Ischemia-reperfusion Injury Group Research Articles

Effect of Aliskiren on Renal Cortical Ischemia/Reperfusion Injury in Albino Rats: A Histological and Immunohistochemical Study

Background: Ischemia/reperfusion injury (IRI) is a foremost reason for acute kidney injury, which is a common clinical complication having high morbidity rate and lacks effective therapy. Renin-angiotensin system (RAS) activation and angiotensin II level elevation are important risk factors in IRI. Aliskiren (ALS) is the first oral direct rennin inhibitor that blocks the first step in RAS and has been approved for treating hypertension.Aim of work: Investigating whether ALS pretreatment protects the renal cortex of adult male albino rats subjected to IRI.Material and Methods: Thirty adult male albino rats were divided into 3 groups: group I (control group), group II (IRI group): were subjected to IRI procedure, group III (ALS group): rats were pretreated with aliskiren (ALS, 100 mg/kg) twice orally 24h and 1h before IRI and left for 24h of reperfusion before scarification. Blood samples were taken to estimate the levels of blood urea nitrogen (BUN), serum creatinine (SCr) and superoxide dismutase enzyme (SOD). Kidneys sections were stained with HandE and PAS stains and immunohistochemical staining against COX-2 and caspase-3. This was followed by morphometric and statistical analysis.Results: IRI group showed deterioration in renal function tests with massive histological alterations in the Malpighian renal corpuscles and the convoluted tubules. There was significant decrease in optical density of PAS reaction, with significant increase in the mean area% of COX-2 and caspase-3 immunoexpression. ALS group showed significant improvement in renal function tests with preserved normal histology of the renal cortex. There was significant increase in the optical density of PAS reaction, with significant decrease in the mean area% of COX-2 and caspase-3 immunoexpression.Conclusion: A protective effect of ALS was detected in IRI-induced renal cortical damage in adult male albino rats. This was evidenced by laboratory results, histological and immunohistochemical evaluation.

The Effect of Nitric Oxide on Remote Ischemic Preconditioning in Renal Ischemia Reperfusion Injury in Rats.

Although remote ischemic preconditioning (RIPC) is an organ-protective maneuver from subsequent ischemia reperfusion injury (IRI) by application of brief ischemia and reperfusion to other organs, its mechanism remains unclear. However, it is known that RIPC reduces the heart, brain, and liver IRI, and that nitric oxide (NO) is involved in the mechanism of this effect. To identify the role of NO in the protective effect of RIPC in renal IRI, this study examined renal function, oxidative status, and histopathological changes using N-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor. Remote ischemic preconditioning was produced by 3 cycles of 5 minutes ischemia and 5 minutes reperfusion. Blood urea nitrogen, creatinine (Cr), and renal tissue malondialdehyde levels were lower, histopathological damage was less severe, and superoxide dismutase level was higher in the RIPC + IRI group than in the IRI group. The renoprotective effect was reversed by L-NAME. Obtained results suggest that RIPC before renal IRI contributes to improvement of renal function, increases antioxidative marker levels, and decreases oxidative stress marker levels and histopathological damage. Moreover, NO is likely to play an important role in this protective effect of RIPC on renal IRI.

Protective Effects of Butyrate on Renal Ischemia-Reperfusion Injury in Rats

Background: Renal ischemia-reperfusion injury (IRI) usually causes acute kidney injury. There is an urgent need to develop an effective agent to prevent renal IRI. This study aimed to examine the effect of butyrate on renal IRI in rats. Materials and Methods: Rats were randomly assigned into 3 groups (10 rats in each group): the sham group, the IRI group, and the butyrate group. Rats were injected intravenously with 300 mg/kg of sodium butyrate in the butyrate group and with a saline solution in the sham group and IRI group 30 min before renal ischemia. After 24 h of reperfusion, renal function and histologic damage were examined. Myeloperoxidase (MPO) activity assay, in situ apoptosis examination, enzyme-linked immunosorbent assay, immunohistochemical assay, and Western blot were performed as well. Results: Butyrate pretreatment significantly reduced renal dysfunction and histologic damage induced by renal IRI. Butyrate pretreatment caused a significant attenuation of neutrophil infiltration, which was reflected by the reduction of renal MPO activity. Butyrate also reduced apoptotic tubular cell death and improved caspase-3 activation. The expression of TNF-α was decreased following butyrate pretreatment. Conclusions: Butyrate pretreatment protects rats from renal IRI by inhibiting inflammation and apoptosis. Therefore, butyrate may be a potential therapeutic agent for preventing renal IRI.

