,molecular mechanisms of microwave radiation are still in a state of ambiguity,which blocks the development and application of anti-microwave agents.Therefore,the effects,characteristics,and mechanisms of natural products need to be illuminated during microwave radiation injury.Additionally,the preliminary study found active lipid ingredients from fermented soybeans,prepared by supercritical CO₂（SC-CO₂）extraction and named FSE-C that possesses the characteristics of effectiveness,availability,and safety and potential development prospect.ObjectiveThis study aims to analyze the chemical composition of FSE-C,especially focusing on the changes of structures and contents in lipid classes after fermentation.Then,the optimization of the SC-CO₂ extraction was performed to improve the lipid classes ratio and biological activities of FSE-C.Additionally,based on microwave-induced biological effects（e.g.cognitive impairment）,we also revealed the characteristics and mechanisms of FSE-C by regulating lipid metabolic pathways,in order to provide novel techniques and screening strategies for the investigation of anti-microwave agents.Methods1.Gas chromatography equipped with a flame ionization detector（GC-FID）and high performance liquid chromatography（HPLC）coupled with evaporative light-scattering detector or atmospheric pressure chemical ionization mass spectrometry were performed to analyze the fatty acid composition and identify the chemical structures of FSE-C,respectively.Then,the changes of lipid classes between FSE-C and unfermented soybean extract（SE-C）were comparatively analyzed.2.The preparation process of FSE-C was optimized by a single-factor experiment combined with response surface methodology.Then,the fatty acid composition,acid value,saponification value,and lipid phytochemicals（i.e.vitamin E,phytosterols,squalene,total flavonoids,and total polyphenols）of FSE-C,prepared by the optimum extraction conditions,were analyzed and determined based on GC,titrimetric analysis,HPLC,and spectrophotometry.3.The rat model of microwave-induced cognitive impairment was established.（1）Morris water maze,hematoxylin-eosin（H&E）staining,organ indexes,serum biochemical analyzer,and peripheral blood cell counter were used to evaluate the effects of FSE-C against microwave-induced cognitive impairment in rats.（2）The lipid metabolic profiles of serum and hippocampus in microwave-exposed rats were performed to analyze the effects of FSE-C on regulating lipid metabolism against microwave-induced cognitive impairment based on the ultraperformance liquid chromatography coupled with the mass spectrometry.4.The analytical methods of colorimetric analysis,fluorescence staining,flow cytometry,quantitative real-time PCR,western blotting,and lipidomics were used to investigate and explore lipid metabolism-associated effects,mechanisms,and differential metabolites of FSE-C in vitro based on the erastin-induced model of PC12cells.Results1.FSE-C mainly consisted of free fatty acids（FFAs）,diacylglycerols（DAGs）,and triacylglycerols（TAGs）,which with higher palmitic acid andα-linolenic acid,and lower oleic acid in the fatty acid composition compared to SE-C.Moreover,the contents of FFAs and DAGs in FSE-C were higher than SE-C,while the content of TAGs was lower,which indicates that FFAs and DAGs might be the potential active material base for guiding the optimization of FSE-C preparation.2.The optimum SC-CO₂extraction of FSE-C was conducted at 35℃,30 MPa,and the moisture content of miso should be lower than 3.0%.（1）Under the optimum parameters,FSE-C was stable and its practical response value was 6.93±0.11 g/100g that was higher than the predicted response value（6.78g/100g）.FFAs,DAGs,and TAGs of FSE-C accounted for 75.73±2.46%,4.84±0.28%,and 16.77±2.54%,respectively.（2）Based on the optimum parameters,the fatty acid composition of FSE-C included 80.04%of unsaturated fatty acid and 14.39%of saturated fatty acid.The acid value and saponification value of FSE-C were 148.90±0.34 mg KOH/g and 197.46±1.34 mg KOH/g,respectively.The total vitamin E,total phytosterols,squalene,total flavonoids,and total polyphenols contents of FSE-C were 162.31±1.87 mg/100 g,654.89±20.09 mg/100 g,13.81±0.16 mg/100 g,9.85±0.90 mg/100 g,and 50.64±4.44 mg/100 g,respectively.3.We established a rat model of microwave-induced cognitive impairment.（1）Morris water maze test showed that the average escape latency（AEL）of rats in the microwave-exposed（MW）group was much longer than the sham-exposed（SH）group at 14 d after microwave radiation.Rats with the treatment of FSE-C showed a shorter AEL in comparison with the MW group（p<0.01）,suggesting reduce cognitive impairment in microwave-exposed rats.（2）H&E staining showed that attenuated triangular or spindle nuclei were observed in rat hippocampal neurons of the MW group at 7 d after radiation.These changes were alleviated in rat hippocampal neurons with the treatment of FSE-C,suggesting that FSE-C could reduce the pathological and morphological changes of rat hippocampal neurons caused by microwave radiation.（3）Organ indexes showed that spleen and thymus indexes of rats had significant differences between the MW and SH groups at 7 d after radiation.Treatment with FSE-C could revert these changes of the spleen（p<0.001）and thymus indexes（p<0.05）in microwave-exposed rats.（4）Serum biochemical analysis showed that serum triglyceride（TG）and high-density lipoprotein（HDL）levels had significant differences between the MW and SH groups at 7 d after radiation.Treatment with FSE-C could modify serum TG（p<0.01）and HDL（p<0.05）in microwave-exposed rats,facilitating their recovery.