| Objective:The current thesis aimed to investigate the protective effect of Crocetin?CRT?on PC12 cells lesion induced by Amyloid beta-peptide?25-35?(A?25-35),and to unravel its possible mechanisms.Methods:?1?The geniposide and crocin-1 were separated and purified from gardenia fruit by macroporous adsorption resin method and silica gel column separation method,and crocetin was prepared by hydrolysis reaction of total crocins from gardenia lutein as raw material.?2?CCK-8 method was used to determine the effect of 0,2,5,10,20,30 and40 mol/L of A?25-35 on the viability of PC12 cells,and to determine the concentration of cell lesion model.?3?To detect the protective effect of geniposide,crocin-1 and crocetin on lesion of PC12 damaged cells induced by A?25-35.?4?PC12 cells were divided into blank group?Control?,Model group(Model,20?mol/L A?25-35),50?mol/L dose of crocetin(CRT-50?mol/L+20?mol/L A?25-35),and 100?mol/L A?25-35 dose of crocetin(CRT-100?mol/L A?25-35+20?mol/L A?25-35).CCK-8 method was used to detect the effect of CRT on the survival rate of PC12 cells challenged by A?25-35.?5?fluorescent microscope was employed to observe the effect of CRT on mitochondrial membrane potential?MMP?of PC12 cells induced by A?25-35.Flow cytometry was used to detect the effect of CRT on the apoptosis of PC12 cells induced by A?25-35.Cell immunofluorescence and Flow Cytometry were used to detect the distribution and expression of autophagy marker protein LC3B in PC12 cells induced by CRT to A?25-35.Western blotting was used to determine the expression levels of CRT on apoptosis-related proteins b-lymphoma cell-2?Bcl-2?,b-lymphokine-related X protein?Bax?,and caspase-3 protein?caspase-3?in PC12 cells induced by A?25-35.Expression levels of autophagy-related proteins beclin-1,light chain3B?LC3B?,SQSTM1/P62 and autophagy-related pathway proteins m-tor,p-mtor,p 70 S6kinase,and p-p 70 S6 kinase were measured.Results:?1?Through chromatographic identification,it was found that the purity of samples of geniposide,crocin-1 and crocetin purified in this study reached the standard and could be used in subsequent experiments.?2?The protective effect of crocetin on PC12cells induced by A?25-35 was stronger than that of geniposide and crocin-1.?3?Compared with the blank group,cell viability in the model group was significantly reduced?P<0.01?,apoptosis rate was significantly increased?P<0.01?,intracellular MMP was significantly reduced,and autophagy protein LC3B expression was increased.Mitochondrial apoptosis pathway protein Bcl-2 was decreased?P<0.05?,the expressions of Bax protein and caspase-3 protein were increased?P<0.05?,the expressions of autophagy protein LC3B and SQSTM1/P62 were increased?P<0.05?,and the expression of autophagy protein beclin-1 was decreased?P<0.05?.Pathway proteins p-mTOR and p-p70 S6 Kinase decreased?P<0.05?,while m-TOR and p70 S6 Kinase increased?P<0.05?.Compared with the model group,the effective dose of CRT can significantly improve the cell viability and apoptosis rate.Cell viability was significantly increased?P<0.01?,apoptosis rate significantly decreased?P<0.01?,LC3B protein expression was decreased,apoptosis-related protein Bcl-2 protein expression was significantly increased?P<0.05?,and Bax protein and caspase3 protein expression was significantly decreased?P<0.01?.The increased MMP in the cell was increased.The protein expression of p-mTOR and p-p70 S6 Kinase increased?P<0.05?.Reduced expression of mTOR,p70 S6 Kinase?P<0.05?.Conclusion:?1?CRT can improve the damage of A?25-35 to PC12 cells and improve the survival rate of PC12 cells.?2?the effective dose of CRT can significantly inhibit the apoptosis of PC12 cells induced by A?25-35,activate autophagy pathway,and improve cell damage.Considering that crocin is mainly converted to crocetin in the body,the above research results show that crocin has the potential for the development of nervous system drugs,and support the empirical use of saffron and gardenia in the treatment and prevention of neuro-related diseases. |
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