| Objective:Intracerebral hemorrhage(ICH)is a subtype of stroke with a very high mortality and disability rate,accounting for 15%-20% of stroke.It brings great economic burden to patient,family and society.Injury after cerebral hemorrhage is mainly divided into primary brain injury resulting from hematoma compression,and secondary brain injury caused by erythrocyte lysis metabolites.Among them,neuroinflammation caused by secondary brain injury plays a critical role on the prognosis of ICH.At present,the clinical treatment methods for ICH are very limited,including conservative medical treatment and surgical treatment,and the prognosis of ICH is poor.Therefore,the study targeting on neuroinflammation after ICH is very important and worthy.In recent years,microRNAs(miRs)have been widely studied in the treatment of neurological diseases such as ICH,and have a good therapeutic prospect.MicroRNA-23b(MiR-23b)is located on human chromosome 9.At present,researchs for miR-23 b mainly focuse on the tumor associated field,and little is known about its research in the neurological field.In recent years,some researchers have pointed out that miR-23 b is significantly down-regulated in patients with autoimmune encephalitis,and can improve neuroinflammation after autoimmune encephalitis.In addition,in our previous research,we found that miR-23 b in peripheral blood and perihematoma tissue of ICH patients was significantly decreased,but its effect on ICH is unclear,needing to be explored.Autophagy,a kind of "self-eating" process in cells,refers to the degradation of damaged organelles and macromolecular substances for maintaining the stability of cellular internal environment.It has been reported that autophagy is over-activated in ICH,but the specific interaction mechanism between autophagy activation and neuroinflammation in ICH is unclear,and whether mi R-23 b can participate in regulating this interaction is also unknown,needing further investigation.In this study,we used collagenase Ⅶ injection for in vivo model and hemin stimulation for in vitro model of ICH,and then over-expressed miR-23 b to further observe the effects of miR-23 b on neuroinflammation,autophagy and neuronal survivalin ICH.It will provide new theoretical basis for the miRs treatment on ICH in the further.Methods:In this study,lentivirus over-expressing miR-23b(LV-miR-23b)was injected into the lateral ventricle of Wistar rats,and two weeks later,ICH model was induced by collagenase Ⅶ.All animal experiments were randomly divided into four group: sham group,ICH group,ICH + lentivirus control group(LV-miR-NC),ICH + lentivirus group over-expressing miR-23b(LV-miR-23b).Real-time qPCR was used to detect the expression of miR-23 b in the perihematoma tissues;the corner test and forelimb placement test were used to observe the neurological function of rats;brain edema was evaluated by brain water content;hematoma area was analyzed by HE staining;microgila activation was evaluated by immunofluorescence of Iba1;the survival of neurons was detected by TUNEL assay and immunofluorescence of NeuN;Western blotting was used to detect iNOS,mcp-1/CCL2,cleaved Caspase-3 and Bcl-2.For in vitro study,hemin was used to stimulate microglial BV2 and hippocampal neuron cell HT22.Elisa assay was used to determine the expression of IL-6,IL-1β,TNF-α and NO content in BV2 cells supernate;and iNOS,mcp-1/CCL2,LC3 B and SQSTM1 / p62 were detected by western blotting;mRFP-GFP-LC3 adenovirus transfection was used to detect the autophagy flux in cells;the apoptosis of neuronal cells HT22 was detected by flow cytometry and CCK-8 assay.Potential target gene of miR-23 b were selected by several gene prediction websites,and IPMK was valiated by RT-PCR and dual-luciferase reporter assay to be a target gene of miR-23 b.Finally,by using the IPMK siRNA and autophagy inhibitors,we observed if mi R-23 b can inhibit autophagy by reducing IPMK expression,thereby attenuate neuroinflammation and exert neuroprotection after ICH.Results:1.miR-23 b was down-regulated in the brain tissue of ICH rats(P <0.05).2.Overexpression of miR-23 b can effectively alleviate cerebral edema of ICH rats(P<0.05),and significantly reduce hematoma area and brain injury.3.Overexpression of miR-23 b can significantly improve neurological function in ICH rats.4.miR-23 b can effectively alleviate the activation of microglia and neuroinflammatory factors expression in ICH rats.5.miR-23 b can reduce perihematoma neuronal apoptosis after ICH.6.Overexpression of miR-23 b can reduce the inflammatory factors secretion of microglial BV2 under hemin treatment and improve the anti-apoptotic ability of co-cultured neuronal cells.7.The overexpression of miR-23 b can inhibit autophagy flux of microglial BV2 cells under hemin treatment.8.miR-23 b can reduce IPMK expression,and reduce the inflammatory response by inhibiting autophagy flux,thus to exert neuroprotective function in vitro.9.The AKT / mTOR pathway was activated in the regulation of miR-23 b onautophagy.Conclusion:1.Overexpression of mi R-23 b can effectively enhance the recovery of neurological function in ICH rats.2.miR-23 b can attenuate neuroinflammation after ICH in rats,and reduce the inflammation level of microglial BV2 cells under hemin treatment in vitro,thus to improve the anti-apoptotic ability of neurons.3.miR-23 b can reduce the autophagy level of BV2.It can reduce IPMK expression by directly binding with the 3’-UTR region of IPMK mRNA,and further activate the AKT / mTOR pathway to inhibit autophagy,thus to alleviate neuroinflammation and exert neuroprotective effect in ICH.miR-23 b can decrease the expression level of IPMK and further activate the AKT /mTOR pathway to regulate the autophagy process in microglial cells,thereby alleviating neuroinflammation and promoting the recovery of neurological function.The study will provid new strategy for the treatment of ICH. |
