ABSTRACT
This study aims to investigate the role of slient mating-type information regulation 2 homolog 1(SIRT1)/tuberous sclerosis complex 2(TSC2)/mammalian target of rapamycin(mTOR) signaling pathways in the Periplaneta americana extract CⅡ-3-induced senescence of human leukemia K562 cells. K562 cells were cultured in vitro and treated with 0(control), 5, 10, 20, 40, 80, and 160 μg·mL~(-1) of P. americana extract CⅡ-3. Cell counting kit-8(CCK-8) and flow cytometry were employed to examine the proliferation and cell cycle of the K562 cells. Senescence-associated β-galactosidase stain kit(SA-β-gal) was used to detect the positive rate of senescent cells. Mitochondrial membrane potential was detected by flow cytometry. The relative mRNA level of telomerase reverse transcriptase(TERT) was determined by fluorescence quantitative PCR. The mRNA and protein levels of SIRT1, TSC2, and mTOR were determined by fluorescence quantitative PCR and Western blot, respectively. The results showed that CⅡ-3 significantly inhibited the proliferation of K562 cells and the treatment with 80 μg·mL~(-1) CⅡ-3 for 72 h had the highest inhibition rate. Therefore, 80 μg·mL~(-1) CⅡ-3 treatment for 72 h was selected as the standard for subsequent experiments. Compared with the control group, CⅡ-3 increased the proportion of cells arrested in G_0/G_1 phase, decreased the proportion of cells in S phase, increased the positive rate of SA-β-Gal staining, elevated the mitochondrial membrane potential and down-regulated the mRNA expression of TERT. Furthermore, the mRNA expression of SIRT1 and TSC2 was down-regulated, while the mRNA expression of mTOR was up-regulated. The protein expression of SIRT1 and p-TSC2 was down-regulated, while the protein expression of p-mTOR was up-regulated. The results indicated that P. americana extract CⅡ-3 induced the senescence of K562 cells via the SIRT1/mTOR signaling pathway.
Subject(s)
Humans , Animals , Periplaneta , Sirtuin 1/genetics , K562 Cells , Signal Transduction , TOR Serine-Threonine Kinases/genetics , RNA, Messenger , MammalsABSTRACT
The aim of this paper was to investigate the effect of SIRT1/TSC_2 signal axis on leukemia stem cell senescence induced by ginsenoside Rg_1. CD34~+CD38~- leukemia stem cells(CD34~+CD38~-LSCs) was isolated by magnetic cell sorting(MACS) and divided into two groups. The control group cells were routinely cultured, 40 μmol·L~(-1) ginsenoside Rg_1 was added to the control group for co-culture in Rg_1 group. The effect of Rg_l to induce CD34~+CD38~-LSCs senescence were evaluated by senescence-associated β-Galactosidase(SA-β-Gal) staining, cell cycle assay, CCK-8 and Colony-Assay. The expression of senescence associated SIRT1, TSC_2 mRNA and protein was examined by Real-time fluorescence quantitative PCR(FQ-PCR) and Western blot. The results showed that the CD34~+CD38~-LSCs could effectively be isolated by MACS, and the purity of CD34~+CD38~-LSCs is up to(95.86±3.04)%. Compared with the control group, the percentage of positive cells expressed SA-β-Gal in the Rg_1 group is increased, the senescence morphological changes were observed in the CD34~+CD38~-LSCs in the Rg_1 group. The proliferation inhibition rate and the number of cells entered G_0/G_1 phase in the Rg_1 group were increased, but the colony-formed ability was decreased, Rg_1 could significantly inhibit the proliferation and self-renewal ability of CD34~+CD38~-LSCs. The expression of SIRT1 and TSC_2 mRNA and protein were down regulated in the Rg_1 group compared with the control group. Our research implied that Rg_1 may induce the senescence of CD34~+CD38~-LSCs and SIRT1/TSC_2 signal axis plays a significant role in this process.
