Role of injury and phenotype shift of liver sinusoidal endothelial cells in the development of portal hypertension of cirrhosis in rats

AIM: To study the role of injury and phenotype shift of liver sinusoidal endothelial cells in the development of portal hypertension of liver cirrhosis in rats. METHODS: The rat liver cirrhosis model was established by peritoneal injection of dimethylnitrosamine (DMN) (at a dose of 10 mg·kg^-1, 3 times a week, for 4 weeks). The dynamic changes of liver cirrhosis were observed at different time points (1 day, 2 days, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks). The pressure of portal vein (Ppv), the expression of CD44, von Willebrand factor (vWF), endothelin-1 (ET-1) mRNA and endothelial nitric oxide synthase (eNOS) mRNA, the serum hyaluronic acid (HA) content and liver ET-1 content were measured. RESULTS: Compared with the normal control rats, CD44 positive staining was weak in the 1 day model rats, and the numbers of fenestrae of sinusoidal endothelial cells (SECs) rapidly decreased, but serum HA content rapidly increased (P<0.05). vWF positive staining in the 2-day model rats was stronger than that in normal control rats (P<0.05). There was a positive correlation between the Ppv and the vWF expression, serum HA content in the DMN-induced liver cirrhosis rats (P<0.05). Compared with the normal control rats, ET-1 mRNA expression increased in the 2-day and 3-day model rats, and ET-1 content lightly increased. eNOS mRNA expression was stronger in the 1-day, 2-day and 3-day model rats than that in normal control rats, meanwhile eNOS always expressed at a low level. CONCLUSION: The injury and phenotype shift of SECs is a pathological basis in the development of portal hypertension of DMN-induced liver cirrhosis in rats. Imbalance of ET-1 and NO production increases intrahepatic resistance, which plays an important role in the development of portal hypertension.     

Expression and significance of angiopoietin-2 and Tie-2 receptors in a rat model of acute lung injury

AIM: To explore the expressions and significance of angiopoietin-2 (Ang-2) and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie-2) receptors in a rat model of acute lung injury (ALI). METHODS: Wistar rats (n=42) were divided into control group (n=12) and cecal ligation and puncture (CLP) group (n=30). Control group underwent sham operation, and CLP group underwent cecal ligation and puncture to make the model of ALI. 12 h after sham operation or CLP, 6 rats in each group were killed, and arterial blood gas analysis and lung coefficient were tested. The expressions of Ang-2 and Tie-2 receptors in lung tissue were observed by immunohistochemical method. Blood samples of the rest rats were collected from vena caudalis, and Ang-2 levels were measured by enzyme linked immunosorbent assay (ELISA). The mortality rate in each group within 36 h was compared. The lung architecture was observed under microscope. RESULTS: The lung architecture in control group was clear and intact. Alveolar septum was thicker, blood capillary was congested, and neutrophils and macrophages were infiltrated in the lung tissue in CLP group. Tie-2 receptors were expressed in bronchial epithelial cells, smooth muscle cells and endothelial cells in control group. Besides the similar expression as control group, high expression of Tie-2 on neutrophils and macrophages in CLP group was observed. In the adhesion location of Tie-2 receptors positive inflammatory cells, there was stronger staining in endothelial cells. Ang-2 was expressed in smooth muscle cells, bronchial epithelial cells and endothelial cells in control group. The Ang-2 level in CLP group were higher than that in control group ［(8.14±1.74) μg/L vs (4.63±0.49) μg/L, P<0.01］, and the Ang-2 level of dead rats was higher than that of survival rats within 36 h in CLP group ［(8.95±1.61)μg/L vs (6.80±0.96)μg/L, P<0.01］. Oxygen partial pressure in control group was lower (P<0.01) and lung coefficient was higher (P<0.01) than that in CLP group. CONCLUSION: Ang-2 and Tie-2 receptors may participate in the pathophysiology of ALI, and Ang-2 level is correlated with mortality.     

