Heat shock preconditioning reduces ischemic tissue necrosis by heat shock protein (HSP)-32-mediated improvement of the microcirculation rather than induction of ischemic tolerance

INTRODUCTION: Supraphysiologic stress induces a heat shock response, which may exert protection against ischemic necrosis. Herein we analyzed in vivo whether the induction of heat shock protein (HSP) 32 improves survival of chronically ischemic myocutaneous tissue, and whether this is based on amelioration of microvascular perfusion or induction of ischemic tolerance. METHODS: The dorsal skin of mice was subjected to local heat preconditioning (n = 8) 24 hours before surgery. In additional heat-preconditioned animals (n = 8), HSP-32 was inhibited by tin-protoporphyrin-IX. Unconditioned animals served as controls (n = 8). A random-pattern myocutaneous flap was elevated in the back of the animals and fixed into a dorsal skinfold chamber. The microcirculation, edema formation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day period using intravital fluorescence microscopy. RESULTS: HSP-32 protein expression was observed only in heat-preconditioned but not in unconditioned flaps. Heat preconditioning induced arteriolar dilation, which was associated with a significant improvement of both arteriolar blood flow and capillary perfusion in the distal part of the flap. Further, heat shock reduced interstitial edema formation, attenuated apoptotic cell death, and almost completely abrogated the development of flap necrosis (4% +/- 1% versus controls: 53% +/- 5%; P[r] < 0.001). Most strikingly, inhibition of HSP-32 by tin-protoporphyrin-IX completely blunted the preconditioning-induced improvement of microcirculation and resulted in manifestation of 72% +/- 4% necrosis. CONCLUSION: Local heat preconditioning of myocutaneous tissue markedly increases flap survival by maintaining adequate nutritive perfusion rather than inducing ischemic tolerance. The protection is caused by the increased arteriolar blood flow due to significant arteriolar dilation, which is mediated through the carbon monoxide-associated vasoactive properties of HSP-32.     

Local heat-shock priming-induced improvement in microvascular perfusion in osteomyocutaneous flaps is mediated by heat-shock protein 32

BACKGROUND: Stress conditioning is thought to improve microvascular free flap survival but the mechanisms of protection are not clear. The aim of this study was to determine whether local induction of heat-shock protein (HSP) 32 improves microvascular perfusion in transferred osteomyocutaneous flaps. METHODS: The hindlimb harvest region of osteomyocutaneous flaps in Wistar rats was subjected to stress conditioning by local heating (30 min, 42.5 degrees C) 24 h before microvascular flap transfer. In a second group of animals, after heat-shock priming, the action of HSP-32 was inhibited by tin protoporphyrin IX. Animals with unconditioned flaps served as controls. After transfer, the microcirculation of the muscle, cutaneous, subcutaneous and periosteal tissue of the flap was analysed quantitatively for 6 h using intravital fluorescence microscopy. RESULTS: Immunohistochemistry revealed that HSP-32 was detectable only after priming and not in unconditioned flaps. Priming did not alter functional capillary density or capillary red blood cell velocity compared with that in unconditioned flaps. However, heat-shock priming induced significant capillary dilatation (P < 0.05) and thus a substantial increase in capillary blood flow volume (P < 0.05) in all tissues of the transferred flaps. Inhibition of HSP-32 by tin protoporphyrin IX completely abolished the priming-induced improvement in capillary perfusion, as indicated by the lack of increased capillary diameters and volumetric blood flow. CONCLUSION: The present study demonstrated that stress conditioning by local heat-shock priming improves nutritive perfusion in osteomyocutaneous flaps by capillary dilatation, probably mediated through the vasoactive action of HSP-32.     

Mechanical deformation induced apoptosis in human proximal renal tubular epithelial cells is caspase dependent

PURPOSE: Ureteral obstruction (UO) results in apoptosis of renal tubular epithelial cells. We postulated that mechanical deformation and inflammation contribute to the cellular loss that occurs as a result of UO and it is mediated through altered heat shock protein 70 (HSP-70) expression and the caspase cascade. MATERIALS AND METHODS: Human HK-2 renal tubular cells were subjected to mechanical stretch. Cell viability and apoptosis were assessed by flow cytometry; HSP-70 and caspase 3 protein expression by Western blotting, and caspase 3 activity by fluorescence substrates. RESULTS: Mechanical stretch caused direct apoptosis induction and it also primed for tumor necrosis factor-alpha induced apoptosis, which was caspase 3 dependent. Although HSP-70 protein expression was increased during mechanical stretch, the protective effects of HSP-70 were only seen after further induction by heat shocking. CONCLUSIONS: Altering HSP-70 expression and manipulating the caspase cell death proteases represent a novel pathway to protect against renal tubular cell apoptosis and the potential for progression to renal failure in UO.     

