MiR-1249 on Endothelial Extracellular Vesicles Mediates Cigarette Smoke-Induced Pulmonary Hypertension by Inhibiting HDAC10 (Histone Deacetylase 10)-NFκB (Nuclear Factor κB)-CaSR (Calcium-Sensing Receptor) Cascade.

BACKGROUND: Overproduction of endothelial extracellular vesicles (eEVs) is correlated with pulmonary hypertension progression, but the precise mechanism remains largely unclear. METHODS: MicroRNA-chip and real-time polymerase chain reaction were conducted to screen and validate microRNA profiles in blood plasma eEVs of rats and human with or without cigarette smoking. Pulmonary artery smooth muscle cells were cultured to study signaling pathways. Pulmonary hypertension phenotypes were evaluated in wild-type and calcium-sensing receptor knockout rats to identify the pathophysiological significance of the microRNA pathway. RESULTS: MicroR-1249 was predominant highly expressed in eEVs from plasma of rats exposed to cigarette smoking, and confirmed in eEVs from plasma of human smokers as well as in eEVs from cigarette smoke extract-treated pulmonary artery endothelial cells, but not in cigarette smoke extract-treated pulmonary artery smooth muscle cells. In cultured pulmonary artery smooth muscle cells, microR-1249 downregulated the expression of histone deacetylase 10, which in turn enhanced the acetylated form of NFκB (nuclear factor κB) level and its nuclear translocation leading to increased expression of calcium-sensing receptor. In rats, the repression of microR-1249 in eEVs by microR-1249 inhibitor, histone deacetylase 10 overexpression, or calcium-sensing receptor knockout profoundly inhibited the proliferative capacities and diminished apoptosis-resistance of pulmonary artery smooth muscle cells and pulmonary hypertension development in rats intravenously administrated with eEVs preparation from cigarette smoke extract-treated pulmonary artery endothelial cells. CONCLUSIONS: Cigarette smoke-enriched microR-1249 in endothelial extracellular vesicles facilitates the hyperproliferative and antiapoptotic status of pulmonary artery smooth muscle cells promoting pulmonary hypertension evolution through the inhibition of histone deacetylase 10-NFκB-calcium-sensing receptor cascade.

Ferrostatin-1 Alleviates White Matter Injury Via Decreasing Ferroptosis Following Spinal Cord Injury.

Spinal cord injury (SCI), a devastating neurological impairment, usually imposes a long-term psychological stress and high socioeconomic burden for the sufferers and their family. Recent researchers have paid arousing attention to white matter injury and the underlying mechanism following SCI. Ferroptosis has been revealed to be associated with diverse diseases including stroke, cancer, and kidney degeneration. Ferrostatin-1, a potent inhibitor of ferroptosis, has been illustrated to curb ferroptosis in neurons, subsequently improving functional recovery after traumatic brain injury (TBI) and SCI. However, the role of ferroptosis in white matter injury and the therapeutic effect of ferrostatin-1 on SCI are still unknown. Here, our results indicated that ferroptosis played a pivotal role in the secondary white matter injury, and ferrostatin-1 could reduce iron and reactive oxygen species (ROS) accumulation and downregulate the ferroptosis-related genes and its products of IREB2 and PTGS2 to further inhibit ferroptosis in oligodendrocyte, finally reducing white matter injury and promoting functional recovery following SCI in rats. Meanwhile, the results demonstrated that ferrostatin-1 held the potential of inhibiting the activation of reactive astrocyte and microglia. Mechanically, the present study deciphers the potential mechanism of white matter damage, which enlarges the therapeutic effects of ferrostatin-1 on SCI and even in other central nervous system (CNS) diseases existing ferroptosis.

Identification of a potentially novel LncRNA-miRNA-mRNA competing endogenous RNA network in pulmonary arterial hypertension via integrated bioinformatic analysis.