Subject(s)
Humans , Cellular Senescence , Ginsenosides , Pharmacology , Leukemia, Myeloid, Acute , Neoplastic Stem Cells , Signal Transduction , Sirtuin 1 , Metabolism , Tuberous Sclerosis Complex 2 Protein , Metabolism , Tumor Cells, CulturedABSTRACT
Aim To study the effect of the Kangfuxin liquid on 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced ulcerative colitis in rats, and to explore its mechanism. Methods Male SD rats were divided into normal control, model control, SASP groups, and Kangfuxin low,medium and high dose groups. In ad-dition to the normal control group, other groups were induced ulcerative colitis with TNBS solution. Disease activity index (DAI), organ index, colon mucosa damage index (CMDI) and histopathological score (HS),the expression levels of IL-4, IL-17 in serum, and MPO, EGF, TGF-β1 in colonic mucosa were de-termined. Results The DAI score showed that the model was successful. Compared with the normal group,the level of IL-4 and IL-17, EGF and TGF-β1 in the model group were reduced significantly, while the CMDI score,HS score,colon index and MPO were elevated significantly. The DAI, CMDI, HS and MPO were reduced (P<0.05 or P<0.01), and the levels of IL-4, IL-17, EGF and TGF-β1 increased signifi-cantly(P <0.01). Conclusions Kangfuxin liquid can effectively alleviate ulcerative colitis induced by TNBS in rats. The mechanism may be related to the down-regulation of MPO expression and up-regulation of IL-4,IL-17,EGF and TGF-β1 levels.
ABSTRACT
With reverse transcriptase PCR, the transcripton of copper homeostasis relative gene Afe0329 in Acidithiobacillus ferrooxians standard strain ATCC23270 was investigated. The further analysis of genes in this transcripton was analyzed employed by Vector NTI, Blast, TMHMM Server, PSORTb software and so on. From the DNA of different strains, the transcripton of Afe0329 was amplified using special primer pairs to identify the universality of it in the genome of A.ferrooxidans strains. The results showed that gene Afe0330 and Afe0331 were cotranscribed with Afe0329, and they were in a single transcripton. Gene Afe0329 was supported to express a P1b3-type ATPase which is a heavy metal ion pumping transmembrane protein, protein AFE0330 which expressed by gene Afe0330 was a cytoplasmic protein, no significant ho- mologous sequences of Afe0330 or Afe0331 had been obtained by Blast analysis. And the transcripton of Afe0329 was universal in genome of A. ferrooxidans strains.
ABSTRACT
Reverse transcriptase-PCR experiments suggest that the two clusters of genes potentially involved in the oxidation of reduced sulfur compounds are organized as operons in strain of the acidophilic, chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans ATCC 23270, the two clusters of genes including such the ORF of putative sulfate-thiosulfate-molybdate binding proteins, the ORF of putative thiosulfate: quinone oxidoreductase and the ORF of the rhodanese-like protein (P21). Bioinformatic analyses have predicted the possible promoters sequences and the possible +1 start site of transcription for the doxDA operons.
ABSTRACT
Metal sulfides are chemically attacked by Fe~ 3+ and H~+, resulting in the formation of elemental sulfur via polysulfides or thiosulfate pathway. Elemental sulfur may aggregate and even form a layer on the metal sulfide surface, which will inhibit leaching metals from the sulfides minerals. Elimination of inert elemental sulfur in a typical acidic environment can exclusively be by way of oxidation of acidophilic sulfur-oxidizing bacteria, such a way includes the attachment, transport and oxidation process of elemental sulfur by acidophilic sulfur-oxidizing bacteria. On the basis of analysis on the pertinent researches, the molecular mechanism of sulfur elimination by the acidophilic bacteria is far away from elucidated, and to attain that target, there are still much work to be done for elucidating the molecular mechanism on the attachment, transport and oxidation process of elemental sulfur by the acidophilic sulfur-oxidizing bacteria.