Effects of betaine on expression of GFAP,glycine and glycine receptor in hippocampus of pentylenetetrazole-induced epileptic rats

AIM：To study the effects of betaine on glial fibrillary acidic protein (GFAP), glycine (Gly) and glycine receptor (GlyR) expression in the hippocampus of rats with epilepsy induced by pentylenetetrazole (PTZ). METHODS：Forty-eight healthy male Wistar rats were randomly divided into control group, PTZ (35 mg·kg-1·d-1, intraperitoneal injection) group, PTZ+betaine (450 mg·kg-1·d-1, intragastric administration) group, PTZ+betaine (225 mg·kg-1·d-1, intragastric administration) group, PTZ+betaine (112.5 mg·kg-1·d-1, intragastric administration) group and PTZ+sodium valproate (200 mg·kg-1·d-1, intragastric administration) group. The rats in control group were intraperitoneally injected with saline at the same volume as PTZ injection, and those in control group and PTZ group received intragastric administration of saline at 1.0 mL·d-1. Rat behavior was recorded. Serum homocysteine (Hcy) level was measured. The expression of GFAP in the hippocampus was measured by immunofluorescence. Hippocampal Gly content was measured by an amino acid analysis system. The expression of GlyR was detected by immunofluorescence and Western blotting. RESULTS：There was no difference in the latency of grand mal seizures among groups (P>0.05). However, betaine treatment significantly decreased the duration of the first grand mal seizure compared with PTZ group (P<0.01). Serum Hcy level in PTZ group was significantly lowered compared with control group (P<0.01), and further decreased after betaine treatment (P<0.05). GFAP in PTZ group was significantly higher than that in control group (P<0.01), and decreased after betaine treatment (P<0.05). Gly in PTZ group was significantly lowered compared with control group (P<0.01), and increased after betaine treatment (P<0.05). The content of GlyR among groups showed the same trend as Gly. CONCLUSION：Betaine treatment shows antiepileptic effect, which may be related to its effects on the metabolites of Hcy and Gly.     

Role of benazepril on extracellular signal-regulated kinase activity and expression of B-type natriuretic peptide in spontaneously hypertensive rats

AIM：To investigate the role of benazepril on extracellular signal-regulated kinase (ERK) activity and expression of B-type natriuretic peptide in spontaneously hypertension rat (SHR).METHODS：Wistar Kyoto rats were used as control group.Twenty one 14-week-age SHR were randomized into 3 groups,7 rats each：benazepril group (10 mg·kg-1·d-1);hydralazine group (10 mg·kg-1·d-1) and sham group.In each group drugs or equal volume of vehicle (0.5% carboxymethyl cellulose) were administered respectively for 10 weeks by gavage.The ratio of left ventricle weight to body weight （LVW/BW） was measured to reflect myocardial hypertrophy.The caudal arterial pressure was measured by tail-cuff.Protein expression of p-ERK in myocardial tissue was detected by Western blotting,BNP mRNA in myocardial tissue was examined by RT-PCR,and protein expression of plasma BNP was detected by ELISA.RESULTS：1.Benazepril and hydralazine lowered the blood pressure after 10 weeks treatment (P<0.01).2.The ratio of LVW/BW in SHR benazepril group was significantly lower than that in SHR hydralazine group and SHR sham group (P<0.01),and similar to that in WKY group (P>0.05).3.The protein expression of p-ERK in myocardial tissue in SHR benazepril group was significantly lower than that in SHR hydralazine group and SHR sham group (P<0.01),and similar to that in WKY group (P>0.05).There was no significant difference of p-ERK expression between SHR hydralazine group and SHR sham group (P>0.05).4.The levels of plasma BNP and BNP mRNA in myocardial tissue in SHR benazepril group were significantly lower than that in SHR hydralazine group and SHR sham group (P<0.01),and similar to that in WKY group (P>0.05).There was no significant difference of plasma BNP and BNP mRNA in myocardial tissue between SHR hydralazine group and SHR sham group (P>0.05).CONCLUSIONS：Benazepril inhibited ERK activation,resulting in regression of myocardial hypertrophy and accompanied by the reduction of BNP level.However,in spite of the effect of lowering blood pressure,hydralazine did not prevent or regress cardiac hypertrophy and did not decrease the level of p-ERK and BNP in SHR.BNP level might serve as a therapeutic index for reversal of myocardial hypertrophy.     

Effects of atorvastatin on iopromide-induced apoptosis of renal tubular epithelial cells in diabetic rats