AIMS: Pulmonary arterial hypertension (PAH) is a fatal cardiovascular disease with a cancer-like phenotype. Competing endogenous RNA (ceRNA) networks extensively involve in its pathological processes. But rare ceRNA networks and profound molecular mechanisms have been revealed in PAH. The aim of this study was to illuminate the ceRNA networks in PAH. MATERIALS AND METHODS: In this work, we have chosen the idiopathic PAH as an example. GSE15197 (mRNA) and GSE56914 (miRNA) from the Gene Expression Omnibus (GEO) were selected to explore key genes and novel ceRNA networks in PAH by a series of integrated bioinformatic analysis. To be more scientific, a part of pairs in identified ceRNA network were detected in hypoxia-induced HPASMCs. And the dual-luciferase assay was performed to certify the relationship between miRNAs and mRNAs. KEY FINDINGS: Totally, 311 differentially expressed genes (DEGs) were identified and functional enrichment analysis illuminated that the majority of DEGs were enriched in proliferation, anti-apoptosis, inflammation and cancer-related pathways. And 10 hub genes were determined via Cytohubba after PPI network construction. Sequentially, with stepwise reverse prediction and pan-cancer co-expression analysis from mRNA to LncRNA in TargetScan, miRNet, ENCORI (Starbase V3.0) databases, a crucially ceRNA network was identified including 14 LncRNAs, 2 miRNAs, and 3 mRNAs. Further, in hypoxia-induced HPASMCs, the alterations of mRNAs, miRNAs and LncRNAs and their relationship were in accordance with the results we identified. SIGNIFICANCE: Consequently, the unique hub genes and ceRNA network we proposed may advance our understanding of the molecular mechanisms in PAH.

Grape seed procyanidin suppresses inflammation in cigarette smoke-exposed pulmonary arterial hypertension rats by the PPAR-γ/COX-2 pathway.

BACKGROUND AND AIM: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling, which is mainly caused by inflammation. Inhibiting inflammation can relieve PAH. Grape seed procyanidin (GSP) possesses remarkable anti-inflammatory property and vascular protective function. In this experiment, we verified the anti-inflammatory property of GSP in cigarette smoke-exposed PAH rats and revealed its molecular mechanism. METHODS AND RESULTS: In vivo, 45 Sprague Dawley (SD) rats were divided into 5 groups randomly, treated with normoxia/cigarette smoke (CS)/GSP + CS/CS + solvent/GSP. After GSP + CS administration, a decrease in mPAP, PVR, RVHI, WT%, and WA% was detected in the rats as compared to those treated with CS. In vitro, the proliferation of pulmonary arterial smooth muscle cells (PASMCs) caused by cigarette smoke extract (CSE) was effectively attenuated with GSP + CSE administration. Furthermore, GSP significantly increased the expression of peroxisome proliferator-activated receptor γ (PPAR-γ) together with the lowered expression level of cyclooxygenase 2 (COX-2) in PASMCs co-incubated with CSE. CONCLUSION: These findings indicate that GSP ameliorates inflammation by the PPAR-γ/COX-2 pathway and finally inhibits the proliferation of PASMCs, which leads to pulmonary vascular remodeling.

[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.

Grape seed proanthocyanidin inhibits monocrotaline-induced pulmonary arterial hypertension via attenuating inflammation: in vivo and in vitro studies.

Inflammation in pulmonary arterioles initiates and maintains pathological processes in pulmonary arterial hypertension (PAH), and inhibition of it attenuates PAH development. Grape seed proanthocyanidin (GSP) is believed to be effective in protecting vascular system via inhibiting inflammation, while its effect on pulmonary circulation remains inconclusive. In this study, we made observations in monocrotaline (MCT)-induced PAH rats and found decreases in mean pulmonary arterial pressure, pulmonary vessel resistance, right ventricular hypertrophy index, percentage of medial wall thickness, percentage of medial wall area, and lung weight of wet and dry tissue ratio after GSP administration in vivo. At the cellular and molecular levels, we also found several effects of GSP on MCT-induced PAH: (a) endothelial nitric oxide synthase expression in lung tissue and plasma NO level were increased; (b) Ca2+ level in pulmonary arterial smooth muscle cell (PASMC) was decreased; (c) transcription of inflammatory factors such as myeloperoxidase, interleukin (IL)-1ß, IL-6 and tumor necrosis factor alpha (TNF-α) was down-regulated in lung tissue; (d) nuclear factor-κB pathway was inhibited as IκBα was less phosphorylated; (e) TNFα-induced PASMC overproliferation could be inhibited. These results indicated a possible mechanism of GSP reversing pulmonary vascular remodeling and vascular contraction by inhibiting inflammation, and it may be useful for preventing PAH development.

Apple polyphenol relieves hypoxia-induced pulmonary arterial hypertension via pulmonary endothelium protection and smooth muscle relaxation: In vivo and in vitro studies.

