Quercetin attenuates the progression of monocrotaline-induced pulmonary hypertension in rats

Pulmonary arterial hypertension （PAH） is a progressive disease associated with increased constriction and remodeling of the pulmonary vasculature. Quercetin is a natural fiavonoid and has a variety of pharmacological effects including improvement of endothelial cell function. However, its pharmacological effects on pulmonary hypertension have been rarely reported. We sought to observe the protective effect of quercetin in rats with monocrotaline induced PAH. We divided 30 male Sprague-Dawley rats randomly into three groups with ten rats in each group： the monocrotaline group, the quercetin group and the control group. We found that, compared with the controls, the mean pulmonary artery pressure （mPAP） and the right ventricular hypertrophy index in the monocrotaline group were significantly higher （P 〈 0.01）. Quercetin caused a significant reduction both in the mPAP and fight ventricular hypertrophy index compared with the monocrotaline group （P 〈 0.01） while no difference was found between the quercefin group and the control group （P 〉 0.05）. Monocrotaline induced a marked increase in the wall thickness （WT） in small and mid-sized pulmonary arteries compared with the controls （P 〈 0.01）. Monocrotaline also induced a marked increase in the wall area （WA） in small [（56.38 ±6.65）% in monocrotaline vs. （19.80±4.63）% in control] and mid-sized [（43.71± 5.38）% in monocrotaline vs. （14.24± 3.66）% in control] pulmonary arteries （P 〈 0.01）. Quercefin treatment markedly reduced monocrotaline induced increase in both WT and WA （P 〈 0.01）, which, however, still remained significantly elevated compared with those of the controls （P 〈 0.01）. Furthermore, compared with controls, proliferating cell nuclear antigen （PCNA） expression in the pulmonary artery tissues was markedly increased by monocrotaline [（45.59± 1.27） in monocrotaline vs. （9.64± 0.69） in controls], which was significantly attenuated by quercetin. Our animal experiment indicated that quercetin could have protective effects on monocrotaline-induced PAH.

Reliable experimental model of hepatic veno-occlusive disease caused by monocrotaline

BACKGROUND:Hepatic veno-occlusive disease(HVOD)is a severe complication of chemotherapy before hematopoietic stem cell transplantation and dietary ingestion of pyrrolizidine alkaloids.Many experimental models were established to study its mechanisms or therapy,but few are ideal.This work aimed at evaluating a rat model of HVOD induced by monocrotaline to help advance research into this disease. METHODS:Thirty-two male rats were randomly classified into 5 groups,and PBS or monocrotaline was administered (100 mg/kg or 160 mg/kg).They were sacrificed on day 7(groups A,B and D)or day 10(groups C and E).Blood samples were collected to determine liver enzyme concentrations.The weight of the liver and body and the amount of ascites were measured.Histopathological changes of liver tissue on light microscopy were assessed by a modified Deleve scoring system.The positivity of proliferating cell nuclear antigen(PCNA)was estimated. RESULTS:The rats that were treated with 160 mg/kg monocrotaline presented with severe clinical symptoms (including two deaths)and the histopathological picture of HVOD.On the other hand,the rats that were fed with 100 mg/kg monocrotaline had milder and reversible manifestations.Comparison of the rats sacrificed on day 10 with those sacrificed on day 7 showed that the positivity of PCNA increased,especially that of hepatocytes.CONCLUSIONS:Monocrotaline induces acute,dose- dependent HVOD in rats.The model is potentially reversible with a low dose,but reliable and irreversible with a higher dose.The modified scoring system seems to be more accurate than the traditional one in reflecting the histopathology of HVOD.The enhancement of PCNA positivity may be associated with hepatic tissue undergoing recovery.

Changes in activity of superoxide dismutase and content of malondialdehyde in rats with monocrotaline-induced pulmonary hypertension

To observe changes in activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) in rats with monocrotaline-induced pulmonary hypertension. Methods: Adult ma1e Sprague-Dawley rats were given a single subcutaneous injection of monocrotaline (MCT, 60 mg/kg) for modeling PH. Activities of SOD and contents of MDA in plasma and pulmonary homogenate were measured by colorimetric analysis. The thickness of the media of pulmonary arterioles (external diameter <100μm) was measured using colour image analysis system. Results: Four weeks after injection of MCT, activities of SOD in venous plasma and pulmonary homogenate for MCT group were 106±45 NU/ml (P<0.05) and 317±59 NU/ml (P<0.01) respectively, whileactivities of SOD for control group were 159±28 NU/ml (P<0.05) and 505±47 NU/ml (P<0.01) respectively.COntents of MDA in venous plasma and pulmonary homogenate for MCT group were 15±5 and 59±14 μmol/L,while contents of MDA for control group were 5. 3±2. 8 and 32±19 ±mol/L. The thickness of the media of pulmonary arterioles increased significantly. Conclusion: The primary cause of PH is the injury of pulmonary vascular endothelial cells by MCT, which decreases the O2 removing ability of the lungs but increases lipid peroxidation,thus inducing PH.

