Two new sesquiterpenes from Sonchus oleraceus and inhibitory mechanism on murine haemangioendothelioma (EOMA) cell lines.

Sonchus oleraceus is a kind of medicinal and edible plant which is widely distributed. In this study, two new sesquiterpenes 1 and 2 along with three known compounds 3-5 were isolated from Sonchus oleraceus by the methods of column chromatography. The structures of the two novel compounds were constructed on the basis of HR-MS and NMR spectra. Cytotoxicities of 1 and 2 were assayed on EOMA cell lines and 1 exhibited no inhibitory effect while 2 elicited moderate inhibitory effect on EOMA cells with IC50 value of 26.5 µM. Western Blot assay indicated that 2 could suppress EOMA cell proliferation by inducing apoptosis through Bax/caspase-3 pathway.

miR-31 from adipose stem cell-derived extracellular vesicles promotes recovery of neurological function after ischemic stroke by inhibiting TRAF6 and IRF5.

Ischemic stroke affects many people in the world, but the underlying mechanism is not completely understood. In this study, we investigated the effect of microRNA (miR)-31 on ischemic stroke. We also determined downstream signaling pathway of miR-31 in recovery of neurological function in ischemic stroke. Middle cerebral artery occlusion (MCAO) in mice was used to mimic human stroke. Foot fault test and mNSS were used to evaluate neurological deficits in mice after stroke. TTC staining in brain tissues was used for determining infarct volume. We extracted and identified extracellular vesicles (EVs) derived from adipose-derived stem cells (ADSCs) to study the impact of miR-31 and TRAF6 by miR-31 overexpression or TRAF6 knockdown on stroke recovery. Primary mouse neuron exposed to oxygen-glucose deprivation (OGD) was used to mimic neuronal ischemic injury. RT-qPCR and Western blot analysis were used for determination of mRNA and protein expression, respectively. MTT assay was used for studying cell survival. TUNEL staining was sued for neuron apoptosis. Starbase website and dual luciferase reporter gene assay were utilized to predicted and verify binding relationship between miR-31 and TRAF6. Neurological functions were improved by miR-31 from ADSC-derived EVs, as suggested by improved foot fault and mNSS. miR-31 from ADSC-derived EVs also reduced infarct volume and neuronal cell apoptosis after stroke in mice. Similarly, in neuronal cell culture, miR-31 from ADSC-derived EVs reduced the expression of apoptosis-related factors cleaved caspase-3 and Bax, increased the survival, and reduced apoptosis of neuronal cells after OGD. miR-31 was found to downregulate the expression of TRAF6 by binding to the 3'-untranslated region (3'-UTR) of TRAF6, which in turn upregulated IRF5 expression. Increased expression of IRF5 led to increased neuron apoptosis after OGD. In conclusion, miR-31 from ADSC-derived EVs can downregulate expression of TRAF6 and IRF5, leading to reduced neuronal damage induced by ischemic stroke.

microRNA-381-3p Confers Protection Against Ischemic Stroke Through Promoting Angiogenesis and Inhibiting Inflammation by Suppressing Cebpb and Map3k8.

Ischemic stroke is a serious disease with limited prevention methods, and various genes and microRNAs (miRNAs) have been found to be dysregulated in the pathogenesis of this disease. This study aims to explore the potential role of miR-381-3p in ischemic stroke, along with its underlying mechanism. A mouse model of ischemic stroke was developed using middle cerebral artery occlusion. Next, the expression of mitogen-activated protein kinase kinase kinase 8 (Map3k8) and CCAAT enhancer binding protein beta (Cebpb) was determined by RT-qPCR. Gain- and loss-of-function approaches were applied to analyze the effects of miR-381-3p, Cebpb and Map3k8 on the biological functions of endothelial progenitor cells (EPCs) with the involvement of the tumor necrosis factor-α (TNF-α) signaling pathway. In addition, dual luciferase reporter gene assay was performed for the analysis of the relationship among miR-381-3p, Map3k8 and Cebpb. Further, rescue experiment was performed with the help of JNK/p38 specific agonist, Anisomycin. Map3k8 and Cebpb were highly expressed in ischemic stroke. Loss-of-function of Map3k8 or Cebpb in EPCs contributed to accelerated proliferation, migration and angiogenesis of EPCs. Next, miR-381-3p downregulated the expression of its two target genes, Map3k8 and Cebpb. miR-381-3p overexpression promoted angiogenesis of EPCs, and inhibited inflammation, which could be reversed by restoration of Map3k8 or Cebpb. Additionally, silencing Map3k8 or Cebpb inhibited the activation of TNF-α signaling pathway. Furthermore, Anisomycin treatment could enhance inflammation and inhibit angiogenesis. Taken together, miR-381-3p downregulates Map3k8 and Cebpb to protect against ischemic stroke, broadening our understanding of the pathogenesis of ischemic stroke.

