Protective effects of histone deacetylase 6 specific inhibitor tubastatin A on subarachnoid hemorrhage in rats and the underlying mechanisms / 中南大学学报(医学版)

OBJECTIVES@#Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease. Early brain injury (EBI) and cerebral vasospasm are the main reasons for poor prognosis of SAH patients. The specific inhibitor of histone deacetylase 6 (HDAC6), tubastatin A (TubA), has been proved to have a definite neuroprotective effect on a variety of animal models of acute and chronic central nervous system diseases. However, the neuroprotective effect of TubA on SAH remains unclear. This study aims to investigate the expression and localization of HDAC6 in the early stage of SAH, and to evaluate the protective effects of TubA on EBI and cerebral vasospasm after SAH and the underlying mechanisms.@*METHODS@#Adult male SD rats were treated with modified internal carotid artery puncture to establish SAH model. In the first part of the experiment, rats were randomly divided into 6 groups: a sham group, a SAH-3 h group, a SAH-6 h group, a SAH-12 h group, a SAH-24 h group, and a SAH-48 h group. At 3, 6, 12, and 24 h after SAH modeling, the injured cerebral cortex of rats in each group was taken for Western blotting to detect the expression of HDAC6. In addition, the distribution of HDAC6 in the cerebral cortex of the injured side was measured by immunofluorescence double staining in SAH-24 h group rats. In the second part, rats were randomly divided into 4 groups: a sham group, a SAH group, a SAH+TubAL group (giving 25 mg/kg TubA), and a SAH+TubAH group (giving 40 mg/kg TubA). At 24 h after modeling, the injured cerebral cortex tissue was taken for Western blotting to detect the expression levels of HDAC6, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining to detect apoptosis, and hematoxylin and eosin (HE) staining to detect the diameter of middle cerebral artery.@*RESULTS@#The protein expression of HDAC6 began to increase at 6 h after SAH (P<0.05), peaked at 24 h (P<0.001), and decreased at 48 h, but there was still a difference compared with the sham group (P<0.05). HDAC6 is mainly expressed in the cytoplasm of the neurons. Compared with the sham group, the neurological score was decreased significantly and brain water content was increased significantly in the SAH group (both P<0.01). Compared with the SAH group, the neurological score was increased significantly and brain water content was decreased significantly in the SAH+TubAH group (both P<0.05), while the improvement of the above indexes was not significant in the SAH+TubAL group (both P>0.05). Compared with the sham group, the expression of eNOS was significantly decreased (P<0.01) and the expressions of iNOS and HDAC6 were significantly increased (P<0.05 and P<0.01, respectively) in the SAH group. Compared with the SAH group, the expression of eNOS was significantly increased, and iNOS and HDAC6 were significantly decreased in the SAH+TubA group (all P<0.05). Compared with the SAH group, the number of TUNEL positive cells was significantly decreased and the diameter of middle cerebral artery was significantly increased in the SAH+TubA group (both P<0.05) .@*CONCLUSIONS@#HDAC6 is mainly expressed in neurons and is up-regulated in the cerebral cortex at the early stage of SAH. TubA has protective effects on EBI and cerebral vasospasm in SAH rats by reducing brain edema and cell apoptosis in the early stage of SAH. In addition, its effect of reducing cerebral vasospasm may be related to regulating the expression of eNOS and iNOS.

Role of SIRT1 in regulating endoplasmic reticulum stress in early brain injury after subarachnoid hemorrhage / 国际生物医学工程杂志

Objective:To investigate the effect of Sirtuin 1 (SIRT1) on subarachnoid hemorrhage (SAH) and its possible mechanism.Methods:A mouse model of SAH was constructed by internal carotid artery puncture. The protein and mRNA expression levels of SIRT1 at 0, 3, 6, 12, 24, 48, and 72 h were detected by Western Blot and qRT-PCR. A Western Blot assay was used to examine SIRT1 and the expression levels of endoplasmic reticulum stress-related markers GRP78, p-PERK/PERK, p-eIF2α/eIF2α, and CHOP after administration of a SIRT1 inhibitor or SIRT1 si-RNA. At 24 h after SAH, subarachnoid hemorrhage volume, neurological function score, brain water content, and blood-brain barrier integrity were measured.Results:The highest expression of SIRT1 protein and mRNA was observed at 24 h compared with other time points, and the differences were statistically significant (all P < 0.001). Inhibition of SIRT1 expression leads to increased expression of endoplasmic reticulum stress-related proteins GRP78, p-PERK/PERK, p-eIF2α/eIF2α, and CHOP, exacerbating hemorrhage and brain water content, disrupting blood-brain barrier integrity, and significantly reducing neurological function scores. Conclusions:Inhibition of SIRT1 expression significantly increased the endoplasmic reticulum response to excitation and exacerbated early brain injury after SAH.