Two methods for modeling of sick sinus syndrome in rats: Ischemia reperfusion and sodium hydroxide induced injury

The Sick Sinus Syndrome (SSS) is a serious life-threatening heart disease. It is important to establish a credible and stable sinus node damage model. In this study, we use two methods to construct an SSS damage model in rats. One is to inject sodium hydroxide to the SSS area through internal jugular vein. Another is to cause ischemia-reperfusion injury on the SSS area. 43 healthy SD rats were randomly divided into 4 groups, namely ischemia-reperfusion injury group (IRIG), inject sodium hydroxide group (ISHG), and propranolol group (PG) and the control group (CG). The achievement ratio of modeling was 67% in the IRIG and 83% in the ISHG. The HR significantly decreased after operation in the IRIG and ISHG compared with pre-operation (P＜0.01). The HR was reduced by above 30% in these 2 groups after modeling, while the reduction was better maintained in IRIG. Additionally, the sinoatrial node recovery time (SNRT) and sinoatrial conduction time (SACT) were significantly prolonged compared with pre-modeling in 2 groups (P < 0.01). Morphology results showed blurry in structure and boundaries with pale cytoplasm. It is speculated that IRIG and ISHG modeling might influence the calcium concentration and damage the sinus node function by decrease the expression of HCN4 and SCN5A, which impaired the driving ability of sinus node and leading to apoptosis. Ischemia reperfusion injury and sodium hydroxide injury could construct stable SSS models which could represent clinic pathological damage. Thus, both methods could be used for further studies of the SSS mechanisms and drugs.

Impacts of Renal Denervation and Vagus Nerve Stimulation on Acute Renal Failure Induced by Renal Ischemia Reperfusion injury in Rat Model

Ischemia-Reperfusion Injury (IRI) is characterized by temporary cessation followed by restoration of blood supply and re-oxygenation of a certain organ. In the kidney, IRI contributes to Acute Kidney Injury (AKI) with rapid kidney damage and high morbidity and mortality rates. A surgical or drug-induced blockage of renal sympathetic nerve prevents, partially, the development of IR-induced AKI. Modulation of the Cholinergic Anti-Inflammatory Pathway (CAP) by Vagal Nerve Stimulation (VNS) has also a delayed but effective impact in renal IRI. However, the combined effect of renal sympathectomy and VNS had not been well investigated. Objectives: This work aimed at investigating the combined effect of VNS and Renal Denervation (RDN) in preventing deleterious effects of IRI in rats compared to the effects obtained by RDN alone and to elucidate the possible mechanisms. Methods: 32 adult male albino rats were equally allocated into four groups, sham group, IRI group, RDN group subjected to RDN before IRI and a group subjected to RDN and VNS before IRI. Results: Compared to the sham group, renal IRI led to the elevation of BUN, serum creatinine and MDA levels, it also elevated TNFα and reduced GPX activity and nitrate levels in the renal tissue. In addition, IRI lowered BCL2 in the immune-histochemical study and caused renal damage as observed by the histological light and electron microscopic examination. On the other hand, RDN demonstrated partial correction while, a combination of RDN and VNS demonstrated nearly optimum recovery of renal functions, oxidant/antioxidant balance, inflammatory markers as well as marked amelioration of immunohistochemical, structural and ultra-structural studies. Conclusion: VNS augmented and accelerated the renoprotective effects of RDN owing to its antioxidant, antiinflammatory as well as anti-apoptotic effects. Additionally, when VNS was combined to RDN, the protection against acute renal failure induced by IRI was rapid and effective.

Hydrogen-rich saline protects against hepatic injury induced by ischemia-reperfusion and laparoscopic hepatectomy in swine

Hydrogen-rich saline (HRS) has antioxidative, anti-inflammatory and anti-apoptotic properties. We investigated the effects of hydrogen on hepatic ischemia-reperfusion (I/R) and laparoscopic hepatectomy in swine. Twenty-one healthy Bama miniature pigs were randomly divided into the sham group, ischemia-reperfusion injury (IRI) group, HRS-5 (5 mL/kg) group, and HRS-10 (10 mL/kg) group. HRS was injected through the portal vein 10 min before reperfusion and at postoperative day 1, 2 and 3. The roles of HRS on oxidative stress, inflammatory response and liver regeneration were studied. Compared with the IRI group, HRS treatment attenuated oxidative stress by increasing catalase activity and reducing myeloperoxidase. White blood cells in the HRS-10 group were reduced compared with the IRI group (P < 0.01). In the HRS-10 group, interleukin-1 beta, interleukin-6 and tumor necrosis factor alpha, C-reactive protein and cortisol were downregulated, whereas interleukin-10 was upregulated. In addition, HRS attenuated endothelial cell injury and promoted the secretion of angiogenic cytokines, including vascular endothelial growth factor, angiopoietin-1 and angiopoietin-2. HRS elevated the levels of hepatocyte growth factor, Cyclin D1, proliferating cell nuclear antigen, Ki-67 and reduced the secretion of transforming growth factor-beta. HRS treatment may exert a protective effect against I/R and hepatectomy-induced hepatic damage by reducing oxidative stress, suppressing the inflammatory response and promoting liver regeneration.

