Supplementary Material for: Up-regulated microRNA-185-3p inhibits the development of hyperlipidemia in rats

Introduction: MicroRNA (miR)-185-3p functions in multiple cancers, while the underlying function of miR-185-3p in hyperlipidemia remained obscure. This research was conducted to unravel its function in hyperlipidemia development via modulating mastermind-like 1 (MAML1). Methods: The hyperlipidemia rat model was established by feeding with high fat diet. MiR-185-3p and MAML1 levels in hyperlipidemia rats were detected. Adenoviral vectors altering miR-185-3p and MAML1 levels were injected into hyperlipidemia rats to examine the levels of serum lipids, oxidative stress, inflammatory cytokine, lipid accumulation and cellular morphology in liver tissues of hyperlipidemia rats. The targeting relation between miR-185-3p and MAML1 was manifested. Results: MiR-185-3p expressed at a low level while MAML1 expressed at high level in hyperlipidemia rats. MiR-185-3p overexpression or MAML1 silencing reduced levels of serum lipids, mitigated oxidative stress and inflammatory response, relieved lipid accumulation and pathological morphology in liver tissues in hyperlipidemia rats; while up-regulated MAML1 reversed the effects of augmented miR-185-3p in hyperlipidemia rats. Mechanically, miR-185-3p targeted MAML1. Conclusion: Up-regulated miR-185-3p represses hyperlipidemia development via modulating MAML1 expression. This research provides novel therapeutic candidates for the treatment of hyperlipidemia

Supplementary Material for: Associations between STAT Gene Polymorphisms and Psoriasis in Northeastern China

<p><b><i>Background:</i></b> Psoriasis is an autoimmune disease with genetic and environmental factors. Based on the roles of STATs (signaling transducers and activators of transcription) in autoimmune diseases, it is assumed STAT gene polymorphisms are associated with psoriasis. <b><i>Objectives:</i></b> To study the association between STAT gene polymorphisms and psoriasis in the northeastern Chinese population. <b><i>Methods:</i></b> Eight single-nucleotide polymorphisms were genotyped: rs2293152, rs3816769, rs4796793, and rs744166 in STAT3, rs7574865 and rs3024866 in STAT4, and rs324011 and rs3024974 in STAT6, using SNaPshot methods. The genotype, allele, and haplotype frequencies were compared between 400 psoriasis patients and 398 healthy individuals in northeastern China. <b><i>Results:</i></b> rs744166GG in STAT3 and rs7574865TT in STAT4 had higher frequencies in the case than the control group, suggesting these 2 genotypes increase the susceptibility to psoriasis (<i>p</i> < 0.05). Three haplotypes (H3, H6, and H7) were found to be associated with psoriasis in the study (<i>p</i> < 0.05). <b><i>Conclusions:</i></b> These results indicate a role of STAT genes in the pathogenesis of psoriasis.</p

Supplementary Material for: Verbascoside Alleviates Atopic Dermatitis-Like Symptoms in Mice via Its Potent Anti-Inflammatory Effect

<b><i>Background:</i></b> Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by the interplay between multiple genetic and environmental factors. The pathogenesis of AD remains incompletely understood. Treatment with topical steroids for chronic AD symptoms has severe side effects and so a new treatment is required.<b><i></i></b> Verbascoside is a hydrophilic phenylethanoid glycoside with antioxidant, anti-inflammatory properties. <b><i>Methods:</i></b> Verbascoside was evaluated in AD-like lesions induced by the repetitive and alternative application of 2,4-dinitrochlorobenzene (DNCB) in BALB/c mice. Overall symptomatic score and serological and molecular changes of the skin lesions were investigated. <b><i>Results:</i></b> Verbascoside relieved the overall AD-like symptoms such as scratching behavior and skin lesion severity. At whole-body level, verbascoside significantly reduced DNCB-induced IgE and Th2 cytokines in the peripheral blood. At the skin lesion site, verbascoside also inhibited DNCB-induced production of proinflammatory cytokine TNF-α, IL-6, and IL-4 mRNA. In a human monocyte THP-1 model, verbascoside could suppress DNCB-induced upregulation of CD86 and CD54 at the cell surface, the secretion of the proinflammatory cytokines TNF-α and IL-6, and the activation of NFκB signaling in a dose-dependent manner. <b><i>Conclusion:</i></b> Our results demonstrate that verbascoside could be a potential therapeutic agent for the treatment of AD

