| With the change of lifestyle,the proportion of glycolipids in the dietary structure of our population has increased,coupled with poor lifestyle and mental stress,people are increasingly vulnerable to diseases related to metabolic disorders,and lipotoxicity due to disorders of lipid metabolism in the body has received more attention.Lipotoxicity refers to the ectopic deposition of free fatty acids(FFAs)and the subsequent oxidative stress damage.It has been found that patients with non-alcoholic fatty liver disease,type 2 diabetes mellitus(T2DM),hyperlipidemia,hyperthyroidism,and cardiovascular disease have abnormally elevated lipid levels.Lipid metabolism and glucose metabolism interact with each other and together determine whether the body has an adequate energy supply.Insulin resistance due to disorders of lipid metabolism can reduce the uptake and utilization of glucose by cells.In studies of neurodegenerative diseases such as Alzheimer’s disease,it has been found that reduced glucose uptake and metabolism can lead to a shortage of brain energy supply,resulting in brain cell dysfunction,toxic protein accumulation,and ultimately cognitive decline and neuropsychiatric symptoms.Improving blood lipid levels,reducing lipotoxic damage,and increasing the ability of body cells to take up glucose are important for the treatment of such diseases.The application of lipid-lowering drugs supplemented with a proper diet is currently a common treatment option to improve blood lipid levels.Widely used lipid-lowering drugs include statins,fibrates,niacin,etc.,which mainly regulate cholesterol,high and low density lipoprotein and triglyceride levels in the blood,while weakly targeting other lipid components such as fatty acids,ceramides,and triglycerides,etc.Increased levels of FFAs are the most important factor in the development of lipotoxicity.There is a clinical need for safe and effective drugs to improve FFAs levels in order to curb lipotoxicity.Cyclodextrin(CDs)is a safe and stable cyclic oligosaccharide compound with the physical property of external hydrophilicity and internal hydrophobicity.They are used in medicine,food,agriculture,textile and other fields.In the pharmaceutical field,they are mostly used as pharmaceutical excipients,which can increase the solubility of drugs,mask the bad odor of drugs,reduce the reaction of gastrointestinal or skin to irritating drugs,etc.In recent years,the potential of cyclodextrin as a drug for disease treatment has been gradually discovered.HP-βCD can be used to treat Niemann-Pick type C disease by intravenous injection,and ycyclodextrin derivatives can be used as antagonists in combination with aminosteroid myorelaxants to reverse profound and moderate muscle relaxation states.Previous studies by our group have shown that the hydrophobic cavities of cyclodextrins can encapsulate lipid-like A chains of lipopolysaccharides and exert anti-sepsis effects.In this study,we investigated the use of the hydrophobic cavity of cyclodextrin to encapsulate lipids to reduce the toxic effect of palmitic acid,a major saturated fatty acid in blood lipids,on HepG2 cells and improve the growth status of hepatocytes.The results and main conclusions obtained in this topic are:1 Binding of cyclodextrins to palmitic acid(PA)The in vitro interactions of α-CD,β-CD,γ-CD,HP-β-CD and cation-CD with palmitic acid were investigated using the competition of palmitic acid and Alizarin Red S(ARS)for the hydrophobic cavity of cyclodextrins.The strongest fluorescence was observed in the absence of palmitic acid,and the fluorescence intensity decreased after the addition of different concentrations of palmitic acid.β-CD was the strongest binding effect with palmitic acid among the five cyclodextrins,followed by γ-CD and α-CD.In view of the high risk of hemolysis of βCD,the safer and more stable γ-CD was selected for the next study.2 Cyclodextrin-inclusive palmitic acid action assay2.1 Thermogravimetric analysis(TGA)Palmitic acid and γ-CD 1:1 inclusion and 1:10 inclusion were prepared by freeze-drying method,and 1:1 physical mixture was prepared in molar ratio.They were subjected to TGA in the range of room temperature to 300℃,respectively.As seen by the thermogravimetric curves,the different ratios of the inclusion complexes did not show any obvious peak in the rate of thermal weight loss(DTG),showing a characteristic thermogravimetric curve different from the rest of each sample,which exhibited the thermodynamic properties of the new substances.2.2 Differential scanning calorimetry(DSC)The same samples were prepared according to the TGA method and then subjected to DSC in the range of room temperature to 300℃.The DSC curves of each group showed that the two exothermic peaks in the 1:1 inclusion group were significantly different from those in the palmitic acid and γ-CD groups,indicating the formation of new substances.while the large broad peak in the 1:10 inclusion group at 88.25℃ was similar to that of γ-CD.In the DSC plot of the 1:1 physical mixture of palmitic acid and γ-CD,the peak characteristics of palmitic acid and γ-CD can be clearly seen,indicating that the physical mixture alone did not change the physical properties of both.2.3 Scanning