| Part 1 Effect of Atorvastatin on endoplasmic reticulum stress-induced neuronal apoptosis after hydraulic brain injuryObjective: To observe the protective effect of atorvastatin on the anti-endoplasmic reticulum stress apoptosis in rats after hydraulic impact brain injury.Methods: Thirty-two healthy adult SD rats(purchased from Animal Center of Hebei Medical University),weighing about 250 g,were made into brain injury models by hydraulic shock method.The rats were randomly divided into 4 groups: sham operation group(Sham group),brain injury group(TBI group),Vehicle group(TBI+Vehicle group),atorvastatin intervention group(Atorv group),each group of 8 only.Sham group only performed craniotomy and no hydraulic impact;TBI group only performed hydraulic impact;TBI + vehicle group opened cranial window to make hydraulic impact,and given equal volume of normal saline;Atorv group performed hydraulic impact and gave 10mg/kg atorvastatin.Atorvastatin was dissolved in physiological saline,and it was administered orally once a day 3 days before surgery until the material was taken.The rats in each group underwent neurological deficit score(mNSS)24 hours after successful modeling,followed by excessive anesthesia execution,brain tissue was quickly retained on the ice plate,brain tissue samples about 100 mg at the front edge of the injury area were selected to measure brain tissue water content by dry-wet weight method;the completely broken brain tissue in the contusion area was removed,and the penumbra cerebral cortex within the range of the contusion area was cut out in a coronal shape,and then quickly frozen in liquid nitrogen,and then stored in a-80 ℃ refrigerator,and then GRP78 and Caspase3 protein were detected by Western Blot.Results:1.Neurological deficit score(mNSS)Rats in each group underwent hydraulic shock showed obvious symptoms of neurological deficits after 24 hours.The main symptoms were increased heart rate,irregular breathing,increased muscle tension in the extremities,and skew to the side of hemiplegia.Sham group rats had no obvious symptoms of neurological deficits.Compared with the Sham group,the neurological deficit symptoms in the TBI group were significantly increased(P<0.05);the neurological deficit symptoms were not significantly different between the TBI group and the Vehicle group(P>0.05);the neurological deficits in Atorv group were lower than those in TBI group(P <0.05).2.Brain water content detectionCompared with the Sham group,the brain water content in the TBI group was significantly increased(P<0.05);there was no significant difference in the brain water content between the TBI group and the Vehicle group(P>0.05);the brain tissue water content in Atorv group was lower than that in TBI group(P<0.05).3.Western BlotGRP78,Caspase3: Compared with the Sham group,GRP78,Caspase3 protein expression in the TBI group was significantly increased(P<0.05);there were no significant differences in the expression of GRP78,Caspase3 protein between the TBI group and the Vehicle group(P>0.05);the expression of GRP78,Caspase3 protein in the Atorv group was lower than that in the TBI group(P<0.05).Conclusions:1.The content of endoplasmic reticulum stress iconic protein GRP78 and apoptosis-related protein Caspase3 were significantly increased after hydraulic impact brain injury in rats,and the symptoms of nerve function defect were significantly aggravated,which indicated that endoplasmic reticulum stress appeared after hydraulic impact brain injury and induced neuronal apoptosis.2.After traumatic brain injury caused by hydraulic shock,atorvastatin can protect against endoplasmic reticulum stress-induced neuronal apoptosis.Part 2 Explore the mechanism of atorvastatin inhibiting neuronal apoptosis induced by endoplasmic reticulum stressObjective:To observe the effect of atorvastatin on Nrf2 after hydraulic shock brain injury in rats,and to explore its neuroprotective effect and its mechanism on endoplasmic reticulum stress and apoptosis induced by hydraulic shock brain injury.Methods: Thirty-two healthy adult SD rats,weighing about 250 g,were made into brain injury models by hydraulic shock method.The rats were randomly divided into 4 groups: sham operation group(Sham group),brain injury group(TBI group),Vehicle group(TBI+Vehicle group),atorvastatin intervention group(Atorv group),each