Implication of IGF1R signaling in the protective effect of Astragaloside IV on ischemia and reperfusion-induced cardiac microvascular endothelial hyperpermeability.

BACKGROUND: Myocardial ischemia-reperfusion (I/R) causes damage to coronary capillary endothelial barrier and microvascular leakage (MVL), aggravating tissue injury and heart dysfunction. However, the effective strategy for protecting endothelium barrier of cardiac vasculature remains limited. PURPOSE: This study aimed to explore the effect of Astragaloside IV (ASIV) on coronary MVL after cardiac I/R and the underlying mechanism. STUDY DESIGN: Sprague-Dawley (SD) rats were used for assessment of the efficacy of Astragaloside IV in protection of myocardial I/R injury, while human cardiac microvascular endothelial cells were applied to gain more insight into the underlying mechanism. METHODS: Sprague-Dawley rats with or without pretreatment by ASIV at 10 mg/kg were subjected to occlusion of left coronary anterior descending artery followed by reperfusion. Endothelial cells were exposed to hypoxia and re-oxygenation (H/R). The distribution of junction proteins was detected by immunofluorescence staining and confocal microscope, the content of junction proteins was detected by Western blot, the level of adenosine triphosphate (ATP) was detected by ELISA, and the signal pathway related to permeability was detected by siRNA infection. The fluorescence intensity of FITC-albumin and FITC-Dextran was measured to evaluate the permeability of endothelial cells. RESULTS: ASIV exhibited protective effects on capillary damage, myocardium edema, albumin leakage, leucocyte infiltration, and the downregulated expression of endothelial junction proteins after I/R. Moreover, ASIV displayed ability to protect ATP from depletion after I/R or H/R, and the effect of ASIV on regulating vascular permeability and junction proteins was abolished once ATP synthase was inhibited. Notably, ASIV activated the insulin-like growth factor 1 receptor (IGF1R) and downstream signaling after reoxygenation. Knocking IGF1R down abolished the effect of ASIV on restoration of ATP, junction proteins and endothelial barrier after H/R. CONCLUSION: ASIV was potential to prevent MVL after I/R in heart. Moreover, the study for the first time demonstrated that the beneficial role of ASIV depended on promoting production of ATP through activating IGF1R signaling pathway. This result provided novel insight for better understanding the mechanism underlying the potential of ASIV to cope with cardiac I/R injury.

Quantitative Proteomics Reveal That Metabolic Improvement Contributes to the Cardioprotective Effect of T89 on Isoproterenol-Induced Cardiac Injury.

BACKGROUND: T89, a traditional Chinese medicine, has passed phase II, and is undergoing phase III clinical trials for treatment of ischemic cardiovascular disease by the US FDA. However, the role of T89 on isoproterenol (ISO)-induced cardiac injury is unknown. The present study aimed to explore the effect and underlying mechanism of T89 on ISO-induced cardiac injury. METHODS: Male Sprague-Dawley rats received subcutaneous injection of ISO saline solution at 24 h intervals for the first 3 days and then at 48 h intervals for the next 12 days. T89 at dose of 111.6 and 167.4 mg/kg was administrated by gavage for 15 consecutive days. Rat survival rate, cardiac function evaluation, morphological observation, quantitative proteomics, and Western blotting analysis were performed. RESULTS: T89 obviously improved ISO-induced low survival rate, attenuated ISO-evoked cardiac injury, as evidenced by myocardial blood flow, heart function, and morphology. Quantitative proteomics revealed that the cardioprotective effect of T89 relied on the regulation of metabolic pathways, including glycolipid metabolism and energy metabolism. T89 inhibited the enhancement of glycolysis, promoted fatty acid oxidation, and restored mitochondrial oxidative phosphorylation by regulating Eno1, Mcee, Bdh1, Ces1c, Apoc2, Decr1, Acaa2, Cbr4, ND2, Cox 6a, Cox17, ATP5g, and ATP5j, thus alleviated oxidative stress and energy metabolism disorder and ameliorated cardiac injury after ISO. The present study also verified that T89 significantly restrained ISO-induced increase of HSP70/HSP40 and suppressed the phosphorylation of ERK, further restored the expression of CX43, confirming the protective role of T89 in cardiac hypertrophy. Proteomics data are available via ProteomeXchange with identifier PXD024641. CONCLUSION: T89 reduced mortality and improves outcome in the model of ISO-induced cardiac injury and the cardioprotective role of T89 is correlated with the regulation of glycolipid metabolism, recovery of mitochondrial function, and improvement of myocardial energy.

Small Nucleolar RNA, C/D Box 16 (SNORD16) Acts as a Potential Prognostic Biomarker in Colon Cancer.

