Discovery and confirmation of protein action site AK1 of ginsenosides in brain based on DARTS technology / 中国中药杂志

This study aims to explore the targets of ginsenosides in brain based on drug affinity responsive target stability(DARTS) technology. Specifically, DARTS technology was combined with label-free liquid chromatography tandem mass spectrometry(LC-MS) to screen out the proteins in the brain that might interact with ginsenosides. Based on the screening results, adenylate kinase 1(AK1) was selected for further confirmation. First, the His-AK1 fusion protein was yielded successively through the construction of recombinant prokaryotic expression vector, expression of target protein, and purification of the fusion protein. Biolayer interferometry(BLI) was employed to detect the direct interaction of Rg_1, Re, Rb_1, Rd, Rh_2, F1, Rh_1, compound K(CK), 25-OH-PPD, protopanaxa-diol(PPD), and protopanaxatriol(PPT) with AK1, thereby screening the ginsenoside monomer or sapogenin that had strong direct interaction with the suspected target protein AK1. Then, the BLI was used to further determine the kinetic parameters for the binding of PPD(strongest interaction with AK1) to His-AK1 fusion protein. Finally, molecular docking technology was applied to analyze the binding properties between the two. With DARTS and LC-MS, multiple differential proteins were screened out, and AK1 was selected based on previous research for target verification. Fusion protein His-AK1 was obtained by prokaryotic expression, and the response(nm) of Re, Rg_1, Rd, Rb_1, Rh_1, Rh_2, F1, PPT, PPD, 25-OH-PPD, and CK with His-AK1 was respectively 0.003 1, 0.001 9, 0.042 8, 0.022 2, 0.013 4, 0.037 3, 0.013 9, 0.030 7, 0.140 2, 0.016 0, and 0.040 8. The K_(on), K_(off), and K_D values of PPD and His-AK1 were determined by the BLI as 1.22×10~2 mol~(-1)·L·s~(-1), 1.04×10~(-2) s~(-1), 8.52×10~(-5) mol·L~(-1). According to the molecular docking result, PPD bound to AK1 with the absolute value of the docking score of 3.438, and hydrogen bonds mainly formed between the two. Thus, AK1 is one of the protein action sites of ginsenosides in the brain. The direct interaction between ginsenoside metabolite PPD and AK1 is the strongest.

Identification of multidrug resistance gene MDR1 associated microRNA of salvianolic acid A reversal in lung cancer / 中国中药杂志

This paper was aimed to investigate the microRNA associated with multidrug resistance gene MDR1 of salvianolic acid A reversal in lung cance. Human lung cancer A549 cells were divided into normal control group and drug group, and the MDR1 expression levels were determined by real-time quantitative PCR. MicroRNA expression profiling of normal control group and drug group were detected by using the latest microRNA microarray. Quantitative RT-PCR was used to validate the differentially expressed miRNA. Forecast of miRNA associated with MDR1 multi-resistant genes of up-regulated miRNA. Experimental results showed that the dosage of MDR1 expression level significantly lowered compared with control group. The miRNA expression spectrum analyses of human lung cancer A549 cells to drug group and the control group were detected by microRNA microarray, 426 differentially expressed miRNA were screened out. Then target prediction were performed for difference up-expression of miRNA and found that there were four obvious increase of miRNA associated with MDR1 multi-resistant genes. Real-time quantitative PCR for 4 microRNA verification, the results were consistent with the chip. So the author considered that salvianolic acid A down lung cancer multidrug resistance gene MDR1 is likely to be affected by the miRNA expression and regulation of target genes, to further clarify the traditional Chinese medicine to reverse multi-drug resistant mechanism provides the experimental basis.

Adrenal mitochondrial injury in early stage of sepsis in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the pathogenesis of adrenal mitochondrial dysfunction in septic rats.</p><p><b>METHODS</b>Thirty SPF rats were randomized into 3 groups, including a normal control group and 2 sepsis groups receiving intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS) and observed at 6 and 24 h after the injection. The adrenal mitochondria were extracted from the rats at the corresponding time points for observation by electronic microscopy. The membrane potential of the mitochondria was detected by flow cytometry. The oxidative stress levels in the mitochondria (activities of NOS and levels of MDA and NO) were assessed.</p><p><b>RESULTS</b>With the progression of sepsis, the serum levels of corticosterone in LPS groups increased significantly as compared with that in the control group. Ultrastructural observation of the adrenal mitochondria showed mild mitochondrial injury in LPS groups in comparison with the control group. The mitochondrial membrane potential was lowered in the LPS groups, but all these changes appeared to be reversible. The activities of NOS and the levels of MDA and NO showed no significant difference between the sepsis groups and the control group.</p><p><b>CONCLUSIONS</b>No obvious adrenal dysfunction occurs in the early stage of sepsis in rats. Mitochondrial injury, which is reversible, occurs in early sepsis without obvious evidence of oxidative stress injury in the adrenal mitochondria, suggesting a strong resistance and capacity of self-repair of the adrenal gland and the mitochondria against sepsis-induced injury.</p>