The influence of local and systemic preconditioning on oxygenation, metabolism and survival in critically ischaemic skin flaps in pigs.

Stress proteins represent a group of highly conserved intracellular proteins that provide adaptation against cellular stress. The present study aims to elucidate the stress protein-mediated effects of local hyperthermia and systemic administration of monophosphoryl lipid A (MPL) on oxygenation, metabolism and survival in bilateral porcine random pattern buttock flaps. Preconditioning was achieved 24h prior to surgery by applying a heating blanket on the operative site (n = 5), by intravenous administration of MPL at a dosage of 35 microg/kg body weight (n = 5) or by combining the two (n = 5). The flaps were monitored with laser Doppler flowmetry, polarographic microprobes and microdialysis until 5h postoperatively. Semiquantitative immunohistochemistry was performed for heat shock protein 70 (HSP70), heat shock protein 32 (also termed haem oxygenase-1, HO-1), and inducible nitrc oxide synthase (iNOS). The administration of MPL increased the impaired microcirculatory blood flow in the proximal part of the flap and partial oxygen tension in the the distal part by approximately 100% each (both P<0.05), whereas both variables remained virtually unaffected by local heat preconditioning. Lactate/pyruvate (L/P) ratio and glycerol concentration (representing cell membrane disintegration) in the distal part of the flap gradually increased to values of approximately 500 mmol/l and approximately 350 micromol/l, respectively (both P<0.01), which was substantially attenuated by heat application (P<0.01 for L/P ratio and P<0.05 for glycerol) and combined preconditioning (P<0.01 for both variables), whereas the effect of MPL was less marked (not significant). Flap survival was increased from 56% (untreated animals) to 65% after MPL (not significant), 71% after heat application (P<0.05) and 78% after both methods of preconditioning (P<0.01). iNOS and HO-1 were upregulated after each method of preconditioning (P<0.05), whereas augmented HSP70 staining was only observed after heat application (P<0.05). We conclude that local hyperthermia is more effective in preventing flap necrosis than systemic MPL administration because of enhancing the cellular tolerance to hypoxic stress, which is possibly mediated by HSP70, whereas some benefit may be obtained with MPL due to iNOS and HO-1-mediated improvement in tissue oxygenation.     

Induction of heme oxygenase-1 and dilatation of hepatic sinusoids by an administration of pyrrolidine dithiocarbamate in rat livers

INTRODUCTION: Inducing heme oxygenase-1 (HO-1) provides the liver with various protective effects against stressful conditions. In this article, we report our use of pyrrolidine dithiocarbamate (PDTC) to induce HO-1 in the liver in vivo and its impact on hepatic microcirculation. MATERIALS AND METHODS: PDTC was injected intramuscularly into rats and the expression of HO-1 in liver tissue was assessed by measuring both mRNA and protein levels. The distribution of induced HO-1 was evaluated immunohistochemically. The effect of PDTC administration on hepatic microcirculation was evaluated using intravital microscopy (IVM). Rats were divided into three groups: PDTC administration (group P), vehicle administration only (group C), and ZnPP-an inhibitor of HO-1-administration after PDTC treatment (group Z). Sinusoidal diameters were measured 24 h after the injections. RESULTS: PDTC administration induced HO-1 strongly in the liver, but not in other organs. HO-1 mRNA expression in liver tissue peaked 3 h after PDTC injection and then gradually decreased. The protein expression reached a maximum level at 24-48 h after the injection, and its expression was dose-dependent with PDTC. Immunohistochemistry revealed that HO-1 was induced not only in Kupffer cells, but also in hepatocytes in the pericentral area. IVM showed that in group P, sinusoidal diameters in zone 3 (21.94 +/- 1.29 microm) were twice as large as those in group C (11.14 +/- 0.28 microm, P < 0.0001). This dilation of sinusoids was completely reversed by ZnPP (10.95 +/- 0.37 microm, P < 0.0001). CONCLUSION: A single administration of PDTC induced HO-1 in the liver with remarkable sinusoidal dilation. PDTC administration, therefore, may be a useful, new strategy in place of other stress preconditioning.     

