Optical Depolarization of DCX-Expressing Cells Promoted Cognitive Recovery and Maturation of Newborn Neurons via the Wnt/ß-Catenin Pathway.

Electrical excitability by membrane depolarization is crucial for survival and maturation of newborn cells in the dentate gyrus of the hippocampus. However, traditional technology for membrane depolarization lacks temporal and spatial precision. Optogenetics can be used to activate channelrhodopsin-2 (ChR2), allowing cationic current to depolarize genetically targeted cells. In this study, we used ChR2-EGFP driven by doublecortin (DCX) to promote survival and maturation of newborn cells in the dentate gyrus after traumatic brain injury (TBI). C57BL/6 mice underwent lateral fluid percussion TBI. TBI mice were transfected with a lentivirus carrying the DCX-ChR2-EGFP gene. We observed that not only immature neurons but also type-2b intermediate progenitor (IPs) and neuroblasts expressed DCX-EGFP, indicating that DCX-expressing newborn cells could provide a long time window for electrical activity regulation. Quantitative results showed that the number of EGFP-expressing cells began to rise at 3 days after TBI and peaked at 9 days after TBI. By optical depolarization of DCX-EGFP-expressing cells between 3 and 12 days, we observed significantly improved cognitive deficits after TBI with enhanced survival and maturation of newborn cells in the dentate gyrus. We also investigated the role of optical depolarization in neural stem cells transfected with a lentivirus carrying the ChR2-DCX-EGFP gene in vitro. By administrating verapamil to block L-type calcium channels, we verified that the up-regulation of MAP2, NeuN, Neurog2, NeuroD1 and GluR2 in newborn cells was mediated by ChR2-elicted depolarization. By using ß-catenin inhibitor Dkk1, we demonstrated that optical depolarization of DCX-EGFP-expressing cells facilitated survival and maturation probably through the Wnt/ß-catenin signaling cascade.

Relationship between high-mobility group box 1 protein release and T-cell suppression in rats after thermal injury.

To study whether high-mobility group box 1 protein (HMGB1) has an effect on T-cell-mediated immunity secondary to burn injury, 96 male Wistar rats weighing 250 to 300 g were randomly divided into three groups as follows:sham burn group, burn group, and burn with ethyl pyruvate treatment group, and they were killed on postburn days (PBDs)1, 3, 5, and 7, respectively, with 8 animals at each time point. Columns of nylon wool were used to isolate splenic T cells. T-Cell proliferation was analyzed with thiazolyl blue and expression of IL-2 receptor alpha (IL-2Ralpha) on the surface of T cell with flow cytometry. Levels of HMGB1 were determined using Western blot analysis. IL-2, soluble IL-2R, IL-4, and interferon-gamma were determined with enzyme-linked immunosorbent assay kits. Gene expressions of HMGB1, IL-2, and IL-2R were assessed using reverse-transcription polymerase chain reaction, and activation of nuclear factor of activated T cell was determined with gel mobility shift assay. The levels of HMGB1 in plasma were significantly elevated on PBDs 1 to 5. Significant proliferation of splenic T cells and IL-2, as well as IL-2Ralpha expression on T cells, were simultaneously suppressed to a certain extent on PBDs 1 to 7. Nuclear factor of activated T-cell activity of splenic T cells was markedly down-regulated on PBDs 1 to 3. Administration of ethyl pyruvate to inhibit HMGB1 can significantly restore proliferative activity, nuclear factor of activated T-cell activity, and expression levels of IL-2 and IL-2Ralpha on T cells. High-mobility group box 1 protein released after major burns might be associated with the pathogenesis of immunosuppression in splenic T lymphocytes in rats.

[Effects of ethyl pyruvate on splenocyte proliferation and apoptosis in burn rats with delayed resuscitation].

OBJECTIVE: To investigate the effects of ethyl pyruvate (EP) on splenocyte proliferation and apoptosis in burn rats with delayed resuscitation, and its potential underlying mechanism. METHODS: Seventy two male Wistar rats were randomly divided into sham-scalded control group (n=24), scald group (n=24), and scald with EP treatment group (n=24). Animals were sacrificed on days 1, 3, and 5 postburn, and spleen samples were collected to determine splenocyte proliferation and apoptosis. RESULTS: Splenic lymphocyte proliferation response to T cell mitogen, concanavalin A (Con A), as significantly depressed from 1 to 5 days after burn injury (all P<0.05). Meanwhile, burn injury resulted in a marked increase in splenic CD3(+)CD4(+)T lymphocyte apoptosis in comparison with that in sham-scalded controls on day 1 postburn (P<0.05). Treatment with EP after burns resulted in a dramatic restoration of lymphocyte proliferation response and reduction of splenic CD3(+)CD4(+)T lymphocyte apoptosis compared with scald group (P<0.05). CONCLUSION: Administration of EP can markedly improve the splenocyte proliferation response and inhibit splenic CD3(+)CD4(+)T lymphocyte apoptosis in thermally injured rats.

