Experimental Study Of MANF On The Mechanism Of Hypoxic Preconditioning Reduce Endoplasmic Reticulum Stress On The Rats After Traumatic Brain Injury

In the conventional weapons of war,The soldiers will be affected by a lot of injuries,Traumatic brain injury is the most deadly damage in wartime and postwar.There will be a variety of secondary damage after traumatic brain injury,Some changes,which characterized by apoptosis will significantly affect the outcome of the disease.MANF which was called endoplasmic reticulum stress pr sensitive protein can enhance the tolerance of cells such as sugar free state and other endoplasmic reticulum stress triggers,can regulate endoplasmic reticulum nyctinastic receptor activity to improve the state of cell survival, reduce apoptosis.Study the MANF expression changes and interventions may become a new target for treatment of traumatic brain injury.Part I :Experimental study of the expression changs of MANF on rats brain cortex after traumatic brain injuryObjective Research of the dynamic expression changes of mesencephalic astrocyte-derived neurotrophic factor in rats brain cortex after traumatic brain injury, and explore the role of apoptosis in the pathogenesis of MANF after traumatic brain injury.Methods 108 Sprague Dawley male rats were randomly divided into TBI group(n=96)and normal control group(n=12). The rats in group TBI group were established traumatic brain injury model with freefall equipment,and no injury was performed in normal control group. The expression of MANF m RNA and protein in the brain tissue was detected by RT-q PCR,Western blot and Immunohistochemistry 1, 3, 6, 12,24 h, and 3, 7, 14 d after injury.Results q RT-PCR,Western blot and Immunohistochemistry results showed that the relative expression of MANF m RNA and protein in the brain cortex increased at 1 h, reached peak at 6 h and then decreased,and decreased to normal 14 d after injury in TBI group. Compared with normal control group, the expression of MANF m RNA and protein was significantly higher from 1h to 7d after injury in TBI group(all P < 0.05). Immunohistochemical staining results showed that the expression MANF protein localized in the cytoplasm, tan or brown, MANF appeared in the cytoplasm at 1h after injury, staining color most obvious at 6h,and had no difference between the control group at 14 d.Conclusion MANF appeared and located in the cytoplasm after traumatic brain injury, The increased expressions of MANF m RNA and protein may be related to activation of an endogenous neuroprotective mechanism of the cells after traumatic brain injury, MANF may be involved in the reduction of apoptosis which was mediated by endoplasmic reticulum stress pathway after brain injury.Part II:Experimental study of MANF on the Mechanism of Hypoxic Preconditioning reduce Endoplasmic Reticulum Stress on the Rats after Traumatic brain injurySection I :Effect of Hypoxic preconditioning on Endoplasmic Reticulum Stress after Traumatic brain injury in RatsObjective To explore the effect of hypoxic preconditioning on endoplasmic reticulum stress after traumatic brain injury in rats.Methods 48 Sprague Dawley rats were randomly divided into HCP group(HPC treated) and a non- HCP group( without pre-treated),with 24 rats in each group. Then,each of the group was further divided into 4 sub-groups with 6 rats:3 sub-groups after TBI for 1, 3 and 7 days(TBI treated,and drawing and observation after various TBI treatment time), respectively, together with a control sub-group(without any treatment).HPC model was made in the low-pressure oxygen chamber 3h daily continuous 3d.The TBI model was established by the modified Freeny’s freely falling equipment.Neurological severity score of rat was recorded after brain injury.Expressions of CHOP m RNA and protein were detected by quantitative real-time PCR(q RT-PCR) and Western blot.TUNEL was used to evaluate the apoptotic rate,and the correlation between the level of CHOP and the apoptotic rate was analyzed.Results m NSS score、the relative expressions of CHOP m RNA and protein、the apoptotic rate of 1, 3 and 7 days subgroups after TBI were higher than control groups(all P<0.05),and peaked at 3d;m NSS score、the relative expressions of CHOP m RNA and protein、the apoptotic rate in HPC group were lower than non- HCP group(all P<0.05);and the correlation analysis showed the expression of CHOP was positive correlated with the apoptotic rate(r=0.667,P<0.05).Conclusion Hypoxic preconditioning can down-regulate the expression of pro-apoptotic protein CHOP which participate in endoplasmic reticulum stress pathway,reduce neuronal apoptosis and improve neurological function.Section II Effect of Hypoxic Preconditioning on the expression of MANF after Traumatic brain injury in RatsObjective Observe the change and influence of hypoxia preconditioning on the expression of MANF and CHOP after traumatic brain injury in rat cortical brain tissuse.To study MANF on the mechanisms of hypoxic preconditioning reduces endoplasmic reticulum stress on rats after traumatic brain injury.Methods 108 Sprague Dawley male rats were randomly divided into HCP group(HPC treated) and a non- HCP group( without pre-treated),with 54 rats in each group. Then, each of the group was further divided into 9 sub-groups with 6 rats:8 sub-groups after TBI for1 h,3h,6h,12 h,24h,3d,7d and 14d(TBI treated,drawing and observation after various TBI treatment time), respectively, together with a control sub-group(without any treatment). Hypoxia preconditioning model was made in the low-pressure oxygen chamber for 3 days(-50 k Pa,3h/d).The TBI model was established by the modified Freeny’s freely falling equipment. The expression of MANF and CHOP in the brain cortex was detected by q RT-PCR and Western blot.Results The relative expressions of MANF m RNA and protein of 1h-14 d subgroups after TBI were higher than control groups(all P<0.05),and peaked at 6h;the relative expressions of MANF m RNA and protein in HPC group were higer than non- HCP group(all P<0.05); the relative expressions of CHOP m RNA and protein 1h-7d subgroups after TBI were higher than control groups(all P<0.05),and peaked at 3d;the relative expressions of CHOP m RNA and protein in HPC group were lower than nonHCP group(all P<0.05);Conclusion Hypoxic preconditioning can up-regulate the expression of MANF; reduce the expression of CHOP in the endoplasmic reticulum stress pathway, reduce apoptosis after traumatic brain injury in rats.