Effect of Qiliqiangxin granule on apoptosis of renal tissues in rats with cardiorenal syndrome

AIM: To investigate the effect of Qiliqiangxin granule on the apoptosis of renal tissues in rats with cardiorenal syndrome (CRS) and its possible mechanism. METHODS: A rat model of CRS was established by ligation of the left anterior descending coronary artery and acute renal ischemia/reperfusion injury. After operation, the rats were divided into 6 groups:2-week sham operation (2w sham) group, 2-week model (2w CRS) group, 2-week drug (2w CRS-Q) group, 4-week sham operation (4w sham) group, 4-week model (4w CRS) group and 4-week drug (4w CRS-Q) group. The rats in 2w CRS-Q group and 4w CRS-Q group were given Qiliqiangxin granule (4 g·kg^-1·d^-1) by gavage for 2 weeks and 4 weeks, respectively. The levels of serum cystatin C (Cys-C), plasma angiotensin Ⅱ (Ang Ⅱ), urine neutrophil gelatinase-associated lipocalin (NGAL) and urine microalbumin (UMA) were measured by ELISA. The serum level of creatinine (Cre) was detected by sarcosine oxidase method. The renal histopathological changes were observed by HE staining. The mRNA and protein expression levels of Ang Ⅱ, Bcl-2 and Bax were evaluated by RT-qPCR and Western blot, respectively. The apoptosis rate of renal cells was assessed by TUNEL staining. RESULTS: The levels of serum Cys-C, serum Cre, plasma Ang Ⅱ, urine NGAL and UMA were significantly increased in 2w CRS group and 4w CRS group compared with 2w sham group and 4w sham group after modeling (P<0.05). The mRNA and protein expression levels of Bax and Ang Ⅱ in the renal tissues of CRS rats were significantly up-regulated (P<0.05), while Bcl-2 was significantly down-regulated (P<0.05) compared with 2w sham group and 4w sham group. Compared with 2w sham group and 4w sham group, the damage of renal tissues in 2w CRS and 4w CRS group was severe, and the apoptotic rates of renal cells were significantly increased. Compared with 2w CRS group and 4w CRS group, Qiliqiangxin granule greatly decreased the levels of Cys-C, Cre, Ang Ⅱ, NGAL and UMA, down-regulated the mRNA and protein expression levels of Bax and Ang Ⅱ in the renal tissues, and up-regulated the expression of Bcl-2 at mRNA and protein levels at 2 and 4 weeks. In addition, Qiliqiangxin granule also greatly attenuated the damage and apoptosis of the renal tissues. CONCLUSION: Qiliqiangxin granule significantly inhibits the apoptosis of renal tissues and improves the renal function of CRS rats, and its mechanism may be related to the inhibition of Ang Ⅱ expression.     

Effect of low-dose paclitaxel on morphology of bladder in rats with infravesical obstruction

AIM:To investigate the effects of low-dose paclitaxel on the morphology of bladder after partial bladder outlet obstruction (BOO) in rats. METHODS:Healthy female Sprague-Dawley rats (n=30) were randomly divided into sham operation group, BOO group and low-dose paclitaxel group. The rats in BOO group and low-dose paclitaxel group received operation to establish an obstruction model, while the rats in sham group underwent sham operation. After operation, the rats in low-dose paclitaxel group received intraperitoneal injection of paclitaxel at a dose of 0.3 mg/kg twice a week for 4 weeks. At the same time, the animals in sham group and BOO group received the same volume of saline by intraperitoneal injection. Four weeks after operation, each rat was sedated and the bladder was weighted. Histological changes of the bladder were observed by HE staining. Collagen deposition in the bladder tissue was observed by Masson staining, and the fibrosis area was measured. The ultrastructure of the detrusor was studied by transmission electron microscopy. RESULTS:Compared with sham operation group, a significant increase in bladder weight (0.376 g±0.052 g vs0.112 g±0.014 g, P<0.05), the muscle hypertrophy, and a decrease in the percentage of collagen area ［collagen/(collagen+muscle), 29.66%±2.69% vs38.94%±3.67%, P<0.05］ was observed in BOO group. Under electron microscope, intracellular connection had more gap junction and desmosomes than intermediate junction. The cell gap widened with a large amount of collagen fiber. Compared with BOO group, low-dose paclitaxel group decreased bladder weight (0.215 g±0.025 g vs0.376 g±0.052 g, P<0.05) and improved the muscle hypertrophy. The percentage of the collagen area was also decreased (19.94%±1.90% vs29.66%±2.69%, P<0.05). The detrusor microstructure showed that the intermediate junction was characterized by a predominance among the intracellular connections, and the intercellular space contained less collagen fibers in low-dose paclitaxel group. CONCLUSION: Low-dose paclitaxel may ameliorate the morphological damage of the bladder and recover bladder function in the rats with BOO by slowing down the process of bladder fibrosis.     

