Abstract:

Objective To discuss the protective effect of fructus mume total flavonoids （FMF） on the injury of the SH-SY5Y cells induced by 1-methyl-4-phenylpyridinium（MPP⁺） through regulating microRNA （miR）-145-3p expression，and to clarify the mechanism. Methods The SH-SY5Y cell model of Parkinson’s disease （PD） was established by MPP⁺ induction. The optimal intervention concentration and action time of MPP⁺ and FMF were screened out by CCK-8 assay. The cells were divided into control group （untreated）， model group （treated with 500 μmol·L^－1 MPP⁺ for 24 h）， MPP⁺+mimics group （transfected with miR-145-3p mimics）， MPP⁺+NC group （transfected with miR-145-3p NC）， MPP⁺+FMF group treated with 0.25 g·L^－1 FMF for 24 h）， MPP⁺+mimics+FMF group （transfected with miR-145-3p mimics and treated with 0.25 g·L^－1 FMF for 24 h），and MPP⁺+NC+FMF group （transfected with miR-145-3p NC，and （treated with 0.25 g·L^－1 FMF for 24 h）. CCK-8 assay was used to detect the proliferation abilities of the cells in various groups； Annexin Ⅴ-FITC/PI staining was used to the detect the apoptotic rates of the cells in various groups； transmission electron microscope was used to observe the ultrastructure morphology of mitochondrial autophagy in the cells in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of miR-145-3p in the cells in various groups； Western blotting method was used to detect the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/ LC3-Ⅰ in the cells in various groups. Results After treated with different concentrations of MPP⁺， the proliferation ability of the cells was significantly decreased（P<0.01）， and the PD cell model was successfully constructed. The optimal concentrations and action time of MPP⁺ and FMF intervention were 500 μmol·L^－1， 24 h， and 0.25 g·L^－1，24 h， respectively.The CCK-8 assay results showed that the proliferation ability of the cells in model group was significantly lower than that in control group （P<0.01）； the proliferation abilities of the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly higher than that in model group（P<0.01）； the proliferation ability of the cells in MPP⁺+mimics+FMF group was significantly higher than that in MPP⁺+FMF group （P<0.01）. The Annexin V-FITC/PI staining results showed that the apoptotic rate of the cells in model group was significantly higher than that in control group （P<0.01）； the apoptotic rates of the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly lower than that in model group （P<0.01）； the apoptotic rate of the cells in MPP⁺+mimics+FMF group was significantly lower than that in MPP⁺+FMF group （P<0.01）. The transmission electron microscope results showed that the cells in control group showed the uniform and intact mitochondrial morphology， the cells in model group showed the enlarged and irregular mitochondria with vacuolar changes，and the cells in MPP⁺+mimics group and MPP⁺+FMF group showed the improved mitochondrial structure， compared with model group， the autophagosome numbers of the cells in MPP⁺+mimics group and MPP⁺+FMF group were increased； the cells in MPP⁺+mimics+FMF group showed more intact mitochondrial structure， compared with MPP⁺+FMF group， the autophagosome numbers in the cells was increased. The RT-qPCR results showed that the expression level of miR-145-3p in the cells in model group was significantly lower than that in control group （P<0.01）； the expression levels of miR-145-3p in the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly higher than that in model group （P<0.01）； the expression level of miR-145-3p in the cells in MPP⁺+mimics+FMF group was significantly higher than that in MPP⁺+FMF group （P<0.01）.The Western blotting results showed that the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/LC3-Ⅰ in the cells in model group were lower than those in control group （P<0.05）； the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/ LC3-Ⅰ in the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly higher than those in model group （P<0.01）； the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/ LC3-Ⅰ in the cells in MPP⁺+mimics+FMF group were significantly higher than those in MPP⁺+FMF group （P<0.01）. Conclusion FMF may promote the autophagy， alleviate the mitochondrial dysfunction， and attenuate the injury of the SH-SY5Y cells induced by MPP⁺， and its mechamism is related to upregulating the miR-145-3p expression.

Key words: Fructus mume total flavone, Micro RNA-145-3p, Dopaminergic toxin 1-methyl-4-phenylpyridinium, Parkinson’s disease, Mitochondrial autophagy