Colon cancer (CC) is considered one of the most common and lethal malignancies occurring both in male and female. Its widespread prevalence demonstrates the need for novel diagnostic and prognostic biomarkers for CC. Emerging evidence has shown that small nucleolar RNAs play critical roles in tumor development. In this study, we investigated the expression profile and functions of SNORD16 in CC. Our data showed that SNORD16, rather than its host gene (RPL4), was upregulated in CC cell lines. Compared to matched adjacent normal tissues, CC tissues showed higher SNORD16 expression levels, and no correlation was found between SNORD16 and RPL4. Patients with high SNORD16 expression levels had a worse prognosis, and multivariate analysis showed the high SNORD16 expression was an independent prognostic factor for CC. In vitro gain- and loss-of-function studies revealed that SNORD16 can promote cell growth, proliferation, migration, and invasion of CC cells by inhibiting apoptosis. These results suggested that SNORD16 has an oncogenic role in CC and might be a novel diagnostic and prognostic biomarker for CC.

Antioxidative system of Deinococcus radiodurans.

Deinococcus radiodurans is famous for its extreme resistance to various stresses such as ionizing radiation (IR), desiccation and oxidative stress. The underlying mechanism of exceptional resistance of this robust bacterium still remained unclear. However, the antioxidative system of D. radiodurans has been considered to be the determinant factor for its unparalleled resistance and protects the proteome during stress, then the DNA repair system and metabolic system exert their functions to restore the cell to normal physiological state. The antioxidative system not only equipped with the common reactive oxygen species (ROS) scavenging enzymes (e.g., catalase and superoxide dismutase) but also armed with a variety of non-enzyme antioxidants (e.g., carotenoids and manganese species). And the small manganese complexes play an important role in the antioxidative system of D. radiodurans. Recent studies have characterized several regulators (e.g., PprI and PprM) in D. radiodurans, which play critical roles in the protection of the bacteria from various stresses. In this review, we offer a panorama of the progress regarding the characteristics of the antioxidative system in D. radiodurans and its application in the future.

A novel traditional Chinese medicine ameliorates fatigue-induced cardiac hypertrophy and dysfunction via regulation of energy metabolism.

Prolonged exercise and exercise training can adversely affect cardiac function in some individuals. QiShenYiQi Pills (QSYQ), which are a compound Chinese medicine, have been previously shown to improve pressure overload-induced cardiac hypertrophy. We hypothesized that QSYQ can ameliorate as well the fatigue-induced cardiac hypertrophy. This study was to test this hypothesis and underlying mechanism with a focus on its role in energy regulation. Male Sprague-Dawley rats were used to establish exercise adaptation and fatigue model on a motorized rodent treadmill. Echocardiographic analysis and heart function test were performed to assess heart systolic function. Food-intake weight/body weight and heart weight/body weight were assessed, and hematoxylin and eosin staining and immunofluorescence staining of myocardium sections were performed. ATP synthase expression and activity and ATP, ADP, and AMP levels were assessed using Western blot and ELISA. Expression of proteins related to energy metabolism and IGF-1R signaling was determined using Western blot. QSYQ attenuated the food-intake weight/body weight decrease, improved myocardial structure and heart function, and restored the expression and distribution of myocardial connexin 43 after fatigue, concomitant with an increased ATP production and a restoration of metabolism-related protein expression. QSYQ upgraded the expression of IGF-1R, P-AMPK/AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α, nuclear respiratory factor-1, P-phosphatidylinositol 3-kinase (PI3K)/PI3K, and P-Akt/Akt thereby attenuated the dysregulation of IGF-1R signaling after fatigue. QSYQ relieved fatigue-induced cardiac hypertrophy and enhanced heart function, which is correlated with its potential to improve energy metabolism by regulating IGF-1R signaling. NEW & NOTEWORTHY Prolonged exercise may impact some people leading to pathological cardiac hypertrophy. This study using an animal model of fatigue-induced cardiac hypertrophy provides evidence showing the potential of QiShenYiQi Pills, a novel traditional Chinese medicine, to prevent the cardiac adaptive hypertrophy from development to pathological hypertrophy and demonstrates that this effect is correlated with its capacity for regulating energy metabolism through interacting with insulin-like growth factor-1 receptor.

Caffeic acid attenuates rat liver injury after transplantation involving PDIA3-dependent regulation of NADPH oxidase.

