| BackgroundCerebrovascular disease is one of the more common and frequentlyoccurring diseases that lead to a high level of disability rate and mortalityand is serious harm to human health. The ischemic cerebrovasculardiseases account for the majority proportion of the cerebrovascular diseases.The neuron injury induced by cerebral ischemia/reperfusion (I/R) is themain cause of nerve function defect. Therefore, promoting nerveregeneration plays an important role after cerebral I/R. Recently numerousresearchs have found that after cerebral ischemia, endogenous neural stemcells (NSCs) spontaneous proliferation in the damaged area. However, theexact molecules involved in the process remain largely unknow. Previousstudies indicated that after cerebral I/R injury, astrocytes are activated andstart to proliferate in the damaged areas. As the major components of theadult neurogenic niche, astrocytes play an important role in regulatingneural stem cell regeneration. Recent studies have identified that after focalcerebral I/R, astrocytes can release high-mobility group box1(HMGB1) to promot endothelial progenitor cell (EPC) proliferation. Studies have alsofound that under the physiology condition, the expression of HMGB1isstrongly associated with the proliferation of neural progenitor cells and thebrain development. However, whether astrocyte released HMGB1canmodulate NS/PC proliferation after focal cerebral I/R remains unknown.This research includes three parts. Firstly, we used an in vitro model ofoxygen glucose deprivation/reperfusion (OGD/R) astrocytes (mimic an invivo I/R condition) to confirm that whether HMGB1can release fromOGD/R astrocyts. And then, we supressed the expression of HMGB1inastrocytes with the technology of RNA interference. Secondly, to confirmthe proliferative effect of HMGB1on NSC proliferation, we used theastrocyte-conditioned medium (ACM) with or without HMGB1and theexogenous HMGB1. Thirdly, we evaluate that HMGB1-induced NSCproliferation was mediated by RAGE-dependent JNK and PI3K/Aktsignaling pathway.Part1: The influence of oxygen glucose deprivation/reperfusion andRNA interference on astrocytes synthesis and secretion of HMGB1Objectives: To observe the oxygen glucose deprivation/reperfusion(OGD/R) on astrocytic HMGB1expression, and RNA interference onastrocytic HMGB1surpression. Methods: Within24h neonatal SD ratswere used for primary astrocyte cultures. After lentiviral-mediated HMGB1 gene silencing, the astrocytes were selected to use in the followingexperiment. The astrocytes were divided into four groups: normal group,OGD/R group, HMGB1shRNA OGD/R group and Control shRNAOGD/R group. HMGB1levels in astrocyte-conditioned medium (ACM)were detected by enzyme-linked immunosorbent assay (ELISA).Double-immunofluoresence labeling and Western blotting assays were usedto detect the expression of HMGB1in astrocytes. Results:①Comparedwith normal ACM, HMGB1protein levels in OGD/R ACM and ControlshRNA OGD/R ACM were significantly increased after OGD for6h andreperfusion for24h (p＜0.001). Compare with OGD/R ACM, HMGB1level was remarkably reduced in HMGB1shRNA ODG/R ACM (p＜0.001).②Western blot analysis of astrocytes lysates show that comparedwith normal astroytes, HMGB1proteins expression were markedlyincreased in OGD/R astrocytes and CoOGD/R astrocytes (P＜0.001).Compared with OGD/R astrocytes, HMGB1protein in RiOGD/Rastrocytes was strongly attenuated (P＜0.001).③Double-immunfluoresence staining of astrocytes show that HMGB1only expressedin the nucleus of normal astroytes. In OGD/R astrocytes and ControlshRNA OGD/R astrocytes, HMGB1protein expression were widelyexpressed throughout the cell. While in HMGB1shRNA OGD/R astrocytes,HMGB1protein in was strongly attenuated both in nucleus and cytoplasm.Conclusions: HMGB1expression is significantly increased in astrocytes when exposure to OGD for6h and reperfusion for24h. And RNAinterference can effectly attenuate HMGB1synthesis and secretion inastrocytes.Part2: The effect of HMGB1released by oxygen glucosedeprivation/reperfusion astrocytes on neural stem cell proliferationObjectives: To observe the HMGB1released by astrocytes on neuralstem cell (NSC) proliferation. Methods: Within24h neonatal SD rats wereused for primary NSC cultures. The NSCs were divided into six groups:control group, normal ACM group, OGD/R ACM group, HMGB1shRNAOGD/R ACM group, Control shRNA OGD/R ACM group and HMGB1group. Each group was further randomly divided into four culturedsubgroups for24h,48h,72h and96h. The NSC proliferative ability wasquantified by using the cell counting kit-8(CCK-8) assay and proliferationindex (PI). Results: There was no significant difference in the OD valuesof CCK-8and PI among all groups up to48h. Compared with the controlgroup, the OD values of CCK-8and PI in OGD/R ACM group, ControlshRNA OGD/R ACM group and HMGB1group had a significant increaseat72h and96h (P＜0.05). Compared with the OGD/R ACM group, theOD values of CCK-8and PI in HMGB1shRNA OGD/R ACM groupobviously decreased at72h and96h (P＜0.05). There was no significantdifference in the OD values of CCK-8and PI between the vehicle group and the nACM group at all four time-points. Conclusion: HMGB1releasedby oxygen glucose deprivation/reperfusion astrocytes can stimulate neuralstem cell proliferation.Part3: The mechanisms of HMGB1-induced NSC proliferationObjectives: To evaluate whether HMGB1-induced NSC proliferationwas mediated by RAGE-dependent JNK and PI3K/Akt signaling pathway.Methods: The experiment consists of three parts:①RAGE receptorpathway experiment: Cells were divided into two groups: IgG group andanti-RAGE group.②JNK signaling pathway experiment: Cells weredivided into four groups according to the different concentrations of JNKpathway inhibitor SP600125. They were normal control group,0μMgroup,1μM group,10μM group.③PI3K/Akt signaling pathwayexperiments: Cells were divided into four groups according to the differentconcentrations of PI3K/Akt pathway inhibitor LY294002. They werenormal control group,0μM group,1μM group,10μM group. After96h, NSC proliferation was evaluated by CCK-8assay, and the p-JNK andp-Akt levels in NSC was evaluated by Western Blotting. Results:①RAGE receptor pathway experiment: After cultured for96h, comparedwith that in IgG group, the absorbance values of CCK-8in anti-RAGEgroup was decreased significantly, and correspondingly the p-JNK andp-Akt protein levels also decreased (P＜0.001);②JNK signal pathway experiment: After cultured for96h, compared with that in control group,the p-JNK levels in0μM group significantly increased and the absorbancevalues of CCK-8increased correspondingly(P＜0.001); compared with thatin0μM group, p-JNK levels and absorbance values of CCK-8in1μMgroup had no significantly difference (P＞0.05); And in10μM group, whenp-JNK levels decreased the absorbance values of CCK-8decreased either(P＜0.001);③PI3K/Akt signaling pathway experiment: After culturedfor96h, compared with that in control group, the p-Akt levels in0μMgroup significantly increased and the absorbance values of CCK-8increased correspondingly(P＜0.001); compared with that in0μM group,p-Akt levels and absorbance values of CCK-8in1μM group had nosignificantly difference (P＞0.05); And in10μM group, when p-Akt levelsdecreased the absorbance values of CCK-8decreased either (P＜0.001).Conclusion: HMGB1-induced NSC proliferation was mediated byRAGE-dependent JNK and PI3K/Akt signaling pathway. |
PDF Full Text Request