Effects of endoplasmic reticulum stress induced by increased expression of calcium-sensing receptor in myocardial hypoxia/re-oxygenation injury

AIM: To observe the effects of endoplasmic reticulum stress induced by the increase in expression of calcium-sensing receptor in myocardial hypoxia/re-oxygenation injury. METHODS: The primarily neonatal rat ventricular cardiomyocytes were incubated for 4-5 d, then randomly divided into 5 groups: (1) sham control group; (2) hypoxia/re-oxygenation group; (3) H/Re+ NiCl2, CdCl2-reperfusion group; (4) H/Re+GdCl3, NiCl2, CdCl2-reperfusion group; (5) H/Re+caffeine,GdCl3, NiCl2, CdCl2-reperfusion group. The neonatal cells were in ischemia-mimetic solution for 3 h, and re-incubated cells in normal culture medium for 9 h to establish a model of H/Re. The activity of LDH was determined, the viability and apoptosis of cardiomyocytes were assayed by MTT, the expressions of CaSR and caspase-12 in each group were analyzed using Western blotting, and the concentration of intracellular calcium was measured by laser confocal scanning microscope. RESULTS: The apoptosis index, the activity of LDH, the concentration of intracellular calcium, and quantitative expression of CaSR and caspase-12 in H/Re and activator groups were significantly higher than those in control group, while the viability and apoptosis of cardiomyocytes were lower than those in control group. CONCLUSION: In myocardial hypoxia/re-oxygenation, CaSR induces endoplasmic reticulum stress by altering the intracellular calcium homeostasis. The induction of apoptosis may be due to the increase in the expreesions of caspase-12 and other proapoptotic proteins.     

Expression of calcium sensing receptor during ischemia/reperfusion myocardial damage and relationship between CaSR and injury of myocardium

AIM: To investigate the expression of calcium sensing receptor(CaSR) during myocardial injury induced by ischemia/reperfusion and disclose the relationship between CaSR and myocardial ischemia/reperfusion. METHODS: The experimental model was established by the 30 min ligating and 1 h, 2 h, 4 h, 6 h reperfusing the left descending coronary artery (LAD) in rats. Wistar rats were randomly divided into 5 groups: sham group, ischemia/reperfusion 1 h, 2 h, 4 h, 6 h groups (I/R 1 h, 2 h, 4 h, 6 h group). CaSR mRNA expression was detected by RT-PCR. Left ventricular function was recorded. The levels of plasma lactate dehydrogenase (LDH), alondialdehyde (MDA) and superoxide dismutase (SOD) were measured. The change of ultrastructure in the ischemia/reperfusion myocardium of rats was observed by electron microscopy. RESULTS: LVSP,±dp/dtmax and SOD activity decreased gradually with the reperfusion time prolonged. LDH and MDA peaked at 2 h. The ultramicro-structural injury at the 1 h and 2 h was more serious than that at 4 h and 6 h. The expression of CaSR increased significantly after reperfusion of 1 h and 2 h, and decreased after 4 h and 6 h. CONCLUSION: The increased expression of CaSR mRNA and serious injure of myocardium were observed. CaSR may be associated with the myocardial ischemia/reperfusion injury.     

Change of calcium sensing receptor expression and apoptotic pathways in myocardial infarction rat induced by isoprel

AIM:To observe the change of calcium sensing receptor (CaSR) expression and apoptotic pathways in myocardial infarction rat induced by isoprel.METHODS: The myocardial infarction rat models were prepared by subcutaneous injection of isoprel (ISO 200 mg·kg-1·d-1 for 2 d). Wistar rats were divided into three groups randomly: ① Control group; ② ISO/1d group; ③ ISO /2d group. The expressions of CaSR, 〖STBX〗bcl-2, bax〖STBZ〗 and caspase-3 mRNA and protein were analyzed by RT-PCR and Western blotting, respectively. Apoptotic cells were measured by TUNEL staining assay. The morphological and ultrastructural changes were observed under optical microscope and electronic microscope. The activity of LDH, CK, SOD and the content of MDA were assayed with ultraviolet spectrophotometer. The level of troponin(cTnT) was observed by chemical immunofluoresence.RESULTS: Compared with control group, the activity of LDH and CK, the content of MDA and cTnT, the apoptosis index and the expression of CaSR, Bax and caspase-3 were reached the highest level, but the SOD activity and Bcl-2 expression were decreased. The myocardial ultrastructural injury was aggravated in the ISO/1d group. The change of above parameters in ISO/2d group was between control and ISO/1d group.CONCLUSION:The increased expression of CaSR is involved in rat myocardial infarction induced by isoprel, which is related with oxidative stress and apoptosis.     

