ObjectiveTo study the changes of levels of α subunits of stimulatory (Gsα) and inhibitory guanine nucleotide binding protein (Giα) in newborn guinea pig (0 2 days old) myocardium undergoing global ischemic reperfusion, and influences on the changes by St.Thomas Ⅱ and cold blood cardioplegic solution.MethodsThirty newborn guinea pigs were randomly assigned to three groups. GroupⅠ ( n = 10): the newborn hearts suffered by hypothermic global ischemia; group Ⅱ( n =10): the newborn hearts arrested by St. Thomas Ⅱ , and group Ⅲ ( n = 10): the newborn hearts arrested by cold blood cardioplegic solution. Levels of Gsα and Giα were investigated with Western blot analysis.ResultsNo differences of levels of Gsα and Giα were found in three groups before ischemia ( P gt;0.05). The level of Gsα after ischemia was significantly decreased than before ischemia in groupⅠand group Ⅱ ( P lt; 0 01), whereas no pronounced changes in group Ⅲ ( P gt;0.05) were noted after ischemia. The level of Gsα in group Ⅲ was not significantly changed after reperfusion compared with before ischemia( P gt;0 05), and it was much higher than those in groupⅠand group Ⅱ ( P lt; 0 01). Level of Giα was found not markedly changed in group Ⅲ after reperfusion compared with that before ischemia, but was notable higher in groupⅠand group Ⅱ( P lt;0.01). ConclusionsSignificant decrease of level of Gsα, whereas marked increase of level of Giα are found in myocardium of newborn guinea pig undergoing hypothermic (20℃) ischemic reperfusion. No impact of St. Thomas Ⅱ on these changes is verified, but recovery to the level of Gsα and Giα before ischemia is achieved by cold blood cardioplegic solution after ischemia and reperfusion. Unbalance between Gsα and Giα is the one of the mechanisms of ischemic reperfusion injury for immature myocardium.
Objective To study the interaction and mechanism of prostaglandin I2 (PGI2) receptor/thromboxane A2 (TxA2) receptor (IP/TP) and cyclooxygenase-2 (COX-2) in ischemia reperfusion injury after liver transplantation of rat. Methods Rats were randomly divided into three groups: control group （n=16), orthotropic liver transplantation group （n=32) and nimesulide intervention group （n=32). The samples were obtained at 3 h, 6 h, 12 h and 24 h after operation. The expressions of COX-2, IP and TP mRNA were detected by RT-PCR. Immunohistochemistry was used to detect the localization and expression of COX-2. Hematoxylin Eosin staining was used to classify the injury extent of liver. Serum ALT and AST levels were detected to evaluate the changes of liver enzyme. Results COX-2 protein expression detected by immunohistochemistry in orthotropic liver transplantation group mainly distributed in the district of liver sinusoidal endothelial cells, liver cells and macrophage cells, which was significantly higher than control group and nimesulide intervention group. Expressions of IP mRNA, TP mRNA and COX-2 mRNA in the orthotropic liver transplantation group were significantly increased than those in control group (P＜0.05), and the ratio of IP/TP increased (P＜0.05). Expressions of IP mRNA and TP mRNA in nimesulide intervention group were significantly lower than that in the orthotropic liver transplantation group at 6 h and 12 h after operation (P＜0.05), and the ratio of IP/TP decreased at 3 h, 6 h and 24 h after operation (P＜0.05). The expression of COX-2 mRNA in nimesulide intervention group was significantly lower than that in the orthotropic liver transplantation group at 6 h, 12 h and 24 h after operation. In orthotropic liver transplantation group liver injury was obvious by HE staining, and more severve than that in nimesulide intervention group. Serum AST (each time) and ALT (3 h, 6 h and 12 h) levels in the orthotropic liver transplantation group were significantly higher than that in control group and nimesulide intervention group (P＜0.05) and peaked at 6 h after operation. Conclusion The balance of IP/TP takes part in and plays an important role in the ischemia reperfusion injury of liver transplantation. Changing imbalance of IP/TP may reduce liver transplantation ischemia reperfusion injury by inhibiting COX-2 expression.
