Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnan...Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnant women were collected.Macrophages were obtained by the density gradient method and then flow cell sorting,then the exosomes were extracted.The structure of the exosomes was observed by transmission electron microscope.The expression of CD63,a marker protein of the exocrine body,was detected by western blot,and the exosomes were identified.CCK-8 was used to detect the effect of exosomes on trophoblast cell viability.Transwell migration experiment was used to detect the influence on migration ability.The expression of miR-146a-5p in exosomes was detected by qPCR.The effect of exosomes on the expression of HIF1αprotein in trophoblasts was detected by western blot and detection of the binding site between miR-146a-5p and HIF1αby double luciferase reporter gene was conducted.Results:The exosomes of macrophages present a"cake"structure with a middle depression about 30-130 nm in diameter,and CD63 is highly expressed,which conforms to the characteristics of exosomes.Compared with the normal group,the exosomes of decidual macrophages in the PE group inhibited the activity and migration of trophoblast cells(P<0.001).The expression of miR-146a-5p in the exosomes of decidual macrophages in the PE decreased significantly,and after exosomes of PE decidual macrophages treating trophoblast cells,the protein expression of HIF1αin trophoblast cells was significantly increased.There are targeted binding sites between miR-146a-5p and HIF1α.Conclusion:PE decidual macrophage exosomes can inhibit the viability and migration of trophoblast cells,which may be related to the decreased expression of miR-146a-5p in exosomes,thus promoting HIF1αprotein expression of trophoblast cells.展开更多
Objective: To observe the effect of total flavonoids of Scutellaria barbata (TF‑SB) on the injury of high glucose induced podocytes (MPC‑5) and the influence of Smad4/PKM2/HIF‑1α pathway. Methods: Firstly, CCK8 was u...Objective: To observe the effect of total flavonoids of Scutellaria barbata (TF‑SB) on the injury of high glucose induced podocytes (MPC‑5) and the influence of Smad4/PKM2/HIF‑1α pathway. Methods: Firstly, CCK8 was used to analyze the safety and efficacy concentration of TF‑SB on MPC‑5 cells. Then, MPC‑5 was then divided into the control group, model group and TF‑SB group. In addition to the control group, model group and TF‑SB group were induced by high glucose to establish MPC‑5 cell injury model. The effects of TF‑SB on ATP, apoptosis and ROS levels of MPC‑5 cells were detected respectively. The contents of IL‑1β, TNF‑α, and MCP‑1 were determined by ELISA, the expression abundance of glycolytic genes (GLU1, PFK1 and HK1) were detected by RT‑PCR. Western blot method was used to detect the expression level of related proteins in Smad4/PKM2/HIF‑1α pathway. Results: Compared with the blank group, ATP content, GLU1, PKF1 and HK1 expression abundance of MPC‑5 cells in the model group decreased significantly, apoptosis, ROS level and IL‑1 β、 TNF‑ α And MCP‑1 significantly increased (P<0.01);Compared with model group, ATP content, GLU1, PKF1 and HK1 expression abundance, apoptosis, ROS level and IL‑1β in TF‑SB group were significantly increased , TNF‑ α The contents of MCP‑1 and MCP‑1 decreased significantly (P<0.01). In addition, compared with the blank group, the model group Smad4 and HIF‑1 α The protein expression and PKM2 expression in nucleus were significantly increased, while PKM2 expression in cytoplasm was significantly decreased (P<0.01);Compared with model group, TF‑SB group Smad4, HIF‑1 α The expression of PKM2 in the nucleus and expression of PKM2 were significantly decreased, while the expression of PKM2 in the cytoplasm was significantly increased (P<0.01). Conclusion: TF‑SB promotes the mitochondrial activity of MPC‑5 cells to induce glycolysis, and then inhibits the secretion of inflammation, which may play a role in treating diabetes nephropathy by inhibiting Smad4/PKM2/HIF‑1α signaling pathway.展开更多
