Rutaecarpine Inhibits Intimal Hyperplasia in A Balloon-Injured Rat Artery Model.

OBJECTIVE: To investigate the effect and potential mechanisms of rutaecarpine (Rut) in a rat artery balloon-injury model. METHODS: The intimal hyperplasia model was established by rubbing the endothelia with a balloon catheter in the common carotid artery (CCA) of rats. Fifty rats were randomly divided into five groups, ie. sham, model, Rut (25, 50 and 75 mg/kg) with 10 rats of each group. The rats were treated with or without Rut (25, 50, 75 mg/kg) by intragastric administration for 14 consecutive days following injury. The morphological changes of the intima were evaluated by hematoxylin-eosin staining. The expressions of proliferating cell nuclear antigen (PCNA) and smooth muscle (SM) α-actin in the ateries were assayed by immunohistochemical staining. The mRNA expressions of c-myc, extracellular signal-regulated kinase 2 (ERK2), MAPK phosphatase-1 (MKP-1) and endothelial nitric oxide synthase (eNOS) were determined by real-time reverse transcription-polymerase chain reaction. The protein expressions of MKP-1 and phosphorylated ERK2 (p-ERK2) were examined by Western blotting. The plasma contents of nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) were also determined. RESULTS: Compared with the model group, Rut treatment significantly decreased intimal thickening and ameliorated endothelial injury (P<0.05 or P<0.01). The positive expression rate of PCNA was decreased, while the expression rate of SM α-actin obviously increased in the vascular wall after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01). Furthermore, the mRNA expressions of c-myc, ERK2 and PCNA were downregulated while the expressions of eNOS and MKP-1 were upregulated (P<0.05 or P<0.01). The protein expressions of MKP-1 and the phosphorylation of ERK2 were upregulated and downregulated after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01), respectively. In addition, Rut dramatically reversed balloon injury-induced decrease of NO and cGMP in the plasma (P<0.05 or P<0.01). CONCLUSION: Rut could inhibit the balloon injury-induced carotid intimal hyperplasia in rats, possibly mediated by promotion of NO production and inhibiting ERK2 signal transduction pathways.

Evodiamine Inhibits Angiotensin II-Induced Rat Cardiomyocyte Hypertrophy.

OBJECTIVE: To investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms. METHODS: Cardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 µmol/L), and Evo (0.03, 0.3, 3 µmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca2+]i) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis. RESULTS: Compared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca2+]i) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 µmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca2+]i concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05). CONCLUSION: Evo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca2+]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

Rutaecarpine inhibits angiotensin II-induced proliferation in rat vascular smooth muscle cells.

OBJECTIVE: To evaluate the effects and possible mechanisms of rutaecarpine on angiotensin II (Ang II)-induced proliferation in cultured rat vascular smooth muscle cells (VSMCs). METHODS: VSMCs were isolated from Male Sprague-Dawley rat aorta, and cultured by enzymic dispersion method. Experiments were performed with cells from passages 3-8. The cultured VSMCs were randomly divided into control, model (Ang II 0.1 µmol/L), and rutaecarpine (0.3-3.0 µmol/L) groups. VMSC proliferation was induced by Ang II, and was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and cell counting. To examine the mechanisms involved in anti-proliferative effects of rutaecarpine, nitric oxide (NO) levels and NO synthetase (NOS) activity were determined. Expressions of VSMC proliferation-related genes including endothelial nitric oxide synthase (eNOS), and c-myc hypertension related gene-1 (HRG-1) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Rutaecarpine (0.3-3.0 µmol/L) inhibited Ang II-induced VSMC proliferation and the best effects were achieved at 3.0 µmol/L. The Ang II-induced decreases in cellular NO contents and NOS activities were antagonized by rutaecarpine (P <0.05). Ang II administration suppressed the expressions of eNOS and HRG-1, while increased c-myc expression (P <0.05). All these effects were attenuated by 3.0 µmol/L rutaecarpine (P <0.05). CONCLUSION: Rutaecarpine is effective against Ang II-induced rat VSMC proliferation, and this effect is due, at least in part, to NO production and the modulation of VMSC proliferation-related gene expressions.

