| Objective: The protective effect of Salidroside（SAL）on anoxic GC-1 cells and its mechanism were investigated by constructing oxidative stress damage model of GC-1 cells in vitro.Methods: 1.GC-1 cells were treated under simulated hypoxia conditions to observe the effect of hypoxia on GC-1 cells.CCK-8 method and Hoechst 33342 staining method were used to screen the optimal molding time.2.Observe the expression of mitochondrial signaling pathway in GC-1 cells under simulated hypoxia conditions.Control group and experimental group were set;reactive oxygen species（ROS）,malondialdehyde（MDA）,lactate dehydrogenase（LDH）,total anti-oxidant capacity（T-AOC）and super oxide dismutase（SOD）were measured in hypoxic GC-1cells,in addition,mitochondrial membrane potential,mitochondrial staining,mitochondrial adenosine triphosphate（ATP）and Cystein-asparate protease-3（Caspase-3）Cytochrome（Cytochrome C,Cyt-C）gene transcription level were detected to investigate the effect of hypoxia on mitochondrial signaling pathway of GC-1 cells.3.GC-1 cells were treated with different concentrations of SAL（15,10,15,20 μM）under simulated hypoxia conditions,Three concentrations of salidroside in low dose group,medium dose group and high dose group were screened by CCK-8method.Control group,model group,salidroside experimental group（SAL1μM group,SAL5μM group,SAL10μM group）were set up.Changes of ROS,MDA,LDH,T-AOC and SOD were used to evaluate the oxidative stress level,mitochondrial membrane potential,mitochondrial staining and mitochondrial ATP were used to evaluate mitochondrial function,expression levels of Caspase-3,Cyt-C,Bcl-2 and Bax were detected,fluorescence intensity of Cleaved Caspase-3 was detected by immunofluorescence method,and apoptosis rate was detected by flow cytometry.Statistical analysis: The experimental data were statistically analyzed by SPSS 25.0data analysis software,and Graphpad Prism 9 was used for graphing.The measure ment data conforming to normal distribution were expressed as mean ± standard deviation（（?）±S）,and comparisons between multiple groups were made by One-Way-Anova analysis,and post hoc tests were conducted by LSD test.Two groups were compared using the t-test.Spearman bivariate correlation analysis was used for correlation analysis.Differences were considered statistically significant at P<0.05.Results: 1.With the extension of hypoxia time,the number of apoptosis of GC-1cells gradually increased,which was consistent with the results of Hoechst 33342 staining,and there was no significant change in cell viability after 24 hours of hypoxia（P>0.05）,cell viability decreased after 30 h of hypoxia（P<0.05）,the viability of GC-1 cells decreased further after 36 h of hypoxia（P<0.01）,cell viability decreased after 48 h of hypoxia（P<0.001）,the anoxia duration was negatively correlated with the viability of GC-1 cells（r =-0.857,P<0.001）.2.Under hypoxic conditions for 36 hours,the levels of ROS,MDA and LDH were increased（P<0.01）and the levels of T-AOC and SOD activity were decreased（P<0.01）in the experimental group compared with the control group.under hypoxic conditions,the mitochondrial membrane potential decreased,the distribution of intracellular mitochondria was altered,their number decreased and the ATP content decreased in the experimental group（P<0.01）;RT-PCR results showed that the expression levels of Caspase-3 and Cyt-C genes were increased in the experimental group（P<0.01）;3.Results of the protective effect of rhodiogenin on hypoxic GC-1 cells under simulated hypoxic conditions: 3.1.Compared with the control group,cell viability decreased（P<0.001）,ROS,MDA and LDH levels increased（P<0.001）,T-AOC and SOD levels decreased（P<0.001）;cell membrane potential levels decreased,mitochondrial number decreased（P<0.05）,ATP content decreased（P<0.01）,Caspase-3 expression level did not change significantly（P>0.05）,Cleaved-Caspase-3,Cyt-C,Bax expression level increased（P<0.001）,Bcl-2 expression level decreased（P<0.001）;apoptosis rate increased（P<0.001）;3.2.Compared to the model group: Cell viability increased in the experimental group（P<0.05）;the differences in ROS levels were not statistically significant in the SAL1μM group（P>0.05）and decreased in the SAL5 μM and SAL10 μM groups（P<0.001）;MDA levels decreased in all three groups（P<0.05）;LDH levels decreased in the SAL1 μM and SAL5 μM groups（P<0.001）,and the change in LDH levels in the SAL10 μM group was not statistically significant（P>0.05）;the change in T-AOC levels in the SAL1 μM and SAL10 μM groups was not statistically significant（P>0.05）,and the change in T-AOC levels in the SAL5 μM group was increased（P<0.001）;SOD level increased in all three groups（P<0.001）;no significant changes in mitochondrial membrane potential,no statistically significant differences in the number and distribution of mitochondria in the SAL1 μM group（P>0.05）,significant changes in mitochondrial membrane potential,increased number and uniform distribution of mitochondria in the SAL5 μM and SAL10 μM groups（P<0.05）.The ATP content increased in the three groups（P<0.001）,the differences in the expression levels of Caspase-3 in the three groups were not statistically significant（P>0.05）,the expression levels of Cleaved-Caspase-3,Cyt-C and Bax decreased in the three groups（P<0.05）,and the expression levels of Bcl-2 increased（P<0.05）;apoptosis rate decreased in all three groups（P<0.001）.Conclusions: 1.The oxidative stress damage model of GC-1 cells was successfully constructed under hypoxia conditions of 3% O2,5% CO2,92% N2 for 36 h.2.Hypoxia can induce oxidative stress in GC-1 cells,reduce mitochondrial function and activate mitochondrial signaling pathway to promote cell apoptosis.3.The effect of low dose salidroside on oxidative stress damage of hypoxic GC-1 cells was not obvious.Medium and high doses of salidroside can relieve oxidative stress in hypoxic GC-1 cells to varying degrees,and reduce apoptosis of hypoxic GC-1 cells by down regulating mitochondrial signaling pathway. |