Arginase-2 protects myocardial ischemia-reperfusion injury via NF-κB/TNF-α pathway.

Arginase-2 exerts an anti-inflammatory potential. However, whether nuclear factor-κB (NF-κB)/TNF-α (tumor necrosis factor-α) pathway is involved in the anti-inflammation effect of arginase-2 has not been fully elucidated. Our study aims to explore the regulatory role of arginase-2 on ischemia-reperfusion injury (IRI) in rats and its underlying mechanism. 24 male Sprague Dawley (SD) rats were randomly assigned into sham group, IRI group and arginase-2 group, with 8 rats in each group. Electrocardiogram was performed in each rat before and after animal procedures. Serum samples and heart samples of each rat were collected 10 days after animal procedures. Serum levels of CK-MB (creatine kinase-MB) and LDH (lactate dehydrogenase) in each rat were detected using the relative commercial kits. Pathological lesions in rat myocardium were observed by hematoxylin and eosin (HE) staining. Cardiomyocyte apoptosis in rat heart was accessed by TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling) staining. Expression levels of NF-κB, TNF-α, VCAM-1, ICAM-1 and MCP-1 in rat myocardium were detected by Western blot and immunohistochemistry. Electrocardiogram showed slower heart rate, lower voltage of QRS wave and longer Q-T interval in rats of IRI group than those of sham group (p < 0.05). A few rats in IRI group even presented arrhythmia. On the contrary, rats in arginase-2 group presented higher heart rate and voltage of QRS wave, as well as shorter Q-T interval compared with those of IRI group (p < 0.05). Rats in arginase-2 group presented lower plasma levels of CK-MB and LDH than those of IRI group. Pathological lesions in rat myocardium and cardiomyocyte apoptosis were alleviated in arginase-2 group in comparison with those of IRI group. Western blot and immunohistochemistry indicated that arginase-2 pretreatment remarkably downregulated expressions of NF-κB, TNF-α, VCAM-1, ICAM-1 and MCP-1 in rat myocardium. Arginase-2 protects myocardial ischemia-reperfusion injury in rats through inhibiting the inflammatory response via suppression of NF-κB/TNF-α pathway.

Complement depletion with cobra venom factor alleviates acute hepatic injury induced by ischemia‑reperfusion.

Increasing evidence has demonstrated that complement activation is required for ischemia-reperfusion injury (IRI)-induced hepatic damage, and cobra venom factor (CVF) can deplete the complement components. The aim of the current study was to investigate the effect and intrinsic mechanism of CVF pretreatment on IRI-induced acute hepatic injury in rats. Acute hepatic injury in rats was induced by bone fracture to simulate trauma, followed by hemorrhage for 90 min, and then the rats were resuscitated for a period of 20 min of reperfusion. The survival times under different CVF treatment doses and schedules for rats with IRI were evaluated. Hepatic tissues and serum samples were analyzed for acute hepatic injury, complement activation, inflammatory mediator release and apoptosis at predetermined times and compared between the IRI group and the CVF pretreatment + IRI groups. Compared to the rats with IRI alone, the survival times were significantly improved among rats with IRI receiving a high-dose or low-dose CVF pretreatment (all P<0.01). Upon histological examination, severe hepatic damage was observed in the rats with IRI, accompanied by liver function deterioration, complement and membrane attack complex activation, inflammatory mediator release and hepatic cell apoptosis. CVF pretreatment significantly attenuated the hepatic injury through depletion of anaphylatoxic C5a and membrane attack complex C5b-9 activation, and subsequent inhibition of inflammatory mediator release and hepatic cell apoptosis (all P<0.05). The results indicated that CVF pretreatment ameliorates IRI-induced acute hepatic injury. However, further studies are required to determine whether this therapy could be a potential agent for the treatment of IRI injuries in clinical settings.

