Protective Effects Of UFH-SPIONs Against The Neuronal Lipotoxicity Induced By Saturated Fatty Acids

In recent years,due to the continuous improvement of people’s living standards,obesity and long-term high-fat diet(HFD)have been increasing.Obesity and HFD lead to saturated fatty acids(SFA)content,and the body’s white fat cell number is limited,when including SFA lipid content over adipose tissue storage capacity and tissue oxidation utilization ability,free SFA in the blood,and deposited in the non-fat cells,causing the tissue cell damage,eventually lead to target organ dysfunction.This dysregulation of cellular homeostasis due to the accumulation of lipids is lipotoxicity.Lipotoxicity mainly affects the liver,skeletal muscle,and pancreatic islet β cells by causing metabolic disorders,inflammation and oxidative stress,and endoplasmic reticulum stress pathway activation,and can trigger cell death.These changes can cause diseases such as type 2 diabetes,heart diseases,and fatty liver disease.The growing number of research have shown that lipotoxicity does the same harm to neurons,leading to the occurrence of disease such as diabetic encephalopathy,Alzheimer’s disease,and depression.At present,targeting the harm of lipotoxicity is still an unsolved clinical problem.Unfractionated heparin(UFH)is a mixture of α-D-glucosamine(N-sulfation,O-sulfate,or N-acetylation),and O-glucuronide(α-L-aduride or β-D-glucuronide)that are alternately connected to form a polymer composed of sugar chains of different molecular weights.UFH has various biological effects including anticoagulation,anti-inflammatory,anti-ferrimodulin,and glycocalyx protection.Because of its strong polarity,large molecular weight,and large negative charge,it cannot cross the blood-brain barrier.Superparamagnetic iron oxide nanoparticles(SPIONs)has unique physical-chemical properties and is widely used in various fields.The asked SPIONs has the disadvantage of short half-life and poor stability,which needs to be modified.Our research group has previously developed the unfractionated heparinmodified superparamagnetic iron oxide nanoparticles(UFH-SPIONs),which is a nanosystem obtained by using UFHas a surface modifier to modify SPIONs by electrostatic interaction.It is found that UFH-SPIONs particle size,good stability,no cytotoxicity,and enzyme-like activity.It can cross the blood-brain barrier in epileptic animal rats,and exert its effects through anti-inflammatory,temporal lobe epilepsy,and inhibitory mechanism of neuronal apoptosis.There are inflammation,oxidative stress,and neuronal apoptosis mechanisms of SFA.so we speculate that UFH-SPIONs may antagonize the lipid toxicity of neurons caused by saturated fatty acids.This topic to palmitic acid(PA)as the representative of SFA,using its effect on neuronal cells to establish neuronal lipotoxicity model from the dysfunction of neuronal energy metabolism,oxidative stress,and neuronal apoptosis to explore the possibility of UFHSPIONs inhibit PA neuron lipotoxicity,for the development of antagonistic SFA neuron lipotoxicity of new drugs.The main contents and results of this research are as follows:1.Preparation,characterization,and cytotoxicity of UFH-SPIONs1.1 Preparation of UFH-SPIONsIn this study,SPIONs and UFH-SPIONs were prepared by chemical coprecipitation.1.2 Determination of iron content in UFH-SPIONsThe total Fe concentration of SPIONs and UFH-SPIONs was measured by phenanthroline spectrophotometry.The total Fe concentration of SPIONs is 28 μg/mL.The total Fe concentration of UFH-SPIONs ranges from 100 to 160 μg/mL.1.3 Determination of UFH content in UFH-SPIONsThe concentration of UFH in UFH-SPIONs was measured by using the toluidine blue method.The UFH concentration of UFH-SPIONs is about 110 μg/mL.1.4Determination of UFH-SPIONs aggregation statusTEM microscopy showed SPIONs were clustered in multi-particle clusters,while UFHmodified SPIONs was dispersed,indicating that UFH-SPIONs had good stability and good dispersion.1.5UFH-SPIONs hydrokinetic diameter,polydispersion coefficient,and Zeta potential determinationThe dynamic light scattering(DLS)method