Expression of calcium-sensing receptor in the rat cardiac tissue at different age and its relation with anoxia-reperfusion injury

AIM：To investigate the changes of expression of calcium-sensing receptor (CaSR) in the rat cardiac tissue at different age and its relation with anoxia -reperfusion (A/R) injury.METHODS：RT-PCR was used to detect the mRNA expression of CaSR.The A/R injury was remodeled in vitro,and the ultrastructure of cardiac tissue was observed under transmission electron microscope.RESULTS：The expression of CaSR mRNA in the rat cardiac tissue increased gradually after birth,and reached to its maximum in the 1st month.In the 2nd to 3rd month,the expression were almost equal to that at birth and then increased again to a higher level.The expression level of CaSR mRNA increased more significantly than those in control groups after 40 min of anoxia and reperfusion of 1 h and 2 h,and decreased after 3 h and 4 h.Moreover,the longer the reperfusion time lasted,the more serious the changes of ultrastructure were observed.CONCLUSION：Our results demonstrate that the CaSR is expressed in the rat cardiac tissue.The mRNA expression of CaSR changes with age and is involved in the anoxia -reperfusion injury.     

Protective effect of allitridi on hippocampus of rats with cerebral ischemia-reperfusion and P53 expression

AIM: To observe the protective effect of allitridi on hippocampal neuron of rats with cerebral ischemia-reperfusion (I/R) injury and to investigate its effects on P53 expression in hippocampus.METHODS: The global cerebral ischemia-reperfusion models were established by 4-vessel occlusion. Allitridi at doses of 10, 20 or 30 mg/kg was injected through rat’s tail vein, half dose at 30 min before brain ischemia and another half dose at 10 min after reperfusion were injected, respectively. The hippocampus of rat was removed 24 h after reperfusion. Toluidine blue staining was applied to estimate morphologic changes. Flow cytometry was used to evaluate neuronal apoptosis rate of hippocampus. Immunohistochemistry was used to observe the expression of P53 protein.RESULTS: Compared with sham group, survival neuronal density in I/R group was significantly depressed. The rate of neuronal apoptosis and the expression of P53 protein were significantly increased. Allitridi significantly increased the number of survival neurons in hippocampus compared to I/R group. Meanwhile, allitridi remarkably inhibited the rate of neuronal apoptosis and the expression of P53 protein.CONCLUSION: Allitridi has protective role against brain ischemia reperfusion injury. The mechanism may be involved in blocking P53 protein expression in hippocampus of rats with ischemia-reperfusion.     

Effect of limb ischemic postconditioning on brain focal ischemia and reperfusion injury in mice

AIM: To establish the mouse model in which the limbic ischemic postconditionning (LIPostC) enhances the tolerance against brain ischemia, and to investigate the effects of LIPostC on the ischemic extent and roles of heat shock protein 70 (HSP70) in ischemia and reperfusion injury. METHODS: The male Kunming mice were used in the study. The brain ischemia reperfusion (I/R) model was made by middle cerebral artery occlusion (MCAO). In the first test, the male mice were randomly divided into 9 groups (n=10): sham group, ischemia/reperfusion (I/R) groups (with ischemia for 0.5 h, 1 h,1.5 h and 2 h) and LIPostC+I/R groups (0.5 h+LIPostC,1 h+LIPostC,1.5 h+LIPostC,2 h+LIPostC). The reperfusion was performed after LIPostC for 24 h. After the neurologic deficit scores were evaluated, the brains were taken out to measure the infarct volume with TTC staining and to observe the pathological changes of cerebral cortex with HE staining. The neuronal apoptosis was determined by TUNEL. In the second test, the male mice were randomized into 4 groups (n=6): sham group, I/R group, LIPostC+I/R group and LIPostC+I/R+quercetin group (2 h ischemia). The neurological deficit scores were evaluated at 24 h after operation. The expression of HSP70 was determined by Western blotting.RESULTS: The duration of brain ischemia was related to the motor behavior and degree of brain injury. The longer the ischemic duration of the brain was performed, the more severe the pathological and behavioral changes were observed. The brain injury in 2 h MCAO mice was more severe than that in 1 h and 1.5 h MCAO mice (P<0.05). Compared to I/R group, each LIPostC group showed lower neurological score, less infarct volume and TUNEL positive neuron. The expression of HSP70 protein was increased and neurological functions were improved significantly in the mice with LIPostC. However, the neuroprotective role of LIPostC was attenuated by treating with quercetin, an inhibitor of HSP70.CONCLUSION: LIPostC promotes the expression of HSP 70, improves the neurological functions and attenuates the ischemia and reperfusion injury in MCAO mice. HSP70 produces a marked effect on the ischemic tolerance induced by LIPostC in MCAO mice.     