Effects of propofol pretreatment on myocardial cell apoptosis and SERCA2 expression in rats with hepatic ischemia/reperfusion

IntroductionHepatic ischemia-reperfusion injury is a common pathophysiological process in liver surgery. Whether Propofol can reduce myocardial ischemia-reperfusion injury induced by hepatic ischemia-reperfusion injury in rats, together with related mechanisms, still needs further studies. ObjectiveTo investigate if propofol would protect the myocardial cells from apoptosis with hepatic ischemia-reperfusion injury. MethodsMale Sprague-Dawley rats (n=18) were randomly allocated into three groups: Sham Group (Group S, n=6), Hepatic Ischemia-reperfusion Injury Group (Group IR, n=6) and Propofol Group (Group P, n=6). Group S was only subjected to laparotomy. Group IR was attained by ischemia for 30min and reperfusion for 4h. Group P was subjected identical insult as in Group IR with the administration of propofol started 10min before ischemia with 120mg.kg−1, following by continuous infusion at 20mg.kg−1.h−1. Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and cysteine-containing aspartic acid cleaved-caspase3 (cleaved-caspase3) were assayed by western blot and Altimeter polymerase chain reaction. ResultsApoptosis rate was increased, with mRNA and protein of SERCA2 down-regulated and cleaved-caspase3 up-regulated in Group IR compared with Group S (p<0.01). Apoptosis rate was decreased, with mRNA and protein of SERCA2 up-regulated and cleaved-caspase3 down-regulated in Group P compared with Group IR (p<0.01). ConclusionsPropofol can reduce hepatic ischemia-reperfusion injury-induced myocardial cell apoptosis, meanwhile, can up-regulate mRNA and protein of SERCA2 in rats.

Effect of Autophagy Inhibition on the Protection of Ischemia Preconditioning against Myocardial Ischemia/Reperfusion Injury in Diabetic Rats.

Background:Ischemia preconditioning (IPC) remains the most powerful intervention of protection against myocardial ischemia/reperfusion injury (IRI), but diabetes can weaken or eliminate its cardioprotective effect and detailed mechanisms remain unclear. In this study, we aimed to explore whether changes of autophagy in the diabetic condition are attributable to the decreased cardioprotective effect of IPC.Methods:Sixty diabetic male Sprague-Dawley rats were randomly divided into the control (C), IRI, rapamycin (R), wortmannin (W), rapamycin + IPC (R + IPC), and wortmannin + IPC (W + IPC) groups. The in vivo rat model of myocardial IRI was established by ligaturing and opening the left anterior descending coronary artery via the left thoracotomy. Durations of ischemia and reperfusion are 30 min and 120 min, respectively. Blood samples were taken at 120 min of reperfusion for measuring serum concentrations of troponin I (TnI) and creatine kinase isoenzyme MB (CK-MB) using the enzyme-linked immunosorbent assay. The infarct size was assessed by Evans blue and triphenyltetrazolium chloride staining. The expressions of LC3-II, beclin-1, phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), and P-Akt/Akt ratio in the ischemic myocardium were assessed by Western blotting.Results:Compared to the IRI group, infarct size (56.1% ± 6.1% vs. 75.4 ± 7.1%, P < 0.05), serum cTnI (0.61 ± 0.21 vs. 0.95 ± 0.26 ng/ml, P < 0.05), and CK-MB levels (6.70 ± 1.25 vs. 11.51 ± 2.35 ng/ml, P < 0.05) obviously decreased in the W + IPC group. Compared with the C group, myocardial expressions of LC3-II (0.46 ± 0.04 and 0.56 ± 0.04 vs. 0.36 ± 0.04, P < 0.05) and beclin-1 (0.34 ± 0.08 and 0.38 ± 0.07 vs. 0.24 ± 0.03, P < 0.05) evidently increased, and myocardial expressions of mTOR (0.26 ± 0.08 and 0.25 ± 0.07 vs. 0.38 ± 0.06, P < 0.05), PI3K (0.29 ± 0.04 and 0.30 ± 0.03 vs. 0.38 ± 0.02, P < 0.05), and P-Akt/Akt ratio (0.49 ± 0.10 and 0.48 ± 0.06 vs. 0.72 ± 0.07, P < 0.05) markedly decreased in the IRI and R groups, indicating an increased autophagy. Compared with the IRI group, myocardial expression of beclin-1 (0.26 ± 0.03 vs. 0.34 ± 0.08, P < 0.05) significantly decreased, and myocardial expressions of mTOR (0.36 ± 0.04 vs. 0.26 ± 0.08, P < 0.05), PI3K (0.37 ± 0.03 vs. 0.29 ± 0.04, P < 0.05), and P-Akt/Akt ratio (0.68 ± 0.05 vs. 0.49 ± 0.10, P < 0.05) increased obviously in the W + IPC group, indicating a decreased autophagy.Conclusions:Increased autophagy in the diabetic myocardium is attributable to decreased cardioprotection of IPC, and autophagy inhibited by activating the PI3K-Akt-mTOR signaling pathway can result in an improved protection of IPC against diabetic myocardial IRI.