Effect of neutrophils on cell pyroptosis in early brain injury following subarachnoid hemorrhage and its mechanism in mice / 中华神经医学杂志

Objective:To investigate the effect of neutrophils on cell pyroptosis and its mechanisms in mice with early brain injury (EBI) following subarachnoid hemorrhage (SAH).Methods:Seventy six male C57BL/6J mice were randomly divided into sham-operated group, SAH group, SAH+vehicle group, and SAH+anti-ly6G group ( n=19). SAH models in the latter 3 groups were established by modified endovascular perforation. Mice in the SAH+vehicle group and SAH+anti-ly6G group received intravenous injection of equal normal saline or anti-ly6G antibody (4 mg/kg) 24 h before SAH. At 24 h after SAH, immunofluorescent staining was used to detect the locations/expressions of neutrophils, S100 calcium binding protein A8 (S100A8) and gasdermin D (GSDMD); FJC staining was performed to assess the neuronal injury; modified Garcia test and rotarod test were used to evaluate the neurological functions, and brain water content test was applied to evaluate the brain edema; Western blotting was used to detect the expressions of S100A8, Toll-like receptor 4 (TLR4), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cleaved cysteinyl aspartate specific proteinase-1 (cleaved-caspase1), and cleaved N-terminal gasdermin D (GSDMD-N). Results:(1) Compared with those in the sham-operated group, neutrophil infiltration at the damaged cortex with highly expressed S100A8 in neutrophils was observed in the SAH group, and increased GSDMD expression at the damaged cortex and GSDMD co-localization in astrocytes, microglia and neurons were observed in the SAH group. (2) Compared with the sham-operated group, the SAH group and SAH+vehicle group had significantly increased numbers of infiltrated neutrophils and FJC-positive neurons, significantly decreased falling latency in the modified Garcia score and rotarod test, significantly increased brain water content, and significantly elevated expressions of S100A8, TLR4, NLRP3, cleaved-caspase1 and GSDMD-N ( P<0.05); the SAH+anti-ly6G group had statistically decreased numbers of infiltrated neutrophils and FJC-positive neurons, statistically increased falling latency in the modified Garcia score and rotarod test, statistically decreased brain water content, and statistically decreased expressions of S100A8, TLR4, NLRP3, cleaved-caspase1 and GSDMD-N compared with the SAH group and SAH+vehicle group ( P<0.05). Conclusion:Inhibition of neutrophils can down-regulate the S100A8 expression after SAH and attenuate TLR4/NLRP3 activation-mediated cell pyroptosis, thereby improving EBI.

sDR5 attenuates DR5-Trail-mediated neuronal apoptosis and improves long-term neurological deficits following subarachnoid hemorrhage / 中华神经医学杂志

Objective:To investigate the role and molecular mechanism of death receptor 5 (DR5) in early brain injury (EBI) after subarachnoid hemorrhage (SAH), as well as the neuroprotective effect of soluble DR5 (sDR5) on SAH.Methods:Experiment 1: SD rats were randomly divided into sham-operated group ( n=6) and SAH group (SAH model was established by carotid artery puncture, n=30), and the SAH group was further subdivided into post-SAH (6 h) group, post-SAH (12 h) group, post-SAH (24 h) group, post-SAH (48 h) group and post-SAH (72 h) group ( n=6); Western blotting was used to detect the expressions of tumor necrosis factor (TNF)-α and DR5; immunofluorescent DR5 and neuronal nuclear antigen (NeuN) double staining was used to evaluate the DR5 expression in neurons. Experiment 2: SD rats were randomly divided into sham-operated group, SAH group, Trail group (injected Trail agonist dordaviprone), and Trail+sDR5 group (injected dordaviprone+sDR5, n=6); at the 24 th h of successfully constructed SAH model, the caspase family protein levels were detected by Western blotting, and Tunel staining and immunofluorescent DR5 and Caspase-3 double staining were performed. Experiment 3: SD rats were divided into sham-operated group, SAH group, Trail group and Trail+sDR5 group ( n=6); long-term motor functions, by modified Gracia score, forelimb placement experiment, rotarod test and misstep experiment, were evaluated 5, 7 and 12 d after successfully constructed SAH model; and long-term learning and memory functions were detected by water maze experiment 14, 16, 18, 20 and 21 d after successfully constructed SAH model. Results:(1) Result of Experiment 1: the expressions of TNF-α and DR5 in sham-operated group, post-SAH (6 h) group, post-SAH (12 h) group, post-SAH (24 h) group, post-SAH (48 h) group and post-SAH (72 h) group were statistically different ( F=837.992, P<0.001; F=503.942, P<0.001), and these expressions peaked 24 h after SAH; immunofluorescent DR5 and NeuN double staining showed that DR5 was located in neurons after SAH. (2) Result of Experiment 2: compared with the SAH group and Trail group, the Trail+sDR5 group had significantly decreased levels of activated caspase-8, tBid and activated caspase-3, significantly decreased numbers of Tunel positive cells and DR5 and activated caspase-3 co-marked positive cells ( P<0.05). (3) Result of Experiment 3: compared with the SAH group and Trail group, the Trail+sDR5 group had significantly increased Garcia scores, decreased failure rate in forelimb placement experiment, prolonged duration of stick rotation, and decreased foot fault rate ( P<0.05), suggesting that sDR5 could improve the long-term motor function deficit after SAH; water maze experiment showed that 21 d after SAH, compared with the SAH group and Trail group, Trail+sDR5 group had significantly increased proportion of escape time in the original platform quadrat in total escape time and increased proportion of movement path in the original platform quadrat in total movement path after platform removal ( P<0.05), suggesting that sDR5 could improve long-term learning and memory impairment after SAH. Conclusion:The sDR5 can inhibit DR5-Trail-mediated neuronal apoptosis and improve long-term neurological functional deficits after SAH.