AIM：To investigate the protective effect of atorvastatin (ATO) against contrast medium (CM)-induced apoptosis of renal tubular epithelial cells in diabetic rats. METHODS：Streptozocin-induced diabetic Wistar rats were fed for 8 weeks and then randomly divided into 5 groups: diabetes mellitus (DM) group, DM with iopromide (a kind of CM) treatment group (DM+CM group), and groups of DM rats treated with ATO at 5 mg·kg-1·d-1 (ATO1 group), 10 mg·kg-1·d-1 (ATO2 group) and 30 mg·kg-1·d-1 (ATO3 group) before iopromide injection. Healthy Wistar rats served as normal controls (N group). Urine creatinine (UCr) and 24-hour urinary albumin (24 h-UAlb) were determined 24 h after iopromide injection. Serum creatinine (SCr) and blood urea nitrogen (BUN) were detected 48 h after iopromide injection, and then creatinine clearance (CCr) and 24-hour urinary albumin excretion rate (24 h-UAER) were calculated. The rats were sacrificed and both kidneys were removed 48 h after iopromide injection. For the left kidney, the morphology by HE staining, the renal tubular apoptosis by TUNEL and the expression of Bax and Bcl-2 by immunohistochemistry were detected. For the right kidney, the expression of Bax and Bcl-2 was measured by Western blotting. RESULTS：Compared with N and DM groups, the levels of SCr, BUN and 24 h-UAER, as well as the expression of Bax in the renal medulla were higher, the levels of Ccr and Bcl-2 expression in the renal medulla were lower and TUNEL-positive cells were more in DM+CM group. Compared with DM+CM group, ATO attenuated these changes, especially in ATO3 group. CONCLUSION: Iopromide could cause renal tubular apoptosis. Early application of ATO could dose-dependently attenuate the development of contrast-induced acute kidney injury, partly due to suppression of iopromide-induced renal tubular apoptosis.     

Panax quinquefolium saponin attenuates ventricular remodeling after acute myocardial infarction in rats by inhibiting endoplasmic reticulum stress-related apoptosis

AIM: To investigate the effect of Panax quinquefolium saponin (PQS) on ventricular remodeling after acute myocardial infarction (AMI) in rats and its mechanism. METHODS: Ninety healthy male SD rats were randomly divided into sham group, AMI group, taurine 300 mg·kg-1·d-1 group, PQS 50 mg·kg-1·d-1 group, PQS 100 mg·kg-1·d-1 group and PQS 200 mg·kg-1·d-1 group. AMI models were produced by ligating the left coronary arteries in SD rats. The rats in each treatment group were gavaged with drugs dissolved in water (10 mL·kg-1·d-1), and the rats in sham group and AMI group received equal volume of water. Four weeks after MI, the left ventricle fractional shortening, ejection fraction and structure were evaluated by echocardiography. Myocardial infarct size was measured by 2,3,5-triphenyltetrazolium chloride staining. The hydroxyproline level was measured by colorimetric method. Apoptosis of the cardiomyocytes was detected by TUNEL. In addition, the expression of endoplasmic reticulum stress-related molecules in the noninfarcted myocardium was determined by Western blotting. RESULTS: Compared with AMI group, the left ventricular end-systolic dimension in PQS 50 mg·kg-1·d-1 group, PQS 100 mg·kg-1·d-1 group and PQS 200 mg·kg-1·d-1 group decreased by 17.2%, 20.3% and 38.8% respectively,and the left ventricular end-diastolic dimension decreased by 8.91%, 8.95% and 17.20%, respectively.The left ventricular end-systolic volume decreased by 31.4%, 38.5% and 67.0%, respectively, and the left ventricular end-diastolic volume decreased by 18.2%, 18.8% and 34.2%, respectively.The left ventricular ejection fraction increased by 44.9%, 60.1% and 118.0%, respectively,and the fractional shortening increased by 55.4%, 71.0% and 148.0%, respectively.The infarction size decreased by 4.6%, 39.5% and 55.8%, respectively,and the hydroxyproline level in noninfarcted myocardium decreased by 34.5%, 35.9% and 48.7%, respectively. Compared with AMI group, the myocardial apoptotic index in PQS 200 mg·kg-1·d-1 group decreased by 27.3%, the protein expression of Bcl-2 increased by 114.0%, and that of Bax, GRP78, CRT and CHOP decreased by 53.1%, 79.9%, 80.8% and 42.5%, respectively. The above mentioned protective effects in PQS 200 mg·kg-1·d-1 group and taurine group were similar. The Spearman correlation analysis revealed that CHOP expression had significant positive correlation with apoptotic index (r=0.797, P<0.01). CONCLUSION: PQS attenuates ventricular remodeling in rats. The underlying mechanism may be associated with the inhibition of CHOP-mediated endoplasmic reticulum stress-related cardiomyocyte apoptosis.     

Effects of metoprolol on phosphorylated connexin 43 expression in myocardial tissues and apoptosis of myocardial cells in heart failure rats