Effects of Ulinastatin on Liver Warm Ischemia Reperfusion Injury in Mice

Objective Ulinastatin, a trpsin inhibitor, has been reported that can inhibits the liver inflammation and improves the liver function. The role of UTI on liver warm ischemia reperfusion injury (IRI) and the underlying mechanisms remain to be further investigated. Method In bred C57BL/6 mice and liver warm IRI model were employed. The ligamentum of hepatoduodenale was clamped for 45mins and followed with reperfusion of 1 hour, 6hour, 24hour, respectively. UTI was treated preoperatively. Serum enzyme release, liver cell histology and apoptosis, and Caspase-3 mRNA & Fas mRNA expression from the liver tissues were examined, respectively. Results ALT/AST levels are remarkably lower in IRI+UTI group than IRI group at reperfusion time of 1hour, 6hour, and 24 hour. Observed under light microscope of 6 hours of reperfusion sample, hepatic cell was significantly edema degeneration, with partial liver cell vacuolization obviously, some patchy necrosis, sinusoidal congestion and become narrow. RT-PCR result shows that expression of Caspase-3mRNA and Fas mRNA are significantly higher in IRI group than that in IRI+UTI group(P<0.05). Compared with SO group, expression of IL-6 and TNF-α of IRI group and UTI group is up-regulated (P<0.05). Compared the latter two group, IRI+UTI group's expression is significantly down regulated. Apotosis of liver cells in IRI+UTI group is relieved in contrast to IRI group(P<0.05), while SO group shows a negative resault. Conclusion Ulinastatin has the ability to inhibit releasing of inflammatory factors by hepatocytes after hepatic ischemia reperfusion, and helps clean ROX and endotoxins. Meanwhile, down regulate expression of Caspase-3 mRNA and Fas mRNA, in this way reduce hepatocytes apoptosis, as a resault significantly prevent liver ischemia reperfusion injury through multiple links. Nature Science Foundation of China (NSFC) (81170416; 81273264); International cooperation fund of Jilin Science & Technology Committee (2016322).

Blockade of the G-CSF Receptor is Protective in a Mouse Model of Renal Ischemia Reperfusion Injury

Background Ischemia-reperfusion injury (IRI) is a complex inflammatory process that detrimentally affects the function of transplanted organs. Several pathways contribute to cell death and microvascular dysfunction in IRI, including activation of the innate and adaptive arms of the immune system. Inhibiting complement, a key component of innate immunity, by C5 blockade has been shown to protect against renal IRI in mice. In this study, we investigated whether targeting neutrophils with VR81, a monoclonal antibody recognising the granulocyte colony stimulating factor receptor (G-CSFR), would reduce inflammation and protect against injury in a mouse model of renal IRI. The anti-C5 antibody BB5.1 was used for comparison. Methods Male 10-12 week old C57BL/6 mice underwent right nephrectomy followed by clamping of the left renal pedicle for 22 min, or right nephrectomy only (Sham), at 37°C. Mice (n = 5-8 per group) were injected i.p. at 24 hr pre-ischemia with VR81 (100, 200 or 500 μg), BB5.1 (100 μg), or isotype-matched antibody (Ab) control (500 μg). Mice were euthanased 24 hr after reperfusion, and serum and plasma samples were collected to assess renal function (creatinine, urea) and complement activation (C5a ELISA). Kidneys were harvested to analyse complement C9 deposition and immune cell infiltration by immunofluorescence. Results Compared to Sham, the Ab control IRI group showed significantly increased creatinine, urea and C5a, deposition of C9, and infiltration of neutrophils and macrophages. As expected, C5 blockade with BB5.1 markedly reduced renal injury and inflammation, reflected in significantly lower creatinine, urea, C5a and C9 deposition versus Ab control. Surprisingly, VR81 was also strongly protective in a dose-dependent manner, with the 500 μg dose significantly reducing creatinine, urea and cellular infiltration. Conclusion Unexpectedly, G-CSFR blockade using VR81 significantly protected against renal IRI with an efficacy similar to that of anti-C5 treatment. Protection was associated with markedly reduced infiltration by neutrophils and macrophages. G-CSFR blockade is thus a promising new approach to reduce IRI and improve organ transplant function.

Research on the protective role of remote ischemic preconditioning in kidney ischemia reperfusion injury

Objective To investigate the protective effects and underlying mechanism of remote ischemic preconditioning (RIPC) in kidney ischemia reperfusion injury (IRI). Methods Sprague-Dawley rats were randomly divided into three groups following right-side nephrectomy: Sham-group, IRI-group, RIPC-group. At 24 h after reperfusion, the blood samples, renal tissues and urine were collected. The degree of renal injury was evaluated by serum creatinine (SCr), blood urea nitrogen (BUN) and histological score. We also detected the expression of cyclooxygenase-2 (COX-2), membrane associated prostaglandin E synthase-1 (mPGES-1) and prostaglandin E-2 (PGE2). Results Rats treated with RIPC before ischemia revealed significant improvements in renal function (P value is 0.031 and 0.004 respectively) and morphology (IRI: 3.63±0.54, RIPC: 2.44±0.66, P=0.001). The results derived from immunohistochemistry revealed that the content of COX-2 and PGE2 were obviously decreased in RIPC group than those in IRI group (P value is 0.000 and 0.034 respectively), however, the expression of mPGES-1 remained no significant change (P=0.367). Conclusion The results suggested that RIPC could attenuate renal IRI and improve kidney function. The COX-2/PGE2 pathway may play a critical role in this process. Key words: Remote ischemic preconditioning; Ischemia reperfusion injury; Cyclooxygenase-2

