Study Of Black Wolfberry Anthocyanin On Apoptosis Of Mouse Macrophage RAW264.7 And The Interaction Mechanism Between Autophagy And Apoptosis

As a characteristic wild medicinal plant on the Qinghai-Tibet Plateau,black wolfberry is known as the"king of anthocyanins"because it is rich in anthocyanins.A large number of studies have found that black wolfberry anthocyanins on the Qinghai-Tibet Plateau have anti-inflammatory,antioxidant,and immune-enhancing properties.power and other biological effects.In this study,mouse macrophage RAW264.7 was used as the research object.1)Flow cytometry,scanning electron microscope,RT-q PCR and Western Blot were used to detect apoptosis rate,cell morphology,the mRNA and protein expression of apoptotic factor and JNK signaling pathway factor after mouse macrophage RAW264.7 were induced by different concentrations of anthocyanin and anthocyanin combined with JNK pathway inhibitor SP600125.2)CCK8 method was used to detect the effect of H₂O₂on mouse macrophage RAW264.7activity and black wolfberry anthocyanins on apoptosis induced by H₂O_2.3)To explore the effect of black wolfberry anthocyanins binding autophagy inhibitor 3-m A on mouse macrophage RAW264.7 apoptosis.4)To study the effect of black wolfberry anthocyanins binding JNK apoptosis pathway inhibitor SP600125 on the autophagy of mouse macrophage RAW264.7.To explore the molecular mechanism of black wolfberry anthocyanins on the apoptosis of mouse macrophage RAW264.7 and the interaction between apoptosis and autophagy based on the cellular level.In order to provide the theoretical basis for the development and utilization of black wolfberry anthocyanins as immune enhancers.The main research contents and results are as follows:1.Effects of different concentrations of black wolfberry anthocyanins on apoptosis of mouse macrophage RAW264.7Compared with the control group,the anthocyanin group can significantly reduced the apoptosis rate of mouse macrophage RAW264.7（P<0.05）;significantly reduced the mRNA and protein expressions of Caspase-3,Bax,Bax/Bcl-2（P<0.01）.significantly increased the mRNA and protein expression of Bcl-2（P<0.01）,indicating that black wolfberry anthocyanins can inhibit the apoptosis of mouse macrophage RAW264.7 in a dose-dependent manner.2.Effects of black wolfberry anthocyanins and it combined with JNK pathway inhibitor SP600125 on apoptosis of mouse macrophages RAW264.7Compared with the control group,the anthocyanin group significantly decreased the mRNA expression of JNK pathway factor JNK and the protein expression of p-JNK（P<0.01）in a dose-dependent manner;The effect of anthocyanin combined with SP600125 on the key factors of JNK pathway,compared with the control group,the anthocyanin group,SP600125group and anthocyanin+SP600125 group significantly reduced the apoptosis rate of mouse macrophage RAW264.7（P<0.01）;the anthocyanin+SP600125 group significantly reduced JNK mRNA expression and p-JNK protein expression of JNK pathway factor（P<0.01）;compared with the SP600125 group,the anthocyanin+SP600125 group significantly reduced the mouse macrophage RAW264.7 apoptosis rate（P<0.01）.The JNK mRNA expression and p-JNK protein expression of pathway factor JNK were significantly reduced（P<0.01）,indicating that black wolfberry anthocyanins inhibited mouse macrophage RAW264.7 apoptosis of through the JNK apoptosis pathway.3.Effects of black wolfberry anthocyanins on apoptosis of mouse macrophage RAW264.7 induced by H₂O₂The results of CCK-8 showed that 50,100,150,200,250μmol/L H₂O₂ group induced mouse macrophage RAW264.7 12,24,36,48,72 h effectively reduced mouse macrophage RAW264.7 activity.The best way for H₂O₂ to establish the apoptosis model of mouse macrophage RAW264.7 is:200μmol/L H₂O₂ induced mouse macrophage RAW264.7 for 24 h.After H₂O₂ induced mouse macrophage RAW264.7,anthocyanins significantly reduced the apoptosis rate of mouse macrophage RAW264.7（P<0.01）.The mRNA and protein expressions of Caspase-3and Bax were significantly decreased（P<0.01）;the mRNA and protein expression of Bcl-2 were significantly increased（P<0.01）;the JNK pathway factors JNK mRNA expression and p-JNK protein expression were significantly decreased（P<0.01）,indicated that black wolfberry anthocyanins inhibited mouse macrophage RAW264.7apoptosis induced by H₂O₂through the JNK pathway.4.Effects of black wolfberry anthocyanins combined with autophagy inhibitor 3-MA on apoptosis of mouse macrophage RAW264.7Compared with the control group,the anthocyanin group,3-MA group and anthocyanin group+3-MA group significantly reduced mouse macrophage RAW264.7 apoptosis rate,Caspase-3,Bax/Bcl-2 ratio,JNK mRNA expression and p-JNK protein expression（P<0.01）;significantly increased Bcl-2 mRNA expression（P<0.01）;compared with 3-MA group,anthocyanin+3-MA group Significantly decreased the mRNA expression of Caspase-3 and JNK（P<0.05）;significantly decreased the apoptosis rate,Bax,Bax/Bcl-2 mRNA expression and p-JNK protein expression in mouse macrophage RAW264.7（P<0.01）;significantly increased Bcl-2mRNA expression（P<0.05）;indicating that inhibit autophagy can enhance the function of black wolfberry anthocyanins inhibit mouse macrophage RAW264.7apoptosis.5.Effects of black wolfberry anthocyanins combined with JNK pathway inhibitor SP600125 on autophagy of mouse macrophage RAW264.7Compared with the control group,the anthocyanin group,SP600125 group and the anthocyanin+SP600125 group significantly decreased the mRNA expression of LC3-II and Beclin-1（P<0.05）,and significantly decreased the protein expression of LC3-II and Beclin-1.Compared with the SP600125 group,the anthocyanin group+SP600125 group significantly decreased the mRNA expression of LC3-Ⅱand Beclin-1（P<0.05）,and significantly decreased the protein expression of LC3-Ⅱ（P<0.01）,indicating that inhibit apoptosis can enhance the function of black wolfberry anthocyanins inhibit mouse macrophage RAW264.7 autophagy.