Roles of PI3K/Akt and JAK2/STAT3 signaling pathways in protection of SO2 against limb ischemia/reperfusion-induced acute lung injury in rats

AIM: To investigate the role of PI3K/Akt and JAK2/STAT3 pathways in the protection of sulfur dioxide (SO₂) against limb ischemia/reperfusion (I/R)-induced acute lung injury (ALI) in rats. METHODS: ALI was induced by limb I/R in the SD rats. Na₂SO₃(0.54 mmol/kg, ip)/NaHSO₃ (0.18 mmol/kg, ip) as SO₂ donor was injected at 20 min before reperfusion. The inhibitors of JAK2/STAT3 and PI3K/Akt pathways, Stattic (3 mg/kg, iv) and LY294002(40 mg/kg, iv), respectively, were injected at 1 h before reperfusion. Peripheral blood and lung tissues were collected for determining the contents of the cytokines, the protein levels of the molecules related to the signaling pathways, apoptosis and histopathologic changes by ELISA, TUNEL and Western blot. RESULTS: Compared with control group, the content of MDA, the activity of MPO, lung coefficient, apoptotic index, cytokine expression, and the protein levels of p-Akt and p-STAT3 in I/R group all increased significantly, and administration of Na₂SO₃/NaHSO₃ attenuated the damage in the lung. Besides, the results of Western blot showed that the rat lung tissues expressed p-STAT3 protein and p-Akt protein. After I/R, the protein levels of p-STAT3 and p-Akt were increased. After using Na₂SO₃/NaHSO₃, p-Akt was increased, but p-STAT3 was decreased (P<0.05). CONCLUSION: Both JAK2/STAT3 and PI3K/Akt pathways are likely involved in the protective effect of SO₂ against limb I/R-induced ALI in rats. The activation of JAK2/STAT3 signaling pathway increases I/R injury. Reversely, the activation of PI3K/Akt signaling pathway reduces I/R injury. Besides, JAK2/STAT3 and PI3K/Akt signaling pathways may have crosstalk during I/R-induced ALI and JAK2/STAT3 pathway may have an impact on the P13K/Akt pathway.     

HBx modulates apoptosis by activating JAK2/STAT3 signaling pathway in human renal proximal tubular epithelial cells

AIM: To investigate the correlation of hepatitis B virus X protein (HBx) with renal tubular epithelial cell apoptosis in hepatitis B virus-associated glomerulonephritis (HBVGN) and the possible signaling mechanism. METHODS: The activation of JAK2/STAT3 signal pathway and the expression of apoptosis-related proteins in human kindey proximal tubular epithelial cells (HK-2 cells) were determined by Western blotting after transfection with HBx eukaryotic expression vector. The cell proliferation was observed by CCK-8 assay. The cell apoptosis was analyzed by the imaging of HO33342 staining, transmission electron microscopy and flow cytometry with Annexin V/PI double staining. RESULTS: After transfection of the target gene HBx, the expression levels of both p-JAK2 and p-STAT3 were significantly increased. At the same time, the cell proliferation was obviously inhibited, and the apoptotic rate was increased. After incubation with AG490, the JAK2/STAT3 signal pathway was partially blocked, and the cell apoptosis induced by HBx was reduced. CONCLUSION：HBx up-regulates the activation of JAK2/STAT3 signal pathway to induce renal tubular epithelial cell apoptosis, which is possibly involved in the pathogenic mechanism that HBV directly damages nephridial tissue.     

