Potential actions of capsaicin for preventing vascular calcification of vascular smooth muscle cells in vitro and in vivo.

Vascular calcification (VC) is an accurate risk factor and predictor of adverse cardiovascular events; however, there is currently no effective therapy to specifically prevent VC progression. Capsaicin (Cap) is a bioactive alkaloid isolated from Capsicum annuum L., a traditional medicinal and edible plant that is beneficial for preventing cardiovascular diseases. However, the effect of Cap on VC remains unclear. This study aimed to explore the effects and related mechanisms of Cap on aortic calcification in a mouse and on Pi-induced calcification in vascular smooth muscle cells (VSMCs). First, we established a calcification mouse model with vitamin D3 and evaluated the effects of Cap on calcification mice using von Kossa staining, calcium content, and alkaline phosphatase activity tests. The results showed that Cap significantly improved calcification in mice. VSMCs were then cultured in 2.6 mM Na2HPO4 and 50 µg/mL ascorbic acid for 7 days to obtain a calcification model, and we investigated the effects and mechanisms of Cap on VSMCs calcification by assessing the changes of calcium deposition, calcium content, and subsequent VC biomarkers. These results showed that Cap alleviated VSMCs calcification by upregulating the expressions of TRPV1. Moreover, Cap reduced the expression of Wnt3a and ß-catenin, whereas DKK1 antagonised the inhibitory effect of Cap on VSMC calcification. This study is the first to offer direct evidence that Cap inhibits the Wnt/ß-catenin signaling pathway by upregulating the expression of the TRPV1 receptor, resulting in the decreased expression of Runx2 and BMP-2, thereby reducing VSMC calcification. Our study may provide novel strategies for preventing the progression of VC. This could serve as a theoretical basis for clinically treating VC with spicy foods.

CN-3 increases TMZ sensitivity and induces ROS-dependent apoptosis and autophagy in TMZ-resistance glioblastoma.

Many glioma patients develop resistance to temozolomide (TMZ) treatment, resulting in reduced efficacy and survival rates. TMZ-resistant cell lines SHG44R and U87R, which highly express O6 -methylguanine DNA methyltransferase (MGMT) and P-gp, were established. CN-3, a new asterosaponin, showed cytotoxic effects on TMZ-resistant cells in a dose- and time-dependent manner via reactive oxygen species (ROS)-mediated apoptosis and autophagy. Transmission electron microscopy and monodansylcadaverine (MDC) staining showed turgidity of the mitochondria and autophagosomes in CN-3-treated SHG44R and U87R cells. The autophagy inhibitor 3-methyladenine was used to confirm the important role of autophagy in CN-3 cytotoxicity in TMZ-resistant cells. The ROS scavenger N-acetyl- l-cysteine (NAC) attenuated the levels of ROS induced by CN-3 and, therefore, rescued the CN-3 cytotoxic effect on the viability of SHG44R and U87R cells by Cell Counting Kit-8 assays and JuLI-Stage videos. MDC staining also confirmed that NAC rescued an autophagosome increase in CN-3-treated SHG44R and U87R cells. Western blotting revealed that CN-3 increased Bax, cleaved-caspase 3, cytochrome C, PARP-1, LC3-Ⅱ, and Beclin1, and decreased P-AKT, Bcl-2, and p62. Further rescue experiments revealed that CN-3 induced apoptosis and autophagy through ROS-mediated cytochrome C, cleaved-caspase 3, Bcl-2, P-AKT, PARP-1, and LC3-Ⅱ. In addition, CN-3 promoted SHG44R and U87R cells sensitive to TMZ by reducing the expression of P-gp, MGMT, and nuclear factor kappa B p65, and it had a synergistic cytotoxic effect with TMZ. Moreover, CN-3 disrupted the natural cycle arrest and inhibited the migration of SHG44R and U87R cells by promoting cyclin E1 and D1, and by decreasing P21, P27, N-cadherin, ß-catenin, transforming growth factor beta 1, and Smad2.

LINGO-1 regulates Wnt5a signaling during neural stem and progenitor cell differentiation by modulating miR-15b-3p levels.