The transplanted liver inevitably suffers from ischemia reperfusion (I/R) injury, which represents a key issue in clinical transplantation determining early outcome and long-term graft survival. A solution is needed to deal with this insult. This study was undertaken to explore the effect of Caffeic acid (CA), a naturally occurring antioxidant, on I/R injury of grafted liver and the mechanisms involved. Male Sprague-Dawley rats underwent orthotopic liver transplantation (LT) in the absence or presence of CA administration. In vitro, HL7702 cells were subjected to hypoxia/reoxygenation. LT led to apparent hepatic I/R injury, manifested by deteriorated liver function, microcirculatory disturbance and increased apoptosis, along with increased PDIA3 expression and nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase activity, and membrane translocation of NADPH oxidase subunits. Treatment with CA attenuated the above alterations. siRNA/shRNA-mediated knockdown of PDIA3 in HL7702 cells and rats played the same role as CA not only in inhibiting ROS production and NADPH oxidase activity, but also in alleviating hepatocytes injury. CA protects transplanted livers from injury, which is likely attributed to its protection of oxidative damage by interfering in PDIA3-dependent activation of NADPH oxidase.

Use of recombinant Lactobacillus sakei for the prevention and treatment of radiation-induced enteritis.

Radiation-induced enteritis is one of the most common complications in patients under radiotherapy at abdominal or pelvic cavity. Up to 50% of patients treated with pelvic radiotherapy has been reported radiation-induced acute enteritis, and half of them developed chronic enteritis. Overproduction of free radicals, activation of inflammatory pathways and vascular endothelial dysfunction were considered as the primary mechanisms of radiation-induced enteritis. Because probiotics have been demonstrated as a promising potential candidate for treating intestinal diseases, it may be a safer and more effective radioprotector for the enteritis compared to conventional chemical agents with inherent toxicities. Here, we propose that a recombinant Lactobacillus sakei would decrease the complications or symptoms significantly through against different pathogenic mechanisms simultaneously. Therefore, application of higher radiation dose for tumor control would be feasible when co-treating with recombinant Lactobacillus sakei.

PTEN expression is a prognostic marker for patients with non-small cell lung cancer: a systematic review and meta-analysis of the literature.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a known tumor suppressor in non-small cell lung cancer (NSCLC). By performing a systematic review and meta-analysis of the literature, we determined the prognostic value of decreased PTEN expression in patients with NSCLC. We comprehensively and systematically searched through multiple online databases up to May 22, 2016 for NSCLC studies reporting on PTEN expression and patient survival outcome. Several criteria, including the Newcastle-Ottawa Quality Assessment Scale (NOS), were used to discriminate between studies. In total, 23 eligible studies with a total of 2,505 NSCLC patients were included in our meta-analysis. Our results demonstrated that decreased expression of PTEN correlated with poor overall survival in NSCLC patients and was indicative of a poor prognosis for disease-free survival and progression-free survival in patients with NSCLC.

[Influence of immunosuppressive therapy on expression of TNF-α/IFN-γ in cytoplasm of peripheral blood lymphocytes of patients with aplastic anemia].

The purpose of this study was to investigate the influence of immunosuppressive therapy on the expression of TNF-α/IFN-γ in cytoplasm of peripheral blood lymphocytes of patients with aplastic anemia (AA). The expression of TNF-α and IFN-γ in cytoplasm of peripheral CD3(+) lymphocytes were measured by flow cytometry in 25 cases of de novo AA patients and 20 cases of AA after immunosuppressive therapy. The results showed that the positive rates of CD3(+)/TNF-α(+) and CD3(+)/IFN-γ(+) in de novo AA patients were (5.97 ± 6.78)% and (15.20 ± 11.28)% respectively, and (1.56 ± 0.87)% and (1.76 ± 0.87)% in normal controls respectively. There was significant difference between de novo AA patients and normal controls (p < 0.05). The positive rates of CD3(+)/TNF-α(+) and CD3(+)/IFN-γ(+) in immunosuppressive therapy group were (1.67 ± 1.26)% and (4.35 ± 4.33)% respectively. The difference between immunosuppressive therapy group and de novo AA group was statistically significant (p < 0.05). It is concluded that the levels of intracellular TNF-α and IFN-γ in AA patients are higher than those in normal controls. Immunosuppressive therapy significantly reduces the expression of intracellular TNF-α and IFN-γ. Its relationship with the clinical treatment is worth further observing.

Analyses of anti-HIV type 1 activity of a small CCR5 peptide antagonist.

We have identified a small peptide (AFDWTFVPSLIL) that specifically binds to CC chemokine receptor 5 (CCR5) expressed on the Chinese hamster ovary (CHO) cell surface. Here, we further investigate its interaction with CCR5 on activated peripheral blood mononuclear cells (PBMCs), and determine whether the peptide inhibits HIV-1 infection mediated by CCR5 in PBMCs. The peptide antagonized the binding of CCR5 ligands, the second extracellular loop-specific monoclonal antibody (mAb) (2D7), regulated on activation of normal expressed and secreted T cells (RANTES), to PBMCs and blocked CCR5-mediated Ca(2+) signaling elicited by RANTES at a concentration of 10 µg/ml. Moreover, the peptide displayed selective inhibition of R5 HIV-1 replication. We conclude that the peptide is a CCR5 antagonist with anti-HIV-1 activity.

