Expression And Interaction With The Receptor Of FGF 21 In Neurons And Brain Endothelial Cells

Part 1:Fibroblast growth factor 21 (FGF21) rapidly down regulate hyperglycemia effect in diabetic miceAims:There are a large number of studies have shown that FGF21 regulation of glucose metabolism, continuous inject FGF21 7-14 days can reduce Hyperglycemia in in type 2 diabetes, and also enhance insulin sensitivity, reduce insulin resistance. But few studies showed FGF21 can rapidly down regulate hyperglycemia. Rapidly down regulate hyperglycemia for the people who have hyperglycemia or hyperglycemia as the main risk factors diseases such as acute trauma, inflammation, and cardiovascular diseases have important significance.Methods:Human recombinant FGF21 protein provided by Wenzhou Medical College, injected in type 2 diabetic mice (T2D mice) 14 days, the mice randomly blood glucose were test by glucose test strips. Elisa used for test insulin levels in mice. After the injection of FGF21 1 hour, detect blood glucose with glucose test strips.Results:The mice were significantly decreased blood glucose and increased insulin sensitivity and decreased insulin resistance. After Injecting FGF21 1 hour, T2D mice’s blood glucose and simulation stress hyperglycemia mice’s blood glucose were significantly decreased. That means FGF 21 may also can rapidly decrease blood glucose.Part 2:FGF21 serum levels of FGF21 and expression levels in the brainAims:Some studies have shown a significant increase in serum levels of FGF21 when suffering from cardiovascular disease, liver disease, hypertension, obesity, diabetes and other diseases. Possible mechanisms is FGF21 signaling occurs obstacles, the sensitivity of FGF21 weakened, the liver only has to overexpression FGF21. Very important point is that studies have shown that FGF21 can pass through the blood brain barrier (BBB). But studies have found that FGF21 through transmembrane protein β-Klotho and fibroblast growth factor receptor (FGFR) binding together and then the receptor can be activated, which makes the role of FGF21 organized selectivity. And whether FGF21 can enter the brain, and in the brain FGF21 receptor andβ-Klotho expression and activation mechanisms have not been studied.Methods:In this study, the detection of non-hyperglycemic mice (ND mice) and type 2 diabetes mice(T2D mice) serum levels of FGF21 by Elisa.Use whole brain protein to do western blot to detect ND mice and T2D mice FGF21 expression levels. After intravenous injection exogenous FGF21 to mice, by western blot to detect the same time in different parts of the brain of mice about FGF21, pFGFRl andβ-Klotho expression levels.Results:Serum FGF21 in mice was significantly higher in T2D mice than in ND mice. FGF21 protein expression in T2D mice brain significantly higher than ND mice. After intravenous injection of exogenous FGF21,collected different time points mouse brain protein, do western blot to detect fibroblast growth factor receptor 1 (FGFR1) phosphorylatio、FGF21 and P-Klotho expression in different parts of the brain. This suggests that increase serum FGF21, in brain FGFR1 receptor phosphorylation increased,too. And in the brain, exogenous FGF21 is induced FGF21 expression and FGFR1 phosphorylation is increased.That means maybe FGF21 can direct enter the brain and induce FGFR1 phosphorylation, or FGF21 induced other factors in FGF family to activated FGFR1. But due to the detection of β-Klotho expression, FGF21 may be able to directly activate receptors.Part 3:FGF21 effects on brain endothelial cellsAims:Neuroprotective not limited to the protection of neuronal cells, the blood brain barrier protection is critical. Elevated serum FGF21 direct contact with the vascular endothelial cells, but whether FGF21 on vascular endothelial cells have a protective effect and protection mechanisms currently no research. The expression of FGF21 in the brain endothelial, in the brain endothelial after injury, form of the receptor complexes, pathways and mechanisms are to be studied.Methods:In this study, use Western blot to detect the expression of FGF21 in brain endothelial cells, oxygen-glucose deprivation (OGD) 4 hours before reoxygenation 20 hours later. And to detect FGFR1 phosphorylation and