Myocardial autophagy variation during acute myocardial infarction in rats: the effects of carvedilol

Background The loss of cardiac myocytes is one of the mechanisms involved in acute myocardial infarction （AMI）-related heart failure. Autophagy is a common biological process in eukaryote cells. The relationship between cardiac myocyte loss and autophagy after AMI is still unclear. Carvedilol, a non-selective α1-and β-receptor blocker, also suppresses cardiac myocyte necrosis and apoptosis induced by ischemia. However, the association between the therapeutic effects of carvedilol and autophagy is still not well understood. The aim of the present study was to establish a rat model of AMI and observe changes in autophagy in different zones of the myocardium and the effects of carvedilol on autophagy in AMI rats. Methods The animals were randomly assigned to a sham group, an AMI group, a chloroquine intervention group and a carvedilol group. The AMI rat model was established by ligating the left anterior descending coronary artery. The hearts were harvested at 40 minutes, 2 hours, 24 hours and 2 weeks after ligation in the AMI group, at 40 minutes in the chloroquine intervention group and at 2 weeks in other groups. Presence of autophagic vacuoles （AV） in the myocytes was observed by electron microscopy. The expression of autophagy-, anti-apoptotic- and apoptotic-related proteins, MAPLC-3, Beclin-1, Bcl-xl and Bax, were detected by immunohistochemical staining and Western blotting. Results AVs were not observed in necrotic regions of the myocardium 40 minutes after ligation of the coronary artery. A large number of AVs were found in the region bordering the infarction. Compared with the infarction region and the normal region, the formation of AV was significantly increased in the region bordering the infarction （P 〈0.05）. The expression of autophagy- and anti-apoptotic-related proteins was significantly increased in the region bordering the infarction. Meanwhile, the expression of apoptotic-related proteins was significantly increased in the infarction region. In the chloroquine intervention group, a large number of initiated AVs （AVis） were found in the necrotic myocardial region. At 2 weeks after AMI, AVs were frequently observed in myocardial cells in the AMI group, the carvedilol group and the sham group, and the number of AVs was significantly increased in the carvedilol group compared with both the AMI group and the sham group （P 〈0.05）. The expression of autophagy- and anti-apoptotic-related proteins was significantly increased in the carvedilol group compared with that in the AMI group, and the positive expression located in the infarction region and the region bordering the infarction. Conclusions AMI induces the formation of AV in the myocardium. The expression of anti-apoptosis-related proteins increases in response to upregulation of autophagy. Carvedilol increases the formation of AVs and upregulates autophagy and anti-apoptosis of the cardiac myocytes after AMI.     

Effect and mechanism of panaxoside Rg1 on neovascularization in myocardial infarction rats

<正>Objective:To investigate the effects and mechanisms of panaxoside Rg1 on the new vessel formation in acute myocardial infarction(AMI) rats.Methods:The AMI model of male Sprague-Dawley(SD) rats was established,and rats were randomly divided into the AMI model group,the treatment group of panaxoside Rg1,the placebo group and the treatment group of panaxoside Rg1 plus rapamycin.Cardiac creatases were determined with 1 mL blood drawn from vena caudalis of the rats 48 h after the model was successfully made. After 4 weeks,Evans blue was injected into the aorta roots of the rats,and then,red tetrazoline was dyed again and the myocardial infarction area was evaluated.The microvessel density(MVD) of infarction area was determined by the immunohistochemistry of CD31;enzyme-linked immunosorbent assay(ELISA) was used to detect the protein content of CD31 and hypoxia inducible factor-1α(HIF-1α) of the infarction area. Results:The MVD in the infarction area and the contents of CD31 and HIF-1αin the Rg1 treatment group were higher than those in the AMI model group significantly(P0.05).The cardiac creatase and infarction area were lower in the Rg1 treatment group than those in the AMI model group significantly(P0.05).The above effects,however,disappeared when rapamycin,the antagonist of mammalian target of rapamycin(mTOR),was administered simultaneously.Conclusions:Panaxoside Rg1 could increase the expression of HIF-1αand CD31 of myocardium and stimulate the angiogenesis.The above mentioned role of panaxoside Rg1 might be related to the excitation of mTOR receptor.     

Over-expression of VEGF in marrow stromal cells promotes angiogenesis in rats with cerebral infarction via the synergistic effects of VEGF and Ang-2

This study explored whether the transplantation of modified marrow stromal cells (MSCs) has angiogenic effects in a left middle cerebral artery occlusion infarction/reperfusion (MCAO I/R) rat model and preliminarily examined the mechanism of angiogenesis following cerebral infarction.MSCs were isolated by using a direct adherent method and cultured.Vascular endothelial growth factor (VEGF) was transfected into MSCs by employing the liposome transfection.The transfection efficiency was measured by the optical density method.The protein expression of VEGF gene before and after transfection was measured by Western blotting.SD rat model of transient occlusion of the left middle cerebral artery was established by using an approach of intra-luminal occlusion.Tetrazolium (TTC) and HE staining were performed to observe the cerebral infarction.ELISAs were used to measure the levels of VEGF in the rat cerebral tissues.The expression patterns of angiopoietin-2 (Ang-2) and CD34 in cells surrounding the area of infarction were immunohistochemistrically oserved.Ang-2 protein expression in the tissue surrounding the area of infarction was measured by Western blotting.VEGF expression in the MSCs increased after transfection at a rate of approximately 28%±3.4%.ELISA showed that the expression of VEGF in the cerebral tissue was significantly increased after induction of infarction,peaking on the 4th day and decreasing to the levels of the sham surgery group (normal) within 7 to 10 days.The VEGF level was significantly higher at each time point in the VEGF-MSC and MSC groups compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group than in the MSC group and stayed relatively high until the 10th day.The immunohistochemical results showed that 10 days after the infarction,the number of Ang-2 and CD34-expressing cells in the area surrounding the infarction was significantly higher in the VEGF-MSC group and the MSC group compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group th     