BACKGROUND: Manipulation of neural stem and progenitor cells (NSPCs) is critical for the successful treatment of spinal cord injury (SCI) by NSPC transplantation, since their differentiation into neurons and oligodendrocytes can be inhibited by factors present in inflamed myelin. In this study, we examined the effects of LINGO-1 on spinal cord-derived NSPC (sp-NSPC) differentiation, the underlying mechanisms of action, and the functional recovery of mice after transplantation of manipulated cells. METHODS: sp-NSPCs were harvested from female adult C57/BL6 mice after SCI induced with an NYU impactor. These cells were infected with lentiviral vectors containing LINGO-1 shRNA sequence or a scrambled control and transplanted into SCI mice. Tuj-1- and GFAP-positive cells were assessed by immunofluorescence staining. Wnt5a, p-JNK, JNK, and ß-catenin expression was determined by Western blot and RT-qPCR. miRNAs were sequenced to detect changes in miRNA expression. Motor function was evaluated 0-35 days post-surgery by means of the Basso Mouse Scale (BMS) and by the rotarod performance test. RESULTS: We discovered that LINGO-1 shRNA increased neuronal differentiation of sp-NSPCs while decreasing astrocyte differentiation. These effects were accompanied by elevated Wnt5a protein expression, but unexpectedly, no changes in Wnt5a mRNA levels. miRNA-sequence analysis demonstrated that miR-15b-3p was a downstream mediator of LINGO-1 which suppressed Wnt5a expression. Transplantation of LINGO-1 shRNA-treated sp-NSPCs into SCI mice promoted neural differentiation, wound compaction, and motor function recovery. CONCLUSIONS: LINGO-1 shRNA promotes neural differentiation of sp-NSPCs and Wnt5a expression, probably by downregulating miR-15b-3p. Transplantation of LINGO-1 shRNA-treated NSPCs promotes recovery of motor function after SCI, highlighting its potential as a target for SCI treatment.

CN-3 induces mitochondrial apoptosis in glioma via Ros-mediated PI3K/AKT pathway.

Recently we isolated CN-3, a new asterosaponin from starfish Culcita novaeguineae, and reported that asterosaponin arrests glioma cell cycle via SCUBE3. However, the multiple mechanisms underlying CN-3 anti-glioma action remains poorly known. Thus, the focus of this study was to evaluate the inhibitory effect of CN-3 on human glioma cells and its underlying molecular mechanisms. U87 and U251 cells were incubated with various concentrations of CN-3, and CCK-8, transmission electron microscopy, ICELLigence, TUNEL, flow cytometry, N-acetyl-L-cysteine, and western blot were conducted. As a result, it was found that CN-3 significantly inhibited U87 and U251 cell viability and proliferation in a time- and dose- dependent manner, and also induced mitochondrial apoptosis. Furthermore, we detected that CN-3 downregulated PI3K, P-Akt, AKT and BCL-2, and upregulated cytochrome C and BAX in U87 and U251 cells. Moreover, ROS triggered the inhibition and cell apoptosis for CN-3 via inactivation of P-Akt and activation of cytochrome C. In conclusion, these findings suggest that CN-3 may be a promising candidate for the development of a therapy of glioma.

Comparison of healing patterns of different side-cut angulations after FS-LASIK.

AIM: To investigate and evaluate healing patterns around flaps made with different side-cut angulations after femtosecond laser in situ keratomileusis (FS-LASIK). METHODS: Thirty-four patients (68 eyes) received a 90° side-cut (n=34) or a 120° side-cut flaps (n=34) made with a femtosecond laser. One day, 1wk, 1 and 3mo postoperatively, side-cut scar was evaluated under slit-lamp photography according to a new grading system (Grade 0=transparent scar, 1=faint healing opacity, and 2=evident healing opacity). In vivo corneal confocal microscopy and anterior segment optical coherence tomography (AS-OCT) were used to observe wound-healing patterns around flap margin in the two groups. Sirius Scheimpflug Analyzer was also used to analyze higher order aberrations 3mo after surgery. RESULTS: There were no significant differences in flap wound-healing patterns at each follow up between the two groups (P>0.05). Three months after surgery, the flap edge scar classified as Grade 0 had excellent apposition and rapid nerve regeneration. At 3 mm and 5 mm pupil diameters, there were significant differences in trefoil aberrations between the two groups (P<0.05), but no statistically significant differences were found in total higher order aberrations (HOAs), spherical aberrations or coma in any of the pupil size conditions (P>0.05). CONCLUSION: Flap edge scars classified as Grade 0 have excellent apposition and rapid nerve regeneration, and 120° side-cut angle flaps induce less trefoil aberrations after FS-LASIK.

