Biglycan Regulates Neuroinflammation By Promoting Ml Microglial Activation In Early Brain Injury After Subarachnoid Hemorrhage In Mice

Objective:To investigate the mechanism and impact of biglycan(BGN)on neuroinflammation during early brain injury(EBI)after subarachnoid hemorrhage(SAH)in mice.To provide a novel strategy for the treatment of neurological deficits after SAH.Methods:1.To investigate the effect of BGN on neuroinflammation during EBI after SAH.(1)Young male C57BL/6J mice were randomly divided into Sham group,SAH 6 h group,SAH 12 h group,SAH 24 h group,SAH 48 h group,and SAH 72 h group(5 mice were used for data collection each group).SAH was induced by endovascular perforation in young male C57BL/6J mice.The protein level of BGN was detected by western blot.(2)Young male C57BL/6J mice were randomly divided into Sham group,Sham control lentivirus(BGN-NC)group and Sham BGN lentivirus(BGN-KD)group(3 mice were used for data collection each group).Control lentiviral and BGN lentiviral vector were administered intracerebroventricularly to knock down BGN.The mRNA level of BGN was detected by quantitative real-time polymerase chain reaction(qRT-PCR).(3)Young male C57BL/6J mice were randomly divided into Sham group,SAH BGN-NC group and SAH BGN-KD group.The methods of SAH model and lentivirus administration were the same as before.The Modified Garcia Score and Beam Balance Tests were conducted to evaluate the neurobehavioral function(6 mice were used for data collection each group);The protein level of BGN was detected by western blot(5 mice were used for data collection each group);The protein levels of TNF-?,IL-1?,and IL-6 were measured by enzyme-linked immunosorbent assay(ELISA)(5 mice were used for data collection each group);The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL)was used to detect neuronal apoptosis(3 mice were used for data collection each group).2.To investigate the mechanism of how BGN regulated neuroinflammation during EBI after SAH.(1)Young male C57BL/6J mice were randomly divided into Sham group and SAH 48 h group(3 mice were used for data collection each group).The method of SAH model was the same as before.Immunofluorescence double staining was conducted to explore the expression of BGN on microglia.(2)Control(CON)group and oxyhemoglobin(HB)group were prepared(3 independent cell culture preparations were used for data collection each group).BV2 cells,a mouse microglia cell line,were challenged by oxyhemoglobin(HB)to mimic SAH in vitro.Immunofluorescence double staining was conducted to explore the co-localization of BGN and toll like receptor 4(TLR4);Co-immunoprecipitation was used to detect the protein-protein binding between BGN and TLR4.(3)HB BGN-NC group and HB BGN-KD group were prepared(3 independent cell culture preparations were used for data collection each group).The method of SAH model in vitro was the same as before.Western blot and qRT-PCR were used to detect the protein and mRNA levels of BGN respectively.(4)CON group,HB BGN-NC group and HB BGN-KD group were prepared(3 independent cell culture preparations were used for data collection each group).The method of SAH model in vitro was the same as before.Western blot was conducted to explore the expression of inducible nitric oxide synthase(iNOS),phosphor-nuclear transcription factor kappa B(p-NF-?B)and nuclear transcription factor kappa B(NF-?B);The protein levels of TNF-?,IL-1?,and IL-6 were measured by ELISA;The expression of CD 16/3 2 was detected by immunofluorescence staining;The expression of CD86 was detected by flow cytometry.(5)Young male C57BL/6J mice were randomly divided into Sham group,SAH BGN-NC group and SAH BGN-KD group(5 mice were used for data collection each group).The methods of SAH model and lentivirus administration were the same as before.Western blot was conducted to explore the expression of iNOS,p-NF-?B and NF-?B.Results:1.(1)The expression of protein and mRNA of BGN were increased in EBI after SAH and peaked at SAH 48 h group,which was statistically different compared with sham group(P<0.05).(2)The mRNA expression of BGN has no significant difference between Sham group and BGN-NC group(P>0.05),while the mRNA expression of BGN was decreased in BGN-KD mice compared with BGN-NC mice(P<0.05).(3)BGN down-regulation improved the Modified Garcia Score and Beam Balance Score and suppressed the production of BGN,TNF-?,IL-1?,and IL-6 as well as attenuated neural apoptosis in SAH 48 h mice(P<0.05).2.(1)BGN was detected in microglia.(2)Protein-protein binding was detected between BGN and TLR4.(3)The protein and mRNA expression of BGN was decreased in HB BGN-KD group compared with HB BGN-NC group(P<0.05).(4)BGN down-regulation suppressed the production of p-NF-?B,TNF-?,IL-1?,IL-6,and M1 microglia marker,iNOS,CD16/32,and CD86,in BV2 cells challenged by HB 48 h(P<0.05).(5)BGN down-regulation suppressed the production of p-NF-?B,TNF-?,IL-1?,IL-6,and M1 microglia marker,iNOS,in SAH 48 h mice(P<0.05).Conclusion:Inhibition of BGN can significantly reduce M1 microglia,activated via TLR4/NF-?B signaling pathway,and attenuated.neurological deficits.This finding provides a novel strategy and basis for the treatment of neurological deficits after SAH.