electron microscope(SEM)observationThe samples of each group were scanned using SEM and photographed at 200 × and 2000 ×magnification,respectively.It was observed that the morphological characteristics of the groups differed significantly under 200× magnification.Under 2000× magnification,the palmitic acid samples showed irregular rocky masses,while γ-CD showed a crystalline structure with smooth surface and sharp edges.1:1 physical mixture showed similar characteristics to the palmitic acid group.1:1 inclusion group samples showed obvious scale-like shape and pore-like cavities could be seen,and their structural characteristics were significantly different from those of the other groups,thus indicating that the production of new substances after the inclusion of palmitic acid with cyclodextrin..The above experiments indicate that γ-CD and palmitic acid can undergo binding to form new inclusion complexes.3 Effect of γ-CD on palmitic acid-induced lipid apoptosis in hepatocytes3.1 Effect of γ-CD and palmitic acid on hepatocyte activity separately and after encapsulationThe effects of γ-CD on HepG2 cell activity at 24h and 48h were investigated by CCK-8 assay.The results showed that the cells maintained more than 80%activity after applying 6.25 mmol/L γ-CD intervention for 48h.The toxic effect concentration of palmitic acid was still investigated by CCK-8 assay,and the results showed that intervention with 400 μmol/L palmitic acid for 24 h resulted in a significant decrease in cell activity.When palmitic acid was encapsulated with γ-CD and applied to HepG2 cells,the high dose group significantly increased the activity of HepG2 cells(P<0.05).The above experiments indicate that γ-CD encapsulated with palmitic acid can significantly reduce the lipotoxicity produced by saturated fatty acids.3.2 Effects of γ-CD and palmitic acid on apoptosis of hepatocytes separately and after encapsulationThe expression of intracellular Bcl-2-Associated X(Bax)and B-cell lymphoma-2(Bcl-2)proteins was detected by enzyme linked immunosorbent assay(ELISA),and the results showed that palmitic acid group Compared with the control group,the content of the pro-apoptotic protein Bax was significantly higher(P<0.01);the content of Bax was significantly lower(P<0.05)in both the low.medium and high dose groups of γ-CD.The anti-apoptotic protein Bcl-2 content in each group showed no significant changes between groups(P>0.05).The expression of Cysteinyl aspartate-specific proteinase-3(Caspase-3)was detected by immunohistochemistry in each group.The red fluorescence was significantly stronger in the palmitic acid group than in all other groups,while the application of γ-CD-coated palmitic acid resulted in a significantly lower fluorescence intensity in the medium-high dose group than in the palmitic acid group.4 Effects of γ-CD on palmitic acid-induced lipid accumulation and oxidative stress in hepatocytes4.1 Effect of γ-CD on palmitic acid-induced lipid accumulation in hepatocytesIntracellular lipid droplets were observed by oil red O staining,and the formation of lipid droplets could visually reflect lipid accumulation.When HepG2 cells were stained with 400μmol/L palmitic acid for 48 h,the palmitic acid group showed obvious reddish-brown round lipid droplets,indicating the formation of lipid deposition.After encapsulation of palmitic acid with γ-CD,all groups showed a decrease in lipid droplet content.For further accurate quantitative analysis of intracellular lipids,intracellular total cholesterol(TC)and triglyceride(TG)contents were measured,and it was found that the TC content in the palmitic acid group was significantly increased compared with the control group(P<0.05),and after the application of γ-CD,the TC content in all groups was reduced,but there was no significant difference(P>0.05).The TG content was significantly higher in the palmitic acid group compared with the control group(P<0.01),and there was a significant decrease in all dose groups of γ-CD compared with the palmitic acid group(P<0.05),and the difference was more significant in the high dose group(P<0.01).4.2 Effect of γ-CD on palmitic acid-induced oxidative stress in hepatocytesThe intracellular malondialdehyde(MDA)content was measured by thiobarbituric acid method,and the results showed that the MDA content increased significantly in palmitic acid group(P<0.01),and decreased significantly in the middle and high dose groups after adding yCD(P<0.01).Detection of reactive oxygen species(ROS)content with the fluorescent probe DCFH-DA showed a significant increase in ROS content in the palmitic acid group compared to the control group(P<0.05),which did not show significant differences in the low and medium dose groups after the application of γ-CD(P>0.05),and a significant decrease in ROS in the high dose group(P<0.05).Intracellular Superoxide dismutase(SOD)activity was detected by applying xanthine oxidation method.The results showed that the SOD enzyme activity was significantly lower in the palmitic acid group(P<0.01),and the SOD enzyme activity tended to increase in all dosing groups,showing significant differences only in the high-dose group(P<0.05).ELISA was performed to detect the level of tumour necrosis factor-α(TNF-α)in