Promotion of Ros-mediated Bax/Cyt-c apoptosis by polyphyllin II leads to suppress growth and aggression of glioma cells.

BACKGROUND: Gliomas remain among the most difficult cancers to treat, with a 5-year overall survival no greater than 5%. Many saponins showed a wide spectrum of anti-cancer activities at low concentration. Polyphyllin II is one of the common saponins from Paris polyphylla. However, the effect of Polyphyllin II on glioma cells has not been evaluated. Objective of the present study was to investigate whether Polyphyllin II have inhibition on glioma cells, and the possible mechanisms. METHODS: The viability of U87 and U251 cells was detected by cell counting kit-8, cell counting real time cellular analysis and cell clone formation methods. Transwell was used to estimate the aggression of U87 and U251. The cell apoptosis rate was tested by flow cytometry. The morphological change was determined by transmission electron microscopy. The levels of AKT, phosphorylation of AKT, Bax, Bcl-2, cytochrome c, and cleaved caspase 3 proteins were assessed by Western blot. N-acetyl-L-cysteine was used to check the role of ROS in polyphyllin II inhibition to glioma cells. RESULTS: Polyphyllin II showed significant suppress to proliferation and aggression of U87 and U251 in a dose- and time- dependent manner. Result of flow cytometry confirmed that Polyphyllin II induced apoptosis to U87 and U251 cells. Transmission electron microscopy observation revealed majority of the glioma cells treated with Polyphyllin II had turgidity of mitochondrion, disarrangement, diminution and vacuolization, those refer to mitochondrial apoptosis. Western blot indicated that Polyphyllin II promoted cyt-c, Bax, caspase 3 and cleaved-caspase 3, and decreased Bcl-2, AKT and p-AKT. Rescue experiments using N-acetyl-L-cysteine, a reactive oxygen species scavenger, reversed the levels of Bax and cyt-c, and the inhibition in Polyphyllin II-treated U87 and U251 cells. CONCLUSIONS: The present findings revealed that polyphyllin II may be a potential drug against glioma.

Reduction of SCUBE3 by a new marine-derived asterosaponin leads to arrest of glioma cells in G1/S.

Many saponins are characterized as exhibiting a wide spectrum of antitumor activities at low concentrations. Most of the previous studies that aimed to understand the mechanisms underlying anticancer saponins have focused on numerous classical signaling pathways. However, at the oncogene level, little is known about the action of saponins, especially asterosaponin. In this study, CN-3, a new asterosaponin isolated from the starfish Culcita novaeguineae, decreased the proliferation of U87 and U251 cells at low doses in a dose- and time-dependent manner. Microarray analysis revealed CN-3 significantly induced the differential expression of 661 genes that are related to its antiglioma effect in U251. Nine downregulated genes (SCUBE3, PSD4, PGM2L1, ACSL3, PRICKLE1, ABI3BP, STON1, EDIL3, and KCTD12) were selected, for further verification of their low expression. Then, shRNA transfection and high-content screening were performed and significantly decreased U251 cell proliferation rate was only observed for the SCUBE3 knockdown. qPCR confirmed SCUBE3 was highly expressed in U251 and U87 cells, and had medium expression levels in U373 cells. Real-time cellular analysis using iCELLigence demonstrated that SCUBE3 is an oncogene in U251 and U87 cells, with knockdown of SCUBE3 inhibiting U251 and U87 cell proliferation while, conversely, SCUBE3 overexpression promoted their proliferation. Afterward, SCUBE3 protein was found to have high expression in primary glioma specimens from patients examined by immunohistochemistry but low expression in normal brain. PathScan ELISA analysis in conjunction with TEM observation demonstrated that the effect of SCUBE3 knockdown in U251 does not appear to be related to the induction of apoptosis. Employing CCK-8, iCELLigence, flow cytometry, western blotting, and shRNA transfection (knockdown and overexpression) experiments, we reveal that the reduction of SCUBE3 expression, induced by CN-3, mediated both inhibition and G1/S arrest of U251 via the Akt/p-Akt/p53/p21/p27/E2F1 pathway.

A novel endoscopic classification for craniopharyngioma based on its origin.

