Shiyu Song

Myricitrin attenuates endothelial cell apoptosis to prevent atherosclerosis: An insight into PI3K/Akt activation and STAT3 signaling pathways.

Blood vessel endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) has been implicated in the pathogenesis of atherosclerosis and vasculopathy. The ox-LDL-elicited reactive oxygen species (ROS) release has been assumed to serve a critical function in endothelial damage. Myricitrin (from Myrica cerifera) is a natural antioxidant that has strong anti-oxidative, anti-inflammatory, and anti-nociceptive activities. However, the protective effect of myricitrin on ROS-induced endothelial cell injury and its related molecular mechanisms have never been investigated. This study demonstrates that myricitrin can inhibit ox-LDL-induced endothelial apoptosis and prevent plaque formation at an early stage in an atherosclerotic mouse model. The administration of myricitrin in vivo decreases the thickness of the vascular wall in the aortic arch of ApoE-/- mice. In vitro study shows that ox-LDL-induced human umbilical vein endothelial cell apoptosis can be reduced upon receiving myricitrin pre-treatment. Treatment with myricitrin significantly attenuated ox-LDL-induced endothelial cell apoptosis by inhibiting LOX-1 expression and by increasing the activation of the STAT3 and PI3K/Akt/eNOS signaling pathways. At the same time, our result demonstrates that myricitrin treatment optimizes the balance of pro/anti-apoptosis proteins, including Bax, Bad, XIAP, cIAP-2, and survivin. Our study suggests that myricitrin treatment can effectively protect cells from ox-LDL-induced endothelial cell apoptosis, which results in reduced atherosclerotic plaque formation. This result indicates that myricitrin can be used as a drug candidate for the treatment of cardiovascular diseases.

Heme oxygenase-1 induction attenuates imiquimod-induced psoriasiform inflammation by negative regulation of Stat3 signaling

Heme oxygenase-1 (HO-1), a stress-inducible protein with a potential anti-inflammatory effect, plays an important role in skin injury and wound healing. However, the function of HO-1 in cutaneous inflammatory diseases, such as psoriasis, remains unknown. The abnormal activation of Stat3, a known transcription factor that induces inflammation and regulates cell differentiation, is directly involved in the pathogenesis and development of psoriasis. Hence, targeting Stat3 is potentially beneficial in the treatment of psoriasis. In this study, HO-1 activation significantly alleviated the disease-related pathogenesis abnormality. To determine the mechanism by which HO-1 exerts immune protection on Th17-related cytokines, IL6/IL22-induced Stat3 activation was significantly suppressed, accompanied by decreased cell proliferation and reversed abnormal cell proliferation. Importantly, HO-1-induced Stat3 suppression was mediated through the activation of protein tyrosine phosphatase SHP-1. Overall, our study provides direct evidence indicating that HO-1 might be a useful therapeutic target for psoriasis. SHP-1-mediated suppression of Stat3 activation after HO-1 activation is a unique molecular mechanism for the regulation of Stat3 activation.

Luteolin selectively kills STAT3 highly activated gastric cancer cells through enhancing the binding of STAT3 to SHP-1

The antitumor effect of luteolin, a plant flavonoid, in gastric cancer (GC) cells has not been fully understood. Here we show that luteolin selectively kills STAT3 overactivated GC cells that are often drug resistant. The treatment of luteolin in these GC cells significantly inhibited STAT3 phosphorylation and reduced the expression of STAT3 targeting gene Mcl-1, Survivin and Bcl-xl. Silencing of SHP-1, a protein tyrosine phosphatase, abolished the inhibitory effect of luteolin on STAT3 and cell apoptosis, suggesting that SHP-1 is crucial in luteolin-mediated cellular function. Moreover, this luteolin effect of STAT3 dephosphorylation by SHP-1 involved in HSP-90, which protected STAT3 phosphorylation by forming HSP-90/STAT3 complex. Thus, luteolin inhibited STAT3 activation through disrupting the binding of HSP-90 to STAT3, which promoted its interaction to SHP-1, resulted in the dephosphorylation of STAT3. The GC cell xenograft mouse model confirmed the effectiveness of luteolin induced inhibition of tumor growth in vivo.

