Polydatin prevents the induction of secondary brain injury after traumatic brain injury by protecting neuronal mitochondria.

Polydatin is thought to protect mitochondria in different cell types in various diseases. Mitochondrial dysfunction is a major contributing factor in secondary brain injury resulting from traumatic brain injury. To investigate the protective effect of polydatin after traumatic brain injury, a rat brain injury model of lateral fluid percussion was established to mimic traumatic brain injury insults. Rat models were intraperitoneally injected with polydatin (30 mg/kg) or the SIRT1 activator SRT1720 (20 mg/kg, as a positive control to polydatin). At 6 hours post-traumatic brain injury insults, western blot assay was used to detect the expression of SIRT1, endoplasmic reticulum stress related proteins and p38 phosphorylation in cerebral cortex on the injured side. Flow cytometry was used to analyze neuronal mitochondrial superoxide, mitochondrial membrane potential and mitochondrial permeability transition pore opened. Ultrastructural damage in neuronal mitochondria was measured by transmission electron microscopy. Our results showed that after treatment with polydatin, release of reactive oxygen species in neuronal mitochondria was markedly reduced; swelling of mitochondria was alleviated; mitochondrial membrane potential was maintained; mitochondrial permeability transition pore opened. Also endoplasmic reticulum stress related proteins were inhibited, including the activation of p-PERK, spliced XBP-1 and cleaved ATF6. SIRT1 expression and activity were increased; p38 phosphorylation and cleaved caspase-9/3 activation were inhibited. Neurological scores of treated rats were increased and the mortality was reduced compared with the rats only subjected to traumatic brain injury. These results indicated that polydatin protectrd rats from the consequences of traumatic brain injury and exerted a protective effect on neuronal mitochondria. The mechanisms may be linked to increased SIRT1 expression and activity, which inhibits the p38 phosphorylation-mediated mitochondrial apoptotic pathway. This study was approved by the Animal Care and Use Committee of the Southern Medical University, China (approval number: L2016113) on January 1, 2016.

SIRT1 plays a neuroprotective role in traumatic brain injury in rats via inhibiting the p38 MAPK pathway.

Traumatic brain injury (TBI) is a major cause of disability and death in patients who experience a traumatic injury. Mitochondrial dysfunction is one of the main factors contributing to secondary injury in TBI-associated brain damage. Evidence of compromised mitochondrial function after TBI has been, but the molecular mechanisms underlying the pathogenesis of TBI are not well understood. Silent information regulator family protein 1 (SIRT1), a member of the NAD+-dependent protein deacetylases, has been shown to exhibit neuroprotective activities in animal models of various pathologies, including ischemic brain injury, subarachnoid hemorrhage and several neurodegenerative diseases. In this study, we investigated whether SIRT1 also exert neuroprotective effect post-TBI, and further explored the possible regulatory mechanisms involved in TBI pathogenesis. A lateral fluid-percussion (LFP) brain injury model was established in rats to mimic the insults of TBI. The expression levels of SIRT1, p-p38, cleaved caspase-9 and cleaved caspase-3 were all markedly increased and reached a maximum at 12 h post-TBI. In addition, mitochondrial function was impaired, evidenced by the presence of swollen and irregularly shaped mitochondria with disrupted and poorly defined cristae, a relative increase of the percentage of neurons with low ΔΨm, the opening of mPTP, and a decrease in neuronal ATP content, especially at 12 h post-TBI. Pretreatment with the SIRT1 inhibitor sirtinol (10 mg/kg, ip) induced p-p38 activation, exacerbated mitochondrial damage, and promoted the activation of the mitochondrial apoptosis pathway. In contrast, pretreatment with the p38 inhibitor SB203580 (200 µg/kg, ip) significantly attenuated post-TBI-induced expression of both cleaved caspase-9 and cleaved caspase-3 and mitochondrial damage, whereas it had no effects on SIRT1 expression. Together, these results reveal that the 12 h after TBI may be a crucial time at which secondary damage occurs; the activation of SIRT1 expression and inhibition of the p38 MAPK pathway may play a neuroprotective role in preventing secondary damage post-TBI. For this reason, both SIRT1 and p38 are likely to be important targets to prevent secondary damage post-TBI.