Effects of Panax Notoginseng Saponins on Homing of C-kit~+ Bone Mesenchymal Stem Cells to the Infarction Heart in Rats

Objective:To investigate the effects of panax notoginseng saponins(PNS) on homing of C-kit+ bone mesenchymal stem cells(BMSCs) to the infarction heart.Methods:The acute myocardial infraction(AMI) model was established in 140 Wistar rats,105 model rats survived after operation,and the model rats were randomly divided into five groups,21 rats in each group:Western medicine group mobilized by subcutaneous injection of human granuloctye colony stimulating factor(G-CSF) 50 μg·kg-1·d-1;sham operation group and a model group treated by subcutaneous injection of normal saline 50 μg·kg-1·d-1;Chinese medicine group mobilized by intraperitoneal injection of Xuesaitong(血塞通)(ingredients of PNS) 150 mg·kg-1·d-1;integrative medicine group mobilized by subcutaneous injection of G-CSF 50 μg·kg-1·d-1 and intraperitoneal injection of Xuesaitong 150 mg·kg-1·d-1.Except for the sham-operated group,each group was divided into three sub-groups by three time points of 1 d,7 d and 14 d.G-CSF was injected once a day for 7 d.Xuesaitong was injected once a day until the rats were killed.The flow cytometry was used for detection of C-kit + cells in the peripheral blood in different time points,and immunohistochemical method was used for detection of the changes of C-kit + cell and Ki-67+ cell numbers in the marginal zone of AMI.Results:Twenty-four hours after the operation,C-kit + cells had a slight increase in the model group compared with the sham operation group(P>0.05).The peripheral blood C-kit+ cells in the integrative group increased significantly compared with the other groups on 7 d and 14 d(all P<0.05).Meanwhile the expression of C-kit + cells and Ki-67+ cells in the marginal zone of AMI in the integrative group increased significantly compared with the Chinese medicine group,the western medicine group and the model group on 1 d,7 d and 14 d(all P<0.05),and the cells in the integrative group decreased significantly on 14 d compared with that on 7 d(P<0.05).Conclusion:PNS can cooperate with G-CSF to mobilize C-kit+ BMSCs from the marrow into the peripheral blood and promote them "homing" to the infarction heart.     

Effect of Quyu Chencuo Formula(去菀陈莝方)on Renal Fibrosis in Obstructive Nephropathy Rats

Objective: To observe the effect of Quyu Chencuo Formula(去菀陈莝方, QCF) on renal fibrosis in rats with obstructive nephropathy. Methods: Twenty-four rats were randomly divided into three groups, 4 for sham operation as the control group, 10 for unilateral ureteral obstruction(UUO) model group, and the rest 10 for QCF treating UUO model group. All rats were sacrificed under 3% pentobarbital(50 mg/kg) anesthesia on the 14 th day after surgery, then the right kidney samples of rats were harvested for hematoxylin eosin(HE) staining and Masson staining to observe the renal pathological changes. Immunohistochemistry and Western blotting were used to examine the expression of transforming growth factor β1(TGF-β1), and real-time polymerase chain reaction(RT-PCR) was employed to examine the expressions of TGF-β1, α-smooth muscle actin(α-SMA) and E-cadherin mRNA. Results: HE and Masson staining showed that the renal interstitial of the rats in the control group had no significant fibrotic lesion; in the model group, there were obvious interstitial fibrosis; for the QCF group, there were epithelial cell necrosis, infiltration of lymphocytes and mononuclear cells, aggravated interstitial fibrosis in varied degrees, but the pathological changes were less in the QCF group than in the model group. The immunohistochemistry and Western blotting results showed that the TGF-β1 expression was increased significantly in the model group, while decreased significantly in the QCF group(P0.05); RT-PCR showed that the mRNA expression of α-SMA and TGF-β1 increased significantly in the model group, while both were significantly decreased in the QCF group compared with the model group(P0.05). The mRNA expression of E-cadherin was decreased significantly in the model group, and it was significantly increased in the QCF group as compared with the model group(P0.05). Conclusion: QCF may improve renal fibrosis by regulating the expressions of TGF-β1, α-SMA and E-cadherin, and prevent the progress of kidney fibrosis.     

Effects of Xuesetong Soft Capsules(血塞通软胶囊) on angiogenesis and VEGF mRNA expression in ischemic myocardium in rats with myocardial infarction

OBJECTIVE:To observe the effects of Xuesetong Soft Capsules(血塞通软胶囊,Notoginseng total saponin) on angiogenesis and vascular endothelial growth factor(VEGF) mRNA expression in ischemic myocardium of rats with myocardial infarction.METHODS:The left coronary artery of rats was ligated to establish the animal model of acute myocardial infarction.Rats were randomly divided into Xuesetong Soft Capsule,Shexiangbaoxin Pill(positive control),model(negative control) and sham operation groups.After 6 weeks,microvessel count(MVC),microvessel density(MVD) and VEGF mRNA expressioninischemicmyoc ardium were evaluated.RESULTS:MVC and MVD in the myocardial infarct border area in model,Shexiangbaoxin Pill and Xuesetong Soft Capsule groups significantly increased compared with those of the sham operation group(P<0.05).MVC and MVD in the myocardial infarct border area in Xuesetong Soft Capsule and Shexiangbaoxin Pill groups significantly increased compared with those of the model group(P<0.05).No significant differences between Xuesetong Soft Capsule and Shexiangbaoxin Pill groups were observed(P>0.05).The model group showed signifi-cantly higher VEGF mRNA expression than that in the sham operation group(P<0.05).Xuesetong Soft Capsule and Shexiangbaoxin Pill groups showed significantly higher VEGF mRNA expression than that of the model group(P<0.05).No significant difference between Xuesetong Soft Capsule and the Shexiangbaoxin Pill groups was observed(P>0.05).CONCLUSION:Xuesetong Soft Capsules promote angiogenesis in ischemic myocardium after myocardial infarction and the mechanism may be associated withVEGF mRNA expression.     