Schisandrin B Inhibits NLRP3 Inflammasome Pathway and Attenuates Early Brain Injury in Rats of Subarachnoid Hemorrhage / 中国结合医学杂志

OBJECTIVE@#To determine whether Schisandrin B (Sch B) attenuates early brain injury (EBI) in rats with subarachnoid hemorrhage (SAH).@*METHODS@#Sprague-Dawley rats were divided into sham (sham operation), SAH, SAH+vehicle, and SAH+Sch B groups using a random number table. Rats underwent SAH by endovascular perforation and received Sch B (100 mg/kg) or normal saline after 2 and 12 h of SAH. SAH grading, neurological scores, brain water content, Evan's blue extravasation, and terminal transferase-mediated dUTP nick end-labeling (TUNEL) staining were carried out 24 h after SAH. Immunofluorescent staining was performed to detect the expressions of ionized calcium binding adapter molecule 1 (Iba-1) and myeloperoxidase (MPO) in the rat brain, while the expressions of B-cell lymphoma 2 (Bcl-2), Bax, Caspase-3, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated specklike protein containing the caspase-1 activator domain (ASC), Caspase-1, interleukin (IL)-1β, and IL-18 in the rat brains were detected by Western blot.@*RESULTS@#Compared with the SAH group, Sch B significantly improved the neurological function, reduced brain water content, Evan's blue content, and apoptotic cells number in the brain of rats (P<0.05 or P<0.01). Moreover, Sch B decreased SAH-induced expressions of Iba-1 and MPO (P<0.01). SAH caused the elevated expressions of Bax, Caspase-3, NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the rat brain (P<0.01), all of which were inhibited by Sch B (P<0.01). In addition, Sch B increased the Bcl-2 expression (P<0.01).@*CONCLUSION@#Sch B attenuated SAH-induced EBI, which might be associated with the inhibition of neuroinflammation, neuronal apoptosis, and the NLRP3 inflammatory signaling pathway.

Effect of biglycan on neural apoptosis in mice with early brain injury after subarachnoid hemorrhage / 中华神经医学杂志

Objective To investigate the effect ofbiglycan (BGN) on neural apoptosis in mice with early brain injury (EBI) after subarachnoid hemorrhage (SAH).Methods SAH models were induced by endovascular perforation in young male C57BL/6J mice.(1) Totally,48 mice were randomly divided into sham-operated group,SAH 6 h group,SAH 12 h group,SAH 24 h group,SAH 48 h group,and SAH 72 h group (n=8);the BGN protein and mRNA expressions were detected by Western blotting and real-time quantitative PCR (qRT-PCR).(2) Totally,16 mice were randomly divided into sham-operated group and SAH 48 h group (n=8);immunofluorescent double staining was conducted to explore the BGN expression in the neurons of brain tissues.(3) Totally,24 mice were randomly divided into sham-operated group,sham+control lentivirus group,and sham+BGN lentivirus group (n=8);BGN lentiviral vector and control lentivirus were administered intracerebroventricularly 7 d before sham-operation;qRT-PCR was performed to explore the BGN mRNA expression.(4) Totally,48 mice were randomly divided into sham-operated group,SAH+control lentivirus group,and SAH+BGN lentivirus group (n=16);BGN lentiviral vector and control lentivirus were administered intracerebroventricularly 7 d before SAH;neurological scores were detected by modified Garcia scale and beam balance tests;TUNEL was used to detect the neuronal apoptosis,and Western blotting was performed to explore the expressions of nuclear transcription factor kappa B (NF-κB) and phosphorylated-(p-) NF-κB.Results (1) Mice in the SAH 48 h group had the highest BGN protein and mRNA expressions,which showed statistical differences as compared with the sham-operated group (P＜0.05).(2) A majority of BGN expressions were detected in the neurons 48 h after SAH.(3) The sham+BGN lentivirus group had statistically lower BGN mRNA expression than the sham+control lentivirus group (P＜0.05).(4) As compared with those in the SAH+control lentivirus group,both scores of modified Garcia scale and beam balance tests were significantly higher in SAH+BGN lentivirus group (6.125±1.246 vs.13.000±1.309;1.125±1.126 vs.2.875±0.835),and neural apoptosis ratio and ratio of p-NF-κB/NF-κB were significantly lower in the SAH+BGN lentivirus group (51.950％±11.166％ vs.31.938％±7.705％;1.161±0.156 vs.0.886±0.142,P＜0.05).Conclusion Inhibition of BGN can effectively reduce neuronal apoptosis in mice with EBI after SAH,and attenuate neurological deficits.

Baicalin Reduces Early Brain Injury after Subarachnoid Hemorrhage in Rats / 中国结合医学杂志

OBJECTIVE@#To evaluate the effect of baicalin on subarachnoid hemorrhage (SAH) in rats and explore the potential mechanisms.@*METHODS@#Sprague-Dawley rats underwent experimental SAH and received treatment with baicalin at 10 or 50 mg/kg after 2 and 12 h of SAH. Neurological scores, brain water content, Evans-blue extravasation, and levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), myeloperoxidase (MPO), and malondialdehyde (MDA) were measured 24 h after SAH. Expression of nuclear factor erythroid-related factor 2 (Nrf2), NAD(P)H: quinone oxidoreductase 1 (NQO1), matrix metalloproteinase-9 (MMP-9), aquaporin 4 (AQP4), occludin, and zonulaoccludens-1 (ZO-1) were detected in the brain by Western blot. Heme oxygenase-1 (HO-1) was detected by quantitative polymerase chain reaction, and tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were assessed by enzyme-linked immunosorbent assay.@*RESULTS@#Baicalin attenuated EBI 24 h after SAH in rats (P<0.05). Baicalin elevated neurological scores, GSH-Px, SOD, and increased the expression of Nrf2, NQO1, HO-1, occludin, and ZO-1 in SAH rats (P<0.05 or P<0.01). Baicalin reduced MPO, MDA, and the expression of MMP-9, AQP4, TNF-α, and IL-1β (P<0.05 or P<0.01).@*CONCLUSION@#Baicalin reduced SAH-induced EBI, partially via activation of the Nrf2/HO-1 pathway and inhibition of MMP-9 and AQP4.