AIM: This study aims to test the effect of apple polyphenol (APP) on hypoxia-induced pulmonary arterial hypertension (PAH) and explore its possible underlying mechanisms. METHODS AND RESULTS: Rats were treated with control, APP, hypoxia (8 h/d), hypoxia + APP. Mean pulmonary arterial pressure (mPAP) and pulmonary vessel resistance (PVR) were examined. Phenylephrine (PE)-pretreated pulmonary vessel rings were prepared for observation of APP administration. eNOS, sGC inhibitors (L-NAME, MB), Ca2+ channel blockers (NiCl2, Calhex231), K+ channel blockers (4-AP, 5-HD, TEA, BaCl2) were applied to pulmonary vessel rings and pulmonary arterial smooth muscle cell (PASMC). Flow cytometry analysis and CCK-8 assay were applied to detect apoptosis of pulmonary artery endothelium cell (PAEC). Caspase-3, NO, eNOS, iNOS were detected in PAEC. APP reversed mPAP and PVR elevation in vivo. Contraction of pulmonary vessel rings with/without endothelium induced by hypoxia were inhibited by APP. APP effect was hindered by L-NAME or MB, and could be reduced by K+channel blockers. Further, APP was found to decrease cytosolic Ca2+ in PASMC and protect PAEC from apoptosis. In PAEC, Caspase-3, iNOS were decreased and NO, eNOS were increased after APP administration. CONCLUSIONS: APP reverses pulmonary vasoconstriction through enzyme expression and cation channel activities, thus has effects of PASMC relaxation and PAEC protection.

Grape seed proanthocyanidin reverses pulmonary vascular remodeling in monocrotaline-induced pulmonary arterial hypertension by down-regulating HSP70.

Heat shock protein 70 (HSP70) is a molecular chaperone which has a low content in cytoplasm under normal physiological conditions. A higher intracytoplasmic HSP70 level can be observed in pulmonary arterial smooth muscle cell (PASMC) in pulmonary arterial hypertension (PAH), and this up-regulation can promote pho-IκBα expression, which is an NF-κB signaling pathway inhibitor. NF-κB signaling pathway up-regulation can promote PASMC proliferation and pulmonary vascular remodeling in PAH, resulting in elevation of pulmonary pressure and the subsequent right heart failure caused by right ventricular hypertrophy. Grape seed proanthocyanidin (GSP) is effective in vascular protection and several tumor treatments, and its effect on PAH treatment remains to be elucidated. In this study, we made observations and contrasts in monocrotaline(MCT) -induced PAH rats, and found decrease in mPAP, PVR and RVHI after GSP administration. Our study also proved GSP's effect on down-regulating the intracytoplasmic HSP70 content both in cellular and animal levels. The results indicate a possible mechanism of GSP reversing pulmonary vascular remodeling by down-regulating HSP70, and this change may influence pho-IκBα expression. Therefore, inhibition of NF-κB signaling pathway caused by GSP can lead to inhibition of PASMC proliferation in PAH.

A novel small molecule compound possesses immunomodulatory properties on bone marrow-derived dendritic cells via TLR7 signaling pathway and alleviates the development of SLE.

Dendritic cells (DCs) play an important role in the development and maintenance of immune tolerance. Activation of TLR7, which is expressed in DCs, is thought to contribute to the complex pathophysiology of systemic lupus erythematosus (SLE). In this study, we analyzed the in vitro and in vivo function of a novel small-molecule compound, FC-99, which was previously reported to have immunomodulatory functions. We found that FC-99 inhibited the expression of CD40 and inflammatory mediators (IL-6, IL-12, and CXCL-10), as well as R848-induced phosphorylation of IκB-α. We also present evidence that FC-99 is remarkably efficacious in the treatment of murine lupus. Interestingly, FC-99 affected the maturation and percentage of DCs in lupus-prone mice. Therefore, FC-99 may serve as a potential drug candidate for treatment of SLE.

A new benzenediamine derivative modulates Toll-like receptors-induced myeloid dendritic cells activation and ameliorates lupus-like syndrome in MRLlpr/lpr mice.

Modulators of the over-activation of myeloid dendritic cells (mDCs) by Toll-like receptors (TLRs) have an advantage in the treatment of systemic lupus erythematosus (SLE). This study was designed to evaluate the effects of FC-99, a novel benzenediamine derivative, on TLR-induced activation of mDCs, and to assess the efficacy of FC-99 in a murine model of SLE. In vitro, FC-99 inhibited the phenotypic (CD40 and MHC-II) and functional activation (IL-12 and CXCL10) of mDCs induced by TLR ligands. In vivo, MRLlpr/lpr mice displayed renal diseases associated with increased levels of proteinuria and immunoglobulin, which were ameliorated by FC-99. Enhanced accumulation and activation of mDCs in lymphoid organs was also impaired by FC-99. Additionally, FC-99 inhibited the activation of IκB-α and upregulated the expression of TNFα-induced protein 3 (TNFAIP3) in vitro and in vivo. These results indicate that FC-99 modulates TLR-induced activation of mDCs and ameliorates lupus-like syndrome in MRLlpr/lpr mice. This effect is closely associated with the inhibition of IκB-α and upregulation of TNFAIP3.

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.

[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.

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.