Long non-coding RNA Gas5 potentiates the effects of microRNA-21 downregulation in response to ischaemic brain injury.

Brain ischaemia, which can cause severe nerve injury, is a global health challenge. Long non-coding RNA (lncRNA) growth-arrest specific 5 (Gas5) has been documented to exert tumour suppressive effects in several cancers. However, its role in cerebrovascular disease still requires further investigation. Therefore, in this study, we focused on the role of lncRNA regulatory signalling related to lncRNA Gas5 in ischaemic brain injury. Middle cerebral artery occlusion (MCAO) was employed as a model of ischaemic brain injury in rats. The expression of lncRNA Gas5 and microRNA-21 (miR-21) was altered in neurons to elucidate their effects in ischaemic brain injury and to identify the interactions among lncRNA Gas5, miR-21 and Pten. The neuronal survival rate, apoptosis and the expression of phosphatidyl inositol 3-kinase (PI3K)/Akt signalling pathway-related genes were also evaluated in vitro to determine the effects of lncRNA Gas5. In the brains of rats subjected to MCAO, the expression of lncRNA Gas5 and Pten was upregulated, while miR-21 was downregulated. LncRNA Gas5 inhibited miR-21 expression, leading to elevated levels of Pten. In vitro experiments revealed that lncRNA Gas5 depletion and miR-21 elevation resulted in the suppression of neuronal apoptosis, thus promoting neuronal survival via the PI3K/Akt signalling pathway. These findings demonstrate that lncRNA Gas5 increases miR-21 and activates Pten, contributing to the development of ischaemic brain injury, supporting the silencing of lncRNA Gas5 as a possible therapeutic target for the treatment of ischaemic brain injury.

Upregulated microRNA-31 inhibits oxidative stress-induced neuronal injury through the JAK/STAT3 pathway by binding to PKD1 in mice with ischemic stroke.

Ischemic stroke (IS), which is characterized by high morbidity, disability, and mortality, is recognized as a major cerebrovascular disease. MicroRNA-31 (miR-31) was reported to participate in the progression of brain disease. The present study was conducted in order to investigate the effect of miR-31 on oxidative stress-induced neuronal injury in IS mice with the involvement of protein kinase D1 (PKD1) and the JAK/STAT3 pathway. C57BL/6J mice were used to establish the middle cerebral artery occlusion (MCAO) model. Astrocytes were transfected with miR-31 mimic, miR-31 inhibitor, si-PKD1, or JAK-STAT3 pathway inhibitor. Following the establishment of an oxygen-glucose deprivation (OGD) model, the astrocytes were cocultured with neuronal OGD. Lower miR-31, higher PKD1 expressions, and activated JAK/STAT3 pathway were found in both the MCAO and OGD models. miR-31 could negatively target PKD1. In an MCAO model, overexpressing miR-31 and silencing PKD1 reduced neuronal injury, cerebral infarct volume, neuron loss, and oxidative stress injury, inhibited the activation of JAK/STAT3 pathway and the expressions of PKD1, interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, malondialdehyde, 4-HNE, 8-HOdG, caspase-3, and Bax, but increased the superoxide dismutase content. In the OGD model, overexpression of miR-31 and silencing of PKD1 attenuated oxidative stress-induced neuronal injury, and diminished the lactate dehydrogenase leakage and reactive oxygen species level, accompanied by elevated neuronal viability. These results indicate that miR-31 alleviates inflammatory response as well as an oxidative stress-induced neuronal injury in IS mice by downregulating PKD1 and JAK/STAT3 pathway.

MicroRNA-150 contributes to ischemic stroke through its effect on cerebral cortical neuron survival and function by inhibiting ERK1/2 axis via Mal.

