Sen Hu

Electroacupuncture at Zusanli (ST36) Prevents Intestinal Barrier and Remote Organ Dysfunction following Gut Ischemia through Activating the Cholinergic Anti-Inflammatory-Dependent Mechanism

This study investigated the protective effect and mechanism of electroacupuncture at ST36 points on the intestinal barrier dysfunction and remote organ injury after intestinal ischemia and reperfusion injury in rats. Rats were subjected to gut ischemia for 30u2009min, and then received electroacupuncture for 30u2009min with or without abdominal vagotomy or intraperitoneal administration of cholinergic α7 nicotinic acetylcholine receptor (α7nAChR) inhibitor. Then we compared its effects with electroacupuncture at nonchannel points, vagal nerve stimulation, or intraperitoneal administration of cholinergic agonist. Cytokine levels in plasma and tissue of intestine, lung, and liver were assessed 60u2009min after reperfusion. Intestinal barrier injury was detected by histology, gut injury score, the permeability to 4u2009kDa FITC-dextran, and changes in tight junction protein ZO-1 using immunofluorescence and Western blot. Electroacupuncture significantly lowered the levels of tumor necrosis factor-α and interleukin-8 in plasma and organ tissues, decreased intestinal permeability to FITC-dextran, and prevented changes in ZO-1 protein expression and localization. However, abdominal vagotomy or intraperitoneal administration of cholinergic α7nAChR inhibitor reversed these effects of electroacupuncture. These findings suggest that electroacupuncture attenuates the systemic inflammatory response through protection of intestinal barrier integrity after intestinal ischemia injury in the presence of an intact vagus nerve.

Pyruvate Ringer's Solution Corrects Lactic Acidosis and Prolongs Survival during Hemorrhagic Shock in Rats

BACKGROUNDnLactic acidosis is a life-threatening complication of hemorrhagic shock. There is no ideal therapy for it in the clinical setting.nnnOBJECTIVEnThis study was designed to investigate whether pyruvate Ringers solution could treat hypoxic lactic acidosis associated with lethal hemorrhagic shock in rats.nnnMETHODSnA total of 54 rats were subjected to hemorrhagic shock with mean arterial pressure (MAP) of 40xa0mm Hg for 1xa0h. They were then randomly divided into three groups (nxa0=xa018 each): Group N had no fluid resuscitation; Group L received lactated Ringers solution infusion; and Group P received pyruvate Ringers solution infusion. The survival rate was investigated after 24xa0h. In addition, a second set of 54 rats was selected for blood sampling, with identical methods for shock and resuscitation being followed, to determine arterial pH, blood gas analysis, lactate, pyruvate, and organs enzyme activities at various time points. The MAP was monitored for 6xa0h in both populations.nnnRESULTSnPyruvate Ringers solution significantly increased the survival rate of rats subjected to fatal shock and receiving pyruvate Ringers solution (Group P) by 1.5 times the survival rate in Group L at 24xa0h after fluid resuscitation (55.6% vs. 22.2%, respectively; pxa0<xa00.05). Pyruvate infusion maintained a higher MAP and fully corrected severe acidosis 1xa0h after resuscitation in comparison to the lactated infusion, and markedly decreased blood lactate levels and the lactate-to-pyruvate ratio 4xa0h after resuscitation. It also significantly improved serum markers of organ dysfunction.nnnCONCLUSIONnPyruvate Ringers solution efficiently treated hypoxic lactic acidosis and significantly increased the survival rate in ratsxa0with lethal hemorrhagic shock. Pyruvate Ringers solution is potentially applicable to clinical resuscitation in humans.