二甲胺四环素在蛛网膜下腔出血中保护作用的研究

Objective To investigate the effect of minocycline on early brain injury (EBI) following subarachnoid hemorrhage(SAH) in rats. Methods SAH was induced by the filament perforation model in male Sprague Dawley rats. SD rats(n=77) were randomly assigned to sham (n=22), SAH+vehicle (n=28), and SAH+minocycline (n=27) groups. Minocycline (135mg/kg) or equal volume of vehicle was administered 1 h after SAH induction. Mortality, neurological scores, brain edema were evaluated 24 h after SAH. Cell apoptosis were examined by TUNEL staining, and the expression of caspase-3 and Bcl-2 was assayed by Western blot at the same time point. Results The mortality was 21.4% in SAH+vehicle group, 18.5% in the SAH+minocycline group, while no death was observed in sham-operated rats; there was no significant difference in mortality between SAH+vehicle and SAH+minocycline groups (P＞0.05), but the mortality in these two groups was much higher than that in shamgroup(P＜0.05). The water content of brain was significantly increased in the SAH+vehicle group (80.00±0.16)% compared with that in sham group [(79.13±0.08)%, P＜0.05]. Minocycline treatment markedly reduced brain water content (79.36±0.07)% compared with that in SAH+vehicle group (P＜0.05). Caspase-3 levels were markedly increased in SAH+vehicle group (1.53±0.24) compared with sham group (1.00±0.21). Minocycline treatment significantly reduced caspase-3 levels, compared to SAH+vehicle group (1.11±0.18, P＜0.05). A significant decrease in Bcl-2 expression was observed in SAH+vehicle group(0.65±0.03) compared with the sham group (1.00±0.12). The treatment of minocycline upregulated the expression of Bcl-2,compared to SAH+vehicle group (0.93±0.13, P＜0.05). TUNEL-positive cells were increased in the cortex of SAH+vehicle rats,compared to sham group [(31.50±3.70)%, P＜0.05]. Minocycline treatment significantly reduced the number of TUNEL positive cells, compared to SAH+vehicle group [(14.25±2.50)%, P＜0.05]. Conclusion Minocycline may reduce early brain injury after subarachnoid hemorrhage in rats by inhibiting cell apoptosis, which is associated with down-regulation of caspase-3 and up-regulation of Bcl-2.     

Transplanted human umbilical cord blood mononuclear cells improve left ventricular function through angiogenesis in myocardial infarction

Background Human umbilical cord blood contains an abundance of immature stem/progenitor cells, which may participate in the repair of hearts that have been damaged by myocardial infarction (MI). This study aimed to evaluate the effects of human umbilical cord blood mononuclear cells (hUCBC) transplantation on cardiac function and left ventricular remodeling in rat model of MI. Methods Forty-five male Wistar rats were randomized into three groups: MI or control group (n=15), MI plus cell transplantation (n=15), and sham group (n=15). Acute myocardial infarction (AMI) was established by ligating the left anterior descending artery, thereafter, hUCBC were implanted into the marginal area of infarcted myocardium. In MI/control group, DMEM was injected instead of hUCBC following the same protocol. Left ventricular function assessment was carried out by echocardiography and invasive hemodynamic measurements one month post MI. All rats were sacrificed for histological and immunochemical examinations. Results The transplanted hUCBC survived and engaged in the process of myocardial repair in the host heart. Echocardiography demonstrated that left ventricular function improved significantly in the rats that underwent cell transplantation. Hemodynamic studies found a significantly decreased left ventricular end-diastolic pressure (LVEDP) [(21.08±8.10) mmHg vs (30.82±9.59) mmHg, P&lt;0.05], increase in +dp/dt(max) [(4.29±1.27) mmHg/ms vs (3.24±0.75) mmHg/ms, P&lt;0.05), and increase in －dp/dt(max) [(3.71±0.79) mmHg/ms vs (3.00±0.49) mmHg/ms, P&lt;0.05] among MI group with hUCBC transplantation when compared with MI/control group. Masson’s trichrome staining revealed that the collagen density in the left ventricle was significantly lower in rats of transplantation group than that in the MI control groups [(6.33±2.69)% vs (11.10±3.75)%, P&lt; 0.01]. Based on immunostaining of α-actin, the numbers of microvessels were significantly (P&lt;0.01) increased at the boundary of infarction site. Similarly higher mRNA expression of vascular endothelial growth factor (VEGF) 164 and VEGF188 were found at 7- and 28-day post cell transplantation in MI group with hUCBC transplantation when compared with MI/ control group.Conclusions Transplanted hUCBC can survive in host myocardium without immunorejection, significantly improve left ventricular remodeling after AMI and promote a higher level of angiogenesis in the infarct zones. All these factors beneficially affect cardiac repair in the setting of MI. Therefore human umbilical cord blood may be potential source for cell-based therapy for AMI.     

