ADENOSINE MONOPHOSPHATE-ACTIVATED PROTEIN KINASE PHOSPHORYLATION MEDIATED BY SIRTUIN 5 ALLEVIATES SEPTIC ACUTE KIDNEY INJURY.

ABSTRACT: Background : Our previous studies have shown that ameliorating mitochondrial damage in renal tubular epithelial cells (RTECs) can alleviate septic acute kidney injury (SAKI). It is reported that AMPK phosphorylation (p-AMPK) could ameliorate mitochondrial damage in renal tissue and Sirtuin 5 (SIRT5) overexpression significantly enhanced the level of p-AMPK in bovine preadipocytes. However, the role of SIRT5-mediated phosphorylation of AMPK in SAKI needs to be clarified. Methods : WT/SIRT5 gene knockout mouse model of cecal ligation and puncture-induced SAKI and a human kidney 2 cell model of LPS-induced SAKI were constructed. An AMPK chemical activator and SIRT5 overexpression plasmid were used. Indexes of mitochondrial structure and function, level of p-AMPK, and expression of SIRT5 protein in renal tissue and RTECs were measured. Results : After sepsis stimulation, the p-AMPK level was decreased, mitochondrial structure was disrupted, and ATP content was decreased. Notably, an AMPK activator alleviated SAKI. Sirtuin 5 gene knockout significantly aggravated SAKI, while SIRT5 overexpression alleviated mitochondrial dysfunction after LPS stimulation, as manifested by the increase of p-AMPK level, the alleviation of mitochondrial structure damage, the restoration of ATP content, the decrease of proapoptotic protein expression, as well as the reduction of reactive oxygen species generation. Conclusions : Upregulation of SIRT5 expression can attenuate mitochondrial dysfunction in RTECs and alleviate SAKI by enhancing the phosphorylation of AMPK.

Flurbiprofen axetil alleviates the effect of formalin-induced inflammatory pain on the cognitive function of rats with mild cognitive impairment through the AMPKα/NF-κB signaling pathway.

Background: Mild cognitive impairment (MCI) as a prestage of dementia shares the most risk factors with dementia. In the present study, we explored the effect of flurbiprofen axetil on reducing the response of the central nervous system to inflammatory factors and anti-inhibiting apoptosis with the aim of developing a formalin-induced inflammatory pain model using MCI rats. Methods: Rats were subjected to sham operation (Sham group) or formalin-induced inflammatory pain, with or without flurbiprofen axetil (10 mg/kg). MCI rats were administered D-galactose (1,000 mg/kg) for 7 days subcutaneously. Thereafter, formalin was injected subcutaneously into the hind paws of rats, while sham group was injected with only normal saline. In the formalin/flurbiprofen group (F/F group), flurbiprofen axetil was injected into the tail vein 15 min before formalin was given, and the formalin/saline group (F/S group) used normal saline instead of the drug for injection. The pain score was recorded, and the time-score curve was drawn. The escape latency time and the number of times crossing the platform were recorded. The expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), AMP-activated protein kinase-α (AMPKα), and nuclear factor-κB p65 (NF-κB p65) in hippocampal tissue was determined. Varying degrees of pathological changes in the hippocampal CA1 region were observed. Results: The II phase pain score of rats in the F/F group was lower than that of the F/S group rats (P<0.05). The evasion incubation period and the number of platform crossings increased in both the F/F group and the F/S group (P<0.05), and were more significant in the F/S group. The relative content of AMPKα increased sequentially in the 3 groups, and the difference between the two comparisons of each group was statistically significant (P<0.05). The relative content of IL-6, TNF-α and NF-κB in the F/S group was greater than that of the F/F group, and the difference was statistically significant (P<0.001). Pathological morphological observations can be seen that the phenomena of nuclear consolidation, deep staining, and neuronal apoptosis occur, and the F/S group is more obvious. Conclusions: Flurbiprofen axetil can reduce the inflammatory response and cognitive function of an inflammatory pain model using MCI rats through the AMPKα/NF-κB signaling pathway.

Melatonin suppresses ferroptosis via activation of the Nrf2/HO-1 signaling pathway in the mouse model of sepsis-induced acute kidney injury.

