Investigation Of The Mechanism Of Endoplasmic Reticulum Stress In Traumatic Brain Injury

ObjectiveTraumatic brain injury(TBI)is one of the leading causes of mortality and disability in the 21 st century.Its main mechanism is closely related to neuronal apoptosis and inflammation after brain injury.Emerging studies have shown that endoplasmic reticulum(ER)stress plays an important role in apoptosis and inflammation following TBI.However,the relationship between endoplasmic reticulum stress and brain injury remains unclear.Based on previous studies,we will explore the changes of endoplasmic reticulum stress following TBI and reveal its relationship with neuronal apoptosis and inflammatory response in mice with traumatic brain injury.Via blocking TBI-induced endoplasmic reticulum stress with tauroursodeoxycholic acid(TUDCA)to further explored the mechanism of endoplasmic reticulum stress in brain injury and provide a new therapeutic target for traumatic brain injury.Methods: 1.We subjected mice to TBI with a controlled cortical impact(CCI)device.Male C57BL/6 mice were randomly divided into Sham group and TBI group.The mice in TBI group were divided into 12h、1D、3D、7D group according to the time of execution.Western Blot and immunofluorescence were used to detect the changes of endoplasmic reticulum stress-related proteins Grp78 in brain tissue of mice at different time points of TBI.2.To explore the role of endoplasmic reticulum stress in brain injury,TUDCA(500mg/kg)was injected intraperitoneally to block the endoplasmic reticulum stress in TBI mice.The experimental animals were divided into sham group,TBI group and TBI + TUDCA group.Western Blot was used to detect the expression levels of p-PERK,PERK,p-eIF2 a,eIF2a and ATF4 to evaluate the inhibitory effect of TUDCA on endoplasmic reticulum stress in TBI mice.Neurological and motor deficits were assessed by modified neurological severity scores(mNSS)and beam balance and beam walking tests.Brain water content was assessed by dry-wet weight method.Evans Blue leakage method was used to evaluate the damage degree of blood-brain barrier in TBI mice.The tight junction protein expression(including ZO-1 and Claudin5)levels were detected by Western Blot to explore the protective effect and possible mechanism of endoplasmic reticulum stress on blood-brain barrier damage after TBI.Immunofluorescence technique was used to detect the activation of microglia and the ELISA was used to detect the release of inflammatory factors such as IL-1beta,TNFalpha and IL-6 after TBI to explore the relationship between endoplasmic reticulum stress and post-TBI inflammatory response.Neuronal cell death was assessed by a terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end-labeling(TUNEL)assay,and CHOP expression in neuronal cells was detected by doubleimmunofluorescence staining.Western Blot was used to detect the expression of Pten,p-Akt,Bax,Bcl-2,Caspase-12 and Chop in brain tissue of TBI mice to explore the mechanism of endoplasmic reticulum stress on neuronal apoptosis.3.The expression of Akt gene was blocked by oral administration of MK2206(60 mg/kg)or intracerebroventricular injection of siRNA-Akt in TBI mice.The recovery of neurological function after Akt signal pathway was blocked was detected by Modified Neurological Severity Scores(mNSS score)and Beam Balance Test.Western Blot and qRT-PCR were used to detect the expression levels of Grp78,p-eIF2α,eIF2α,Bcl-2,Bax,CHOP,p-Akt,Akt,Pten and Caspase-12 to further explore the neuroprotective mechanism of TUDCA.Results: 1.(1)The expression of GRP78,an ER stress marker,was significantly elevated at 6 h following TBI and peaked on the 72 h after TBI.(2)Compared with Sham group,the CHOP expression in neuronal cells subjected to TBI was significantly elevated.2.(1)TUDCA can effectively block the activation of PERK-ATF4-CHOP signaling pathway.(2)Blocking endoplasmic reticulum stress can significantly improve the neurological prognosis of TBI mice.(3)Blocking endoplasmic reticulum stress can significantly decrease brain water content and reduce blood-brain barrier leakage in TBI mice.(4)Blocking endoplasmic reticulum stress can significantly reduce the expression of endoplasmic reticulum stress-related apoptotic protein in neurons and reduce neuronal apoptosis.(5)Blocking endoplasmic reticulum stress can effectively reduce the excessive activation of microglia and the release of inflammatory factors after TBI,and alleviate the immune inflammatory response 3.(1)The ratio of p-Akt/Akt was decreased at 6 h after TBI and reached its lowest level on the 72 h after TBI.(2)TUDCA can effectively activate Akt signaling pathway while inhibit the expression of Pten gene and alleviate the apoptosis of brain tissue induced by TBI via regulating the expression of downstream apoptotic protein.(3)Both MK2206(60 mg/kg)and siRNA-Akt could abolish the neuroprotective effect of TUDCA on TBI mice,further verifying that the protective effect of TUDCA is dependent on the activation of Akt signaling pathway.Conclusion: 1.TBI can induce endoplasmic reticulum stress and promote the expression of endoplasmic reticulum stress-related apoptotic protein in neurons and which reach the peak at 72 hours after TBI.2.Blocking endoplasmic reticulum stress can effectively promote the repair of bloodbrain barrier,promote angiogenesis and reduce blood-brain barrier leakage in TBI mice.3.Blocking endoplasmic reticulum stress can effectively alleviate neuroinflammation,reduce neuronal apoptosis and improve the prognosis of TBI.4.TUDCA can effectively block the expression of Pten,promote the activation of Akt signaling pathway and improve the prognosis of TBI by regulating the expression of apoptosis-related proteins such as Bax and Bcl-2.5.Akt inhibitors can abolish the anti-apoptotic effect of TUDCA and promote the repair of blood-brain barrier which suggesting that the neuroprotective effect of TUDCA depends on the activation of Akt signaling pathway.