OBJECTIVETo investigate the fetotoxicity of monocrotaline.

METHODMouse whole embryo culture (WEC) was applied. Post-implantation (8.5 d) mouse embryos were isolated from their mothers and put into the medium of immediately centrifuged serum (ICS) prepared from rats. Different concentrations of monocrotaline (100, 50, 25, 12.5 mg x L(-1)) were added into the WEC. Development (yolk sac diameter, crown-rump length, head length, somite number) and organic morphodifferentiation (yolk sac circulation, allantois, embryonic flexion, heart, brain, optic-otic-olfactory organ, branchial arch, maxillary, mandible, bud) of embryos were observed at 48 h after treatment.

RESULTObvious fetotoxicity could be observed in various monocrotaline treatment groups in a dose-dependent manner. Development of embryos was delayed significantly at dose 12.5-100 mg x L(-1). Malformations were shown in all organic morphodifferentiation indice, especially in opti-otic organ, mandible and bud.

CONCLUSIONMonocrotaline had obvious fetotoxicity in vitro WEC, indicating that exposure of pregnant mice to monocrotaline may have potential risk on fetus.

Animals ; Cell Differentiation ; drug effects ; Culture Media ; Embryo, Mammalian ; drug effects ; physiology ; Female ; Male ; Mice ; Monocrotaline ; toxicity

OBJECTIVETo modify the empirical method of precision-cut liver slice technique, and study the hepatotoxicity of monocrotaline by this technique.

METHODLiver slices were prepared by the domestic shaking slicer. The technique of precision-cut liver slice was established by detecting MTT reduction used as the slice viability under different culture medium, thickness of slices, pH and culture temperature. After monocrotaline and liver slices co-culture for 6, 24 h, the slice viability, enzyme activity of GPT, GOT, LDH, GGT and protein concentration were detected by MTT reduction, enzyme kinetics method and BCA protein assay method, respectively.

RESULTWhen the thickness of slices was 200 microm and pH of medium was 6.8, culture temperature was 37 degrees C, BPM culture medium, the viability of slices could maintain on a steady level. LDH leakage was significantly increased and protein content was obviously decreased after monocrotaline co-culture for 24 h with final concentration 0.02, 0.1 and 0.5 g x L(-1). No statistically significant difference between control group and monocrotaline 3 dose groups was observed in the slice viability and the content of GPT, GOT, LDH, GGT and protein after monocrotaline co-culture for 6 h.

CONCLUSIONThe slice viability could retain 24 h in modified BPM medium surroundings; monocrotaline displayed liver toxicity in some degree after co-culture for 24 hours in 0.02, 0.1 and 0.5 g x L(-1) concentration.

Animals ; Hydrogen-Ion Concentration ; L-Lactate Dehydrogenase ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Monocrotaline ; toxicity ; Rats ; Rats, Sprague-Dawley ; Temperature ; Toxicity Tests

OBJECTIVETo observe the effect of total ginsenosides (TG) on right ventricular hypertrophy induced by monocrotaline (MCT) in rats, and study its relationship with the nitric oxide pathway.

METHODMale Sprague Dawley rats were randomly divided into the control group, the MCT model group, TG-treated (20, 40, 60 mg kg-1 d-1) groups, and the L-arginine (L-arg) th NO release, T + L-N and L-a + L-N groups were wi th NOS into study TG's effect 200 mg kg-1 d-1 group. Besides, and its relationship wi also set, intraperitoneally injected with TG 40 mg kg-1 d -1 and L-arg 200 mg kg-1 - d-1, and orally administered hibitor L-NAME 20 mg kg-1 d-1. After all of the groups were given drugs for 18 d, their right ventricular peak systolic pressure (RVSP) ventricular hypertrophy index (RVHI) and RVW/BW were determined. Ultra-structure of myocardial cells was observed with transmission electron microscope. The NO2 -/NO3 - content in myocardial tissues were detected with the nitrate reduction method. ANF and eNOS mRNA expressions in right ventricle tissues were detected by using real-time RT-PCR.

