[Panax notoginseng saponins improve monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting ADAM10/Notch3 signaling pathway].

In this study, we investigated the effects of Panax notoginseng saponins (PNS) on pulmonary vascular remodeling and ADAM10/Notch3 pathway in pulmonary arterial hypertension (PAH). PAH rat model was established, and male Sprague Dawley (SD) rats were randomly divided into control group, monocrotaline (MCT) group and MCT+PNS group, with 10 rats in each group. Rats in the control group were intraperitoneally injected with equal volume of normal saline. Rats in the MCT group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with the same volume of normal saline every day. Rats in the MCT+PNS group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with 50 mg/kg PNS every day. The modeling time of each group lasted for 21 days. After the model was established, the mean pulmonary artery pressure (mPAP) was measured by right heart catheterization technique, the right ventricular hypertrophy index (RVHI) was calculated, the microscopic morphology and changes of pulmonary vascular wall were observed by HE and Masson staining, and the expressions of ADAM10, Notch3, Hes-1, P27, PCNA, Caspase-3 proteins and mRNA in pulmonary vascular tissue of rats were detected by Western blot and qPCR. The expression and localization of Notch3 and α-SMA were detected by immunofluorescence staining. The protein expression of ADAM10 was detected by immunohistochemical staining. The results showed that compared with the control group, mPAP, RVHI, pulmonary vessels and collagen fibers in the MCT group were significantly increased, the expressions of ADAM10, Notch3, Hes-1, and PCNA protein and mRNA were significantly increased, while the expressions of P27 and Caspase-3 protein and mRNA were decreased significantly. Compared with the MCT group, mPAP and RVHI were significantly decreased, pulmonary vessels were significantly improved and collagen fibers were significantly reduced, the expressions of protein and mRNA of ADAM10, Notch3, Hes-1, and PCNA were decreased in MCT+PNS group, but the expressions of protein and mRNA of P27 and Caspase-3 were increased slightly. The results of immunofluorescence showed that Notch3 and α-SMA staining could overlap, which proved that Notch3 was expressed in smooth muscle cells. The expression of Notch3 in the MCT group was increased significantly compared with that in the control group, while PNS intervention decreased the expression of Notch3. Immunohistochemical staining showed that compared with the control group, the amount of ADAM10 in the MCT group was increased significantly, and the expression of ADAM10 in the MCT+PNS group was decreased compared with the MCT group. These results indicate that PNS can improve the PAH induced by MCT in rats by inhibiting ADAM10/Notch3 signaling pathway.

Compositional and functional alterations of gut microbiota in patients with stroke.

BACKGROUND AND AIMS: There is accumulating evidence that gut microbiota plays a key role in cardiovascular diseases. Gut bacteria can transform dietary choline, l-carnitine, and trimethylamine N-oxide (TMAO) into trimethylamine, which can be oxidized into TMAO again in the liver. However, the alterations of the gut microbiota in large artery atherosclerotic (LAA) stroke and cardioembolic (CE) stroke have been less studied. METHODS AND RESULTS: We performed a case-control study in patients with LAA and CE types of strokes. We profiled the gut microbiome using Illumina sequencing of the 16S ribosomal RNA gene (V4-V5 regions), and TMAO was determined via liquid chromatography-tandem mass spectrometry. Our results showed that the TMAO levels in the plasma of patients with LAA and CE strokes were significantly higher than those in controls (LAA stroke, 2931 ± 456.4 ng/mL; CE stroke, 4220 ± 577.6 ng/mL; healthy control, 1663 ± 117.8 ng/mL; adjusted p < 0.05). The TMAO level in the plasma of patients with LAA stroke was positively correlated with the carotid plaque area (rho = 0.333, 95% CI = 0.08-0.55, p = 0.0093). Notably, the composition and the function of gut microbiota in the LAA stroke group were significantly different from those in the control group (FDR-adjusted p-value < 0.05). There was no significant association between gut microbiota and CE stroke in our study. CONCLUSION: This study provides evidence for significant compositional and functional alterations of the gut microbiome in patients with LAA stroke. Gut microbiota might serve as a potential biomarker for patients with LAA stroke.