Nitrosoglutathione improves blood perfusion and flap survival by suppressing iNOS but protecting eNOS expression in the flap vessels after ischemia/reperfusion injury

BACKGROUND: The effects of nitric oxide (NO) on the microcirculation and free tissue survival remain controversial. With the use of a rat inferior epigastric artery flap as an ischemia/reperfusion injury (I/R) model, we investigated whether exogenous NO donation regulates endogenous NO synthase (NOS) expression in the flap vessels and promotes flap survival. METHODS: Thirty minutes before flap reperfusion, normal saline (1 ml), nitrosoglutathione (GSNO 0.2, 0.6, 3 mg/kg), or N(G)-nitro-L-arginine-methyl ester (L-NAME, 450 mg/kg), was injected intravenously into 20 rats. Total plasma NOx (NO(2)-/NO(3)-) was measured to reflect NO production. Immunohistochemical staining was investigated for the endothelin-1 (ET-1) and NOS isoforms expression on the flap vessels. NOS isoforms expression was evaluated by Western blot. Laser-Doppler flowmetry monitored flap perfusion. Survival areas were assessed by gross examination at 7 days postoperatively. RESULTS: Flap ischemia at 12 hours followed by reperfusion resulted in endothelial cell damage, as demonstrated by induction of iNOS and ET-1 expression in the flap vessels. An optimal dose of nitrosoglutathione (0.6 mg GSNO/kg) significantly increased plasma NOx levels (P=.027) and improved flap perfusion by laser Doppler measurement (P=.014), and increased the flap viability area (P<.001). Additionally, it selectively suppressed iNOS induction, but enhanced eNOS expression and decreased ET-1 deposition in the flap vessels. In contrast, an NOS inhibitor, N(G)-nitro-L-arginine methyl ester, inhibited both iNOS and eNOS expression in the flap vessels, decreased endogenous NOx production, and compromised flap viability. CONCLUSION: This study indicates that intravenous administration of exogenous GSNO can appropriately donate NO to suppress iNOS induction and enhance eNOS expression in pedicle vessels, resulting in better blood perfusion and a higher flap survival after I/R injury.     

Inhibition of heme oxygenase-1 protects against tissue injury in carbon tetrachloride exposed livers

BACKGROUND/AIMS: During the metabolism of the hepatotoxin carbon tetrachloride (CCl(4)) by cytochrome P450, heme, and free radicals are released. Heme oxygenase (HO-1) is an enzyme that is induced by heme as well as oxidative stress and has been reported to be involved in mediating protection against toxic liver injury. The purpose of the present study was to specify the role of HO-1 in CCl(4)-hepatotoxicity. METHODS AND RESULTS: We could demonstrate an up-regulation of HO-1 protein in CCl(4)-exposed liver tissue that reaches its maximum after 6 to 12 h, along with intrahepatic leukocyte accumulation and tissue injury. When animals were pretreated with hemin for augmentation of HO-1 expression, CCl(4)-exposure was associated with a reduction of intrahepatic leukocyte accumulation, while inhibition of CCl(4)-induced HO-1 expression by tin protoporphyrin-IX (SnPP-IX) enhanced leukocytic response. Of interest, however, liver morphology, transaminases, and bile flow as parameters of hepatocellular integrity and excretory function did not concur with reduced leukocyte numbers in the hepatic microcirculation, and revealed best organ function and tissue preservation in case of HO-1 inhibition by SnPP-IX. In contrast, hemin-treated CCl(4)-exposed livers demonstrated pathologic enzyme release and cholestasis. CONCLUSIONS: Taken together, inhibition of HO-1 in CCl(4)-hepatotoxicity protected the liver, while higher HO-1 activity harmed liver tissue, most probably due to interference of the HO-1 pathway with CCl(4)-dependent metabolism via cytochrome P450 and heme overload-associated toxicity.     

