HSFAS mediates fibroblast proliferation, migration, trans-differentiation and apoptosis in hypertrophic scars via interacting with ADAMTS8.

Hypertrophic scar (HS) is one of the most common sequelae of patients, especially after burns and trauma. The roles of regulatory long noncoding RNAs (lncRNAs) in mediating HS remain underexplored. Human hypertrophic scar-derived fibroblasts (HSFBs) have been shown to exert more potent promoting effects on extracellular matrix (ECM) accumulation than normal skin-derived fibroblasts (NSFBs) and are associated with enhanced HS formation. The purpose of this study is to search for lncRNAs enriched in HSFBs and investigate their roles and mechanisms. LncRNA MSTRG.59347.16 is one of the most highly expressed lncRNAs in HS detected by lncRNA-seq and qRT-PCR and named as hypertrophic scar fibroblast-associated lncRNA (HSFAS). HSFAS overexpression significantly induces fibroblast proliferation, migration, and myofibroblast trans-differentiation and inhibits apoptosis in HSFBs, while knockdown of HSFAS results in augmented apoptosis and attenuated proliferation, migration, and myofibroblast trans-differentiation of HSFBs. Mechanistically, HSFAS suppresses the expression of A disintegrin and metalloproteinase with thrombospondin motifs 8 (ADAMTS8). ADAMTS8 knockdown rescues downregulated HSFAS-mediated fibroblast proliferation, migration, myofibroblast trans-differentiation and apoptosis. Thus, our findings uncover a previously unknown lncRNA-dependent regulatory pathway for fibroblast function. Targeted intervention in the HSFAS-ADAMTS8 pathway is a potential therapy for HS.

Inhibition of autophagy via 3-methyladenine alleviates the progression of preeclampsia.

Autophagy is a cellular mechanism for self-renewal that involves the breakdown of cytoplasmic proteins or organelles within lysosomes. Although preeclampsia (PE) exhibits several characteristics that could imply disrupted autophagy, there is limited evidence supporting the notion that impaired placental autophagy directly causes PE, as indicated by differential expression profiling of whole placental tissue. In this study, we aim to explore the significance of autophagy in maintaining pregnancy and its association with PE. First, the RNA-seq results show that 218 genes are differentially expressed in placentas from preeclamptic pregnancies. Notably, KEGG pathway analysis reveals significant enrichment of genes related to autophagy-related signaling pathways, including the PI3K-Akt signaling pathway, the AMPK signaling pathway, and the mTOR signaling pathway. Additionally, our findings indicate an increase in autophagy in placentas from pregnancies complicated by preeclampsia as well as in trophoblasts subjected to hypoxic conditions. Next, we examine the impact of 3-methyladenine (3-MA), a targeted inhibitor of autophagy, on the progression of PE. The administration of 3-MA profoundly alleviates the severity of PE-like symptoms in rats subjected to reduced uterine perfusion pressure (RUPP). The findings from our study suggest that inhibiting autophagy may serve as a promising approach for adjuvant chemotherapy for PE.

Homocysteine accelerates hepatocyte autophagy by upregulating TFEB via DNMT3b-mediated DNA hypomethylation.

Autophagy plays a critical role in the physiology and pathophysiology of hepatocytes. High level of homocysteine (Hcy) promotes autophagy in hepatocytes, but the underlying mechanism is still unknown. Here, we investigate the relationship between Hcy-induced autophagy level and the expression of nuclear transcription factor EB (TFEB). The results show that Hcy-induced autophagy level is mediated by upregulation of TFEB. Silencing of TFEB decreases the level of autophagy-related protein LC3BII/I and increases p62 expression level in hepatocytes after exposure to Hcy. Moreover, the effect of Hcy on the expression of TFEB is regulated by hypomethylation of the TFEB promoter catalyzed by DNA methyltransferase 3b (DNMT3b). In summary, this study shows that Hcy can activate autophagy by inhibiting DNMT3b-mediated DNA methylation and upregulating TFEB expression. These findings provide another new mechanism for Hcy-induced autophagy in hepatocytes.

LncRNA FPASL suppresses fibroblast proliferation through its DNA methylation via DNMT3b in hypertrophic scar.