Application of Precision-Cut Rat Liver Slice to Study the Influence of Monocrotaline, Tussilago farfara Alkaloids on the Expression of Cytochrome P450 Enzymes

Precision-cut liver slice has been successfully used to study the mechanism of drug-induced hepatotoxicity, the prediction of liver toxicity, the discovery of early hepatic toxicity biomarker and the metabolism of drug in liver. We detected the expression of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 in precision-cut liver slice after co-cultured with monocrotaline or Tussilago farfara alkaloids to investigate the hepatotoxicity mechanism of those drugs. After co-culturing with monocrotaline or Tussilago farfara alkaloids for 6 hours, the expression of CYP3A4 in the microsome of precision-cut liver slices was detected by Western blot, and the expressions of CYP2B1 + CYP2B2 and CYP2E1 were detected by immunofluorescence. The results showed that monocrotaline induced the expression of CYP3A4 and CYP2B1 + CYP2B2, and Tussilago farfara alkaloids obviously up-regulated the expression of CYP2E1 and CYP3A4. Thus, we conclude that the up-regulation of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 may be one of the toxic mechanisms of liver injury of those drugs.

3-Bromopyruvate alleviates the development of monocrotalineinduced rat pulmonary arterial hypertension by decreasing aerobic glycolysis,inducing apoptosis,and suppressing inflammation

Background:Pulmonary arterial hypertension(PH)is a progressive disease with limited therapeutic options,ultimately leading to right heart failure and death.Recent findings indicate the role of the Warburg effect(aerobic glycolysis)in the development of PH.However,the effect of the glycolysis inhibitor 3-bromopyruvate(3-BrPA)on the pathogenesis of PH has not been well investigated.This study aimed to determine whether 3-BrPA inhibits PH and its possible mechanism.Methods:PH was induced in adult Sprague-Dawley rats by a single intraperitoneal injection of monocrotaline(MCT).3-BrPA,or phosphate-buffered saline(PBS)was administered via intraperitoneal injection every other day from the first day of MCT-injection to 4 weeks of follow-up,and indices such as right ventricular systolic pressure(RVSP),right ventricular hypertrophy index(RVHI),pulmonary arteriolar remodeling indicated by percent media thickness(%MT),lactate levels and glucose consumption,were evaluated.Pulmonary arteriolar remodeling and right ventricular hypertrophy were observed in hematoxylin-eosin-stained lung sections.Western blotting,immunohistochemistry,and/or immunofluorescence analyses were used to measure the expression of relevant proteins.A cytochrome C release apoptosis assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining were used to measure cell apoptosis.Results:MCT-induced PH showed a significant increase in glucose consumption(0 vs.4 weeks:0.87±0.23 vs.2.94±0.47,P=0.0042)and lactate production(0 vs.4 weeks:4.19±0.34 vs.8.06±0.67,P=0.0004).Treatment with 3-BrPA resulted in a concomitant reduction in glucose consumption(1.10±0.35 vs.3.25±0.47,P=0.0063),lactate production(5.09±0.55 vs.8.06±0.67,P=0.0065),MCT-induced increase in RVSP(39.70±2.94 vs.58.85±2.32,P=0.0004),pulmonary vascular remodeling(%MT,43.45%±1.41%vs.63.66%±1.78%,P<0.0001),and right ventricular hypertrophy(RVHI,38.57%±2.69%vs.62.61%±1.57%,P<0.0001)when compared with those of the PBS-treated group.3-BrPA,a hexokinase 2 inhibitor,exerted its beneficial effect on PH by decreasing aerobic glycolysis and was also associated with inhibiting the expression of glucose transporter protein-1,inducing apoptosis,and suppressing inflammation.Conclusions:3-BrPA might have a potential beneficial effect on the PH treatment.