基金Hainan Provincial Natural Science Foundation Project(821MS128,822MS164)Hainan Provincial People's Hospital National Natural Science Foundation Cultivation Project(530)(2021MSXM04)。
文摘Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnant women were collected.Macrophages were obtained by the density gradient method and then flow cell sorting,then the exosomes were extracted.The structure of the exosomes was observed by transmission electron microscope.The expression of CD63,a marker protein of the exocrine body,was detected by western blot,and the exosomes were identified.CCK-8 was used to detect the effect of exosomes on trophoblast cell viability.Transwell migration experiment was used to detect the influence on migration ability.The expression of miR-146a-5p in exosomes was detected by qPCR.The effect of exosomes on the expression of HIF1αprotein in trophoblasts was detected by western blot and detection of the binding site between miR-146a-5p and HIF1αby double luciferase reporter gene was conducted.Results:The exosomes of macrophages present a"cake"structure with a middle depression about 30-130 nm in diameter,and CD63 is highly expressed,which conforms to the characteristics of exosomes.Compared with the normal group,the exosomes of decidual macrophages in the PE group inhibited the activity and migration of trophoblast cells(P<0.001).The expression of miR-146a-5p in the exosomes of decidual macrophages in the PE decreased significantly,and after exosomes of PE decidual macrophages treating trophoblast cells,the protein expression of HIF1αin trophoblast cells was significantly increased.There are targeted binding sites between miR-146a-5p and HIF1α.Conclusion:PE decidual macrophage exosomes can inhibit the viability and migration of trophoblast cells,which may be related to the decreased expression of miR-146a-5p in exosomes,thus promoting HIF1αprotein expression of trophoblast cells.
基金Heilongjiang Traditional Chinese Medicine Research Project (No.ZHY19?058)。
文摘Objective: To observe the effect of total flavonoids of Scutellaria barbata (TF‑SB) on the injury of high glucose induced podocytes (MPC‑5) and the influence of Smad4/PKM2/HIF‑1α pathway. Methods: Firstly, CCK8 was used to analyze the safety and efficacy concentration of TF‑SB on MPC‑5 cells. Then, MPC‑5 was then divided into the control group, model group and TF‑SB group. In addition to the control group, model group and TF‑SB group were induced by high glucose to establish MPC‑5 cell injury model. The effects of TF‑SB on ATP, apoptosis and ROS levels of MPC‑5 cells were detected respectively. The contents of IL‑1β, TNF‑α, and MCP‑1 were determined by ELISA, the expression abundance of glycolytic genes (GLU1, PFK1 and HK1) were detected by RT‑PCR. Western blot method was used to detect the expression level of related proteins in Smad4/PKM2/HIF‑1α pathway. Results: Compared with the blank group, ATP content, GLU1, PKF1 and HK1 expression abundance of MPC‑5 cells in the model group decreased significantly, apoptosis, ROS level and IL‑1 β、 TNF‑ α And MCP‑1 significantly increased (P<0.01);Compared with model group, ATP content, GLU1, PKF1 and HK1 expression abundance, apoptosis, ROS level and IL‑1β in TF‑SB group were significantly increased , TNF‑ α The contents of MCP‑1 and MCP‑1 decreased significantly (P<0.01). In addition, compared with the blank group, the model group Smad4 and HIF‑1 α The protein expression and PKM2 expression in nucleus were significantly increased, while PKM2 expression in cytoplasm was significantly decreased (P<0.01);Compared with model group, TF‑SB group Smad4, HIF‑1 α The expression of PKM2 in the nucleus and expression of PKM2 were significantly decreased, while the expression of PKM2 in the cytoplasm was significantly increased (P<0.01). Conclusion: TF‑SB promotes the mitochondrial activity of MPC‑5 cells to induce glycolysis, and then inhibits the secretion of inflammation, which may play a role in treating diabetes nephropathy by inhibiting Smad4/PKM2/HIF‑1α signaling pathway.