Long noncoding RNAs (lncRNAs) are increasingly being implicated as key regulators of cell proliferation, apoptosis, and differentiation. However, the molecular mechanisms of specific lncRNAs in the context of hypertrophic scar remain largely unclear. Here, we find that the lncRNA FPASL (fibroblast proliferation-associated LncRNA) is downregulated in HS, and FPASL reduces fibroblast proliferation and colony formation and blocks cell cycle progression. Using GO annotation enrichment analysis along with AZC (a specific inhibitor of DNA methylation), we identify that DNA methylation is responsible for downregulating FPASL in hypertrophic scar. Subsequent studies demonstrate that high expression of DNMT3b inhibits FPASL expression in HS. Mechanistic study reveals a significant increase in fibroblast proliferation after transfection with LNA-FPASL, which is further inhibited by knockdown of DNMT3b. Thus, our study reveals that DNMT3b mediates hypermethylation of the lncRNA FPASL promoter and the downregulation of lncRNA FPASL promotes fibroblast proliferation in hypertrophic scar.

DNA Hypomethylation of miR-30a Mediated the Protection of Hypoxia Postconditioning Against Aged Cardiomyocytes Hypoxia/Reoxygenation Injury Through Inhibiting Autophagy.

BACKGROUND: Ischemic postconditioning (IPostC) is an endogenous protective mechanism to reduce ischemia-reperfusion (I/R) injury. However, whether IPostC protects aged cardiomyocytes against I/R injury is not fully understood. Considering the protective function of microRNA 30a (miR-30a) against ischemia-induced injury in H9C2 cells, its role in the protective effects of IPostC on I/R injury of aged cardiomyocytes was investigated further.Methods and Results:To mimic I/R and IPostC in vitro, the aged cardiomyocyte model for hypoxia postconditioning (HPostC) treatment was established by 9 days of incubation with 8 mg/mL D-galactose and then followed by exposure to hypoxic environment. HPostC significantly alleviated hypoxia/reoxygenation (H/R) injury and reduced autophagy of aged cardiomyocytes, as evidenced by decreased LC3B-II expression and increased p62 by Western blot. Quantified by quantitative real-time polymerase chain reaction (qRT-PCR), miR-30a was increased in aged cardiomyocytes treated with HPostC compared with I/R injury group. Overexpression of miR-30a by LV3-rno-miR-30a mimic promoted cardioprotective effect of HPostC in aged cardiomyocytes by suppressing BECN1-mediated autophagy, all of which was abrogated by knockdown of miR-30a expression. Epigenetic analyses demonstrated that HPostC reduced DNA methyltransferase 3b-mediated DNA hypomethylation levels at miR-30a promoter, leading to upregulation of miR-30a. CONCLUSIONS: HPostC protected aged cardiomyocytes survival against H/R injury via DNMT3b-dependent activation of miR-30a. miR-30a could be a potential therapeutic target for ischemic myocardial infarction.

Aberrant MFN2 transcription facilitates homocysteine-induced VSMCs proliferation via the increased binding of c-Myc to DNMT1 in atherosclerosis.

It is well-established that homocysteine (Hcy) is an independent risk factor for atherosclerosis. Hcy can promote vascular smooth muscle cell (VSMC) proliferation, it plays a key role in neointimal formation and thus contribute to arteriosclerosis. However, the molecular mechanism on VSMCs proliferation underlying atherosclerosis is not well elucidated. Mitofusin-2 (MFN2) is an important transmembrane GTPase in the mitochondrial outer membrane and it can block cells in the G0/G1 stage of the cell cycle. To investigate the contribution of aberrant MFN2 transcription in Hcy-induced VSMCs proliferation and the underlying mechanisms. Cell cycle analysis revealed a decreased proportion of VSMCs in G0/G1 and an increased proportion in S phase in atherosclerotic plaque of APOE-/- mice with hyperhomocystinaemia (HHcy) as well as in VSMCs exposed to Hcy in vitro. The DNA methylation level of MFN2 promoter was obviously increased in VSMCs treated with Hcy, leading to suppressed promoter activity and low expression of MFN2. In addition, we found that the expression of c-Myc was increased in atherosclerotic plaque and VSMCs treated with Hcy. Further study showed that c-Myc indirectly regulates MFN2 expression is duo to the binding of c-Myc to DNMT1 promoter up-regulates DNMT1 expression leading to DNA hypermethylation of MFN2 promoter, thereby inhibits MFN2 expression in VSMCs treated with Hcy. In conclusion, our study demonstrated that Hcy-induced hypermethylation of MFN2 promoter inhibits the transcription of MFN2, leading to VSMCs proliferation in plaque formation, and the increased binding of c-Myc to DNMT1 promoter is a new and relevant molecular mechanism.