Objective To determine whether the different durations and times of the ischemic preconditioning affect the effectiveness of the ischemic preconditioning. Methods Ninety male Wistar rats were randomly divided into the control group and the eight preconditioned groups of 10 rats each. A transverse rectus abdominis musculocutaneous flap (TRAM) was elevated in each rat. The flaps were preconditioned by clamping the pedicle and reperfusing for 5 or 10 minutes per cycle. This was repeated for one or two cycles. The controls were simply perfused for 30 minutes. Each flap was then subjected to 4 hours of the global ischemia. Three rats in each group were killed for anestimate of the water content in the muscle and for observation on the muscularstructure under microscope. The flap surface survival areas of the other rats were calculated on the 7th postoperative day by the computerized video planimetry. Results The water content in the muscle was evidently reduced. The mean survival area of the flap in every preconditioned group increased by2-3 times compared with that of the controls(P＜0.001). The different proceduresof the ischemic preconditioning produced different protective effects. Conclusion The ischemic preconditioning is an available means to alleviate an ischemiareperfusion injury to the transverse rectus abdominis musculocutaneous flap in rats. The effect of the ischemic preconditioning is affected by the duration and time of the ischemic preconditioning.
This prospective animal study was designed to investigate the changes of plasma endothelin (ET) levels in acute necrotizing pancreatitis (ANP). Sprague-Dawley rats were randomly devided into 3 groups: acute necrotizing pancreatitis (ANP) group in which ANP was induced by infusion of 5% sodium taurocholate (STC) into biliopancreatic duct, sham operation (SO) group and platelet activating factor antagonist BN50739 (BN) group. Blood levels of ET and platelet activating factor (PAF) were detected. Pancreatic microcirculatory blood flow was measured and pancreatic histological scores were evaluated. Results showed that the pancreatic microcirculatory blood flow in ANP group was decreased to a great extent immediatly after induction of ANP and soon began to rise slowly for 3 hours and again decreased steadily after that. The blood levels of ET, PAF and histological scores in ANP group were significantly higher than those in SO group. In BN group, the blood flow was significantly improved and the levels of blood ET, PAF and histological scores were all significantly lower as compared to those in ANP group. It is concluded that ischemia/ reperfusion is present in the initiation of acute necrotizing pancreatitis induced by STC in the rat. This leads to injuries of endothelial cells and increase in the production of ET and PAF. I/R lesions,and interaction of ET and PAF lead to a vicious circle, thus augmenting the pathological changes in the pancreas.
Objective To investigate the mechanism of AMP-activated protein kinase (AMPK) in hepatic ischemia-reperfusion injury (HIRI). Methods ① Grouping. Forty-two mice were randomly divided into Sham group, 4 ischemia reperfusion (IR) group of different times (2, 6, 12, and 24 h), Compound C group, and Compound C+repamycin (Rapa) group, each group enrolled in 6 mice. Compound C group: mice were modeled at 1 h after intraperitoneal injection of Compound C (25 mg/kg). Compound C+Rapa group: mice were modeled at 1 h after intraperitoneal injection of rapamycin (1 mg/kg) and Compound C (25 mg/kg). Mice of 4 IR groups, Compound C group, and Compound C+Rapa group were used to prepare HIRI model. Mice of Sham group were treated only for laparotomy, freeing the first portal hepatis and closing peritoneal. ② To filter the best IR time. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice in Sham group and IR groups of 4 different reperfusion time points were measured. The pathological changes of liver tissues were observed by HE staining, and the expressions of related proteins in liver tissue of mice were detected by Western blot. Considering the results of blood biochemical test, HE staining, and Western blot together to determine the best IR point. ③ The exploration of signal pathway for AMPK. The expressions of proliferating cell nuclear antigen (PCNA) were observed by immunohistochemical staining in the liver tissues of IR-12 h group, Compound C group (12 h after IR) and compound C+Rapa group (12 h after IR). The mitochondrial damage was observed by rhodamine 123 staining, and the apoptotic status of liver cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (TUNEL). Results ① The 12 h after IR was the best observation time point. Compared with IR-12 h group, the levels of ALT and AST in Sham group, IR-2, 6, and 24 h groups were lower (P<0.05). HE staining showed that liver tissue destruction in IR-12 h group was the most severe. Western blot showed that, expressions of AMPKα, phosphorylated adenylate activated protein kinase α (p-AMPKα), Nip3-like protein X (Nix), BCL-2 homologous water-soluble protein (Bax), as well as ratio of autophagy microtubule-associated protein light chain 3 (LC3)Ⅱto LC3Ⅰof Sham group, IR-2, 6, and 24 h group were all lower than those of IR-12 h group (P<0.05), but the expressions of phosphorylated mammalian target of Rapa (p-mTOR) of Sham group, IR-2, 6, and 24 h group were all higher (P<0.05). Therefore, 12 h after IR was the best time to observe. ② Compared with IR-12 h group, the expression level of PCNA protein in liver tissue of Compound C group was lower (P<0.05), the mitochondrial luminescence intensity was weaker and the apoptotic cells were more. Compared with Compound C group, the expression of PCNA protein in the liver tissue of the Compound C+Rapa group was higher (P<0.05), the mitochondrial intensity was stronger and the apoptotic cells were less. ③ Compared with IR-12 h group, the expressions of Nix and p-AMPKα, and ratio of LC3Ⅱ to LC3Ⅰ in liver tissue of Compound C group decreased (P<0.05), while the expressions of p-mTOR, Caspase-3, and Cleaved Caspase-3 increased (P<0.05). Compared with Compound C group, the expressions of p-AMPKα and Nix in the liver tissue of Compound C+Rapa group increased (P<0.05), while the expressions of p-mTOR, Caspase-3, and Cleaved Caspase-3 decreased (P<0.05). Conclusion During the HIRI in mouse, AMPK regulates mitophagy and apoptosis through the mTOR/Nix pathway.