Exogenous hydrogen sulfide attenuates obstructive kidney injury in mice

AIM:To investigate the protective effect of exogenous hydrogen sulfide (H₂S) on obstructive renal injury in mice, and to explore the possible potential mechanisms involved in this animal model. METHODS:Male C57BL/6 mice (8 weeks old) were randomly divided into sham group, operation group and H₂S group, with 5 rats in each group. The model of obstructive renal injury was induced by unilateral ureteral obstruction (UUO). The mice in H₂S group were intraperitoneally injected with NaHS daily, while the mice in sham group and operation group were administered with the same volume of saline intraperitoneally. After 7 d, the mice were executed and the renal tissues were taken out for experiments. RNA was extracted to detect the mRNA expression of H₂S catalytic enzymes in the mice of 3 groups. HE staining was performed to observe the structural changes of renal tissues in the mice. Renal fibrosis in the mice of 3 groups was evaluated by Masson staining. The content of cystatin C in the plasma was detected to reflect glomerular filtration ability. The protein expression of LC3, beclin-1 and fibronectin (FN) in the mice of 3 groups was determined by Western blot. RESULTS:Compared with sham group, the mRNA expression of cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) in operation group decreased significantly. The collagen fiber content in operation group was increased significantly, while collagen fiber content in H₂S group was decreased significantly as compared with operation group. Compared with sham group, the protein expression of FN in operation group was increased significantly, while the protein expression of FN in H₂S group was decreased significantly as compared with operation group. Compared with sham group, the protein expression of LC-Ⅱ and beclin-1 in operation group was increased significantly, while the protein expression of LC-Ⅱ and beclin-1 in H₂S group was increased significantly as compared with the operation group. CONCLUSION:Exogenous H₂S possibly mitigates renal fibrosis in UUO mice by up-regulating autophagy.     

Astragaloside IV promotes angiogenesis in rats with myocardial infarction via PKD1-HDAC5-VEGF pathway

AIM: To investigate the angiogenic effect and mechanisms of astragaloside IV (AS-IV) in rats with myocardial infarction via protein kinase D1 (PKD1)-histone deacetylase 5 (HDAC5)-vascular endothelial growth factor (VEGF) signaling pathway. METHODS: The classic model of myocardial infarction by ligation of the left anterior descending coronary artery was replicated, and the rats were randomly divided into model group, AS-IV group, and AS-IV+CID755673 (PKD1 inhibitor) group. The sham operation control group and DMSO control group were also set up. All the rats were given intravenous injection via caudal vein. The rats were sacrificed 4 weeks later, and segmental heart samples were used for HE staining and Masson staining. The expression of PKD1, HDAC5 and VEGF was analyzed by immunohistochemistry, RT-PCR and and Western blot. RESULTS: Compared with sham operation group and DMSO group, the myocardium in model group showed disordered arrangement, accompanied with necrotic myocardial cells and obvious fibrosis tissue. After treatment with AS-IV, the morphological changes of myocardium were obviously improved, and the number of new blood vessels increased significantly. However, after treatment with AS-IV+CID755673, the myocardial tissues of the rats became disordered again, with increased necrotic cells and some closed vessels. The mRNA and protein expression of PKD1, HDAC5 and VEGF in myocardial tissue in model group was significantly lower than that in sham operation and DMSO groups (P<0.05). The expression in AS-IV group was significantly higher than that in model group (P<0.01), while that in AS-IV+ CID755673 group was significantly lower than that in AS-IV group (P<0.05). CONCLUSION: AS-IV promotes the angiogenesis of myocardial tissues in the rats after myocardial infarction partly by regulating the PKD1-HDAC5-VEGF signaling pathway.     

Effects of monocrotaline-induced pulmonary hypertension on expression of Hippo signaling pathway-related molecules in rat lung

AIM:To investigate the expression of Hippo signaling pathway-related molecules in the lung tissues of the rats with pulmonary hypertension induced by monocrotaline for exploring the significance of Hippo signaling pathway in the development of pulmonary hypertension. METHODS:SD rats (n=45) were randomly divided into control group (n=15) and model group (n=30). The rats in model group was given neck subcutaneous injection of monocrota-line at 60 mg/kg to establish pulmonary hypertension model, and the rats in control group was injected with the same volume of normal saline. Four weeks later, right ventricular systolic pressure (RVSP) was measured by right cardiac catheterization, and right ventricular hypertrophy index (RVHI) and right ventricular mass index (RVMI) were calculated. The remodeling of the pulmonary arterioles was observed by HE staining, and medial thickness/external diameter (M/E%) was evaluated. The fibrosis of lung tissues was detected by Masson staining. The protein expression of Yes-associated protein (YAP), tafazzin (TAZ) and TEAD was detected by immunohistochemistry, and the protein and mRNA levels of YAP, TAZ and TEAD in lung tissues were determined by Western blot and RT-qPCR. RESULTS:Compared with control group, the vascular wall in model group was thickened significantly, the M/E% was increased (P<0.01), the pulmonary fibrosis was obvious, and the RVSP and RVHI in model group were significantly higher than those in control group (P<0.01). The immunohistochemical staining showed that the protein expression of YAP, TAZ and TEAD in the pulmonary arterioles in model group was significantly higher than that in control group. The YAP, TAZ and TEAD protein and mRNA levels in the lung tissues were also higher than those in control group (P<0.05). CONCLUSION:The activation of Hippo signaling molecules may promote the remodeling of pulmonary arterioles and further regulate the development of monocrotaline-induced pulmonary hypertension.     