AIM：To explore the in vivo effects of metoprolol on the expression of phosphorylated connexin 43 (p-Cx43) in myocardial tissues and the apoptosis of myocardial cells in rats with heart failure (HF).METHODS：One hundred Sprague-Dawley rats were randomly divided into 5 groups (each n=20): sham group, HF group, low-dose (1.25 mg·kg-1·d-1) metoprolol treatment (MetoA) group, middle-dose (5 mg·kg-1·d-1) metoprolol treatment (MetoB) group and high-dose (20 mg·kg-1·d-1) metoprolol treatment (MetoC) group.The rats in HF group and metoprolol treatment groups were subject to abdominal aortic ligation, and different doses of metoprolol were given 4 weeks later till 8 weeks after operation.Echocardiography was conducted to monitor the hemodynamic parameters at the 4th and 8th weeks, and the rat hearts were taken at the 8th week after operation.The morphological changes and the proliferation of collagen fibers in myocardial tissues were observed by HE and Masson staining, respectively.The expression level of p-Cx43 was detected by Western blotting and the apoptosis of myocardial cells was assessed by TUNEL method.The relationship between p-Cx43 expression level and apoptotic index was analyzed by Pearson’s correlation.RESULTS：(1) Echocardiography showed that metoprolol could effectively improved cardiac hemodynamics in HF rats, and pathological findings suggested that metoprolol could effectively reverse HF-induced cardiac remodeling in a dose-dependent manner within the therapeutic dose range.(2) Western blotting showed that p-Cx43 expression in HF group was significantly higher than that in sham group (P<001), and that in all metoprolol treatment groups was significantly decreased compared with HF group (P<005 or P<001), among which pairwise comparisons also showed significant differences (P<001).(3) The myocardial apoptotic index in HF group ［(51.17±6.94)%］ was significantly increased compared with sham group ［(4.62±160)%, P<001］.Compared with HF group, myocardial apoptotic indexes in MetoA group ［(40.60±4.15)%］, MetoB group ［(30.66±4.00)%］ and MetoC group ［(22.24±5.69)%］ were significantly decreased (P<001), among which pairwise comparisons also showed significant differences (P<001).(4) The expression level of p-Cx43 was positively correlated with the apoptotic index (r=0.905, P<001).CONCLUSION: The mechanism of metoprolol against HF-induced myocardial apoptosis may be related to inhibition of p-Cx43 expression.     

Effects of Shensongyangxin capsule on ventricular electrical properties,structural remodeling and cardiac function in diabetic rats

AIM：To determine the effects of Shensongyangxin capsule (SSYX) on the ventricular electrical properties, structural remodeling and cardiac function in the rats with diabetes mellitus (DM). METHODS：Male SD rats (n=45) were randomly divided into control group (n=15), DM group (n=15) and SSYX group (n=15). The rats in DM group and SSYX group were injected with streptozotocin (60 mg/kg, ip), while the rats in control group were given normal saline (1 mL/kg, ip). The blood samples were collected 72 h after treatment for determining the blood glucose levels in DM group and SSYX group. The model rats in SSYX group were administered with SSYX (1 g·kg-1·d-1, ig) for 6 weeks, while the other rats received normal saline (2 mL·kg-1·d-1, ig). The echocardiography was used to assess the cardiac function, and the lead II electrocardiogram was also recorded in all the animals. The radioimmunoassay and Masson trichrome staining were used to measure the plasma levels of endothelin-1 (ET-1) and the collagen deposition in the ventricles, respectively. A whole Langendorff-perfused heart model was used to conduct the electrophysiologic study. The monophasic action potential (MAP) and the ventricular effective refractory period (VERP) were recorded in the left anterior free wall (LAF), and the burst pacing was used to induce ventricular arrhythmia (VA). RESULTS：Compared with control group, the VERP, action potential duration (APD), QT interval, incidence of VA, degree of myocardial fibrosis and plasma level of ET-1 were increased, while the cardiac function was declined in DM group. Compared with DM group, the VERP, APD, QT interval, incidence of VA, degree of myocardial fibrosis and plasma level of ET-1 were all decreased, while the cardiac function was improved in SSYX group. CONCLUSION：SSYX attenuates the electrical and structural remodeling and improves the cardiac function in DM rats.     

Expression of connexin 40 and connexin 45 in renal tissue of rats with chronic renal failure and effect of treatment with Astragalus polysaccharide and stachydrine combination

AIM：To explore the expression of connexin 40 (Cx40) and connexin 45 (Cx45) in chronic renal failure rats, and to investigate the effect of an Astragalus polysaccharide and stachydrine combination on the expressions of the 2 proteins, and the treatment effect of the combination on chronic renal failure. METHODS：Wistar rats were randomly divided into 4 groups:control group (6 rats), chronic renal failure model group (8 rats), low-dose Astragalus polysaccharide (200 mg·kg-1·d-1) and stachydrine (2.5 mg·kg-1·d-1) combination group (8 rats), and high-dose Astragalus polysaccharide (400 mg·kg-1·d-1) and stachydrine (5 mg·kg-1·d-1) combination group (8 rats). The rat model of chronic renal failure was induced by adenine (200 mg·kg-1·d-1) gavage. After 7-week administration, the expression of Cx40 and Cx45 in the renal tissue was determined by immunohistochemical staining. The renal function, electrolyte levels, and pathological changes of the kidneys were also analyzed. RESULTS：The expression of Cx40 and Cx45 was increased in the renal tissue of chronic renal failure rats. The combination of Astragalus polysaccharide and stachydrine reduced the kidney weight and index, attenuated the pathological renal damage in the model rats, decreased the expression of Cx40 and Cx45, maintained the stability of serum electrolytes, and decreased the levels of serum creatinine and blood urea nitrogen. CONCLUSION:Chronic renal failure caused the increase in the expression of Cx40 and Cx45 in the rat renal tissue. The combination of Astragalus polysaccharide and stachydrine reduces the expression of Cx40 and Cx45 in the renal tissue, and improves the renal function, thus playing a role in protecting the kidneys.     

