| Royal jelly(RJ)is a milky or yellowish,slightly sweet and tart,milky substance secreted by the subpharyngeal and mandibular glands of the head of 5-15 day worker bees.RJ is rich in protein,carbohydrates,lipids,mineral salts and vitamins.More and more studies have shown that the active substances in natural food can interact with cell proteins,nucleic acids and lipids through chemical modification or physical binding to regulate human health.Alzheimer’s disease is the most common type of dementia and has a huge impact on the public health of the society and the national economy.Aging is the main cause of Alzheimer’s disease;therefore,with the serious tendency of aging population and the increasing number of patients,Alzheimer’s disease has triggered widespread attention.SH-SY5 Y is a neuroblastoma cell from the metastasis of bone tumor,which has the morphological characteristics of neurons and is widely used in the study of the pathogenesis of nervous system diseases and the mechanism of drug action.Moreover,tau protein,protein kinase and protein phosphatase that regulate the phosphorylation of tau protein are high in content.Okadaic acid(OA)is a kind of protein phosphatase inhibitor,have been used to establish the model of Alzheimer’s disease,it can improve the degree of phosphorylation of microtubular-related tau protein and cause oxidative damage to cells.Hyperphosphorylation of tau is a major component of double helix fibers in the brains of Alzheimer’s patients and large amounts of aggregation and precipitation can form nerve fiber tangles.Therefore,the injury of SH-SY5 Y cells by OA can be used to establish the cell model of Alzheimer’s disease.The pathological causes of Alzheimer’s disease are complicated,and are considered to be related to neurofibrillary tangles,amyloid-β plaques,inflammation and oxidative stress.Royal jelly has the functions of anti-inflammation,anti-oxidation,regulating blood lipid and so on;thus,long-term use of royal jelly may have positive effects on the prevention and treatment of Alzheimer’s disease.The main research contents and results of this paper are as follows:(1)Selection of royal jelly species: SH-SY5 Y cells were injured by 20 nmol/L OA for 24 h,culture with different concentrations(10、5、4、2、1、0.5、0.25、0.125、0.0625 mg/mL,31.25、15.625、7.8125、3.91、1.95、0.977、0.488、0.244、0.122、0.061、0.0305、0.015 μg/mL)of vitex royal jelly solution 1 and vitex royal jelly solution 2 for 24 h,48 h and 72 h.The cell viability was detected,and it was found that the two royal jelly solutions had the same trend of influence on the cell viability within the range of test concentration.According to the OD value,the result of vitex royal jelly solution 1 was slightly higher than that of vitex royal jelly solution 2,so vitex royal jelly 1 was selected for the subsequent test.After data analysis,there was a significant difference in cell viability between 1 mg /mL and 10 mg/mL of royal jelly compared with the control group,so the concentration range of royal jelly in subsequent experiments will include this concentration range for further experiments.(2)Selection of OA concentration and processing time: SH-SY5 Y cells were cultured with OA solution of different concentrations(30、40、50、60、70 nmol/L)for 12 h and 24 h,cell viability was then measured.The results showed that the cell viability of SH-SY5 Y cells injured by OA at different concentrations for 24 h was significantly lower than that of the control group,and the cell viability was the lowest at the OA concentration of 40 nmol/L.The results showed that the effect of 40 nmol/L OA on SH-SY5 Y cells was the best after 24 h.(3)The antioxidant effect of royal jelly on the OA-induced SH-SY5 Y cell model of alzheimer’s disease: SOD,MDA and ROS were used to evaluate the antioxidant effect of royal jelly on the OA-induced SH-SY5 Y cell model of Alzheimer’s disease.SH-SY5 Y cells were cultured,and the appropriate density of SH-SY5 Y cells were selected for slab-laying.After 24 h in an incubator,cells were damaged with 40 nmol/LOA for 24 h,followed by 24 h,48 h and 72 h incubation with royal jelly of different concentrations.The cells were collected,and the contents of SOD,MDA and ROS were detected with related kits and compared with the control group.The results showed that within the range of test concentration,SOD content in cells treated with royal jelly at the concentration of 2 mg/mL for 48 h was significantly different from that in the control group;MDA content was inversely proportional to the use concentration of royal jelly,decreased with the increase of the use concentration of royal jelly,and the effective time and concentration range are wide;after 48 h,low concentration of royal jelly will reduce the ROS content.These results show that royal jelly has a good antioxidant effect on the SH-SY5 Y cell model of Alzheimer’s disease.(4)The effect of royal jelly on the apoptosis of SH-SY5 Y cell model of Alzheimer’s disease induced by OA: SH-SY5 Y cells were cultured,and the appropriate density of SH-SY5 Y cells were selected for slab-laying.After 24 h in an incubator,cells were damaged with 40 nmol/L OA for 24 h,followed by 24 h,48 h and 72 h incubation with royal jelly of different concentrations.The cells were collected,aoptosis was detected by flow cytometry using AV/PI apoptosis detection kit.The results showed that all cells were in the stage of apoptosis after OA injury,but the royal jelly of 0.125-8 mg/mL cultured 24 h,the royal jelly of 0.125-1 mg/mL cultured 48 h,the royal jelly of 0.125 mg/mL cultured 72 h could increase the proportion of cells in the early apoptosis stage and decrease the proportion in the late apoptosis stage,which could delay the further cell membrane damag and cell death.(5)The study of royal jelly on the genes expression related to the SH-SY5 Y cell model of Alzheimer’s disease induced by OA: The SH-SY5 Y cells damaged by 40 nmol/L OA were incubated with royal jelly solution of 2 mg/mL for 24 h.RNA was extracted from the cells and reversely transcribed into cDNA.The expressions of late onset risk factors CLU,PICALM,early onset risk factors SORL1,amyloid plaque formation factor RELN,and related genes of CSNK1 D and DPYSL2 were analyzed with the help of fluorescence quantitative PCR.The results showed that the expressions of CSNK1 D and PICALM genes were up-regulated,the expressions of DPYSL2 and RELN genes were down-regulated,and there was no significant difference in the expressions of the genes of CLU and SORL1 compared with the control group.Therefore,royal jelly may protect damaged nerve cells by stimulating the cells to produce casein kinase I to enhance the cells’ self-repair ability,increase endocytosis to maintain cell homeostasis,reduce the amount of protein that can be further phosphorylated by reducing the production of proteins associated with DPYSL2 and reduce the secretion of extracellular matrix proteins. |
PDF Full Text Request