Homocysteine inhibits endothelial progenitor cells proliferation via DNMT1-mediated hypomethylation of Cyclin A.

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.

Neuroprotection of Cytisine Against Cerebral Ischemia-Reperfusion Injury in Mice by Regulating NR2B-ERK/CREB Signal Pathway.

The aim of the study was to elucidate the therapeutic effects of Cytisine (CYT) on cerebral ischemia-reperfusion injury in mice. Male ICR mice were pretreated with reagents (drug), and then subjected to 2 h focal cerebral ischemia and 24 h reperfusion. Morphologically, the histopathological impairment were estimated by the TTC, HE and TUNEL staining. The expression of GluN2B-containing NMDA receptor, phosphorylation of extracellular regulated protein kinases, total ERK, phosphorylation of cAMP-response element binding protein and total CREB were determined by immunofluorescence and Western blot assay, respectively. The mRNA expression of NR2B, ERK and CREB were quantified by the real-time RT-PCR. CYT significantly diminished the infarct size and neuronal apoptosis. Additionally, it ameliorated histopathological lesion dramatically. CYT promoted the phosphorylation of ERK, CREB and their mRNA expression. In contrast, the expression of NR2B was suppressed in concomitant with the down-regulation of genes. The overall results thus far suggest that CYT confers the neuroprotection against cerebral I/R injury by regulating the NR2B-ERK/CREB signal pathway.

Mechanism of Lycium barbarum polysaccharides on primary cultured rat hippocampal neurons.

Lycium barbarum polysaccharides (LBP) have been reported to have a wide range of beneficial effects including neuroprotection, anti-aging and anticancer. However, the anti-inflammation mechanism of LBP on primary cultured rat hippocampal neurons injured by oxygen-glucose deprivation/reperfusion (OGD/RP) is incompletely understood. We investigate the neuroprotective effects of LBP on neonatal rat primary cultured hippocampal neurons injured by OGD/RP with different approaches: MTT assay was used to detect cell viability, lactate dehydrogenase leakage was used to detect neuronal damage, formation of reactive oxygen species was determined by using fluorescent probe DCFH-DA. Hoechst 33,342 staining and TUNEL staining were used to determine the cell apoptosis. JC-1 was used to evaluate loss of mitochondrial membrane potential (MMP). The fluorescence intensity of [Ca2+]i in hippocampal neurons was determined by laser scanning confocal microscopy. The expression of various apoptotic markers such as TLR4, IκB, IL-6 and NF-κB were investigated by RT-PCR and western blot analysis. Results from each approach demonstrated that LBP increased the cell abilities and decreased the cell morphologic impairment. Furthermore, LBP increased MMP but inhibited [Ca2+]i elevation and significantly suppressed overexpression of NF-κB, IL-6 TLR4 and increased IκB expression.

Endoplasmic reticulum oxidoreductin 1α mediates hepatic endoplasmic reticulum stress in homocysteine-induced atherosclerosis.