A study on the relationship between placental inflammation, prenatal corticosteroids and early brain injury of preterm infants / 中国新生儿科杂志

Objective To study the correlation between histological chorioamnionitis (HCA),prenatal corticosteroids and early brain injury of preterm infants.Method From December 2014 to December 2016,preterm infants with gestational age ≤ 34 weeks admitted to our hospital and umbilical cord blood samples taken immediately after birth were reviewed.According to the results of pathological examination of their mother's placenta and the use of glucocorticoids (GCs),they were assigned into HCA+ GCs + group,HCA + GCs-group,HCA-GCs-group,and HCA-GCs +group.The levels of lnterleukin-6 (IL-6),hepcidin,erythropoietin (EPO),human activin A (ACV-A),S-100β protein,and CC-chemokine ligand 18 (CCL18) in premature infants' umbilical cord blood in each group were tested using ELISA method.The incidences of premature infants' early brain injury and the correlation with inflammatory factors in each group were analyzed.The ROC curve was used to analyze the sensitivity and specificity of S-100β protein and IL-6 level in predicting early brain injury in preterm infants with placenta inflammation.Result A total of 343 infants with gestational age ≤ 34 weeks and their umbilical cord blood samples were tested.Among the 343 premature infants,47.1％ suffered from early brain injury (98/208) in the HCA+ group;while 27.4％ suffered from early brain injury (37/135) in the HCA-group,the difference was statistically significant between the two groups (P ＜ 0.001).A total of 142 cases received prenatal GCs treatment,and 41 (28.9％) cases had early brain injury.201 cases didn't receive prenatal GCs treatment,and 94 (46.8％) had early brain injury.The differences between the two groups were also statistically significant (P=0.001).The incidence of early brain injury in the HCA+GCs-group was significantly higher than the HCA+GCs+ group,HCA-GCs-group and HCA-GCs+ group(P＜0.05).The S-100β protein and IL-6 level in umbilical cord blood of the HCA+GCs-group and HCA+GCs+ group were higher than the HCA-GCs-group(P＜0.05).The area under the ROC curve for IL-6 predicting early brain injury in preterm infants was 0.732 (95％CI 0.675～0.789,P＜0.05).The cut-off value of 213.45 pg/ml of IL-6 (was selected to predict the risk of early brain injury with the sensitivity of 41.9 ％ and the specificity 99.0 ％.The area under the ROC curve for S-100β protein was 0.511 (95％CI 0.449～0.574,P=0.723).Conclusion Placental inflammation and insufficient prenatal glucocorticoids treatment are closely related to the occurrence of early brain injury in preterm infants.S-100β protein and IL-6 in umbilical cord blood may play an important role in early brain injury of premature infants.The IL-6 level has a higher predictive value for early brain injury,while S-100β protein level has a less predictive value.

Effect of Gastrodin on Early Brain Injury and Neurological Outcome After Subarachnoid Hemorrhage in Rats / 神经科学通报·英文版

Gastrodin is a phenolic glycoside that has been demonstrated to provide neuroprotection in preclinical models of central nervous system disease, but its effect in subarachnoid hemorrhage (SAH) remains unclear. In this study, we showed that intraperitoneal administration of gastrodin (100 mg/kg per day) significantly attenuated the SAH-induced neurological deficit, brain edema, and increased blood-brain barrier permeability in rats. Meanwhile, gastrodin treatment significantly reduced the SAH-induced elevation of glutamate concentration in the cerebrospinal fluid and the intracellular Ca overload. Moreover, gastrodin suppressed the SAH-induced microglial activation, astrocyte activation, and neuronal apoptosis. Mechanistically, gastrodin significantly reduced the oxidative stress and inflammatory response, up-regulated the expression of nuclear factor erythroid 2-related factor 2, heme oxygenase-1, phospho-Akt and B-cell lymphoma 2, and down-regulated the expression of BCL2-associated X protein and cleaved caspase-3. Our results suggested that the administration of gastrodin provides neuroprotection against early brain injury after experimental SAH.

