Small Molecule Compound Me6 Promotes Hematopoietic And Intestinal Stem And Progenitor Cells Proliferation

Acute radiation syndrome is caused by nuclear terrorist attacks, sudden nuclear power plant accident or nuclear war. And it can even lead to death. Ionizing radiation can damage the body in various tissues. But on a certain dose, due to the different radiation sensitivity of tissue and cells, the reaction of each organ is also different. The hematopoietic tissue and intestinal tissue is more sensitive to radiation. And radiation can damage the hematopoietic and gastrointestinal system. The rapid development of nuclear technology has also been a good application in the medical field. For example, radiotherapy has become an important method for the treatment of malignant tumors. Although radiotherapy can target tumor and kill them, however, application of this technology will still have side effects causing damage to normal tissues, such as intestinal radiation injury. These side effects also greatly limits the use of effective dose of radiotherapy. It is still the current hot research that how to promote tissue regeneration after radiation damage. Recent studies show that the stem cells is an ideal seed cells in tissue regeneration. Stem cell has the capacity to self-renewal and differentiate into specialized cell types. In addition, it also have the ability to migrate to the injured tissue, differentiate into daughter cells and secrete cytokine. Therefore, we can regulate proliferation and directional differentiation of stem cells in vitro, or modulate the activity of stem cells in vivo, to better play the role of seed cells. Thus it can accelerate the repair of tissue after radiation injury.In previous work, our group used a mouse model for screening a series of small molecule compounds which have the ability to regulate bone marrow (BM) stem and progenitor cells. Our group discovered a novel small molecule compound that is Me6TREN, three (2-dimethylaminoethyl) amine. Me6 can mobilize mouse bone marrow hematopoietic stem and progenitor cells (HSPCs) to peripheral blood rapidly and effectively after a single subcutaneous injection. These HSCPs can speed up the recovery of the hematopoietic system when transplanted it into mice after radiation injury. What is the mechanism of Me6 mobilize HSPCs? Whether it related to hematopoietic stem cell proliferation? In order to understand this problem, we carried out the following experiment:First, we divide the mice into Con group and Me6 group. Mice in Me6 group were injected subcutaneously with Me6 at a dose of 5 mg/kg. After 12 hours, we isolated murine bone marrow cells. By CFU assay and flow cytometry analysis, we observed the effects of Me6 on mouse bone marrow HSPCs. It was found that, Me6 increased the numbers of CFU and HPP-CFU at about 1.2 times and 1.3 times compared with Con group. And Me6 increased the percentage of lin-Sca-l+c-Kit+HSPCs at about 1.7 times compared with Con group. These results suggest that Me6 can promote the mouse bone marrow HSPCs proliferation.Then we further observed the effect of Me6 on promote HSPCs expansion in vitro. The mononuclear cells (MNCs) were isolated from murine bone marrow and cultured in vitro with or without Me6 for 4 days. The concentration of Me6 is 100μM. By cell counting, CFU assay and flow cytometry analysis, we detect the effect of Me6 on HSPCs in mononuclear cells. It was found that, Me6 increased the numbers of CFU and HPP-CFU at about 1.4 times and 1.3 times compared with Con group. And Me6 increased the percentage of Ki-67+cells in lin-Sca-1+c-Kit+ HSPCs at about 1.5 times compared with Con group, it means that the proliferative ability of HSPCs in Me6 group was also significantly increased compared with the Con group. These results suggest that Me6 can promote HSPCs expansion in vitro.To further prove whether the small molecule compounds Me6 can promote hematopoietic cells proliferation after radiation injury. The mononuclear cells were isolated from murine bone marrow and radiated with 5Gy. The cells were cultured in vitro with or without Me6 and the concentration of Me6 is 100Mm. After 7 days, we collected the cells. By flow cytometry analysis and CFU-S12 assay, we detect the effect of Me6 on HSPCs in mononuclear cells. The results