Effects of panax notoginseng saponins on pneumocyte apoptosis and Fas/FasL expression in rabbits with lung ischemia/reperfusion injury

AIM: AIM: To explore the relationship between apoptosis in the lung tissues and lung ischemia/reperfusion injury, and observe effects of panax notoginseng saponins (PNS) on apoptosis in lung ischemia/reperfusion injury. METHODS: Single lung in situ ischemia/reperfusion animal model was used. Eighty four Japanese white rabbits were randomly divided into control group (control), ischemia/reperfusion 1 h group (IR1h), IR3h, IR5h, Panax Notoginseng Saponins 1 h group (PNS1h), PNS3h and PNS5h. TUNEL, immunocytochemistry and in situ hybridization techniques were used to observe apoptosis and Fas/FasL expression in various phases of lung ischemia/reperfusion. RESULTS: Cell apoptosis in lung tissues were significantly high, Fas/FasL mRNA and its protein were up-regulated in lung tissues of lung ischemia/reperfusion injury compared with control (all of P<0.01). The PNS suppressed apoptosis as well as expression of Fas/FasL mRNA and its protein (P<0.05 or P<0.01, respectively). There was a significant correlation between expression of Fas/FasL protein, Fas/FasL mRNA and cell apoptosis (r=0.540，0.658，0.668，0.686；all P<0.01). CONCLUSIONS: Activation of Fas/FasL system and its initiating cell apoptosis of lung tissues may contribute to the pathogenesis of lung ischemia/reperfusion injury. The protective effects of PNS include suppressing the activation of Fas/FasL system and blocking apoptosis in lung tissues in lung ischemia/reperfusion injury.     

Effects of Salvia miltiorrhiza Bunge.f.alba. on mitochondrial damage and apoptosis induced by cerebral ischemia and reperfusion

AIM: To observe the effects of Salvia miltiorrhiza Bunge.f.alba. (Sal) on the mitochondrial ultra-structure, oxidative stress and apoptosis induced by ischemia injury in a rat model of focal cerebral ischemia and reperfusion.METHODS: The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established by a modified Longa occlusion method. Adult male SD rats were randomly divided into control group, simple ischemia reperfusion group, Sal with ischemia reperfusion group and butylphthalide with ischemia reperfusion group. To study the protective effects of Sal and its mechanism, the intervention of Sal was given and the ultra-structure of mitochondria, functions of mitochondria under oxidative stress and the incidence of apoptosis of brain cells were determined.RESULTS: Many electron dense toxic granulation and vacuolus in mitochondria were observed in the rat brain of focal cerebral ischemia and reperfusion. Under the condition of ischemia and reperfusion, the mitochondria membrane was disaggregative, and the tubular cristae of mitochondrion disappeared. MDA content was obviously increased and the activity of glutathione peroxidase decreased significantly. The apoptosis of brain cells were observed in a great quantity. The changes of ultra-structure of mitochondria and the activity of GSH-Pxase were significantly improved by the treatment of Sal. Furthermore, treatment with Sal delayed the decrease of GSH-Pxase activity, and inhibited the increase in MDA content in brain tissue after ischemia and reperfusion. The incidence of apoptosis of brain cells was also decreased.CONCLUSION: Sal protects the brain tissue from ischemia injury.     