The role of toll-like receptor 4 in early brain injury in rats with subarachnoid hemorrhage and its effect on the autophagy of neurons in the hippocampus / 西安交通大学学报(医学版)

Objective: To investigate the role of Toll-like receptor 4 (TLR4) in early brain injury in rats with subarachnoid hemorrhage and its effect on autophagy in hippocampus CA1 area. Methods: Totally 192 SD rats were randomly divided into sham operation group, SAH model group (model group), SAH+DMSO solvent group (solvent group), and SAH+TLR4 inhibitor group (CLI-095 group). Each group was divided into 24 h and 48 h 2 time points. The SAH model was established by internal carotid artery puncture. The drug-administered group was injected with 10 μL of DMSO solution or 100 μg/mL of CLI-095 solution of 10 μL before the preparation of SAH model. The behavioral changes of the rats were detected by Garcia score; the cerebral edema was detected by wet and dryweight method. The morphology of hippocampal CA1 neurons was observed by HE staining. Immunohistochemistry and immunoblotting were used to detect the expressions of TLR4, LC3 and Beclin1 in the hippocampus CA1 area. Results: Compared with the sham group, Garcia score was reduced at each time point (P<0.05). The degree of brain edema was increased and the number of viable neurons was reduced (P<0.05). The expressions of TLR4, LC3 and Beclin1 were increased in the hippocampus CA1 area in the model group (P<0.05). Compared with the model group, the Garcia score was increased at each time point (P<0.05). The degree of brain edema was reduced and the number of viable neurons was increased (P<0.05). The expressions of TLR4, LC3 and Beclin1 were reduced in the hippocampus CA1 area in the CLI-095 group (P<0.05). Conclusion: TLR4 may participate in the regulation of SAH-induced autophagy and aggravate the process of secondary brain injury in SAH.

Involvement of phosphatidylinositol 3-kinase/protein kinase B signaling pathway in early brain injury after subarachnoid hemorrhage and its mechanism / 中华神经医学杂志

Objective To explore the role of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway in early brain injury after subarachnoid hemorrhage (SAH) and its possible mechanism in rats. Methods Ninety healthy male SD rats were randomly divided into 5 groups (n=18) according to the random number table, namely sham-operated group, SAH group, vehicle control group, SAH+PI3K/Akt pathway agonist group (group A), and SAH+PI3K/Akt pathway inhibitor group (group I, n=18). Rat SAH models of the later 4 groups were established by intravascular puncture; rats in the vehicle control group, group A, and group I were given 10μL dimethyl sulfoxide (DMSO), insulin-like growth factor (IGF-1, 1μg/10 μL), and Ly294002 (25μg/10μL) solution, respectively, before model making. After 24 h of intravascular puncture, modified Garcia nerve function scale was used to evaluate the motor function, dry-wet weight method was used to detect the water content of the brain tissues, and immunofluorescence staining was employed to observe the β-amyloid precursor protein (β-APP)/ βtubulin III positive expressions in the subcortical white matter, and Western blotting was employed to detect theβ-APP, and total (t)-and phosphorylated (p)-PI3K and Akt protein expressions in the brain tissues; one month after modeling, HE staining was used to observe the changes of cell structure after cerebral edema. Results As compared with the sham-operated group, SAH model group had significantly lower modified Garcia nerve function scale scores, significantly increased brain water content, significantly larger number ofβ-APP/βtubulin III positive cells, statistically increasedβ-APP protein expression, and significantly increased t-PI3K, and t-Akt protein expressions (P<0.05). As compared with the SAH model group, group A had significantly higher modified Garcia nerve function scale scores, significantly decreased brain water content, significantly smaller number of β-APP/ βtubulin III positive cells, statistically decreasedβ-APP protein expression, and significantly increased p-PI3K and p-Akt protein expressions (P<0.05). As compared with group A, group I had significantly lower modified Garcia nerve function scale scores, significantly increased brain water content, significantly larger number ofβ-APP/βtubulin III positive cells, statistically increasedβ-APP protein expression, and significantly decreased p-PI3K and p-Akt protein expressions (P<0.05). HE staining showed serious morphological damage. Conclusion PI3K/Akt signaling pathway plays an important role in early brain injury after SAH in rats;early stimulation of PI3K/Akt signaling pathway activation can alleviate early brain injury after SAH, and the mechanism may be related to regulation of axonal injury in white matter after SAH.

Role of hesperitin in regulating inflammatory response in early brain injury after subarachnoid hemorrhage / 中华神经医学杂志

Objective To investigate the role of hesperitin in regulating inflammatory response in early brain injury after subarachnoid hemorrhage (SAH). Methods A total of 96 adult male SD rats were divided into sham-operated group, SAH group, solvent group and intervention group (n=24) by random number table method. The SAH rat models in the latter three groups were prepared by carotid artery puncture method; the rats in the intervention group were given oral administration of hesperidin solution, which was dissolved in 5％ dimethyl sulfolide (DMSO), with a concentration of 1 mg/100 μL and a dosage of 40 mg/kg within 30 min after operation; rats in the solvent group were given oral administration of an equal volume of 5％ DMSO solution. Modified Garcia behavioral scale was used to evaluate the neurobehavior of rats, and the wet/dry weight method was used to measure the water content in the brain tissues of the left and right hemispheres of the rats 24 h after SAH. Immunofluorescence staining was used to detect the microglia activation, Fluoro-dyed Jade C staining was used to assess the brain neuron degeneration, enzyme-linked immunosorbent assay (ELISA) was employed to detect the inflammatory factors interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) content in the brain tissues, and Western blotting was used to detect the nuclear factor-κB (NF-κB) and phosphorylated (p) -NF-κB protein expressions. Results As compared with the solvent group, intervention group had significantly increased improved modified Garcia behavioral scale scores (10.08±1.73 vs. 13.83±1.70), and significantly decreased water content of brain tissues in the left and right hemispheres ([81.44 ± 1.05]％ vs. ([79.14±0.82]％; [80.55±1.55]％ vs. [78.79±1.02]％), significantly smaller number of CD68+ and Iba1+ microglias (30.17±1.04 vs. 10.67±0.75; 29.33±1.16 vs. 12.00±0.41), significantly smaller number of degenerate neurons (53.21±0.94 vs. 31.33±0.28), significantly reduced levels of inflammatory cytokines IL-1β, IL-6 and TNF-α ([429.88±106.32] pg/mL vs. [221.50±48.80] pg/mL; [1015.50±221.80] pg/mL vs. [448.11±93.40] pg/mL; [1021.75±149.17] pg/mL vs. [595.71±190.81] pg/mL), and significantly lower p-NF-κB/NF-κB ratio (1.13±0.07 vs. 0.71±0.02, P<0.05). Conclusion Hesperitin may reduce the inflammatory response mediated by microglia after subarachnoid hemorrhage by inhibiting NF-κB pathway, thereby improving the neurological dysfunction of rats.