Expression of Heme Oxygenase-1 in Human Livers Before Transplantation Correlates with Graft Injury and Function After Transplantation

Upregulation of heme oxygenase-1 (HO-1) has been proposed as an adaptive mechanism protecting against ischemia/reperfusion (I/R) injury. We investigated HO-1 expression in 38 human liver transplants and correlated this with I/R injury and graft function. Before transplantation, median HO-1 mRNA levels were 3.4-fold higher (range: 0.7-9.3) in donors than in normal controls. Based on the median value, livers were divided into two groups: low and high HO-1 expression. These groups had similar donor characteristics, donor serum transaminases, cold ischemia time, HSP-70 expression and the distribution of HO-1 promoter polymorphism. After reperfusion, HO-1 expression increased significantly further in the initial low HO-1 expression group, but not in the high HO-1 group. Postoperatively, serum transaminases were significantly lower and the bile salt secretion was higher in the initial low HO-1 group, compared to the high expression group. Immunofluorescence staining identified Kupffer cells as the main localization of HO-1. In conclusion, human livers with initial low HO-1 expression (<3.4 times controls) are able to induce HO-1 further during reperfusion and are associated with less injury and better function than initial high HO-1 expression (>3.4 times controls). These data suggest that an increase in HO-1 during transplantation is more protective than high HO-1 expression before transplantation.     

Improvement of nutritive perfusion after free tissue transfer by local heat shock-priming-induced preservation of capillary flowmotion

BACKGROUND: Capillary flowmotion protects pedicled flaps during critical perfusion conditions. However, free tissue transfer, causing ischemia-reperfusion and surgical trauma, have been shown to blunt these protective blood flow fluctuations. Because heat shock priming protects tissue after transfer, we herein studied whether heat shock protein expression is capable to preserve critical perfusion-induced capillary flowmotion in transferred composite flaps. METHODS: In Sprague Dawley rats (n = 16), osteomyocutaneous flaps were subjected to critical perfusion after harvest and 1 h and 4 h after free transfer. In eight animals additional heat shock priming was induced 24 h before flap harvest. Microcirculation including capillary flowmotion was analyzed using intravital fluorescence microscopy. RESULTS: After harvest, critical perfusion induced capillary flowmotion in skeletal muscle tissue of all flaps. By this, functional capillary density (FCD), an indicator of nutritive perfusion, was maintained not only in muscle but also in periosteum, subcutis, and skin. In contrast, 1 h after flap transfer muscle capillary flowmotion was completely abrogated, resulting in a significant decrease of FCD in all tissues. Heat shock-priming completely restored capillary flowmotion, and, by this, maintained tissue FCD. CONCLUSIONS: The loss of muscle capillary flowmotion after free tissue transfer-associated ischemia-reperfusion can be prevented by heat shock-priming. This may represent the mechanism of protection by local heat application.     

Pravastatin attenuates subsequent ischemia reperfusion injury in the rat kidney and the molecular mechanism

Objective To investigate the effect of pretreatment with pravastatin on subsequent ischemia reperfusion (IR) injury in rat kidney. Methods Health Sprague-Dawley rats were randomized into three subgroups: ⑴ group sham (n＝5); ⑵ group IR (n＝8); ⑶group pravastatin＋IR(n＝8). IR injury were induced by clamping both renal arteries for 45 min. Rats were pretreated with pravastatin (20 mg·kg-1·d-1) for 7 d before IR injury and killed 24 h later. Renal tissues were stained with periodic acid Schiff (PAS) to measure the tubular necrosis and the heat shock protein 70 (HSP-70) expression was measured by immunohistochemistry. TUNEL assay and Western blotting were used to measure apoptotic cell death and apoptosis-related protein (Bcl-2, Bax and active caspase-3) expression. Results Normal rats treated with pravastatin displayed a significant increase in HSP-70 expression [(173.9±19.2)% vs (100.9±13.7)%, P﹤0.01], which mainly located at inner cortex and outer medulla of rat kideys. Compared to group sham, the levels of BUN and Scr, tubular necrosis index [(36.0±1.2)% vs 0] and the number of TUNEL-positive cells (295.4±28.3 vs 1.4±1.1) in group IR were increased (P﹤0.01). In the molecular level, IR injury significantly inhibited the ratio of Bcl-2/Bax protein [(57.3±7.3)% vs (100.0±5.4)%, P﹤0.01], but increased active caspase-3 [(385.2±38.7)% vs (96.9±3.1)%, P﹤0.01] expression. Pre-treatment with pravastatin improved all of above parameters (all P﹤0.05). Interestingly, there was no significant difference in serum lipid levels between experimental groups. Conclusion Pravastatin prevents against subsequent IR injury in rat kidney, and the effect is closely associated with induction of HSP-70 which is hypolipidemia-independent.     