Effects of Tongxinluo on connexin 43 remodeling and ventricular arrhythmia after myocardial infarction in rats

AIM: To determine the effects of Tongxinluo(TXL) on connexin 43(Cx43) remodeling and ventricular arrhythmia(VA) after myocardial infarction(MI) in rats. METHODS: Male SD rats were randomly divided into sham-operated(sham) group(n=25) and operation group(n=75). The left anterior descending(LAD) was ligated in operated group, while the rats in sham group only underwent pericardiotomy. The rats in operation group which survived for 3 d after operation were randomly assigned to TXL group and MI group. The rats in TXL group was administrated with TXL(2 g·kg^-1·d^-1, intragastric administration) for 4 weeks, while normal saline was applied to the rats in sham group and MI group. The levels of interleukin-1β(IL-1β) and endothelin-1(ET-1) in the tissue from the border zone were measured by ELISA after treatment. The distribution and the mRNA and protein expression of Cx43 were detected by immunohistochemical staining, RT-PCR and Western blotting, respectively. The burst pacing was used to induce ventricular arrhythmia(VA). RESULTS: Compared with sham group, the levels of IL-1β and ET-1 and the incidence of VA were significantly increased, while the mRNA and protein expression of Cx43 was markedly reduced with irregular distribution in MI group(P<0.05). Compared with MI group, the levels of IL-1β and ET-1 and the incidence of VA were significantly reduced, while the expression of Cx43 at mRNA and protein levels was markedly increased with augmented linear distribution in the myocardial cell intercalated disc in TXL group(P<0.05). CONCLUSION: TXL reduces the incidence of VA after MI via inhibiting the Cx43 remodeling.     

Changes of liver sinusoidal endothelial cells and basement membrane in early stage of liver fibrosis induced by carbon tetrachloride in rats

AIM：To explore the development of hepatic sinusoidal capillarization in the early stage of liver fibrosis induced by carbon tetrachloride (CCl4) in rats. METHODS：Clean SD rats were randomly divided into normal control group (group N, n=6) and liver fibrotic model group (group M, n=32). The rats in group N were intraperitoneal injected with saline and the rats in group M were intraperitoneal injected with CCl4 (2 mL/kg, twice a week for 4 weeks). At the end of the 3rd day and the 1st, 2nd and 4th weeks, all rats were killed and then the samples were collected. The pathological changes in the livers were observed by HE staining and Masson straining. The development of hepatic sinusoidal capillarization was observed by transmission electron microscopy (TEM) and immunohistochemical staining. The cell surface expression of vascular endothelium-associated marker CD31, collagen type Ⅳ (Col IV) and laminin (LN) was determined. RESULTS：HE and Masson staining showed the formation of liver fibrosis after treatment with CCl4 for 4 weeks. TEM showed that the fenestrate diameter of liver sinusoidal endothelial cells (LSECs) grew down, the fenestrate numbers of LSECs were decreased along with the development of liver fibrosis, and the consecutive basement membrane was formed at the end of the experiment. The expression of CD31 was significantly increased along with the development of defenestration, and the expression of Col IV and LN was significantly increased after the treatment with CCl4 for 2 weeks and 4 weeks, respectively. CONCLUSION：The typical hepatic sinusoidal capillarization was detected in the early stage of liver fibrosis, and the deposition of LN in the liver sinusoidal walls was the mainly factor of formation of the consecutive basement membrane.     