Inhibition of JAK2/STAT3 to ameliorate early brain injury following subarachnoid hemorrhage through downregulating expression and nuclear translocation of high mobility group box 1 / 中华神经医学杂志

Objective To investigate the role of JAK2/STAT3 signaling pathway in regulating the expression and nuclear-cytoplasm translocation of high mobility group box 1 (HMGB1) in early brain injury (EBI) after subarachnoid hemorrhage (SAH).Methods Ninety SD rats were divided into a sham group (15 rats),an SAH group (altogether 45 rats,with 15 ones for each time point of 6 h,1 d,and 3 d),an SAH+AG490 (JAK2/STAT3 inhibitor) group (15 rats) and an SAH+dimethyl sulfoxide (DMSO)group (15 rats).The SAH models in the later 3 groups were established by endovascular perforation;the blood vessels were not perforated in the sham group but the other operations were the same as in the SAH groups.(1) Western blotting was used to detect the expression of HMGB1 and phosphorylated JAK2/STAT3 (p-JAK2/p-STAT3) in the 4 groups (at different time points in the SAH group) and compared the expression changes between the 4 groups after AG490 intervention.(2)Immunofluorescence confocal microscopy was used to detect HMGB1 nuclear translocation in the 4 groups.(3) TUNEL staining was used to detect apoptosis in the 4 groups.(4) Brain water contents and neurobehavioral scores in the 4 groups were measured.Results (1) Western blotting showed that the expression levels ofp-JAK2 and p-STAT3 were significantly increased at 6 h,1 d,and 3 d after SAH,and there were significant differences between the sham group and the SAH group (P＜0.05).HMGB1 total protein,cytoplasmic HMGB1 and nucleus HMGB1 also increased significantly at different time points after SAH,and statistically significant differences existed between the sham group and the SAH group (P＜0.05).The expression levels ofp-JAK2/p-STAT3,HMGB1 and cytoplasm and nucleus HMGB1 in the SAH+AG490 group were significantly lower than in the SAH group and SAH+DMSO group(P＜0.05).(2) The immunofluorescence staining showed that HMGB1 staining was positive in the SAH group while the positive staining of HMGB1 was present mainly in the nucleus but not in the cytoplasm in the sham and SAH+AG490 groups,suggesting that AG490 might inhibit the nucleus-cytoplasm transposition of HMGB1.(3) Compared with the SAH and SAH+DMSO groups,the TUNEL staining positive cells in the SAH+AG490 group were significantly decreased (P＜0.05).(4) Compared with the SAH and SAH+DMSO groups,the brain water contents in the SAH+AG490 group decreased significantly and the neurobehavioral scores increased significantly (P＜0.05).Conclusions JAK2/STAT3 signaling pathway is involved in the pathological process of early brain injury after SAH,and its mechanism may be related to the regulation of HMGB1 expression and nuclear-cytoplasm transposition.The regulation of JAK2/STAT3 may contribute to the neuroprotection dependent of HMGB 1.

Expression profile of circular RNA in mouse cortex after subarachnoid hemorrhage / 中华神经医学杂志

Objective To explore the altered expression of circular RNA (circRNA) and mRNA in the mouse cortex in the early phase of subarachnoid hemorrhage (SAH) and possible biological functions of the circRNA in early brain injury (EBI).Methods A total of 18 C57BL/6J male mice were randomly divided into a sham and a SAH group (n=9).SAH models were prepared by endovascular perforation.Total RNAs of brain samples were extracted to construct the cDNA library 24 h after operation.RNA-sequencing (RNA-seq) was carried out by HiSeqTM 2500 User Guide and followed by RT-qPCR for confirmation.Reads were aligned to the mouse transcriptome to obtain expression profiles ofcircRNA and mRNA.Bioinformatic study included GO analysis,KEGG pathway analysis and forecast of targeted miRNA of circRNA.Results A total of 26 circRNA (6 up-regulated and 20 down-regulated) and 804 mRNA (396 up-regulated and 408 down-regulated) were significantly changed.These altered mRNA were mainly related to regulation of neuronal synaptic plasticity,inflammatory and immune response.Bioinformatics showed that some significantly altered circRNA contained binding sites for many miRNA.The RT-qPCR analysis of 4 randomly selected circRNA (circFoxj3,circSetbp1,circArpp21 and circ2010111 I01Rik) confirmed the accuracy of RNA-seq.Conclusions SAH alters the expression of circRNA in mouse cortex and the differentially expressed circRNA may be involved in regulation of EBI following SAH,promising a potential therapeutic target for the diagnosis,treatment and prognosis of SAH.