[Alteration in intestinal epithelial permeability and its role in the pathogenesis of burn shock].

The intestinal epithelial barrier serves a dual role: to keep harmful external agents out of the body and to allow beneficial nutrients to enter the body. Tight junction (TJ) is of crucial importance for the barrier function. Over the past 15 years, some of the molecular events underlying the epithelial barrier regulation have been described. This forum introduces briefly the molecular structure of TJ and its regulation in gut barrier. It was shown that gut barrier function was impaired as early as 5 minutes post burn and became worst by 4 hours. In this forum the mechanism of gut barrier injury in burns is described, and it includes 4 aspects: the phosphorylation of TJ protein and perijunctional actin-myosin ring, the reduction of TJ proteins expression, the endocytosis of TJ proteins, and the apoptosis and necrosis of the epithelial cells. It is well known that the increase in gut permeability promotes bacterial translocation in burns. Moreover, a new auto-digestion theory of gut in shock and MODS was recently raised. Therefore, protection against gut barrier damage has again been recognized as a therapeutic target in shock and MODS treatment.

[Effect of limited volume resuscitation on hemodynamic changes in pregnant rabbit with hemorrhagic shock].

OBJECTIVE: To determine the effects of two fluid resuscitation strategies on the changes of hemodynamic variables, serum concentration of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in a clinically relevant model of uncontrolled hemorrhagic shock in pregnant rabbits. METHODS: Hemorrhagic shock was induced by bleeding via carotied artery, followed by transection of a medium vessel in gestational sac. Experimental design consisted of three phases, shock phase (0-30 min), prehospital phase (30-90 min) and hospital phase (90-180 min). Twenty pregnant rabbits were randomly divided into two groups (n=10 /group), aggressive fluid resuscitation group (PNL group) and limited volume resuscitation group (PLH group). In the shock phase, animals were hemorrhaged by blood withdrawal to mean arterial pressure (MAP) of 40-45 mm Hg (1 mm Hg=0.133 kPa) via carotid artery. In the prehospital phase, a medium vessel in the gestational sac was transected, then the animals in the PNL group and PLH group were resuscitated with 0.9% normal saline (NS) and shed blood to MAP of 80, 60 mm Hg respectively. In the hospital phase, bleeding was controlled by surgical intervention and all the animals were reinfused with shed blood and NS to MAP 80 mm Hg. Hemodynamic variables and respiration rate were monitored and blood samples were collected for TNF-alpha and IL-6 measurement, and finally subsequent volume resuscitation and survival rate were recorded. RESULTS: (1) At 120 min, the respiration rate and heart rate in the animals assigned to PLH group was (66+/-16) bpm, (235+/-41) bpm respectively, which were significantly lower than those in PNL group (P<0.01), while MAP and central venous pressure in the PLH group was (80.4+/-7.2) mm Hg, (8.0+/-4.4) cm H2O, respectively, which were significantly higher than those in PNL group (P<0.01); (2) The serum concentration of TNF-alpha, IL-6 of all the animals were markedly increased after hemorrhagic shock, and peak at 24 min. The serum concentration of TNF-alpha, IL-6 in animals assigned to PLH group were (105+/-67) ng/L, (118+/-51) ng/L respectively, which were significantly lower than those in PNL group (P<0.01). The serum concentration of TNF-alpha, IL-6 in the animals assigned to PLH group were decreased to normal at 480 min; (3) The subsequent blood transfusion volume and NS resuscitation volume in PLH group in prehospital phase were (16.0+/-2.2) ml, (39.0+/-5.5) ml respectively, while those in hospital phase were (28.0+/-6.7) ml, (90.0+/-7.1) ml respectively, which were significantly lower than those in PNL group (P<0.05); (4) The 24 and 72 hours survival rate in the animals assigned to PLH group were 100%, 90% respectively; which were significantly higher than those in PNL group (P<0.01). CONCLUSION: Limited volume resuscitation improves thermodynamic changes of pregnant rabbit, attenuates the increase of serum concentration of TNF-alpha, IL-6, and results in higher survival rate. Limited volume resuscitation is an ideal means for hemorrhagic shock resuscitation in pregnant rabbit.

[Effect of Bay K 8644 on arteriole smooth muscle cell membrane potential in rats with severe hemorrhagic shock].