Endoscopic endonasal approach for craniopharyngioma (CP) resection provides a wide view and direct observation of hypothalamus and origin of tumor. Under endoscopy, 92 CPs were classified into 2 types: Peripheral and Central, according to its relation to pituitary stalk. Peripheral type was further divided into 3 subtypes: Hypothalamic stalk, Suprasellar stalk and Intrasellar stalk CP, according to the different origin site along hypothalamus-pituitary axis. Peripheral type arisen from the stalk but expanded and grown laterally in an exophytic pattern, accounting for 71.7% of all CPs, preservation rate of stalk was higher (76.0%). Central type grew within and along pituitary stalk and located strictly in the midline. The pituitary stalk was hardly preserved (only15.4%). Hypothalamic stalk CPs (n = 36, 54.6%) developed from the junction of hypothalamus and stalk, hypothalamus damage was found in all of this subtype after surgery. Suprasellar stalk CPs (n = 14, 21.2%) originated from the lower portion of stalk and displaced hypothalamus upward rather than infiltrated it. Intrasellar stalk CPs (n = 16, 24.2%) arose from the subdiaphragma portion of the stalk, with less hypothalamus damage. Recoginzing the origin of CP is helpful to understand its growth pattern and relation to hypothalamus, which is critical in planning the most appropriate surgical approach and degree of excision.

Coxsackievirus B3 induces viral myocarditis by upregulating toll-like receptor 4 expression.

In the present study, we investigated the potential pathogenesis of coxsackievirus B3 (CVB3)-induced viral myocarditis and the promising protective effect of silencing RNA (small interfering RNA, siRNA). One hundred and twenty mice were included in the study, and 30 mice were intraperitoneally inoculated with CVB3 to establish an acute viral myocarditis model. The survival rate was observed for the CVB3-infected mouse model (MOD), and myocardial injury was examined by HE (hematoxylin and eosin) staining assay. Real-time PCR (RT-PCR) and Western blot assay were selected to detect the toll-like receptor 4 (TLR4) expression in myocardial tissues. The TLR4 gene was silenced for the MOD mice, and the effects of this treatment were observed. The results indicate that the expression of TLR4 mRNA and the protein significantly and persistently increased during the progression of CVB3-induced myocarditis. The activities of cardiac enzymes including CK, LDH, AST, and CK-MB were also enhanced in CVB3-induced myocardial tissues. Interestingly, when the TLR4 gene was silenced, the CVB3-induced TLR4 production was significantly decreased and the severity of myocarditis was significantly lessened. In conclusion, CVB3 may induce viral myocarditis by upregulating toll-like receptor 4 expression. The viral myocarditis can be ameliorated by silencing the TLR4 gene in the CVB3 viral myocarditis model, which may be a feasible therapeutic method for treatment of viral myocarditis.

Prognostic value of LGR5 in colorectal cancer: a meta-analysis.

OBJECTIVE: Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) has recently been reported to be a marker of cancer stem cells (CSCs) in colorectal cancer (CRC), and the prognostic value of LGR5 in CRC has been evaluated in several studies. However, the conclusions remain controversial. In this study, we aimed to evaluate the association between the expression of LGR5 and the outcome of CRC patients by performing a meta-analysis. METHODS: We systematically searched for relevant studies published up to February 2014 using the PubMed, Web of Science, EMBASE and Wangfang databases. Only articles in which LGR5 expression was detected by immunohistochemistry were included. A meta-analysis was performed using STATA 12.0, and pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were used to estimate the strength of the association between LGR5 expression and the prognosis of CRC patients. RESULTS: A total of 7 studies comprising 1833 CRC patients met the inclusion criteria, including 6 studies comprising 1781 patients for overall survival (OS) and 3 studies comprising 528 patients for disease-free survival (DFS). Our results showed that high LGR5 expression was significantly associated with poor prognosis in terms of OS (HR: 1.87, 95% CI: 1.23-2.84; P = 0.003) and DFS (HR: 2.44, 95% CI: 1.49-3.98; P<0.001). Further subgroup analysis revealed that many factors, including the study region, number of patients, follow-up duration and cutoff value, affected the significance of the association between LGR5 expression and a worse prognosis in patients with CRC. In addition, there was no evidence of publication bias, as suggested by Begg's and Egger's tests. CONCLUSIONS: The present meta-analysis indicated that high LGR5 expression was associated with poor prognosis in patients with CRC and that LGR5 is an efficient prognostic factor in CRC.