Ischemic stroke, caused by the blockage of blood supply, is a major cause of death worldwide. For identifying potential candidates, we explored the effects microRNA-150 (miR-150) has on ischemic stroke and its underlying mechanism by developing a stable middle cerebral artery occlusion (MCAO) rat model. Gene expression microarray analysis was performed to screen differentially expressed genes associated with MCAO. We evaluated the expression of miR-150 and Mal and the status of ERK1/2 axis in the brain tissues of MCAO rats. Then the cerebral cortical neurons (CCNs) were obtained and introduced with elevated or suppressed miR-150 or silenced Mal to validate regulatory mechanisms for miR-150 governing Mal in vitro. The relationship between miR-150 and Mal was verified by dual luciferase reporter gene assay. Besides, cell growth and apoptosis of CCNs were detected by means of MTT assay and flow cytometry analyses. We identified Mal as a downregulated gene in MCAO, based on the microarray data of GSE16561. MiR-150 was over-expressed and negatively targeted Mal in the brain tissues obtained from MCAO rats and their CCNs. Increasing miR-150 blocked the ERK1/2 axis, resulting in an inhibited cell growth of CNNs but an enhanced apoptosis. Furthermore, MiR-150 inhibition was observed to have effects on CNNs as opposed to those inhibited by miR-150 promotion. The key findings of this study support the notion that miR-150 under-expression-mediated direct promotion of Mal protects CNN functions through the activation of the ERK1/2 axis, and underscore the concept that miR-150 may represent a novel pharmacological target for ischemic stroke intervention.

CXCL8 gene silencing promotes neuroglial cells activation while inhibiting neuroinflammation through the PI3K/Akt/NF-κB-signaling pathway in mice with ischemic stroke.

OBJECTIVE: Ischemic stroke is known as a neurodegenerative disorder, which induces long-period tissue damage. Chemokine (C-X-C motif) ligand 8 (CXCL8) is involved in acute inflammation and tumor progression through the phosphoinositide-3-kinase/protein kinase B/nuclear factor-κB (PI3K/Akt/NF-κB)-signaling pathway. In this study, we aimed to explore the mechanism of CXCL8 in ischemic stroke in relation to the PI3K/Akt/NF-κB-signaling pathway. METHODS: Microarray-based gene expression profiling of peripheral blood mononuclear cells was used to identify ischemic stroke-related differentially expressed genes and explore role of CXCL8 in ischemic stroke. Next, the ischemic mice model was successfully established, with transfection efficiency detected. After that, deflection index, recovery of nervous system, infarct sizes, ischemia-induced apoptosis, and neuroinflammatory response in ischemic stroke were measured. At last, the content of inflammatory factors as well as the expression of CXCL8, caspase-3, caspase-9, Bad, interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), Akt, PI3K, and NF-κB were determined. RESULTS: Comprehensive gene expression profiling analysis identified that CXCL8 might affect the development of ischemic stroke through regulating the PI3K/Akt/NF-κB-signaling pathway. CXCL8 silencing significantly reduced deflection index and infarct size, improved neurological function, and suppressed neuroglial cell loss and apoptosis index. In addition, glial fibrillary acidic portein (GFAP) and ionized calcium-binding adapter molecule 1 (IBA-1) expressions were decreased following CXCL8 suppression, suggesting CXCL8 affected neuroglial activation. Importantly, we also found that CXCL8 silencing activated neuroglial cell and suppressed inflammatory cytokine production in ischemic stroke mice. CONCLUSION: Taken together, these findings highlight that functional suppression of CXCL8 promotes neuroglial activation and inhibits neuroinflammation by regulating the PI3K/Akt/NF-κB-signaling pathway in mice with ischemic stroke, which might provide new insight for ischemic stroke treatment.

Roles of Nrf2/HO-1 and HIF-1α/VEGF in lung tissue injury and repair following cerebral ischemia/reperfusion injury.