The Protective Effect of NecroX-5 on Flap Survival and Ischemic Reperfusion Injury Inflammation in Rat Model

Background NecroX-5 has been evaluated to have anti-necrotic potential on ischemia reperfusion injury (IRI) in skin flaps. Nevertheless, there is no study focusing on the underlying mechanism involved in this protective effect. We used NecroX-5 to assess the preventive effect and regulatory mechanisms on flap survival in rat model. Methods Twenty male Sprague-Dawley rats were allocated into two groups with 10 each: control IRI group and IRI with 30 mg/kg of NecroX-5-treated group. On postoperative day 7, viable area of the skin flap was measured and specimens were used for immunohistochemical analysis. Results The necrotic area of NecroX-5 group was significantly lower than those of control group (NecroX-5 group: 6±0.019%; control group: 38±0.11%; P<0.05). Furthermore, levels of interleukin-1β and tumor necrosis factor-α were markedly lower in NecroX-5 groups than in control groups and NecroX-5 groups resulted in an apparent decrease of infiltrating immune cells. Conclusion We suggest that NecroX-5 acts as a protective factor during IRI of skin flaps by inducing a negligible amount of proinflammatory cytokines. Key Words: NecroX-5; Ischemia; Reperfusion; Reperfusion injury; Rats; Cytokines; Surgical flap.

Protective effects of Epigallocatechin gallate on liver ischemia reperfusion injury in rats

Objective To investigate the protective roles of Epigallocatechin gallate (EGCG) on li-ver ischemia reperfusion injury (IRI) in rats. Methods 30 healthy male SD rats were selected and equally and randomly divided into 3 groups. Sham group, IRI group and IRI-EGCG group were established to construct 70% liver IRI rat model. Drinking water with 0.4 mg/ml EGCG was administered for 2 weeks before the experiment in IRI-EGCG group. HE staining was performed to evaluate the injury. Transaminases in serum were investigated to assess liver injury. p-p85 and p-AKT was detected by Western-blot assay. qPCR was carried out to study the mRNA expression of TNF-α, IL-6 and IL-1β in liver tissue. The secretion of TNF-α, IL-6 and IL-1β in serum was examined with ELISA assay. Results EGCG pretreatment reduced AST and ALT in serum [AST: (550.0±66.5) IU/L vs. (220.0±63.5) IU/L; ALT: (376.0±25.7) IU/L vs. (158.0±33.1) IU/L, all P<0.05] and mitigated liver tissue damage. p-p85 and p-AKT increased due to liver IRI, and IRI-EGCG group showed higher expression of p85 and AKT. The proinflammatory cytokines of TNF-α, IL-6 and IL-1β exhibited a relatively lower mRNA expression in IRI-EGCG group comparing with IRI group. IRI-EGCG group also revealed a decreased secretion of TNF-α, IL-6 and IL-1β in serum [TNF-α: (398.0±33.4) ng/L vs. (211.0±23.6) ng/L; IL-6: (341.0±27.3) ng/L vs. (187.0±19.6) ng/L; IL-1β: (486.0±43.7) ng/L vs. (352.0±31.5) ng/L; all P<0.05]. Conclusion EGCG pretreatment can enhance IRI-induced activation of PI3K/AKT signaling and reduce the release of proinflammatory cytokines to exert liver protective effects. Key words: Liver; Ischemia reperfusion injury (IRI); Epigallocatechin gallate (EGCG); Signal transduction, PI3K/AKT; Inflammatory cytokine

S-Adenosylmethionine attenuates bile duct early warm ischemia reperfusion injury after rat liver transplantation