Histone deacetylase inhibitors suppress RSV infection and alleviate virus-induced airway inflammation

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and young children. However, the majority of RSV-infected patients only show mild symptoms. Different severities of infection and responses among the RSV-infected population indicate that epigenetic regulation as well as personal genetic background may affect RSV infectivity. Histone deacetylase (HDAC) is an important epigenetic regulator in lung diseases. The present study aimed to explore the possible connection between HDAC expression and RSV-induced lung inflammation. To address this question, RSV-infected airway epithelial cells (BEAS-2B) were prepared and a mouse model of RSV infection was established, and then treated with various concentrations of HDAC inhibitors (HDACis), namely trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). Viral replication and markers of virus-induced airway inflammation or oxidative stress were assessed. The activation of the nuclear factor-κB (NF-κB), cyclo-oxygenase-2 (COX-2), mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling pathways was evaluated by western blot analysis. Our results showed that RSV infection in airway epithelial cells (AECs) significantly decreased histone acetylation levels by altering HDAC2 expression. The treatment of RSV-infected AECs with HDACis significantly restricted RSV replication by upregulating the interferon-α (IFN-α) related signaling pathways. The treatment of RSV-infected AECs with HDACis also significantly inhibited RSV-induced pro-inflammatory cytokine release [interleukin (IL)-6 and IL-8] and oxidative stress-related molecule production [malondialdehyde (MDA), and nitrogen monoxide (NO)]. The activation of NF-κB, COX-2, MAPK and Stat3, which orchestrate pro-inflammatory gene expression and oxidative stress injury, was also significantly inhibited. Our in vivo study using a mouse model of RSV infection validated these results. Treatment with HDACis alleviated airway inflammation and reduced in vivo RSV replication. Our data demonstrated that RSV reduced histone acetylation by enhancing HDAC2 expression. Treatment with HDACis (TSA/SAHA) significantly inhibited RSV replication and decreased RSV-induced airway inflammation and oxidative stress. Therefore, the inhibition of HDACs represents a novel therapeutic approach in modulating RSV-induced lung disease.

Puerarin acts on the skeletal muscle to improve insulin sensitivity in diabetic rats involving μ-opioid receptor

Abstract Puerarin, a major active isoflavone extracted from the root of Pueraria lobate, significantly increases plasma &bgr;‐endorphin and insulin levels and improves impaired insulin signaling in diabetic animals. However, the target tissues and underlying mechanisms in and through which puerarin functions to ameliorating insulin resistance remains largely unclear. In this study, we showed that puerarin enhanced &mgr;‐opioid receptor expression and phosphorylation, and increased insulin‐stimulated glucose transporter 4 translocation to the plasma membrane in the skeletal muscle of diabetic rats, which were recaptured by a direct application of puerarin in the palmitate‐induced insulin‐resistant L6 myotubes. Naloxone, an antagonist of &mgr;‐opioid receptor, blocked these functions of puerarin. No &bgr;‐endorphin was detected either in the muscle of diabetic rats or in the palmitate‐induced insulin‐resistant L6 cells. Furthermore, we presented the evidence to show the interaction between &mgr;‐opioid receptor and insulin receptor substrate 1 in the muscle tissues and cells. These results suggested that puerarin improved insulin sensitivity in the skeletal muscle at least in part by its local effects involving &mgr;‐opioid receptor function.

STAT3 degradation mediated by calcineurin involved in the neurotoxicity of isoflurane.

Isoflurane, a commonly used volatile anesthetic, causes widespread neuronal apoptosis in the developing brain of rodents. Signal transducer and activator of transcription-3 (STAT3) signaling is crucial for cell survival during the neural network establishment period. The aim of this study was to determine whether isoflurane would target STAT3 to deliver its neurotoxicity. Mice at postnatal day 7 and primary cortical neurons cultured for 5 days were treated with isoflurane. Our data showed that isoflurane exposure downregulated the STAT3 survival pathway in the brain of mice and in primary neurons, whereas the mRNA levels of STAT3 remained unchanged after isoflurane exposure. We found that inhibiting the activity of calcineurin, which specifically promotes STAT3 degradation, alleviated isoflurane-induced neural apoptosis. Further studies showed that isoflurane increased calcineurin activity and that the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel was involved in these isoflurane-induced molecular cascades. These findings suggest that isoflurane-induced neurotoxicity may stem from STAT3 degradation, partially through the activation of calcineurin.