Objective Isoflurane has an acute preconditioning effectiveness against ischemia in kidney, but this beneficial effectiveness can only last for 2-3 hours. To investigate whether isoflurane produces delayed preconditioningagainst renal ischemia/reperfusion (I/R) injury, and whether this process is mediated by hypoxia inducible factor 1α(HIF- 1α). Methods A total of 52 male C57BL/6 mice were randomly assigned to 4 groups (n=13 in each group): the controlgroup (group A), PBS/isoflurane treated group (group B), scrambled small interference RNA (siRNA)/isoflurane treated group (group C), and HIF-1α siRNA/isoflurane treated group (group D). In groups C and D, 1 mL RNase-free PBS containing 50 μg scrambled siRNA or HIF-1α siRNA was administered via tail vein 24 hours before gas exposure, respectively. Equivalent RNasefree PBS was given in groups A and B. Then the mice in groups B, C, and D were exposed to 1.5% isoflurne and 25%O2 for 2 hours; while the mice in group A received 25%O2 for 2 hours. After 24 hours, 5 mice in each group were sacrificed to assesse the expressions of HIF-1α and erythropoietin (EPO) in renal cortex by Western blot. Renal I/R injury was induced with bilateral renal pedicle occlusion for 25 minutes followed by 24 hours reperfusion on the other 8 mice. At the end of reperfusion, the serum creatinine (SCr), the blood urea nitrogen (BUN), and the histological grading were measured. Results The expressions of HIF-1α and EPO in groups B and C were significantly higher than those in group A (P lt; 0.01). The concentrations of SCr and BUN in groups B and C were significantly lower than those in group A, as well as the scores of tubules (P lt; 0.01), and the injury of kidney was amel iorated noticeably in groups B and C. The expressions of HIF-1α and the concentrations of SCr and BUN in group D were significantly lower than those in group A (P lt; 0.01). Compared with groups B and C, the expression of HIF- 1α and EPO in group D decreased markedly (P lt; 0.01), the concentrations of SCr and BUN were increased obviously, as well asthe scores of tubules (P lt; 0.01), and the renal injury was aggratived significantly. Conclusion Isoflurane produces delayed preconditioning against renal I/R injury, and this beneficial effectiveness may be mediated by HIF-1α.
【Abstract】Objective To observe the synthesis of TLR2 protein and its mRNA expression in Kupffer cells (KCs) and sinusoidal endothelial cells(SECs).Methods Thirty-two BALB/c mice divided into two groups (operation group and false operation group) were used to prepare the model of partial hepatic ischemia/reperfusion (I/R) injury. After injury KCs and SECs were isolated with twosteps situ perfusion technique. And these cells were dyed by rat anti-mouse TLR2 IgG and anti-rat IgG2b labeled with flurescein isothiocyanate (FITC). The sysnthesis of TLR2 protein were determined by flow cytometric (FCM) analysis and real time reverse transcription polymerase chain reaction (Real-Time RT-PCR) analysis for gene expression.Results As for KCs: TLR2 expression was significant higher in operation group, compared with false operation group 〔protein expression: （9.19±1.07）% vs （1.52±0.21）%, P＜0.01; gene expression: 0.54±0.77 vs 2.62±2.19, P＜0.05〕. But there were no significant differences with expression in SECs. Conclusion Synthesis of TLR2 protein and its gene expression increased in KCs in the mouse partial hepatic ischemia-reperfusion injury.