Supplementary Material for: Elevated Plasma Level of Pentraxin-3 Predicts In-Hospital and 30-Day Clinical Outcomes in Patients with Non-ST-Segment Elevation Myocardial Infarction Who Have Undergone Percutaneous Coronary Intervention

<b><i>Objectives:</i></b> This investigation explored the short-term prognostic value of pentraxin-3 (PTX3) levels in patients with non-ST-segment elevation myocardial infarction (NSTEMI) treated by percutaneous coronary intervention (PCI). <b><i>Methods:</i></b> We measured plasma levels of PTX3 and other biomarkers in 525 PCI-treated NSTEMI patients (mean age, 57.7 years; 328 males). The associations of PTX3 levels with cardiac events and cardiac deaths occurring within 30 days of discharge were evaluated with multivariable Cox proportional hazard models. <b><i>Results:</i></b> Renal function, diabetes prevalence, systolic blood pressure, heart rate and ejection fraction differed significantly in the high PTX3 (≥3.0 ng/ml, n = 107) and low PTX3 (<3.0 ng/ml, n = 418) groups (all p < 0.05). Plasma PTX3 levels were correlated with high-sensitivity C-reactive protein, troponin T and N-terminal pro-B-type natriuretic peptide in NSTEMI patients (all p < 0.05). Kaplan-Meier analysis showed in-hospital and 30-day cardiac events and deaths were higher in the high PTX3 group (both p < 0.01). Elevated PTX3 was an independent predictor of 30-day cardiac events (95% CI 1.09-1.68; p = 0.006) and mortality (95% CI 1.18-2.15; p = 0.002). <b><i>Conclusions:</i></b> An elevated plasma level of PTX3 predicts 30-day cardiac events and mortality in PCI-treated NSTEMI patients

Supplementary Material for: Febuxostat Prevents Renal Interstitial Fibrosis by the Activation of BMP-7 Signaling and Inhibition of USAG-1 Expression in Rats

<b><i>Background:</i></b> Renal interstitial fibrosis (RIF) is a common pathology associated with end-stage renal diseases. The activation of bone morphogenetic protein-7 (BMP-7)-Smad1/5/8 pathway seems to alleviate RIF. Uterine sensitization-associated gene-1 (USAG-1), a kidney-specific BMPs antagonist, is associated with the development and prognosis of several renal diseases. Febuxostat is a xanthine oxidase inhibitor that can attenuate the renal dysfunction of patients. The purpose of this study was to investigate the effects of febuxostat on renal fibrosis and to clarify the mechanisms underlying these effects. <b><i>Methods:</i></b> Rats were randomly divided into 6 groups termed a sham-operated group, a unilateral ureteral obstruction (UUO) group, 3 doses of febuxostat groups (low, intermediate and high doses) and a sham group treated with high-dose febuxostat. After 14 days, renal function, relative kidney weight, accumulation of glycogen and collagens were examined by different methods. Expression of α-SMA, transforming growth factor-β1 (TGF-β1), BMP-7 and USAG-1 was detected by western blotting and RT-PCR, respectively. The phosphorylation level of Smad1/5/8 was also quantified by western blotting. <b><i>Results:</i></b> The renal function was declined, and large amounts of glycogen and collagens were deposited in the kidneys of UUO rats compared with the rats in the sham group. Besides, expression of α-SMA and USAG-1 in these kidneys was elevated, and the TGF-β1 was also activated, while the BMP-7-Smad1/5/8 pathway was inhibited. Febuxostat reversed the changes stated earlier, exhibiting protective effects on RIF induced by UUO. <b><i>Conclusion:</i></b> Febuxostat was able to attenuate RIF caused by UUO, which was associated with the activation of BMP-7-Smad1/5/8 pathway and the inhibition of USAG-1 expression in the kidneys of UUO rats

PowerPoint Slides for: Superagonistic CD28 Protects against Renal Ischemic Injury by Expansion of Regulatory T-Cell