Protective effect of all-trans retinoic acid pretreatment against rat intestinal injury induced by hepatic inflow occlusion

AIM：To investigate the effect of all-trans retinoic acid (ATRA) on the intestinal injury induced by hepatic inflow occlusion (HIO) and its mechanisms. METHODS：Thirty-two male Sprague-Dawley rats were randomly divided into four groups: sham group, HIO group, dimethyl sulfoxide (DMSO) + HIO group and ATRA (15 mg·kg-1·d-1) + HIO group. The hepatoduodenal ligament of the rats in the latter three groups was occluded (Pringle manoeuvre) by clamp for 30 min. After reperfusion for 2 h by release of the clamp, samples of distal ileum and serum were collected. Histological changes and Chiu’s scores of the ileac mucosa were evaluated under light microscope. Serum content of diamine oxidase (DAO), and ileac tissue levels of malonaldehyde (MDA) and superoxide dismutase (SOD) were analyzed by colorimetry. Serum concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α were evaluated by enzyme-linked immunosorbent assay method. Expression of manganese superoxide dismutase (MnSOD) in cytoplasm and nuclear factor kappa B (NF-κB) p65 in nucleus was assessed by Western blotting. RESULTS：Compared with sham group and DMSO+HIO group, ATRA significantly reduced the mucosal Chiu’s scores, the serum content of DAO and the tissue level of MDA, enhanced the serum activity of SOD and the protein expression of MnSOD, and decreased the content of NF-κB p65 in nucleus (all P<0.05). Subsequently, ATRA significantly reduced the levels of TNF-α and IL-1β in serum (P<0.05). CONCLUSION：ATRA can attenuate rat intestinal injury induced by HIO through improving the antioxidant capacity of tissue, inhibiting the activation of NF-κB and suppressing the overexpression of pro-inflammatory factors.     

Protective effect of catalpol on diabetic rat aorta and its antioxidant mechanisms

AIM：To investigate the protective effect of catalpol on Goto-kakizaki (GK) rat aorta and to explore its antioxidant mechanisms. METHODS：Six-month-old GK rats (n=45) were randomly divided into diabetic model group, metformin (100 mg·kg-1·d-1) group, and high-dose (100 mg·kg-1·d-1), medium-dose (50 mg·kg-1·d-1) and low-dose (10 mg·kg-1·d-1) catalpol groups. The healthy male Wistar rats (n=10) were used as control group. The rats in control and model groups were given a same volume of saline. All reagents were administered by oral gavage for 12 weeks. Blood glucose and lipids were detected by an automatic biochemical analyzer. Serum reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) levels were detected by commercial kits. The expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the thoracic aorta was determined by Western blotting. The pathological changes of the thoracic aorta were observed by HE staining. The ultrastructural changes of the thoracic aorta were observed under electron microscope. RESULTS：After catalpol treatment, the levels of blood glucose and blood lipids were decreased significantly, and serum levels of ROS and MDA were significantly decreased, but the activity of SOD and T-AOC were significantly enhanced. The protein expression of Nrf2 and HO-1 in the thoracic aorta were significantly increased, the thoracic aortic lesions indicated by HE staining significantly reduced, and the thoracic aortic damage under ultrastructural observation was attenuated slightly. CONCLUSION：Catalpol effectively protects GK rat thoracic aorta, which may be associated with decreasing blood lipids, reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathways     

Effects of Panax quinquefolium saponin on mitochondrial membrane potential and cardiomyocyte apoptosis in ischemia/reperfusion rats

AIM：To investigate whether mitochondrial membrane potential (ΔΨm) and the mitochondrial apoptotic pathway are involved in the protective mechanism of Panax quinquefolium saponin (PQS) against cardiomyocyte apoptosis after ischemia/reperfusion (I/R) injury in rat myocardium. METHODS：Ninety healthy male SD rats were randomly divided into sham group, I/R group, PQS (200 mg·kg-1·d-1) +I/R group, cyclosporine A (CsA) group, CsA (10 mg·kg-1) +I/R group and PQS +CsA +I/R group. The model of myocardial I/R injury in vivo was established by ligating the left anterior descending artery (LAD) for 30 min followed by 120 min of reperfusion in the rats. The serum activity of lactate dehydrogenase (LDH) was measured by automatic chemistry analyzer. The myocardial infarct size was measured by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cardiomyocyte apoptosis was detected by in situ TDT-mediated dUTP nick end labeling (TUNEL). The protein levels of Bcl-2, Bax, cleaved caspase-3 and cytosolic cytochrome C were determined by Western blotting. ΔΨm was measured by laser scanning confocal microscopy and fluorescence microplate reader. RESULTS：Compared with I/R group, the serum content of LDH,the infarction size in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group and the myocardial apoptotic index were decreased. Compared with I/R group, the fluorescence intensity of mitochondria after JC-1 staining was enhanced in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, and the relative fluorescence units (RFU) of ΔΨm were improved in those 3 groups. In PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, the protein expression of Bcl-2 was increased, and that of Bax was decreased compared with I/R group. Moreover, in those 3 groups, the protein levels of cleaved-caspase-3 and cytosolic cytochrome C were decreased compared to I/R group, respectively. CONCLUSION：PQS attenuates myocardial injury and cardiomyocyte apoptosis during I/R, and the protective mechanisms of PQS were associated with the modulation of ΔΨm and the inhibition of mitochondrial apoptosis pathway.     