RESULTLow, middle and high doses of TG and L-arg preventive administration could significantly reduce RVSP, RVHI, RVW/BW and ANF mRNA expressions (P < 0. 05) , and ameliorate cellular mitochondrial swelling and degeneration. L-NAME could prevent the effect of L-arg on above indexes, whereas L-NAME of the same dose could not impact the reducing effect of TG 40 mg kg -1 on above indexes. TG 60 mg kg -1 could raise eNOS mRNA expression, but TG 20 mg kg-1 and 40 mg kg-1 showed no effect.

CONCLUSIONTG can significantly attenuate MCT-induced right cardiac hypertrophy in rats. Its anti-hypertrophic effect is partially realized through NO.

Animals ; Ginsenosides ; therapeutic use ; Hypertrophy, Right Ventricular ; chemically induced ; drug therapy ; metabolism ; Male ; Monocrotaline ; toxicity ; Nitric Oxide ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVEPulmonary hypertension is a proliferative vascular disease characterized by pulmonary vascular structural remodeling. Until now, the pathogenesis of pulmonary hypertension is still not fully understood. Although considerable progress has been made, there is, to date, no cure for advanced pulmonary vascular disease. Recently, a number of studies suggest that endogenous vascular elastase (EVE) play a role in the vascular changes associated with pulmonary hypertension. The purpose of the study was to determine whether an elastase inhibitor might reverse advanced pulmonary vascular disease produced in rats by injection of monocrotaline.

METHODSOne hundred and twenty male Sprague-Dawley rats were used in this study. The rats were divided into three groups: control, model and ZD-0892 groups. In the model and ZD-0892 groups, the rats were subjected to a single subcutaneous injection of monocrotaline (60 mg/kg) in the hind flank, while the rats in control group received an equivalent volume of 0.9% saline. From day 21, the rats in the ZD-0892 and model groups received twice-daily gavage tube feedings of either ZD-0892 at a dose of 240 mg/kg per day or its administration vehicle, while the rats in control group were subjected to an equivalent volume of 0.9% saline. On days 21, 28 and 35 post-injection, the elastolytic activity was measured with a fluorescence microplate reader and pulmonary artery pressure was detected via catheterization. Meanwhile, the lungs were evaluated morphologically, using the barium-gelatin perfusion technique.

RESULTSThe injection of monocrotaline led to severe pulmonary hypertension in rats 21 days later and pulmonary artery elastolytic activity increased remarkably. A 1-week treatment with ZD-0892 resulted in declines in elastase activity. This was associated with significant declines in pulmonary artery pressure, decreases in muscularization of peripheral arteries and reductions in medial hypertrophy. After 2 weeks, elastase activity returned to normal level. Pulmonary artery pressure and structure were normalized.

CONCLUSIONIncreased elastase activity is important in the development of vascular changes and progressive pulmonary hypertension. ZD-0892 can suppress the elastase activity and completely reverse the fatal pulmonary hypertension induced by monocrotaline in rats.

Animals ; Hypertension, Pulmonary ; chemically induced ; drug therapy ; Male ; Monocrotaline ; toxicity ; Pancreatic Elastase ; antagonists & inhibitors ; Pulmonary Artery ; drug effects ; pathology ; physiopathology ; Pyrroles ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sulfonamides ; pharmacology

In this study, we used a rat model of pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT) to investigate the role and mechanism of angiotensin (Ang)-(1-7) in regulating pulmonary artery diastolic function. Three weeks after subcutaneous injection of MCT or normal saline, the right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) of rats were detected using a right heart catheter. Vascular endothelium-dependent relaxation was evaluated by acetylcholine (ACh)-induced vasodilation. The relaxation function of vascular smooth muscle was evaluated by sodium nitroprusside (SNP)-induced vasodilation. Human pulmonary artery endothelial cells (HPAECs) were incubated with Ang-(1-7) to measure nitric oxide (NO) release levels. The results showed that compared with control rats, RVSP and RVHI were significantly increased in the MCT-PAH rats, and both ACh or SNP-induced vasodilation were worsened. Incubation of pulmonary artery of MCT-PAH rats with Ang-(1-7) (1 × 10^-9-1 × 10^-4 mol/L) caused significant vaso-relaxation. Pre-incubation of Ang-(1-7) in the pulmonary artery of MCT-PAH rats significantly improved ACh-induced endothelium-dependent relaxation, but had no significant effect on SNP-induced endothelium-independent relaxation. In addition, Ang-(1-7) treatment significantly increased NO levels in HPAECs. The Mas receptor antagonist A-779 inhibited the effects of Ang-(1-7) on endothelium-dependent relaxation and NO release from endothelial cells. The above results demonstrate that Ang-(1-7) promotes the release of NO from endothelial cells by activating Mas receptor, thereby improving the endothelium-dependent relaxation function of PAH pulmonary arteries.
Rats ; Humans ; Animals ; Vasodilation ; Pulmonary Arterial Hypertension ; Monocrotaline/toxicity* ; Rats, Sprague-Dawley ; Hypertension, Pulmonary/chemically induced* ; Endothelial Cells ; Pulmonary Artery ; Endothelium ; Acetylcholine/pharmacology* ; Nitroprusside/pharmacology*