An in vitro study on interaction of anisodine and monocrotaline with organic cation transporters of the SLC22 and SLC47 families

Current study systematically investigated the interaction of two alkaloids, anisodine and monocrotaline, with organic cation transporter OCT1, 2, 3, MATE1 and MATE2-K by using in vitro stably transfected HEK293 cells. Both anisodine and monocrotaline inhibited the OCTs and MATE transporters. The lowest IC50 was 12.9 μmol·L-1 of anisodine on OCT1 and the highest was 1.8 mmol·L-1 of monocrotaline on OCT2. Anisodine was a substrate of OCT2(Km = 13.3 ± 2.6 μmol·L-1 and Vmax = 286.8 ± 53.6 pmol/mg protein/min). Monocrotaline was determined to be a substrate of both OCT1(Km = 109.1 ± 17.8 μmol·L^-1, Vmax = 576.5 ± 87.5 pmol/mg protein/min) and OCT2(Km = 64.7 ± 14.8 μmol·L^-1, Vmax = 180.7 ± 22.0 pmol/mg protein/min), other than OCT3 and MATE transporters. The results indicated that OCT2 may be important for renal elimination of anisodine and OCT1 was responsible for monocrotaline uptake into liver. However neither MATE1 nor MATE2-K could facilitate transcellular transport of anisodine and monocrotaline. Accumulation of these drugs in the organs with high OCT1 expression(liver) and OCT2 expression(kidney) may be expected.

Experimental animal models of pulmonary hypertension:Development and challenges

Pulmonary hypertension(PH) is clinically divided into 5 major types, characterized by elevation in pulmonary arterial pressure(PAP) and pulmonary vascular resistance(PVR), finally leading to right heart failure and death. The pathogenesis of this arteriopathy remains unclear, leaving it impossible to target pulmonary vascular remodeling and reverse the deterioration of right ventricular(RV) function. Different animal models have been designed to reflect the complex mechanistic origins and pathology of PH, roughly divided into 4 categories according to the modeling methods: noninvasive models in vivo, invasive models in vivo, gene editing models, and multi-means joint modeling. Though each model shares some molecular and pathological changes with different classes of human PH, in most cases the molecular etiology of human PH is poorly known. The appropriate use of classic and novel PH animal models is essential for the hunt of molecular targets to reverse severe phenotypes.

The potential therapeutic effect DL0805-2 on experimental pulmonary hypertensive rats and the underlying mechanisms

Aim DL0805-2 is a novel Rho-kinases inhibitor which has been found to have potent cardiovascular effects. In the present research, we aimed to study the potential of DL0805-2 in the treatment of pulmonary arterial hypertension （PAH） and discuss the underlying mechanisms preliminarily. Methods A classical PAH animal model was used, which was established by single injection of 50 mg · kg^-1 monocrotaline （MCT）. One week later, the rats were administrated with 1, 3, 10 mg · kg^-1 DL0805-2 via intraperitoneal injection for 18 days. At the end of the experiment, the body weight and survival rate were recorded. Meanwhile, the respiration function, heart function, blood pressure and pulmonary artery pressure were detected. Serum was collected for biochemical index analysis. The weight of vital organs was used to calculate the organ index. Histopathology examination was em-ployed to observe the subtle changes in hearts, vessels and lungs. Furthermore, the mechanisms were studied main- ly by the method of western blotting. Results DL0805-2 did not show significant influence on body weight of PAH rats. But the survival rate of PAH rats treated with 3 and 10 mg · kg^-1 DL0805-2 was increased up to 90. 9% com- pared with the model group （68.2%）. DL0805-2 improved the pulmonary artery blood flow especially the maximal -1 -1 velocity （PV max） from 397.2 cm · s^-1 to 506.5, 540. 1 and 574.0 cm · s^-1 respectively. The results of echocar- diography and electrocardiogram show that DL0805-2 had little effect on left ventricle and systemic circulation but attenuated right ventricle injury and decreased the right ventricle pressure from 73.73 mmHg to 47.80, 42.64 and 46.45 mmHg respectively after DL0805-2 intervention. Disease markers of PAH including NT-proBNP in serum and ET-1 in lung tissue homogenate and serum biochemical indicators, ALT, AST and LDH, were reduced by DL0805-2. DL0805-2 also relieved edema of lungs and decreased inflammatory cytokines production. Through the examination on histopathologic slide of pulmonary main artery, right ventricle and lung, DL0805 derivatives were found to have significant protection effect on structural changes of organs induced by pulmonary hypertension. Ac- cording to the preliminary study on the mechanisms of DL0805-2 in PAH, Rho/ROCK pathway was significantly in- hibited by DL0805 derivatives. In addition, DL0805 derivatives showed effect on BMPRII/p-Smad pathway and ap- optosis related pathway. Conclusion DL0805-2 has showed potent treatment effect on the PAH rats. And the un- derlying mechanisms studies also indicated that RhoA/ROCK and BMPRII pathways were involved. This work will provide basis experimental data for the further research and development of DL0805-2.