Preliminary Study On The Dose-effect Relationship And Mechanism Of 5-hydroxymethylfurfural On PC12 Cells And Caenorhabditis Elegans

5-hydroxymethylfurfural（5-HMF）is an intermediate product produced by the Maillard reaction of sugary foods.It is found at different levels in different types of food.It is found at low levels in fresh,unprocessed foods;the amount of 5-HMF in dairy products varies depending on the amount of sugar and the processing methods;the content in liquor and juice is 0.2 g/L;coffee contains more than 1.0 g/kg of 5-HMF.In daily life,baked foods such as bread and coffee are one of the main ways for the human body to ingest 5-HMF.Previously our laboratory found that a certain concentration of 5-HMF has a negative effect on the nervous system of zebrafish,but the mechanism and the dose-effect relationship remain unclear.Therefore,on this basis,this study selected the rat adrenal medullary pheochromocytoma cell line PC12 cells with neuron-like function and morphology,and the model organism Caenorhabditis elegans（C.elegans）,which is often used to predict the development of mammals,to investigate the effects of different concentrations of 5-HMF on the body,as well as the relevant dose-effect relationship,and to initially explore its pathway of action.Firstly,the bidirectional modulatory effects of 5-HMF on PC12 cells were studied in vitro.The influence of 5-HMF in the concentration range of 0.03125 mg/m L to 3mg/m L on the activity of PC12 cells was determined by Cell counting Kit-8（CCK-8）Kit,and the dose-effect relationship fitting curve between concentration of 5-HMF and survival rate of PC12 cells was established.The results showed that the cell activity increases significantly when the concentration of 5-HMF is lower than 0.0625 mg/m L.When the concentration is in the range of 0.125 mg/m L to 0.5 mg/m L,there is no significant difference in cell activity compared with the control group.When the concentration is higher than 1 mg/m L,the cell activity shows a significant downward trend.By the fitting equation,the maximum non-lethal concentration（MNLC）and semi-inhibitory concentration(IC₅₀)of 5-HMF on PC12 cells were calculated to be about 0.392 mg/m L and 2.314 mg/m L,respectively.Secondly,according to the results of CCK above,based on the concentration of5-HMF,the experimental groups were set as low concentration group（0.0625 mg/m L）,medium concentration group（0.5 mg/m L）and high concentration group（2 mg/m L,2.5mg/m L,3 mg/m L）in subsequent experiments,in order to explore the bidirectional modulatory effects of different concentrations of 5-HMF on oxidative stress and apoptosis-related indicators of PC12 cells.The morphological changes of cells were observed by a microscope;reactive oxygen species（ROS）,malondialdehyde（MDA）,superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）were determined by a microplate reader;cell apoptosis was analyzed by a flow cytometry;andreal time fluorescence quantitative polymerase chain reaction（qRT PCR）was used to detect apoptosis-related genes:deacetylated apoptosis factor（p53）,B-cell lymphoma 2（Bcl-2）,apoptosis promoting genes（Bax and Bim）in the Bcl-2 gene family,important apoptosis inhibiting genes（Bcl-xl）in the Bcl-2 family,and cysteine protease 3（Caspase-3）,oxidative stress-related signaling pathway Keap-1/Nrf2-related genes:Nuclear factor erythroid 2 related factor 2（Nrf2）,Kelch-like Ech-associated protein-1（Keap-1）,Heme oxygenase 1（HO-1）,NAD（P）H:quinone oxidoreductase 1（NQO1）,and axon-oriented genes:transmembrane protein receptor（Roundabout 1,Robo1）and its ligand gene Slit2.The results showed that low concentration of 5-HMF（0.0625mg/m L）could maintain the normal morphology of cells,reduce the oxidative stress level of cells,up-regulate the expression of anti-apoptotic genes and down-regulate the expression of pro-apoptotic genes,so as to improve cell activity and inhibit cell apoptosis;on the contrary,high concentration of 5-HMF（2 mg/m L,2.5 mg/m L,3mg/m L）could cause the cells to shrink and beome round,and cause the pseudopods to disappeare and no longer extend to the periphery,so as to damage the cells,enhance the oxidative stress level of cells,downregulate the expression of anti-apoptotic genes and up-regulate the expression of pro-apoptotic genes,so as to reduce the