Warm ischemia reperfusion injury (IRI) plays a key role in biliary complication, which is a substantial vulnerability of liver transplantation. The early pathophysiological changes of IRI are characterized by an excessive inflammatory response. S-Adenosylmethionine (SAM) is an important metabolic intermediate that modulates inflammatory reactions; however, its role in bile duct warm IRI is not known. In this study, male rats were treated with or without SAM (170 μmol/kg body weight) after orthotopic autologous liver transplantation. The histopathological observations showed that bile duct injury in the IRI group was more serious than in the SAM group. The alanine aminotransferase (ALT), alkaline phosphatase (ALP) and direct bilirubin (DBIL) levels in the serum of the IRI group were significantly increased compared to the SAM group (P < .05). Simultaneously, SAM effectively improved the survival of the transplant recipients. Furthermore, the H2O2 and malondialdehyde (MDA) of the IRI group were much higher compared to the SAM group (P < .05). The GSH/GSSG ratio in the SAM group was significantly increased by SAM treatment compared to the IRI group (P < .05). SAM administration significantly inhibited macrophage infiltration in liver and bile duct tissues, down-regulated TNF-α levels and up-regulated IL-10 expression in bile duct tissues compared to the IRI group (P < .05). The number of apoptotic biliary epithelial cells and caspase-3-positive cells in IRI rat livers were much higher compared to those in SAM-treated rats at 24 h after liver transplantation (P < .05). These data suggested that SAM protected bile ducts against warm IRI by suppressing oxidative stress, inflammatory reactions and apoptosis of biliary epithelial cells after liver transplantation.α

Protective effects of N(2)‑L‑alanyl‑L‑glutamine mediated by the JAK2/STAT3 signaling pathway on myocardial ischemia reperfusion.

To explore the protective effect of N(2)-L-alanyl-L-glutamine (NLAG) on myocardial ischemia-reperfusion injury (IRI), and observe the influence of NLAG on the Janus activated kinase signal transducer 2 and activator of transcription 3 (JAK2/STAT3) signaling pathway-associated molecules. Wistar rats were randomly divided into three groups: Sham, IRI and NLAG. In the IRI rat model, the cardiac hemodynamics, the maximum rate of left ventricular pressure (+dP/dtmax) and the left ventricular end-diastolic pressure (LVDP) were recorded. Hematoxylin-eosin and Masson staining were used to detect myocardial histological changes. The levels of plasma interleukin (IL)-1β and −6, tumor necrosis factor (TNF)-α, lactase dehydrogenase (LDH), troponin (cTn)I, creatine kinase (CK), heart type fatty acid binding protein (hFABP), malondialdehyde (MDA) and succinate dehydrogenase (SDH) were determined with ELISA. The protein expression levels of B-cell lymphoma (Bcl)-2, Bcl2-associated X protein (Bax), Caspase-3, JAK2, phosphorylated (p)-JAK2, STAT3 and p-STAT3 were detected by western blot analysis. The IRI model demonstrated notable myocardial injury; myocardial cells were arranged disorderly with some nuclei disappearing, and cardiac muscular fibers were degenerated. Following 60 min of reperfusion, LVDP, HR and +dP/dtmax were 31.3±4.53 mmHg, 239.17±8.45 beats/min and 615.17 mmHg/sec, respectively. Compared with the Sham group, the levels of LDH, cTnI, CK, hFABP release, inflammatory factors (IL-1β, IL-6 and TNF-α) and oxygen free radical (MDA and SDH) levels were increased in the IRI group. In the NLAG group, myocardial injury was improved, the concentrations of LDH, cTnI, CK, hFABP, IL-1β, IL-6, TNF-α, MDA were decreased, and SDH release was increased compared with the IRI group. In addition, NLAG significantly increased Bcl-2, JAK2, p-JAK2, STAT3 and p-STAT3 protein expression, and decreased Bax protein expression compared with the IRI group. In conclusion, myocardial ischemia-reperfusion can lead to myocardial cell apoptosis and myocardial injury and NLAG attenuates the IRI-induced mitochondrial oxidative stress injury and apoptosis by activating the JAK2/STAT3 signaling pathway, thus exerting protective effects against IRI.

Role of short-term starvation in alleviating hepatic ischemia-reperfusion injury in mice and possible mechanism of action