Expression and function of calcium-sensing receptor in mouse embryonic stem cells

AIM: To observe the functional expression of calcium-sensing receptor (CaSR) in the mouse embryonic stem cells (mESCs). METHODS: The expression and distribution of CaSR were detected by Western blotting and immunofluorescence observation in 129 mouse ES-D3 cells. The intracellular concentration of free calcium (［Ca2+］i) was determined by confocal laser scanning microscopy. The cell viability was analyzed by MTT assay and flow cytometry. RESULTS: CaSR protein was expressed in mESCs. Extracellular calcium or neomycin significantly increased the expression of CaSR and ［Ca2+］i. Neomycin increased the cell viability, up-regulated the protein expression of p-ERK2. These effects of neomycin were inhibited by NPS2390. CONCLUSION: CaSR is expressed in mESCs. The activation of CaSR is involved in the proliferation of mESCs.     

Effects of ischemic preconditioning on oxidative stress and mitochondrial function in young and old rat myocardium with ischemia/reperfusion

AIM: To study the protective effect of ischemia preconditioning (IPC) on ischemia/reperfusion (IR)-damaged myocardium in young and old rats. METHODS: Male Wistar rats aged at 3 months (young) and 20 months (old) were used to establish myocardial IPC model and IR model with the method of Langendorff heart perfusion. The rats were divided into young ischemia/reperfusion (YIR) group, young ischemic preconditioning (YPC) group, old ischemia/reperfusion (OIR) group and old ischemic preconditioning (OPC) group. Transmission electron microscopy was used to observe the ultrastructural changes of myocardial tissue and myocardial mitochondria. The myocardial infarction area was determined by TTC staining. The lactate dehydrogenase (LDH) content in coronary effluent fluid and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in myocardial tissues were detected by the method of colorimetry. The levels of nitrated and carbonylated proteins in myocardial tissue were measured by ELISA. The myocardial cell apoptosis was analyzed by TUNEL assay. The mitochondrial respiratory function and mitochondrial permeability transition pore opening induced by calcium load were evaluated by oxygen electrode method. RESULTS: Compared with YIR group, the myocardial infarction area in YPC group was obviously smaller, SOD activity in myocardial tissues increased, LDH activity in coronary effluent fluid and the content of MDA decreased, and the levels of nitrated and carbonylated proteins in the cardiac tissues reduced. In YPC group, the mitochondrial membrane structure appeared intact, cristae of the mitochondria showed close arrangement, and the matrix was compressed under the electron microscope. Myocardial mitochondrial respiratory control rate, state Ⅲ oxygen consumption and the P/O ratio in YIR group all significantly increased, proton leak decreased, mitochondrial swelling induced by calcium distinctly reduced, and myocardial apoptosis rate declined. No significant difference of the above indexes between OIR group and OPC group was observed. Compared with YPC group, myocardial ultrastructural damage increased clearly, cardiac oxidative stress increased, mitochondrial respiratory function declined, and cell apoptosis and necrosis increased in OPC group. CONCLUSION: Ischemic preconditioning has protective effect against myocardial IR injury in young rat hearts, while old rat hearts were less sensitive to ischemic preconditioning, leading to bluntness of cardioprotection with IPC in aging hearts. This may be related to mitochondrial injury and severe cellular apoptosis caused by increase of cardiac oxidative stress levels in the aging ischemic preconditioning heart.     

Effects of human thioredoxin on pneumocyte apoptosis and Bcl-2/Bax expression in rats with lung ischemia/reperfusion injury

