| Objective:To investigate the protective functions and mechanisms of fibroblast growth factor10 on cerebral cortical neurons induced by oxygen-glucose deprivation injury,including:(1)the first part:To observe the effect of the recombinant protein FGF10 on vitro cultured cerebral cortical neurons in hypoxia-hypoglycemic conditions of cell survival.To investigate the changes of oxidative stress and apoptotic signaling pathway in cerebral cortical neurons under hypoxia-hypoglycemia.(2)the second part:to explore the FGF10 through HO-1 play a neuroprotective molecular mechanism.(HIF-1α)AMP-activated protein kinase(AMPK),extracellular regulatory protein kinase(ERK1/2,heme oxygenase 1(HO-1)FGF10 treatment,the clear FGF10 protective neurons,against OGD damage in the key molecular mechanisms.Methods:(1)The pregnant rats were sacrificed at the 17th day or 18th day of pregnancy.After removal of the fetal cortex from the cortex,the cortical neurons were obtained by isolation and primary culture.The cells were cultured in a balanced saline solution(EBSS)without glucose and incubated for 2-4 h in a cell incubator containing less than 1%oxygen to establish oxygen-glucose deprivation(OGD,also known as hypoxia-hypoglycemia)Model;while the control group was added 25mM glucose containing EBSS solution into the normal culture environment.(2)FGF10 treatment group was added FGF10 in OGD model group,the final concentration was 0,1,10,100 and 1000 ng/ml,respectively.(3)The cell viability of the control group,OGD treated group and FGF10 treated group were measured by CCK-8 Kit.(4)Lactate Dehydrogenase Kit(LDH)was used to determine the cytotoxicity of the control group,OGD treatment group and FGF10 treatment group.(5)Dichloro-dihydro-fluorescein diacetate(DCFH),superoxide anion radical Kit(Superoxide O2~-Kits)and manganese superoxide dismutase(Mn-SOD Kits)kit,were used to detect the levels of ROS,MDA and oxidation products Mn-SOD.(6)Morphological changes of neurons in control group,OGD treatment group and FGF10 treatment group were observed by immunocytochemical staining.use(7)Apoptosis of the control group,OGD treatment group and FGF10 treatment group were measured by using flow cytometry combined with Annexin V-FITC kit.(8)Western blotting was used to detect the expression and activity of the marker Caspase-3 in the cascade of the apoptotic signal.(9)The activity of Caspase-3,Caspase-8 and Caspase-9 was detected using Caspase-3,-8,-9 activity kit.(10)The expression of HO-1 and HIF-1 and the phosphorylation levels of AMPK and ERK1/2 were detected by immunoblotting.Results:Part 1:protective effect of FGF10 on primary neuronal OGD model.(1)FGF10 can improve OGD-induced neuronal cell damage.Immunocytochemistry showed that the neurons in the cortical neurons of the mice were significantly decreased(P<0.05),and the survival rate of the neurons was significantly decreased after OGD treatment compared with the normal group,and 100 ng/ml and 1000 ng/Ml of FGF10,the cell status was significantly improved,neuronal synapses increased significantly(P<0.05).(P<0.05),and the cytotoxicity was significantly increased(P<0.05),while the effect of FGF10 on the cell viability was significantly higher than that of the control group(P<0.05),and the cell viability was significantly decreased(P<0.05)(P<0.05),and the cytotoxicity was significantly decreased(P<0.05).The cell viability was significantly increased at 100 ng/ml and 1000 ng/ml.(2)FGF10 can reduce oxidative stress induced by OGD in neurons.(P<0.05),the content of Mn superoxide dismutase was significantly decreased(P<0.05),and the content of Mn superoxide dismutase(P<0.05)was significantly increased(P<0.05)(P<0.05),and the levels of ROS(P<0.05)in neurons were significantly decreased in the FGF10 treatment group at 100 ng/ml and 1000 ng/ml,and the superoxide anion(P<0.05)was significantly increased The content of Mn-SOD in neurons(P<0.05).(3)FGF10 can inhibit the activation of OGD on neuronal apoptosis and apoptotic signaling pathway.Annexin V-FITC flow cytometry showed that the apoptosis of neurons increased significantly(P<0.05)after OGD treatment,while 100 ng/ml FGF10could significantly decrease the apoptosis of cells.In addition,we found that the expression of Caspase-3 did not change significantly after OGD treatment compared with the normal group,but the activity of Caspase-3(Caspase-3)was significantly increased(P<0.05).At the same time,the activities of Caspase-3,Caspase-8 and Caspase-9 were significantly increased(P<0.05).FGF10 significantly reduced the Caspase-3 shear(P<0.05)and significantly reduced the activity of Caspase-3,Caspase-8 and Caspase-9(P<0.05).Part Ⅱ:Molecular mechanism of FGF10 acting as a neuroprotective agent by HO-1(1)By immunoblotting,we found that OGD significantly increased the expression of HIF-1αand HO-1(P<0.05)compared with the normal group,and also significantly activated the AMPK signaling pathway(p-AMPK and t(P>0.05),but the activation of ERK1/2 pathway(ie,the ratio of p-ERK1/2 to t-ERK1/2)did not change significantly(P>0.05).The addition of FGF10 did not alter HIF-1 and AMPK signaling pathway activation(p-AMPK/t-AMPK ratio),but significantly increased the expression of HO-1(P<0.05).(2)In PC12 neurons,the protective effect of FGF10 on cell viability was eliminated after knockdown of HO-1 gene by si RNA interference technique,and the protective effect of FGF10 on oxidative stress was also eliminated(P<0.05).Conclusion:(1)Hypoxia and glucose deprivation can cause significant cell damage,oxidative stress and neuronal cell apoptosis in primary cultured neurons in vitro,whereas FGF10can significantly improve hypoxia-hypoglycemia-induced neuronal damage,reduce oxidative stress and inhibit apoptosis.(2)FGF10 can reduce the oxidative stress by HO-1,promote the survival of neurons,reduce the apoptosis,and play the role of neuroprotection.Innovation:(1)A new neuroprotective factor FGF10 was found for the first time,which can be used to protect cerebral ischemia in vitro OGD model(2)FGF10 has been shown to exert neuroprotective effects by up regulation of HO-1 for the first time... |
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