High-frequency ultrasound evaluation of effects of early treatment with metoprolol on myocardial inflammatory cytokine expression in rats with acute myocardial infarction

This study evaluated the effects of early treatment with β-adrenergic blocker metoprolol on ventricular remodeling and function after acute myocardial infarction (AMI) by using high frequency ultrasound.The relationship between the efficacy and the expression level of cardiac myocardial inflammatory cytokine was examined in rats.The rat model of AMI was induced by ligating the left ante-rior descending artery.The surviving rats were randomly assigned to two experimental groups:MI control (MI) group and MI metoprolol (MI-B) group,with the rats undergoing sham operation serving as normal control (Sham).MI-B group was given metoprolol for 4 weeks (refer to the CCS-2 protocol) and the other two groups received equal volume of saline via intragastric (i.g.) administation.The ventricular remodeling and function were evaluated by high frequency ultrasound 4 weeks after the treatment.Then all rats were sacrificed for pathological examination and immunohistochemistrical detection of inflammatory cytokines,including IL-1β,IL-6,IL-10 and TNF-α.Compared with the MI group,the left ventricular end-systolic dimension,end-diastolic dimension,end-systolic volume and end-diastolic volume of the MI-B group were significantly decreased (P<0.01),while the left ventricular anterior wall end-diastolic thickness,ejection fraction and fractional shortening were obviously increased (P<0.01).The conspicuous improvement in the left ventricular morphology and function was coincident with the markedly reduced TNF-α and IL-1β expression and the increased IL-10 expression.We are led to conclude that early metoprolol treatment for AMI can regulate myocardial inflammatory cytokine expression to improve cardiac function and the underlying mechanism might be that it decreases the level of pro-inflammatory cytokines and increases the level of its anti-inflammatory counterparts in cardiac myocytes.Our study also showed that echocardiography is a useful technique for the structural and functional assessment of left ventricle after acute my     

Electro-acupuncture combined with transcranial magnetic stimulation improves learning and memory function of rats with cerebral infarction by inhibiting neuron cell apoptosis

This study examined the effect of electro-acupuncture (EA) combined with transcranial magnetic stimulation (TMS) therapy at different time windows on learning and memory ability of rats with cerebral infarction and the underlying mechanism.Two hundred SD rats were randomly divided into four groups:normal group,sham-operated group,model group and EA+TMS group,and each group was then divided into five sub-groups in terms of the different time to start treatment post operation:6,12,24,48 and 72 h.Cerebral infarction models were established in the model and the EA+TMS groups by left middle cerebral artery occlusion/reperfusion (MCAO/R).After treatment for 14 d,the Morris water maze test was applied to examine the spatial learning and memory abilities of rats.In infarcted area,the expression of caspase-3 was immunohistochemically detected,and real-time fluorescent quantitative PCR was used to measure the expression of Bcl-2 mRNA.The results showed that in EA+TMS group compared with model group at the same treatment time windows,the escape latency was substantially shortened,the expression of caspase-3 was considerably decreased and the expression level of Bcl-2 mRNA significantly increased (P<0.05).In the EA+TMS sub-groups,the escape latency was shortest,the expression level of caspase-3 lowest,and the expression level of Bcl-2 mRNA highest at the treatment time window of 24 h.It was concluded that EA combined with TMS can promote neurological function of rats with cerebral infarction by increasing the expression level of Bcl-2 mRNA and decreasing the expression of caspase-3.The best time window is 24 h after perfusion treatment to ischemia.     

Fuzheng Huayu Formula(扶正化瘀方) Prevents Rat Renal Interstitial Fibrosis Induced by HgCl_2 via Antioxidative Stress and Down-regulation of Nuclear Factor-Kappa B Activity

Objective:To investigate the mechanism of action of Fuzheng Huayu Formula(扶正化瘀方,FZHY)against renal interstitial fibrosis(RIF)relating to oxidative injury and nuclear factor-kappa B(NF-κB)activity.Methods:Thirty-two Sprague-Dawley rats were randomly divided into 3 groups:normal group,model group and FZHY treatment group.The RIF model was induced by oral administration of HgC l2 at a dose of 8 mg/kg body weight once a day for 9 weeks.Meanwhile,rats in FZHY treatment group orally took FZHY at a dose of4.0 g/kg rat weight for 9 weeks.The content of hydroxyproline(Hyp)and collagen deposition in kidney were observed.The activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),the content of glutathione(GSH)and malondialdehyde(MDA)of kidney were tested.The expressions of inhibitor-κappa B(IκB),phospho-IκB(p-IκB),tumor necrosis factor-α(TNF-α),matrix metalloproteinase-2(MMP-2)andα-smooth muscle actin(α-SMA)were analyzed by Western blot.α-SMA expression was also observed by immunofluorescent staining.MMP-2 activity was measured by gelatin zymography.NF-κB activation was determined by electrophoretic mobility shift assay.Results:Renal interstitial fibrosis was induced by Hg Cl2,demonstrated by remarkably increased Hyp contents and excessive collagen deposition in kidney(P0.01).FZHY significantly inhibited renal interstitial collagen deposition and reduced Hyp content of the Hg Cl2-treated rats(P0.01).GSH content decreased obviously,and MDA content increased significantly in HgC l2-treated rats compared with that of normal rats(P0.01).FZHY significantly increased GSH content and decreased MDA content in the model rats(P0.01).The expressionα-SMA was increased in model rats compared with that of normal rats,FZHY significantly decreased its expression(P0.01).The expressions of p-IκB and TNF-αand MMP-2,MMP-2 activity,and NF-κB activation were increased in model group compared with that in normal group(P0.01),FZHY significantly decreased NF-κB activation,MMP-2 activity and p-IκB and TNF-αexpressions(P0.01).Conclusions:FZHY could protect kidney from oxidative injury intoxicated by Hg Cl2,and antagonized oxidative stress-stimulated NF-κB activity through inhibition of IκB phosphorylation in the interstitial fibrotic kidney,these effects importantly contributed to FZHY action mechanism against renal interstitial fibrosis.     