is used to detect the hydrodynamic diameter,Polymer dispersity index(PDI),and Zeta potential of SPIONs and UFH-SPIONs.The hydrodynamic diameter and PDI of UFH-SPIONs are smaller than SPIONs.The absolute value of Zeta potential is significantly higher than that of SPIONs,indicating that UFH-SPIONs have better stability and uniform particle size.1.6UFH-SPIONs infrared spectroscopy characterizationA Fourier transforms Infrared spectrometer(FT-IR)is used to scan SPIONs and UFHSPIONs in the wavelength range of 2000-600 cm-1 and record infrared spectra.The results indicated that UFH combined with SPIONs successfully.1.7 The cytotoxicity of UFH-SPIONs on neuronsThe CCK-8 method was used to detect the effects of UFH and UFH-Spions on mouse hippocampal neurons.The results showed that both 50 μg/mL and 100 μg/mL UFH showed inhibitory effects on cell proliferation in mice hippocampal neuron cells(P<0.01),and the cell activities were 68.24%and 52.59%,while.UFH-SPIONs(50 μg/mL and 100 μg/mL)promoted neuronal cell proliferation in mice hippocampal neuron cells(P<0.01),and the cell activity was 124.92%and 119.74%.2.Effect of UFH-SPIONs on neuronal energy metabolism dysfunction induced by palmitate2.1 Lipid accumulationPA was used to establish a lipotoxic cell model of mouse hippocampal neurons HT22 cells,and oil red O staining was used to stain the lipids.The results showed that the cell state of the model group was poor,the morphology was triangular or irregular,the membrane kernel boundary was not clear,and there was a large amount of lipid deposition in the cell.Compared with the model group,the cells of the UFH-SPIONs group were in a good state with regular morphology and only very few cellular lipid deposits,while the cells of the UFH group were in a good state with regular morphology but had abundant lipid deposits in the cells.2.2 Neuronal membrane fluidityPA was used to establish a lipotoxic cell model of mouse hippocampal neurons HT22 cells,the membranes were labeled with NBD-C6-HPC cell membrane dye solution,and the membrane fluidity of mouse hippocampal neurons was detected using a fluorescence bleaching technique.The results showed that compared with the control group,the membrane fluidity of hippocampal neurons in the model group was significantly decreased(P<0.01).Compared with the model group,the UFH-SPIONs group had significantly increased membrane fluidity(P<0.01),but the UFH group had no significant effect on the decrease in cell membrane fluidity of mouse hippocampal neurons induced by PA(P>0.05).2.3 Studies of neuronal glucose uptakePA was used to establish a lipotoxic cell model of mouse hippocampal neurons HT22 cells,and 2-NBDG was used as a glucose uptake tracer.The results showed that glucose uptake significantly decreased in the model group compared with the control group(P<0.01).Compared with the model group,glucose uptake in hippocampal neurons of the UFH-SPIONs group and the UFH group was significantly increased(P<0.0.1);The UFH-SPIONs group promoted glucose uptake more significantly than the UFH group(P<0.01).2.4 Studies of ATP production of neuronsPA was used to establish a lipotoxic cell model of mouse hippocampal neurons HT22 cells,and the ATP content was detected by enzyme-linked immunosorbent assay.Compared with the control group,ATP content produced by hippocampal neurons in the model group was significantly decreased(P<0.01).Compared with the model group,ATP production in hippocampal neurons of the UFH-SPIONs group and the UFH group was significantly increased(P<0.01);The UFH-SPIONs group protected the neuronal energy metabolism system more significantly compared with the UFH group(P<0.05).3.Effect of UFH-SPIONs on neuronal oxidative stress by palmitate3.1 Study on the oxidative stress pathway of P-JNK/NADH dehydrogenasePA was used to establish the lipotoxic cell model of mouse hippocampal neurons HT22 cells,to detect the content of phosphorylated c-JNK amino-terminal kinase(P-JNK)and NADH dehydrogenase,and the intracellular fluorescence intensity was detected by using DCFH-DA fluorescent probe to label reactive oxygen