BACKGROUND: Ferroptosis is a regulated form of cell death. At present, the role of ferroptosis in sepsis-induced acute kidney injury (SAKI) has not been studied. Melatonin (MEL) has been reported to be an effective ferroptosis inhibitor, but it is unclear whether Melatonin can regulate ferroptosis in SAKI and whether its downstream mechanism correlates with the Nrf2/HO-1 pathway. METHODS: The cecal ligation and puncture (CLP) method and LPS injection were used to induce SAKI in mouse model. Ferroptosis markers, including malondialdehyde (MDA) and glutathione peroxidase 4 (GPX4), were assessed. The ferroptosis inhibitor ferrostatin-1 (Fer-1) was used to explore the role of ferroptosis in SAKI. The GPX4 inhibitor RSL3, the HO-1 inhibitor zinc protoporphyrin(ZnPP), and the Nrf2 inhibitor ML385 were used to explore the specific mechanism of MEL in alleviation of SAKI. RESULTS: The ferroptosis level was increased in the renal tissue of CLP- and LPS-induced septic mice. Both Fer-1 and MEL administration could suppress ferroptosis and attenuate kidney injury upon sepsis challenge. RSL3 partially blocked MEL's beneficial renal-protective effects. MEL up-regulated Nrf2 and HO-1 in CLP mice, and both ZnPP and ML385 blocked the MEL-mediated effects of ferroptosis inhibition and renal protection. CONCLUSIONS: Ferroptosis aggravates SAKI. Melatonin treatment suppresses ferroptosis and alleviates kidney injury in the context of experimental sepsis by upregulating Nrf2/HO-1 pathway.

The effects of dexmedetomidine on the cognitive function of mild cognitive impairment (MCI) rats.

Background: The study sought to investigate the effects of dexmedetomidine (DEX) on cognitive function after anesthesia and to examine its actual mechanism. Methods: A total of 48 rats were injected with d-galactose (D-gal) 1,000 mg·kg-1·d-1 and normal saline at the neck and back for 1 week to establish rats with mild cognitive impairment (MCI) and conduct behavioral tests. Sevoflurane was inhaled and DEX was pumped into each group respectively. Morris water maze (MWM) test was conducted 24 hours later. The inflammatory factors interleukin (IL)-1, interleukin (IL)-6, and a tumor necrosis factor (TNF)-α in brain homogenate were quantitatively measured by enzyme-linked immunosorbent assay (ELISA) on the next day. The apoptosis of hippocampal cells was observed by hematoxylin-eosin staining (HE staining). Results: In relation to the model establishment, we found that there was no significant difference in body weight and swimming speed before and after modeling. There was no statistically significant difference in the escape latency between Groups A, B, C, and D before modeling. After modeling, there was no statistical difference in the escape latency between Groups A, B, and C, but the difference was statistically significant when compared to Group D (P<0.05). In relation to the DEX intervention, we found that compared to Group C, MWM test performance in Groups A and B was considerably worse longer escape latencies and fewer platform crossings within 90 seconds), and were more significant in Group A. Compared with Group D, the levels of inflammatory cytokines of the brain homogenates were elevated, and this elevation was highest in Group A, followed by Group B; the pathological changes were consistent with changes in behavioral tests. In Group A, there were obvious disorders of glial cell arrangement, apoptosis and deletion. There was no significant change in Group D. And the changes of vertebral cells in Group B and Group C were slight, with orderly arrangement and intact cell structure. Conclusions: DEX inhibits the apoptosis of hippocampal cells and reduces the cognitive dysfunction of rats with MCI induced by D-gal via the inhibition of the release of inflammatory cytokines.

Drp-1 as Potential Therapeutic Target for Lipopolysaccharide-Induced Vascular Hyperpermeability.

Mitochondria-dependent apoptotic signaling has a critical role in the pathogenesis of vascular hyperpermeability (VH). Dynamin-related protein-1- (Drp-1-) mediated mitochondrial fission plays an important role in mitochondrial homeostasis. In the present study, we studied the involvement of Drp-1 in resistance to VH induced by lipopolysaccharide (LPS). To establish the model of LPS-induced VH, LPS at 15 mg/kg was injected into rats in vivo and rat pulmonary microvascular endothelial cells were exposed to 500 ng/ml LPS in vitro. We found that depletion of Drp-1 remarkedly exacerbated the mitochondria-dependent apoptosis induced by LPS, as evidenced by reduced apoptosis, mitochondrial membrane potential (MMP) depolarization, and activation of caspase-3 and caspase-9. Increased FITC-dextran flux indicated endothelial barrier disruption. In addition, overexpression of Drp-1 prevented LPS-induced endothelial hyperpermeability and upregulated mitophagy, as evidenced by the loss of mitochondrial mass and increased PINK1 expression and mitochondrial Parkin. However, the mitophagy inhibitor, 3-Methyladenine, blocked these protective effects of Drp-1. Furthermore, inhibition of Drp-1 using mitochondrial division inhibitor 1 markedly inhibited LPS-induced mitophagy and aggravated LPS-induced VH, as shown by increased FITC-dextran extravasation. These findings implied that Drp-1 strengthens resistance to mitochondria-dependent apoptosis by regulating mitophagy, suggesting Drp-1 as a possible therapeutic target in LPS-induced VH.