AIM: To explore the relationship between apoptosis in the lung tissues and lung ischemia/reperfusion injury, and to observe the effects of human thioredoxin (hTrx) on apoptosis in lung ischemia/reperfusion injury. METHODS: The single lung in situ ischemia/reperfusion animal model was used. Eighty four Wistar rats were randomly divided into control group (control), groups of ischemia for 1 h and reperfusion for different times (IR1h, IR3h, IR5h), and groups of IR+human thioredoxin treatment (IR1h +hTrx, IR3h +hTrx and IR5h +hTrx). Transmission electron microscope (TEM), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immunocytochemistry techniques were used to observe apoptosis, apoptosis signal-regulating kinase 1 (ASK1) and expression of Bcl-2 and Bax in various phases of lung ischemia/reperfusion. RESULTS: Cell apoptosis in lung tissues was significantly high, ASK1, Bcl-2 and Bax protein were up-regulated in lung tissues of lung ischemia/reperfusion injury as compared to control (all P<0.01). Compared to IR group, hTrx suppressed apoptosis as well as expression of ASK1 and Bax protein (P<0.01), Bcl-2 protein and the ratio of Bcl-2/Bax were up-regulated in lung tissues (all P<0.05 or P<0.01). There was a significant correlation between the expression of ASK1, Bax protein and cell apoptosis (r=0.775, r=0.814, respectively; all P<0.01). There was a negative correlation between cell apoptosis and Bcl-2/Bax protein (r=-0.275, P<0.05). CONCLUSION: Initiating cell apoptosis by the activation of Bcl-2/Bax system in lung tissues may contribute to the pathogenesis of lung ischemia/reperfusion injury. The protective effects of hTrx include suppressing the expression of ASK1, down-regulating the ratio of Bcl-2/Bax and blocking apoptosis in lung tissues in lung ischemia/ reperfusion injury.     

Role of glycine receptor in protection of glycine against anoxia/reoxygenation injury in cardiomyocytes

AIM:To investigate whether glycine receptor is involved in the protection of glycine against anoxia/reoxygenation injury in cardiomyocytes by detecting oxygen free radical metabolism, apoptosis and intracellular calcium overload. METHODS:The neonatal rat cardiomyocytes were cultured and exposed to anoxia and reoxygenation (A/R) in the presence of glycine receptor antagonist, glycine or in free chloride buffer. The superoxide dismutase (SOD) activity, the contents of malondialdehyde (MDA) and nitric oxide (NO), the intracellular free calcium concentration and the apoptotic rate in the cardiomyocytes were determined. RESULTS:SOD activity and NO content in cardiomyocytes were lower, but MDA content, intracellular free calcium concentration and apoptotic rate in cardiomyocytes were higher in A/R group than those in control. Pretreatment with glycine inhibited the above changes caused by A/R, which was reversed by strychnine treatment and in the free chloride medium. CONCLUSIONS:Glycine inhibits free radical production, attenuates calcium overload, decreases apoptotic rate and increases SOD activity and NO release in cardiomyocytes exposed to A/R. These findings suggest that glycine exerts a protective effect against A/R injury via glycine receptor and glycine protects the neonatal rat cardiomycytes from A/R-induced injury in a chloride-dependent manner.     

Inhibition of calcineurin is involved in cardioprotection induced by ischemic postconditioning in rats

AIM: To demonstrate the mechanisms underlying cardioprotection induced by ischemic postconditioning (I-postC) via studying the alteration of calreticulin (CRT)/calcineurin (CaN) signaling pathway in rat heart subjected to ischemia/reperfusion (I/R).METHODS: The model of myocardial I/R injury in vivo was made by occluding the left anterior descending artery for 45 min followed by 24 h of reperfusion in Wistar rats.Hemodynamics and activity of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in plasma were measured.Myocardial infarct size was measured by 2,3,5- triphenyltetrazolium chloride (TTC) staining and cardiomyocyte apoptosis was detected using in situ TDT-mediated dUTP nick end labeling (TUNEL).The activity of CaN,the expressions of CaN and CRT in myocardium were detected by enzyme reaction phosphorus measurement and Western blotting analysis,respectively.RESULTS: Cyclosporin A,the inhibitor of CaN,limited significantly myocardial infarct size and cardiomyocyte apoptosis induced by I/R,but had no significant effect on cardiac function.I-postC ameliorated significantly the cardiac dysfunction induced by I/R.Compared with those in I/R group,the myocardial infarct size,the LDH and CK-MB activities in plasma and the cardiomyocyte apoptotic index were significantly reduced in I-postC group.In addition,I/R-induced upregulation of CaN activity,CaN and CRT expression were relieved by I-postC.No significant difference was found between I-postC and ischemic preconditioning groups.I-postC had stronger protective effect on the reperfused heart compared with cyclosporin A.CONCLUSION: The findings indicate that I-postC protects myocardium against I/R injury,at least in part,via inhibiting the CRT/CaN signaling pathway.     