Xuebijing relieves testicular ischemia/reperfusion injury in rats by activating PI3K/Akt/mTOR signaling pathway

AIM: To investigate the effect of Xuebijing on testicular ischemia/reperfusion (I/R) injury in rats and its related mechanisms. METHODS: Male Sprague-Dawley rats (n=45) were randomly divided into control group, I/R group, low-dose Xuebijing group, high-dose Xuebijing group and dexamethasone group (n=9 in each group). Except for the rats in control group, the rats in other groups underwent testicular torsion, and after the operation, the rats were treated with 0.5 mL·kg^-1·d^-1 Xuebijing, 2 mL·kg^-1·d^-1 Xuebijing and 0.5 mL·kg^-1·d^-1 dexamethasone in low-dose Xuebijing group, high-dose Xuebijing group and dexamethasone group, respectively. On the 3rd, 7th, and 14th days after treatment, the left testis in the rats of each group was taken. The histopathological changes of the testis were observed by hematoxylin-eosin staining. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), endothelin-1 (ET-1) and nitric oxide (NO) in the testicular tissue were detected by biochemical methods. The protein levels of cell cycle-related molecules, apoptosis-related proteins and PI3K/Akt/mTOR signaling pathway-related proteins were determined by Western blot. RESULTS: Xuebijing significantly attenuated the testicular damage in I/R rats, significantly increased the activity of SOD in the testis of I/R rats, reduced the content of MDA, ET-1 and NO, inhibited oxidative stress in I/R-injured tissues, mediated the protein expression of cell cycle-related factors and apoptosis-related factors, and significantly increased the protein levels of p-PI3K, p-AKT, p-mTOR and p-S6K in the testis of I/R rats (P<0.05). These effects were time-dependent and dose-dependent. CONCLUSION: Xuebijing reduces testicular I/R injury of rats by mediating the expression of cell cycle-related and apoptosis-related proteins and activating PI3K/Akt/mTOR signaling pathway in dose-dependent and time-dependent manners.     

Effects of astragaloside IV on autophagy in rats with cerebral ischemia/reperfusion injury

AIM: To investigate the effects of astragaloside IV (AS-IV) on autophagy in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: The focal cerebral ischemia/reperfusion of rat left middle cerebral artery occlusion (MCAO) was induced by suture method. Male SD rats (n=70) were randomly divided into sham operation group, I/R group, solvent control group, AS-IV group, AS-IV+autophagy inhibitor (3-methyladenine, 3-MA) group, 3-MA group and autophagy activator (rapamycin, Rapa) group. Except for sham operation group, the rats in other groups were subjected to ischemia for 2 h and reperfusion for 24 h. The rats with successful modeling were selected according to Zea Longa scoring criteria. The volume of cerebral infarction was measured by TTC staining. The morphological changes of nerve cells in the rats were observed with Nissl staining. The phenomenon of autophagy was observed under transmission electron microscope. The protein expression of beclin-1 and LC3-Ⅱ was determined by Western blot. RESULTS: No neurological deficit in sham operation group was observed, and the cerebral infarction was not found. Compared with sham operation group, obvious cerebral infarction was observed, the Nissl bodies were small in size and number and stained light, typical autophagosomes were observed, and the protein expression of beclin-1 and LC3-Ⅱ was increased in I/R group (P<0.05). Compared with I/R group, the volume of cerebral infarction was decreased obviously, neurological deficit restored significantly, and the number of autophagosomes and the protein expression of beclin-1 and LC3-Ⅱ were increased in AS-IV group and Rapa group (P<0.05). However, no significant difference between solvent control group and I/R group was observed (P>0.05). Compared with AS-IV group, the neurological deficit was serious, the volume of cerebral infarction and the number of autophagosomes were increased, while the expression of beclin-1 and LC3-Ⅱ was decreased in AS-IV+3-MA group and 3-MA group (P<0.05). CONCLUSION: Astragaloside IV may play an important role in atte-nuating cerebral ischemia/reperfusion injury by activating autophagy.     

