Effects Of Glycyrrhizin On The Expression Of High - Migratory Protein B1 After Traumatic Brain Injury In Rats

PartⅠExpression and clinical significance of HMGB1 in the plasma of patients with acute traumatic brain injuryObjective:To explore the expression and clinical significance of high mobility cluster protein B1 (HMGB1) in the plasma of patients with traumatic brain injury. Methods: 142 patients with acute traumatic brain injury were chosen in this study between January 2012 and June 2012. There were 68 cases in mild group (GCS13～15),48 cases in moderate group (GCS9-12), and 26 cases in severe group (GCS3-8). Upper limb venous blood of all patients were collected in 4-6 hours after the injury, and the expressions of HMGB1 were detected in serum by enzyme linked immunosorbent assay.15 cases of normal healthy volunteers were chosen as a control group over the same period. The expressive levels of HMGB1 in brain trauma group were compared with control group. And the risk factors of mortality within 1 months and poor prognosis within 6 months were analyzed in patients with acute traumatic brain injury. Results:The expressions of HMGB1 in plasma of patients with acute traumatic brain injury were significantly higher than those in the control group (P<0.05); The plasma HMGB1 levels were significantly negatively correlated with GCS scores at admission (P<0.05).Single factor Logistic regression analysis showed that plasma HMGB1 levels were the risk factors of mortality within one month and of poor prognosis within six months in patients with acute traumatic brain injury. ROC curve analysis showed that the plasma HMGB1 levels had significant predictive values for mortality within one month and poor prognosis within six months in patients with acute traumatic brain injury. Conclusions:The plasma HMGB1 expression in patient with acute traumatic brain injury is increased, and the higher level is positively correlated with the severity of traumatic brain injury. The plasma HMGB1 level may be an important indicator to evaluate the patient prognosis of traumatic brain injury.Part IIExpression of HMGB1 in rat with acute traumatic brain injuryObjective:To investigatethe expression of HMGB1, the brain edema and apoptosis in blood and brain tissue after acute traumatic brain injury in rats. Methods:Male Sprague-Dawley rats were randomly divided into seven groups:sham group, TBI 2h group, TBI 6h group, TBI 12h group, TBI 24h group, TBI 48h group and TBI 72h group (n=12 per group). Rat TBI model was made by the modified Feeney’s method. The expressions of HMGB1 in the serum were detected by ELISA. HMGB1 positive cells were observed by immunohistochemical staining. Brain water contents were determined by wet-dry weight way. Apoptotic cells were detected by TUNEL staining. Results:The expressions of HMGB1 in plasma were 0.83±0.15ng/ml in the sham group,3.50± 0.57ng/ml in TBI 2h group (P<0.01 compared with sham group), 7.87±0.60ng/ml in TBI 6h group (P<0.01 compared with TBI 2h group), 7.85±0.59ng/ml in TBI 12h group,7.61±0.66ng/ml in TBI 24h group (P>0.05 compared between TBI 6h group, TBI 12h group and TBI 24h group), 4.63±0.68ng/ml in TBI 48h group (P<0.01 compared with TBI 24h group) and 3.11±0.66ng/ml in TBI 72h group (P<0.01 compared with TBI 48h group). HMGB1 positive cells were 7.49±1.76% in the sham group,36.54±5.92% in TBI 2h group (P<0.01 compared with sham group),65.58±5.46% in TBI 6h group (P<0.01 compared with TBI 2h group),65.18±4.83% in TBI 12h group,64.68±4.09% in TBI 24h group (P>0.05 compared between TBI 6h group, TBI 12h group and TBI 24h group),43.17±4.48% in TBI 48h group (P<0.01 compared with TBI 24h group) and 36.50±4.92% in TBI 72h group (P<0.01 compared with TBI 48h group). Brain water contents of injured sides were 79.21±0.96% in the sham group,79.27±0.92% in TBI 2h group (P>0.05 compared with sham group),81.00±0.85% in TBI 6h group (P<0.01 compared with TBI 2h group),82.26±0.95% in TBI 12h group (P<0.05 compared with TBI 6h group),83.54±0.93% in TBI 24h group (P<0.05 compared with TBI 12h group),82.31±0.50% in TBI 48h group (P<0.05 compared with TBI 24h