the cell supernatant.The results showed that the content of inflammatory factor TNF-α was significantly higher in the palmitic acid group(P<0.05),and the content of TNF-α decreased in the groups with the addition of γ-CD,and a significant difference was observed in the middle and high dose groups compared to the palmitic acid group(P<0.05).4.3 Effect of γ-CD on palmitic acid induced endoplasmic reticulum stress(ERS)The expression of intracellular protein kinase R-like endoplasmic reticulum kinase(PERK)and Activating Transcription Factor 4(ATF4)were labeled by immunofluorescence.The results showed that the expression of PERK and ATF4 was enhanced in the palmitic acid group compared to the control group,while the addition of different doses of γ-CD significantly weakened these two indicators of ERS.This part of the results indicates that palmitic acid intervention in HepG2 cells can produce significant lipid deposition and lipotoxicity,including the imbalance of intracellular redox system,the release of inflammatory factor TNF-α and ERS;the application of γ-CD-coated palmitic acid improved all of these indicators,indicating that γ-CD-coated palmitic acid can reduce the series of oxidative stress and inflammatory responses caused by palmitic acid.5 Effect of γ-CD on palmitic acid-induced hepatocyte membrane fluidity and glucose metabolism5.1 Effect of γ-CD encapsulated palmitic acid on fatty acid exchange and uptake in hepatocyte membranesThe effect of γ-CD co-acting with FITC-palmitic acid on cells was first photographed by laser copolymer microscopy and then analyzed by Image J processing.There was no significant fluorescence in the control group,a stronger fluorescence in the palmitic acid group,and the medium to high dose γ-CD group presented a significant decrease in the mean fluorescence intensity(P<0.05).It indicates that γ-CD blocked the uptake of palmitic acid by the cells.5.2 Detection of hepatocyte membrane fluidityFluorescence recovery after photobleaching(FRAP)was applied to detect the fluidity of the cell membrane.and the fluorescence recovery rate(M)was used to express the membrane fluidity.Data processing and analysis showed that palmitic acid significantly reduced membrane fluidity(P<0.01),and the application of γ-CD-coated palmitic acid showed some improvement in membrane fluidity,and significant differences were observed in the medium and high dose groups compared with the palmitic acid group(P<0.05,P<0.01).5.3 Effect of γ-CD-coated palmitic acid on hepatocyte response to sugar metabolism5.3.1 Hepatocyte sugar uptake assay and glucose consumption assay in cell culture medium2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy glucose(2-(N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose,2-NBDG)is a fluorescent glucose analogue and was assayed by flow analyzer uptake of 2-NBDG by cells.In the experiments without insulin,the palmitic acid group showed significantly lower uptake of 2-NBDG compared to the control group(P<0.05),while the medium-dose γ-CD significantly improved this situation(P<0.05)and the high-dose group showed increased uptake.The overall 2-NBDG uptake was increased in the experimental groups with insulin compared to those without insulin,indicating that insulin promoted the cellular utilization of sugar.Sugar uptake was significantly lower in the palmitic acid group compared to the control group(P<0.01),and cellular uptake of 2-NBDG was significantly higher in each group after application of γ-CD-coated palmitic acid(P<0.05,P<0.01).Glucose consumption experiments revealed that sugar consumption was significantly reduced in the palmitic acid group compared to normal cells(P<0.01),and γ-CD did not appear significantly elevated in the low and medium dose groups compared to the palmitic acid group(P>0.05),and was elevated significantly in the high dose group(P<0.01).Both results suggest that palmitic acid acting on hepatocytes can inhibit the uptake of glucose by hepatocytes.5.3.2 Assay of hepatocyte ATP contentThe intracellular ATP was significantly lower in the palmitic acid group than in the control group(P<0.05).There was a trend of elevation in all dose groups of applied γ-CD,with significant differences in the medium and high dose groups compared to the palmitic acid group(P<0.05).This part of the results shows that palmitic acid acting on hepatocytes decreases the fluidity of cell membranes,which impedes the membrane related sugar uptake and utilization,thus putting hepatocytes in a low energy metabolic state.The application of γ-CD-coated palmitic acid not only improved the membrane fluidity to some extent,but also increased the sugar uptake and utilization by hepatocytes,restoring the normal energy metabolism of hepatocytes.6 ConclusionThis study confirmed that γ-CD can encapsulate palmitic acid,and through this action,it can attenuate or block palmitic acid-induced lipotoxicity in hepatocytes,including lipid apoptosis,oxidative stress,endoplasmic reticulum stress,decreased cell membrane fluidity and concomitant impaired glucose metabolism,showing that γ-CD has the potential to be developed as a new lipid-lowering drug targeting free fatty acids. |
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