Apoptosis in rat hepatocytes with ischemia/reperfusion injury

AIM: To investigate the distributive rules of apoptosis index (AI) in liver with ischemia/reperfusion (I/R) injury and evaluate the factors related to the hepatocyte apoptosis. METHODS: Sixty SD rats in specific pathogen free grade were randomly divided into three groups: control group (n=18), sham operation group (n=18) and I/R group (n=24). In I/R group, liver injury was induced by blocking blood inflow in rat liver for 20 min, then reperfusion for 22 h. Rats in the control group didnt receive any management. Rats in the sham operation group only subjected sham operation. All rat blood samples and livers were obtained for determination. Blood serum ALT, AST, TBIL, TNF-α, IFN-γ, IL-4, plasma endotoxin concentration, MDA level and SOD activity in liver were detected. Hepatic histological analysis was conduced through HE staining. Apoptosis was detected by TUNEL methods. RESULTS: Focal necrosis occurred in six rats livers in I/R group, in control group and sham group no necrosis cell was found in livers. The hepatic AI of I/R group was significantly increased compared with other groups. The AI in region under hepatic amicula was higher than that in central veins region and portal area. The necrotic regions contained apoptotic cells and AI was higher than that of other regions. Hepatic AI was significantly associated with ALT, AST, TNF-α, IFN-γ and SOD/MDA. CONCLUSION: In liver with I/R injury, the apoptotic cells in the region under hepatic amicula and the focus of necrosis are significantly higher than those in other regions, apoptotic cells and necrosis cells co-exist in the same zone. Hepatic AI may be significantly associated with ALT, AST, TNF-α and SOD/MDA.     

Effect of PKC activition on cardiac myocyte apoptosis and Bcl- 2 expression during myocardial ischemia/reperfusion in rats

AIM: To explore the effect of PKC activition on cardiac myocyte apoptosis and expression of bcl- 2 during myocardial ischemia/reperfusion(I/R) in rats. METHODS: TUNEL,immunohistochemistry and in situ hybridization were used. RESULTS: The TUNEL data showed that the numbers of positive cardiac myocyte nucleus and the percentage of positive cardiac myocyte nucleus in PMA+IR_{3 h} group decreased significantly(P＜0.05,P＜0.01), compared to those in IR_3h group. The number of Bcl-2 protein positive cardiomyocytes and the percentage of Bcl-2 protein positive cardiomyocytes in PMA+IR_3h group were higher than those in IR_3h group (P＜0.01) bcl- 2 mRNA expression showed the same changes in PMA+IR_0h group compared to IR_1h group.CONCL USIONS:Activation of PKC decreased cardiomyocyte death during I/R.Upregulation of bcl-2 gene expression in cardiomyocytes during I/R may be one of the mechanisms of decreasing cardiomyocyte death by PCK activating during I/R.     

Effects of curcumin on actinomycin D/TNF-α-induced injury in PC12 cells and rat hippocampal neurons

AIM: To study the possible mechanism of curcumin on actinomycin D (ActD)/tumor necrosis factor α (TNF-α)-induced injury in PC12 cells and rat hippocampal neurons. METHODS: PC12 cells were divided into control group, TNF-α group, ActD group, curcumin group, ActD/TNF-α group and curcumin+ActD/TNF-α group. The cells were cultured for 24 h. Inverted fluorescence microscopy was used to observe the morphological changes of the cells in each group. Annexin V/PI double staining was applied to analyze the apoptosis of PC12 cells. The level of intracellular Ca²⁺ was detected by Fluo-3 AM staining. Rat hippocampal slices were prepared and divided into the same groups as the PC12 cells. Extracellular microelectrode recording technique was used to observe and calculate the changes of long-term potentiation (LTP) in different groups. RESULTS: Apoptosis of PC12 cells was induced by ActD/TNF-α. Curcumin protected the PC12 cells from ActD/TNF-α-induced apoptosis (P<0.05). ActD/TNF-α increased the intracellular Ca²⁺ concentration. Curcumin significantly reduced ActD/TNF-α-induced apoptosis by decreasing the intracellular Ca²⁺ concentration (P<0.05), inversed the effect of ActD/TNF-α on LTP in hippocampal slices, and improved the synaptic plasticity (P<0.05). CONCLUSION: Curcumin protects ActD/TNF-α-induced neuronal damage by depressing the intracellular Ca²⁺ concentration and maintaining the homostasis of intracellular calcium.     