Recombinant human growth hormone secreted from tissue-engineered bioartificial muscle improves left ventricular function in rat with acute myocardial infarction

Background Experimental studies and preliminary clinical studies have suggested that growth hormone （GH） treatment may improve cardiovascular parameters in chronic heart failure （CHF）. Recombinant human GH （rhGH） has been delivered by a recombinant protein, by plasmid DNA, and by genetically engineered cells with different pharmacokinetic and physiological properties. The present study aimed to examine a new method for delivery of rhGH using genetically modified bioartificial muscles （BAMs）, and investigate whether the rhGH delivered by this technique improves left ventricular （LV） function in rats with CHE Methods Primary skeletal myoblasts were isolated from several Sprague-Dawley （SD） rats, cultured, purified, and retrovirally transduced to synthesize and secrete human rhGH, and tissue-engineered into implantable BAMs. Ligation of the left coronary artery or sham operation was performed. The rats that underwent ligation were randomly assigned to 2 groups： CHF control group （n=6） and CHF treatment group （n=6）. The CHF control group received non-rhGH-secreting BAM （GFP-BAMs） transplantation, and the CHF treatment group received rhGH-secreting BAM （GH-BAMs） transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups： sham control group （n=6） and sham treatment group （n=6）. The sham control group underwent GFP-BAM transplantation, and the sham treatment group underwent GH-BAM transplantation. GH-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats with ligation of the left coronary artery or sham operation was performed. Eight weeks after the treatment, echocardiography was performed, hGH, insulin-like growth factor-1 （IGF-1） and TNF-a levels in rat serum were measured by radioimmunoassay and ELISA, and then the rats were killed and ventricular samples were subjected to immunohistochemistry. Results Primary rat myoblasts were retrovirally transduced to secrete rhGH and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted 1 to 2 lug of bioactive rhGH per day. When implanted into syngeneic rat, GH-BAMs secreted and delivered rhGH. Eight weeks after therapy, LV ejection fraction （EF） and fractional shortening （FS） were significantly higher in CHF rats treated with GH-BAMs than in those treated with GFP-BAMs （（65.0i-6.5）% vs （48.1±6.8）%, P 〈0.05）, （（41.3±7.4）% VS （26.5±7.1）%, P 〈0.05）. LV end-diastolic dimension （LVEDD） was significantly lower in CHF rats treated with GH-BAM than in CHF rats treated with GFP-BAM （P 〈0.05）. The levels of serum GH and IGF-1 were increased significantly in both CHF and sham rats treated with GH-BAM. The level of serum TNF-α decreased more significantly in the CHF treatment group than in the CHF control group.Conclusions rhGH significantly improves LV function and prevents cardiac remodeling in rats with CHF. Genetically modified tissue-engineered bioartificial muscle provides a method delivering recombinant protein for the treatment of heart failure.     

Fuzheng Huayu Formula (扶正化瘀方) Prevents Rat Renal Interstitial Fibrosis Induced by HgCl2 via Antioxidative Stress and Down-regulation of Nuclear Factor-Kappa B Activity

Objective: To investigate the mechanism of action of Fuzheng Huayu Formula (扶正化瘀方, FZHY) against renal interstitial fibrosis (RIF) relating to oxidative injury and nuclear factor-kappa B (NF-κB) activity. Methods: Thirty-two Sprague-Dawley rats were randomly divided into 3 groups: normal group, model group and FZHY treatment group. The RIF model was induced by oral administration of HgCl2 at a dose of 8 mg/kg body weight once a day for 9 weeks. Meanwhile, rats in FZHY treatment group orally took FZHY at a dose of 4.0 g/kg rat weight for 9 weeks. The content of hydroxyproline (Hyp) and collagen deposition in kidney were observed. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), the content of glutathione (GSH) and malondialdehyde (MDA) of kidney were tested. The expressions of inhibitor-κappa B (IκB), phospho-IκB (p-IκB), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-2 (MMP-2) and α-smooth muscle actin (α-SMA) were analyzed by Western blot. α-SMA expression was also observed by immunofluorescent staining. MMP-2 activity was measured by gelatin zymography. NF-κB activation was determined by electrophoretic mobility shift assay. Results: Renal interstitial fibrosis was induced by HgCl2, demonstrated by remarkably increased Hyp contents and excessive collagen deposition in kidney (P<0.01). FZHY significantly inhibited renal interstitial collagen deposition and reduced Hyp content of the HgCl2-treated rats (P<0.01). GSH content decreased obviously, and MDA content increased significantly in HgCl2-treated rats compared with that of normal rats (P<0.01). FZHY significantly increased GSH content and decreased MDA content in the model rats (P<0.01). The expression α-SMA was increased in model rats compared with that of normal rats, FZHY significantly decreased its expression (P<0.01). The expressions of p-IκB and TNF-α and MMP-2, MMP-2 activity, and NF-κB activation were increased in model group compared with that in normal group (P<0.01), FZHY significantly decreased NF-κB activation, MMP-2 activity and p-IκB and TNF-α expressions (P<0.01). Conclusions: FZHY could protect kidney from oxidative injury intoxicated by HgCl2, and antagonized oxidative stress-stimulated NF-κB activity through inhibition of IκB phosphorylation in the interstitial fibrotic kidney, these effects importantly contributed to FZHY action mechanism against renal interstitial fibrosis.     