cell activity and promote cell apoptosis.Thirdly,the in vivo model of Caenorhabditis elegans was selected for study.The effects of different concentration groups of 5-HMF on life span,oviposition and basic movement index of Cryptostrongylus elegans were studied,and the dose-effect relationship between different concentrations of 5-HMF and life span was determined by nonlinear curve fitting.It was found that the high concentration group（1 mg/m L）and medium concentration group（0.1 mg/m L）of 5-HMF could significantly shorten the life cycle of C.elegans（P<0.05）,while the low concentration group（0.01 mg/m L）of 5-HMF could prolong the life cycle of C.elegans（P<0.05）.The oviposition of nematodes was studied,and it was found that 5-HMF in low concentration group has no significant effect on the number of oviposition,but when the concentration increases to a medium or a high level,the number of oviposition of nematodes is reduced to varying degrees.The basic physiological indexes such as pharyngeal pump frequency,head swing and body bending were measured.It was found that 5-HMF in high and medium concentration groups has significant effects on these indexes（P<0.05）,but there is no statistical significance in low concentration group（P>0.05）.According to the expression of the dose-effect relationship of average longest life span and longest life span,,it can be seen that 0.035 mg/m L and 0.056 mg/m L of 5-HMF can be used as the critical concentration value of average longest life span and longest life span of C.elegans,that is,when the concentration of 5-HMF is less than 0.035 mg/m L,the average longest life span of C.elegans can be extended,when the concentration of5-HMF is less than 0.056 mg/m L,the longest life span of C.elegans can be extended.According to the life curve of C.elegans,the median survival time（the time required for the cumulative survival of 50%of C.elegans）changes significantly after treatment with 5-HMF in different concentration groups.Compared with the control group,the median survival time in the low concentration group increases from 11.0±0.7 days to13.0±0.8 days.In the medium and high concentration groups,the value is shortened to5.0±0.5 days and 3.0±0.4 days,respectively.Finally,qRT-PCR was used to determine the related gene expression of the signaling pathway related to the lifespan of C.elegans,insulin/insulin-like growth factor signaling pathway（IIS）.It was found that 5-HMF in low concentration group could significantly reduce the expression level of Insulin receptor like gene（Daf-2）,and increase the expression level of nuclear transcription factor（Daf-16）,superoxide dismutase 3（Sod-3）and yeast homologous genes（Sir-2.1）.（P<0.05）;in high and medium concentration groups,5-HMF significantly increases the expression of Daf-2gene,and decreases the expression of Daf-16,Sod-3 and Sir-2.1 genes（P<0.05）,thusly affecting the life cycle of C.elegans.The dose-effect relationship analysis of the expression results of the above four genes showed that the critical concentrations of5-HMF bidirectional regulation the expression levels of Daf-2,Daf-16,Sod-3 and Sir-2.1 genes are 0.067 mg/m L,0.067 mg/m L,0.075 mg/m L and 0.052 mg/m L,respectively.In conclusion,the positive and negative effects of 5-HMF on PC12 cells in vitro are closely related to the concentration of 5-HMF,with bidirectional regulatory effect and there is a dose-effect relationship(MNLC=0.392 mg/m L,IC₅₀=2.314 mg/m L).The bidirectional modulatory effects of 5-HMF on experimental model Caenorhabditis elegans in vivo are closely related to the concentration of 5-HMF,and the critical concentration of 5-HMF affecting the average longest life span and longest life span of C.elegans is 0.035 mg/m L and 0.056 mg/m L.This result is combined with the results of the critical concentrations（0.067 mg/m L,0.067 mg/m L,0.075 mg/m L and 0.052mg/m L,respectively）affecting four life related genes of nematode Daf-2,Daf-16,Sod-3 and Sir-2.1,which can preliminarily calculate the critical concentrations affecting the life of nematode between 0.01 mg/m L and 0.1 mg/m L.The research results and conclusions of this topic provide a preliminary reference for the further study on the safety of 5-HMF,and provide a theoretical basis for toxicological analysis and safe application of 5-HMF.