Dexmedetomidine reduces renal injury induced by lung ischemia/reperfusion in mice through inhibiting endoplasmic reticulum stress response

AIM:To investigate the effect of dexmedetomidine (DEX) on renal injury induced by lung ischemia/reperfusion (I/R) in mice and its relationship with endoplasmic reticulum stress response.METHODS:Healthy SPF male C57BL/6J mice,weighing 20~24 g,aged 8~10 weeks,were randomly divided into 5 groups (n=10 each):sham operation group (sham group),I/R group,atipamezole (Atip) group,DEX group,and DEX+Atip group.In vivo lung I/R model was established by occlusion of the left pulmonary artery for 30 min followed by 180 min of reperfusion in the mice.The Atip (250 μg/kg),DEX (20 μg/kg) and DEX+Atip were intraperitoneally infused into the mice before left pulmonary hilus was blocked in Atip group,DEX group and DEX+Atip group,and other operations were the same as I/R group.After experiment,the mice were killed,and the renal tissues were harvested to observe the morphological changes.The enzymatic activity of caspase-3,serum creatinine and blood urea nitrogen,and cell apoptotic index of the renal cells were also analyzed.The expression of c-Jun N-terminal kinase (JNK),caspase-12,CCAAT/enhancer-binding protein homdogous protein (CHOP) and glucose-regulated protein 78(GRP78) at mRNA and protein levels in the renal tissues was determined by RT-PCR and Western blot.RESULTS:Compared with sham group,the enzymatic activity of caspase-3,serum creatinine and blood urea nitrogen,renal cell apoptotic index,and the mRNA and protein levels of JNK,caspase-12,CHOP and GRP78 in I/R group were significantly increased (P<0.01),and the renal tissues had obvious damage under light microscope.Compared with I/R group,Atip group and DEX+Atip group,the enzymatic activity of caspase-3,serum creatinine and blood urea nitrogen,renal cell apoptotic index,and the mRNA and protein levels of JNK,caspase-12 and CHOP in DEX group were significantly decreased,and the expression level of GRP78 significantly increased (P<0.01).Furthermore,the renal tissue damage was obvious reduced.CONCLUSION:DEX effectively relieves the renal injury induced by lung I/R in mice,which may be associated with exciting α₂-adrenergic receptor and inhibiting endoplasmic reticulum stress response.     

Effects of propofol on lung injury and PI3K/Akt pathway in rats after liver ischemia and reperfusion

AIM：To study the effect of propofol on phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway in the model of rat lung injury after hepatic ischemia and reperfusion (IR). METHODS：Sixty-six SD rats were randomly divided into 4 groups：sham operation group (n=6), IR group (n=24), propofol group (n=24) and propofol+wortmannin group (n=12). The rats in IR group and propofol group were further divided into 4 subgroups according to the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The rats in propofol+wortmannin group were also divided into 2 subgroups according to the time points of 3 h and 6 h after reperfusion. The rats in sham group were only dissected porta without ligation. The ligation of hepatic pedicle in the rats in IR group was performed to induce liver ischemia for 30 min and then reperfusion was conduced. The rats in propofol group were given slow injection of propofol (20 mg/kg) from the caudal vein 10 min before ischemia, and then propofol was continuously pumped at dose of 20 mg·kg-1·h-1 until rats' death. Other procedures were the same as the rats in IR group. The rats in propofol+wortmannin group were injected with wortmannin, a blocker of PI3K, at dose of 15 μg/kg before ischemia, and also given the injection of propofol as the rats in propofol group. The lung tissues of the rats were collected at the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The protein levels of total Akt (t-Akt), phosphorylated Akt (p-Akt) and Bcl-2 in the lung tissues were detected by Western blotting. The apoptotic rate was analyzed by flow cytometry with annexin V-FITC/PI staining. RESULTS：Compared with sham group, the protein levels of p-Akt and Bcl-2, and the cell apoptotic rate in the lung tissues in IR group, propofol group and propofol+wortmannin group increased. Compared with IR group, the protein levels of p-Akt and Bcl-2 increased, and cell apoptotic rate of the lung tissues decreased in propofol group. Compared with propofol group, the protein levels of p-Akt and Bcl-2 decreased, and cell apoptotic rate in the lung tissues increased in propofol+wortmannin group. CONCLUSION：Propofol reduces the lung injury in rats induced by liver ischemia and reperfusion, and its mechanism may be involved in PI3K/Akt signaling pathway.     