group of 8 only.The rats in each group underwent neurological deficit score 24 hours after successful modeling,followed by excessive anesthesia execution,brain tissue was quickly retained on the ice plate,brain tissue samples about 100 mg at the front edge of the injury area were selected to measure brain tissue water content by dry-wet weight method;remove the completely broken brain tissue in the contusion area,cut the cortex of the penumbra in the area of the contusion into two parts,divide one part into liquid nitrogen and freeze it quickly,then store it in-80 ℃refrigerator,and then use GRP78,P-PERK,P-eif2α,ATF6,P-IRE1α,P-JNK,CHOP,Caspase3,Caspase12,Nrf2 nuclear protein and cytosolic protein were detected by Western Blot.Another was performed immediately by flow cytometry.Results:1.Neurological deficit scoreRats in each group underwent hydraulic shock showed obvious symptoms of neurological deficits after 24 hours.The main symptoms were increased heart rate,irregular breathing,increased muscle tension in the extremities,and skew to the side of hemiplegia.Sham group rats had no obvious symptoms of neurological deficits.Compared with the Sham group,the neurological deficit symptoms in the TBI group were significantly increased(P<0.05);the neurological deficit symptoms were not significantly different between the TBI group and the Vehicle group(P>0.05);the neurological deficits in Atorv group were lower than those in TBI group(P<0.05).2.Brain water content detectionCompared with the Sham group,the brain water content in the TBI group was significantly increased(P<0.05);there was no significant difference in the brain water content between the TBI group and the Vehicle group(P>0.05);the brain tissue water content in Atorv group was lower than that in TBI group(P<0.05).3.The early apoptosis rate by flow cytometryCompared with the Sham group,the early neuronal apoptosis rate in the TBI group was significantly increased(P<0.05);there was no significant difference in early neuronal apoptosis rate between the TBI group and the Vehicle group(P>0.05);the early apoptotic rate of neurons in Atorv group was lower than that in TBI group(P<0.05).4.Western BlotGRP78,P-PERK,P-eif2α: Compared with the Sham group,GRP78,P-PERK,and P-eif2α protein expression in the TBI group was significantly increased(P<0.05);there were no significant differences in the expression of GRP78,P-PERK,and P-eif2α protein between the TBI group and the Vehicle group(P>0.05);the expression of GRP78,P-PERK,and P-eif2α protein in the Atorv group was lower than that in the TBI group(P<0.05).P-IRE1α,P-JNK: Compared with the Sham group,the expressions in the TBI group,Vehicle group and Atorv group were significantly increased(P<0.05);between TBI group,TBI + vehicle group and Atorv group There were no significant differences in the expression of-IRE1α and P-JNK(P> 0.05).ATF6,Caspase12,CHOP,Caspase3: Compared with the Sham group,the expression of ATF6,Caspase12,CHOP,and Caspase3 in the TBI group was significantly increased(P<0.05);there was no significant difference in the expression of ATF6,Caspase12,CHOP,and Caspase3 in the TBI group and Vehicle group(P>0.05);the expressions of ATF6,Caspase12,CHOP and Caspase3 in Atorv group were lower than those in TBI group(P<0.05).Nrf2: Compared with the Sham group,the expression of Nrf2 nuclear protein in the TBI group was significantly increased(P<0.05);there was no significant difference in the expression of Nrf2 nuclear protein between the TBI group and the Vehicle group(P>0.05);the expression of Nrf2 nuclear protein in Atorv group was higher than that in TBI group(P<0.05).Compared with the Sham group,the expression of Nrf2 cytosolic protein in the TBI group was significantly reduced(P<0.05);there was no significant difference in the expression of Nrf2 cytosolic protein between the TBI group and the Vehicle group(P>0.05);the expression of Nrf2 cytosolic protein in Atorv group was lower than that in TBI group(P<0.05).Conclusions:1.Atorvastatin can increase Nrf2 activity after intervention.2.After traumatic brain injury caused by hydraulic shock,atorvastatin can protect nerve cells by inhibiting the expression of proteins related to the PERK,ATF6 and Caspase12 apoptotic pathways,regardless of the IRE1 pathway.Part 3 Explore the mechanism of atorvastatin up-regulated Nrf2 expression inhibits