Cannabidiol alleviates hemorrhagic shock-induced neural apoptosis in rats by inducing autophagy through activation of the PI3K/AKT pathway.

Recently, several studies have reported that the pharmacological effects exerted by cannabidiol (CBD) are partially related to the regulation of autophagy. Increasing evidence indicates that autophagy provides protection against ischemia-induced brain injury. However, the protective effect of CBD against mitochondrial-dependent apoptosis in hemorrhagic shock (HS)-induced brain injury has not been studied. In the present study, we observed the protective effects of CBD against neural mitochondrial-dependent apoptosis in a rat model of HS. In addition, CBD increased Beclin-1 and LC3II expression and reduced P62 expression, which were indicative of autophagy. CBD treatment attenuated the neural apoptosis induced by HS, as reflected by restoring mitochondrial dysfunction, downregulation of BAX, neuro-apoptosis ratio and NF-κB signaling activation, and upregulation of BCL2 in the cerebral cortex. Such protective effects were reversed by 3-Methyladenine, a specific autophagy inhibitor, indicating that the protective effects of CBD treatment involved autophagy. LY294002, a PI3K inhibitor, significantly inhibited CBD-induced autophagy, demonstrating that PI3K/AKT signaling is involved in the CBD's regulation of autophagy. Furthermore, we found that CBD treatment upregulated PI3K/AKT signaling via cannabinoid receptor 1. Therefore, these findings suggested that CBD treatment protects against cerebral injury induced by HS-mediated mitochondrial-dependent apoptosis by activating the PI3K/AKT signaling pathway to reinforce autophagy.

Role of Parkin-mediated mitophagy in the protective effect of polydatin in sepsis-induced acute kidney injury.

BACKGROUND: We have reported that polydatin (PD) alleviates mitochondrial dysfunction in rat models of sepsis-induced acute kidney injury (SI-AKI), but the mechanism is not well understood. Here, we investigated the role of Parkin-mediated mitophagy in the protective effects of PD in SI-AKI in mice. METHODS: Sepsis was induced in the mice by caecal ligation and puncture. Mitophagy was determined by mitochondrial mass. NLRP3 inflammasome activation was determined by NLRP3, ASC and caspase-1. Mitophagy was blocked by treatment with mitochondrial division inhibitor-1 and Parkin knockout. KEY RESULTS: PD treatment increased the sepsis-induced loss of mitochondrial mass, indicating the upregulation of mitophagy. Furthermore, PD treatment mediated Parkin translocation from the cytoplasm to the mitochondria. This suggests that Parkin-mediated mitophagy is an underlying mechanism. This was confirmed by the suppression of PD-induced mitophagy in Parkin-/- mice and in mice that were treated with a mitophagy inhibitor. PD-induced Parkin translocation and mitophagy were blocked by inhibiting SIRT1; thus, activation of SIRT1 might be an important molecular mechanism that is triggered by PD. Additionally, PD treatment protected against sepsis-induced kidney injury. These effects were blocked by inhibition of Parkin-dependent mitophagy. Furthermore, PD also protected against mitochondrial dysfunction and mitochondria-dependent apoptosis, and the effect was blocked when Parkin-dependent mitophagy was inhibited. Finally, PD suppressed NLRP3 inflammasome activation that was also dependent on Parkin-mediated mitophagy. CONCLUSIONS: These findings indicate that Parkin-mediated mitophagy is important for the protective effect of PD in SI-AKI, and the underlying mechanisms include the inhibition of mitochondrial dysfunction and NLRP3 inflammasome activation.

Genipin attenuates mitochondrial-dependent apoptosis, endoplasmic reticulum stress, and inflammation via the PI3K/AKT pathway in acute lung injury.