[Effects of ethyl pyruvate on cell-mediated immune function in rats with delayed resuscitation after burn injury].

OBJECTIVE: To investigate the effects of ethyl pyruvate (EP) on cell-mediated immune function in rats with delayed resuscitation after burn injury, and its potential regulatory mechanism. METHODS: Wistar rats were subjected to 30% full-thickness scald injury with delayed resuscitation. One hundred and three male rats were randomly divided into normal controls (n=7), sham scald group (n=32), scald group (n=32) in which 40 ml/kg normal saline was infused peritoneally 6 hours after scald, and EP treatment group (n=32) in which 40 mg/kg EP was injected peritoneally 6 hours after scald. Animals were sacrificed on postburn day 1, 3, 5, and 7, and spleen was collected to determine splenocyte proliferation, IL-2 production and cell-surface IL-2 receptor (IL-2R) expression. RESULTS: Splenic lymphocyte proliferation responses to T cell mitogen, concanavalin A (Con A), were depressed from 1 to 7 days after scald injury (all P<0.05). Meanwhile, in comparison with sham scald group, burn injury resulted in a significant decrease in splenic production of IL-2 on postburn day 1, 3, as well as 5, and a marked suppression of IL-2R expression on days 1 and 3 postburn (all P<0.05). Treatment with EP after burn injury showed a dramatic restoration of lymphocyte proliferation rate and increased production of IL-2 at various time points (all P<0.05). However, treatment with EP did not affect IL-2R expression compared with scalded rats (all P>0.05). CONCLUSION: Treatment with EP could markedly elevate splenic T lymphocyte proliferation response and increase production of IL-2 following burn injury, thereby improving cell-mediated immunity in thermally injured rats with delayed resuscitation.

[The role of Janus kinase-signal transducer and transcription activator pathway in the regulation of synthesis and release of lipopolysaccharide-induced high mobility group box-1 protein].

OBJECTIVE: To investigate the role of Janus kinase-signal transducer and transcription activator (JAK-STAT) pathway in the regulation of synthesis and release of lipopolysaccharide-induced high mobility group box-1 protein (HMGB1). METHODS: Peritoneal macrophages harvested from male Wistar rats were incubated for 3 days before the experiment. The activation of Janus kinase-2 (JAK2), signal transducer and activator of transcription-1 (STAT1) and STAT3 was observed before and 10, 30, 60 and 120 mins after LPS stimulation (4 determinations at each time point) and it was expressed as A value (absorption). In addition, the cells were divided into normal control, LPS stimulation, JAK2 inhibition (with AG490 treatment 2 hours before LPS stimulation), STAT1 inhibition (with fludarabine treatment 2 hours before LPS stimulation) and STAT3 inhibition (with rapamycin treatment 2 hours before LPS stimulation) groups. The cells in all groups except control group were stimulated with LPS 3 days after culture. The expression of HMGB1 gene and its protein release in each group were determined for 4 times and were expressed as A value. RESULTS: LPS could activate JAK2, STAT1 and STAT3 within 2 hours, especially the activation of STAT3 appeared more quickly, peaking at 10 minutes after LPS stimulation (7.47 +/- 0.56). Pretreatment with the inhibitors of JAK-STAT pathway could markedly reduce the expression of HMGB1 mRNA (P < 0.01), but exerted no effect on HMGB1 release. CONCLUSION: JAK-STAT pathway can be activated early during endotoxin challenge, and it may play a role in the regulation of HMGB1 synthesis.

[High mobility group box-1 protein activates Janus kinase-signal transducer and activator of transcription pathway in rat peritoneal macrophages].

OBJECTIVE: To investigate the potential signal transduction mechanism in high mobility group box-1 protein (HMGB1)-induced inflammatory response in rat peritoneal macrophages. METHODS: Peritoneal macrophages obtained from male Wistar rats were incubated for 3 days before they were stimulated by HMGB1 (10 microg/ml). At various time points after HMGB1 stimulation, macrophages were denatured directly in cell culture flasks to detect activation of Janus kinase-2 (JAK2), signal transducer and activator of transcription-1 (STAT1) and STAT3 by immunoprecipitation, Western blotting and electrophoretic mobility shift assay, respectively. RESULTS: HMGB1 stimulation could activate STAT1 and STAT3 in peritoneal macrophages in 2 hours, among them the activation of STAT3 appeared to be the quickest, peaking as early as 10 minutes after stimulation. But no marked change in JAK2 activity was observed within 2 hours following HMGB1 stimulation. CONCLUSION: These data suggest that Janus kinase-signal transducer and activator of transcription pathway might be involved in regulation of HMGB1-induced inflammatory response in peritoneal macrophages.