RESUMO
AIM: To investigate whether α-ketoglutarate dehydrogenase complex (α-KGDHC) has protective effects on adaptive reperfusion after ischemic stroke in rats, and whether its mechanism is related to inhibition of apoptotic pathways. METHODS: A model of middle cerebral artery occlusion (MCAO) ischemia/reperfusion (I/R) was established, and the volume of cerebral infarction was assessed by TTC staining following computer analysis. The α-KGDHC activity was detected based on chemical reaction, to evaluate the change trend of α-KGDHC activity with cerebral ischemia time, and compare the difference between normal reperfusion group and adaptive reperfusion group; treatment of B35 and SH-SY5Y with CoCl2 to mimic cell hypoxia, and inhibition of α-KGDHC activity with E1K siRNA to detect the expression of apoptosis-related proteins Bcl-2/Bax/Caspase 3 by Western blot. RESULTS: In vivo experiments: In MCAO model, adaptive reperfusion further reduced cerebral infarction volume compared with normal reperfusion (P<0.05), and the expression of Caspase 3 was the lowest; α-KGDHC activity in cerebral cortex and hippocampal brain tissue decreased as ischemic time prolonged (P<0.05), and adaptive reperfusion inhibited the rate of decrease in α-KGDHC activity (P<0.05). In vitro experiments: Inhibiting α-KGDHC activity by interfering E1K expression led to downregulation of Bcl-2 (P<0.05) and upregulation of Bax (P<0.05) and Caspase 3 (P<0.05). CONCLUSION: α-KGDHC is an important factor in the protection of adaptive reperfusion after cerebral ischemia, and it may exert protective effect by inhibiting the activation of apoptotic pathways.
RESUMO
Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.