OBJECTIVETo observe the effects and related mechanisms of fasudil on monocrotaline-induced pulmonary arterial hypertension (PAH) in rats.

METHODSA total of 56 healthy male Sprague-Dawley rats were randomly divided into 5 groups: 4 weeks control group (N4), 4 weeks PAH group (M4), 8 weeks control group (N8), 8 weeks PAH group (M8), 8 weeks PAH and fasudil group (F8). PAH was induced by subcutaneous injection of monocrotaline (50 mg/kg). Animals in F8 group received intraperitoneal injection of fasudil hydrochloride (15 mg×kg(-1)×d(-1)) from the end of the 4th week to the end of the 8th week. Rats in control groups and PAH groups received equal volume saline injection. Polyethylene catheters were inserted into the RV through the jugular vein for right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) measurements after various treatment protocols. RV hypertrophy index [RV/(LV+S)] was also measured. Arteries of 50 to 150 µm were evaluated for the median wall thickness and wall area by HE staining as follows: percent wall thickness (WT%) = [(medial thickness×2/external diameter)]×100 and percent wall area (WA%) = (wall area/total area)×100%. The mRNA expression of ROCK-1 in lung tissue was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The protein expressions of ROCK-1 and MYPT-1 in lung tissue were analyzed by Western blot and MYPT-1 phosphorylation, respectively.

RESULTSForty-one rats survived and mortality rate was zero in N4, N8 and M4 groups. Survival rate was significantly higher in F8 group compared to M8 group (75.00% vs. 31.25%, P < 0.05). At the end of the 4th week, RVSP [(62.25 ± 3.24) vs. (31.33 ± 2.35) mm Hg(1 mm Hg = 0.133 kPa)], mPAP [(36.38 ± 2.31) vs.(20.32 ± 1.81) mm Hg], [RV/(LV+S)] (0.5648 ± 0.0580 vs. 0.3458 ± 0.0455), WT% [(25.63 ± 5.35)% vs.(13.38 ± 3.45)%], WA% [(60.36 ± 2.51)% vs. (38.42 ± 2.84)%] were all significantly higher in M4 group than in N4 group (all P < 0.01). RVSP [(54.64 ± 4.11) vs. (67.37 ± 4.68) mm Hg], mPAP [(26.25 ± 2.32) vs. (39.83 ± 1.83) mm Hg], and markedly relieve [RV/(LV+S)] (0.3985 ± 0.0210 vs. 0.7600 ± 0.0341), WT% [(15.64 ± 2.81)% vs. (28.26 ± 4.38)%], WA% [(40.35 ± 2.82)% vs. (68.83 ± 1.63)%] were all significantly lower in F8 group than in M8 group (all P < 0.05) while the expression of ROCK-1 mRNA (1.2139 ± 0.1778 vs. 1.6839 ± 0.3251, P < 0.01), and the protein expression of ROCK-1 and MYPT-1 as well as the extent of MYPT-1 phosphorylation were all downregualted in F8 group compared to M8 group (all P < 0.01).