Toxicology evaluation of realgar-containing niu-huang-jie-du pian as compared to arsenicals in cell cultures and in mice.

Niu-Huang-Jie-Du Pian (NHJD) is a widely used traditional Chinese medicine containing realgar (As4S4). Realgar has been included in many traditional medicines, but is often taken as arsenite for risk assessment. To evaluate true risk of realgar and realgar-containing NHJD, their toxicity was compared with common arsenicals. In cultured cells, the LC50 for NHJD (1200 µM) and realgar (2000 µM) was much higher than arsenite(35 µM), arsenic trioxide (280 µM), and arsenate (400 µM). Acute toxicity in mice showed more severe liver and kidney injury after arsenite or arsenate, but was mild after realgar and NHJD, corresponding to cellular and tissue arsenic accumulation. The expressions of arsenic-sensitive stress gene metallothionein-1 were increased 3-7-folds after arsenite or arsenate, but were unaltered after NHJD and realgar. Thus, realgar and NHJD are much less toxic than arsenite and arsenate. The use of total arsenic to evaluate the safety of realgar and realgar-containing NHJD is inappropriate.

Protective effects of icariin on cognitive deficits induced by chronic cerebral hypoperfusion in rats.

1. Icariin is a major constituent of flavonoids derived from the Chinese medicinal herb Epimedium revicornum Maxim. The aim of the present study was to investigate whether icariin has protective effects on learning ability and memory in a rat model of chronic cerebral hypoperfusion. 2. Chronic cerebral hypoperfusion was induced by permanent ligation of the common carotid artery in Wistar rats for 4 months. One month after permanent artery occlusion, rats were adminitered icariin at doses of 0, 30, 60 or 120 mg/kg per day, p.o., for 3 months. Neurobehavioural and neurobiochemical parameters were examined to evaluate the effects of icariin on cognitive deficits induced by chronic cerebral hypoperfusion. 3. The Morris water maze test revealed that learning ability and memory were severely impaired in untreated rats, but were significantly improved in icariin-treated rats. Icariin treatment also ameliorated chronic cerebral hypoperfusion-induced oxidative stress in the brain, as evidenced by reduced malondialdehyde formation and maintained superoxide dismutase activity. In addition, the decreased hippocampal levels of acetylcholine, acetylcholinesterase and choline acetyltransferase associated with chronic cerebral hypoperfusion were significantly prevented by icariin treatment. 4. In conclusion, icariin protects against cognitive deficits induced by chronic cerebral hypoperfusion in rats. These effects appear to be mediated through its anti-oxidant effects, as well as its effects on the circulatory and cholinergic systems.

Effects of isorhynchophylline on angiotensin II-induced proliferation in rat vascular smooth muscle cells.

Proliferation of vascular smooth muscle cells (VSMCs) is a crucial event in cardiovascular diseases. Isorhynchophylline, an alkaloid from a traditional Chinese medicine Gambirplant, has been used to treat cardiovascular diseases. The aim of this study was to investigate the effects of isorhynchophylline on angiotensin II (Ang II)-induced proliferation of rat VSMCs. VSMCs were isolated from rat artery and cultured for 14 days before experimentation. The effect of isorhynchophylline on Ang II-induced proliferation was evaluated by cell number, MTT assay and flow cytometry, and nitric oxide (NO) content and activity of NO synthase (NOS) were measured. The expression of proto-oncogene c-fos, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) mRNAs was measured by real-time RT-PCR. VSMC cultures were verified by morphology and immunostaining with alpha-smooth muscle actin. Isorhynchophylline (0.1-10.0 microM) was not toxic to VSMCs, but markedly decreased Ang II (1.0 microM)-enhanced cell number and MTT intensity, and blocked cell transition from G(0)/G(1) to S phase. Furthermore, isorhynchophylline increased the NO content and NOS activity, and suppressed Ang II-induced over-expression of c-fos, OPN and PCNA. Thus, isorhynchophylline was effective against Ang-II induced cell proliferation, an effect that appears to be due, at least in part, to increased NO production, regulation of the cell cycle, and depressed expression of c-fos, OPN and PCNA related to VMSC proliferation.