Effects of catestatin on ventricular arrhythmia in isolated rat hearts with chronic heart failure

AIM:To determine the effects of catestatin (CST) on ventricular arrhythmia (VA) in isolated rat hearts with chronic heart failure (CHF). METHODS:Fifty-one male rats were randomly divided into 2 groups: control (CTL) group (n=17) and CHF group (n=34), which were injected with 0.9% normal saline (1 mL·kg-1·d-1, ip) and isoproterenol (ISO, 5 mg·kg-1·d-1, ip) for 7 d,respectively. The echocardiography was used to assess the cardiac functions 2 weeks after the end of modeling in both groups. The CHF rats were divided into non-treatment group (n=17) and CST treatment group (CST group, n=17). The rats in CST group was given CST (2 nmol·kg-1·d-1, ip) for 3 weeks, while 0.9% normal saline (1 mL·kg-1·d-1, ip) was applied to the rats in non-treatment group. To all the whole Langendorff-perfused hearts, the monophasic action potential (MAP) and the ventricular effective refractory period (VERP) were recorded and measured in left anterior free wall (LAF). The programmed electrical stimulation and burst pacing were used to induce action potential duration (APD) alternans (ALT) and VA in the LAF, respectively. The car-diac myocytes of LAF were enzymatically isolated and the technique of whole-cell patch clamp was used to record L-type Ca2+ current (ICa-L). RESULTS:Compared with CTL group, the peak ICa-L density, 90% of MAP duration (MAPD90), VERP, median of maximum pacing cycle length (PCLmax) inducing APD-ALT and incidence of VA (83.33% vs 1667%) were significantly increased in non-treatment group (all P<0.01). Compared with non-treatment group, the peak ICa-L density, MAPD90, VERP, median of PCLmax inducing APD-ALT and incidence of VA were significantly decreased in CST group (all P<0.05). CONCLUSION: Treatment with CST reduces the incidence of VA in CHF rats, which might be associated with the inhibition of ICa-L.     

Therapeutic effect of neuregulin-1β on pressure overload-induced myocardial hypertrophy in rats

AIM：To investigate the therapeutic effect and the mechanism of neuregulin-1β (NRG-1β) on the rat model of myocardial hypertrophy induced by pressure overload．METHODS：Eight weeks after coarctation of abdominal aorta, the Wistar rats were randomly divided into 4 groups: myocardial hypertrophy (model) group, sham operation (sham) group, NRG-1β treatment group (intravenous injection of NRG-1β at dose of 10 μg/kg daily for 7 d) and NRG-1β+Herceptin (HERCE) treatment group ［intravenous injection of NRG-1β (10 μg/kg) plus HERCE (10 μg/kg) daily for 7 d］. The characteristics of heart functions were evaluated by the methods of hemodynamics and echocardiography. Masson staining was employed to observe the pathological changes of myocardial tissues. The concentration of angiotensin II (Ang II) in myocardial tissues was measured by radioimmunoassay. The level of tumor necrosis factor α (TNF-α) in myocardial tissues was detected by ELISA. The mRNA expression of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2-associated X protein (bax) in the myocardium was determined by RT-PCR. RESULTS：The left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were higher, while the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were smaller in NRG-1β group than those in model group. The left ventricular end-systolic pressure (LVESP) and maximal rate of increase/decrease in left ventricular pressure (±dp/dtmax) were higher, and left ventricular end-diastolic pressure (LVEDP) was significantly lower in NRG-1β group than those in model group. Compared with model group, treatment with NRG-1β decreased collagen volume fraction (CVF), reduced the Ang II and TNF-α, increased bcl-2 mRNA expression, and decreased bax mRNA expression in myocardial tissues. No difference of the above parameters between model group and NRG-1β+HERCE treatment group was observed. CONCLUSION：NRG-1 reduces the expression of Ang II and TNF-α in myocardial tissues in pressure-overload rats, thus reducing Ang II and TNF-α mediated myocardial interstitial remodeling. Increase in the mRNA expression of bcl-2 and decrease in the mRNA expression of bax by NRG-1 inhibit myocardial cell apoptosis, which is responsible for its role of improving cardiac function of myocardial hypertrophy induced by pressure overload.     

Regulatory effect of RXR/VDR agonists on atherosclerosis and NF-κB expression in diabetic ApoE knockout mice