Electroacupuncture improves gut barrier dysfunction in prolonged hemorrhagic shock rats through vagus anti-inflammatory mechanism

AIMnTo investigate whether electroacupuncture (EA) at Zusanli (ST36) prevents intestinal barrier and remote organ dysfunction following prolonged hemorrhagic shock through a vagus anti-inflammatory mechanism.nnnMETHODSnSprague-Dawley rats were subjected to about 45% of total blood volume loss followed by delayed fluid replacement (DFR) with Ringer lactate 3h after hemorrhage. In a first study, rats were randomly divided into six groups: (1) EAN: EA at non-channel acupoints followed by DFR; (2) EA: EA at ST36 after hemorrhage followed by DFR; (3) VGX/EA: vagotomy (VGX) before EA at ST36 and DFR; (4) VGX/EAN: VGX before EAN and DFR; (5) α-bungarotoxin (α-BGT)/EA: intraperitoneal injection of α-BGT before hemorrhage, followed by EA at ST36 and DFR; and (6) α-BGT/EAN group: α-BGT injection before hemorrhage followed by EAN and DFR. Survival and mean arterial pressure (MAP) were monitored over the next 12 h. In a second study, with the same grouping and treatment, cytokine levels in plasma and intestine, organ parameters, gut injury score, gut permeability to 4 kDa FITC-dextran, and expression and distribution of tight junction protein ZO-1 were evaluated.nnnRESULTSnMAP was significantly lowered after blood loss; EA at ST36 improved the blood pressure at corresponding time points 3 and 12 h after hemorrhage. EA at ST36 reduced tumor necrosis factor-α and interleukin (IL)-6 levels in both plasma and intestine homogenates after blood loss and DFR, while vagotomy or intraperitoneal injection of α-BGT before EA at ST36 reversed its anti-inflammatory effects, and EA at ST36 did not influence IL-10 levels in plasma and intestine. EA at ST36 alleviated the injury of intestinal villus, the gut injury score being significantly lower than that of EAN group (1.85 ± 0.33 vs 3.78 ± 0.59, P < 0.05). EA at ST36 decreased intestinal permeability to FITC-dextran compared with EAN group (856.95 ng/mL ± 90.65 ng/mL vs 2305.62 ng/mL ± 278.32 ng/mL, P < 0.05). EA at ST36 significantly preserved ZO-1 protein expression and localization at 12 h after hemorrhage. However, EA at non-channel acupoints had no such effect, and abdominal vagotomy and α-BGT treatment could weaken or eliminate the effects of EA at ST36. Besides, EA at ST36 decreased blood aminotransferase, MB isoenzyme of creatine kinase and creatinine vs EAN group at corresponding time points. At the end of 12-h experiment, the survival rate of the EA group was significantly higher than that of the other groups.nnnCONCLUSIONnEA at ST36 attenuates the systemic inflammatory response, protects intestinal barrier integrity, improves organ function and survival rate after hemorrhagic shock via activating the cholinergic anti-inflammatory mechanism.

Valproic acid treatment inhibits hypoxia-inducible factor 1α accumulation and protects against burn-induced gut barrier dysfunction in a rodent model.

Objective Burn-induced gut dysfunction plays an important role in the development of sepsis and multiple organ dysfunction. Emerging evidence suggests that hypoxia-inducible factor-1α (HIF-1α) is critical in paracelluar barrier functions via regulating vascular endothelial growth factor (VEGF) and myosin light chain kinase (MLCK) expression. Previous studies have also demonstrated that histone deacetylase inhibitors (HDACIs) can repress HIF-1α. This study aims to examine whether valproic acid (VPA), a HDACI, protects against burn-induced gut barrier dysfunction via repressing HIF-1α-dependent upregulation of VEGF and MLCK expression. Methods Rats were subjected to third degree 55% TBSA burns and treated with/ without VPA (300mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and histologic evaluation. Histone acetylation, tight junction protein zonula occludens 1 (ZO-1), VEGF, MLCK and HIF-1α were measured. In addition, CaCO2 cells were transfected with siRNA directed against HIF-1α and were stimulated with CoCl2 (1mM) for 24 hours with/without VPA (2mM) followed by analysis of HIF-1α, MLCK, VEGF and ZO-1. Results Burn insults resulted in a significant increase in intestinal permeability and mucosal damage, accompanied by a significant reduction in histone acetylation, ZO-1, upregulation of VEGF, MLCK expression, and an increase in HIF-1α accumulation. VPA significantly attenuated the increase in intestinal permeability, mucosa damage, histone deacetylation and changes in ZO-1 expression. VPA also attenuated the increased VEGF, MLCK and HIF-1α protein levels. VPA reduced HIF-1α, MLCK and VEGF production and prevented ZO-1 loss in CoCl2-stimulated Caco-2 cells. Moreover, transfection of siRNA directed against HIF-1α led to inhibition of MLCK and VEGF production, accompanied by upregulation of ZO-1. Conclusions These results indicate that VPA can protect against burn-induced gut barrier dysfunction. These protective effects may be due to its inhibitory action on HIF-1α, leading to a reduction in intestinal VEGF and MLCK expression and minimizing ZO-1 degradation.