[Effects of combined use of total alkaloids of Uncaria rhynchophylla and Coryadlis ambailis migo on cerebral ischemia-reperfusion injury in rats].

OBJECTIVE: To study the effects of combined use of total alkaloids (TA) of Uncaria rhynchophylla (UR) and Coryadlis ambailis migo (CAM) on cerebral ischemia/reperfusion injury in rats. METHODS: Rat model of middle cerebral artery ischemia/reperfusion was established, the changes of neurological state was scored before and after treatment with the two kinds of TA, single or combined, and the changes of cerebral infarcted volume, cerebral water content, activities of NOS and SOD and content of MDA in rats' brain were estimated as well. RESULTS: After being treated with the combination of both TA, the average neurological score, cerebral infracted volume, cerebral water content, activity of NOS and content of MDA in the model rats significantly decreased, and the activity of SOD was significantly increased (all P < 0.05). The effect of combined use of the two TA was higher than that of use TA of UR or CAM alone (P <0.05). Moreover, the central nervous system inhibitory effect induced by combined TA was significantly weaker than that of UR. CONCLUSION: Combined use of TA of UR and CAM may facilitate the protection against cerebral ischemia/reperfusion damage, the action mechanism might be relevant to reducing the lipid peroxidation injury of brain cells through inhibiting the NOS activity and increasing the SOD activity.

Protective effect of Ginkgo biloba leaf extract on learning and memory deficit induced by aluminum in model rats.

OBJECTIVE: To examine the protective effect of Ginkgo biloba leaf extract (GbE) on learning and memory deficit induced by aluminum chloride (AlCl(3)), and explore its mechanisms. METHODS: The rat models with learning and memory deficit were induced by administering via gastrogavage and drinking of AlCl(3) solution. And the model rats were treated with GbE at the dose of 50, 100, 200 mg/kg every day for 2 months accompanied with drinking of AlCl(3) solution, respectively. Their abilities of spatial learning and memory were tested by Morris water maze, and the acetylcholinesterase (AChE) activity in serum was assayed with chemical method, the AChE expression in hippocampus was observed by immunohistochemistry assay, and then quantitative analysis was done by BI 2000 image analysis system. RESULTS: Learning and memory deficit of rats could be induced by AlCl(3) solution (P < 0.01), and AChE expressions in rats hippocampus were increased (P < 0.01); GbE ameliorated learning and memory deficit and reduced AChE expression in rats hippocampus in a dose-dependent manner, while GbE significantly increased serum AChE activity at the dose of 200 mg/kg each day (P < 0.05). CONCLUSION: GbE can ameliorate learning and memory deficit induced by AlCl(3), which may be due to its inhibition of the AChE expression in hippocampus.

Protective effects of Ginkgo biloba leaf extract on aluminum-induced brain dysfunction in rats.

This study examined the protective effects of Ginkgo biloba extract (GbE) on the learning and memory function in aluminum-treated rats and potential mechanisms. Wistar rats were given daily aluminum chloride 500 mg/kg, i.g, for one month, followed by continuous exposure via the drinking water containing 1600 ppm aluminum chloride for up to 5 months. The ability of spatial learning and memory was tested by Morris water maze. Aluminum administration significantly increased escape latency and searching distance, indicative of brain dysfunction. GbE treatment (50-200 mg/kg, i.g) significantly protected against aluminum-induced brain dysfunction, as evidenced by decreased escape latency and searching distance compared with the Al alone group. To examine the mechanisms of the protection, the expressions of amyloid precursor protein (APP) and caspase-3 in brain regions were examined by immunohistochemistry. GbE treatment reduced the contents of APP and caspase-3 in hippocampus of aluminum-treated rats in a dose-dependent manner. At the highest dose of GbE (200 mg/kg), the immunostain for APP and caspase-3 was returned to normal levels. In summary, this study demonstrates that GbE is effective in improving the ability of spatial learning and memory of aluminum-intoxicated rats. This protection appears to be due to a decreased expression of APP and caspase-3 in rat brain, resulting in a decrease in the production of insoluble fragments of Abeta-amyloid.

Effects of protopine on intracellular calcium and the PKC activity of rat aorta smooth muscle.

