Frehmaglutin F and frehmaglutin H ameliorate hypoxy-induced PASMCs injury via SIRT3/HIF-1α/PKM2 signal pathway

To explore the intervention mechanism of the new compounds Frehmaglutin F (Fre F) and Frehmaglutin H (Fre H) in Rehmannia glutinosa Libosch. (RG) on pulmonary hypertension (PH). An in vitro model of PH was established by using hypoxia-induced aberrant proliferation of Pulmonary Arterial Smooth Muscle Cells (PASMCs). Firstly, the activity and safety of Fre F and Fre H compounds were evaluated. Subsequently, the PH gene expression profile chip dataset GSE244830 and GSE113439 were downloaded from the gene expression omnibus (GEO) database to analyze the differential genes. Last, The cell migration and proliferation ability, the level of oxidative stress, mitochondrial damage, indicators related to the SIRT3/HIF-1α/PKM2 pathway, and the level of glycolysis were examined by adding the SIRT3 inhibitor (3-TYP) or silencing the SIRT3 gene to explore the the mechanism of action of Fre F and Fre H in improving PH. The SIRT3 gene was significantly reduced in both patients and mice with PH, and further molecular docking showed that the binding energies of Fre F and Fre H to the SIRT3 protein receptor were - 12.4 kJ/mol and - 11.2 kJ/mol. This result indicated that Fre F and Fre H had better binding activity to SIRT3 protein. The results of in vitro experiments showed that Freh F and Fre H significantly inhibited the proliferative and migratory abilities of PASMCs, decreased the intracellular levels of ROS, regulated mitochondrial damage, modulated the expression of SIRT3, HIF-1α, PKM2, and inhibited the glycolytic ability of PASMCs, but the above effects were completely or partially reversed upon the addition of 3-TYP and the silencing of SIRT3. Fre F and Fre H may exert anti-PH effects by reducing glycolysis levels via SIRT3/HIF-1α/PKM2 signal pathway.