OBJECTIVE: To investigate the effect of Ca(2+) influx through L-type Ca(2+) channels on normal and hyperpolarized membrane potential of arteriole smooth muscle cells (ASMCs) in rats. METHODS: The ASMCs isolated from normal rats and those with severe hemorrhagic shock were labeled with DiBAC4 (3) for membrane potential detection. RESULTS: Ca(2+) influx caused hyperpolarization of the membrane potential in the normal ASMCs but depolarization in the cells from rats with hemorrhagic shock, and this effect could be inhibited by TEA. CONCLUSION: Ca(2+)-activated potassium channels activated by Ca(2+) influx through L-type Ca(2+) channels in normal ASMCs to cause hyperpolarization but leads directly to membrane potential depolarization in ASMCs from rats with severe hemorrhagic shock. This finding can be meaningful for treatment of vascular hyporeactivity in advanced stage of severe shock.

[Role of Rho kinase in reorganization of the vascular endothelial cytoskeleton induced by rat burn serum].

OBJECTIVE: To investigate the changes in endothelial cytoskeletal reorganization and the role of Rho in the signal transduction pathway. METHODS: ECV304 cells were cultured and randomly divided into following groups: i.e. sham (with normal rat serum treatment), burn (with burn rat serum treatment), Y (with 30 micromol/L Rho kinase inhibitor Y-27632 treatment), burn plus Y (pretreatment of cells with burn serum before treated with 30 micromol/L Y-27632), Y plus burn (pretreatment of cells with Y-27632 for 1 hour before treated with burn serum), LPA (with normal rat serum and 13 micromol/L LPA), and LPA plus Y (pretreatment of cells with LPA before treated with Y-27632) groups. The indices were examined at 6, 7 and 8 posttreatment hours (PTH) in all groups except in Y group. The endothelial morphology was observed with HE staining. Endothelial cytoskeleton was observed by dual-fluorescence labeling of filamenta (F) with Rhodamine-phalloidin and monomer (G) with oregon green labeled DNAase. The actin content in the cells in all groups was measured with flow cytometry. RESULTS: In sham and control group, the cells were in fusiform or polygonal shape, with satisfactory growth; filamentous actin (F-actin) was mainly distributed in the peripheral site of the cytoplasm and formed peripheral filamental band. The cells became confluent to form a single layer with reticular structure. Globular actin (G-actin) was concentrated in the nucleus and per nucleus. In burn group, after 6 hours of burn serum treatment, the ability of cells to adhere to vessel wall was weakened, and a striking reorganization of the actin cytoskeleton and the formation of the stress fibers were found. Furthermore, the fluorescent intensity of the peripheral filament bands was weakened, and dispersed actin monomers were seen in the cytoplasm. This reaction was enhanced along with elapse of stimulation time. In burn plus Y or Y plus burn group, the cells grew and adhered well to the wall of culture vessel. The distribution of the filamentous actin was the same as the sham group, while the stress fiber decreased in amount obviously. The structure of globular actin was condensed with little G-actin in the cytoplasm. The changes in actin cytoskeleton in LPA group was similar to that in burn group. The effects of LPA on actin reorganization could also be reversed by Y-27632. The content of F-actin in burn group at 6 PTH (0.63 +/- 0.07) was lower than that in sham group (0.75 +/- 0.08), while the content of G-actin in burn group (1.28 +/- 0.27) was higher than that in sham group (1.16 +/- 0.16, P > 0.05). CONCLUSION: Burn serum induces vascular endothelial actin cytoskeleton reorganization in endothelial cells via the Rho-dependent signal pathway. Similar to the effect of LPA, this effect could be reversed by Y-27632.

Effects of lipopolysaccharide on actin reorganization and actin pools in endothelial cells.