[Effects of panax notoginseng saponins on pulmonary vascular remodeling in rats with pulmonary hypertension and its mechanisms].

Objective: To investigate the effects of panax notoginseng saponins (PNS) on pulmonary vascular remodeling and SIRT1/FOXO3a/p27 pathway in pulmonary arterial hypertension (PAH) rats. Methods: Male SD rats weighing 200~250g were randomly divided into control group, monocrotaline group (MCT) and monocrotaline + panax notoginseng saponins group (MCT+PNS), with 10 rats in each group. The rats in control group were injected intraperitoneally with normal saline 3 ml/kg on the first day, then injected intraperitoneally with normal saline 2.5 ml/kg every day. The rats in MCT group were injected intraperitoneally with MCT 60 mg/kg on the first day, followed by daily injection of normal saline 2.5 ml/kg. In MCT+PNS group, 60 mg/kg MCT was injected intraperitoneally on the first day, and 50 mg/kg PNS was injected intraperitoneally every day. The above models were fed conventionally for 4 weeks. After the modeling was completed, the mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) of rats in each group were detected by right heart catheter method, weighed and calculated right ventricular hypertrophy index (RVHI), and the pulmonary vascular structure and morphological changes were observed by HE and Masson staining. The protein and gene expressions of SIRT1, FOXO3a, p27, PCNA and Caspase-3 were detected by qPCR and Western blot. Results: Compared with control group, mPAP, RVSP and RVHI in MCT group were increased significantly (P＜0.01), pulmonary vessels were thickened significantly and collagen fibers were increased, protein and gene expressions of SIRT1, FOXO3a, p27 and Caspase-3 were decreased (P＜0.05 or P＜0.01). The protein and gene expressions of PCNA were increased (P＜0.05). Compared with MCT group, the levels of mPAP, RVSP and RVHI in MCT+PNS group were decreased significantly (P＜0.05 or P＜0.01), pulmonary vascular thickening was alleviated and collagen fibers were reduced. The protein and gene expressions of SIRT1, FOXO3a, p27 and Caspase-3 were increased (P＜0.05 or P＜0.01), while the protein and gene expressions of PCNA were decreased (P＜0.05 or P＜0.01). Conclusion: Panax notoginseng saponins can relieve pulmonary vascular remodeling in rats with pulmonary hypertension by activating SIRT1/FOXO3a/p27 pathway.

[Effects of apple polyphenols on monocrotaline-induced pulmonary vascular remodeling in rats and its mechanism].

OBJECTIVE: To investigate the effects of apple polyphenols on pulmonary vascular remodeling in rats with pulmonary arterial hypertension and its mechanism. METHODS: Rats were randomly divided into 4 groups:control (Con) group, monocrotaline (MCT) group, apple polyphenol (APP) group,monocrotaline + apple polyphenol (MCT+APP) group. In Con group, rats received a subcutaneous injection of physical saline. In APP group, rats received intraperitoneal injection of 20 mg/kg APP, every other day. In MCT group, rats received a single subcutaneous injection of MCT(60 mg/kg). In MCT+APP group, rats received subcutaneous injection of 60 mg/kg MCT followed by an intraperitoneal injection of 20 mg/kg APP every other day. All the disposal lasted 3 weeks. Then the PAH-relevant indicators, such as mean pulmonary artery pressure(mPAP), pulmonary vascular resistance(PVR), right ventricular hypertrophy index (RVHI) ,wall thickness (WT%) and wall area (WA%) were tested. After that, the inflammatory pathway related indicators, such as interleukin1(IL-1),interleukin1(IL-6), tumor necrosis factor α(TNF-α), cyclooxygenase 2(COX-2) and myeloperoxidase(MPO) in pulmonary tissue and free intracellular Ca2+ in pulmonary smooth muscle cell(PASMC), content of eNOS and NO in endothelial cells were determined. RESULTS: Compared with the control group, the levels of mPAP, PVR, RVHI, WA%, WT%, and IL-1, IL-6, TNF-α, COX-2, MPO in tissue and the expression of Ca2 + in PASMC of MCT group were increased significantly, while the contents of eNOS and NO in endothelial cells were decreased significantly (P＜0.05). Compared with the MCT group, the apple polyphenol treatment could improve the above mentioned situation, and the COX-2 and Ca2+ indicators of the apple polyphenol treatment group were decreased significantly (P＜0.05). CONCLUSION: MCT can increase COX-2 expression and intracellular Ca2+ in pulmonary artery smooth muscle cells, decrease the contents of eNOS and NO in endothelial cells, while apple polyphenols can significantly inhibit these effects.