Relationship between histone H3K36 trimethylation and acute renal injury induced by ischemia/reperfusion

AIM: To observe the expression change of H3K36 trimethylation (H3K36me3) in acute kidney injury (AKI) induced by ischemia/reperfusion (IR) and analyze its correlation with SMYD2 and renal injury, and to provide a new target for clinical treatment of IR-AKI. METHODS: The ICR mice (n=30) were randomly divided into IR group (n=15) and sham operation group (sham group, n=15). The AKI model was established by clamping bilateral renal pedicles for 45 min. The serum and renal tissues of the mice were collected after the model was established for 24 h. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by biochemical method. The pathological changes of the kidney were observed by HE staining. The changes of NGAL and cleaved caspase-3 were observed by immunohistochemical (IHC) staining. The protein levels of NGAL, SMYD2, H3K36me3, p-P53, P53, cleaved caspase-3, Bax, Bcl-2, STAT3, p-STAT3, JNK and p-JNK1/2/3 in the renal tissues were determined by Western blot. The relationship between H3K36me3 and SMYD2/NGAL was analyzed by Pearson correlation method. RESULTS: Compared with sham group, BUN and SCr levels of the mice in IR group were significantly increased (P<0.05). HE staining showed significant edema, abscission and necrosis in renal tubular epithelial cells of the micc in IR group, and abscission of brush border and large amount of cell debris in the lumen were also observed. The IHC staining results showed that the protein levels of NGAL and cleaved caspase-3 in IR group were significantly increased. The results of Western blot showed that the protein levels of NGAL, SMYD2, H3K36me3, p-P53, cleaved caspase-3, Bax, STAT3, p-STAT3, JNK and p-JNK1/2/3 in IR group were significantly up-regulated (P<0.05), while the Bcl-2 levels were significantly down-regulated (P<0.05). The correlation analysis showed that H3K36me3 was positively correlated with the levels of SMYD2 and NGAL. CONCLUSION: H3K36me3 is closely related to SMYD2 and renal injury in IR-AKI mice, and the up-regulated expression of H3K36me3 may be involved in the regulation of IR-AKI occurrence and development together with the activation of STAT3 and JNK signaling pathways.     

Riboflavin preconditioning alleviates hepatic ischemia/reperfusion injury in rats

AIM: To explore the protective effect of riboflavin preconditioning on hepatic ischemia/reperfusion injury in rats. METHODS: Twenty-four Sprague-Dawley rats wererandomly divided into 3 groups (n=8): sham group, ischemia/reperfusion (I/R) group and riboflavin preconditioning (R+I/R) group. The rats in sham group and I/R group received a standard chow,while the rats in R+I/R group received a chow supplemented with riboflavin. After 4 weeks, portal vein and hepatic artery supplying the middle and left hepatic lobes were clamped with a traumatic vascular clip for induction of partial hepatic ischemia in the rats in I/R group and R+I/R group. After 1 h of ischemia, 1 h of reperfusion was conducted by removal of the clip. The activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum,the activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in serum and liver were measured. Western blotting was employed to examine the protein expression of heme oxygenase-1(HO-1) in the liver. RESULTS: The results showed that ischemia/reperfusion injury markedly increased the activity of AST and ALT in serum, decreased the activity of SOD, and elevated the level of MDA and the activity of HO-1 in the liver as compared with sham group (P<0.01). The riboflavin pretreatment significantly decreased the activity of AST and ALT in serum, increased the activity of SOD and decreased the levels of MDA in serum and liver as compared with I/R group (P<0.01). In addition, the protein expression of HO-1 and the activity of HO-1 were elevated in R+I/R group (P<0.01). Cytoplasmic vacuolation and swelling of the hepatocytes were observed in I/R group. Treatment with riboflavin markedly alleviated the changes of liver structure. CONCLUSION: Riboflavin preconditioning has protective effect on hepatic ischemia/reperfusion injury. The mechanism may be correlated with enhancing the anti-oxidation and alleviating the reaction of lipid peroxidation.     