Inhibiting Effect of Electroacupuncture at Zusanli on Early Inflammatory Factor Levels Formed by Postoperative Abdominal Adhesions

We observed the inhibitive effect of electroacupuncture (EA) at Zusanli on inflammatory mediators of postoperative intra-abdominal adhesions to find out the relationship between EA and the cholinergic anti-inflammatory pathway. Sixty-four rats were divided into 8 groups (A–H, each = 8): A = sham control; B = abdominal adhesions model; C = abdominal adhesions plus EA; D = sham acupoint control; E = abdominal adhesions plus vagotomy; F = abdominal adhesions plus EA after vagotomy; G = abdominal adhesions plus α-bungarotoxin (BGT); and H = abdominal adhesions plus EA after α-BGT. α-BGT (1u2009μg/kg) was injected into the abdominal cavity after surgery, and the bilateral celiac vagotomy was done during the surgery. On the third day the levels of inflammatory mediators (TNF-α, nitric oxide (NO), and nitric oxide synthase (NOS)) in tissues were evaluated. The abdominal adhesion groups developed obvious edema. Compared with sham control, the abdominal adhesion resulted in a significant elevation of inflammatory mediators. EA lowered the elevated levels of inflammatory mediators significantly; EA plus α-BGT and vagotomy showed less anti-inflammatory effects. The activation of the cholinergic anti-inflammatory pathway might be one of the mechanisms of EA at Zusanli acupoints to exert the anti-inflammatory effects.

Pyruvate Is Superior to Citrate in Oral Rehydration Solution in the Protection of Intestine via Hypoxia-Inducible Factor-1 Activation in Rats With Burn Injury

BACKGROUNDnRecent studies have suggested that pyruvate-enriched oral rehydration solution (Pyr-ORS) may be superior to the standard bicarbonate-based ORS in the protection of intestine from ischemic injury. The aim of this study was to compare the effects of Pyr-ORS with citrate-enriched ORS (Cit-ORS) on the intestinal hypoxia-inducible factor-1 (HIF-1)-erythropoietin (EPO) signaling pathway for enteral rehydration in a rat model of burn injury.nnnMETHODSnRats were randomly assigned to 4 groups (N = 20, 2 subgroups each: n = 10): scald sham (group SS), scald with no fluid resuscitation (group SN), scald and resuscitation with enteral Cit-ORS (group SC), and scald and resuscitation with enteral Pyr-ORS (group SP). At 2.5 and 4.5 hours after a 35% total body surface area (TBSA) scald, intestinal mucosal blood flow (IMBF), contents of HIF-1, EPO, endothelial nitric oxide synthase (eNOS), nitric oxide (NO), barrier protein (ZO-1), levels of serum diamine oxidase (DAO), and intestinal mucosal histology injury score were determined.nnnRESULTSnSerum DAO activities in the scalded groups were significantly elevated, but less raised in group SP than in group SC, at 2.5 hours and at 4.5 hours after the scald. Further, group SP more profoundly preserved intestinal HIF-1 expression compared with group SC at the 2 time points. Compared with group SC, group SP had markedly elevated intestinal EPO, eNOS, and NO levels at the same time points, respectively (P < .05). Similarly, IMBF and ZO-1 levels were significantly higher in group SP than in group SC. Intestinal mucosal histopathological scores were statistically higher at 2.5 hours and 4.5 hours after scalding but were more attenuated in group SP than in group SC (P < .05). Immunofluorescence expression of intestinal mucosal ZO-1 was consistent with the above changes. The above parameters were also significantly different between groups SC and SN (all P < .05).nnnCONCLUSIONnPyr-ORS provides a superior option to Cit-ORS for the preservation of intestinal blood flow and barrier function and the attenuation of histopathological alterations in enteral resuscitation of rats with burn injury. Its underlying mechanism may be closely related to the pyruvate in activation of intestinal HIF-1-EPO signaling cascades.