Effects of isoflurane on ICAM-1 expression and neutrophils infiltration in rats with liver ischemia and reperfusion injury

Objective： To establish a rat model of warm partial hepatic ischemia-reperfusion （IR）, and investigate the protective and anti-inflammatory effects of isoflurane on warm hepatic ischemia-reperfusion injury （IRI） in rats. Methods： Thirty-two female Sprague-Dawley rats were divided equally into 4 groups （n-8）： PB-Sham group in which the rats were anesthetized by intraperitoneal injection of pentobarbital sodium （1.0%, 40 mg/kg, PB） and received a sham operation without occlusion of liver blood flow; PB-IR group whose rats underwent partial hepatic IR after anesthesia; Iso-Sham group in which inhalation of 1.0 MAC isoflurane and sham operation was performed; Iso-IR group in which 1.0 MAC isoflurane was inhaled for 4 h and IR was performed. Rat model of warm partial hepatic IR was established by clamping the hepatic arteries and hilar vessels distributing to the left and median lobes to induce partial hepatic ischemia （70%） for 60 rain followed by reperfusion for 3 h. The rats were killed 3 h after declamping, and specimens of liver tissue and blood were obtained. The serum ALT and AST were detected as liver damage markers. Viability of myeloperoxidase （MPO） in liver was measured. The protein level of ICAM-1 in the liver was detected by immunohistochemistry and Western blotting. Results： Rats treated with 1.0 MAC isoflurane during warm partial （70%） hepatic ischemia 60 rain and 3 h reperfusion had significantly lower serum ALT and AST compared with rats anesthetized with pentobarbital sodium subjected to hepatic IRI. The expression of ICAM-1 in hepatic tissue was significantly increased by hepatic IRI after pentobarbital sodium anesthesia. Isoflurane significantly inhibited protein expression of ICAM-1 in hepatic IR injury compared with pentobarbital sodium anesthesia. Viability of liver MPO was significantly increased by hepatic IRI after pentobarbital sodium anesthesia; Isoflurane can significantly inhibit MPO alteration in rat liver ischemia-reperfusion injury compared with rats anesthetized with pentobarbital sodium. Conclusion： Isoflurane anesthesia can attenuate liver IR injury in rats that maybe by inhibiting ICAM-I expression and reducing the infiltration of neutrophils.     

Ventricular remodeling and intervention with losartan following rabbit chronic hibernating myocardium

Objectives To explore the changes of myocyte and the interstitial collagen in a model of chronic hibernating myocardium (CHM) in rabbits, and to determine whether these alterations affect the cardiac function, and to further observe the effects of losartan on ventricular remodeling. Methods A left anterior descending (LAD) coronary artery stenosis was created and maintained for 4 weeks to create a CHM model in rabbits. Thirty-six rabbits were assigned to the following three groups(12 rabbits per group): CHM for 4 weeks(CHM group), low-dose of losartan intervention group for 4 weeks(LTl group, 10 mg·kg-1·d-1), high-dose of losartan intervention group for 4 weeks (LT2 group, 30 mg·kg-1·d-1); and 12 sham-operated rabbits served as controls (SO group). A microscopic imaging system (Image-Pro Plus, Olympus) was used to assess the Interstitial collagen volume fraction (ICVF) in myocardial sections with picrosirius-red staining, and a polarized light microscopy to qualitatively analysis the changes in the type and the proportion of collagen fibers, to semi-quantitatively score the proportion of collagen I to collagen III(PI/III). The expression of MMP-2, 9 and TIMP-2 was assessed by immunohistochemistry and western bloting. The myocyte apoptosis rate (Rapo) was calculated with TUNEL-staining. And echocardiography was performed to measure left ventricular end-systolic and end-diastolic diameter (LVESD, LVEED), and left ventricular short-axis fraction shortening (LVFS) and ejection fraction (LVEF). Results The animal model of CHM was induced successfully in 36 out of 39-rabbits and maintained for 28 days. Compared with the sham group, ICVF) was significantly increased (P<0.01) in CHM group; compared with CHM group, ICVF was significantly decreased (P<0.01,each) in LTl group and LT2 group, and the change were more remarkable in LT2 group, compared to LTl group(P<0.05). Compared with sham group, PI/IH was significantly increased (P < 0.01) in CHM group; compared with CHM group, PI/III was significantly decreasedin the losartan intervention groups (P<0.01,each), and the change was more remarkable in LT2 group compared to LTl group (P<0.05). Compared with sham group, the expression of MMP-2 and MMP-9 were greatly increased (P<0.01) in CHM group, while TIMP-2 were greatly decreased(P< 0.01); compared with CHM group, the expression of MMP-2 and MMP-9 were significantly decreased (P<0.01,each) .while TIMP-2 were significantly increased (P<0.01,each) in the losartan intervention groups, and the change was more remarkable in LT2 group than in LT1 group (.P<0.05). Compared with sham group, myocyte Rapo was markedly increased (P<0.01) in CHM group; compared with CHM group, myocyte Rapo was significantly decreased (P<0.01,each) in the losartan intervention groups, and the change was more remarkable in LT2 group than in LT1 group (P<0.05). Compared with sham group, LVEF and LVFS were significantly reduced in CHM group (P <0.01), compared with CHM group, LVEF and LVFS were higher (P<0.01,each) in the losartan intervention groups ,and the changes were more remarkable in LT2 group than in LTl group (P<0.05). Conclusions CHM underwent interstitial collagen proliferation and myocyte apoptosis, leading to ventricular remodeling and ventricular functional impairment, Losartan intervention reduces myocyte apoptosis and interstitial collagen proliferation, and improve ventricular function.     