The protective effects of genipin against lipopolysaccharide (LPS)-induced acute lung injury (ALI) have been reported; however, the mechanism is unclear. Genipin performs its pharmacological effects via activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. In the present study, we aimed to determine whether the PI3K/AKT pathway is involved in the protective effects of genipin against mitochondrial-dependent apoptosis, endoplasmic reticulum stress (ERS), and inflammation in ALI. We constructed in vivo and in vitro models of LPS-induced ALI. PI3K/AKT signaling was inhibited using LY294002. Pretreatment with genipin increased AKT phosphorylation, indicating that PI3K/AKT signaling was upregulated. Genipin pretreatment prevented LPS-induced histopathological deterioration, increased pulmonary edema, and decreased oxygenation index, all of which were inhibited using LY294002. In addition, genipin pretreatment attenuated LPS-mediated mitochondrial apoptosis, as indicated by improved mitochondrial dysfunction, downregulation of BAX (BCL2 associated X, apoptosis regulator), upregulation of BCL2 (BCL2 apoptosis regulator), inhibited the release of cytochrome c, activation of caspase-3, and cell apoptosis. Genipin pretreatment inhibited the LPS-induced upregulation of AF4/FMR2 family member 4 (CHOP), glucose-regulated protein, 78 kDa (GRP78), and X-box binding protein 1 (XBP1) levels, indicating ERS suppression. Moreover, genipin pretreatment alleviated LPS-induced inflammation, indicating by blockade of nuclear factor kappa b (NF-κB) signaling activation and reduced tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, and IL-6 levels in the lung and bronchoalveolar lavage fluid. LY294002 could inhibit these genipin-induced protective effects against apoptosis, ERS, and inflammation. Thus, genipin significantly activates PI3K/AKT signaling to ameliorate mitochondria-dependent apoptosis, ERS, and inflammation in LPS-induced ALI.

Evidence for SIRT1 Mediated HMGB1 Release From Kidney Cells in the Early Stages of Hemorrhagic Shock.

BACKGROUND: This study is to explore the effect of SIRT1 deacetylating inactivation on organ-derived high mobility group box 1 (HMGB1) during the development of severe hemorrhagic shock (HS). METHODS: Hemorrhagic shock model was reproduced in rats by blood shedding and mimicked in HK-2 cells by hypoxia-reoxygenation (H/R) treatment. The level and acetylation of HMGB1 and the expression and activity of SIRT1 were detected in tissue, serum and cultured cells and supernatant. The deacetylated sites of HMGB1 was determined by Co-IP. RESULTS: Serum HMGB1 in HS rats was increased but were reduced in multiple organs, especially in kidney tissue, with enhanced HMGB1 acetylation, and reduced deacetylase SIRT1 activity in isolated RTECs. HMGB1 in suspension of H/R-treated HK-2 cells was increased, accompanying with enhanced HMGB1 acetylation, and nuclear-plasma translocation. SIRT1 down-regulated by siRNA aggravated acetylation of HMGB1 and nucleus-to-cytoplasm translocation and resulted in increased HMGB1 in cultured HK-2 suspension. Immunoprecipitation data suggested that SIRT1-indcuced deacetylated sites of HMGB1 were K90 and K177 following H/R. SIRT1 overexpression inhibited the acetylation of HMGB1 and reduced the content of extracellular HMGB1 in H/R-treated HK-2 cells. Inhibiting mutation of SIRT1 restored the acetylation of HMGB1 and HMGB1 content in extracellular suspension. In HS rat model, the neutralization of HMGB1 with antibody could reduce serum HMGB1 and pro-inflammatory cytokine contents, but had no effect on SIRT1 protein expression and activity. Polydatin (PD), a potential SIRT1 agonist, up-regulated SIRT1 activity and inhibited nucleus-to-cytoplasm translocation of HMGB1 in RTECs. Moreover, PD also attenuated RTEC apoptosis, protected renal function, and prolonged survival in HS rats. These beneficial effect of PD is largely blocked by specific inhibition of SIRT1 with Ex527. CONCLUSION: The acetylation of HMGB1 in K99 and K177 is enhanced during HS due to the downregulation of SIRT1. The nucleus-to-cytoplasm translocation and the release of acetylated HMGB1 from RTECs of kidney might exacerbate the pro-inflammatory effects of HMGB1 during the development of HS.

Flexible video endoscope versus Macintosh laryngoscope for orotracheal tracheal intubation in the lateral position: a study protocol for a randomized controlled trial.