<p><b><i>Background:</i></b> Regulatory T (Treg) cells are a highly suppressive subset of CD4⁺ lymphocytes and have recently been proved to be crucial to suppress the inflammatory responses of ischemic kidney injury. CD28 superagonists (CD28sa) are monoclonal antibodies that preferentially expand Treg cells without a T-cell receptor and a costimulatory signal. This study aims to test the protection and discover the mechanisms of CD28sa treatment against renal ischemia-reperfusion (IR) injury (IRI). <b><i>Methods:</i></b> Male C57BL/6N mice were treated with CD28sa via peritoneal injection (0.1 mg) 6 days before the induction of IRI, or with 18-min ischemic precondition (IPC). IRI was induced by bilateral clamping of renal pedicles for 35 min followed by reperfusion. The role of Treg expansion in renal protection conferred by CD28sa treatment was examined using anti-CD25 antibody. <b><i>Results:</i></b> CD28sa treatment alone significantly increased the percentage of Treg cells in the spleen (18.10 ± 2.00 vs. 6.64 ± 0.86%, <i>p</i> < 0.01), peripheral blood (16.43 ± 5.94 vs. 2.57 ± 1.09%, <i>p</i> < 0.01), and kidney (2.69 ± 0.90 vs. 0.53 ± 0.14%, <i>p</i> < 0.01) of C57BL/6N mice 6 days after the administration. Mice pretreated with CD28sa or IPC had less renal injury at 24 h after IRI with attenuation of renal tubular damage and lower serum creatinine compared with the mice that underwent renal IRI alone. The number of infiltrating macrophages in the kidney and IFN-γ secreting CD4⁺ T cells in peripheral blood were diminished in the CD28sa-IR group and the IPC-IR group. The renal protection bestowed by CD28sa or IPC was abolished by anti-CD25 antibody administration. <b><i>Conclusions:</i></b> Treg expansion induced by CD28sa ameliorated renal IRI.</p

Supplementary Material for: Primary Malignant Melanoma of the Endometrium: A Case Report

Introduction: Malignant melanoma most commonly occurs in the skin. Primary malignant melanoma of endometrium is quite rare. Its diagnosis depends on clinical characteristics and pathological examination. It usually exhibits high degree of tumor histology, early onset of distant metastases, and unfavorable prognoses. Case Presentation: In this paper, we report a case of a 73-year-old woman with primary malignant melanoma of endometrium. This patient denied a history of nevus removal or any family medical history of cancer. She was admitted to the hospital for irregular vaginal bleeding after menopause and performed an endometrial biopsy. Pathological of the scrapings suggested malignant melanoma. She subsequently underwent a radical surgery. The final pathology diagnosis was primary malignant melanoma of endometrium, and BRAF gene mutation was detected. The tumor staged as IVB according to the International Federation of Gynecology and Obstetrics (FIGO) classification. Thus, she then started adjuvant chemotherapy. This patient is currently on oral targeted therapy and is still being followed up. Conclusion: Mucosal melanoma is infrequent, and primary malignant melanoma of endometrial is a rare subtype. To the best of our knowledge, malignant melanoma originating from endometrium has never been reported before. It has a high degree of malignancy and is prone to early metastasis. Further investigations are warranted to explore its underlying pathogenesis, management, and outcomes

Supplementary Material for: hCLCA1 DNA Vaccine Suppresses Cell Hyperplasia and Mucin Expression of Goblet Cells in vitro

<b><i>Background:</i></b> Excessive airway mucus secretion is a remarkable trait of asthma. Mucus overproduction mainly resulted from an increase in goblet cell numbers, which causes considerable damage to health. However, effective therapeutic treatments are still lacking for mucus hypersecretion. Human calcium-activated chloride channel 1 (hCLCA1) has been identified to be predominantly responsible for mucus hypersecretion. <b><i>Objectives:</i></b> In this study, we investigated the effects of an hCLCA1 DNA vaccine on the control of mucus production and goblet cell proliferation using an in vitro model goblet cell line (NCI-H292). <b><i>Methods:</i></b> The effect of the hCLCA1 DNA vaccine on cell viability and proliferative activity of NCI-H292/hCLCA1 was analyzed by electron microscopy, MTT assay, and flow cytometry. Expression of mucins and MUC5AC, a major member of the mucin gene family in airway goblet cells, was assessed under hCLCA1 DNA vaccine challenges by periodic acid-Schiff staining, quantitative real-time PCR and Western blot, respectively, and the expression profile of granulocyte-macrophage colony-stimulating factor (GM-CSF), a critical cytokine in airway inflammation, was also examined by real-time PCR and immunocytochemistry. <b><i>Results:</i></b> Results showed that hCLCA1 overexpression caused high cell proliferation and mucin expression, whereas the hCLCA1 DNA vaccine could effectively reverse these abnormal effects. In addition, GM-CSF expression was highly induced by hCLCA1 overexpression and efficiently suppressed by hCLCA1 DNA vaccine. <b><i>Conclusions:</i></b> These results illustrate that the hCLCA1 DNA vaccine effectively inhibits cell hyperplasia and mucin gene expression of goblet cells, suggesting that the hCLCA1 DNA vaccine has potential value in the treatment of human asthma