Effects of puerarin on neurological function,and synaptic morphological structure and parameter of cerebral cortex in cerebral ischemia-reperfusion rats

AIM: To investigate the protective effect of puerarin (Pue) on the neural function in a rat model of cerebral ischemia-reperfusion (IR) injury by observing synaptic morphological structural alterations and the parameter variations in brain cortex. METHODS: Male SD rats (n=69) were randomly divided into sham operation group (sham group, n=15), model group (IR group, n=18), Pue group (n=18) and nimodipine (NIM) positive control group (NIM group, n=18). The model of cerebral IR injury was established by a suture method. After ischemia for 2 h and reperfusion for 24 h, the rats in Pue group and NIM group were injected with Pue at 8 mg·kg^-1·d^-1 and NIM at 1 mg·kg^-1·d^-1, respectively, while the rats in sham group and IR group were injected with the same volume of saline. The modified neurological severity scores were evaluated at 3, 7 and 14 d, the ultrastructural changes of the synapses at the ischemic side of cerebral cortex were observed under transmission electron microscope, and the postsynaptic density was analyzed by Quantiment-520 image analysis software. RESULTS: Compared with IR group, the scores of neurological deficit were both reduced in Pue group and NIM group (P<0.05), and the difference between Pue group and NIM group was not obvious. The presynaptic and postsynaptic membranes and synaptic cleft were indistinct under electron microscope in IR group, the number of synaptic vesicles in presynaptic component was reduced, and the density of postsynaptic dense zone was decreased. The synapses were dense in Pue group and NIM group compared with IR group, the dented synapses were increased, 2 active synapses were often presented, postsynaptic density was incrassated and the synaptic cleft was clear. The curvature of synaptic interface was increased, the postsynaptic density was thickened, and the synaptic cleft was narrow in ischemic penumbra (P<0.05). The parameters of synaptic morphological structure at 7 d and 14 d were better than those at 3 d (P<0.05). CONCLUSION: The treatment with Pue promotes neural functional recovery against cerebral IR injury in the rats, which may be related to restore synaptic structure.     

Abnormal expression of angiopoietins in glomerulus following podocyte injury and its role in the development of progressive glomerulosclerosis in rats

AIM: To study the potential pathological role of abnormal expression of endogenous angiopoietins in progressive glomerulosclerosis. METHODS: 80 male Wistar rats were randomly allocated into sham operation group (sham, n=25), unilateral nephrectomy group (UNx, n=25) and UNx＋daunorubicin (DRB)group (n=30). The rats in DRB group were intravenously injected with DRB (5 mg/kg) on the seventh and the fourteenth day respectively after excising one kidney. Then, at week 1, 2, 4, 6 and 8, 5, male Wistar rats from each group were taken randomly for determining 24 h urinary protein quantitative measurement (24hUPQ), BUN, Scr, and the kidneys were examined by electronic microscope, PAS staining, immunohistochemical staining and in situ hybridization histochemistry. RESULTS: There was a trend towards an increase respectively in levels of 24hUPQ, Bun, Scr, GSI in DRB from week 2 to week 8. Electronic microscope revealed that podocyte injury presented in DRB group. Expression of Ang1 mRNA and protein in glomerulus in DRB group decreased, while expression of Ang2 protein in glomeruli in DRB group increased. In DRB group, expression of Ang1 protein had a negative correlation with 24hUPQ, BUN, Scr, GSI, expression of Ang2 protein and CoIV protein. Expression of Ang2 protein had a positive correlation with 24hUPQ, BUN, Scr, GSI,expression of CoIV protein. CONCLUSION: Podocyte injury may lead to glomeruli abnormally express angiopoietins. A decrease in expression of Ang1, and upregulation in expression of Ang2 may facilitate progressive glomerulosclerosis in the rat.     

Effects of catestatin on calcium handling abnormalities and ventricular arrhythmia after myocardial infarction in rats

AIM: To determine the effects of catestatin (CST) on calcium handling abnormalities and ventri-cular arrhythmia (VA) after myocardial infarction (MI) in rats. METHODS: The adult male SD rats (n=85) were randomly divided into sham group (n=20) and operation group (n=65). MI was induced by ligation of the left anterior descending coronary artery in operation group. The rats in sham group underwent pericardiotomy but without ligating the artery. The rats survived for 1 week after operation were randomly assigned to MI group and CST group. The rats in CST group was treated with CST (30 mg·kg^-1·d^-1, intraperitoneal administration) for 4 weeks, while saline was applied to the rats in sham group and MI group. The calcium imaging study was performed by loading isolated ventricular cardiomyocytes with Fura-2 AM. In the whole Langendorff-perfused hearts, the programmed electrical stimulation was used to induce action potential duration (APD) alternans and VA. The protein levels of ryanodine receptor 2 (RyR2), phosphorylated RyR2 (p-RyR2), calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylated CaMKII (p-CAMKII) were determined by Western blot. RESULTS: Compared with sham group, the protein levels of p-RyR2 and p-CaMKII, the diastolic intracellular Ca²⁺ concentrations and the inducibility of VA were significantly increased, whereas the thresholds of Ca²⁺ transient (CaT) and APD alternans and the CaT amplitude were markedly decreased in MI group (P<0.01). Compared with MI group, the protein levels of p-RyR2 and p-CaMKII, the diastolic intracellular Ca²⁺ concentration and the inducibility of VA were significantly decreased, while the thresholds of CaT and APD alternans and the CaT amplitude were markedly increased in CST group (P<0.01). No significant difference of the protein expression of RyR2 and CaMKII among the 3 groups was observed (P>0.05). CONCLUSION: CST reduces the susceptibility to VA after MI via preventing calcium handling abnormalities.     