BACKGROUND: Tracheal intubation with the patient in the lateral position is difficult because the laryngeal view is compromised during direct laryngoscopy. Flexible video endoscopes may facilitate intubation even when laryngeal views are poor on direct laryngoscopy because the patients are positioned laterally. Thus, this trial aims to compare the efficacy of flexible video endoscopes to Macintosh laryngoscopes for orotracheal intubation in the lateral position and to investigate their feasibility, i.e., whether the use of the two devices in combination can secure the airway when endotracheal intubation in the lateral position has failed using one device. METHODS: This will be a prospective, randomized, single-center, clinical trial. One hundred and seventy-four patients aged 18-65 years, who have been scheduled to undergo tracheal intubation under uniform general anesthetic techniques for elective kidney surgery in the lateral decubitus position will be randomly divided into the flexible video endoscope and the Macintosh laryngoscope groups. Primary outcomes include intubation time and intubation success rate. Secondary outcomes include overall user satisfaction (graded from 1 to 10 (1 = very poor, 10 = excellent)) and perioperative side effects and complications, such as frequency of esophageal intubation, lip or dental injury, sore throat, and hoarseness. DISCUSSION: The trial will clarify the efficacy of intubation with a Macintosh laryngoscope and a flexible video endoscope in the lateral position, and whether the two devices could be used in combination to secure the airway in cases where endotracheal intubation in the lateral position has failed with one device. TRIAL REGISTRATION: Chinese Clinical Trial Register, ChiCTR- IOR-15007175 . Registered on 6 October 2015.

Polydatin mediates Parkin-dependent mitophagy and protects against mitochondria-dependent apoptosis in acute respiratory distress syndrome.

Mitophagy removes dysfunctional mitochondria and is known to play an important role in the pathogenesis of several diseases; however, the role of mitophagy in acute respiratory distress syndrome (ARDS) remains poorly understood. While we have previously demonstrated that polydatin (PD) improves lipopolysaccharide (LPS)-induced ARDS, the specific mechanism remains unclear. In present study, we aimed to determine whether PD activates Parkin-dependent mitophagy to protect against LPS-induced mitochondria-dependent apoptosis and lung injury. To establish the ARDS model, C57BL/6 mice were intratracheally injected with LPS (5 mg/kg) in vivo and Beas-2B cells were exposured to 0.5 mM LPS in vitro. Our results indicate that PD facilitates Parkin translocation to mitochondria and promotes mitophagy in ARDS-challenged mice and LPS-treated Beas-2B cells. However, PD-induced mitophagy was suppressed in Parkin-/- mice and Parkin siRNA transfected cells, indicating that PD activates Parkin-dependent mitophagy. Furthermore, the protective effects of PD against LPS-induced mitochondria-dependent apoptosis and lung injury were suppressed when Parkin was depleted both in vivo and in vitro. The inhibition of mitophagy with mitophagy inhibitor mitochondrial division inhibitor-1 in vivo and silencing of autophagy-related gene 7 in vitro also blocked the protective effects mediated by PD. Our data suggest that Parkin-dependent mitophagy induced by PD provides protection against mitochondria-dependent apoptosis in ARDS.

SIRT1-mediated HMGB1 deacetylation suppresses sepsis-associated acute kidney injury.

Sepsis is the leading cause of death in the intensive care unit and continues to lack effective treatment. It is widely accepted that high-mobility group box 1 (HMGB1) is a key inflammatory mediator in the pathogenesis of sepsis. Moreover, some studies indicate that the functions of HMGB1 depend on its molecular localization and posttranslational modifications. Our previous study confirms that sirtuin 1, silent information regulator 2-related enzyme 1 (SIRT1), a type III deacetylase, can ameliorate sepsis-associated acute kidney injury (SA-AKI). We explored the effect and mechanism of SIRT1 on HMGB1 using a mouse model of cecal ligation and puncture-induced sepsis and LPS-treated human kidney (HK-2) cell line. We found that HMGB1 is elevated in the serum but is gradually reduced in kidney cells in the later stages of septic mice. The acetylation modification of HMGB1 is a key process before its nucleus-to-cytoplasm translocation and extracellular secretion in kidney cells, accelerating the development of SA-AKI. Moreover, SIRT1 can physically interact with HMGB1 at the deacetylated lysine sites K28, K29, and K30, subsequently suppressing downstream inflammatory signaling. Thus the SIRT1-HMGB1 signaling pathway is a crucial mechanism in the development of SA-AKI and presents a novel experimental perspective for future SA-AKI research.