Cerebral ischemia/reperfusion injury (CIRI) leads to injury in distant organs, most commonly the lungs, although limited studies have examined self-protective mechanisms during CIRI-induced lung injury. Here, we investigated self-protective mechanisms that attenuate stress-related injury and promote the angiogenetic repair of epithelial function during CIRI-induced lung injury by measuring nuclear factor erythroid-related factor 2 (Nrf2) and hypoxia-inducible factor-1α (HIF-1α) levels. A CIRI model was established in male Sprague-Dawley rats by blocking the middle cerebral artery. Rats were divided into five subgroups based on the reperfusion time (6, 12, 24, 48, and 72 hr). Lung injury was assessed using a semiquantitative score and a thiobarbituric acid-based method of determining malonaldehyde production. Lung tissue angiogenesis was detected by CD34 and CD31 immunolabeling. Changes in Nrf2, heme oxygenase-1 (HO-1), HIF-1α, vascular-endothelial growth factor (VEGF), phosphatidylinositol 3-kinase (PI3K), extracellular-regulated kinase1/2 (ERK1/2), and phospho-ERK1/2 ( p-ERK1/2) protein- and mRNA-expression levels were measured by immunohistochemistry and reverse transcription polymerase chain reactions, respectively. Oxidative stress induced by cerebral ischemia/reperfusion (CI/R) caused lung injury. Expression of the Nrf2/HO-1 antioxidative stress pathway in lung tissues increased following CI/R, peaking after 24 hr. PI3K, ERK, and p-ERK1/2, which act upstream of Nrf2/HO-1, were expressed at higher levels in the CI/R-model group, consistent with the general trends observed for Nrf2/HO-1. Within 72 hr post-CI/R, HIF-1α, and VEGF expression significantly increased versus the sham group. Thus, during CIRI-induced lung injury, the body may upregulate antioxidative stress activities and promote angiogenesis to repair the endothelial barrier through the Nrf2/HO-1 and HIF-1α/VEGF signaling pathways, enabling self-protection.

Cerebral ischemia/reperfusion injury induces airway mucus hypersecretion in rats and activates IL­13­associated inflammatory mechanisms.

The majority of patients that suffer a stroke have excessive sputum, which accelerates the development of pulmonary complications. However, it is unclear whether cerebral ischemia and reperfusion (I/R) injury induces mucus hypersecretion, and the potential role of inflammation remains unknown. In the present study, the reversible middle cerebral artery occlusion model was applied in rats to induce cerebral I/R injury. The rats were grouped according to the duration of reperfusion (6, 12, 24, 48 and 72 h). Neurological dysfunction was evaluated by Longa scoring and lung dry­to­wet weight (dw/ww) ratios were determined to reflect the degree of mucus secretion. Inflammatory factor interleukin­13 (IL­13) and tumor necrosis factor­α (TNF­α) levels in serum and bronchoalveolar lavage fluid (BALF) were determined by enzyme­linked immunosorbent assay. Pulmonary levels of mucin 5AC (MUC5AC) and key molecules involved in nuclear factor­κB (NF­κB) signaling were determined by western blotting and immunohistochemistry. Rats with cerebral I/R had impaired neurological function, which was associated with the length of reperfusion time. In addition, the dw/ww lung ratio decreased and the pulmonary expression of MUC5AC increased with the increase in severity of neurological dysfunction, indicating that cerebral I/R may induce mucus hypersecretion in a reperfusion time­dependent manner. IL­13 and TNF­α levels in serum and BALF, as well as the nuclear translocation of NF­κB p65 in pulmonary tissues, significantly increased following cerebral I/R, which suggests that the activation of IL­13 and NF­κB inflammatory pathways may be involved. The present study concluded that cerebral I/R injury may induce airway mucus hypersecretion by activating IL­13 and NF­κB inflammatory pathways.

Saw Palmetto Extract Inhibits Metastasis and Antiangiogenesis through STAT3 Signal Pathway in Glioma Cell.

Signal transducer and activator of transcription factor 3 (STAT3) plays an important role in the proliferation and angiogenesis in human glioma. Previous research indicated that saw palmetto extract markedly inhibited the proliferation of human glioma cells through STAT3 signal pathway. But its effect on tumor metastasis and antiangiogenesis is not clear. This study is to further clear the impact of saw palmetto extract on glioma cell metastasis, antiangiogenesis, and its mechanism. TUNEL assay indicated that the apoptotic cells in the saw palmetto treated group are higher than that in the control group (p < 0.05). The apoptosis related protein is detected and the results revealed that saw palmetto extract inhibits the proliferation of human glioma. Meanwhile pSTAT3 is lower in the experimental group and CD34 is also inhibited in the saw palmetto treated group. This means that saw palmetto extract could inhibit the angiogenesis in glioma. We found that saw palmetto extract was an important phytotherapeutic drug against the human glioma through STAT3 signal pathway. Saw palmetto extract may be useful as an adjunctive therapeutic agent for treatment of individuals with glioma and other types of cancer in which STAT3 signaling is activated.

