The Role Of GM130 In The Formation Of Brain Edema After Intracerebral Hemorrhage

Background:The prognosis of intracerebral hemorrhage(ICH)is closely related to the degree and development speed of brain edema.Dehydration and diuresis are the main treatment for cerebral edema after intracerebral hemorrhage,while the curative effect is not good.As an important subcellular organelle,Golgi apparatus may be involved in the pathological process of brain edema after intracerebral hemorrhage.In order to find a new target treatment with neuroprotective effect,this study focused on the role of Golgi apparatus and Golgi matrix protein 130(GM130)in the formation of brain edema after intracerebral hemorrhage.Objective:1.The effect of GM130 on neuroinflammation and brain edema by regulating the expression of Aquaporin2(AQP2)was investigated after ICH.2.The possible mechanism of GM130 affecting the integrity of tight junctions between endothelial cells and brain edema via autophagy pathway after ICH.Methods:1.To explore the regulation effect of GM130 on AQP2 and inflammation in rat immortal astrocyte cell line CTX-TNA2 ICH model and SD rat ICH model:(1)Hemin was used to induce rat astrocytes(CTX-TNA2)damage to establish ICH model in vitro.To investigate the role of GM130 in astrocytes via over-expressing or silencing GM130 in CTX-TNA2.Western Blotting(WB)was used to measure the expression of AQP2,Glial fibrillary acidic protein(GFAP)and Interleukin-1β(IL-1β).Immunofluorescence(IF)was used to observe the distribution of GFAP.(2)To investigate the role of AQP2 in astrocytes and microglia via over-expressing or silencing AQP2 in CTX-TNA2 ICH model.To explore the possible mechanism on the regulation role of AQP2 on astrocytes and microglia via using Toll-like receptor 4(TLR4)inhibitor after AQP2 overexpression in CTX-TNA2.WB was used to measure the expression of AQP2,GFAP,IL-1β,TLR4 and nuclear factor kappa-B(NF-κB).IF was used to observe the expression of AQP2 and GFAP.The rat microglial(HAPI)was cultured with the medium extracted from astrocytes was used to measure the expression of CD206,CD68 and CD86,and the ratio of M1/M2(CD68/CD206 or CD86/CD206)was calculated.(3)Collagenase was stereotactically injected into the basal ganglia of SD rats to establish the ICH model in vivo.The expression of GM130,AQP2 and inflammatory factors were observed in the ICH group.Thirty-six SD rats were divided into four groups,including sham group,12 h ICH group,24 h ICH group and 48 h ICH group.WB was used to determinate the expression of GM130,AQP2,GFAP and IL-1(n=6).The expression of AQP2 was observed by immunohistochemistry(IHC)or IF staining(n = 3).(4)To investigate the effect of GM130 on AQP2 and inflammatory factors in ICH model of SD rats,twenty-four SD rats were divided into four groups,including sham group,ICH group,AAV-NC + ICH group and AAV-GM130+ICH group.The expressions of GM130,AQP2,GFAP and IL-1β were measured by WB after ICH in 48h(n = 6).Nine SD rats were divided into three groups: sham group,AAV-NC + ICH group and AAV-GM130+ICH group.AQP2 and GFAP were co-labeled via IF to evaluate the expression of AQP2 in astrocytes between the three groups(n = 3).2.To explore the effect of regulating GM130 on autophagy formation and tight junction in mice brain microvascular endothelial cells(b End.3)ICH model and SD rat ICH model:(1)Hemin was used to induce b End.3 damage to establish ICH model in vitro.To investigate the role of GM130 on autophagy formation and tight junction via over-expressing or silencing GM130 in b End.3 cells.To explore the possible mechanism on the regulation role of GM130 on b End.3 cells by using autophagy inhibitor 3-Methyladenine(3-MA)after GM130 silenced in b End.3 cells.WB was used to measure the expression of GM130,LC3 B,p62,Occludin and ZO-1,IF was used to observe the continuity of Occludin in b End.3 cells,transmission electron microscope(TEM)was used to observe Golgi morphology and autophagosome formation.(2)To explore the expression of GM130,autophagy level and tight junction protein in ICH model of SD rats,thirty SD rats were divided into5 groups: sham,12 h ICH,24 h ICH,48 h ICH and 72 h ICH groups.The expressions of GM130,lc3 b,p62,occludin and ZO-1 were measured by WB(n=6).(3)To study the effect of GM130 overexpression on autophagy level and tight junction,twenty-four SD rats were divided into four groups,including sham group,ICH group,AAV-NC + ICH group and AAV-GM130+ICH group.The expressions of GM130,LC3 b,p62,occludin and ZO-1 were measured by WB after ICH in 48h(n=6).Nine SD rats were divided into three groups: sham group,AAV-NC+ICH group and AAV-GM130+ICH group.The continuity of occludin on blood vessels was observed by IF(n=3);endothelial cells and LC3 B were co-labeled via IF to observe the alteration of autophagy flux in endothelial cells(n=3).Eighteen SD rats were divided into two groups:AAV-NC+ICH group and AAV-GM130+ICH group.The permeability of blood-brain barrier was measured by Evans blue staining(n=3),the neurological function was measured by Garcia score and water content of brain was measured by dry-wet weight(n=6).