The IL-6 From MSCs Suppresses The Proliferation Of Hippocampal Astrocytes In The Neonatal HIBD Rats Through Gp130/Ampk/mTor Pathway

PartⅠMSCs transplatation repressed the proliferation of astrocytes in the hippocampus of neonatal HIBD ratsObjective:To investigate the effect of mesenchymal stem cells（MSCs）and its endogenous interleukin-6（IL-6）on the proliferation of injured astrocytes and its possible mechanism in the neonatal rats with hypoxic-ischemic brain damage（HIBD）.Methods:Seven-days-postnatal SD rats were permanently ligated the left carotid artery and treated with hypoxia 2.5 h to establish HIBD model.After five days,the HIBD rats were intracerebroventricularly injected with MSCs.The therapeutic effects of MSCs transplantation on learning and memory function of the neonatal rats subjected to HIBD were assessed by morris water maze test and object-in-place task.The proliferation of hippocampal astrocytes were dynamically recorded in the hippocampus of neonatal HIBD rats using immunofluorescence staining,and enzyme-linked immuno sorbent assay（ELISA）was used to detect to the level of IL-6 expression in the hippocampus.Rat primary astrocytes were cultured,subjected to oxygen glucose deprivation（OGD）injury and then immediately separatedly co-cultured with MSCs.Cell cycle analysis was performed in the OGD-injured astocytes by flow cytometry.Changes in protein expressions levels of IL-6,IL-6 receptor glycoprotein（gp）130,phospho-mammalianAMP-activatedproteinkinase（AMPK）α,phospho-mammalian target of rapamycin（p-mTOR）and its downstream targets in the HIBD hippocampal tissues or OGD-injured astrocytes were examined with ELISA or western blot.Co-immunoprecipitation（Co-IP）and immunofluorescence staining were used to test the interactions between the proteins gp130 and p-AMPKαor p-mTOR.Results:（1）The rats of HIBD+MSCs groups required less time to find the platform than that in the HIBD group(^**P（27）0.01)and entering the platform region was significantly more than that of the HIBD rats（^*P（27）0.05）in morris maze water test;Meanwhile the object-in-place task results demonstrated that HIBD+MSCs rats spent significantly more time exploring the toys whose position had been changed than that in the HIBD group(^**P（27）0.01).The above results suggest that MSCs transplantation promotes the restoration of learning and memory ability in the neonatal HIBD rats.（2）Double immunostaining further revealed that MSCs transplantation significantly reduced the number of GFAP⁺/ki67⁺double-labeled cells in the hippocampus of HIBD rats on the day 7-14 after HIBD（^*P<0.05）.（3）The percentage of S phase cells in the OGD+MSCs group was significantly decreased compared with that in the OGD group(^**P<0.01).（4）MSCs transplantation significantly increased IL-6 secretion levels both in the hippocampus of rats following 7,14,and 21 days of HIBD(^**P_7d<0.01,^***P_14d<0.001,^***P_21d<0.001)and in the co-culture culture medium in vitro(^**P<0.01).（5）MSCs treatment in vitro or in vivo induced the protein expressions levels of gp130 and p-AMPKαin the hippocampus or OGD-injured astrocytes,while reduced the expression levels of p-mTOR,p-p7S6K,and p-4E-BP1(^***P_gp130<0.001,^***P_p-AMPK<0.001,^*P_p-mTOR<0.05,^***P_p-p7S6K<0.001,^***P_p-4E-BP1<0.001).（6）MSCs seperated coculture facilitated the interaction between gp130 and p-AMPKαin the OGD-injured astrocytes.Conclusions:MSCs transplantation in vivo or co-culture in vitro facilitated the secrection level of IL-6 in the injured microenvironment,activated the AMPK/mTOR signaling pathway in the injured hippocampal astrocytes,and suppressed the proliferation of hippocampal astrocytes following damage for 14 days to improve the learning-memory ability restoration of HIBD rats.Part Ⅱ The regulating mechanism of endogenous IL-6 from MSCs to suppress the proliferation of hippocampal astrocytes in neonatal HIBD ratsChapterⅠ Silence of endogenous IL-6 impairs repressing effect of MSCs on the proliferation of hippocampal astrocytes in neonatal HIBD ratsObjective: To verify the pivotal role of endogenous IL-6 from MSCs in inhibiting the proliferation of hippocampal astrocytes