P-Klotho expression after treated with recombinant FGF21 protein, to find whether there is a time-dependent and dose-dependent with FGF21. Using RNA interference (SiRNA) technology, after transfection FGF21 SiRNA detected by Western blot analysis to confirm the FGF21 expression in brain endothelial cells. After P-Klotho SiRNA transfection by Western blot and immunofluorescence analysis to observe when the β-Klotho gene silencing, whether FGFR1 could be FGF21 activated and FGF21/phosphorylation FGFR1/ P-Klotho is co-localized in the brain endothelial cells. MTT assay with exogenous FGF21 on brain endothelial cells can produce a protective role in the OGD 4 hours and reoxygenation 20 hours of injury. FGF21 permeability protective effect was detected by FITC under high glucose and inflammatory injury on endothelial cells. Use western blot to detect FGF21 increase which protein to have the protective effect.Results:The brain endothelial cells can express FGF21 and FGF21 expression can be induced by OGD-reperfusion-injury. FGF21 in brain endothelial cells can activate FGFR1 phosphorylation and simultaneously induce expression of β-Klotho. And FGF21/phosphorylation FGFR1/ β-Klotho in the cells are co-localization. But after β-Klotho gene silencing FGF21 could not activate FGFR1. This result suggests that in brain endothelial cells, FGF21 sinaling pathway is through the formation of FGF21/FGFR1/β-Klotho trimeric complex, activated FGFR1 phosphorylation. Exogenous FGF21 in brain endothelial cells can produce a protective role in the OGD 4 hours and reoxygenation 20 hours of injury. And exogenous FGF21 has protective effects to cerebral vascular endothelial cells in OGD reperfusion injury and high sugar merger inflammatory injury. FGF21 also reduced endothelial cells permeability in high glucose merger inflammatory injury, one of the possible role is increase PPARy. FGF21 can also increase cell junction proteins ZO-1, VE-Cadherin to reduce cell permeability.Part 4:FGF21 effects on neuronal cellsAims:FGF21 can cross through the blood-brain barrier and neurons as the most important function cells of nervous system, if FGF21 has protective effect on neurons has not been studied. And if FGF21 express in neuronal cells, or whether it can respond FGF21 stimulation, as well as what FGF21 in neuronal cell receptors and signaling pathways are needed further study.Methods:In this study, use Western blot analysis to detect FGF21 expression changes in primary cultured cortical neurons under normal conditions and after hypoxia, and mouse primary cultured cortical neurons were treated with recombinant FGF21 protein, if phosphorylation of fibroblast growth factor receptor 1 (FGFR1) and β-Klotho expression induced, and whether has time-dependent and dose-dependent with FGF21. Use immunofluorescence staining to detect phosphorylated FGFR1 and β-Klotho whether could be induced by FGF21 and co-localization with FGF21. By Co-IP and Western blot analysis to detect whether FGF21 and FGFR1 and β-Klotho form a trimeric complex. Use LDH assay to detect whether exogenous FGF21 protein could have protective effects in mice during primary neuronal damage induced by hypoxia or hyperglycemia.Results:The mouse primary cutured cortical neurons under normal circumstances, the amount of FGF21 expression is very low, but after hypoxic injury expression of FGF21 can be induced. In primary cultured mouse cortical neurons can be seen in FGF21/phosphorylated FGFR1/ β-Klotho is co-localization, and phosphorylation of FGFR1 was activated by FGF21. Under normal conditions of mouse primary cultured neurons, FGF21 expression levels are too low, and do not form FGF21/FGFR1/ β-Klotho trimeric complex, FGFR1/β-Klotho does not form a dimer complex,either. But after primary cultured mouse cortical neurons treated with recombinant FGF21 protein, can be observed FGF21/FGFR1 /β-Klotho form a trimer complex. These results suggested that in the mouse primary cultured cortical neurons, FGF21 signaling pathway like the liver and fat cells, through the formation of FGF21/FGFR1/ (3-Klotho trimeric complex, activated FGFR1 phosphorylation. Exogenous FGF21 in mouse primary cultured cortical neurons under hypoxia and hyperglycemia injury has protective effects...