species(ROS).Result display,compared with the control group,the P-JNK content,and intracellular ROS fluorescence intensity in hippocampal neurons in the model group were significantly decreased(P<0.01),Meanwhile,the NADH dehydrogenase content decreased significantly(P<0.01),Compared to the model group,Compared with model group,UFH-SPIONs group,and UFH group significantly decreased the content of P-JNK(respectively P<0.01 and P<0.05)and intracellular ROS fluorescence intensity(respectively P<0.01 and P<0.05),while significantly increased NADH dehydrogenase(respectively P<0.01 and P<0.05).The results showed that UFH-SPIONs inhibited oxidative stress by regulating P-JNK/NADH dehydrogenase pathway.3.2 Study on the protective effect of UFH-SPIONs on oxidoreductasePA was used to establish a lipotoxic cell model of mouse hippocampal neurons HT22 cells,and an enzyme-linked immunosorbent experiment was used to detect superoxide dismutase(SOD),nitric oxide synthase(NOS 1),and peroxidase(POD).The results showed that compared with the control group,both SOD enzyme content and POD enzyme content in hippocampal neurons decreased in the model group(P<0.01),and the content of the NOS1 enzyme was increased(P<0.01).Compared with the model group,the SOD enzyme content(P<0.01 or P<0.05)and POD enzyme content(P<0.01)were significantly increased in the UFH-SPIONs group and UFH group,and the NOS1 enzyme contents were also significantly decreased(P<0.01)in UFH-SPIONs group and UFH group.This suggests that UFH-SPIONs can protect the antioxidant enzymes of hippocampal neurons and can resist oxidative stress.4.Effect of UFH-SPIONs on palmitate-induced neuronal apoptosisPA was used to establish the lipotoxic cell model of mouse hippocampal neurons HT22 cells,the effects of UFH-SPIONs and UFH on the cellular activity of the hippocampus neurons in lipotoxic mice were measured by the method of CCK-8,the cytochrome C(Cyt-C)was detected by ELISA,A Fluo-4 fluorescent probe was used to label intracellular calcium ions,Intracellular mitochondrial membrane potential was detected using JC-1 fluorescent probe,Result display,PA had cytotoxic effects on hippocampal neurons(P<0.01);the effect of the PA on HT22 cells also caused a significantly increased in intracellular calciumion content and CytC content(P<0.01);Compared with the control group(380.16%),the JC-1 polymer/monomer fluorescence intensity ratio(14.67%)was significantly decreased(P<0.01),Mitochondria are mainly green fluorescent and exhibit very low mitochondrial membrane potential;UFHSPIONs had a significant inhibitory effect on PA-induced cytotoxicity in mouse hippocampal neuronal cells,showing a significant effect on promoting the value added of HT22 cells(P<0.01),Compared with the model group,the intracellular calcium content and Cyt-C content of UFH-SPIONs group were significantly decreased(P<0.01);UFH-SPIONs could significantly increase the decreased mitochondrial membrane potential caused by palmitic acid,and the fluorescence intensity ratio of JC-1 polymer/monomer was significantly increased(183.65%,P<0.01).The UFH group showed no protection against PA-induced hippocampal neuronal cytotoxicity in mice,showing further inhibitory effects on neuronal proliferation(P<0.01),Compared with the model group,the contents of calcium ion and Cyt-C in UFH group were significantly reduced(P<0.01),UFH significantly restored the decreased mitochondrial membrane potential caused by palmitic acid,and the JC-1 polymer/monomer fluorescence intensity ratio increased significantly(69.63%,P<0.01).In conclusion,UFH-SPIONs with uniform particle size,good stability,and non-neuronal cytotoxicity were prepared by the chemical coprecipitation method in this study.UFH-SPIONs were confirmed to play an antagonistic role against PA-induced neuronal lipid toxicity by regulating three pathways of energy metabolism disorder,oxidative stress,and cellular activity.These results indicate that the potential application of UFH-SPIONs for diseases in the presence of lipotoxic damage of neurons,such as diabetic encephalopathy,and senile dementia deserves further investigation.