Endoplasmic reticulum (ER) stress is emerging as an important modulator of different pathological process and as a mechanism contributing to homocysteine (Hcy)-induced hepar injury. However, the molecular event that Hcy-induced ER stress in the hepar under the atherosclerosis background is currently unknown. Endoplasmic reticulum oxidoreductin 1α (ERO1α) plays a crucial role in maintaining ER stress function. In this study, we determined the expression of ERO1α in the hepar in hyperhomocysteinemia and the effect of ERO1α in hepacytes ER stress in the presence of Hcy. HHcy model was established by feeding the methionine diet in apolipoprotein-E-deficient (ApoE-/-) mice, and the hepatocytes were incubated with folate and different concentrations of Hcy. Our results showed that Hcy triggered ER stress characterized by an increased contents of glucose-regulated protein 78 (GRP78), protein kinase RNA-like ER kinase (PERK), activating transcription factor (ATF) 6 and X-box binding protein-1 (XBP-1). The ERO1α expressions in HHcy mice and Hcy-treated hepatocytes were decreased compared with those in ApoE-/- group and control hepacytes (P < 0.05), respectively. Knocking-down the expression of ERO1α with small-interfering RNA significantly augmented Hcy-induced ER stress. Meanwhile, the expressions of ER stress-related factor including GRP78, PERK, ATF6 and XBP-1, were significantly decreased when the ERO1α gene was over-expressed in hepacytes. Our results suggested that ERO1α may be involved in Hcy-induced hepar ER stress, and the inhibition of ERO1α expression can accelerate this process.

Neuroprotective effects of oxysophocarpine on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion.

CONTEXT: Oxysophocarpine (OSC), a quinolizidine alkaloid extracted from leguminous plants of the genus Robinia, is traditionally used for various diseases including neuronal disorders. OBJECTIVE: This study investigated the protective effects of OSC on neonatal rat primary-cultured hippocampal neurons were injured by oxygen-glucose deprivation and reperfusion (OGD/RP). MATERIALS AND METHODS: Cultured hippocampal neurons were exposed to OGD for 2 h followed by a 24 h RP. OSC (1, 2, and 5 µmol/L) and nimodipine (Nim) (12 µmol/L) were added to the culture after OGD but before RP. The cultures of the control group were not exposed to OGD/RP. MTT and LDH assay were used to evaluate the protective effects of OSC. The concentration of intracellular-free calcium [Ca(2+)]i and mitochondrial membrane potential (MMP) were determined to evaluate the degree of neuronal damage. Morphologic changes of neurons following OGD/RP were observed with a microscope. The expression of caspase-3 and caspase-12 mRNA was examined by real-time quantitative PCR. RESULTS: The IC50 of OSC was found to be 100 µmol/L. Treatment with OSC (1, 2, and 5 µmol/L) attenuated neuronal damage (p < 0.001), with evidence of increased cell viability (p < 0.001) and decreased cell morphologic impairment. Furthermore, OSC increased MMP (p < 0.001), but it inhibited [Ca(2+)]i (p < 0.001) elevation in a dose-dependent manner at OGD/RP. OSC (5 µmol/L) also decreased the expression of caspase-3 (p < 0.05) and caspase-12 (p < 0.05). DISCUSSION AND CONCLUSION: The results suggested that OSC has significant neuroprotective effects that can be attributed to inhibiting endoplasmic reticulum (ER) stress-induced apoptosis.

Oxysophoridine protects against focal cerebral ischemic injury by inhibiting oxidative stress and apoptosis in mice.

Our previous studies have demonstrated that oxysophoridine (OSR) has protective effects on cerebral neurons damage in vitro induced by oxygen and glucose deprivation. In this study, we further investigated whether OSR could reduce ischemic cerebral injury in vivo and its possible mechanism. Male Institute of cancer research mice were intraperitoneally injected with OSR (62.5, 125 and 250 mg/kg) for seven successive days, then subjected to brain ischemia induced by the model of middle cerebral artery occlusion. After reperfusion, neurological scores and infarct volume were estimated. Morphological examination of tissues was performed. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining. Oxidative stress levels were assessed by measurement of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. The expression of various apoptotic markers as Caspase-3, Bax and Bcl-2 were investigated by immunohistochemistry and Western-blot analysis. OSR pretreatment groups significantly reduced infract volume and neurological deficit scores. OSR decreased the percentage of apoptotic neurons, relieved neuronal morphological damage. Moreover, OSR markedly decreased MDA content, and increased SOD, GSH-Px activities. Administration of OSR (250 mg/kg) significantly suppressed overexpression of Caspase-3 and Bax, and increased Bcl-2 expression. These findings indicate that OSR has a protective effect on focal cerebral ischemic injury through antioxidant and anti-apoptotic mechanisms.

Relaxative effect of core licorice aqueous extract on mouse isolated uterine horns.