AIM：To explore the effect of retinoid X receptor (RXR) agonist bexarotene (Bex) and vitamin D receptor (VDR) agonist calcitriol (Cal) on the expression of nuclear factor-kappa B (NF-κB) and the development of atherosclerosis in streptozotocin-induced diabetic apolipoprotein E knockout (STZ-ApoE-/-) mice. METHODS：Male mice were treated for 12 weeks as follows: (1) C57+vehicle; (2) ApoE-/-+vehicle; (3) STZ-ApoE-/-+vehicle; (4) STZ-ApoE-/-+Bex (10 mg·kg-1·d-1); (5) STZ-ApoE-/-+Cal (10 μg/kg, twice a week); (6) STZ-ApoE-/-+Bex (10 mg·kg-1·d-1)+Cal (10 μg/kg, twice a week). Intraperitoneal injection of STZ was performed to establish the diabetic animal model. Western blotting and immunohistochemical method was used to detect NF-κB level in the thoracic aorta. Plaque area in the thoracic aorta was measured using HE staining. RESULTS：Compared with the C57 mice, the fasting blood glucose in the ApoE-/- mice was not remarkably changed. The levels of total cholesterol (TC) and low-density lipoprotein (LDL) were greatly increased. The fasting blood glucose and lipid levels in STZ-ApoE-/-group were much higher than those in ApoE-/- group. Compared with STZ-ApoE-/- group, the fasting blood glucose and lipid levels in Bex group and Cal group were not significantly changed. Compared with the C57 mice, the protein expression of NF-κB in the ApoE-/- mice and the STZ-ApoE-/- mice was remarkably increased. Compared with STZ-ApoE-/- group, the levels of NF-κB in Bex group, Cal group and combination group were greatly decreased.Compared with STZ-ApoE-/- group, the thoracic artery plaque areas in Bex group and Cal group were inhibited (both P<005). Compared with Bex group, the plaque area of the thoracic artery in combination group was significantly decreased (P<005). CONCLUSION：Bexarotene or calcitriol decreases the development of atherosclerosis in streptozotocin-induced diabetic ApoE-/- mice. Bexarotene combined with calcitriol affords greater protection than monotherapy. The mechanism may be involved in down-regulating the expression of NF-κB.     

Effects of TNF-α-mediated insulin resistance on INSIG2 and SCAP expressions in vivo

AIM: To investigate the effects of TNF-α induced insulin resistance (IR) on INSIG1, INSIG2, SCAP and SREBP expressions in mice. METHODS: Male C57BL/6J mice were randomly divided into 4 groups. The mice were given an intraperitoneal injection of TNF-α (6 μg·kg-1·d-1; 3 μg·kg-1·d-1 and 1 μg·kg-1·d-1) and saline (NC group) twice daily for 7 d. The insulin sensitivity and glucose metabolism in awaken mice were evaluated by intravenous glucose tolerance test (IVGTT). The mRNA expression and protein levels of gene were measured by RT-PCR and Western blotting. RESULTS: After TNF-α treatment, fasting blood glucose (FBG), plasma insulin and free fatty acids (FFA) were significantly elevated in TNF-α (6 μg·kg-1·d-1) group compared to NC, TNF-α (1 μg·kg-1·d-1) and TNF-α (3 μg·kg-1·d-1) groups (P<0.01 and P<0.05, respectively). There was a lower glucose tolerance in TNF-α (6 μg·kg-1·d-1) group than that in other three groups during IVGTT. In TNF-α (6 μg·kg-1·d-1) group, the insulin release of glucose-stimulation was higher than that in NC and TNF-α (1 μg·kg-1·d-1) groups (P<0.01 and P<0.05). The INSIG2 mRNA expression of adipose tissues in TNF-α (6 μg·kg-1·d-1) group was significantly increased compared with NC group (P<0.01), and INSIG2 protein levels were also increased (P<0.05). In TNF-α treatment mice, SCAP mRNA level in adipose tissues was significantly up-regulated than that in the controls (P<0.05). The mRNA expressions of INSIG1 and SREBP1 in two groups were not significantly changed (P>0.05). CONCLUSION: In TNF-α induced insulin resistance, INSIG2 and SCAP may be involved in the pathways of lipid metabolism.     

Effect of atorvastatin on cardiac function and HGF/c-Met signaling pathway after acute myocardial infarction in diabetic rats

AIM: To investigate the effect of atorvastatin on myocardial apoptosis, ventricular remodeling and cardiac function after acute myocardial infarction (AMI) in diabetic rats, and to explore whether the effect is mediated by hepatocyte growth factor (HGF)/c-Met signaling pathway. METHODS: Diabetes in 70 male SD rats was induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg). After 8 weeks, AMI was induced by the ligation of the left anterior descending coronary artery in the diabetic rats, and 32 surviving rats were divided into AMI group (n=16) and AMI＋atorvastatin group (n=16, 20 mg·kg-1·d-1) at random. The similar surgical procedure was completed in sham group (n=11) without coronary ligation. Atorvastatin was given daily by gavage from the first day after AMI. Two weeks later, the cardiac function, pathological changes of myocardial tissues, myocardial apoptosis, and the expression of HGF and c-Met were compared among groups. RESULTS: AMI significantly reduced cardiac function, increased collagen volume fraction (CVF) and myocardial apoptotic index, and up-regulated the expression of HGF and c-Met at mRNA and protein levels in AMI control group (P<0.05). The cardiac function was improved, and CVF and myocardial apoptotic index were reduced by the treatment with atorvastatin, which also up-regulated the expression of HGF and c-Met (P<0.05). CONCLUSION: Atorvastatin significantly attenuates myocardial apoptosis and cardiac remodeling, and improves cardiac function after AMI in diabetic rats by further enhancing the activation of HGF/c-Met pathway.     

