Showing papers in "Molecular and Cellular Biochemistry in 2019"

Exosomes: cell-created drug delivery systems.

Abstract: Exosomes are 40- to 100- nm cell-originated vesicles derived from endocytic compartments that are released into almost all biological fluids. Exosomes are cell-created vesicles that inherit identical phospholipid membrane, explaining a wide application of electroporation as a technique for exosomes loading with exogenous cargoes. Another way of loading exosomes with therapeutic cargo is to overexpress a certain gene in exosome-donor cells or treat cell line with drug of interest that later will be gently enveloped into vesicles based on the process of EV biogenesis. Similarly, to visualize siRNA loading into exosomes as well as the exosomal product delivery to recipient cells, we have conducted an experiment where chemical-based exosome transfection was used. In this review, we discuss different ways of extracellular vesicle loading with exogenous cargoes and their advantages/limitations as well as novel alternative techniques of substance incorporation into nanoparticles.

Role of miRNA-210, miRNA-21 and miRNA-126 as diagnostic biomarkers in colorectal carcinoma: impact of HIF-1α-VEGF signaling pathway

Abstract: Colorectal cancer (CRC) is a major cause of death worldwide. Novel non-invasive, high diagnostic value screening test is urgently needed to improve survival rate, treatment and prognosis. Stable, small, circulating microRNA (miRNA) offers unique opportunities for the early diagnosis of several diseases. It acts as tumor oncogenes or suppressors and involve in cell death, survival, and metastasis. Communication between miRNA and carcinogenesis is critical but it still not clear and needs further investigation. The aim of our study is to evaluate the role of miR-210, miR-21, miR-126, as non-invasive diagnostic biomarkers for screening, early detection of CRC, studying their correlation with prognostic variables, and clarifying the roles of miRNAs on HIF-1α-VEGF signaling pathway. The expression of miR-210, miR-21 and miR-126 was performed using qRT-PCR in adenocarcinoma (no = 35), adenomas (no = 51), and neoplasm free controls (no = 101). Serum levels of VEGF and HIF-1α was determined by ELISA Kit. The results show that the expression of miR-210, miR-21, VEGF, HIF-1α was significantly up-regulated while that miRNA-126 was down-regulated in both adenocarcinoma and adenomas compared with controls (p < 0.001 for each). No significant difference was noted comparing patients with adenocarcinoma and adenomas. The three miRNAs correlated with VEGF, HIF-α. The miR-210 and miR-21 associated with TNM classification and clinical staging of adenocarcinoma (p < 0.001) and they show high diagnostic value with sensitivity and specificity 88.6%, 90.1% and 91.4%, 95.0% respectively. Our study revealed that circulating miR-210, miR-21 were up-regulated while miR-126 was down-regulated in CRC and adenomas patients, they all correlated with TNM staging and they had high diagnostic value. HIF-1α VEGF signaling pathways regulated by miRNAs played a role in colon cancer initiation. To the best of our knowledge, this is the first study of this miRNAs panel in CRC in our community. These data suggested that these biomarkers could be a potential novel, non-invasive marker for early diagnosis, screening and predicting prognosis of CRC. Understanding the molecular functions by which miRNAs affect cancer and understanding its roles in modulating the signaling output of VEGF might be fruitful in reducing the incidence and slowing the progression of this dark malignancy.

Melatonin and its ubiquitous anticancer effects

Abstract: Melatonin (N-acetyl-5-methoxy-tryptamine), which is generally considered as pleiotropic and multitasking molecule, secretes from pineal gland at night under normal light or dark conditions. Apart from circadian regulations, Melatonin also has antioxidant, anti-ageing, immunomodulation and anticancer properties. From the epidemiological research, it was postulated that Melatonin has significant apoptotic, angiogenic, oncostatic and anti-proliferative effects on various oncological cells. In this review, the underlying anticancer mechanisms of Melatonin such as stimulation of apoptosis, Melatonin receptors (MT1 and MT2) stimulation, paro-survival signal regulation, the hindering of angiogenesis, epigenetic alteration and metastasis have been discussed with recent findings. The Melatonin utilization as an adjuvant with chemotherapeutic drugs for the reinforcement of therapeutic effects was also discussed. This review precisely emphasizes the anticancer effect of Melatonin on various cancer cells. This review exemplifies the epidemiology and anticancer efficiency of Melatonin with prior attention to the mechanisms of actions.

Exosomes derived from microRNA-101-3p-overexpressing human bone marrow mesenchymal stem cells suppress oral cancer cell proliferation, invasion, and migration.