in HIBD rats by the silence of IL-6 expression in MSCs with si RNA interference technology.Methods: After 5 days of HIBD injury in the 7-day-old postnatal rats,they were intracerebroventricularly injected by si IL-6 MSCs or si RNA-control MSCs,respectively.The levels of IL-6 expression in the hippocampal tissue were detected using an ELISA kit,and the number of GFAP+/ki67+ double-labeled proliferation cells in the hippocampus from the two treatment groups were observed using immunofluorescence staining at 7 days,14 days and 21 days after HIBD.When the rats of the two groups reached four or five weeks of age,they were subjected to morris water maze or object-in-place task,respectively.After the rat primary astrocytes injured by OGD were sepretedly cocultured with si IL-6 MSCs or si RNA-control MSCs for 24 h,flow cytometer technology was used to analyze percentage of S phase in the OGD-injured astrocytes.The levels of IL-6 release in the culture medium from the two treatment groups were detected with ELISA,and the protein expressions levels of gp130,p-AMPKα,p-m TOR,p-p70S6 K and p-4E-BP1 were examined in the hippocampus of HIBD rats and OGD astrocytes following si IL-6 MSCs treatment by western blot.Results:（1）When compared to rats of the HIBD+si RNA-control MSCs group,si IL-6 MSCs-transplantion significantly reduced IL-6 expresion levels in the hippocampus at 7,14,and 21 days following HIBD（**P7d<0.01,***P14d<0.001,***P21d<0.001）.（2）The Morris water maze test displayed that the escape latency to find the platform was statistically longer（***P<0.001）and the number of passes through the platform region in the spatial probe test was also significantly lessened（**P<0.01）in the HIBD+si IL-6 MSCs group compare with those in the HIBD+si RNA-control MSCs group.In the object-in-place test,the discrimination score of rats in the HIBD+si IL-6 MSCs group was significantly decreased compared to that in the HIBD rats treated with the si RNA-control MSCs（*P<0.05）.These indicate that the silence of IL-6 in MSCs is disadvantaged to improve learning and memory function in HIBD rats.（3）The number of GFAP+/ki67+ double-labeled cells in the hippocampus of HIBD rats transplanted with si IL-6 MSCs were significantly greater than those with si RNA-control MSCs transplantation on the day 7-14 after HIBD（*P<0.05）.（4）The percentage of S phase cells in the OGD astrocytes following coculture with si IL-6 MSCs was significantly higher than that with the si RNA-control MSCs（*P<0.05）.（5）A significant reduction of IL-6 secretion was seen in the si IL-6 MSCs co-culture system（***P<0.001）,and the protein expressions levels of gp130 and p-AMPKα in the OGD-injured astrocytes were also decreased accordingly following coculture with si IL-6 MSCs,while the protein expression levels of p-m TOR,p-p7S6 K,and p-4E-BP1 were increased（**Pgp130<0.01,*Pp-AMPK<0.05,***Pp-m TOR<0.001,**Pp-p7S6 K <0.01,*Pp-4E-BP1<0.05）.The changes of gp130,p-AMPKα,m TOR,p-p7S6 K,and p-4E-BP1 protein expressions levels in the hippocampal tissue of HIBD rats transplanted by si IL-6 MSCs were the same as observed in vitro.Conclusions: Both si IL-6 MSCs transplantion in vivo and seperated coculture in vitro reduced IL-6 expression levels in the injury microenviroment.The silence of IL-6 expression impaired the effects of MSCs on suppressing the reactive astrocyte proliferation and improving the spatial learning and memory function in HIBD rats,which possible regulating mechanism is through AMPK/m TOR pathway.ChapterⅡ Blocking m TOR signal inhibits the proliferation of hippocampal astrocytes in neonatal HIBD ratsObjective: To confirm the regulation effect of m TOR on the proliferation of hippocampal astrocytes in HIBD rats through the intervention of m TOR specific inhibitor rapamycin.Methods: The rapamycin（m TOR specific inhibitor）was given intraperitioneally once daily for 7 days in the neonatal HIBD rats.The levels of m TOR protein expression in hippocampal tissue of HIBD rats were detected by western blot.The learning and memory function of the HIBD rats treated by rapamycin was conducted by morris water maze and object-in-place