OBJECTIVE: To investigate the dose and time-dependent effects of lipopolysaccharide (LPS) on cytoskeletal F-acitn and G-actin reorganizations by visualizing their distribution and measuring their contents in human umbilical vein endothelial cell line ECV-304. METHODS: F-actin was labeled with rhodamine-phalloidin and G-actin with deoxyribonuclease I (DNase I)conjugated with fluorescein isothiocyanate (FITC). Contents of cytoskeletal proteins were obtained by flow cytometry. RESULTS: F-actin was mainly distributed peripherally in endothelial cells under normal conditions. LPS stimulation caused the formation of stress fibers and filopodia. G-actin was normally seen in perinuclear and nuclear areas in control ECV-304 cells. Under LPS stimulation, G-actin dots appeared in the cytoplasmic region. The actin disorganization was accompanied by the time- and dose- dependent decrease in F-actin pool and increase in G-actin pool. CONCLUSIONS: LPS can induce characteristic morphological alterations of actin cytoskeleton and formation of intercellular gap in endothelial cells, accompanied by changes in F-actin and G-actin pools.

Effect of SOD and NaHCO3 on the vascular hyporeactivity of rats after severe hemorrhagic shock.

OBJECTIVE: To investigate the effect of SOD and NaHCO3 on the vascular hyporeactivity after severe hemorrhagic shock in rats. METHODS: Twenty eight Sprague-Dawley rats were divided randomly into 4 groups of 7 each: SOD-treated group, NaHCO3-treated group, SOD+NaHCO3-treated group and normal saline-treated control group. The responses of arterioles to norepinephrine (NE), the effects of dopamine on blood pressure, blood flow velocity in arterioles, the mean arterial pressure (MAP), and the survival time within 24 h were measured. RESULTS: Two hours after shock, the arteriolar vasoreactivity was significantly reduced and threshold concentration of NE was increased by 24 to 27 folds. After beting treated separately with SOD, NaHCO3 and SOD+NaHCO3, arteriolar vasoreactivity was restored to some extent, among which the SOD+NaHCO3 group obtained a most significant effect with decreased threshold concentration of NE to 21% of its normal level. In SOD+NaHCO3-treated group, injection of dopamine indicated a most significant effect on MAP with 1.9 times more than that in control group, and the enhanced MAP was maintained at a level of more than 13.33 kPa after reinfusion of shed blood. The blood flow in arteriole was 2.54-fold higher than that in control group after 2 h resuscitation. The average survival time in SOD+NaHCO3- treated group was 2.9 times longer than that in control group. CONCLUSIONS: SOD and NaHCO3 could both recover the lower vasoreactivity and increase the enhancing effect of dopamine on blood pressure of rats in severe hemorrhagic shock, suggesting that coadministration of SOD and NaHCO3 could be a new approach to the treatment of severe hemorrhagic shock.

[Properties of large-conductance calcium-activated potassium channel in rat mesenteric arteriole smooth muscle cells].

OBJECTIVE: To investigate the electrophysiological properties of large-conductance calcium-activated potassium channel (BKCa) in mesenteric arteriole smooth muscle cells of rat. METHOD: Mesenteric arteriolar smooth muscle cells from rats were isolated and inside-out patch clamp technique was used to study BKCa. RESULTS: The single channel conductance of BKCa recorded was 221+/-6 pS and the reversal potential was -0.12 mV in the presence of high potassium in the bathing and pipette solutions. The open probability (NPo) of the channel was both voltage- and intracellular calcium dependent, whereas the amplitude of the channel was not calcium-dependent. Prolonged closed state could be observed at intracellular calcium concentrations of 1 micromol/L or above, known as calcium-dependent inactivation. CONCLUSION: BKCa in rat mesenteric arterial smooth muscle cells has the properties of large conductance, high potassium selectivity, steep voltage dependence and high calcium sensitivity. The high sensitivity to calcium of BKCa may be related to the compensatory regulation of the smooth muscles in pathophysiological conditions.

[Construction of vector of his-tagged cytoplasmic fragment of human toll like receptor 4 and its expression in E.coli].

OBJECTIVE: To construction of vector of his-tagged cytoplasmic fragment of human Toll like receptor 4 (hTLR4) and its expression in E.coli. METHODS: hTLR4 cytoplasmic cDNA codon domain was amplified by polymerase chain reaction (PCR) and cloned into pET-DsbA2.0 plasmid expressing His-DsbA fusion protein. After being identified by the assay of restrictional enzyme and sequencing, His-Dsb A fusion proteins were induced with isopropy-beta-D-thiogalactoside (IPTG) and further purified. RESULTS: A fusion protein with molecular weight of 42 kd was obtained. CONCLUSION: hTLR4 which was constructed and expressed successfully under nondenaturing conditions provides a tool for further studies.