Abstract: Dysregulation of microRNAs (miRNAs) has been found to disrupt the progression of oral cancer. However, which miRNAs are most effective against oral cancer and how these miRNAs should be delivered are major unanswered problems. We aimed at investigating if human bone marrow mesenchymal stem cells (hBMSCs)-derived exosomes affect oral cancer development, and the potential regulatory mechanism associated with COL10A1 and miR-101-3p. COL10A1 was upregulated, while miR-101-3p was downregulated in oral cancer, and miR-101-3p targeted COL10A1 as verified by dual-luciferase reporter gene assay. Meanwhile, exosomes derived from hBMSCs were isolated and then co-cultured with oral cancer cells to identify the role of exosomes, and the results suggested that hBMSCs-derived exosomes overexpressing miR-101-3p inhibited oral cancer progression. Furthermore, tumorigenicity assay in nude mice further confirmed the inhibitory effects of hBMSCs-derived exosomes, loaded with miR-101-3p, on oral cancer, which provides a new theoretical basis in the treatment of oral cancer.

AGE-RAGE stress: a changing landscape in pathology and treatment of Alzheimer's disease.

Abstract: Numerous hypotheses including amyloid cascade, cholinergic, and oxidative have been proposed for pathogenesis of Alzheimer's disease (AD). The data suggest that advanced glycation end products (AGEs) and its receptor RAGE (receptor for AGE) are involved in the pathogenesis of AD. AGE-RAGE stress, defined as a balance between stressors (AGE, RAGE) and anti-stressors (sRAGE, AGE degraders) in favor of stressors, has been implicated in pathogenesis of diseases. AGE and its interaction with RAGE-mediated increase in the reactive oxygen species (ROS) damage brain because of its increased vulnerability to ROS. AGE and ROS increase the synthesis of amyloid β (Aβ) leading to deposition of Aβ and phosphorylation of tau, culminating in formation of plaques and neurofibrillary tangles. ROS increase the synthesis of Aβ, high-mobility group box 1(HMGB1), and S100 that interacts with RAGE to produce additional ROS resulting in enhancement of AD pathology. Elevation of ROS precedes the Aβ plaques formation. Because of involvement of AGE and RAGE in AD pathology, the treatment should be targeted at lowering AGE levels through reduction in consumption and formation of AGE, and lowering expression of RAGE, blocking of RAGE ligand binding, increasing levels of soluble RAGE (sRAGE), and use of antioxidants. The above treatment aspect of AD is lacking. In conclusion, AGE-RAGE stress initiates, and Aβ, HMGB1, and S100 enhance the progression of AD. Reduction of levels of AGE and RAGE, elevation of sRAGE, and antioxidants would be beneficial therapeutic modalities in the prevention, regression, and slowing of progression of AD.

LncRNA H19 promotes lung cancer proliferation and metastasis by inhibiting miR-200a function

Abstract: Lung cancer is the major cause leading to cancer mortality, and the 5-year survival rate for patients with lung cancer still remains low. It is urgent to fully understand the development and progression of lung cancer to discover new therapeutic targets and develop new therapeutic approaches. H19 was documented to be upregulated in lung cancer and related to cell proliferation. However, it is still unclear if H19 has other functions in lung cancer. The mRNA levels of genes were detected by qRT-PCR, and the cell proliferation rate and cell viability were measured through cell count assay and MTT assay. Transwell assays were applied to detect cell abilities to migration and invasion, while luciferase reporter assay and RNA pull-down assay were used to examine interaction between H19 and miR-200a. H19 expression was elevated in the lung cancer tissues and cell lines, while H19 overexpression promoted the lung cancer cell growth, cell migration and invasion, as well as the epithelial mesenchymal transition (EMT). Meantime, RNA pull-down assay showed that H19 interacted with miR-200a, and miR-200a inhibited the activity of H19-fused luciferase. Furthermore, H19 overexpression inhibited miR-200a function and thereby upregulated miR-200a target genes, ZEB1 and ZEB2.H19 sponged and inhibited miR-200a to de-repress expression of ZEB1 and ZEB2, and thereby enhanced lung cancer proliferation and metastasis.

Is there any evidence that AGE/sRAGE is a universal biomarker/risk marker for diseases?