Roles of PI3K/Akt/mTOR signaling pathway in macrophage autophagy and atherosclerotic plaque instability

AIM：To investigate whether selective inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway stabilizes the vulnerable atherosclerotic plaques by promoting macrophage autophagy.METHODS：In in vitro study, casodex (20 μmol/L), rapamycin (10 nmol/L) or mTOR-siRNA (30 nmol/L) was used to treat mouse macrophage cell line RAW 264.7. Inflammation-related cytokines secreted by macrophages were measured by means of ELISA. Ultrastructural changes of the macrophages were examined by transmission electron microscopy. The mRNA and protein expression levels of Akt, mTOR and autophage-related protein Beclin 1 were assayed by real-time fluorescence quantitative RT-PCR and Western blotting. The expression of autophagy-related indicator LC3-II was detected by immunofluorescence and Western blotting. In in vivo study, 24 New Zealand white rabbits underwent balloon-induced abdominal aortic wall injury and were fed with a diet of 1% cholesterol for 8 weeks. The rabbits were randomly divided into control group (n=8), casodex group (1.0 mg·kg-1·d-1, n=8) and rapamycin group (0.5 mg·kg-1·d-1, n=8). Four weeks after drug administration, intravascular ultrasound (IVUS) was carried out to observe the plaque imaging. Ultrastructural changes of the macrophages and the protein expression of Akt, mTOR and LC3-II in the macrophages were also measured.RESULTS：In in vitro study, more typical autophagosomes were detected in casodex-, rapamycin- or mTOR-siRNA-treated cells. The expression level of LC3-II increased, but Beclin 1,p-Akt and p-mTOR significantly decreased in the 3 treatment groups. The concentration of IL-10 decreased while IFN-γ significantly increased in the treatment groups. In in vivo study, IVUS found that external elastic membrane area (EEMA),plaque area(PA) and plaque burden (PB) significantly decreased in casodex and rapamycin treatment groups. Expression of LC3-II increased significantly in the 2 treatment groups. The staining of RAM-11 and p-mTOR in the macrophages was significantly reduced as compared with control group.CONCLUSION：Selective inhibition of PI3K/Akt/mTOR signaling pathway reduces the infiltration of macrophages and stabilizes the vulnerable atherosclerotic plaques by promoting macrophage autophagy.     

Comparison between mobilization and transplantation of bone marrow stem cells for the therapy of myocardial infarction in rabbits

AIM: To compare bone marrow stem cell mobilization with bone marrow-derived mononuclear cells (BMCs) transplantation for the therapy of myocardial infarction (MI) in rabbits, and to explore more effective and practical stem cell therapeutic strategy for MI. METHODS: In mobilization group (M, n=10), granulocyte-colony stimulating factor (G-CSF) (30 μg·kg-1·d-1) was injected subcutaneously 3 hours after MI and every 24 hours for 5 days. On the 5th day, the BMCs from 10 mL peripheral blood were labeled with bromodeoxyuridine (BrdU) for 24-48 hours, then reinjected intravenously. In transplantation group (T, n=10), BMCs transplantation was performed 5-7 days after MI. After being obtained from bone marrow (3-5 mL) of iliac crest and labeled with BrdU for 24-48 hours, BMCs were transplanted into infracted myocardium through intramyocardial injection. Control animals (C, n=10) did not receive any treatment after MI. Echocardiography were performed for the evaluation of cardiac function 1 week and 5 weeks after MI. Hemodynamic studies and histological study were performed 5 weeks after MI. RESULTS: LV ejection fraction increased significantly in group M, had no change in group T, and decreased 1 week and 5 weeks after MI in group C. Group M and group T had higher LV max +dp/dt and max -dp/dt, lower LV end-diastolic pressure compared with group C 5 weeks after MI. Histological studies revealed that there were BrdU positive cells in the infarcted area in group M and group T. The vascular density of group M and group T in the infarcted area was significantly greater in comparison with group C. No regeneration of smooth muscle cells and cardiomyocytes were found in the infarcted area. CONCLUSION: Bone marrow stem cell mobilization with G-CSF and transplantation of BMCs both significantly improve the cardiac function for the therapy of MI through vascular genesis in the infarcted area. Bone marrow stem cell mobilization may offer a new and non-invasive therapeutic strategy for MI.     

Overexpression of integrin-linked kinase improves survival and proliferation of cardiac c-Kit+ cells