CONTEXT: Primary dysmenorrhea is one of the most frequent gynecological disorders in young women. Chinese herbal medicine has the advantage in terms of multi-targeting efficacy, lower toxicity, as well as lower cost. Core licorice is the hard and atropurpureus heart part in root and rootstock of Glycyrrhiza uralensis Fisch (Leguminosae), having a therapeutic effect on dysmenorrhea. OBJECTIVE: This experiment indicated the spasmolytic effect of core licorice aqueous extract (CLE) on spontaneous rhythmic contractions and spasmogen-provoked contractions of stilbestrol primed, estrogen-dominated, non-pregnant mouse isolated uterine horns and its spasmolytic mechanism. MATERIALS AND METHODS: We investigated the spasmolytic effect of CLE (0.025-0.1 mg/mL) on spontaneous contractions and potassium chloride (KCl, 40 mM), acetylcholine (ACh, 5 µg/mL), carbachol (CCh, 5 µg/mL), oxytocin (OT, 2 U/L) or bradykinin (5 ng/mL)-provoked contractions of mouse isolated uterine horns. Contractions were recorded by tension force transducers using Biolap 420F software on a PC. RESULTS: Our present study showed that graded, escalated concentrations of CLE (0.025-0.1 mg/mL) significantly inhibited the amplitude of spontaneous phasic contractions (15.03-55.10%), as well as the contractions produced by KCl (40 mM; 20.16-53.99%), ACh (5 µg/mL; 14.65-48.32%), CCh (5 µg/mL; 38.40-76.70%), OT (2 U/L; 21.53-58.49%) or bradykinin (5 ng/mL; 58.01-79.44%) of the estrogen-dominated isolated mice uterine horn preparations in a concentration-related manner. DISCUSSION AND CONCLUSION: The spasmolytic effect of CLE observed in the present study lends pharmacological support to the traditional use of core licorice in the management, control and treatment of primary dysmenorrhea.

miR-195-3p alleviates homocysteine-mediated atherosclerosis by targeting IL-31 through its epigenetics modifications.

Atherosclerosis is a serious age-related disease, which has a tremendous impact on health care globally. Macrophage inflammation is crucial for the initiation and progression of atherosclerosis, and microRNAs (miRNAs) recently have emerged as potent modulators of inflammation, while the underlying mechanisms of its involvement in homocysteine (Hcy)-mediated macrophage inflammation of atherosclerosis remain largely unknown. Here, we demonstrated that elevated Hcy inhibits the expression of miR-195-3p, which in turn enhances IL-31 expression and thereby causes the secretion of macrophages pro-inflammatory factors IL-1ß, IL-6 and TNF-α and accelerate atherosclerosis. Furthermore, we identified that Hcy can induce DNA hypermethylation and H3K9 deacetylation of miR-195-3p promoter due to the increased the binding of DNMT3a and HDAC11 at its promoter. More importantly, Sp1 interacts with DNMT3a suppressed the binding of HDAC11 at miR-195-3p promoter and promoted its transcription. In summary, our results revealed a novel mechanism that transcriptional and epigenetic regulation of miR-195-3p inhibits macrophage inflammation through targeting IL-31, which provides a candidate diagnostic marker and novel therapeutic target in cardiovascular diseases induced by Hcy.

TGFB3-AS1 promotes Hcy-induced inflammationof macrophages via inhibiting the maturityof miR-144 and upregulating Rap1a.

It has been demonstrated that homocysteine (Hcy) can cause inflammatory diseases. Long noncoding RNAs (lncRNA) and microRNAs (miRNAs) are involved in this biological process, but the mechanism underlying Hcy-induced inflammation remains poorly understood. Here, we found that lncRNA TGFB3-AS1 was highly expressed in macrophages treated with Hcy and the peripheral blood monocytes from cystathionine beta-synthase heterozygous knockout (CBS +/-) mice with a high-methionine diet using lncRNA microarray. In vivo and in vitro experiments further confirmed that TGFB3-AS1 accelerated Hcy-induced inflammation of macrophages through the Rap1a/wnt signaling pathway. Meanwhile, TGFB3-AS1 interacted with Rap1a and reduced degradation of Rap1a through inhibiting its ubiquitination in macrophages treated with Hcy. Rap1a mediated inflammation induced by Hcy and serves as a direct target of miR-144. Moreover, TGFB3-AS1 regulated miR-144 by binding to pri-miR-144 and inhibiting its maturation, which further regulated Rap1a expression. More importantly, we found that high expression of TGFB3-AS1 was positively correlated with the levels of Hcy and proinflammatory cytokines in serum of healthy individuals and patients with HHcy. Our study revealed a novel mechanism by which TGFB3-AS1 promoted inflammation of macrophages through inhibiting miR-144 maturation to stay miR-144 regulated inhibition of functional Rap1a expression.