Abstract: Advanced glycation end products (AGE) and its cell-bound receptor called receptor for AGE (RAGE) are implicated in the pathogenesis of numerous diseases. Soluble receptor for AGE (sRAGE) counteracts the adverse effects of AGE-RAGE interaction by competing with RAGE for binding with AGE. Low levels of serum sRAGE have been proposed as a biomarker for diseases. However, the serum levels of sRAGE in diabetes and end-stage renal disease (ESRD) are elevated. Thus, low levels of sRAGE cannot be a universal biomarker. An elevated ratio of AGE/sRAGE was then proposed as a universal biomarker. However, evidence was not provided for this new biomarker. The objective of this paper is to provide evidence in support of elevated AGE/sRAGE being a universal biomarker. The data for serum levels of AGE, sRAGE, and ratio of AGE/sRAGE were collected from patients with low serum sRAGE [non-ST-elevation myocardial infarction (NSTEMI), hyperthyroidism (HT), thoracic aortic aneurysm (TAA),and hypercholesterolemia (HC)], and with high serum levels of sRAGE [type-2 diabetes (T2D) and ESRD], and control subjects. The serum levels of AGE and ratio of AGE/sRAGE were higher in all types of patients irrespective of low or high serum sRAGE as compared to control subjects. Reasons are provided as to why AGE or sRAGE individually cannot be considered as a universal biomarker. In conclusion, the evidence supports the validity of the high ratio AGE/sRAGE as a universal biomarker/risk marker for diseases.

Novel insights of elevated systemic levels of bisphenol-A (BPA) linked to poor glycemic control, accelerated cellular senescence and insulin resistance in patients with type 2 diabetes.

Abstract: There is a striking interaction of genes and environment in the etiology of type 2 diabetes mellitus (T2DM). While endocrine disrupting chemicals (EDCs) like bisphenol-A (BPA) have received special attention for their mechanistic role in metabolic disruption, there is a lack of clinically relevant data on BPA levels in Asian Indians, a population which is more susceptible to type 2 diabetes mellitus (T2DM) and cardiovascular diseases. Therefore, we measured systemic levels of BPA in patients with T2DM compared to individuals with normal glucose tolerance (n = 30 each). Serum BPA levels were estimated using ELISA kit, and biochemical determinations were done by standard protocols. Peripheral blood mononuclear cells (PBMCs) were used to profile the gene expression alterations with special reference to inflammation, estrogen receptors, and cellular senescence in these subjects. Serum levels of BPA were significantly higher in patients with T2DM compared to control individuals and positively correlated to poor glycemic control and insulin resistance. Patients with T2DM exhibited significantly elevated mRNA levels of senescence (GLB1, p16, p21, and p53) and inflammatory (IL6 and TNF-α) markers, shortened telomeres as well as elevated levels of estrogen-related receptor gamma (ERRγ), a recently identified receptor for BPA. BPA levels were positively correlated to senescence indicators, inflammatory markers and ERRγ and negatively correlated to telomere length. Our study is the first data in the clinical diabetes setting to demonstrate an association of increased BPA levels with cellular senescence, proinflammation, poor glycemic control, insulin resistance, and shortened telomeres in patients with T2DM.

Estradiol attenuates ischemia reperfusion-induced acute kidney injury through PPAR-γ stimulated eNOS activation in rats

Abstract: We investigated the involvement of peroxisome proliferator activated receptor-γ (PPAR-γ)/endothelial nitric oxide synthase (eNOS) pathway in estradiol mediated protection against ischemia reperfusion (I/R)-induced acute kidney injury (AKI) in rats To induce AKI, rats underwent 40 min of bilateral renal ischemia followed by 24 h of reperfusion I/R-induced kidney damage was quantified by measuring serum creatinine, creatinine clearance, urea nitrogen, uric acid, potassium, fractional excretion of sodium, microproteinuria, and renal oxidative stress (thiobarbituric acid reactive substances, superoxide anion generation, and reduced glutathione) Hematoxylin eosin stain demonstrated renal histology, while renal expression of apoptotic markers (Bcl-2, Bax), PPAR-γ and eNOS were quantified by immunohistochemistry Estradiol (1 mg/kg, ip) was administered 30 min before I/R in rats In separate groups, PPAR-γ antagonist, BADGE (30 mg/kg, ip), and NOS inhibitor, l-NAME (20 mg/kg, ip) were administered prior to estradiol treatment, which was followed by I/R in rats I/R caused significant renal damage as demonstrated by biochemical (serum/urine), renal oxidative stress and histological changes alongwith increased expression of Bax and decreased levels of Bcl-2, PPAR-γ and eNOS, which were prevented by estradiol Pre-treatment with BADGE and l-NAME abolished estradiol mediated renoprotection Notably, I/R + estradiol + BADGE group revealed decreased expression of PPAR-γ and eNOS in renal tissues In I/R + estradiol + l-NAME group, eNOS expression was reduced while PPAR-γ levels remained unchanged These results suggest that estradiol modulates PPAR-γ which consequently regulates eNOS expression in rat kidneys We conclude that estradiol protects against I/R-induced AKI through PPAR-γ stimulated eNOS activation in rats

LncRNA ZEB2-AS1 contributes to the tumorigenesis of gastric cancer via activating the Wnt/β-catenin pathway.