Regulation of retinoid X receptor-mediated-autophagy pathway in rat pulmonary ischemia/reperfusion injury

AIM: To investigate the regulation of retinoid X receptor (RXR)-mediated autophagy pathway in rat pulmonary ischemia/reperfusion (I/R) injury. METHODS: The male SPF-grade SD rats were randomly divided into 7 groups (n=10):normal control (C) group, sham (S) group, sham plus 9-cis-retinoic acid (SRA) group, sham plus HX531 (SH) group, I/R group, I/R plus 9-cis-retinoic acid (RA) group and I/R plus HX531 (HX531) group. The model of pulmonary I/R injury was established by clamping the left hilum of lung for 30 min followed by 180 min of reperfusion. The animals in C group didn't receive any treatment. Only sternotomy was performed for the rats in S group, the hilum of lung was not clamped, and the rats were mechanically ventilated for 210 min. The clamping of the left hilum of lung for 30 min followed by 180 min of reperfusion was performed in I/R group. In SRA, SH, RA and HX531 groups, 9-cis-re-tinoic acid (5 mg/kg) was intraperitoneally injected at 90 min before establishment of the model. In SH group and HX531 group, HX531 at 5 mg/kg was intraperitoneally injected at 30 min before establishment of the model. Left lung tissues were removed after 180 min of reperfusion for determing the index of quantitative assessment (IQA) of alveolar damage. The pathological changes of the lung were observed by HE staining, and immunofluorescence staining was used to observe the changes of RXRα in various lung tissues. The mRNA expression of autophagy-associated molecules LC3, beclin 1 and mTOR was detected by RT-PCR. The protein levels LC3-Ⅱ, beclin 1 and p-mTOR in each group were determined by Western blot. RESULTS: Compared with C group, the lung IQA, the mRNA expression of LC3 and beclin 1, and the protein levels of LC3 -Ⅱ and beclin 1 in I/R, RA and HX531 groups were increased significantly, the mTOR mRNA and p-mTOR protein levels were decreased (P<0.05), and the morphological structure of the lung was also impaired. Compared with I/R group, the lung IQA and the expression of LC3 and beclin 1 at mRNA and protein levels were significantly decreased, the mRNA expression of RXRα and mTOR, and the protein level of p-mTOR were increased in RA group (P<0.05), and the structural damage of the lung tissue was also significantly reduced. No statistically significant difference was observed between I/R group and HX531 group. Compared with RA group, the lung IQA and the expression of LC3 and beclin 1 at mRNA and protein levels was increased significantly, the mRNA expression of RXRα and mTOR, and the protein level of p-mTOR were decreased in HX531 group (P<0.05), and the morphological damage of the lung tissue was increased. CONCLUSION: The activation of RXR effectively alleviates the pulmonary I/R injury in rats. The protective role of RXR in lung tissue may be related to the inhibition of autophagy pathway.     

Quercetin reduces microvascular permeability induced by ischemia/reperfusion injury through SIRT1 signaling