[Changes of Foxp3(+); regulatory T cells in patients with acute coronary syndrome].

AIM: To investigate the changes of Foxp3(+); (including CD4(+);Foxp3(+); and CD4(+);CD25(+);Foxp3(+);) regulatory T cells (Tregs) in patients with acute coronary syndrome (ACS). METHODS: Forty-four ACS patients, twenty patients with stable angina (SA) and twenty-four control patients were enrolled. The percentages of the peripheral Tregs, Foxp3 mRNA and plasma level of TGF-ß1 were measured by flow cytometry, real-time quantitative PCR and ELISA. RESULTS: The percentages of CD4(+);CD25(+);Foxp3(+); Tregs and CD4(+);Foxp3(+); Tregs, Foxp3 mRNA and plasma level of TGF-ß1 significantly decreased in the ACS group as compared with those in the SA group and the controls (P<0.05), while there was no significant difference in the percentage of CD4(+);CD25(+); Tregs among all three groups. CONCLUSION: There are less Foxp3(+);Tregs, perhaps with impaired function, in ACS patients, which might contribute to plaque destabilization.

CD58 expression of liver tissue in patients with chronic hepatitis B virus infection.

BACKGROUND: Several kinds of intercellular adhesion molecules closely relate to hepatitis B. The complex of CD(2) and CD58 plays an important role in enhancing the adhesion of T lymphocytes to target cells, hyperplasia and activation of T lymphocytes. In this study, we explored the relationship between the expression of CD58 in liver tissue and chronic hepatitis B infection. METHODS: We determined the expression of the CD58 molecule on the surface of hepatocytes by using immunohistochemistry and the levels of serum HBV DNA from patients with HBV infection and from normal controls. The biochemical parameters of hepatic function were analyzed as well. RESULTS: CD58 expression in hepatocytes significantly increased with the severity progression of chronic HBV infection. The IOD levels (log10) of CD58 in the control, mild, moderate, and severe chronic HBV infection groups were 0, (7.20+/-4.64) x 10(3), (25.63+/-7.41) x 10(3) and (37.47+/-11.17) x 10(3) respectively (P<0.05 compared with the control group, respectively). CONCLUSION: CD58 probably increases cell mediated immunity to eliminate hepatitis B virus and leads to damage of hepatocytes.

Investigation on correlation between expression of CD58 molecule and severity of hepatitis B.

AIM: To investigate the correlation between expression of CD58 and severity of hepatitis B. METHODS: The level of soluble CD58 (sCD58) in serum of patients with hepatitis B was detected by enzyme-linked immunosorbent assay. The level of expression of membrane CD58 molecule in PBMC was detected by direct immunofluorescence. The levels of serumal TBIL, DBIL, IBIL, ALT and AST were detected by the automated biochemistry analyzer as well. RESULTS: The levels of sCD58 in serum and membrane CD58 molecule in PBMC of patients with hepatitis B were significantly higher than that in normal controls (P < 0.05). Level of CD58 was related to the levels of serumal TBIL, DBIL, IBIL, ALT and AST. CONCLUSION: The level of CD58 molecule (in both serum and PBMC form) of patients with hepatitis B is related to the degree of liver damage.

Study on the relationship between level of CD58 expression in peripheral blood mononuclear cell and severity of HBV infection.

BACKGROUND: As one of the intercellular adhesion molecules, CD58 plays important roles in promotion of the adhesion between T cells and target cells, hyperplasia, activation of T cells and natural killer cells, and balance between Th1 and Th2. We studied the relationship between the levels of CD58 expression in peripheral blood mononuclear cells (PBMCs) and severity of HBV infection. METHODS: The levels of CD58 mRNA in PBMCs were detected using quantitative reverse transcription PCR. The percentage of CD58 positive cells was detected by flow cytometry in patients and healthy controls. RESULTS: The levels of CD58 mRNA and the percentage of CD58 positive cells in patients infected with HBV were significantly higher than that in the control. Based on severity of HBV infection, the patients were classified into four groups. The expression of CD58 increased significantly in an order from mild chronic, moderate chronic, severe chronic to severe hepatitis groups. The levels of CD58 mRNA and the percentage of CD58 positive cells in PBMCs from patients with HBV infection were both positively correlated with serum levels of ALT and AST. CONCLUSION: The level of CD58 expression is related with the severity of HBV infection and the degree of liver damage.