neuronal apoptosis induced by endoplasmic reticulum stressObjective: To determine whether Nrf2 participates in the anti-apoptotic effect of atorvastatin on brain injury induced by hydraulic shock in mice by measuring the neurological function score,brain tissue water content,Nrf2 and marker protein expression levels of various apoptosis pathways.Methods: The model of TBI was established by hydraulic percussion in 8～12 weeks old adult male wild type ICR mice Nrf2(+/+)and Nrf2 knockout mice Nrf2(-/-)(donated by Hebei key Laboratory of Neuroscience,second Hospital of Hebei Medical University)in the background of ICR.Under the condition of 22-25 °C and 50% humidity in animal center of the second Hospital of Hebei Medical University,inbred lines were used for reproduction,light / dark circulation for 12 hours,and water intake was free.Thirty-two male mice weighing 30 ±2 g were randomly divided into two groups: Nrf2(-/-)(n=16)and Nrf2(+/+)(n=16).Nrf2(-/-)and Nrf2(+/+)were randomly divided into two groups:TBI+Vehicle group(Vehicle group,n=8)and TBI+Atorv group(Atorv group,n=8).All groups received hydraulic shock.The TBI+Atorv group received 10 mg / kg atorvastatin,and the Vehicle group received an equal volume of normal saline.Atorvastatin was was administered orally once a day 3 days before surgery until the material was taken.Results:1.Neurological deficit scoreMice in each group showed obvious symptoms of neurological deficits after 24 hours.The main symptoms were increased heart rate,irregular breathing,increased muscle tension in the extremities,and skew to the side of hemiplegia.The neurological deficit symptoms of Nrf2(+/+)mice in the Atorv group were significantly lighter than those in the other groups(P<0.05);the Nrf2(-/-)mice in the Atorv group were more significant than the Vehicle group Nrf2(-/-)mice decreased(P<0.05),but were higher than that of Nrf2(+/+)mice in the Atorv group(P<0.05).2.The water content of brain tissueThe brain water content of Nrf2(+/+)mice in Atorv group was significantly less than that of other groups,the difference was statistically significant(P<0.05);the brain water content of Nrf2(-/-)mice in Atorv group was lower than that of Nrf2(-/-)mice in Vehicle group(P<0.05),but higher than that of Nrf2(+/+)mice in Atorv group(P<0.05).3.The degree of neuronal apoptosisThe early apoptotic rate of Nrf2(+/+)mouse nerve cells in Atorv group was significantly lower than that in the other groups,and the difference was statistically significant(P<0.05);the early apoptosis rate of Nrf2(-/-)mice in Atorv group was lower than that of Nrf2(-/-)mice in Vehicle group(P<0.05),but higher than that of Nrf2(+/+)mice in Atorv group(P<0.05).4.Western BlotNrf2: 24 hours after brain injury,the Nrf2 nuclear protein expression of Nrf2(+/+)mice in the Atorv group was higher than that of the Nrf2(+/+)mice in the Vehicle group(P<0.05);in contrast,Nrf2(+/+)mice in the Atorv group had lower expression of Nrf2 cytoplasmic proteins than Nrf2(+/+)mice in the Vehicle group(P<0.05);Nrf2(-/-)mice had no significant Nrf2 expression in each group.GRP78,P-PERK and P-eif2α: The content of GRP78,P-PERK,and P-eif2α in Nrf2(+/+)mice in the Atorv group was significantly lower than those in the other groups,and the differences were statistically significant(P <0.05);the protein content of GRP78,P-PERK and P-eif2α in Nrf2(-/-)mice in the Atorv group was lower than that of the Nrf2(-/-)mice in the Vehicle group(P<0.05),but higher than the Nrf2(+/+)mice in the Atorv group(P <0.05).ATF6,Caspase12,Caspase3 and CHOP: The contents of ATF6,Caspase12,Caspase3,and CHOP in Nrf2(+/+)mice in the Atorv group were significantly lower than those in the other groups,and the differences were statistically significant(P<0.05);the content of ATF6,Caspase12,Caspase3 and CHOP protein in Nrf2(-/-)mice in Atorv group was lower than that of Nrf2(-/-)mice in Vehicle group(P<0.05),but it was higher than that of Nrf2(+/+)mice in Atorv group(P<0.05).Conclusions:1.After brain injury caused by hydraulic shock,Nrf2 has a brain protective effect,which can inhibit endoplasmic reticulum stress-induced neuronal apoptosis.2.Atorvastatin can protect neurons through inhibiting the expression of PERK,ATF6,and Caspase12-related apoptosis pathway genes by activating the Nrf2 signaling pathway. |
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