Effect of Resveratrol on Motor Dysfunction in Unilateral 6-hydroxydopamine Parkinson’s Model Rats and its Underlying Mechanisms

Objective：The present study was designed to investigate the effect of resveratrol（Res） on the motor function of Parkinson’s model rats induced by 6-hydroxydopamine （6-OHDA） and to explore its underlying mechanism. Methods：After a week of adaptive feeding,50 male Sprague-Dawley （SD） rats were randomly divided into fi ve groups：sham-operated group,normal group （Res,30 mg·kg-1）,model group （6-OHDA,8 μg）,6-OHDA+Res low-dose group （15 mg·kg-1,6-OHDA+Res high-dose group （30 mg·kg-1）. 6-OHDA 8 μg （2 μg·μL-1） were injected into the substantia nigra of the rats to establish the model of dopaminergic neuronal damage,while the rats of sham group were injected with volume-matched saline with 0.2% Vitamin C.Rats were pretreated with Res for 1 day before 6-OHDA treatment. Model and sham groups were administered with volume-matched vehicle for 36 days.Rotarod test was applied to evaluate motor function of rats on 7 th,14 th and 21 th day after surgery,open fi eld experiment and grid-walking test applied on 33 th and 35 th day,respectively. Then the rats were sacrificed.Immunohistochemistry was used to detect the number of TH-positive cells in substantia nigra pars compacta （SNc）;the protein expression of TH,Bax,Bcl-2,pro-caspase-3, active-caspase-3,p-PDK1 and p-Akt in midbrain were detected by Western blot. Results：The body weight,motor function,number of dopaminergic neurons in SNc and the protein expression of TH in midbrain of model group were significantly decreased compared with sham group;the protein of Akt phosphorylation levels were decreased while it showed no effect on PDK1,and the expression of apoptosis-related proteins Bax/Bcl-2,active-caspase-3 were significantly increased,pro-caspase-3 decreased. However,compared with model,the body weight,motor function,number of dopaminergic neurons in SNc and the protein expression of TH in midbrain of Res high-dose group were markedly increased;the protein of Akt phosphorylation levels were increased while it showed no effect on PDK1,the protein level of BDNF and TrkB were signifi cantly decreased and the expression of apoptosis-related proteins Bax/Bcl-2,active-caspase-3 were significantly decreased,and pro-caspase-3 were increased.Conclusion：Under the experimental conditions,6-OHDA-induced motor dysfunction and apoptosis in dopaminergic neuronal cells of rats is signifi cantly attenuated by Res,and its potential mechanism may be related to up-regulation of Akt phosphorylation at Ser473 and inhibit the expression of apoptosisrelated proteins.     

Effects of glycine and methylprednisolone on hemorrhagic shock in rats

Background Methylprednisolone (MP), a synthetic glucocorticosteroid, has been broadly studied in experiments on endotoxin-induced shock and septic shock. This study was designed to ascertain whether glycine and MP can protect against organ injury and death caused by hemorrhagic shock, and to elucidate the underlying mechanisms of these protective effects in rats.Method To establish a shock model, Wistar rats were bled to maintain mean arterial pressure at 30-50 mmHg for 1 hour and subsequently resuscitated with the shed blood and normal saline. Just prior to resuscitation, the rats were randomly assigned to four groups: sham group (operation performed without inducing shock), shock group, shock+glycine group (glycine injected at the beginning of resuscitation) and shock+MP group (MP injected at the beginning of resuscitation).Results ① Seventy-two hours after resuscitation, the survival rate of rats from the shock group had decreased to 20%, while the survival rates of rats from the shock+glycine and shock+MP groups were 77.8% and 80%, respectively. The difference was significant (P&lt;0.05). ② Eighteen hours after resuscitation, pathological alterations in the organs of the rats were apparent. In rats from the shock group, edema, interstitial leukocyte infiltration, and cellular degeneration occurred in the liver, lungs, kidneys, and heart. Glycine and MP reduced these pathological changes significantly. ③ Eighteen hours after resuscitation, the levels of creatine phosphokinase, transaminases, and creatine were elevated significantly in rats from the shock group, indicating injury to the heart, liver, and kidneys, while these levels were elevated only slightly in the shock+glycine and shock+MP groups. The differences were significant (P&lt;0.01). ④ There were significant increases in intracellular calcium and production of tumor necrosis factor (TNF-α) by isolated Kupffer cells stimulated by endotoxin after hemorrhagic shock. These changes were completely prevented by glycine and MP (P&lt;0.01). Conclusion Glycine and MP reduce organ injury and mortality caused by hemorrhagic shock by preventing increase of intracellular calcium levels in Kupffer cell, suppressing Kupffer cell activation, decreasing the production of TNF-α by Kupffer cells, and blocking systemic inflammatory responses.     

Tojapride Reverses Esophageal Epithelial Inflammatory Responses on Reflux Esophagitis Model Rats