AIM To investigate whether quercetin (Que) attenuates microvascular injury after myocardial ischemia/reperfusion (I/R), and whether its mechanism is related to the up-regulation of silent information regulator 1 (SIRT1). METHODS In vivo, SD rats (220~250 g) were randomly divided into sham group (only threading without ligation), I/R group (ischemia 30 min/reperfusion 60 min), I/R+Que group (10 mg/kg Que injected intravenously 15 min before reperfusion) and I/R+Que+EX527 group (1 mg/kg EX527 injected intravenously 15 min before Que). The protein expression of SIRT1 in each group was determined by Western blot. The effect of Que on the morphological changes of myocardial microvascular was estimated by HE staining. The contents of endothelial damage markers and inflammatory factors in peripheral blood serum of the rats in each group were measured by ELISA. In vitro, Transwell chamber was used to culture H5V cells and then the influence of Que on the permeability of endothelial cells was evaluated. The expression of myosin light chain phosphatase(MLCP), zonula occludens-1 (ZO-1) and cytoskeleton was determined by Western blot and immunofluorescence method. RESULTS The results of Western blot showed that SIRT1 was significantly reduced by I/R in the myocardial tissue. Que increased the expression of SIRT1 after I/R, whereas EX527 significantly inhibited this effect (P<0.05). Que reduced the incidence of I/R arrhythmia, alleviated microvascular damage and inhibited the infiltrating of inflammatory cells,as well as decreased levels of the endothelial injury markers (E-selectin and VCAM-1) and proinflammatory factors (PAF, IL-1α and IL-6). While EX527 significantly reduced these effects of Que by inhibiting SIRT1. Cultured H5V cells showed that hypoxia/reoxygenation resulted in increased permeability of endothelial cells and decreased expression of ZO-1 and MLCP. However, Que, which up-regulated expression of SIRT1, significantly reduced the permeability of endothelial cells significantly and increased the expression of ZO-1 and MLCP. Meanwhile, the cytoskeleton remodeling basically disappeared. EX527, which down-regulated expression of SIRT1, significantly inhibited the above effects of Que. CONCLUSION Que attenuates myocardial I/R induced microvascular injury. The up-regulation of SIRT1 is involved in this mechanism.     

Proteomic study of myocardial mitochondria with ischemia/reperfusion injury and pinacidil postconditioning in isolated rat hearts

AIM: To investigate the protective effect of pinacidil postconditioning on rat myocardium suffering ischemia/reperfusion injury by mitochondrial proteomics. METHODS: Langendorff apparatus was used to establish the model of myocardial ischemia/reperfusion injury. Sprague-Dawley rats were randomly divided into 2 groups: pinacidil postconditioning group (Pina group) and ischemia/reperfusion injury group (I/R group). After 20 min of perfusion with K-H solution, the perfusion was suspended for 40-min (global ischemia) follow by 60 min of reperfusion in I/R group. In Pina group at the end of 40 min global ischemia, the isolated hearts were perfused with K-H solution containing pinacidil (50 μmol/L) for 2 min followed 58-min perfusion with regular K-H solution. Total proteins extracted from the mitochondria were applied to the two-dimensional gel electrophoresis (2-DE). The differentially expressed protein spots over 2 times were evaluated by a software. Then they were subjected to in-gel digestion, and analyzed by spectrometry. RESULTS: The expression levels of NDUFA10, NDUFS2 and NDUFV2 were elevated but those of IDHA and ECH1 were decreased in Pina group compared with I/R group. Interestingly, 2 spots in the 2-DE map were identified as ATPase subunit δ. The expression levels of one spot was elevated, while the other was decreased. CONCLUSION: Pinacidil postconditioning may decrease the degree of increased expression levels of NDUFA10, NDUFS2 and NDUFV2, promote the expression of IDHA and ECH1, and induce the phosphorylation of ATPase subunit δ, which may be related to the protective mechanism of pinacidil postconditioning.     

Inhibitory effect of ginkgo-dipyridamole injection on ischemia/reperfusion injury in rat hearts in vitro