(4)To study the effect of GM130 silence on autophagy level and tight junction,thirty-six SD rats were divided into six groups,including sham group,ICH group,ICH + vehicle-1 group,ICH + vehicle-2 group,ICH + si GM130 group and ICH + si GM130 + 3-MA group.The expressions of GM130,LC3 b,p62,occludin and ZO-1 were measured by WB after ICH at 48h(n=6).Twelve SD rats were divided into four groups:sham group,ICH group,ICH + vehicle group and ICH + si GM130 group.The continuity of occludin on blood vessels was observed by IF(n=3);endothelial cells and LC3 B were co-labeled via IF to observe the alteration of autophagy flux in endothelial cells(n=3).Twenty-seven SD rats were divided into three groups: sham group,ICH + vehicle group,ICH + si GM130 group.The permeability of blood-brain barrier was measured by Evans blue staining(n=3),the neurological function was measured by Garcia score and water content of brain was measured by dry-wet weight(n=6).3.All statistical analyses were performed using Statistical Package for Social Sciences software.When continuous data were normally distributed,one-way analysis of variance(ANOVA)was used to identify the differences between multi groups;otherwise,differences were examined using the Mann–Whitney U-test.A P-value <0.05 was considered statistically significant.Results:1.The regulation effect of GM130 on AQP2 and inflammation in CTX-TNA2 ICH model and SD rat ICH model:(1)GM130 silencing significantly promotes the expression of AQP2,GFAP and IL-1β in CTX-TNA2 ICH model compared with the control ICH group(P < 0.05,with statistical significance).While GM130 overexpression significantly reduces the expression of AQP2,GFAP and IL-1β in CTX-TNA2 ICH model(P<0.05,with statistical significance).(2)AQP2 overexpression significantly increases the expression of TLR4,NF-κB,GFAP and IL-1β in CTX-TNA2 ICH model compared with the control ICH group(P<0.05,with statistical significance),and indirectly promotes HAPI polarization to pro-inflammatory M1phenotype;while AQP2 silencing induces the opposite effect.TLR4 inhibitor was administrated in CTX-TNA2 after overexpression of AQP2,the expression of TLR4,NF-κB,p-NF-κB,GFAP and IL-1β were significantly lower than that in AQP2 overexpression plasmid transfection group(P<0.05,with statistical significance).(3)GM130 was decrease while AQP2,GFAP and IL-1β were increase in SD rats ICH model compared with the sham group(P<0.05,with statistical significance).(4)GM130 overexpression significantly reduces the expression of AQP2,GFAP and IL-1β in hemorrhaged brain in SD rats compared with the control ICH model(P<0.05,with statistical significance).2.The regulation effect of GM130 on autophagy formation and tight junction in b End.3 ICH model and SD rat ICH model:(1)GM130 overexpression significantly increases the expression of Occludin,ZO-1 and p62,and reduces the conversion ratio of LC3 BI to LCBII in b End.3 ICH model compared with the control ICH group(P<0.05,with statistical significance);TEM showed that GM130 overexpression reduces Golgi morphology alteration and inhibits autophagosome formation in b End.3 ICH model.While GM130 silencing significantly reduces the expression of Occludin,ZO-1 and p62,and increases the conversion ratio of LC3 BII to LCBI in b End.3 cells compared with the control group(P<0.05,with statistical significance);TEM showed that GM130 silencing induces Golgi morphology alteration and promotes autophagosome formation in b End.3 cells.3-MA was administrated in b End.3 after GM130 silencing,the expression of Occludin and ZO-1 were significantly higher than those in si GM130 transfection group(P<0.05,with statistical significance).(2)GM130 overexpression significantly increases the expression of Occludin,ZO-1 and p62,and reduces the conversion ratio of LC3 BI to LCBII in SD rat ICH model compared with the control ICH group(P<0.05,with statistical significance).(3)Compared with the control ICH group,the brain water content significantly reduced in GM130 overexpression ICH group(82.4±0.70 vs.76.82±2.03;P < 0.05,with statistical significance),the Evans blue content significantly reduced in GM130 overexpression ICH group(3.89±0.51 vs.2.76±0.49;P<0.05,with statistical significance)and the Garcia score significantly increased in GM130 overexpression ICH group(11±1.22 vs.13.80±0.84;P<0.05,with statistical significance).(4)Silencing GM130 presents a contrary effect to that observed in GM130 overexpression.3-MA was administrated in SD rat ICH model after GM130 silencing,the expression of Occludin and ZO-1 were significantly higher than those in the si GM130 transfection ICH group(P<0.05,with statistical significance).Conclusion:1.AQP2 exerts a positive regulation effect on neuroinflammation.GM130 pose a negative regulation effect on neuroinflammation and brain edema by decreasing AQP2 expression in astrocytes after ICH.2.GM130 exerts a protective effect on tight junction integrity and reduces brain edema by inhibiting autophagy formation in endothelial after ICH.