Dopamine inhibits proliferation, induces differentiation and apoptosis of K562 leukaemia cells.

BACKGROUND: Dopamine exerts its effects mainly in nervous system through D1, D2 or D3 receptors. There are few reports dealing with the effects of dopamine on leukaemia cells. However, some dopamine agonists or antagonists do show biological effects on some types of leukaemia cells. Here, we report the effects of dopamine on the proliferation, differentiation and apoptosis of K562 leukaemia cells. METHODS: Proliferation was determined by MTT assay and cell counting both in liquid and semisolid cultures. Differentiation was verified by morphology, benzidine staining and flow cytometry. Apoptosis was checked by Hoechst 33258 staining and flow cytometry. The two groups were untreated group and treated group (dopamine 10(-9) mol/L - 10(-4) mol/L). RESULTS: In liquid culture, MTT assay and colony assay, dopamine inhibited proliferation of K562 cells. Inhibition rate was 29.28% at 10(-6) mol/L and 36.10% at 10(-5) mol/L after culture for 5 days in MTT assay. In benzidine staining and CD71 expression, dopamine induced K562 cells toward erythroid differentiation by increased 155% at 10(-6) mol/L and by 171% at 10(-5) mol/L after culture for 5 days in benzidine staining. In Hoechst 33258 staining and flow cytometry, dopamine induced K562 cells toward apoptosis. The sub G1 peak stained by PI was 14.23% at 10(-4) mol/L dopamine after culture for 3 days compared with the control (0.81%) in flow cytometry. CONCLUSION: Dopamine inhibites proliferation and induces both differentiation and apoptosis of K562 leukaemia cells.

[Mechanism of apoptosis-inducing effects of dopamine on K562 leukemia cells].

OBJECTIVE: To investigate the mechanism of the apoptosis-inducing effects of dopamine on K562 leukemia cells. METHODS: K562 cells were treated with DP2785, the dopamine receptors were detected with fluorescence spectrophotometer, UV spectrophotometer and fluorescence microscope; the contents of cAMP in K562 cells were measured; and the subtypes of dopamine receptor on K562 cells were analyzed by receptor blocking. RESULT: The existence of dopamine receptors in K562 cells was demonstrated by fluorescence microscopy, UV spectrophotometer and fluorescence spectrophotometer. Dopamine enhanced the contents of cAMP in K562 cells. Dopamine receptors were blocked by both D1 and D2 antagonists. CONCLUSION: D1 and D2 dopamine receptors may be involved in dopamine-induced apoptosis of K562 cells, and dopamine can also increase the contents of cAMP in K562 cells.

Astragalus Polysaccharides Attenuate Monocrotaline-Induced Pulmonary Arterial Hypertension in Rats.

Astragalus polysaccharides (APS) have been shown to possess a variety of biological activities including anti-oxidant and anti-inflammation functions in a number of diseases. However, their function in pulmonary arterial hypertension (PAH) is still unknown. Rats received APS (200[Formula: see text]mg/kg once two days) for 2 weeks after being injected with monocrotaline (MCT; 60[Formula: see text]mg/kg). The pulmonary hemodynamic index, right ventricular hypertrophy, and lung morphological features of the rat models were examined, as well as the NO/eNOS ratio of wet lung and dry lung weight and MPO. A qRT-PCR and p-I[Formula: see text]B was used to assess IL-1[Formula: see text], IL-6 and TNF-[Formula: see text] and WB was used to detect the total I[Formula: see text]B. Based on these measurements, it was found that APS reversed the MCT-induced increase in mean pulmonary arterial pressure (mPAP) (from 32.731[Formula: see text]mmHg to 26.707[Formula: see text]mmHg), decreased pulmonary vascular resistance (PVR) (from 289.021[Formula: see text]mmHg[Formula: see text][Formula: see text] min/L to 246.351[Formula: see text]mmHg[Formula: see text][Formula: see text][Formula: see text]min/L), and reduced right ventricular hypertrophy (from 289.021[Formula: see text]mmHg[Formula: see text][Formula: see text][Formula: see text]min/L to 246.351 mmHg[Formula: see text][Formula: see text][Formula: see text]min/L) ([Formula: see text]0.05). In terms of pulmonary artery remodeling, the WT% and WA% decreased with the addition of APS. In addition, it was found that APS promoted the synthesis of eNOS and the secretion of NO, promoting vasodilation and APS decreased the MCT-induced elevation of MPO, IL-1[Formula: see text], IL-6 and TNF-[Formula: see text], reducing inflammation. Furthermore, APS was able to inhibit the activation of pho-I[Formula: see text]B[Formula: see text]. In couclusion, APS ameliorates MCT-induced pulmonary artery hypertension by inhibiting pulmonary arterial remodeling partially via eNOS/NO and NF-[Formula: see text]B signaling pathways.