group) and 81.22±0.88% in TBI 72h group (P<0.05 compared with TBI 48h group). Apoptotic cells were 6.62±1.38% in the sham group,8.73±1.52% in TBI 2h group (P>0.05 compared with sham group),35.11±4.72% in TBI 6h group (P<0.01 compared with TBI 2h group),43.23±4.61% in TBI 12h group (P<0.05 compared with TBI 6h group),52.29±4.76% in TBI 24h group (P<0.01 compared with TBI 12h group),39.91±4.66% in TBI 48h (P<0.01 compared with TBI 24h group) group and 32.56±4.85% in TBI 72h group (P<0.05 compared with TBI 48h group). Conclusions: The HMGB1 level in the plasma was elevated at 2h after rate traumatic brain injury, reached peak value at 6h after injury, persisted for 24h, and began to decrease significantly at 48h after injury; HMGB1 began to transfer from the nucleus to the cytoplasm at 2h after rate traumatic brain injury, reached its peak at 6h after injury, lasted for 24h, and began to decline obviously at 48h after injury; Brain water content of injured side began to increase at 6h after rate traumatic brain, reached the peak at 24h after injury,and began to ease at 48h after injury; apoptotic cells were gradually increased at 6h after rate traumatic brain, reached the maximum at 24h after injury, and gradually reduced at 48h after injury.Part IIIGlycyrrhizin’s Effect of HMGB1 after Traumatic Brain Injury in the Rat and its mechanismObjective:To investigate the neuroprotective effects of glycyrrhizin (Gly) as well as its effect on expression of high-mobility group box 1 (HMGB1) in rats after traumatic brain injury (TBI). Methods:Male Sprague-Dawley rats were randomly divided into three groups:sham group, TBI group, and TBI+Gly group (n=36 per group). Rat TBI model was made by the modified Feeney’s method. In TBI+Gly group, Gly was administered intravenously at a dosage of 10 mg/kg 30 min after TBI. At 24 h after TBI, motor function and brain water content were evaluated. Meanwhile, HMGB1/ HMGB1 receptors including toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE)/nuclear factor-κB (NF-κB) signaling pathway and inflammatory cytokines in the injured brain tissues were detected using reverse transcription-polymerase chain reaction, western blot, electrophoreticmobility shift assay and enzyme-linked immunosorbent assay. Furthermore, HMGB1, RAGE and TLR4 immunohistochemistry and apoptosis were analyzed. Results:Motor function impairment caused by TBI was evident in TBI group (P<0.01 compared with sham group), and motor function impairment was dicined in TBI+Gly group (P<0.01 compared with TBI group); Brain water contents of injured sides were 79.97±0.82% in the sham group,80.97±0.49% in TBI group (P<0.01 compared with sham group) and 80.97±0.49% in TBI+Gly group (P<0.01 compared with TBI group); The expressions of HMGB1/HMGB1 receptors (TLR4 and RAGE)/NF-κB DNA-binding activity were significantly increased in brain tissue of traumatic brain injury area (P<0.01 compared with sham group), and the over-expressions of HMGB1 /HMGB1 receptors (TLR4 and RAGE)/NF-κB DNA-binding activity were inhibited after Gly treatment (P<0.05 compared with TBI group); The contentratons of IL-1β, TNF-a and IL-6 around the injured brain were respectively 9.48±5.67pg/mg, 10.52±1.53pg/mg,5.38±0.82pg/mg in sham group,79.57±5.17pg/mg, 18.94±1.45pg/mg,16.08±1.06pg/mg in TBI group (all P<0.01 compared with sham group) and 62.02±5.54pg/mg,15.25±1.52pg/mg,11.60±0.90pg/mg in TBI+Gly (all P <0.01 compared with TBI group); The percentages of HMGB1, RAGE and TLR4-positive cells and apoptotic cells were respectively 7.98±1.44%,5.60±1.12%, 7.60±1.29%,8.19±1.46% in sham group,58.37±5.06%,54.15±4.65%,65.50±4.83%, 52.02±4.63% in TBI group (all P<0.01 compared with sham group) and 39.99±4.99%, 34.87±5.02%,43.33±4.54%,37.84±5.16% in TBI+Gly group (all P<0.01 compared with TBI group). Conclusions:Gly can reduce secondary brain injury and improve outcomes in rat following TBI by down-regulation of HMGB1/HMGB1 receptors (TLR4 and RAGE)/NF-κB mediated inflammatory responses in the injured rat brain. Our research showed that glycyrrhizin would provide neuroprotective effects following traumatic brain injury.