Electroacupuncture at Zusanli Prevents Severe Scalds-Induced Gut Ischemia and Paralysis by Activating the Cholinergic Pathway

Severe burn injuries may result in gastrointestinal paralysis, and barrier dysfunction due to gut ischemia and lowered vagus excitability. In this study we investigate whether electroacupuncture (EA) at Zusanli (ST36) could prevent severe scalds-induced gut ischemia, paralysis, and barrier dysfunction and whether the protective role of EA at ST36 is related to the vagus nerve. 35% burn area rats were divided into six groups: (a) EAN: EA nonchannel acupoints followed by scald injury; (b) EA: EA at ST36 after scald injury; (c) VGX/EA: vagotomy (VGX) before EA at ST36 and scald injury; (d) VGX/EAN: VGX before EAN and scald injury; (e) atropine/EA: applying atropine before scald injury and then EA at ST36; (f) atropine/EAN: applying atropine before scald injury and then EA at nonchannel acupoints. EA at the Zusanli point significantly promoted the intestinal impelling ratio and increased the amount of mucosal blood flow after scald injury. The plasma diamine oxidase (DAO) and intestinal permeability decreased significantly after scald injury in the EA group compared with others. However, EA after atropine injection or cervical vagotomy failed to improve intestinal motility and mucosa blood flow suggesting that the mechanism of EA may be related to the activation of the cholinergic nerve pathway.

Effect of carbachol on intestinal mucosal blood flow, activity of Na+-K+-ATPase, expression of aquaporin-1, and intestinal absorption rate during enteral resuscitation of burn shock in rats.

We investigated the effect of carbachol (CAR, a cholinergic agent) on intestinal mucosal blood flow (IMBF), activity of Na+-K+-ATPase, expression of aquaporin (AQP)-1, and intestinal absorption rate during enteral resuscitation of a 35%TBSA scald in rats with a glucose electrolyte solution (GES). One hundred male Wistar rats were randomly divided into five groups: sham scald (N group); scald without fluid resuscitation (S group); scald resuscitated with enteral GES alone (GES group); scald resuscitated with enteral CAR alone (CAR group); and scald resuscitated with enteral CAR plus GES (GES/CAR group). The rats were inflicted 35%TBSA third degree of scald injury on the back with boiling water (100°C, 15 seconds) in all groups, except the sham scald group. A catheter was inserted into the proximal duodenum (5 cm distal to pylorus) and distal ileum (5 cm proximal to cecum), of each rats through laparotomy, thus a segment of intestine was virtually isolated to form a loop for inlet and outlet of introduced fluid. In N, GES, and GES/CAR groups, fluids were introduced 30 minutes after scald injury. The speed of fluid infusion was 4 ml/kg/1%TBSA for 4 hours. CAR (60 μg/kg) was injected into the intestinal lumen at 30-minute after injury in CAR and GES/CAR groups. At 2 and 4 hours after scald, intestinal absorption rate of water and Na+, and IMBF were determined, respectively. Then, animals were killed, and specimens of intestinal tissue were obtained for the determination of the activity of Na+-K+-ATPase, hematoxylin-eosin coloring, and expression of AQP-1. The intestinal absorption rate was reduced markedly in GES group compared with sham scald group at 2 and 4 hours after scald, and absorption rate of small intestine in GES/CAR was significantly higher than that in GES group (P < .05). It was also found that there was significant decrease in IMBF, activity of Na+-K+-ATPase, and expression of AQP-1 in scald group compared with the sham group. However, in GES/CAR group, the levels of these parameters were significantly increased compared with scald groups (P < .05). The results indicate that CAR promotes intestinal absorption rate of water and Na+ by improving IMBF, ATPase activity, and AQP-1 expression in gut mucosa during resuscitation with enteral GES of burn shock in rats.