SIRT1 Activation Ameliorates Aldara-Induced Psoriasiform Phenotype and Histology in Mice

TO THE EDITOR Sirtuin 1 (SIRT1), an NAD-dependent deacetylase (Imai et al., 2000), acts as a metabolic sensor that functions on both histone and non-histone proteins (Leibiger and Berggren, 2006; Li, 2013). In this study, we find that SIRT1 activation in animals could inhibit Aldarainduced psoriasiform lesions. Moreover, SIRT1–STAT3 interaction may serve as an important mechanism that underlies this anti-psoriasis process in keratinocytes. A psoriatic mouse model (The protocols were approved by the Animal Care and Use Committee at Nanjing University) using Aldara cream (5% imiquimod, 3 M Pharmaceuticals) on shaved back skin exhibited signs of erythema, scaling, and thickening, a psoriasiform phenotype (van der Fits et al., 2009; Walter et al., 2013). Histologic examination showed epidermal hyperplasia and parakeratosis (Figure 1a and b). In addition, the marker for cellular proliferation Ki-67 and the marker for abnormal differentiation of keratinocytes Keratin 17 (Fu and Wang, 2012) were significantly increased in the lesion epithelia (Figure 1d and e). The manifestations closely resembled the characteristics of psoriatic pathology (Supplementary Figure S1 online). Interestingly, the severity of the skin lesion was significantly reduced, when the mice were treated with an SIRT1 activator resveratrol, before and during Aldara administration. This resulted in smoother and thinner skins with decreased scales and erythemas, compared with the mice treated with Aldara only. To determine whether SIRT1 functioned in this process, a SIRT1 inhibitor EX527 was applied to the mice in the same manner. EX527 treatment exacerbated the psoriasiform symptoms (Figure 1a). The score of psoriasis area and the severity index showed a consistent change (Figure 1c). Histologically, skin lesions of resveratrol-treated mice showed reduced epidermal hyperplasia. In comparison, increased epidermal hyperplasia and acanthosis were observed in EX527-treated mice (Figure 1b, Supplementary Figure S2 online). The changes in Ki-67 and Keratin 17 levels in the epithelia were consistent with the histological alterations (Figure 1d and e, Supplementary Figure S3A–S3C online). Furthermore, increased CD4 immunocyte infiltration was observed in the Aldara-induced lesional skins, which resembled one of the characteristics of human psoriatic skin tissues. The infiltration of CD4 immunocytes was reduced in the resveratrol group and increased in the EX527 group (Figure 1f). Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor that regulates cell growth and differentiation in response to cytokines (Sano et al., 2008). Excessive activation of STAT3 has a key role in psoriasis pathogenesis (Sano et al., 2005; Andres et al., 2013). In our study, Aldara treatment also increased STAT3 phosphorylation in Tyr705 (PY-STAT3) in the epithelia of mouse lesion skin. We previously demonstrated that SIRT1 suppresses STAT3 phosphorylation by deacetylating STAT3 key lysine sites in hepatic gluconeogenesis in mice (Nie et al., 2009). Therefore, we asked whether this mechanism is involved in the epithelia in the Aldara-induced psoriasis model. We found that Aldara treatment decreased SIRT1 expression and upregulated acetylated STAT3 (Ac-STAT3), as well as PY-STAT3, in the lesion skin of mice. Furthermore, the epithelia of skin lesion in resveratrol-treated mice showed a significant decrease in Ac-STAT3 and PY-STAT3, indicating a downregulation of STAT3 activity in keratinocytes. By contrast, EX527 treatment upregulated acetylation and phosphorylation of STAT3 (Figure 1e and g, Supplementary Figure S3D online). Both resveratrol and EX527 act to regulate SIRT1 function by modulating its deacetylation enzyme activity (Howitz et al., 2003; Napper et al., 2005). Consistently, no changes in SIRT1 expression were detected after resveratrol and EX527 treatment in this study. To test the association of STAT3 with SIRT1 in the keratinocytes, the extracts of mice skin epithelia were analyzed through co-immunoprecipitation. STAT3 was detected in complexes immunoprecipitated with anti-SIRT1 antibody. However, the binding of STAT3 to SIRT1 was significantly reduced in the Aldara-induced psoriatic lesions (Figure 1h, Supplementary Figure S3E online). Overall, these observations suggest that SIRT1 counteracts the pathologic effect of Aldara, presumably through its deacetylation of STAT3 in keratinocytes. Next, we studied the role of SIRT1 in human keratinocytes in vitro. First, the cytokine-induced STAT3 acetylation and phosphorylation in the HaCaT cells were significantly reduced in response to pretreatment of SIRT1 activators, resveratrol, and SRT1720. Conversely, administration with SIRT1 inhibitor EX527 enhanced STAT3 acetylation and phosphorylation (Figure 2a, Supplementary LETTER TO THE EDITOR

Persistent mitoKATP Activation Is Involved in the Isoflurane-induced Cytotoxicity.