CONCLUSIONSFasudil can effectively reverse the MCT-induced PAH in rats via downregulating ROCK-1 and MYPT-1.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; therapeutic use ; Animals ; Disease Models, Animal ; Familial Primary Pulmonary Hypertension ; Hypertension, Pulmonary ; chemically induced ; drug therapy ; Male ; Monocrotaline ; toxicity ; Protein Phosphatase 1 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Treatment Outcome ; rho-Associated Kinases ; metabolism

The aim of the present study is to investigate the protective effect of chrysin (5,7-dihydroxyflavone) on right ventricular remodeling in a rat model of monocrotaline-induced pulmonary arterial hypertension (PAH). PAH rats were induced by a single injection of monocrotaline (60 mg x kg(-1), sc) and were administered with chrysin (50 or 100 mg x kg(-1) x d(-1)) for 4 weeks. At the end of experiment, the right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored via the right jugular vein catheterization into the right ventricle. Right ventricle (RV) to left ventricle (LV) + septum (S) and RV to tibial length were calculated. Right ventricular morphological change was observed by HE staining. Masson's trichrome stain was used to demonstrate collagen deposition. The total antioxidative capacity (T-AOC) and malondialdehyde (MDA) levels in right ventricle were determined according to the manufacturer's instructions. The expressions of collagen I, collagen III, NADPH oxidase 4 (NOX4) and nuclear factor-kappa B (NF-κB) were analyzed by immunohistochemisty, qPCR and (or) Western blot. The results showed that chrysin treatment for 4 weeks attenuated RVSP, mPAP and right ventricular remodeling index (RV/LV+S and RV/Tibial length) of PAH rats induced by monocrotaline. Furthermore, monocrotaline-induced right ventricular collagen accumulation and collagen I and collagen III expression were both significantly suppressed by chrysin. The expressions of NOX4, NF-κB and MDA contents were obviously decreased, while the T-AOC was significantly increased in right ventricule from PAH rats with chrysin treatment. These results suggest that chrysin ameliorates right ventricular remodeling of PAH induced by monocrotaline in rats through its down-regulating of NOX4 expression and antioxidant activity, and inhibiting NF-κB expression and collagen accumulation.
Animals ; Blotting, Western ; Collagen ; metabolism ; Disease Models, Animal ; Flavonoids ; pharmacology ; Heart Ventricles ; drug effects ; metabolism ; Hypertension, Pulmonary ; chemically induced ; metabolism ; Monocrotaline ; toxicity ; NADPH Oxidase 4 ; NADPH Oxidases ; metabolism ; NF-kappa B ; metabolism ; Rats ; Ventricular Remodeling ; drug effects

Pulmonary artery hypertension (PAH) causes right ventricular failure and possibly even death by a progressive increase in pulmonary vascular resistance. Bone marrow-derived mesenchymal stem cell therapy has provided an alternative treatment for ailments of various organs by promoting cell regeneration at the site of pathology. The purpose of this study was to investigate changes of pulmonary haemodynamics, pathology and expressions of various genes, including ET (endothelin)-1, ET receptor A (ERA), endothelial nitric oxide synthase (NOS) 3, matrix metalloproteinase (MMP) 2, tissue inhibitor of matrix metalloproteinase (TIMP), interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in monocrotaline (MCT)-induced PAH rat models after bone marrow cell (BMC) transfusion. The rats were grouped as the control (C) group, monocrotaline (M) group, and BMC transfusion (B) group. M and B groups received subcutaneous (sc) injection of MCT (60 mg/kg). BMCs were transfused by intravenous injection at the tail 1 week after MCT injection in B group. Results showed that the average RV pressure significantly decreased in the B group compared with the M group. RV weight and the ratio of RH/LH+septum significantly decreased in the B group compared to the M group. Gene expressions of ET-1, ERA, NOS 3, MMP 2, TIMP, IL-6, and TNF-alpha significantly decreased in week 4 in the B group compared with the M group. In conclusion, BMC transfusion appears to improve survival rate, RVH, and mean RV pressure, and decreases gene expressions of ET-1, ERA, NOS 3, MMP 2, TIMP, IL-6, and TNF-alpha.
Animals ; Bone Marrow Cells/*cytology ; *Bone Marrow Transplantation ; Cytokines/genetics/metabolism ; Enzymes/genetics/metabolism ; Gene Expression Regulation ; Hypertension, Pulmonary/chemically induced/*metabolism/pathology ; Lung/metabolism ; Male ; Monocrotaline/toxicity ; Pulmonary Artery/physiology ; Rats ; Rats, Sprague-Dawley ; Survival Rate ; Ventricular Function/physiology