Kinetics of plasma heat shock protein HSP-70 release in coronary artery surgery: on-pump versus off-pump

AIM: Heat shock protein HSP-70 is known as protective chaperone molecule synthetized in response following ischemia and stress agents. It is detected in the myocardium and endothelium as well as in the circulation. Damaged as well as viable but exposed to stress cells contribute to the release of HSP-70 into the circulation. The aim of the study was to investigate if cardiopulmonary bypss (CPB) leads to more circulating HSP-70, on the basis of comparison dynamics of plasma concentration HSP-70 in 8 men undergoing procedures with the use of CPB (coronary artery bypass grafting, CABG group) and 8 men undergoing off-pump surgery (OPCAB group). METHODS: Blood samples were taken preoperatively, twice intraoperatively, immediately after surgical procedure (1 h) and 24-hours thereafter. The concentration of plasma HSP-70 was measured by means of immunoassay. The derived results were compared statistically with the frequency of incidence postoperative atrial fibrillation (AF). RESULTS: In CABG group was observed continuous gradual increase of plasma HSP-70 concentration during the operation with the peak 1 h after surgery (P<0.01), in striking contrast to OPCAB group, in which was detected small, but non statistically significant increase of HSP-70 1 h after operation. Significantly more of circulating HSP-70 it was detected in CABG group during the operation and 1 h after surgery (CABG vs OPCAB, respectively P<0.015 and P<0.028). In both groups among patients witch AF it was found higher postoperative values of circulating HSP-70 compared with the non-AF group (P=0.0415). CONCLUSIONS: The use of CPB leads to significant more release of HSP-70 into the circulation. According to our findings high plasma concentration of HSP-70 may be the measure of operative cellular stress, ischemia or injury and may be related with greater onset of postoperative AF. High circulating HSP-70 levels is connected with higher incidence of postoperative AF after open heart surgery.     

Induction of heme oxygenase-1 in kidneys during ex vivo warm perfusion

BACKGROUND: Reperfusion injury plays a pivotal role in the occurrence of delayed graft function and chronic rejection. Heme oxygenase-1 (HO-1), an inducible heat shock protein, is known to have cytoprotective effects against reperfusion injury. We report on the potential for ex vivo induction of HO-1 expression during acellular warm perfusion of canine kidneys, using cobalt protoporphyrin (CoPP) as an HO-1 inducer and zinc protoporphyrin as an HO-1 inhibitor. METHODS: Canine kidneys were used to evaluate HO-1 increase after exposure to warm ischemia (WI), hypothermic perfusion (mechanical perfusion [MP], 4 degrees C), warm perfusion (exsanguineous metabolic support [EMS], 32 degrees C), or various combinations. RESULTS: WI alone, MP, or EMS with or without WI, had no effect on HO-1 activity. However, the presence of CoPP during EMS perfusion resulted in a significant increase in kidney HO-1 activity, whereas zinc protoporphyrin reduced HO-1 activity. The presence of CoPP during MP did not induce elevated HO-1 expression. The results of our study demonstrate that sufficient metabolism supporting new protein synthesis resulting in the expression of the protective gene, HO-1, can be accomplished during an acellular near-normothermic perfusion using CoPP. Most importantly, the time required for ex vivo HO-1 induction with this method is compatible with the current time frame for which organs are preserved clinically.CONCLUSIONS: The ability to induce HO-1 expression ex vivo eliminates the need for donor therapy to induce HO-1 increase before retrieving organs and also prevents the potential of decreasing HO-1 enzyme activity that is known to occur with temperature-mediated inhibition of oxidative metabolism during hypothermic organ storage.     

In vivo analysis of the microcirculation of osteomyocutaneous flaps using fluorescence microscopy.

