Baicalein Induces Mitochondrial Autophagy to Prevent Parkinson's Disease in Rats via miR-30b and the SIRT1/AMPK/mTOR Pathway

Abstract

Parkinson's disease (PD) is a prevailing neurodegenerative disorder. Baicalein has neuroprotective effects on PD animals, but its mechanism is not clarified. We explored baicalein effects on PD rats. PD rat models were established by injecting 6-hydroxydopamine into the striatum of substantia nigra on the left side of the rat brain and treated with baicalein. Dopamine (DA) content, neuronal apoptosis, neuronal injury, neuronal mitochondria, and autophagy were assessed. Baicalein-treated PD rats were treated with autophagy inhibitor 3-methyladenine to identify the role of autophagy in PD. PD rats were injected with AgomiR-30b-5p or sh-SIRT1 plasmids and treated with baicalein. PD rats elicited decreased neurological score and DA secretion of the striatum, increased neuronal apoptosis, and injury, and reduced number of mitochondria and autophagy, whereas baicalein alleviated neuronal injury and partly recovered mitochondrial dysfunction, 3-methyladenine inhibited the protection of baicalein. miR-30b-5p was elevated and SIRT1 was diminished in PD rats and inhibited by baicalein. miR-30b-5p targeted SIRT1. miR-30b-5p overexpression or SIRT1 silencing annulled the protection of baicalein. The phosphorylation level of AMPK in the substantia nigra of PD rats was decreased and mTOR was increased, whereas baicalein annulled these trends. Briefly, baicalein activated mitochondrial autophagy via miR-30b-5p and the SIRT1/AMPK/mTOR pathway, thus protecting PD rats.

Keywords: AMPK/mTOR pathway; Parkinson's disease; SIRT1; baicalein; miR-30b-5p; mitochondrial autophagy; striatum.

Figure 1

Baicalein played a protective role by promoting mitochondrial autophagy. PD rats were jointly intervened by baicalein and autophagy inhibitor 3-MA. (A) The ultrastructure of mitochondria was observed using TEM; (B,C) the activities of mitochondrial complex I and ATP were detected using kits; (D) the levels of autophagy marker proteins LC3-II/I and p62 were detected by WB. N = 6. The data in the figure are all measurement data and expressed as mean ± standard deviation; one-way ANOVA was used for variance analysis; Tukey's multiple comparisons test was used for the post hoc test.

Figure 2

Upregulation of miR-30b-5p reversed the promotive effect of baicalein on mitochondrial autophagy. The agomiR-30b-5p was transfected into PD rats. (A) The expression of miR-30b-5p was detected by RT-qPCR; (B) the ultrastructure of mitochondria was observed using a TEM; (C,D) the activities of mitochondrial complex I and ATP were detected using kits; (E) the levels of autophagy marker proteins LC3-II/I and p62 were detected by WB. N = 6. The data in the figure are all measurement data and expressed as mean ± standard deviation; one-way ANOVA was used for variance analysis among multigroups; Tukey's multiple comparisons test was used for the post hoc test.

Figure 3

miR-30b-5p targeted SIRT1. (A) The binding sites between miR-30b-5p and SIRT1 were predicted by TargetScan website; (B) the target binding relationship between miR-30b-5p and SIRT1 was verified by dual-luciferase assay; (C) the expression of SIRT1 was detected by RT-qPCR. Three independent repeated cell tests were performed. N = 6. The data in the figure are all measurement data, expressed as mean ± standard deviation, and analyzed by one-way ANOVA and Tukey's multiple comparisons test.

Figure 4

Downregulation of SIRT1 reversed baicalein-induced mitochondrial autophagy. The effect of SIRT1 on baicalein-induced mitochondrial autophagy was observed by inhibiting the expression of SIRT1 in PD rats by sh-SIRT1. (A) The expression of SIRT1 was detected by RT-qPCR; (B) the ultrastructure of mitochondria was observed using a TEM; (C,D) the activities of mitochondrial complex I and ATP were detected using kits; (E) the levels of autophagy marker proteins LC3-II/I and p62 were detected by WB. N = 6. The data in the figure are all measurement data and expressed as mean ± standard deviation; one-way ANOVA was used for variance analysis among multigroups; Tukey's multiple comparisons test was used for the post hoc test.

Figure 5

Baicalein mediated mitochondrial autophagy via miR-30b-5p/SIRT1 and the AMPK/mTOR signaling pathway. The effect of miR-30b-5p on the AMPK/mTOR signaling pathway was observed by transfecting agomir-30b-5p in PD rats. The phosphorylation levels of AMPK and mTOR were detected by WB. N = 6. The data in the figure are all measurement data and expressed as mean ± standard deviation; one-way ANOVA was used for variance analysis among multigroups; Tukey's test was used for the post hoc test.