OBJECTIVE: To explore the therapeutic mechanism of tanshinone IIA in the treatment of pulmonary arterial hypertension (PAH) in rats. METHODS: A total of 100 male SD rats were randomized into 5 groups (n=20), and except for those in the control group with saline injection, all the rats were injected with monocrotaline (MCT) on the back of the neck to establish models of pulmonary hypertension. Two weeks after the injection, the rat models received intraperitoneal injections of tanshinone IIA (10 mg/kg), phosphatidylinositol 3 kinase (PI3K) inhibitor (1 mg/kg), both tanshinone IIA and PI3K inhibitor, or saline (model group) on a daily basis. After 2 weeks of treatment, HE staining and α-SMA immunofluorescence staining were used to evaluate the morphology of the pulmonary vessels of the rats. The phosphorylation levels of PI3K, protein kinase B (PKB/Akt) and endothelial nitric oxide synthase (eNOS) in the lung tissue were determined with Western blotting; the levels of eNOS and NO were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of HE staining and α-SMA immunofluorescence staining showed that tanshinone IIA effectively inhibited MCT-induced pulmonary artery intimamedia thickening and muscularization of the pulmonary arterioles (P < 0.01). The results of Western blotting showed that treatment with tanshinone IIA significantly increased the phosphorylation levels of PI3K, Akt and eNOS proteins in the lung tissue of PAH rats; ELISA results showed that the levels of eNOS and NO were significantly decreased in the rat models after tanshinone IIA treatment (P < 0.01). CONCLUSION Treatment with tanshinone IIA can improve MCT-induced pulmonary hypertension in rats through the PI3K/Akt-eNOS signaling pathway.
Abietanes ; Animals ; Hypertension, Pulmonary/drug therapy* ; Male ; Monocrotaline/toxicity* ; Nitric Oxide Synthase Type III/therapeutic use* ; Phosphatidylinositol 3-Kinase/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Pulmonary Artery ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

The purpose of this study was to investigate the therapeutic effects of small hairpin RNA (shRNA) targeting endothelin-converting enzyme (ECE)-1 in monocrotaline (MCT)-induced pulmonary hypertensive rats. Ninty-four Sprague-Dawley rats were divided into three groups: control (n = 24), MCT (n = 35) and shRNA (n = 35). Four-week survival rate in the shRNA group was significantly increased compared to that in the MCT group. The shRNA group showed a significant improvement of right ventricular (RV) pressure compared with the MCT group. The MCT and shRNA groups also showed an increase in RV/(left ventricle + septum) ratio and lung/body weight. Plasma endothelin (ET)-1 concentrations in the shRNA group were lower than those in the MCT group. Medial wall thickness of pulmonary arterioles were increased after MCT injection and was significantly decreased in the shRNA group. The number of intra-acinar muscular pulmonary arteries was decreased in the shRNA group. The mRNA expressions of ET-1 and ET receptor A (ETA) were significantly decreased in the shRNA group in week 4. The protein levels of ETA were decreased in the shRNA group in week 2. The protein levels of tumor necrosis factor-alpha and vascular endothelial growth factor were decreased in the shRNA group in week 4. In conclusion, the gene silencing with lentiviral vector targeting ECE-1 could be effective against hemodynamic, histopathological and gene expression changes in pulmonary hypertension.
Animals ; Aspartic Acid Endopeptidases/*antagonists & inhibitors/blood/genetics ; Body Weight ; Heart Ventricles/physiopathology ; Hypertension, Pulmonary/chemically induced/*enzymology/mortality ; Lentivirus/genetics ; Lung/anatomy & histology/metabolism/pathology ; Male ; Metalloendopeptidases/*antagonists & inhibitors/blood/genetics ; Monocrotaline/toxicity ; Pulmonary Artery/drug effects/physiopathology ; RNA, Small Interfering/*metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/genetics/metabolism ; Survival Rate ; Tumor Necrosis Factor-alpha/metabolism ; Vascular Endothelial Growth Factor A/metabolism