shows that Me6 increased the percentage of Ki-67+and the numbers of CFU-S12 at about 1.8 times and 2.2 times compared with Con group. These means that can promote hematopoietic cells proliferation after radiation injury. These result further support that the small molecule compounds have the ability to recovery the hematopoietic after radiation injury.The previous work in our group shows that small molecule compounds Me6 can improve the survival in irradiated mice. It also shows the effect of Me6 on promote intestinal tissue recovery after radiation injury. Although intestinal stem cells (ISCs) and HSPCs have different origins, but they have some similarities regulated signaling pathways, such as Wnt and Notch pathways. Whether the recovery of intestinal tissue after radiation injury is related to the regulation of Me6 on intestinal stem cell?IEC-6 cells (rat intestinal epithelial cells, is characterized by undifferentiated intestinal epithelial crypt cells) were irradiated with 5Gy by X-ray radiation and established a radiation injury model in IEC-6 cell. Through CCK-8 assay, flow cytometry BrdU incorporation assay, colony formation assay, to observe the effect of Me6 on IEC-6 cell. But also AnnexinV/PI assay was used to detect the situation of cell apoptosis. The results showed that, Me6 reduce the proportion of irradiated IEC-6 cell apoptosis and promote the proliferation. In addition, Me6 increase the proportion of BrdU incorporation at about 1.5 times compared with Con group. Me6 can significantly increase the number of clones formed in irradiated IEC-6 cells is about 2.6 times higher than Con group. These results indicate that Me6 maybe can promote ISCs proliferation.In order to determine whether the small molecules can promote the proliferation of intestinal stem cells (ISCs), we isolated small intestinal crypt cells. The research shows that the crypt cells isolated in vitro contain ISCs. Through the establishment of three-dimensional culture system (cultured in Matrigel, added the medium and cytokine), ISCs can be successfully cultured in vitro. Crypt cells budding represents the proliferation of ISCs. And the number of budding is used for assess the proliferation of ISCs.We isolated and cultured mouse intestinal crypt cells in vitro. After 7 days, we observe the effect of Me6 on the budding numbers of crypt cells by count and the proportion of Lgr5+ cells by flow cytometry. The results showed that, Me6 promotes the formation of budding body of crypt cells, and increases the number of crypt cell buddings about 2.5 times compared with the Con group. Me6 increased the percentage of Lgr5+ cell, surface markers of intestinal stem cell in crypt, at about 2.3 times. To explore whether a small molecule compound Me6 regulate the expression of genes related to stem cells in the small intestine, we detect the expression of some genes in crypt cell by real-time quantitative PCR. The result shows that Me6 increased the expression of proliferation-related gene such as CyclinDl, Myc, Jun, and increased the expression of intestinal stem cell-related gene such as Lgr5,Olfm4, Ascl2, Bmi1, reduced the expression of apoptosis-related gene such as P53, Puma, Bax. These results suggest that Me6 promote the proliferation of small intestinal crypt cells and reduce the apoptosis.By gene chip analysis of mouse intestinal crypt cells, and by western blot verified the expression of related proteins in IEC-6 cells and mouse crypt cells, we focused on β-catenin signaling pathway. Then we transiently transfected IEC-6 cells with β-catenin siRNA, and detected the effect of Me6 on promote cell proliferation. The results showed that the differentially expressed genes in two groups cells, intestinal stem cell-related genes and β-catenin related genes have partially overlapping. So we speculate Me6 is probably play the role of promoting small intestinal crypt cell proliferation by β-catenin signaling pathway. Me6 increase the expression of p-β-catenin in crypt cells, promote the phosphorylation of β-catenin. And increase the expression of p-β-catenin and p-GSK-3β in IEC-6 cells. After reduced the expression of β-catenin in IEC-6 cells, the effect of Me6 on promote IEC-6 cell proliferation was inhibited. These shows Me6 may play a role by β-catenin signaling pathway.