Isoflurane exposure induces apoptosis in cultured cells and in the developing brain, while the underlying mechanism remains largely unclarified. This study was designed to determine whether the disruption of mitoKATP-mediated ATP balance was involved in the cytotoxicity of isoflurane. Human neuroglioma cells U251 and 7-day-old mice were treated with isoflurane. A specific mitoKATP antagonist 5-HD was used, and the cellular ATP levels, NAD+/NADH ratios, and mitochondrial transmembrane potential (ΔΨm) were measured. Our data showed that the blockage of mitoKATP by 5-HD mitigated the isoflurane-induced ΔΨm disruption, reactive oxygen species (ROS) accumulation, and apoptosis in U251 cells. Moreover, we found that the toxic effect of isoflurane was not observed in the first 2-h exposure; instead, the cellular ATP levels and NAD+/NADH ratios were markedly increased. The reduction of ATP levels and NAD+/NADH ratios was only detected after this initial phase. This dynamical effect of isoflurane was blocked by 5-HD. In contrast, a ROS scavenger NAC sustained the isoflurane-induced ATP elevation. Similar results were observed in animal studies. And again, 5-HD attenuated isoflurane-induced cognitive disorders in the Intellicage test, a system that assesses place learning behavior in a social environment. Our study uncovered a potential mechanism underlying isoflurane’s toxicity with a therapeutic future.

Effect of puerarin in promoting fatty acid oxidation by increasing mitochondrial oxidative capacity and biogenesis in skeletal muscle in diabetic rats

BackgroundType 2 diabetes is characterized by dyslipidemia and the accumulation of lipids in non-adipose tissue, including skeletal muscle. Puerarin, which is a natural isoflavonoid isolated from the root of the plant Pueraria lobata, has been shown to have antidiabetic activity. However, the lipid-reducing effect of puerarin, in particular in skeletal muscle, has not yet been addressed.MethodsWe examined the effect of puerarin on mitochondrial function and the oxidation of fatty acids in the skeletal muscle of high-fat diet/streptozotocin-induced diabetic rats.ResultsPuerarin effectively alleviated dyslipidemia and decreased the accumulation of intramyocellular lipids by upregulating the expression of a range of genes involved in mitochondrial biogenesis, oxidative phosphorylation, the detoxification of reactive oxygen species, and the oxidation of fatty acids in the muscle of diabetic rats. Also, the effect of puerarin on mitochondrial biogenesis might partially involve the function of the μ-opioid receptor. In addition, puerarin decreased the trafficking of fatty acid translocase/CD36 to the plasma membrane to reduce the uptake of fatty acids by myocytes. In vitro studies confirmed that puerarin acted directly on muscle cells to promote the oxidation of fatty acids in insulin-resistant myotubes treated with palmitate.ConclusionsPuerarin improved the performance of mitochondria in muscle and promoted the oxidation of fatty acids, which thus prevented the accumulation of intramyocellular lipids in diabetic rats. Our findings will be beneficial both for elucidating the mechanism of the antidiabetic activity of puerarin and for promoting the therapeutic potential of puerarin in the treatment of diabetes.

Simultaneous induction of Graves’ hyperthyroidism and Graves’ ophthalmopathy by TSHR genetic immunization in BALB/c mice

Background Graves’ disease is the most common form of autoimmune thyroid disorder, characterized by hyperthyroidism due to circulating autoantibodies. To address the pathological features and establish a therapeutic approach of this disease, an animal model carrying the phenotype of Graves’ disease (GD) in concert with Graves’ Ophthalmopathy (GO) will be very important. However, there are no ideal animal models that are currently available. The aim of the present study is to establish an animal model of GD and GO disease, and its pathological features were further characterized. Methods A recombinant plasmid pcDNA3.1- T289 was constructed by inserting the TSHR A-subunit gene into the expression vector pcDNA3.1, and genetic immunization was successfully performed by intramuscular injection of the plasmid pcDNA3.1-T289 on female 8-week-old BALB/c mice. Each injection was immediately followed by in vivo electroporation using ECM830 square wave electroporator. Morphological changes of the eyes were examined using 7.0T MRI scanner. Levels of serum T4 and TSHR antibodies (TRAb) were assessed by ELISA. The pathological changes of the thyroid and orbital tissues were examined by histological staining such as H&E staining and Alcian blue staining. Results More than 90% of the immunized mice spontaneously developed goiter, and about 80% of the immunized mice manifested increased serum T4 and TRAb levels, combined with hypertrophy and hyperplasia of thyroid follicles. A significantly increased synthesis of hyaluronic acid was detected in in the immunized mice compared with the control groups. Conclusion We have successfully established an animal model manifesting Graves’ hyperthyroidism and ophthalmopathy, which provides a useful tool for future study of the pathological features and the development of novel therapies of the diseases.