We have previously shown that the vasodilator effect of protopine (Pro) on rabbit aorta is related to the elevations of cAMP and cGMP. In the present study, the vasodilator mechanisms of Pro were further explored by recording the isotonic contraction of the rat aortic strips, detecting directly the intracellular free Ca(2+) concentration ([Ca(2+)](i)) with Fura-2/AM loaded vascular smooth muscle cells (VSMCs) of rat aorta, and determining the activity of protein kinase C (PKC) in rat aortic tissue with radioactive isotope gamma-32P -ATP-catalyzing assay. By recording the aortic strips contraction induced by noradrenaline (NA) and high potassium (K(+)), Pro shifted nonparallelly the concentration-response curves of NA and high K(+) to right, in which the maximal response was depressed in the presence of Pro (30 and 100 micromol/L), and the values of pD'(2) were 3.70-/+0.25 and 3.97-/+0.15 for NA and high K(+), respectively. In the Fura-2/AM loaded VSMCs, Pro (50 and 100 micromol/L) could not produce any significant change on the resting [Ca(2+)](i), but significantly decreased the [Ca(2+)](i) elevated by NA and high K(+). Pro (30 and 100 micromol/L) had no significant effect on the activity of the cytosolic and membrane PKC in the aortic strips inpretreated by NA. However, in the aortic strips pretreated by NA, the activity of membrane PKC was significantly increased and the activity of cytosolic PKC tended to be decreased by Pro, while the activity of total PKC did not change. These results suggest that Pro seems to promote the translocation of PKC from the cytosol to the membrane in the presence of NA, its vasodilator effect may be the comprehensive result of its decreasing effect on the [Ca(2+)](i) and the increasing effect on cAMP and cGMP, as well as its influence on the PKC.

[Effects of Corydalis ambailis migo total alkaloids on experimental cerebral ischemia].

OBJECTIVE: To observe the protective effects of Corydalis ambailis migo total alkaloids (COAMTA) on cerebral ischemia/reperfusion injury in rats and to investigate its mechanism. METHODS: The effects of COAMTA on decapitated gasping mouse model and rat model of middle cerebral artery ischemia (2 h)/reperfusion (22 h) were observed. The neurological scale, cerebral infarcted volume and cerebral water content subjected to cerebral middle artery ischemia/reperfusion in rats were recorded. The activities of nitric oxide synthase (NOS) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the ratso brain were measured. Cell apoptosis in ischemic penumbral area was observed with light microscope in the method of terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). RESULTS: The average gasping time of the mice (6.0 mg/kg or 9.0 mg/kg COAMTA) was significantly prolonged, the cerebral infarcted volume and cerebral water content of the rats (5.0 mg/kg or 7.5 mg/kg COAMTA) were significantly decreased, as compared with the control groups. The average activity of SOD in cerebral tissue of the rats (5.0 mg/kg or 7.5 mg/kg COAMTA) was significantly higher than that of the control groups, meanwhile, the average activity of NOS and the content of MDA declined significantly. The cell apoptosis in ischemic penumbral area of the rats (5.0 mg/kg COAMTA) was significantly inhibited as compared with the control groups. CONCLUSION: COAMTA can facilitate the protection against cerebral ischemia/reperfusion damage. The mechanism is related to inhibiting the activity of NOS and lipoperoxidation, increasing the activity of SOD and decreasing the neuronal apoptosis.

Ginkgo biloba leaf extract enhances levels of caspase-3 and amyloid precursor protein in normal rat hippocampus.

AIM: To explore the effect of Ginkgo biloba extract (GbE) on the levels of caspase-3 and amyloid precursor protein (APP) in normal rats' hippocampus. METHODS: Immunohistochemistry method was used for qualitative analysis of the expressions of caspase-3 and APP, and an image analysis method was used for the quantification of the levels of caspase-3 and APP after GbE was administered to rats of different ages for 14 d. RESULTS: The mean absorbance of staining of caspase-3 and APP was markedly higher in GbE group than that in control groups. The expressions of caspase-3 and APP were intensified in the hippocampus of rats after GbE administration. CONCLUSION: GbE can raise the levels of caspase-3 and APP in the hippocampus of normal rats.