Objective: To investigate the role of short-term starvation (STS) in alleviating hepatic ischemia-reperfusion injury in mice and possible mechanism of action. Methods: Wild-type male C57BL/6 mice aged 8 weeks were randomly divided into 75% hepatic ischemia-reperfusion injury group (IR group), STS+75% hepatic ischemia-reperfusion injury group (STS group), and sirtinol+STS+75% hepatic ischemia-reperfusion injury group (SIR group), using a random number table, and sham-operation groups (IR-Sham group, STS-Sham group, and SIR-Sham group) were also established. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and the histomorphological changes of the liver were observed, as well as the expression of Sirt1, LC3B, and P62 proteins in liver tissue and the results of LC3B fluorescence staining. An analysis of variance was used for comparison of data between multiple groups, and the t-test was used for comparison of data between two groups. Results: Compared with the IR group, the STS group had significant reductions in the serum levels of ALT (3 152.7 ± 735.6 U/L vs 8 414.2 ± 1 052.2 U/L, P < 0.01) and AST (3 577.0 ± 714.0 U/L vs 10 845.8 ± 1 145.7 U/L, P < 0.01) and significant alleviation of liver pathological injury (Suzuki score: 1.50±0.55 vs 3.50±0.55, P < 0.01). Compared with the STS group, the SIR group had significant increases in the serum levels of ALT (7 002.7 ± 1 485.2 U/L vs 3 152.7 ± 735.6 U/L, P < 0.01) and AST (8 980.7 ± 1 739.1 U/L vs 3 577.0 ± 714.0 U/L, P < 0.01) and significant exacerbation of liver pathological injury (Suzuki score: 3.33 ± 0.52 vs 1.50 ± 0.55, P < 0.01). Compared with the IR group and the IR-Sham group, the STS group and the STS-Sham group had significant increases in the mRNA and protein expression of Sirt1 and the protein expression of LC3B and a significant reduction in the protein expression of P62, as well as a significant increase in the percentage of LC3B-positive cells in liver tissue (22.83% ± 5.19% / 22.17% ± 4.83% vs 10.16% ± 3.06% / 10.83% ± 1.94%, both P < 0.01). Compared with the STS group and the STS-Sham group, the SIR group and the SIR-Sham group had significant reductions in the expression of Sirt1 and LC3B proteins and a significant increase in the expression of P62 protein, as well as a significant reduction in the percentage of LC3B-positive cells in liver tissue (11.83% ± 9.24% / 14.67% ± 4.68% vs 22.83% ± 5.19% / 22.17% ± 4.83%, both P < 0.01). Conclusion: STS can effectively alleviate hepatic ischemia-reperfusion injury, and its protective effect may be associated with increasing the expression of Sirt1, inducing and promoting hepatocyte autophagy, and reducing hepatocyte death.

Carnosol protects against renal ischemia-reperfusion injury in rats.

Acute kidney injury, which is caused by renal ischemia-reperfusion injury (IRI), occurs in several clinical situations and causes severe renal damage. There is no effective therapeutic agent available for renal IRI at present. In this study, we performed an experiment based on an in vivo murine model of renal IRI to examine the effect of carnosol. Thirty Sprague-Dawley rats were randomized into three groups (10 rats in each group): the sham, IRI, and carnosol groups. Rats in the carnosol group were injected intravenously with 3 mg/kg of carnosol, and those in the sham and IRI groups were injected intravenously with 10% dimethyl sulfoxide 1 h before ischemia. Rats were sacrificed after 24 h of reperfusion. The blood and kidneys were harvested, renal function was assessed, and histologic evaluation was performed to analyze renal injury. A renal myeloperoxidase activity assay, in-situ apoptosis examination, enzyme-linked immunosorbent assay, immunohistochemical assay, and western blot were also performed. Carnosol pretreatment significantly reduced renal dysfunction and histologic damage induced by renal IRI. Carnosol pretreatment suppressed renal inflammatory cell infiltration and pro-inflammatory cytokine expression. In addition, carnosol markedly inhibited apoptotic tubular cell death, caspase-3 activation, and activation of the p38 pathway. Carnosol pretreatment protects rats against renal IRI by inhibiting inflammation and apoptosis. Although future investigation is needed, carnosol may be a potential therapeutic agent for preventing renal IRI.

A Novel Inhibitor of Homodimerization Targeting MyD88 Ameliorates Renal Interstitial Fibrosis by Counteracting TGF-β1-Induced EMT in Vivo and in Vitro

Background/Aims: The TLR/MyD88/NF-κB signaling pathway has been successfully used to treat renal interstitial fibrosis (RIF). However, the exact therapeutic mechanism is still unknown. Here, we assessed the therapeutic efficacy of TJ-M2010-2, a small molecular compound that inhibits MyD88 homodimerization, in RIF induced by ischemia reperfusion injury (IRI). Methods: In vivo, RIF was induced in mice by IRI, and the mice were prophylactically treated with TJ-M2010-2. In vitro, HK-2 cells were incubated with TGF-β1 to induce EMT, and the cells were pretreated with TJ-M2010-2. Results: We found that, compared with the IRI group, the TJ-M2010-2 group showed marked attenuation of RIF and renal function injury; decreased expression of TGF-β1, α-SMA, vimentin, MMP2 and MMP9; and increased E-cadherin expression. Furthermore, TGF-β1-induced EMT was blocked by TJ-M2010-2 in HK-2 cells, as evidenced by blocked morphologic transformation, restored E-cadherin expression and inhibited α-SMA expression. In addition, compared to the TGF-β1 group, the TJ-M2010-2 group showed profound inhibition of the expression of TRAF6, p65 and Snail and upregulation of the expression of IκBα. Conclusion: This MyD88 inhibitor may be a potential therapeutic agent to ameliorate RIF.