| Objectives: To study the expression changes of the S100B/RAGE signal-related proteins(S100B,s RAGE,RAGE),Smad 3 signal proteins(p-Smad 3,Smad 3),different phenotypic reactive astrocytes(type A1 and type A2)and inflammatory and trophic factors in neonatal mouse astrocytes after hypoxic ischemic brain injury.To study the moderating relationship between S100B/RAGE and Smad 3.To explore the protective mechanism of gastrodin in hypoxic ischemic brain injury through S100B/ RAGE-Smad 3 signal,and provide a new idea for the prevention and treatment of hypoxic ischemic brain injury in neonates.Methods: In this study,neonatal 10 d C57BL/6J mice were used to replicate HIBD model in vivo and TNC-1 astrocytes were used to construct OGD models in vitro.Part Ⅰ: 86 mice were randomly divided into three groups: Sham operation group(Sham),HIBD model group(HIBD),HIBD+gastrodin intervention(100 mg/kg)group(HIBD+GAS).Zea-Longa score,grip test and geotaxis test were used to detect neurological deficits.The protein expressions of S100 B and s RAGE in brain homogenate and plasma were determined by ELISA.The protein expressions of RAGE,C3,S100A10,TNF-α,BDNF,Smad 3 and p-Smad 3 in astrocytes of each group were detected by Western blot and immunofluorescence double-labeled staining.To study the expression of S100B/RAGE-Smad 3 signal-related proteins on astrocytes after HIBD and the effect of gastrodin on them.Part Ⅱ: TNC-1 astrocytes were divided into Control group(Control),oxygen glucose deprivation group(OGD),oxygen glucose deprivation group+gastrodin(0.34 m M)intervention group(OGD+GAS),and gastrodin intervention group(GAS).The expressions of RAGE,C3,S100A10,TNF-α,BDNF and p-Smad 3 in TNC-1 cells of each group were detected by Western blot and immunofluorescence double-labeled staining,and to study the effects of OGD stimulation on RAGE signal,reactive astrocytes of different phenotypes and inflammation-related proteins.Part Ⅲ: TNC-1 cells were divided into six groups: Control group(Control),oxygen glucose deprivation group(OGD),oxygen glucose deprivation + FPS-ZM1(100 n M)intervention group(OGD+FPS-ZM1),oxygen glucose deprivation+gastrodin(0.34 m M)intervention group(OG D+GAS),oxygen glucose deprivation + gastrodin +FPS-ZM1 intervention group(OGD+GAS+FPS-ZM1).Western blot was used to detect the expressions of p-Smad3/Smad 3,TNF-α,BDNF,C3 and S100A10 in each group,so as to study the effects of RAGE signal on Smad 3 protein and different phenotype of reactive astrocytes and the influence of gastrodin intervention on them.Results: Part Ⅰ:(1)TTC staining and behavioral test results showed that compared with the Sham group,the HIBD group had obvious infarction lesions,increased Zea-Longa score(P <0.05),decreased grasp test score,and prolonged negative ground test time(P <0.05).After gastrodin treatment,the infarct area was decreased,Zea-Longa score was significantly decreased(P <0.05),gripping test score was increased,and geotaxis test time was shortened(P <0.05).(2)ELISA showed that the contents of S100 B and s RAGE in brain homogenate of HIBD group were significantly higher than those of Sham group(P <0.05),and the contents were significantly decreased in HIBD+GAS group(P <0.05).S100 B content in serum was significantly increased in HIBD group(P <0.05),and was significantly decreased in HIBD+GAS group,while s RAGE content in HIBD+GAS group was significantly increased compared with HIBD group(P <0.05).(3)Western-blot and immunofluo rescence double staining showed that astrocytes were activated after HIBD,and the expressions of RAGE,C3,S100A10,TNF-α and BDNF were significantly increased compared with those of Sham group(P <0.05),and the phosphorylation level of Smad3 was increased(P <0.05).The expressions of RAGE,C3 and TNF-α were significantly decreased(P <0.05),and the phosphorylation level of Smad 3 was increased(P<0.05),while the expressions of S100A10 and BDNF were significantly increased(P<0.05).Part Ⅱ: Western-blot and immunofluorescence double staining showed that the expressions of RAGE,p-Smad 3,C3 and S100A10 in OGD-activated astrocytes were significantly higher than those in Control group(P <0.05).Compared with the OGD group,the expressions of RAGE,p-Smad 3 and C3 were significantly decreased in the OGD+GAS group(P <0.05),but the expressions of S100A10 were significantly increased in them(P <0.05).Part Ⅲ: Western-blot showed that FPS-ZM1 blocked RAGE signal,inhibited the activation of OGD-induced TNC-1astrocyte Smad 3,inhibited the expression of TNF-α,C3 and S100A10(P <0.05),and promoted the expression of BDNF.Compared with OGD+GAS+FPS-ZM1 group,the combination of FPS-ZM1 blocker and gastrodin further inhibited the expression of C3(P <0.05),but there were no significant differences in the expressions of TNF-α,BDNF and S100A10(P >0.05).Conclusion:(1)S100B/RAGE and Smad 3 signaling were activated in reactive astrocytes in the corpus callosum of neonatal rats after HIBD,and the expression of inflammatory factor TNF-α and neurotrophic factor BDNF were increased.(2)S100B/RAGE can regulate the expression of Smad 3 signaling and inflammatory and trophic factors in activated astrocytes.(3)Gastrodin can inhibit the expression of A1 astrocytes and TNF-α and promote the expression of A2 astrocytes and BDNF by inhibiting the S100B/RAGE-Smad 3 signal,thus improving the nerve function injury. |
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