Effects of capsaicin on cognitive impairment and mitochondria-associated endoplasmic reticulum membranes in rats with chronic cerebral hypoperfusion

AIM:To observe the effects of capsaicin on cognitive impairment and mitochondria-associated endoplasmic reticulum membranes (MAMs) of hippocampal CA1 area in the rats with chronic cerebral hypoperfusion (CCH), and to investigate the underlying molecule mechanism of cognitive defects induced by ischemia. METHODS:Healthy male Sprague-Dawley (SD) rats(n=48) were randomly divided into sham operation (sham) group,CCH model (CCH) group, capsaicin group,and solvent group, 12 rats in each group. Capsaicin at 2.5 mg/kg was intraperitoneally injected twice a week for 4 weeks, starting on the 7th day after surgery. The rats in solvent group were given the same amount of solvent at the same time and under the same conditions. Morris water maze, object recognition test and open field test were conducted to analyze the cognitive related behavior performance on the 4th week after surgery. The changes of MAMs in the hippocampal CA1 region were observed under transmission electron microscope, the co-localization of the MAMs was observed by immunofluorescence double-labeling, and the expression of mitofusin 2 (Mfn2) in the hippocampal tissue was determined by Western blot.RESULTS:Four weeks after the operation, the behavior tests showed that the cognitive function of CCH rats was impaired compared with sham operation group. Compared with solvent group, spatial learning and memory in capsaicin group was improved significantly. The results of transmission electron microscope and confocal microscope showed that the distance of MAMs in the hippocampal CA1 area of CCH rats was increased compared with sham operation group, and the co-localization of the contacts was decreased (P<0.05). Compared with solvent group, the correlation between the mitochondria and ER in capsaicin group was increased (P<0.05). The protein level of Mfn2 in CCH group was significantly lower than that in sham group (P<0.05). Compared with solvent group, the protein level of Mfn2 in capsaicin group was higher (P<0.05). CONCLUSION:CCH rats showed decreased cognitive function and loosen MAMs. Capsaicin improves the cognitive behavior of CCH rats by up-regulation of MAMs.     

Hyperhomocysteinemia induces endothelial dysfunction and aggravates microcirculation dysfunction and microthrombosis

AIM: To establish a microthrombus model by carrageenan (Ca)/ lipopolysaccharides (LPS) intraperitoneal injection in rats with hyperhomocysteinemia (HHcy) and endothelial dysfunction induced by L-methionine intake.METHODS: ① Male Sprague Dawley rats were randomly divided into 2 groups: control and endothelial dysfunction (HHcy) groups. L-methionine was administered by gavage in HHcy group for total 4 weeks.Purified water was administered by gavage in control rats.Plasma Hcy,NO and vWF were examined and the thoracic aorta were excised after 4 weeks of L-methionine treatment to evaluate endothelial function.② Male Sprague Dawley rats were randomly divided into 3 groups to establish a microthrombus formation model with Ca/ LPS: control,microthrombus formation (Ca/LPS) and endothelial dysfunction plus mitoarothrombus formation (HHcy＋Ca/LPS) groups.Control rats were injected with normal saline (NS).Ca/LPS rats were intraperitoneally injected with carrageenan (Ca) and followed by lipopolysaccharides (LPS) 16 h later.HHcy＋Ca/LPS rats were intragastric gavaged by L-methionine for total 4 weeks,and then were injected with Ca/LPS in the same way as Ca/LPS group.Cruor parameters and platelet count were detected at 20 h after LPS or NS injection and the mesentery microcirculation was monitored.Plasma NO and vWF were also detected at 24 h after LPS or NS injection.RESULTS: ① Plasma Hcy concentrations and vWF level were significantly increased in HHcy group,while plasma NO content was significantly decreased compared with that in control group.Endothelial dependent relaxation (EDR) of aortic rings was significantly decreased in HHcy group,suggesting endothelial damage/dysfunction was induced by HHcy.② Mesentery capillary was obviously blocked by microthrombus in Ca/LPS rats and was blocked more seriously in HHcy＋Ca/LPS rats.Cruor parameter results suggested that Ca/LPS rats were in hypercoagulable phase and HHcy＋Ca/LPS rats were in hypocoagulable phase at 20 h after LPS injection.Platelet count and plasma NO content in HHcy＋Ca/LPS group were significantly decreased,while plasma vWF level was significantly increased compared with Ca/LPS group.CONCLUSION: L-methionine intake induces severe HHcy and causes endothelial dysfunction in rats.Microcirculation dysfunction and microthrombosis can be caused by Ca/LPS intraperitoneal injection and may be aggravated by endothelial dysfunction.     