The Influence of Zusanli and Nonmeridian Acupuncture Points on the Survival Rate and Intestinal Tissue Features after Fatal Hemorrhagic Shock in Rats

Sixty Sprague-Dawley rats were divided into 5 groups: (a) control group (HS); (b) Immediate rehydration group (IFR); (c) ST36 electroacupuncture (EA) delay rehydration group (EA/DFR): EA at ST36 immediately after blood loss with infusion 3u2009h later; (d) EA nonmeridian rehydration group (SEA/DFR): EA at nonacupuncture sites with rehydration similar to EA/DFR; (e) ST36 EA group (EA): EA at ST36 immediately after blood loss with no rehydration. Forty-five percent of the entire blood volume was taken out to make lethal hemorrhagic shock models. We recorded the survival rate, intestinal tissue DAO content, and microcirculation. The survival rate of the EA/DFR group and the IFR group was significantly higher than that of the other three groups (P < 0.05). Twelve hours after blood loss, intestinal tissue DAO content of the EA/DFR group and the IFR group was significantly higher than that of the SEA/DFR group, EA group, and HS group (P < 0.05 and P < 0.01). The mucosal blood flow of the EA/DFR group and the IFR group was significantly higher than the other groups (P < 0.05 each). We conclude that EA improves the blood pressure and raises the early survival rate of hemorrhagic shock rats, maintains the intestinal barrier function, and improves the degree of intestinal ischemia.

Carbachol alleviates rat cytokine release and organ dysfunction induced by lipopolysaccharide.

BACKGROUNDnTo observe the influence of carbachol on inflammatory cytokine release and its protective role on organ function in rat endotoxemia model, and, furthermore, to investigate its receptor mechanism in rat peritoneal macrophages in vitro.nnnMETHODSnIn the animal experiments, Wistar rats were subjected to lipopolysaccharide (LPS) injection (5 mg/kg body weight) to establish an endotoxemia animal model, and carbachol/nicotine was given 15 minutes after LPS injection. Serum contents of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were determined with enzyme-linked immunosorbent assay 4 hours after LPS injection. Plasma alanine aminotransferase, creatine kinase-MB, and diamine oxidase contents were detected 24 hours after LPS injection. In cell experiments, rat peritoneal macrophages were collected and initially pretreated with atropine (muscarinic cholinergic receptor antagonist) or α-Bungarotoxin (an antagonist that specifically binds α7 subunit of nicotinic cholinergic receptor), then with carbachol or nicotine, and finally stimulated with LPS. Contents of TNF-α, IL-6, and IL-10 in supernatant were assayed by enzyme-linked immunosorbent assay. Furthermore, macrophages were exposed to nicotine and carbachol of high concentration and then stained with fluorescein isothiocyanate-labeled α-bungarotoxin and observed with fluorescent confocal microscopy.nnnRESULTSnCarbachol inhibited expression of TNF-α and IL-6 after LPS injection and had no significant effect on IL-10 in rat endotoxemia model. It also inhibited the increase of plasma alanine aminotransferase and creatine kinase-MB contents whereas restored the inhibited plasma diamine oxidase activity. Cell experiments also showed that increases of TNF-α and IL-6 after LPS stimulation could be significantly inhibited by carbachol or nicotine, whereas IL-10 was not apparently altered. Atropine did not downregulate the inhibitive effects of both carbachol and nicotine, whereas α-bungarotoxin significantly downregulated these effects. Fluorescent confocal microscopy showed that nicotine and carbachol pretreatment markedly reduced the intensity of binding between fluorescein isothiocyanate-labeled α-bungarotoxin and macrophages.nnnCONCLUSIONnThe results suggested that both carbachol and nicotine play a role in the anti-inflammatory process and organ function protection through the α7 subunit of nicotinic cholinergic receptor.