A new 2-aminosteroid induces cellular differentiation and upregulates the expression of MafB and Egr-1 genes respectively in HL-60 and K562 leukemia cells.

BACKGROUND: In previous work, we suggested that some 2-aminosteroids inhibited proliferation and induced differentiation of both human and murine leukemia cells. Here, we reported the actions of another new 2-aminosteroid designated as H89712 on human leukemia cells. METHODS: Cell colony counting and MTT assay were used to determine proliferation. Cell morphology, histochemical staining, UV detection and cytometry were used to determine differentiation. RT-PCR was used to detect gene expression. Standard statistical method was used to analyze data. RESULTS: H89712 inhibited proliferation of HL-60 leukemia cells and the inhibition percentage in MTT assay was 18% at the dose of 10(-8) mol/L and 65% at the dose of 10(-5) mol/L, respectively. The inhibition for HL-60 in colony assay was 23% at the dose of 10(-8) mol/L and 96% at the dose of 10(-5) mol/L, respectively. H89712 also induced HL-60 cells toward macrophage-like differentiation. It was verified by flow cytometry that the percentage of positive CD14 expression in differentiated HL-60 cells was about 9 times higher than that of the control at the dose of 10(-8) mol/L and 20 times higher than that of the control at the dose of 10(-5) mol/L respectively, and this action involved upregulation of MafB gene in HL-60 leukemia cells. On the other hand, H89712 inhibited proliferation of K562 leukemia cells and the inhibition of K562 leukemia cells in MTT assay was shown by 34% at the dose of 10(-8) mol/L and 88% at the dose of 10(-5) mol/L respectively. The inhibition of K562 leukemia cells in colony assay was 53% at the dose of 10(-8) mol/L and 100% at the dose of 10(-5) mol/L respectively. H89712 also induced K562 cells toward erythroid-like differentiation and it was verified by flow cytometry that the percentage of positive CD71 expression in differentiated K562 cells was about 9 times higher than that of the control at the dose of 10(-8) mol/L and 16 times higher than that of the control at the dose of 10(-5) mol/L respectively. This action was related to upregulation of Egr-1 gene in K562 leukemia cells. CONCLUSIONS: Our results showed the important roles played by MafB in macrophage differentiation and Egr-1 in erythroid differentiation of human myeloid leukemia cells.

[Differentiation-inducing effects of perphenazine on K562 leukemia cells].

OBJECTIVE: To determine the differentiation-inducing effects of perphenazine on K562 leukemia cells. METHODS: Differentiation-Inducing effects of a phenothiazine perphenazine were evaluated by proliferation, morphology and function of K562 cells. We evaluated the effects of perphenazine on K562 cells proliferation by cellular enumeration in liquid culture assay, MTT assay and clony formation assay, the morphology by Wight-Gimesa staining, and the function by detecting CD71 through flow cytometry. RESULTS: Perphenazine enhanced the expression of CD71 on K562 cells and increased Hb content in K562 cells, while inhibited the proliferation of K562 cells. K562 cells showed differentiation morphology after the drug treatment. CONCLUSION: Perphenazine possessed differentiation-inducing effects on K562 cells.