AIM：To investigate the effect of ginkgo-dipyridamole injection (GD) on ischemia/reperfusion (I/R) injury in rat hearts in vitro and its possible mechanism. METHODS：Forty male Sprague-Dawley rats were randomly divided into 5 groups (n=8): normal control (NC) group, I/R group, ischemic preconditioning (IPC)+I/R group, GD+I/R group and GD+LaCl3+I/R group. Cardiac function indexes, including heart rate (HR), left ventricular systolic pressure (LVSP) and the maximal rise/fall rate of left ventricular pressure (±dp/dtmax), were detected at 5 time points, including stabilizing point, 30 min after ischemia, and 5, 30 and 60 min after reperfusion. The activity of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent at the five time points was assayed. The concentration of Ca2+ and the content of α-ketoglutarate dehydrogenase (α-OGDH) in myocardial mitochondria were determined at the end of the whole experiment. RESULTS：Compared with I/R group, the cardiac function indexes in IPC+I/R and GD+I/R groups were improved at the reperfusion period (P<0.05), the activity of LDH and CK in coronary effluent and the concentration of Ca2+ in mitochondria were significant reduced (P<0.01), and the content of α-OGDH was increased (P<0.05). However, the protective effect of GD was inhibited by LaCl3 (P<0.05). CONCLUSION：GD protects rat hearts against I/R injury by inhibiting calcium overload and improving mitochondrial enzyme activity to stabilize mitochondrial energy metabolism.     

Effects of spermine on myocardial ischemia/ reperfusion injury in rats

AIM: To observe the effect of exogenous spermine (low concentration) on myocardial ischemia/reperfusion injury in rats.METHODS: 40 Wistar rats were randomly divided into 4 groups: sham- operation group (Sham), ischemic reperfusion group (I/R), spermine group (Sp) and natural saline group (NS). The model of ischemic/reperfusion injury was established by ligating rat coronary artery. In Sp group, spermine (0.5 mmol/L, 2 mL/kg) was injected slowly into rat vein. During the process, we recorded the electrocardiogram and the LV functional parameters, assayed the levels of SOD, LDH, NO and MDA in serum, and examined the ultrastructure of the myocardium. RESULTS: In I/R group, the incidence of arrhythmia was 90%, myocardial ultrastructure was injured seriously, values of LVSP and ±dp/dtmax decreased, levels of LDH, NO and MDA increased while SOD activity decreased (P<0.05 or P<0.01, compared with Sham group). Compared with I/R and NS group, all those indexes in Sp group changed significantly (P<0.05 or P<0.01). CONCLUSION: Exogenous spermine alleviates myocardial ischemia/ reperfusion injury in rats. The mechanism may be related to its antioxidant effect and relieving the injury caused by oxygen free radical.     

Effect of pretreatment with 11,12-EET on myocardial ischemia/reperfusion injury in rats

AIM: To examine the effect of pretreatment with low-concentration of 11, 12-epoxyeicosatrienoic acid(EET) on myocardial ischemia/reperfusion injury in rats. METHODS: After tracheotomy, myocardial ischemia/reperfusion was produced by occlusion and release of the left anterior descending artery(LAD) of the rats. Ischemic preconditioning(IP) was made by two times of ischemia(5 min)/reperfusion(5 min). The experiment was conducted in three groups: control,IP and pretreatment with 11,12-EET(6.24×10^-8 mol/L), and each group was subdivided into two subgroups:A,the rats were subjected to ischemia(10 min)/reperfusion(10 min) and arrhythmias during the whole periods were monitored; The rats in B were subjected to ischemia(60 min)/reperfusion(30 min) and arrhythmias, cardiac funtion and myocardial infarction size were documented. RESULTS: Both IP and pretreatment with 11,12-EET could protect the heart against arrhythmias, cardiac disfunction and myocardial infarction. CONCLUSION: Pretreatment with 11,12-EET had protective effect on myocardium in case of ischemia/reperfusion, which was similar to ischemic preconditioning.     