Role of p38 mitogen-activated protein kinase in lipopolysaccharide-induced expression of inducible nitric oxide synthase in human endothelial cells.

OBJECTIVE: To understand the role of p38 mitogen-activated protein kinase (p38MAPK) in the expression of inducible nitric oxide synthase (iNOS) and NO production in human endothelial cells under the stimulation by lipopolysaccharide (LPS). METHODS: NO level in the supernatant of the cell culture media was measured with Griess method, and iNOS protein and mRNA expressions by the cells were detected with immunofluorescence analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively. Immunoprecipitation assay was employed to examine p38 MAPK activity. RESULTS: It was shown that in comparison with the basal level of iNOS expression in cultured endothelial cells line ECV304, iNOS mRNA and protein expressions were significantly increased in the cells after LPS stimulation. In response to LPS treatment, obvious enhancement of p38 MAPK activity in ECV304 took place after the stimulation, with the peak level occurring at 15 min that maintained for approximately 45 min before gradual decline. When treated with SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole], a highly specific inhibitor of p38 MAPK, significant inhibition of LPS-induced iNOS protein and mRNA expressions was observed. CONCLUSIONS: p38 MAPK plays an important role in iNOS expression and NO production in ECV304 cells, and, inhibition of the signal transduction pathway may consequently be an effective approach to reduce the production of iNOS and other cytokines for the treatment of septic shock.

[Expression of intercellular adhesion molecule-1 in different organs of the mice with endotoxic shock induced by lipopolysaccharide].

To investigate and compare the expression of intercellular adhesion molecule-1 (ICAM-1) in different organs of the mice with endotoxic shock induced by lipopolysaccharide (LPS), protein and mRNA of ICAM-1 were measured by Western blotting and RT-PCR respectively in different organs of BALB/c mice administered intraperitoneally with 5 mg/kg LPS. The results showed that the constitutive expression of ICAM-1 protein and mRNA was the greatest in the lungs, followed by the spleen, kidney and intestine. After LPS stimulation, the upregulation of ICAM-1 was still greatest in the lungs, followed by the liver, spleen, heart, kidney and intestine. Compared with the normal mice, the expression of ICAM-1 protein in endotoxic shocked mice increased by 4.5-fold in the lungs, 3.0-fold in the kidney, 1.5-fold in the spleen; the expression in the liver and heart was negative under normal condition and changed into positive during endotoxic shock; but ICAM-1 expression in the intestine did not change significantly. The expression of ICAM-1 mRNA also increased consistently. These data highlight that LPS can up-regulate ICAM-1 protein and mRNA expression in different tissues of the mice with endotoxic shock. The difference in ICAM-1 expression among the organs may lead to different sensitivity of organ damage in endotoxic shock. This suggests that inhibition of ICAM-1 expression may be a useful principle for prevention and treatment of endotoxic shock.

Signal Transduction in TNF-alpha-induced c-jun Gene Expression.

Our previous studies demonstrated that p38 mitogen-activated protein (MAP) kinase regulated the c-jun protein expression through phosphorylation of transcription factors of myocyte enhancer factors 2 (MEF2) family. There was a MEF2 binding site in the promoter of c-jun gene. Members of the MEF2 family of trans-cription factors bound as homo- and heterodimers to this MEF2 binding site. Here the potential role of the p38 and BMK1 MAP kinases in the regulation of c-jun expression induced by TNF-alpha was examined. It was shown that p38 MAP kinase up-regulated the transcription activity of MEF2A, while BMK1 MAP kinase up-regulated not only the transcription activity of MEF2A, but also MEF2D. The p38 and BMK1 MAP kinases had coordinated effect on the regulation of c-jun transcription. TNF-alpha induced the formation of MEF2A/MEF2D hete-rodimer. Over-expression of homodimer of MEF2 proteins inhibited c-jun transcription induced by TNF-alpha, while over-expression of heterodimer MEF2A/MEF2D enhanced c-jun transcription induced by TNF-alpha. Phosphorylation of MEF2A and MEF2D by p38 and BMK1 respectively appeared very important in TNF-alpha induced MEF2A/MEF2D heterodimer formation to enhance c-jun gene expression.