[Screening of proteins binding to FXR1P using yeast two-hybrid technique].

OBJECTIVE: To screen the proteins interacting with FXR1P for functional investigation of FXR1P. METHODS: The yeast strain AH109 transformed with the recombinant expression vector pGBKT7/FXR1 was mated with the yeast strain Y187 pretransformed with human fetal brain cDNA library. The positive clones were screened and identified by sequence analysis. RESULTS: The recombinant expression vector pGBKT7/FXR1 was constructed successfully. Five proteins binding to FXR1P were screened from human fetal brain cDNA library using the yeast two-hybrid system, including CMAS, FTH1, GOLGA4, HSD17B1 and CSH1. CONCLUSIONS: These results provide new clues for investigating the biological functions of FXR1P and the pathogenesis of Fragile X syndrome.

[Preparation and identification of monoclonal antibody against human thrombomodulin.].

To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.

[Alu family and its biological significance].

The Alu family of short (approximately 300bp)interspersed elements (SINEs) is one of the most successful mobile genetic elements, having arisen to a copy number in excess of 500,000 within primate genomes in the last 65 million years. The proliferation of these elements had a significant impact on the architecture of primate genomes. Now the functions of Alu elements are still unclear. It is speculated that Alu elements are involved in aspects of gene regulation, e.g. gene rearrangement, CpG methylation, alternative splicing of hnRNA, binding sites of transcription factors and hormone receptors, etc. At the same time, Alu family is an important research method in human population genetics, forensic, oncology, etc. Additionally, Alu insertion, deletion and recombination led to many ancestor genetic diseases and cancers.

[Effect of 3' exonuclease activity of polymerase on extension of phosphorothioate-modified primers].

OBJECTIVE: To determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase. METHODS: Two-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated. RESULTS: Exo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase. CONCLUSION: These data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.

[Gene expression profile of peripheral blood monocyte in patients with fulminant hepatitis B by cDNA microarray].

OBJECTIVE: To investigate peripheral blood monocyte (PBMC) gene expression profile in patients with fulminant hepatic failure (FHF) by cDNA microarray. METHODS: Microarrays consisting of 8,192 human cDNAs and labelled cDNAs prepared from PBMC in both 10 FHF patients and 10 asymtomatic surface antigen carriers (ASC) were applied to analyze gene expression. Relative ratios of gene expression in individuals were obtained by comparing the hybridization results, by GenePix 4000B scanning and by ImaGene3.0 software analysis, of Cy5-labelled cDNA from FHF patients with those of Cy3-labelled cDNA from ASC. RESULTS: 249 genes out of 8,192 were identified differently, at least two times. Most of the genes (79%) involved in cell signaling transduction, cell cycles, metabolism, inflammatory response and apoptosis, whose mRNAs were differently regulated. CONCLUSIONS: These results suggest that HBV infection alters a broad range of cellular genes expression during developing into FHF and provide a framework for future functional study on the genes expressed differently.

[Study of the inhibiting effect of anti-NKG2D polyclonal antibody on cytotoxicities of NK and LAK cells].

AIM: To investigate the effect of anti NKG2D polyclonal antibody(pAb) on cytotoxicities of NK and LAK cells. METHODS: Peripheral blood mononuclear cells(PBMCs) were separated by centrifugation on Ficoll-Hypaque gradients. LAK cells were induced from PBMCs by PHA (10 mg/L) and rhIL-2 (1x10(6)U/L). Then NK cells were sorted by flow cytometry(FCM). The cytotoxicities of NK and LAK cells were analyzed by MTT colorimetry after NKG2D molecule on NK and LAK cells were blocked with anti-NKG2D pAb. RESULTS: FCM analysis proved that both purity and activity of obtained NK cells were high.The anti-NKG2D pAb could inhibit significantly cytotoxicities of NK and LAK cells to K562 and HepG2 cells, for NK cells,having decreased 82.9% and 75.6%, for LAK cells,having decreased 52.8% and 50.2%, respectively.The anti-NKG2D pAb had no effect on cytotoxicities of NK and LAK cells to CNE cells. CONCLUSION: The anti-NKG2D pAb can inhibit cytotoxocities on tumor cells by NK and LAK cells through NKG2D molecule on two effector cells.