Tristetraprolin attenuates subarachnoid hemorrhage-induced early brain injury in rats / 中国病理生理杂志

AIM:To explore the expression level of tristetraprolin(TTP)in rats after subarachnoid hemor-rhage(SAH)as well as the potential role of TTP in the early brain injury(EBI)after SAH in rats.METHODS:In the first experiment setting,total 56 adult male SD rats were randomly divided into sham group and SAH group.The SAH mod-el was performed by endovascular perforation.The brain tissues were taken out after SAH at 5 different time points(0,12, 24,48,72 h and 1 week).The expression of TTP in the brain tissues was detected by Western blot.In the second experi-ment,a total of 60 SD rats were divided into 4 groups: sham group, SAH group, SAH +vector group and SAH +TPP group.Neurological score,brain water content and blood-brain barrier were evaluated at 48 h after SAH.TUNEL staining was performed to detect cell apoptosis in the rat brain tissue.ELISA method was used for quantitative detection of interleu-kin-6(IL-6)and tumor necrosis factor-α(TNF-α).The protein levels of TTP,Bax,Bcl-2 and cleaved caspase-3 in the rat brain tissue were detected by Western blot.RESULTS:The protein expression of TTP in the brain was downregulated markedly from 12 h after SAH,reached the lowest level at 48 h,and then had an upward trend.After modeling for 48 h, Garcia neurological score was significantly reduced,and brain water content and Evans blue(EB)content of the brain tis-sue of the rats in SAH group were significantly higher than those in sham group(P<0.05).SAH induced significant in-creases in IL-6 and TNF-αlevels in the brain tissue(P<0.05).The number of TUNEL-stained cells was increased in the subcortical brain region after SAH compared with sham group.In addition,a lower level of Bcl-2 and higher levels of Bax and cleaved caspase-3 in the rat brains were observed at 48 h after SAH.However,the neurological deficit score was signif-icantly increased,and the brain water content and EB content in the rat brains were significantly reduced in SAH +TTP group in comparison with SAH +vector group(P<0.05).Over-expression of TTP dramatically suppressed the levels of IL-6 and TNF-αin the rat brains,and reduced the number of TUNEL positive cells.Furthermore,upregulation of TTP signifi-cantly decreased the levels of cleaved caspase-3 and Bax, and evidently enhanced the expression of Bcl-2(P<0.01). CONCLUSION:The expression of TTP is significantly decreased in early period after SAH, and enhancing the level of TTP effectively inhibits EBI following SAH in rats.

The influence of U0126 of ERK signaling pathway inhibitor on early brain inj ury and neurons autophagy in SAH rats / 西安交通大学学报(医学版)

ABSTRACT:Objective To explore role of U0126,the specific inhibitor of ERK signaling pathway,in early brain injury (EBI)and the autophagy of nerve cells in hippocampus area in subarachnoid hemorrhage (SAH). Methods A total of 48 male adult SD rats were randomly divided into control group,SAH group,DMSO+SAH group,and U0126+SAH group,with 12 in each.We established SAH rat model by the puncture of internal carotid artery.The same amount of saline water,DMSO and U0126 solution of 0.5 mL per rat was injected respectively into the rats of different groups 30 min before modeling.The rats were killed at 24 h.To measure brain water content by Wet and dry method after 24 h,the morphological changes of hippocampus CA1 neural cells were observed by microscopy;the expression levels of ERK,Beclin-1 and LC3 were detected by using immunohistochemical method. Results Compared with that in sham group,brain water content increased obviously in SAH model group.The density of surviving neurons in SAH group was significantly lower than that in control group (P<0 .0 5 ).ERK signaling pathway was activated obviously,the expressions of Beclin 1 and LC3-Ⅱ were significantly higher than those in control group (P<0.05).Compared with SAH model group,in U0126 group brain water content increased obviously.Compared with those in SAH group,the density of surviving neurons was significantly lower (P<0.05), ERK signaling pathway was suppressed,the expressions of Beclin-1 and LC3-Ⅱ were significantly lower (P<0.05). Conclusion The U0126,the ERK signaling pathway inhibitor,can inhibit neuron autophagy and increase EBR of SAH.

Protective effect of mitochondria-targeted antioxidant SS31 on early brain injury following subarachnoid hemorrhage in rats / 中南大学学报(医学版)

Objective:To evaluate protective effects of SS31 on early brain injury (EBI) induced by subarachnoid hemorrhage (SAH) in rats.Methods:A total of 96 Sprague-Dawley rats were randomly divided into 4 groups:A sham group,an SAH group,an SAH+vehicle group (SAH+V),and an SAH+SS31 group.The SAHinduced prechiasmatic cistern rat model was established in this study.Neurological deficit scores were evaluated at 24 h after SAH.The SS31 (5 mg/kg) as well as equal volume of vehicle were administrated intraperitoneally at 2 h after SAH.The neurological scores,brain edema,blood-brain barrier (BBB) permeability,apoptosis,malondialdehyde (MDA),glutathione peroxidase (GPx) activity,superoride dismutase (SOD) activity,and the expression ofcytosolic cytochrome c (Cyt C) and Bax were analyzed at 24 h after SAH.Results:Treatment with SS31 could significantly reduce MDA levels,and restored the activities of GPx and SOD in the cortex following SAH when compared with the SAH+V group.In addition,Bax SS31 trearment increased or decreased the levels of mitochondrial Cyt C or Bax,respectively.Moreover,SS31 treatment ameliorated brain edema and Evans blue dye extravasation,improved neurological deficits,and decreased neuronal apoptosis at 24 h after SAH.Conclusion:SS31 could alleviate EBI after SAH through its antioxidant property and ability in inhibition of neuronal apoptosis.