task.Immunofluorescence staining was used to observe the number of hippocampal proliferative astrocytes in the treated HIBD rats.After rapamycin intervention in vitro,the OGD injured astrocytes proliferation rate was monitored by CCK-8 kit at 6 h,24 h,48 h,96 h,120 h after rapamycin treatment.The S phase cell proportion of OGD astrocytes was analyzed at 24 h following intervention using flow cytometer,and the change of Ca2+ levels was determined by calcium imaging system in the OGD-injured astrocytes.The levels of gp130,p-AMPKα,p-m TOR,p-4E-BP1 and p-p70S6 K protein expressions in the OGD-injured astrocytes with rapamycin treatment were detected by western blot.Results:（1）Rapamycin effectively suppressed the expression level of p-m TOR in the hippocampal tissues of HIBD rats.（2）Results of the Morris water maze test revealed that the exploration time to find the platform by the HIBD rats treated by rapamycin was significantly shorter than that in the HIBD+vehicle control rats（***P<0.001）;and the times of the rapamycin treated rats passing through the platform region on the day 6 was also statistically increased（***P<0.001）.Meanwhile,the exploring ability of fresh affairs in the rats treated by rapamycin was also significantly enhanced compared with that of the HIBD+vehicle rats（*P<0.05）,suggesting that inhibition of m TOR can improve the learning and memory function of HIBD rats.（3）Rapamycin intervention significantly decreased the number of GFAP+/ki67+ double-labeled cells（**P<0.01）and the GFAP protein expression level in the hippocampus of HIBD rats,attenuated the proliferation rate of OGD injured astrocytes,decreased the number of S phase cells（**P<0.01）,and suppressed the intracellular Ca2+ concentration induced by ATP in the injured astrocytes（***P<0.001）.（4）Rapamycin effectively reduced the expression levels of p-m TOR,p-p7S6 K,and p-4E-BP1 proteins in the OGD-injured astrocytes,but didn’t impact on the expression levels of gp130 or p-AMPKα（***Pp-m TOR<0.001,**Pp-p7S6 K <0.01,***Pp-4E-BP1<0.001）.Conclusion: Blocking of m TOR expression suppresses the cell proliferation in the HIBD hipocamppus or OGD injured-astrocytes,decreases the levels of p-m TOR and its downstream targets p-p7S6 K,p-4E-BP1 protein expressions levels to enhance the learning and memory function ability in HIBD rats.Part III Activation of NFκB/IL-10 signal in the OGD-injured astrocytes suppresses neuronal apoptosisObjective: To investigate the biological function on neurons and the mechanism of IL-10 secretion in the astrocytes following the oxygen and glucose deprivation（OGD）injury.Methods: Primary astrocytes were injured by OGD,and then the immunofluorescence assay was used to observe expression of p-NFκB p65 in the injured astrocytes.The apoptosis in the neurons and astrocytes injured by OGD were detected following exogenous IL-10 intervention through the Annexin V-FITC/PI double staining.Luciferase reproter plasmids containing different length of rat’s IL-10 promoter were constructed to determine the binding site of p-NFκB p65 and the IL-10 promoter.The 7-day-postnatal rats were injured by HIBD,and then immunofluorescence double-labeling staining were performed in the hippocampus of HIBD rats to evoluate location of TLR2 or IL-10 in the astrocytes,microglia cells or neurons.Results:（1）Exogenous recombinant IL-10 treatment alleviated the apoptosis of OGD-injured neurons（**PFITC+<0.01,**PPI+<0.01）,but no affacted the apoptosis of OGD-injured astrocytes.（2）OGD induced the expression of p-NFκB p65 in the nuclear of the astrocytes.（3）p-NFκB p65 could bind to the-1700/-1000 b.p.proximal region of the IL-10 gene promoter in the OGD-injured astrocytes and this interaction could be controlled by OGD treatment（***P-1700/-1500<0.001,*P-1500/-1000<0.05）.（4）HIBD induced a marked increase of both TLR2 and IL-10 expression in the hippocampal astrocytes but not neurons and microglia（*P<0.05）.Conclusion: These data combined with our previous study results suggest that IL-10 secretion from astrocytes through TLR2/NFκB signaling pathway was activated by OGD to exert an anti-apoptotic effect on the injured neurons.