Serenoa Repens Induces Growth Arrest, Apoptosis and Inactivation of STAT3 Signaling in Human Glioma Cells.

Serenoa repens, the extract of berry in Southeastern United States, is one of several phytotherapeutic agents available for the treatment of Benign prostatic hyperplasia (BPH). In this study, we found for the first time that Serenoa repens effectively inhibited the growth of human U87 and U251 glioma cells. Flow cytometry assay showed that Serenoa repens induced apoptosis of U87 and U251 glioma cells in a dose-dependent manner. Also, Serenoa repens increased the expression of cleaved-PARP, Caspase-3 or p27 protein in these two cell lines, respectively. In addition, we found that Serenoa repens down-regulated basal level of phosphorylated form of signal transducer and activator of transcription 3 (STAT 3) in both U87 and U251 glioma cells. Furthermore, it was discovered that a Janus family of tyrosine kinase (JAK) inhibitor AG490 inhibit the growth of human U87 and U251 glioma cells and AG490 enhanced the ability of Serenoa repens to inhibit the growth of U87 and U251 glioma cells as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. These results indicate that Serenoa repens reduces the growth, causes apoptosis of Glioma cells and inhibits STAT 3 signaling. In addition, it might also be useful for the treatment of individuals with glioma.

Serenoa repens induces growth arrest and apoptosis of human multiple myeloma cells via inactivation of STAT 3 signaling.

Serenoa repens, a palm species native to the Southeastern United States, is one of the widely used phytotherapeutic agents in benign prostatic hyperplasia. In this study, we found for the first time that Serenoa repens induced growth arrest of a variety of human leukemia cells including U266 and RPMI 8226 multiple myeloma cells as measured by mitochondrial-dependent conversion of the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. TUNEL assays showed that Serenoa repens induced apoptosis of U266 cells in a time- and dose-dependent manner. Serenoa repens also increased the expression of cleaved-PARP or p27 protein in different human leukemia cell lines. In addition, we found that Serenoa repens down-regulated basal level of phosphorylated form of signal transducer and activator of transcription 3 (STAT 3) and Interleukin-6 induced level of phosphorylated form of STAT 3 and extracellular signal-related kinase (ERK) were also reduced after Serenoa repens treatment in U266 cells. Furthermore, we found that inhibition of STAT 3 signaling by Serenoa repens or Janus family of tyrosine kinase (JAK) inhibitor of AG490 enhanced the ability of docetaxel to inhibit the growth of U266 and RPMI 8226 cells, as measured by trypan blue exclusion test. These results indicate that Serenoa repens might be useful for the treatment of individuals with multiple myeloma.

Improving nerve regeneration of acellular nerve allografts seeded with SCs bridging the sciatic nerve defects of rat.

The objective of the paper is to evaluate the effect of acellular nerve allografts (ANA) seeded with Schwann cells to promote nerve regeneration after bridging the sciatic nerve defects of rats and to discuss its acting mechanisms. Schwann cells were isolated from neonatal Wistar rats. In vitro Schwann cells were microinjected into acellular nerve allografts and co-cultured. Twenty-four Wistar rats weighing 180-220 g were randomly divided into three groups with eight rats in each group: ANA seeded with Schwann cells (ANA + SCs), ANA group and autografts group. All the grafts were, respectively, served for bridging a 10-mm long surgically created sciatic nerve gap. Examinations of regeneration nerve were performed after 12 weeks by transmission electron microscope (TEM), scanning electron microscope (SEM), and electrophysiological methods, and then analyzed statistically. The results obtained indicated that in vitro Schwann cells displayed the feature of bipolar morphology with oval nuclei. Compared with ANA group, the conduction velocity of ANA + SCs group and autograft group was faster after 12 weeks, latent period was shorter, and wave amplitude was higher (P < 0.05). The difference between ANA + SCs group and autograft group is not significant (P > 0.05). Regeneration nerve myelinated fiber number, myelin sheath thickness, and myelinated fibers/total nerves (%) in both ANA + SCs group and autograft group are higher than that in ANA group; the difference is significant (P < 0.05). The difference between the former two is not significant (P > 0.05). In conclusion, ANA seeded with SCs could improve nerve regeneration and functional recovery after bridging the sciatic nerve gap of rats, which offers a novel approach for the repair peripheral nerve defect.