Study On Effect Of Royal Jelly On The Proliferation,Anti-oxidative Stress Of Mammalian Cells And Its Mechanism

Royal jelly（RJ）is a secretion of worker bees pharyngeal gland of Chinese bee（Apis cerana cerana Fabricius）and Italian bee（Apis mellifera ligustica）.Its composition is very complex.It is used as a kind of natural nutrients.There have been research reports on its antitumor and anti-aging effect in vivo.There have been few research reports on effect of RJ on normal mammalian cells or tumor cells in vitro.It has only been found a research on anti-senescence effect of main RJ protein on human embryonic lung fibroblast（HFL-I）cell line.In this research,we try to explore the effect of RJ on proliferation of Hela and HepG2 cells,and anti-oxidative stress on NIH-3T3 cell and its mechanism.1.Effect of RJ on the proliferation and apoptosis of Hela and HepG2 cellsIn this study,CCK-8 was used to detect the inhibition of different concentrations of cisplatin on HepG2 and Hela cells.According to the result,50μg/mL of cisplatin was selected as the control.The inhibition of different concentrations of royal jelly（1、5、10、20、40、60、8、100μg/mL）on HepG2 and Hela cells were tested with CCK-8.The results showed that the inhibitions of 10μg/mL of RJ on Hela and 25μg/mL of RJ on HepG2 are about 55%and 45%respectively.The inhibitions of higher or lower concentration of RJ on these cells are lower.The inhibitions are significantly lower than that of cisplatin,which is about 80%.2.Anti-oxidative stress effect of royal jelly（RJ）on NIH-3T3 cellsIn this study,the effects of different concentrations of H₂O₂ on the activity of mouse embryonic fibroblasts（NIH-3T3）were tested with CCK-8.According to the result,20μg/mL of H₂O₂ was selected to establish a cell oxidative stress model of NIH-3T3.The effects of different concentrations of RJ on proliferation of model cell were detected with CCK-8,and morphological change of the cells were observed.The result showed that 5,10 and 20μg/mL of RJ have promotion on proliferation of model cells at different times.Therefore,The effects of the three concentration’s RJ on passage time interval,life-span and aging degree（β-galactose glucoside enzyme positive cells rate）of model cells were tested.The results showed that the passage time interval of 5,10 and 20μg/mL of RJ are significantly decreased,and the 10μg/mL group is the most decreased.It means that RJ can reverse the slowing down of cell growth caused by H₂O_2.It is found that the cell life-spans（PDLs）of the RJ group（10,20μg/mL）were prolonged.The results of SA-β-gal staining showed that RJ could improve the oxidative stress senescence induced by H₂O_2.These effects were the most obvious at 10μg/mL.3.Study on the anti-oxidative stress mechanism of royal jelly（RJ）on NIH-3T3 cellsIn this study,the model cells established with H₂O₂ were treated with RJ.The apoptosis of the cells was observed with Hoechst33342/PI double staining.The activity of glutathione peroxidase（GSH-Px）of the cells was tested.The life cycle of the cells was observed with flow cytometry.The mRNAs of p16,p21 and p53 gene were quantified with real-time fluorescence quantitative polymerase chain reaction（qRT-PCR）.The result showed that RJ can inhibit cell apoptosis caused by H₂O_2,but have little effect on necrosis caused by H₂O_2.RJ can enhance the activity of GSH-Px.It can enhance the ability to decompose peroxides,and protect cells from oxidative stress.The results of life cycle of the cells showed that H₂O₂ can reduce the S phase of cells significantly,retard the cells in the G1 phase.RJ can significantly increase cells in the S phase.It means that RJ can improve the G1 retardant of cells induced by H₂O₂,let more cells to enter the S phase,and promote the cell division.RJ could reduce the expression of mRNA of p16,p21 and p53 in the model cells.It means that improvement of RJ on G1 retardant of model is relevant to inhibiting express of p16,p21 and p53 before translation,regulating the life cycle of the cells by p16-cyclinD/CDK-RB、p53/P21 pathway...