Supplementary Material for: Association between Toll-Like Receptor 4 T399I Gene Polymorphism and the Susceptibility to Crohn’s Disease: A Meta-Analysis of Case-Control Studies

<b><i>Background/Aims:</i></b> This article was undertaken to investigate the association of toll-like receptor 4 (TLR4) polymorphism (Thr399Ile) and risk of Crohn’s disease (CD) by performing a meta-analysis.<b><i> Methods:</i></b> Articles were chosen based on PubMed, Embase, China National Knowledge Internet, and Chinese Wanfang databases (up to 12th October 2016). Specific inclusion criteria were used to evaluate articles. Meta-analysis was performed by using a random or fixed effect model. Fifteen eligible case-control studies were finally included into this meta-analysis. We estimated the summary OR with its corresponding 95% CI to assess the association.<b><i> Results:</i></b> Summary results of this meta-analysis showed a moderate association between the TLR4 T399I polymorphism and the risk of CD (allele model: OR 1.26, 95% CI 1.06–1.50, <i>p</i> = 0.009; heterozygote model: OR 1.36, 95% CI 1.11–1.66, <i>p</i> = 0.003; dominant model: OR 1.35, 95% CI 1.10–1.64, <i>p</i> = 0.004; homozygote model: OR 1.08, 95% CI 0.44–2.64, <i>p</i> = 0.866; recessive model: OR 0.97, 95% CI 0.40–2.35, <i>p</i> = 0.946). Stratified analysis on geographical area, ethnicity, and genotypic methods suggested that the polymorphism was associated with increased risk of CD in Asia and Asians, and “T” allele only moderately increased CD risk within polymerase chain reaction-restricted fragment length polymorphism. <b><i>Conclusions:</i></b> Our meta-analysis suggests that TLR4 T399I polymorphism is moderately associated with susceptibility to CD, and more studies are needed to confirm our conclusion

Supplementary Material for: Soy Consumption with Risk of Coronary Heart Disease and Stroke: A Meta-Analysis of Observational Studies

<b><i>Background:</i></b> The association of soy product consumption with the relative risk of cardiovascular disease remains controversial. This meta-analysis aimed at investigating whether an association exists between soy consumption and the risk of stroke and coronary heart disease (CHD) in observational studies. <b><i>Methods:</i></b> A systematic search of the PubMed and EMBASE databases was performed for case-control and cohort studies that assessed soy consumption and the risk of stroke and CHD. Summary relative risks (SRRs) and 95% CIs were combined by using a random-effects model. <b><i>Results:</i></b> Of a total of 1,266 abstracts, 5 prospective cohort and 6 case-control studies met our inclusion criteria, and comprised 4,954 stroke and 7,616 CHD events. Based on the high vs. low analyses, combining cohort studies showed no association between soy intake and risk of stroke (SRR 0.92; 95% CI 0.70-1.10; P_{heterogeneity} = 0.236; I² = 29.4%) or CHD (SRR 0.97; 95% CI 0.74-1.27; P_{heterogeneity} = 0.020; I² = 62.7%), although a significantly inverse association between soy intake and the risk of stroke (SRR 0.54; 95% CI 0.34-0.87; P_{heterogeneity} = 0.001; I² = 79.3%) and CHD (SRR 0.66; 95% CI 0.56-0.77; P_{heterogeneity} = 0.421; I² = 0) was observed in case-control studies. No association between soy isoflavone intake and the risk of stroke and CHD was identified. <b><i>Conclusion:</i></b> There was limited evidence to indicate that soy consumption was inversely associated with the risk of stroke and CHD, although further studies, with prospective designs that use validated questionnaires and control for important confounders, are warranted