Effect of goal-directed fluid therapy on early cognitive function in elderly patients with spinal stenosis: A Case-Control Study.

PURPOSE: To explore effect of goal-directed fluid therapy (GDFT) on early cognitive function in elderly patients with spinal stenosis. METHODS: 83 elderly patients with spinal stenosis were randomly classified into two groups: control group (n = 40) and GDFT group (n = 43). The Montreal Cognitive Assessment (MoCA) score, IL-6 and S100ß levels, hemodynamic parameters, cerebral oxygen saturation (rSO2), arterial lactic acid values, output of surgery, operation time and cases of hypotension, intraoperative complications within 7 days were recorded for all patients. RESULTS: The incidence of postoperative cognitive dysfunction (POCD) was about 21.67% in this study. The MoCA scores, inflammatory mediators, perfusion indexes (rSO2 and lactic acid)and intraoperative hemodynamics(HR, MAP, and CI)were not all the same at different time points (P < 0.05). The levels of inflammatory mediators (IL-6 and S100ß) in GDFT group were lower than those in the control group (P < 0.05). Total intake, amount of lactated Ringer's solution and cases of hypotension in GDFT group were significantly lower than control group (P < 0.05), but amount of voluven was higher than control group(P < 0.05). Compared with control group, the incidence of postoperative delirium, nausea and vomiting, and hypotension in GDFT group was lower (P < 0.05). CONCLUSIONS: GDFT can maintain the stability of perioperative hemodynamics in the prone position of elderly patients with spinal stenosis, improve the balance between perfusion of tissue and organ and supply and demand of oxygen, reduce the inflammatory response, and reduce the incidence of early POCD in this type of surgery.

SIRT1/3 Activation by Resveratrol Attenuates Acute Kidney Injury in a Septic Rat Model.

Sepsis often results in damage to multiple organ systems, possibly due to severe mitochondrial dysfunction. Two members of the sirtuin family, SIRT1 and SIRT3, have been implicated in the reversal of mitochondrial damage. The aim of this study was to determine the role of SIRT1/3 in acute kidney injury (AKI) following sepsis in a septic rat model. After drug pretreatment and cecal ligation and puncture (CLP) model reproduction in the rats, we performed survival time evaluation and kidney tissue extraction and renal tubular epithelial cell (RTEC) isolation. We observed reduced SIRT1/3 activity, elevated acetylated SOD2 (ac-SOD2) levels and oxidative stress, and damaged mitochondria in RTECs following sepsis. Treatment with resveratrol (RSV), a chemical SIRT1 activator, effectively restored SIRT1/3 activity, reduced acetylated SOD2 levels, ameliorated oxidative stress and mitochondrial function of RTECs, and prolonged survival time. However, the beneficial effects of RSV were greatly abrogated by Ex527, a selective inhibitor of SIRT1. These results suggest a therapeutic role for SIRT1 in the reversal of AKI in septic rat, which may rely on SIRT3-mediated deacetylation of SOD2. SIRT1/3 activation could therefore be a promising therapeutic strategy to treat sepsis-associated AKI.

Neuroprotective effects of polydatin against mitochondrial-dependent apoptosis in the rat cerebral cortex following ischemia/reperfusion injury.

The neuroprotective effect of polydatin (PD) against hemorrhagic shock-induced mitochondrial injury has been described previously, and mitochondrial dysfunction and apoptosis were reportedly involved in ischemic stroke. In the present study the neuroprotective effect of PD in preventing apoptosis was evaluated following induction of focal cerebral ischemia by middle cerebral artery occlusion (MCAO) in rats. PD (30 mg/kg) was administered by caudal vein injection 10 min prior to ischemia/reperfusion (I/R) injury. 24 h following I/R injury, ameliorated modified neurological severity scores (mNSS) and reduced infarct volume were observed in the PD treated group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Annexin V/propidium iodide assays demonstrated the anti-apoptotic effect of PD in the ischemic cortex. In addition, PD improved I/R injury­induced mitochondrial dysfunction, reflected by morphological observations and measurements of mitochondrial membrane potential and intracellular ATP measurement. Western blot analysis revealed an increase in B­cell lymphoma 2 apoptosis regulator (Bcl-2) expression, and a decrease in Bcl­2­associated protein X apoptosis regulator expression in the PD group in comparison with the vehicle treated group. PD treatment also prevented the release of cytochrome c from mitochondria into the cytoplasm, and blunted the activities of caspase­9 and caspase­3. Furthermore, PD treatment decreased the levels of reactive oxygen species in neurons isolated from the ischemic cortex. The findings of this study, therefore, suggest that PD has a dual effect, ameliorating both oxidative stress and mitochondria­dependent apoptosis, making it a promising new therapy for the treatment of ischemic stroke.