Panax quinquefolium saponins protect rat myocardium from ischemia/reperfusion injury through attenuating excessive endoplasmic reticulum stress

AIM: To investigate whether excessive endoplasmic reticulum stress (ERS) is involved in the protective mechanism of Panax quinquefolium saponins (PQS) against ischemia/reperfusion (I/R) injury in rat myocardium. METHODS: The model of myocardial I/R injury in vivo was made by ligating the left anterior descending artery for 45 min followed by 24 h of reperfusion in SD rats. The hemodynamics and serum content of cardiac troponin T (cTnT) were measured. The myocardial infarct size was measured by Evans blue and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Cardiomyocyte apoptosis was detected using in situ TDT-mediated dUTP nick end labeling (TUNEL). The protein levels of glucose-regulated protein 78 (GRP78), calreticulin (CRT), C/EBP homologous protein (CHOP), caspase-12, apoptosis-associated proteins Bax and Bcl-2 were determined by Western blotting. RESULTS: Compared with I/R group, the mean arterial pressure in PQR+IR group was decreased by 32.0%, and left ventricular±dp/dtmax was increased by 64.0% and 35.0%, respectively.The serum content of cTnT was decreased by 53.3%, the percentage of area of necrosis (AN)/area at risk (AAR) was reduced by 65.5% and the apoptosis rate was decreased by 54.9%.The myocardial pathological changes were improved. Bcl-2 protein expression was increased by 110.0% and that of Bax was decreased by 47.8%. CRT protein expression was decreased by 43.4 %, CHOP protein expression and the protein level of cleaved caspase-12 were decreased by 38.6% and 23.7% in PQS+I/R group. CONCLUSION: PQS alleviates I/R injury in myocardium by inhibition of excessive ERS.     

Effect of Shenmai injection on cardiomyocyte apoptosis after acute anoxia-reoxygenation

AIM: To study the effects of Shenmai injection on cardiomyocytes apoptosis after acute anoxia-reoxygenation (A/R) and the possible mechanism. METHODS: In this experiment, cultured cardiomyocytes isolated from neonatal rat were used. Model of myocardial anoxia-reoxygenation injury was produced by depriving oxygen for 5 min and then restoring oxygen for 15 min. The apoptotic cells was detected by flow cytometry to detect labbled Annexin V-FITC/PI. The intracellular calcium level was observed by laser scanning confocal microscopy markered Fluo-3/AM. RESULTS: In anoxia-reoxygenation group, the percentage of apoptotic cells and fluorescent intensity of intracellular calcium were both prominently higher than those in control group (P<0.01). The apoptotic rate in Shenmai injection group was notably less than that in A/R group and the intracellular calcium overload was also less obvious in Shenmai injection group than that in A/R group (P<0.01). CONCLUSION: Shenmai injection has notable effects on attenuating apoptotic rate after acute anoxia-reoxygenation in cardiomyocytes, which may be partly due to its alleviating intracellular calcium overload.     

Role of nitric oxide in ischemia/reperfusion injury and ischemic preconditioning

AIM: To clarify the role of nitric oxide(NO) in ischemic preconditioning(IP) and its effects on apoptosis. METHODS: Seventy-two male Wistar rats were divided into the following six groups:ischemia/reperfusion (IR) group,IP group,IR+L-arg group,IP+L-arg group,IR+L-NAME group and IP+L-NAME group,The following changes were measured:cardiac hemodynamic parameters,infarct size,PMNs counting myocardial MPO activity and TUNEL staining.RESULTS: ①L-arg significantly attenuated ischemia/reperfusion-induced heart injury,reduced PMNs infiltration and cardiomyocyte apoptosis.②L-NAME also significantly reduced infarct size,PMNs infiltration and cardiomyocyte apoptosis compared with IR group,however,L-NAME aggravated ischemia/reperfusions-induced cardiac functional injury.③L-arg or L-NAME did not significantly alter the protective effect of ischemic preconditioning. CONCLUSION: Increased production of endogenous NO before prolonged ischemic period can protect hearts and inhibit apoptosis.L-NAME can inhibit iNOS activity and ONOO^- production in reperfusion period to protect heart.