Neuroprotective effects of protease-activated receptor-1 antagonist, SCH79797, on early stage of brain injury and expression patterns of brain-derived neuro trophic factor and nerve growth factor after subarachnoid hemorrhage in rats / 中华神经医学杂志

Objective To investigate the neuroprotective effects of protease-activated receptor (PAR)-1 antagonist SCH79797 on early stage of brain injury and expression patterns of brain-derived neuro trophic factor (BDNF) and nerve growth factor (NGF) after subarachnoid hemorrhage (SAH) in rats. Methods Fifty-four clean male SD rats were randomly divided into three groups:sham-operative group, SAH group and treatment group (n=18). SAH rat models in the later two groups were induced by single cisterna magna blood injection. Rats in the treatment group were given SCH79797 (25μg/kg) after SAH modeling. Neurologic function was assessed and scored after SAH for 24 h. Then the rats were sacrificed, and then, the brains were collected for determination of brain water content. The expression patterns of BDNF and NGF in cerebral tissues were tested by enzyme linked immunosorbent assay and immunofluorescence method. Results As compared with rats in the SAH group, rats in the treatment group had significantly increased neurological deficit scale scores (12.11 ±2.62 vs. 14.59 ±2.24), statistically decreased water content in brain tissues (83.01%±0.38%vs. 79.79%±0.44%), with significant difference (P<0.05). As compared with those in the SAH group, the expressions of BDNF and NGF in treatment group were significantly higher ([100.15±59.13] pg/mL vs. [368.00±137.52] pg/mL; [33.44± 2.21] pg/mL vs. [37.49±2.29] pg/mL, P<0.05). As compared with those in the SAH group, the immunofluorescent staining intensities of BDNF and NGF in treatment group were significantly higher (41.65±1.50 vs. 91.40±1.30;23.50±1.70 vs. 30.65±1.80, P<0.05). Conclusion SCH79797 given early may reduce the degree of brain edema and increase the expressions of BDNF and NGF, which has obvious neuroprotective effect.

Expressions of brain derived neurotrophic factor and tropomyosin-related kinase B and their significances in rats with early brain injury after subarachnoid hemorrhage / 中华神经医学杂志

Objective To observe the expressions of brain derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) in rats with early brain injury (EBI) after subarachnoid hemorrhage,and study the neuroprotective effects of BDNF and TrkB on EBI.Methods Male Sprague-Dawley rats (n=56),weighing 280-320 g,were randomly divided into sham-operated group and SAH group;SAH models were established by endovascular perforation ofinternal carotid artery.At 24 and 72 h after modeling,neurological scale scores were recorded;brain water content was measured;immunohistochemical staining and ELISA were used to observe the dynamical expressions of BDNF and TrkB in the brain.Results At 24 and 72 h after modeling,the neurological function scores and brain water content of SAH rats were higher than those of sham-operated group.The expression scores of BDNF in the SAH rats were 1.33±0.52 and 1.67±0.52,and the expression levels were (12.11±0.44) mg/mL and (15.82±0.89) mg/mL;the expression scores of TrkB were 1.17±0.75 and 2.00±0.00,and the expression levels were (18.89±0.38) mg/mL and (25.18±0.68) mg/mL.The expression scores of BDNF in the sham-operated group were 0.33±0.52 and 0.17±0.41,and the expression levels of BDNF in the sham-operated group was (4.92±0.16) mg/mL and (4.93±0.20) mg/mL;the expression scores of TrkB were 0.17±0.41 and 0.33±0.52,and the expression levels were (8.52±0.41) mg/mL and (8.08±0.34) mg/mL.There were significant differences in BDNF and TrkB expressions between the two groups at 24 and 72 h after modeling (P＜0.05).Conclusion The expressions of BDNF and TrkB increase significantly after SAH,and BDNF and TrkB play protective effect on EBI after SAH.

Effect of Extracellular Regulated Protein Kinases Signaling Pathway on Early Brain Injury and Neurons Autoph-agy in Rats with Subarachnoid Hemorrhage / 中国康复理论与实践

Objective To explore the effect of extracellular regulated protein kinases (ERK) signaling pathway on early brain injury and autophagy of nerve cell in hippocampus area in rats with subarachnoid hemorrhage (SAH). Methods Forty-eight adult male Sprague-Daw-ley rats were randomly divided into sham group, SAH group, SAH+dimethyl sulfoxide (DMSO) group and SAH+U0126 group, with 12 rats in each group. The SAH model was established with puncture of internal carotid artery. The SAH+U0126 group was injected with U0126 0.05 mg/kg;the sham group and SAH group were injected with normal saline, and the SAH+DMSO group was injected with DMSO 30 min-utes before modeling. They were sacrificed 24 hours after modeling. The brain water content was measured with wet and dry method. The morphology changes of neural cells in hippocampus CA1 were observed by HE staining. The expression of phosphorylation ERK (p-ERK), Beclin-1 and LC3-Ⅱwere detected with immunohistochemical method and Western blotting. Results Compared with the sham group, the brain water content increased (P0.05). Conclusion The activation of ERK signaling pathway may alleviate early brain injury after SAH by regulation of autophagy.