Objective To study the dynamic expression and distribution of high mobility group box 1 (HMGB-1)in diffuse axonal injury (DAI)in rats and to clarify its involvement in the inflammatory reaction after DAI in rats,in order to provide new targets for the clinical treatment of DAI.Methods A DAI model was established using a coronal rotation device and evaluated by HE,Glees-Marsland silver staining,and Mallory phosphotungstic acid hematoxylin staining.Immunohistochemistry,Western blot and RT-PCR were used to detect the expression and distribution of HMGB-1 in the cortex of DAI rats at 6 h,1 d,3 d and 7 d.And TUNEL was used to examine the apoptosis of neurons in DAI rats.Results Immunohistochemical results showed that at 6 h and 1 d after DAI,the number of HMGB-1-positive cells decreased,but at 3 and 7 d it began to increase.Western blot also showed that during the early stage after DAI (6 h and 1 d),the level of HMGB-1 protein in the cortex was significantly lower than that in the control group,but at the late stage (3 and 7 d)after DAI it significantly increased compared with that in the control group until 7 d.RT-PCR showed that at 6 h after DAI there was no significant increase in the level of HMGB-1mRNA,but at 1 d there was a slight increase compared with the control group;at 3 and 7 d,it showed an obvious significance.TUNEL staining indicated that the significant neuronal apoptosis appeared as early as 6 h after DAI,and reached the peak at 3 d;it started to decrease at 7 d but still remained at a relatively high level.Conclusion The dynamic expression and distribution of HMGB-1 showed significant changes with the time course after DAI in rats.They decreased at the early stage but increased at the late stage.At the early stage, HMGB-1 is mainly passively released by the necrotic neurons,and at the late stage it may be actively secreted by the active inflammatory cells.HMGB-1 may mediate the post-DAI neural cell apoptosis by inducing the inflammatory reaction.

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.

Objective To investigate effect of Dahuang Fuzi decoction on alveolaur epithelial barrier in rats with lung injury with severe acute pancreatitis. Method Ninty-six health SD rats were randomly divided into three groups: sham operation group, SAP-ALI group, Dahuang Fuzi decoction group, and then according to the time point of sacrifice after operation, each group was subdivided into 3,6,12,24 hour subsets ( each, n = 8). After the belly of a rat in the sham operation group was cut open, the pancreas was flipped several times,and then a stoma was made in the jejunum to form its fistula. In the SAP-ALl group,1 mL/kg sodium taurocholate was reversely injected into the pancreatobile duct to establish the model of SAP, and then the jejimum fistula was performed. The SAP-ALI model in Dahuang Fuzi decoction group was treated by injection of 10ml of Dahuang Fuzi decoctionon into the fistula respectively. Blood was collected from heart to detect serum amvlase and endotoxin (ET) levels before the rat being executed. The lung histopathologic changes, pulmonary injury scores and wet/dry weight(W/D) ratios were observed after the rats were executed. The alveolar liquid clearance rate(ALCR), total lung water content (TLW), extravascular lung water content(EVLW) and alveolar epithelial permeability (AEP) were examined in 3,6, 12,24 h after injury.Results There was continuous increase of AEP,TLW and EVLW,as well as progressive reduction of ALCR compared with sham operation group at 3,6,12,24 h after operation. Compared with SAP-ALI group, there was continuous decrease of AEP,TLW and EVLW, and elevated of ALCR at 3,6,12,24 h after operation.Conclusions Dahuang Fuzi decoction can significantly reduce alveolaur epithelial barrier and degree of lung tissue of SAP-ALI rats by inhibiting the elevation of LPS and inflammation reaction.