Role of apelin in rat myocardial ischemic preconditioning and ischemia/ reperfusion injury

AIM: To study the alteration and role of apelin in myocardial ischemic preconditioning and ischemia/reperfusion injury of rats.METHODS: Forty-five SD rats were randomly divided into three groups: ischemia/reperfusion group (IR),ischemia pre-conditioning group (IP) and sham operation group.ECG was continuously used to evaluate the score of arrhythmias.The protein levels of apelin-36 in myocardium and plasma were detected by radioimmunoassay.The expression of apelin was observed by immunohistochemistry.RESULTS: (1) The scores of arrhythmias in IP group (2.1±0.5) was only 58.3% of IR group (3.6±0.8) ( P<0.01).(2) The apelin-36 protein level of plasma and myocardium in IR group were respectively lower by 36.1% and 45.6% than those in SH group (P<0.01),and those in IP group were lower by 23.8% and 24.7% than those in SH group (P<0.01),but higher than those in IR group (18.9% and 38.5%,respectively,P<0.05).(3) The staining absorbance of apelin in IR,SH and IP group was (7.87±2.41),(22.53±2.54) and (14.23±2.15),respectively.There were significant differences between IR and SH (P<0.01) and between IP group and SH group (P<0.05).(4) The scores of arrhythmias in IP and IR were negatively correlated with the protein level of apelin-36 in myocardium (r= 0.847,P <0.01).CONCLUSION: The down-regulation of apelin-36 in the plasma and myocardium of rats indicates that apelin has an important role in myocardial ischemic preconditioning and ischemia/reperfusion injury.     

Effects of sodium valproate on RPMI8226 and U266 cell proliferation and IL-6/JAK/STAT signaling pathway

AIM: To investigate the effects of sodium valproate (VPA) on the proliferation of multiple myeloma cell lines RPMI8226 and U266 and the regulation of IL-6/JAK/STAT signaling pathway. METHODS: The cells were treated with different concentrations of VPA for 12 h and 24 h. The growth of RPMI8226 cells and U266 cells was detected by MTT assay. Apoptotic rates and cell cycle were analyzed by flow cytometry. The mRNA expression of STAT3, STAT5 and STAT target genes Bcl-xL, Mcl-1, c-Myc, CCND1 and VEGF was measured by RT-PCR. Western blotting analysis was used to determine the total proteins and protein phosphorylation levels of JAK2 and STAT5. RESULTS: VPA inhibited the growth and induced the apoptosis of RPMI8226 cells and U266 cells in a concentration- and time-dependent manner. The levels of IL-6 in the culture supernatants of RPMI8226 cells and U266 cells treated with VPA were significantly higher than that in negative control group. VPA down-regulated the mRNA expression of STAT3, STAT5, Bcl-xL, Mcl-1, c-Myc, CCND1 and VEGF. After treated with VPA, the protein levels of p-JAK2, JAK2, p-STAT5 and STAT5 in RPMI8226 cells and U266 cells were significantly lower than those in control group. CONCLUSION: VPA inhibits the proliferation of PRMI8226 cells and U266 cells in vitro. The modulation of IL-6/JAK/STAT signaling pathway may be involved in its potential mechanisms.     

Hypoxia-inducible factor-1α attenuates myocardial inflammatory injury induced by ischemia and reperfusion in rats

AIM:To investigate the role of hypoxia-inducible factor-1α (HIF-1α) stable expression in myocardial inflammatory injury induced by ischemia and reperfusion (I/R) in rats. METHODS:Male Wistar rats were randomly divided into 4 groups:sham operation (sham) group, I/R group, HIF-1α stabilizer dimethyloxalyl glycine (DMOG)+I/R group and HIF-1α inhibitor YC-1+I/R group. The protein expression of myocardial Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was determined by Western blot. The mRNA levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, TLR4 and NF-κB were detected by real-time PCR. The myeloperoxidase (MPO) activity in the myocardial tissues was measured. HE staining was used to observe the infiltration of inflammatory cells. RESULTS:HIF-1α decreased the infiltration of inflammatory cells, the MPO activity, and the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the myocardial tissues. HIF-1α also reduced the expression of TLR4 and NF-κB at mRNA and protein levels (P<0.05). CONCLUSION:The stable expression of HIF-1α has an anti-inflammatory effect on the myocardial tissues after I/R injury in rats. The mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.