Protective Effects of 18ß-Glycyrrhetinic Acid on Monocrotaline-Induced Pulmonary Arterial Hypertension in Rats.

Pulmonary arterial hypertension (PAH) is a destructive and rare disorder characterized by a progressive increase in pulmonary artery pressure and vasoconstriction, ultimately leading to right ventricular failure and death. 18ß-Glycyrrhetinic acid (18ß-GA) is an active ingredient in the commonly used Chinese herbal medicine radix glycyrrhizae, and it possesses antioxidant, anti-inflammatory, anti-tumor, and other pharmacological properties. This study aimed to determine whether 18ß-GA has protective effects against monocrotaline (MCT)-induced PAH and whether it is associated with oxidative stress. The PAH of rats was induced by MCT (60 mg/kg) and oral administration of 18ß-GA (100, 50, or 25 mg/kg/day), sildenafil (30 mg/kg), or saline for 21 consecutive days. The development of PAH was evaluated by hemodynamic parameters and right ventricular hypertrophy index. Hematoxylin and eosin staining, Masson trichrome staining, and electron microscopy were used to determine the degree of vascular remodeling and proliferation in lung tissue. Moreover, the antioxidant capacity and malondialdehyde levels in the lungs were measured according to the instructions provided by the test kits, and the expression levels of nicotinamide adenine dinucleotide phosphate oxidase-2 (Nox2) and Nox4 were detected through Western blot analysis. Results of our study indicated that 18ß-GA treatment significantly improved the hemodynamic and pathomorphological data of the rats, reduced the changes in oxidative stress biomarkers, and inhibited Nox2 and Nox4 expression. Our research indicated that 18ß-GA has a protective effect against MCT-induced PAH by inhibiting oxidative stress in rats.

Protective effects of 18ß-glycyrrhetinic acid on pulmonary arterial hypertension via regulation of Rho A/Rho kinsase pathway.

PURPOSE: Excessive proliferation, migration and anti-apoptosis of pulmonary artery smooth muscle cells (PASMCs) are the basis for the development of pulmonary vascular remodeling, and it is the driving force for pulmonary arterial hypertension (PAH). 18ß-glycyrrhetinic acid (18ß-GA) is the main active substance extracted from Chinese herbal medicine licorice, with outstanding anti-inflammatory, anti-oxidation and anti-proliferative effects. Our team found in previous studies that 18ß-GA has protective effects on monocrotaline-induced PAH in rats. However, the anti-angiogenic effect of 18ß-GA on PAH remains unclear. Therefore, in order to further investigate whether the beneficial effects of 18ß-GA on PAH are related to its antiproliferative effect, we conducted experiments in vivo and in vitro. METHODS AND RESULTS: In vivo, 18ß-GA relieved mean pulmonary arterial pressure, right ventricular systolic pressure, and right ventricular hypertrophy index, improving pulmonary remodeling. In vitro, 18ß-GA significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of HPASMCs, blocking the progression of G0/G1 to S phase of the cell cycle. Furthermore, after treatment with 18ß-GA, the expression of Rho A, ROCK1, ROCK2 was decreased and ROCK activity was inhibited in HPASMC. In addition, 18ß-GA also attenuated PDGF-induced changes in p27kip1, Bax and Bcl-2. CONCLUSIONS: In summary, these results indicate that 18ß-GA regulates the activity of RhoA-ROCK signaling pathway, inhibits the proliferation of HPASMCs, and has potential value in the treatment of PAH.

Protective effects of aloperin on monocroline-induced pulmonary hypertension via regulation of Rho A/Rho kinsase pathway in rats.