Astragalus polysaccharides improve chronic heart failure by promoting myocardial FFA metabolism via AMPK pathway

AIM: To investigate the effect of Astragalus polysaccharides (APS) on chronic heart failure and its mechanism. METHODS: Male SD rats (n=32) were randomly divided into control group, sham group, model group and APS group (8 rats in each group). The left coronary artery ligation in the rats was conducted to establish myocardial infarction heart failure model. After modeling, the rats in APS group were given APS (3 g·kg^-1·d^-1) by intragastric administration for 6 weeks. Left ventricular diastolic diameter (LVD), left ventricular systolic diameter (LVS), left ventricular ejection fraction (LVEF) and fractional shortening (FS) were detected by echocardiography. HE staining was used to observe the pathological changes. The concentrations of free fatty acid (FFA) in the serum and myocardium were observed by the method of acetyl coenzyme A synthetase and acetyl coenzyme A oxidase (ACS-ACOD). The protein levels of total AMP-activated protein kinase (AMPK), phosphorylated AMP-activated protein kinase (p-AMPK), fatty acid translocase (FAT/CD36) and carnitine palmitoyltransferase I (CPT-1) were measured by Western blotting. RESULTS: No significant difference in each index between sham group and control group was observed. Compared with control group, LVEF and FS in model group was significantly decreased, while LVD and LVS was significantly increased (P<0.05). The LVEF and FS in APS group were significantly improved compared with model group (P<0.05), and there was no significant difference between APS group and control group. LVD and LVS in APS group were obviously improved compared with mo-del group (P<0.05), and the difference was significant compared with control group (P<0.05). Compared with control group, focal myocardial necrosis increased, and residual myocardial cells reduced in model group, while those was much better in APS group as compared with model group (P<0.05). The FFA concentrations in the serum and myocardium in model group increased significantly compared with control group (P<0.05), while those decreased significantly in APS group as compared with model group (P<0.05). The protein levels of p-AMPK, CPT-1, and cell membrane FAT/CD36 in model group decreased significantly compared with control group (P<0.05), and those in APS group increased obviously compared with control group (P<0.05). CONCLUSION: APS improves chronic heart failure by activating the AMPK pathway and promoting myocardial ingestion and utiliation of FFA.     

Effect of Shenshuguanxin granula on coronary circulation in rats with myocardial infarction

AIM：To explore the effect of traditional Chinese medicine Shenshuguanxin granula on coronary circulation in a rat model of myocardial infarction (MI). METHODS：SD rats (n=50, SPF grade) were randomly divided into 5 groups (n=10):sham group, MI group, and high-dose, middle-dose and low-dose Shenshuguanxin granula treatment groups. The rat MI model was established by ligation of the coronary artery. The cardiac markers, small and medium-sized blood vessels ［microvessel count (MVC) value］ in the infarct zone, and platelet endothelial cell adhesion mo-lecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) expression in the infarct border zone were measured. RESULTS：After 4 weeks of coronary artery ligation, the significant increases in MVC in the infarct zone, and the expression of PECAM-1 and VEGF in the infarct border zone were detected compared with sham group (P<0.05). The differences of cardiac markers between MI group and other groups were insignificant (P>0.05). CONCLUSION:Shenshuguanxin granula improves coronary circulation in the rats with myocardial infarction by increasing the expression of PECAM-1 and VEGF, and promoting small and medium-sized angiogenesis.     