Previous studies have indicated that freely transferred osteomyocutaneous flaps may fail despite anastomotic patency. While microvascular dysfunction is thought to be one of the major causes for this type of flap failure, little is known of its underlying mechanisms, probably due to the lack of adequate experimental models allowing detailed intravital microcirculatory analysis. Herein we report quantitative analysis of the microcirculation of periosteum, muscle, subcutis and skin by intravital fluorescence microscopy using an osteomyocutaneous free flap model in the hindlimb of rats. The microcirculation of the different tissues was studied after microanastomotic transfer (free flap), and was compared to that after solely elevating the tissue, mimicking a pedicled osteomyocutaneous flap. Transferred flaps, which were exposed to 1 h of ischaemia during the anastomotic procedure, showed a slight but significant decrease (P< 0.05) of functional capillary density in muscle, subcutis and skin when compared with the microcirculation of pedicled flaps, while capillary diameters, red blood cell velocity and blood flow of perfused capillaries remained almost unaffected. The decrease of functional capillary density was associated by a significant (P< 0.05) inflammatory response, as indicated by the increased number of leukocytes adherent to the endothelial lining of postcapillary venules. While the functional capillary density of periosteum was not affected by the free transfer procedure, the inflammatory response was found similar when compared with that observed in muscle and subcutis. Thus, our study indicates that even after a short 1-h ischaemic time period, capillary perfusion failure and leukocyte-endothelial cell interaction are the main events, characterising microvascular dysfunction after free transfer of osteomyocutaneous flaps. Using the model described herein, intravital microscopic analysis of the microcirculation proved an appropriate tool to study the individual microvascular response after free tissue transfer, and may thus be used to evaluate the effectiveness of novel therapeutic regimens which aim at counteracting microcirculatory dysfunction in free osteomyocutaneous flaps.     

Trauma centre experience: flap reconstruction of traumatic lower limb injuries

BACKGROUND: Decision-making in the management of combined major skeletal and soft tissue trauma to the lower limb is a complex process made more difficult by the uncertainty surrounding outcomes. The aim of this study was to review and present our experience with flap reconstruction of traumatic lower limb defects, with particular reference to in-hospital complications and outcomes related to timing, choice of flap and pre-existing complications. METHODS: Retrospective review of all lower limb flap reconstructions carried out by the Plastic and Reconstructive Surgery Unit at the Alfred Hospital from 1 July 2001 to 20 October 2005 (51-month period) was carried out. RESULTS: Sixty-four patients had 83 flap reconstructions (35 free and 48 local) of 70 separate lower limb injuries. Internal skeletal fixation was followed by earlier soft tissue coverage and lower deep metal infection rates. Twenty-seven flaps (32.5%) developed soft tissue infections, and 16 fixation devices (25.8%) were complicated by deep metal infection. There were six (12.5%) local flap partial necrosis and four (11.4%) free flap failures. Limbs in which flaps were carried out after day 5 were more likely to develop deep metal infection (P = 0.04) and suffer free flap failure or local flap partial necrosis (P = 0.02). Three patients underwent secondary amputation during their initial admission. CONCLUSIONS: The current study presented our experience with flap reconstruction of complex lower limb injuries at a major trauma centre. Thorough wound debridement, internal fixation and early soft tissue coverage (within 5 days of injury) were associated with lower infection rates and optimal outcomes.     

Heat shock-induced protection of renal proximal tubular epithelial cells from cold storage and rewarming injury

Cold storage and reperfusion injury to transplanted kidneys contributes to increased incidence of delayed graft function and may have a negative impact on graft survival. This study examined the mechanisms by which previous heat shock protects against cell death in an in vitro model of kidney storage. Cold storage is mimicked by incubating human renal proximal tubular epithelial (HK-2) cells in University of Wisconsin solution at 4 degrees C with and without subsequent rewarming. Heat shock was induced by incubation of cells at 42 degrees C for 1 h. Altered protein expression was measured by Western blot, and cell viability and apoptosis were measured by propidium iodide DNA staining using flow cytometry. The specific role of heat-shock protein 70 (HSP-70) was determined both by siRNA knockdown and by stable overexpression approaches. Cold storage and rewarming-induced cell death was associated with decreased expression of HSP-70, HSP-90, HSP-27, and Bcl-2. Previous heat shock significantly reduced HK-2 cell death after cold storage and rewarming and was associated with the maintenance of HSP-70, HSP-27, and Bcl-2 protein levels. Blocking heat stress-induced HSP-70 with siRNA did not significantly block the protective effect of heat stress against cold storage and rewarming cell death; however, overexpression of HSP-70 protected HK-2 cells from this stress. It is concluded that previous heat shock protects HK-2 cells from cold storage and rewarming injury. siRNA inhibition of HSP-70 induction did not block the protective effect of heat shock, indicating that HSP-70 is not essential to the heat stress-induced protective effect reported in this study.     