（5）Peripheral blood cell counter showed that white blood cell,lymphocyte,neutrophil percentage,monocyte percentage,and hemoglobin had significant differences between the MW and SH groups at 7 d after radiation.Treatment with FSE-C could promote these differential peripheral blood cells returning to normal levels in microwave-exposed rats.4.（1）Based on untargeted lipid metabolic analysis in rat serum at 7 d after radiation,68 disturbed endogenous lipids,including phosphatidylcholines（PCs）,phosphatidylethanolamines（PEs）,lysophosphatidylcholines（LPCs）,lysophosphatidyl-ethanolamines（LPEs）,TGs,and FFAs,were identified,which indicates that microwave radiation could lead to abnormal lipid metabolism.The important lipid metabolic pathways were involved in linoleic acid metabolism,glycerophospholipid metabolism,glycerolipid metabolism.Moreover,43 differentially expressed lipids in rat serum were reversed with the treatment of FSE-C.Numerous TG metabolites were downregulated by FSE-C and well-matched with its effect on attenuating TG levels based on the serum biochemical analysis.FSE-C also modified levels of differential plasmalogens,PE（20:4/18:0）,and LPE（0:0/18:0）and likely linked to alleviate cognitive impairment caused by microwave radiation.（2）There were 30 differential lipid metabolites,including FA（26:0）,2 PCs,11sphingomyelins（SMs）,and 16 TGs,identified between the MW and SH group based on hippocampal lipidomics in rats at 14 d after radiation.Furthermore,FSE-C positively regulated and facilitated FA（26:0）,2 PCs,4 SMs,and 11 TGs of rat hippocampus returning to levels of the SH group,suggesting reduces cognitive impairment induced by microwave radiation.Among them,FSE-C obviously modified the disturbances of TGs in rat hippocampus.5.We also established the erastin-induced model of PC12 cells.（1）There was no cytotoxic activity or proliferating inhibition in PC12 cells treated by FSE-C,but FFAs,DAGs,and TAGs could cause cell death or damage assayed by the CCK-8 and LDH kits.（2）The cell viability of erastin-induced cells was improved by FSE-C in a dose-dependent manner,and it indicated that FSE-C inhibits erastin-induced ferroptosis in PC12 cells.FFAs and DAGs at high concentrations also raised the viability of erastin-induced cells.（3）FSE-C could significantly suppress the enhancement of Fe²⁺ion levels,reduce lipid peroxidation levels,decrease the production of reactive oxygen species,and improve glutathione concentration in erastin-induced cells.These findings indicate that FSE-C can protect from erastin-induced ferroptosis.（4）FSE-C significantly upregulated both m RNA levels of GPx4 and SLC7A11 and protein expression of GPx4 in erastin-induced cells.In addition,FSE-C significantly downregulated the m RNA expression of ACSL4 and LPCAT3 in erastin-induced cells.These findings suggest that the potential mechanisms of action treated by FSE-C are closely linking to promote GSH synthesis and block lipid peroxidation levels.（5）FSE-C positively regulated 80 differentially expressed lipid metabolites（i.e.6FFAs,30 PCs,5 PEs,34 TGs,and 5 SMs）and potently reversed the levels of differential polyunsaturated fatty acids（PUFAs）and PUFA-containing phospholipids,which strongly supports that FSE-C downregulates the m RNA expression of ACSL4and LPCAT3 and modifies lipid metabolism of cells against erastin-triggered ferroptosis.Conclusions1.The optimization of SC-CO₂ extraction was accomplished,and FSE-C is stable by using the optimum parameters.The chemical profiles of FSE-C were achieved,which shows that FSE-C as a class of active lipid ingredients consists of FFAs,DAGs,TAGs,and minor lipid phytochemicals.2.FSE-C can attenuate microwave-induced cognitive impairment in rats,characterized by reducing AEL in MWM test,alleviating pathological changes of rat hippocampus,facilitating the recovery of TG and HDL levels based on the serum biochemical analysis.3.This study demonstrated that microwave radiation could lead to disturbances in lipid metabolism for the first time,which helps to provide a new insight into mechanisms of action induced by microwave radiation.FSE-C effectively reversed differentially expressed lipid metabolites in rat serum induced by radiation.Importantly,the differential plasmalogens and PE（20:4/18:0）are regarded as potential biomarkers,which provides novel screening strategies for the development of anti-microwave agents.4.The lipid metabolism of the hippocampus from microwave-exposed rats was further analyzed to investigate the relationship between microwave-induced cognitive impairment and neural lipids.FSE-C significantly facilitated the recovery of neural TGs,which indicates that metabolites of TGs are acted as a kind of key biomarker in microwave-induced cognitive impairment and a new target for the development of anti-microwave agents.5.FSE-C can protect PC12 cells from erastin-induced ferroptosis,characterized by increasing cell viability and glutathione concentrations,attenuating levels of intracellular Fe²⁺ion,lipid peroxidation,and reactive oxygen species,as well as modifying gene/protein expression（i.e.GPx4,SLC7A11,ACSL4,and LPCAT3）and lipid metabolism（i.e.differential PUFAs and PUFA-containing phospholipids）.The above results will contribute to further investigations of FSE-C on revealing the mechanisms of action against microwave-induced cognitive impairment.