Objective To investigate the protective effects of intra-peritoneal fluid resuscitation on small intestinal mucosa in rats with hemorrhagic shock. Method Fifty Sprague-Dawley (SD) rats were randomly (random number) divided into five groups, namely sham operation group (group I ), hemorrhagic shock group (group Ⅱ ), intra-venous fluid resuscitation group (group Ⅲ ) . intravenous fluid resuscitation plus intra-peritoneal saline resuscitation (group Ⅳ ) and intravenous fluid resuscitation plus intra-peritoneal PD-2 solution resuscitation group (group Ⅴ ). The rats of 5 groups were processed with cannulations of right common carotid artery, right femoral vein and left femoral artery with systemic heparinization. The rat models of hemorrhagic shock were established with modified Wigger' s method by which the blood exsanguinated from left femoral artery. The rats of group Ⅲ were resuscitated with shed blood plus twice equal volume of Ringer's solution after modeling of hemorrhagic shock.The rats of group Ⅳ and group Ⅴ were administered intra-peritoneally with 30 mL saline and 30 mL of 2.5% PD-2 solution, respectively as adjuncts to those used in the group Ⅲ . The specimens of blood and small intestine of rats of all groups were collected 60-120 minutes after modeling and resuscitation. The activity of plasma diamine oxidase (DAO) was determined with chromatometry, the level of plasma D-lactic acid (D-LA) with spectorophotometry and the level of plasma lipopolysaccharide (LPS) with nephelometry. The histopathological and ultrastructure changes of small intestine tissue of rats were observed under light microscope and electronic microscope. Results There were remarkable differences in activity of DAO, and the levels of D-LA and IPS in rats between those ingroup Ⅱ and group I (P <0.01), and between those in group V and groups Ⅱ , Ⅲ or Ⅳ (P <0.05 or P < 0.01) The pathomorphology and ultra-structure of small intestine tissues were severely damaged in group Ⅱ compared with those in group Ⅰ , and those markedly lessened in group V compared with groups Ⅱ , Ⅲ and Ⅳ . Conclusions Intraperitoneal fluid resuscitation with PD-2 solution can significantly protect the integrity of intestinal mucosa and the normal permeability of intestinal wall, and blunts the histopathological changes, and restrains bacterial translocation from gut and reduces the level of plasma endotoxin.

Objective To investigate the role of AMP-activated protein kinase (AMPK) signal path in cell autophagy activation at early brain injury in rats after subarachnoid hemorrhage (SAH).Methods Adult male SD rats (weighting 300-350 g) were divided into five groups (n=12):sham-operated group,SAH group,and SAH+AICAR group,SAH+Compound C group and SAH+vehicle group.SAH models in the later four groups were established by endovascular perforation technique,and rats in the later three groups were performed left intracerebroventricular injection of AMPK agonist AICAR,AMPK inhibitor Compound C or normal saline 30 min before modeling;animals were subsequently sacrificed at 24 h after modeling.Immunohistochemical method was used to detect the phosphorylated mammalian target of rapamycin (p-mTOR) expression.Expressions of cortex autophagy related proteins LC3,AMPK and phosphorylated AMPK (p-AMPK) were observed by Western blotting.Loeffler's method was used to evaluate the neurologic behavior scores.Results As compared with those in the sham-operated group,the p-AMPK level,p-mTOR expression level and LC3Ⅱ/LC3Ⅰ ratio were significantly increased,while the behavioral deficit scores were significantly lower in the SAH group,with statistical differences (P＜0.05);the p-mTOR mainly expressed at cortex surrounding the hemorrhage areas,and integration areas of deep cortex and brain white matter.As compared with the sham-operated group and SAH+vehicle group,SAH+AICAR group had significantly increased p-AMPK level,decreased p-mTOR expression level,increased LC3Ⅱ/LC3Ⅰ ratio,and decreased behavioral deficit scores (P＜0.05);as compared with the sham-operated group and SAH+vehicle group,SAH+Compound C group had significantly decreased p-AMPK level,decreased LC3Ⅱ/LC3Ⅰ ratio,and decreased behavioral deficit scores (P＜0.05).Conclusion AMPK is involved in the process ofautophagy activation after SAH through regulating mTOR,and the regulation of AMPK may contribute to neuroprotection related to autophagy.