Pulmonary hypertension (PH) is fatal disease which closely involves Rho A/ Rho kinsase (ROCK) pathway. Aloperine is a main active alkaloid extracted from Sophora alopecuroides, which is a traditional Chinese herbal medicine that has been used widely. However, the effects of this alkaloid on pulmonary hypertension and its mechanisms remain unclear. Therefore, this study is designed to investigate whether aloperine has protective effects on PH induced by monocrotaline, whether these effects may be related to regulation of RhoA/ROCK pathway in rats. Pulmonary hypertension was induced by monocrotaline (60mg/kg), and subsequently oral administration of aloperine (25, 50, 100mg/kg/day) for 21 days. At the end of the experiment, rats were underwent hemodynamic and morphologic assessments. At same time, the expression of Rho A, ROCK1, ROCK2, as well as activities of ROCK in the lung of rat has been detected. Afterwards, the expression of p27kip1, Bax, Bcl-2, which was the downstream proliferation and apoptosis factors of ROCK, were tested. The result indicted that aloperine treatment showed significantly improvement in hemodynamic and pathomorphologic data. Moreover, the reduction in expression of Rho A, ROCK1, ROCK2, and suppression in activities of ROCK were found in rat lungs after aloperine treatment. Furthermore, aloperine also alleviated the MCT-induced changes of p27kip1, Bax and Bcl-2. In summary, this study indicates that aloperine have protective effects on monocrotaline-induced PH. And these effects may be partially related to RhoA/ROCK pathway. Thus, aloperine could be considered a possible therapeutic strategy for PH.

Protective effects of aloperine on monocrotaline-induced pulmonary hypertension in rats.

Pulmonary hypertension (PH) is serious, fatal disease which is promoted by oxidative stress. Aloperine have antioxidation effects, which effects on pulmonary arteries remain unclear. Therefore, this study is designed to investigate whether aloperine has protective effects on PH induced by monocrotaline and whether these effects are associated with oxidative stress. PH was induced by monocrotaline (60mg/kg), and subsequently oral administration of aloperine (25, 50, 100mg/kg/day). At the end of the experiment, hemodynamic, pathomorphologic, electrocardiographic and echocardiographic data from the rats were obtained. At same time, oxidative stress biomarkers (superoxide dismutase, malonyldialdehyde, catalase, glutathione peroxidase, total antioxidant capacity) and the protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-2, NOX-4 in the lung of rat has been detected. The result shows that aloperine treatment showed significantly improvement in hemodynamic, pathomorphologic, electrocardiographic and echocardiographic data. Moreover, aloperine treatment can alleviate the changes of oxidative stress biomarkers and suppress the expression levels of NOX-2, NOX-4. In summary, this study indicates that aloperine have protective effects on monocrotaline-induced PH. And these effects may be related to inhibit oxidative stress.

Homocysteine­induced oxidative stress through TLR4/NF­κB/DNMT1­mediated LOX­1 DNA methylation in endothelial cells.

Atherosclerosis (AS) is a progressive disease of multifactorial origin, which occurs in response to endothelial injury. Increased homocysteine (Hcy) is considered a major cause of endothelial dysfunction, oxidative stress and DNA methylation; however, the mechanisms remain to be fully elucidated. The aim of the present study was to investigate whether Hcy causes injury to endothelial cells (ECs) by the effect of lectin­like oxidized­low density lipoprotein receptor­1 (LOX­1) DNA methylation through toll­like receptor 4(TLR4)/nuclear factor (NF)­κB/DNA methyltransferase (DNMT)1. The ECs were treated with different concentrations of Hcy, and it was found that Hcy promoted the expression of TLR4, leading to EC injury. The effect of oxidative stress was analyzed by measuring superoxide dismutase, malondialdehyde and hydrogen peroxide in the ECs. In addition, the association between NF­κB and DNMT1 was examined by treatment of the ECs with pyrrolidine dithiocarbamate (PDTC). The results suggested that Hcy induced LOX­1 DNA hypomethyaltion to promote the expression levels of LOX­1. Taken together, Hcy injured the ECs through the effect of methylation and trans­sulfuration metabolism of LOX­1 through TLR4/NF­κB/DNMT1. Following injury to the ECs, lipids, particularly ox­LDL, accumulated in the sub­endothelial layer to promote the formation of AS.