Polydatin Protecting Kidneys against Hemorrhagic Shock-Induced Mitochondrial Dysfunction via SIRT1 Activation and p53 Deacetylation.

OBJECTIVES: To ascertain if mitochondrial dysfunction (MD) of kidney cells is present in severe hemorrhagic shock and to investigate whether polydatin (PD) can attenuate MD and its protective mechanisms. RESEARCH DESIGN AND METHODS: Renal tubular epithelial cells (RTECs) from rat kidneys experiencing HS and a cell line (HK-2) under hypoxia/reoxygenation (H/R) treatment were used. Morphology and function of mitochondria in isolated RTECs or cultured HK-2 cells were evaluated, accompanied by mitochondrial apoptosis pathway-related proteins. RESULT: Severe MD was found in rat kidneys, especially in RTECs, as evidenced by swollen mitochondria and poorly defined cristae, decreased mitochondrial membrane potential (ΔΨm), and reduced ATP content. PD treatment attenuated MD partially and inhibited expression of proapoptotic proteins. PD treatment increased SIRT1 activity and decreased acetylated-p53 levels. Beneficial effect of PD was abolished partially when the SIRT1 inhibitor Ex527 was added. Similar phenomena were shown in the H/R cell model; when pifithrin-α (p53 inhibitor) was added to the PD/Ex527 group, considerable therapeutic effects were regained compared with the PD group apart from increased SIRT1 activity. CONCLUSIONS: MD is present in severe HS, and PD can attenuate MD of RTECs via the SIRT1-p53 pathway. PD might be a promising therapeutic drug for acute renal injury.

Drag-reducing polyethylene oxide improves microcirculation after hemorrhagic shock.

BACKGROUND: Despite resuscitation after trauma, microcirculatory abnormalities are known to persist in post-shock multiorgan dysfunction. The high-molecular weight polymer polyethylene oxide (PEO) (>10(6) Da), a classic drag-reducing polymer, can improve hemorrhagic shock (HS)-induced hemodynamic abnormalities in rats. MATERIALS AND METHODS: We examined the effects of PEO on microcirculation and on changes in multiple organs after shock. After the spinotrapezius muscle was prepared, HS was induced in Sprague-Dawley rats. Drug administration (normal saline or PEO) was performed 2 h after shock followed by infusion of shed blood. RESULTS: The velocity, blood flow, and functional capillary density in the shock + PEO group were significantly higher than those in the shock + normal saline group. Moreover, the kidney, liver, and lung function was improved, resulting in prolonged survival time. Our findings indicate that intravenous infusion of PEO can ameliorate shock-associated organ dysfunction and prolong survival time in severe HS, which may be a result of increased arteriolar blood velocity, blood flow, and functional capillary density. CONCLUSIONS: PEO could have potential clinical application in the treatment of shock-induced multiorgan dysfunction.

Polydatin Inhibits Mitochondrial Dysfunction in the Renal Tubular Epithelial Cells of a Rat Model of Sepsis-Induced Acute Kidney Injury.

BACKGROUND: Mitochondrial injury is a major cause of sepsis-induced organ failure. Polydatin (PD), a natural polyphenol, demonstrates protective mitochondrial effects in neurons and arteriolar smooth muscle cells during severe shock. In this study, we investigated the effects of PD on renal tubular epithelial cell (RTEC) mitochondria in a rat model of sepsis-induced acute kidney injury. METHODS: Rats underwent cecal ligation and puncture (CLP) to mimic sepsis-induced acute kidney injury. Rats were randomly divided into sham, CLP + normal saline, CLP + vehicle, and CLP + PD groups. Normal saline, vehicle, and 30 mg/kg PD were administered at 6, 12, and 18 hours after CLP or sham surgery via the tail vein. Mitochondrial morphology, metabolism, and function in RTECs were then assessed. Serum cytokines, renal function, survival, and histologic changes in the kidney were also evaluated. RESULTS: CLP increased lipid peroxide content, lysosomal instability, and opening of the mitochondrial permeability transition pore and caused mitochondrial swelling. Moreover, mitochondrial membrane potential (ΔΨm) was decreased and ATP levels reduced after CLP. PD inhibited all the above effects. It also inhibited the inflammatory response, improved renal function, attenuated histologic indicators of kidney damage, and prolonged survival. CONCLUSIONS: PD protects RTECs against mitochondrial dysfunction and prolongs survival in a rat model of sepsis-induced acute kidney injury. These effects may partially result from reductions in interleukin-6 and oxidative stress.