Roxadustat promotes hypoxia-inducible factor-1α/vascular endothelial growth factor signalling to enhance random skin flap survival in rats

Random skin flaps have limited clinical application as a broad surgical reconstruction treatment because of distal necrosis. The prolyl hydroxylase domain-containing protein inhibitor roxadustat (RXD) enhances angiogenesis and reduces oxidative stress and inflammation. This study explored the function of RXD in the survival of random skin flaps. Thirty-six male Sprague–Dawley rats were randomly divided into low-dose RXD group (L-RXD group, 10 mg/kg/2 day), high-dose RXD group (H-RXD group, 25 mg/kg/2 day), and control group (1 mL of solvent, 1:9 DMSO:corn oil). The proportion of surviving flaps was determined on day 7 after surgery. Angiogenesis was assessed by lead oxide/gelatin angiography, and microcirculation blood perfusion was evaluated by laser Doppler flow imaging. Specimens in zone II were obtained, and the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as indicators of oxidative stress. Histopathological status was evaluated with haematoxylin and eosin staining. The levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and the inflammatory factors interleukin (IL)-1β, IL-6, and tumour necrosis factor-α (TNF-α) were detected by immunohistochemistry. RXD promoted flap survival and microcirculatory blood perfusion. Angiogenesis was detected distinctly in the experimental group. SOD activity increased and the MDA level decreased in the experimental group. Immunohistochemistry indicated that the expression levels of HIF-1α and VEGF were increased while the levels of IL-6, IL-1β, and TNF-α were decreased after RXD injection. RXD promoted random flap survival by reinforcing vascular hyperplasia and decreasing inflammation and ischaemia-reperfusion injury.     

Ergothioneine modulates proinflammatory cytokines and heat shock protein 70 in mesenteric ischemia and reperfusion injury

BACKGROUND AND AIM: Ergothioneine (EGT) is a natural compound that is synthesized by soil bacteria in fungal substrates and exhibits antioxidant functions in many cell models. The purpose of this study was to investigate the effect of EGT on mesenteric ischemia and reperfusion injury. MATERIALS AND METHODS: Rats were supplemented with or without l-ergothioneine (10 mg/kg/d) for 15 days prior to intestinal ischemia. Animals were subjected to ischemia induced by clamping the superior mesenteric artery for 60 min followed by reperfusion. Serum tumor necrosis factor (TNF)-alpha and interleukin-1beta (IL-1beta) levels, tissue malondialdehide (MDA), myleoperoxidase (MPO), and heat shock protein (HSP) 70 levels, as well as histological findings, were evaluated after 1, 2, and 4 h of reperfusion. RESULTS: Serum TNF-alpha and IL-1beta levels, and tissue MDA and MPO activities at 1, 2 and 4 h after reperfusion in the EGT group, were significantly lower than the control group (P < 0.05). Tissue HSP-70 levels of the study group were significantly greater than the control group at any time point of reperfusion. No significant differences in tissue damage including morphological changes ranging from villous denudation to focal necrosis, ulceration, hemorrhage, and architectural disintegration at 1 and 2 h after reperfusion exist between the two groups; however, after 4 h of reperfusion, the tissue damage based on histopathologic scores by Chiu was considerably lower in the study group (P < 0.05). After 4 h of reperfusion, focal epithelial lifting and occasional areas of denuded villi could be seen in the samples of the treated animals, thus preserving villous height and mucosal architecture. CONCLUSION: EGT attenuates mesenteric ischemia reperfusion injury in rat intestine by increasing tissue HSP-70 and decreasing TNF-alpha, IL-1beta, MDA, and MPO levels. EGT also improves morphological alterations, which occurred after IR injury after prolonged periods of reperfusion.