AIM: To investigate the effects of integrin-linked kinase (ILK) overexpression on survival and proliferation of cardiac c-Kit⁺ cells, and the role of ILK-overexpressing c-Kit⁺ cell transplantation in cardiac function in a rat myocardial infarction (MI) model.METHODS: Cardiac c-Kit⁺ cells were isolated from the hearts of neonatal Sprague-Dawley (SD) rats and cultured to prepare the ILK-c-Kit⁺ cells by infected with recombinant adenoviral vector harboring human wild-type ILK cDNA. The survival and proliferation of cardiac c-Kit⁺ cells were detected by cell counting and CCK-8 assay at 48 h after infection, respectively. The protein levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) in the cardiac c-Kit⁺ cells were examined by Western blot. MI was induced by coronary artery ligation in 40 adult rats. After 15 min, ILK-c-Kit⁺ cells were transplanted into the hearts by myocardial injection at 3 different sites in the infracted zone and border zone. All rats were randomly divided into 4 groups:sham group, MI plus saline injection group (MI group), MI plus null vector-infected cardiac c-Kit⁺ cell injection group (Ad-null-c-Kit⁺ cell group), and MI plus ILK-overexpressing cardiac c-Kit⁺ cells injection group (ILK-c-Kit⁺ cell group), with 10 rats in each group. At 2 weeks after MI, the protein levels of c-Kit in MI hearts were investigated by immunohistochemical assay. At 4 weeks, left ventricular function was examined by hemodynamic measurement.RESULTS: The survival and proliferation of cardiac c-Kit⁺ cells and the protein levels of cyclin D1 and PCNA were enhanced by ILK overexpression compared with Ad-null group. In MI rat model, the number of c-Kit⁺ cells was increased by ILK-c-Kit⁺ cell injection compared with Ad-null-c-Kit⁺ cell group at 2 weeks after MI. Cardiac function was significantly improved in ILK-c-Kit⁺ cell-transplanted rats.CONCLUSION: ILK overexpression improves survival and proliferation of cardiac c-Kit⁺ cells by increasing the protein levels of cyclin D1 and PCNA. ILK-c-Kit⁺ cell transplantation enhances the therapeutic efficiency of cardiac c-Kit⁺ cells in the post-MI hearts of rats.     

Effects of irbesartan and perindopril on the myocardial expression of connexin 43, desmin and cardiac troponin T in rat cardiac hypertrophy induced by pressure overload

AIM: To investigate the effects of angiotensinⅡ receptor type Ⅰ antagonist irbesartan and angiotensin-converting enzyme inhibitor perindopril on the myocardial expression of connexin 43 (CX43), desmin and cardiac troponin T (cTnT) in the pressure overload-induced rat cardiac hypertrophy. METHODS: 40 male adult Sprague-Dawley rats were divided into 5 groups (8 animals for each): sham operation group and other four groups with ventricular hypertrophy caused by banding aortic artery. Drugs were given one week after operation as follows: sham operation group, normal saline (2 mL·kg-1·d-1 ig) was given; Operative groups: animals with ventricular hypertrophy were treated with normal saline 2 mL·kg-1·d-1 ig; Treatment groups: animals with ventricular hypertrophy were treated with perindopril 2 mg·kg-1·d-1 ig, irbesartan 20 mg·kg-1·d-1 ig or irbesartan 20 mg·kg-1·d-1 ig plus perindopril 2 mg·kg-1·d-1 ig, respectively. Left ventricular mass index (LVMI), transverse diameter of myocardial cell (TDM), and myocardial expression of CX43, desmin and cTnT by immunohistochemistry were performed at the end of 8 weeks of drug intervention. RESULTS: LVMI, TDM were remarkably decreased after drug intervention, compared to animals of operative group (P<0.05). Left ventricular hypertrophy induced by aortic banding in rats were associated with marked disorganization of gap junction distribution. In hypertrophied myocytes, CX43 immunolabeling was dispersed over the entire cell surface rather than confined to the intercalated disks. The CX43 were mainly distributed in the intercalated disks in irbesartan group, perindopril group and their combined group. The myocardial expression of CX43, desmin and cTnT in the operative group was lower than that in irbesartan group, perindopril group and their combined group (P<0.05). CONCLUSION: These data indicate that irbesartan and perindopril play beneficial roles in the myocardial CX43, desmin and cTnT expression and their distribution, and the restoration of myocardial cell structure and gap junction in pressure-overload myocardium hypertrophy.     

Intranasal insulin treatment ameliorates Alzheimer disease-like changes in hippocampus in a rat model of type 2 diabetes

AIM： To investigate Alzheimer disease (AD)-like changes and 2 key components of the insulin signaling pathway in the brain of a rat model of type 2 diabetes (T2D) after insulin treatment. METHODS：The rat model of T2D was established by feeding a high-protein, high-glucose and high-fat diet followed by intrasubcutaneous injection of streptozocin. Intranasal insulin treatment (T2D+I-I) and subcutaneous insulin injection (T2D+S-I) were applied to elevate the insulin level in the brain. The insulin levels in plasma and cerebrospinal fluid as well as the concentration of plasma glucose were measured. Total tau level, the phosphorylation level of tau at some phosphorylation sites, and the activation of GSK-3β and Akt in subcutaneous of the rats were also analyzed by Western blotting.RESULTS：AD-like changes, decreased Akt activation and over-activation of GSK-3β in the hippocampus of the T2D rats were observed. Intranasal insulin treatment for 4 weeks normalized the levels of Akt and GSK-3β, as well as reduced the AD-like changes in the hippocampus of the T2D rats, whereas the treatment with insulin by subcutaneous injection for 4 weeks had minimal effects on the levels of GSK-3β and tau phosphorylation in the hippocampus. CONCLUSION： Intranasal insulin treatment, but not subcutaneous insulin treatment, might decrease the risk of AD in T2D rats by reducing AD-like changes and up-regulating the impaired insulin signaling pathway in the hippocampus,indicating the potential use of intranasal insulin delivery for treatment of AD.