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Background: Our research team has identified a biological active component, panaxadiol saponins component (PDS-C) isolated from total saponins of panax ginseng as a potential targeted drug for treating hemocytopenia. PDS-C possesses dual activities, namely that of promoting hematopoiesis and regulating immune function. Our study is to observe effects of PDS-C on promoting hematopoiesis in normal and aplastic anemia (AA) mice, furthermore, to explore its possible mechanism., Methods: Bone marrow nucleated cells were cultured for colony forming assay of CFU-GM, CFU-E and CFU-MK in the presence of PDS-C at different concentration. The proliferation and differentiation-related genes expression profile was analyzed with DNA membrane microarray. The mRNA expression levels and protein phosphorylated state of GATA-1, GATA-2 transcription factors and AKT-1, MAPK14 protein kinases were detected by RT-qPCR and Western blot, the DNA binding activity and components of GATA-DNA complex were analyzed by EMSA and antibody gel supershift assay., Results: In response to PDS-C at 10, 25 and 50 mg/L, the bone marrow colony numbers of CFU-GM, CFU-E and CFU-MK increased significantly by 25.7%±3.1% to 42.4%±4.5% respectively in normal mice, and 29.7%±3.7% to 53.2%±7.1% in AA mice. The gene microarray profile initiated by PDS-C provided the up-regulated genes by more than 3 times, which can be classified into 11 categories according to their functions, including GATA-1, GATA-2, and AKT-1, MAPK14. The mRNA expression levels of GATA-1, GATA-2 were consistent with their gene microarray profile in PDS-C treated erythroid and megakaryocytic hematopoietic cells. Meanwhile, PDS-C could not only up-regulate expression levels of GATA-1, GATA-2 proteins, but also enhance phosphorylated activity state. Furthermore, PDS-C obviously enhanced binding activity of GATA protein with DNA in erythroid and megakaryocytic cells, and the main composition of GATA-DNA complex was GATA-2 and GATA-1., Conclusions: PDS-C displays the role to promote proliferation and induce differentiation for hematopoietic cells. Its action mechanism may involve in GATA-1, GATA-2 transcription factors, including up-regulating mRNA and protein expression, enhancing DNA binding activity, phosphorylated functional activity and up-regulating AKT-1, MAPK14 protein kinases as the upstream signaling molecule for activation GATA-1, GATA-2 respectively in hematopoietic cells., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-21-4800/coif). The authors have no conflicts of interest to declare., (2022 Annals of Translational Medicine. All rights reserved.)

Objective: To explore the proliferation inhibition and apoptosis effects of polysaccharides extracts from Hedyotis diffusa (PEHD) on multiple myeloma (MM) cell line RPMI 8226 cells in vitro, so as to provide experimental theory for the clinical application in the treatment of MM., Methods: MTT assay was used to examine the effects of PEHD on cell growth. The apoptotic cells were analyzed by flow cytometry with AnnexinⅤ/PI staining. Hoechst staining was used to observe the morphological changes of RPMI 8226 cell apoptosis. The expression levels of caspase-3,-8,-9, PARP, nucleoprotein NF-κB protein and other channel protein were assayed by Western blotting method., Results: The growth of RPMI 8226 cells were suppressed after treatment with PEHD, the highest inhibition rate reached to 92.3%, the results in the doses from 1 to 4 mg/ml showed a dose-and-time-dependent manner. The proportion of apoptotic cells in 1, 2 and 3 mg/ml PEHD treatment groups for 24 h were 22.52%, 62.31% and 69.94%, respectively, and significantly higher than that of control 8.93%. After treated with PEHD, apoptotic body appeared in RPMI 8226 cells nucleus and the number of apoptotic body increased in a dose-dependent manner. With the increasing of PEHD concentration, the expression of caspase-8,-9,-3 and PARP protein increased. The expression of Mcl-1, Bcl-xl, Bid and Bim protein decreased gradually, but the expression of Bax, Bak and Bad protein increased, and the expression of p-AKT protein (60 kDa) and NF-κB obviously decreased., Conclusion: PEHD could inhibited the growth of RPMI 8226 cells and displayed a dose-and-time-dependent manner, its mechanism may involve cell apoptosis induction, which was associated with the activation of caspase-8, caspase-9, and caspase-3 protein and the down-regulation of p-AKT and NF-κB protein expression.

Objective: To construct eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 (HIF-1alpha) and study their expressions in human microvascular endothelial cells (HMVECs)., Methods: Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Pro564 (ccc) in HIF-1alpha into gcc (Ala) in pcDNA3.1(+)-HIF-1alphato obtain single-site-mutated vector pcDNA3.1(+)-HIF-1alpha-564Ala, which was then subjected to a second site-directed mutagenesis to convert the codons for Asn803 into that of Ala (gct) to acquire double-site-mutated pcDNA3.1(+)-HIF-1alpha-564Ala-803Ala. After lipofectin-mediated transient transformation of HMVECs with the 3 recombinant plasmids including the two plasmids containing the mutations and the one without mutation, respectively, the expression levels of HIF-1alpha mRNA and protein were determined using RT-PCR, immunofluorescent staining and Western blotting., Results: DNA sequence analysis demonstrated success of the two-step mutagenesis and the two plasmids of pcDNA3.1+-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha-564Ala- 803Ala were obtained, both of which could produce HIF-1alpha protein resistant to oxidation degradation in HMVECs as compared with the non-mutated one., Conclusion: The recombinant plasmids pcDNA3.1(+)-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha- 564Ala-803Ala have been successfully constructed with efficient expressions in HMVECs.

Objective: To construct the eukaryotic expression vector for human hypoxia-inducible factor-1alpha (HIF-1alpha) gene and examine its expression., Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on the total RNA extracted from HT29 cells to obtain the cDNA of HIF-1alpha, which was inserted into T vector pUC18. DNA sequencing was performed before the amplified products were cloned into the eukaryotic expression vector pcDNA3.1+ identified by endonuclease digestion. This recombinant vector was transfected into HEK293 cells by means of liposome and its expression examined., Results: The amplified products were confirmed as the cDNA of HIF-1alpha by DNA sequencing, and pcDNA3.1+ -HIF-1alpha obtained was verified by endonuclease digestion, being capable of expression in HEK293 cells., Conclusion: We have successfully constructed the eukaryotic expression vector for HIF-1alpha, which can be expressed in HEK293 cells.