Ifenprodil reduces brain edema and cortical apoptosis after subarachnoid hemorrhage in rats

AIM:To explore the effect of ifenprodil, a negative allosteric modulator of GluN2B N-methyl-Daspartate (NMDA) receptor, on neurological deficit, permeability of blood-brain barrier, brain edema and cortical cell apoptosis after subarachnoid hemorrhage (SAH) in rats. METHODS:Adult healthy male Sprague-Dawley rats (n=90) were divided into 3 groups:sham group (n=18), SAH+vehicle group (n=36), and SAH+ifenprodil group (n=36). The rat model of SAH was established by intracranial endovascular puncture. The rats received vehicle or ifenprodil at 2 h, 24 h and 48 h after surgery by intraperitoneal injection. The modified Garcia neurological scoring, dry and wet weight method, Evans blue dye extravasation method, TUNEL staining and Western blot were used to evaluate neurological deficit, brain water content, blood-brain barrier permeability and cortical cell apoptosis at 72 h after SAH. RESULTS:The neurological score and Bcl-2 expression were decreased, while the brain water content, the Evans blue dye extravasation, TUNEL-staining positive cells, and Bax and activated caspase-3/9 protein levels in the basal cortex were increased significantly in SAH+vehicle group as compared with sham group (P<0.05). However, ifenprodil treatment significantly inhibited these SAH-induced changes (P<0.05). CONCLUSION:Ifenprodil attenuates neurological deficits and brain edema, and reduces blood-brain barrier permeability and cortical apoptosis after SAH.     

Influence of ginsenoside Re on vascular intima hyperplasia and NF-κB p65 signaling pathway in balloon-injured rats

AIM: To investigate the inhibitory effect of ginsenoside Re on intimal hyperplasia induced by balloon-injury and to explore the role of NF-κB p65 signaling pathway in the process. METHODS: SD rats(n=40) were divided into 5 groups randomly: sham operation group, model group, low-dose ginsenoside Re group, middle-dose ginsenoside Re group and high-dose ginsenoside Re group. The carotid artery intima injury model was established by 2F balloon catheters in all groups except the sham operation group. The day after modeling, the animals in model group and sham operation group were administered intragastrically with distilled water, and the rats in low-dose, middle-dose and high-dose ginsenoside Re groups were given ginsenoside Re at doses of 12.5 mg/kg, 25mg/kg and 50 mg/kg, respectively. After 14 continuous days, the morphological changes of the injured arteries were observed by HE staining and the lumen area, intima area and media area as well as the ratio of intimal area/media area were determined. The expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) were detected by real-time PCR. The proliferating cell nuclear antigen(PCNA) and nuclear factor-kappa B(NF-κB) p65 were examined by immunohistochemistry.RESULTS: Compared with sham operation group, the vessel cavity was narrowed(P<0.01), the mRNA levels of TNF-α and IL-1β, and the protein expression of PCNA and NF-κB p65 were increased in model group(P<0.05). Compared with model group, the vascular intimal hyperplasia was alleviated obviously(P<0.05), and the mRNA levels of TNF-α and IL-1β, and protein expression of PCNA and NF-κB p65 were decreased in medium and high-dose ginsenoside Re groups(P<0.05). CONCLUSION: Ginsenoside Re inhibits the vascular neointimal hyperplasia induced by balloon-injury in rats, and the molecular mechanism may be related to the inhibition of NF-κB p65 signaling pathway.     

Salvia extract promotes angiogenesis of myocardium in rats with myocardial infarction

AIM: To determine the effect of salvia extract on angiogenesis of the myocardium in the rats with myocardial infarction (MI) and to analyze its possible mechanism. METHODS: Left coronary artery of Sprague-Dawley rats was ligated to establish a MI model. The rats were randomly divided into MI model group, 3 different dose groups of salvia (10, 20 and 40 mg·kg^-1·d^-1), and sham operation group. Each group consisted of 8 rats. The rats in all treatment groups were orally administered with the salvia extract, and the rats in MI group and sham operation group were fed with the same volume of saline. The rats were sacrificed 4 weeks later. The hemodynamic changes of the rats were determined, and the segmental heart samples were used for morphological observation by hematoxylin and eosin staining, Masson staining, or electron microscopic analysis. The expression of vascular endothelial growth factor (VEGF) and cluster of differentiation 34 (CD34) was analyzed according to immunohistochemistry. RESULTS: Compared with sham operation group, the morphological changes of the myocardium in MI group were disordered, part of myocardial cell outline disappeared, and obvious fibrosis in the necrosis myocardial tissue and fuzzy or disappearing microvascular ultrastructure were also observed. Compared with MI group, the number of new microvessels in all the treatment groups increased obviously, and the morphological changes of the endothelial cells were relatively complete according to electron microscopy. Compared with sham operation group, the protein expression of VEGF and CD34 in the cytoplasm of the myocardial tissues in MI group increased only a little. Compared with MI group, the protein expression of VEGF and CD34 in the cytoplasm of the myocardial tissues in all treatment groups increased significantly (P<0.01). CONCLUSION: Salvia extract obviously promotes angiogenesis of the myocardial tissues in the rats after myocardial infarction.