Ulinastatin mediates protection against vascular hyperpermeability following hemorrhagic shock.

OBJECT: Recent studies have suggested that intrinsic apoptotic signaling cascade is involved in endothelial barrier dysfunction following hemorrhagic shock (HS), which results in vascular hyperpermeability. Our previous study demonstrated that ulinastatin (UTI) inhibits oxidant-induced endothelial hyperpermeability and apoptotic signaling. In present study, we hypothesized that UTI would improve HS-induced vascular hyperpermeability by regulating the intrinsic apoptotic signaling cascade. METHODS: Hemorrhagic shock was induced in rats by withdrawing blood to reduce the mean arterial pressure to 40-45 mmHg for 60 min, followed by reperfusion. Mesenteric postcapillary venules were examined for changes in hyperpermeability by intravital microscopy. In vitro, Rat lung microvascular endothelial cells (RLMVECs) were exposed in hemorrhagic shock serum for 120 min, followed by transendothelial electrical resistance (TER) estimation. Mitochondrial release of cytochrome c and caspase-3 activation was estimated in vivo. In vitro, ratio of cell apoptosis was evaluated by Annexin-V/PI double stain assay; mitochondrial membrane potential (∆Ψm) was determined with JC-1; intracellular ATP content was assayed by a commercial kit; reactive oxygen species (ROS) was measured by DCFH-DA; adherens junction protein ß-catenin was detected by immunofluorescense staining. RESULTS: In vivo, UTI attenuated HS-induced vascular hyperpermeability versus the HS group (P < 0.05); In vitro, UTI attenuated shock serum induced RLMEC monolayer hyperpermeability (P < 0.05). In vivo, UTI inhibited HS-induced cytochrome c release and caspase-3 activation (P < 0.05). In vitro, shock serum induced cell apoptosis, low ATP level, ∆Ψm depolarization, ROS increase were improved by UTI pre-treatment (P < 0.05). UTI improved shock serum induced disruption of endothelial cell adherens junction. CONCLUSIONS: UTI inhibits vascular hyperpermeability following HS. UTI regulates oxidative stress and intrinsic apoptotic signaling following HS.

Polydatin: a new therapeutic agent against multiorgan dysfunction.

BACKGROUND: Polydatin (PD), a monocrystalline and polyphenolic drug isolated from a traditional Chinese herb (Polygonum cuspidatum), is protective against mitochondrial dysfunction and has been approved for clinical trials in the treatment of shock. However, whether the administration of PD has a therapeutic effect on multiple-organ dysfunction syndrome (MODS) requires investigation. MATERIAL AND METHODS: MODS was induced in Sprague-Dawley rats via hemorrhage and ligation and puncture of cecum-induced sepsis. The rats were divided into three groups as follows: MODS + PD, MODS + normal saline, and a control group (no treatment). Survival time, blood biochemical indexes, and histopathologic changes in various organs were evaluated; serum oxidative stress (advanced oxidative protein products [AOPPs]) and proinflammatory cytokines (tumor necrosis factor-α, interleukin 1ß, and interleukin 6) were assayed using enzyme-linked immunosorbent assay. Apoptosis-related protein expression (B-cell lymphoma-2 [Bcl-2] and Bax) was assayed by immunohistochemical and Western blotting methods, whereas caspase-3 activity was assayed by spectrophotometry. RESULTS: PD improved organ function, prolonged survival time, and reduced MODS incidence and serum levels of AOPPs and proinflammatory cytokines. It also decreased Bax levels and caspase-3 activity and increased Bcl-2 levels in the kidney and liver. CONCLUSIONS: PD may serve as a potential therapeutic for MODS, as it suppresses oxidative stress, inhibits inflammatory response, attenuates apoptosis, and protects against mitochondrial dysfunction.