Objective: To investigate the mechanism of Tojapride, a Chinese herbal formula extract, on strengthening the barrier function of esophageal epithelium in rats with reflux esophagitis (RE). Methods: Ten out of 85 SD rats were randomly selected as the sham group (n=10), and 75 rats were developed a reflux esophagitis model (RE) by the esophageal and duodenal side-to-side anastomosis. Fifty successful modeling rats were divided into different medicated groups through a random number table including the model, low-, medium-, and high dose of Tojapride as well as omeprazole groups (n=10). Three doses of Tojapride [5.73, 11.46, 22.92 g/(kg?d)] and omeprazole [4.17 mg/(kg?d)] were administrated intragastrically twice daily for 3 weeks. And the rats in the sham and model groups were administered with 10 mL/kg distilled water. Gastric fluid was collected and the supernatant was kept to measure for volume, pH value and acidity. Esophageal tissues were isolated to monitor the morphological changes through hematoxylin-eosin (HE) staining, and esophageal epithelial ultrastructure was observed by transmission electron microscopy. The expressions of nuclear factor kappa-light-chain-enhancer of activated B cells p65 (NF-κ Bp65), κ B kinase beta (IKKβ ), occludin, and zonula occludens-1 (ZO-1) in the esophageal tissues were measured by immunohistochemistry and Western blot, respectively. Results: The gastric pH value in the model group was significantly lower than the sham group (P<0.05). Compared with the model group, gastric pH value in the omeprazole and medium-dose of Tojapride groups were significantly higher (P<0.05). A large area of ulceration was found on the esophageal mucosa from the model rats, while varying degrees of congestion and partially visible erosion was observed in the remaining groups. Remarkable increase in cell gap width and decrease in desmosome count was seen in RE rats and the effect was reversed by Tojapride treatment. Compared with the sham group, the IKKβ levels were significantly higher in the model group (P<0.05). However, the IKKβ levels were down-regulated after treatment by all doses of Tojapride (P<0.01 or P<0.05). The occluding and ZO-1 levels decreased in the model group compared with the sham group (P<0.01 or P<0.05), while both indices were significantly up-regulated in the Tojapride-treated groups (P<0.01 or P<0.05). Conclusions: Tojapride could improve the pathological conditions of esophageal epithelium in RE rats. The underlying mechanisms may involve in down-regulating the IKKβ expression and elevating ZO-1 and occludin expression, thereby alleviating the inflammation of the esophagus and strengthening the barrier function of the esophageal epithelium.     

EFFECTS OF PHYCOCYANIN ON EXPRESSION OF CytC mRNA AND CASPASE-3 mRNA AFTER FOCAL CEREBRAL ISCHEMIA／REPERFUSION IN RATS

Objective To study the effects of phycocyanin on the expression of Cytochrome C (CytC) genes and Caspase-3 genes after focal cerebral ischemia/reperfusion in rats. Methods A rat middle cerebral artery occlusion (MCAO)/reperfusion model was produced using the intraluminal filament method. The rats were divided into three groups: sham operation group, model control group and phycocyanin group. After MCAO, the neurobehavioral testing of all rats was made. The infarction area was evaluated with the method of 2,3,7-triphenylt-etrazolium chloride (TTC) staining. The expression of CytC mRNA and Caspase-3 mRNA were determined by in situ hybridization. Results In the sham operation group and the model control group, there was only a few CytCpositive cells were seen in the normal cerebral tissue. In the model control group, the upregulation of CytC mRNA began 6h after ischemia, reached a maximum at 12h (cortex)-24h (striatum) , then subsided gradually, but still in high level. In the phycocyanin group, CytC-positive cells were also mainly in cortex and striatum, but the number of the cells was significantly lower than the number of the model control group. The time-phase pattern of CytC mRNA in the phycocyanin group was similar to the pattern of the model control group. In the sham operation group and the model control group, there was only a few Caspase-3-positive cells were seen in the normal cerebral tissue. In the model control group, the upregulation of Caspase-3 mRNA began 6h after ischemia, reached a maximum at 24h and subsided at 48h, but still in high level. In the phycocyanin group, Caspase-3-positive cells were also mainly in the penumbral area, but the number of the cells were significantly lower than the number of the model control group. The time-phase pattern of Caspase-3 mRNA in the phycocyanin group was similar to the pattern of the model control group. Conclusion The over-expression of CytC mRNA and Caspase-3 mRNA might play a key role in ischemic cerebral injury after MCAO. Phycocyanin could inhibit the over-expression of CytC mRNA and Caspase-3 mRNA in the cerebral cortex, and might play an important role in the protection of ischemic neurons.     

Monocyte chemotactic protein 1 increases homing of mesenchymal stem cell to injured myocardium and neovascularization following myocardial infarction

Objective：To investigate the effect of MCP-1 on mesenchymal stem cells（MSCs） homing to injured myocardium in a rat myocardial infarction（MI） model. Methods：Rat myocardial infarction model was established by permanent left anterior descending branch ligation. Mesenchymal stem cells from donor rats were cultured in IMDM and labeled with BrdU. The Rats were divided into two groups. Monocyte chemotactic protein I（MCP-1） expression were measured by in situ hybridization and immunohistochemistry in the sham operated or infarcted hearts at 1, 2, 4, 7, 14 and 28 days post operation in MCP-1 detection group. The rats were injected with MCP-1, anti-MCP-1 antibody or saline 4 days after myocardial infarction in intervention group. Then, a total of 5 × 10^6 cells in 2.5 ml of PBS were injected through the tail vein. The number of the labeled MSCs in the infarcted hearts was counted 3 days post injection. Cardiac function and blood vessel density were assessed 28 days post injection. Results：Self-generating MCP-1 expression was increased at the first day, peaked at the 7^th day and decreased thereafter post MI and remained unchanged in sham operated hearts. The MSCs enrichment in the host hearts were more abundant in the MI groups than that in the non-MI group（P= 0.000）, the MSCs enrichment in the host hearts were more abundant in the MCP-1 injected group than that in the anti-MCP-1 antibody and saline injected groups （P = 0.000）. Cardiac function was improved more in MCP-1 injected group than anti-MCP-1 antibody and saline injected groups（P= 0.000）. Neovascularization in MCP-1 injected group significantly increased compared with that of other groups（P = 0.000）. Conclusion： Myocardial MCP-1 expression was increased only in the early phase post MI. MCP-1 may enhance MSCs homing to the injured heart and improve cardiac function by promoting neovascularization.