Abstract: Studies have shown that long noncoding RNA Zinc finger E-box-binding homeobox 2 antisense RNA 1 (ZEB2-AS1) is involved in the progression of lung cancer, bladder cancer, and hepatocellular carcinoma. However, its role in the pathogenesis of gastric cancer remains unknown. The Wnt/β-catenin pathway contributes to the development of gastric cancer. ZEB2-AS1 expression was firstly detected in the gastric carcinoma tissue samples as well as in gastric cancer cells. Knockdown of ZEB2-AS1 was performed by ZEB2-AS1-shRNA, and the viability, migration, invasion, and apoptosis of gastric cancer cells were determined by CCK-8, scratch assay, transwell, and flow cytometry, respectively. Furthermore, levels of Ki-67, PCNA, VEGF, MMP9, epithelial-mesenchymal transition (EMT) markers (E-cadherin, Vimentin and ZEB2), cleaved caspase 3/8/9 and PARP, active β-catenin, c-Myc, cyclinD1, and AXIN2 were assayed by Western blot or real-time PCR. Additionally, the role and mechanism of ZEB2-AS1 were confirmed in a xenograft nude mouse model. We found ZEB2-AS1 expression was increased in gastric carcinoma samples, and it was correlated with tumor progression. Also, its expression was elevated in gastric cancer cells. Knockdown of ZEB2-AS1 reduced the proliferation, migration, invasion, and EMT, but increased the apoptosis of gastric carcinoma cells. Furthermore, ZEB2-AS1 downregulation remarkably suppressed the expression of Ki-67, PCNA, VEGF and MMP9, and the activation of Wnt/β-catenin signaling, whereas elevated the levels of cleaved caspase 3/8/9 and PARP in gastric cancer cells. And ZEB2 overexpression reversed the effects of ZEB2-AS1 downregulation on the proliferation, EMT and inactivation of Wnt/β-catenin signaling. Additionally, ZEB2-AS1 knockdown inhibited tumor growth, Ki-67 staining, and the expression of VEGF, MMP9, active β-catenin, c-Myc, cyclinD1, and AXIN2 in mice. In conclusion, ZEB2-AS1 promotes the tumorigenesis of gastric carcinoma that is related to the upregulation of ZEB2 and the activation of the Wnt/β-catenin pathway.

Inhibition of sonic hedgehog and PI3K/Akt/mTOR pathways cooperate in suppressing survival, self-renewal and tumorigenic potential of glioblastoma-initiating cells.

Abstract: Since PI3K/Akt/mTOR and sonic hedgehog (SHH) signaling pathways are highly activated in glioblastoma-initiating cells (GICs), we examined the effects of inhibiting these pathways on GIC characteristics and tumor growth in mice. NVP-LDE-225 (inhibitor of Smoothened) inhibited the expression of Gli1, Gli2, Smoothened, Patched1, and Patched2, and induced the expression of SuFu, whereas NVP-BEZ-235 (dual inhibitor of PI3K and mTOR) inhibited the expression of p-PI3K, p-Akt, p-mTOR, and p-p70S6K. NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting the self-renewal capacity of GICs, expression of pluripotency maintaining factors (Nanog, c-Myc, Oct4, and Sox2), Musashi1, cyclin D1, and Bcl-2, and transcription and expression of Gli, and in inducing the expression of cleaved caspase-3, cleaved PARP and Bim. Additionally, NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting epithelial-mesenchymal transition. Finally, the combination of NVP-LDE-225 and NVP-BEZ-235 was superior in inhibiting tumor growth, regulating the expression of pluripotency promoting factors, stem cell markers, cell cycle, and cell proliferation, and modulating EMT compared to single agent alone. In conclusion, the combined inhibition of PI3K/Akt/mTOR and SHH pathways was superior to single pathway inhibition in suppressing glioblastoma growth by targeting GICs.

Bitter taste receptors are expressed in human epithelial ovarian and prostate cancers cells and noscapine stimulation impacts cell survival

Abstract: Bitter taste receptors (Tas2Rs) are a subfamily of G-protein coupled receptors expressed not only in the oral cavity but also in several extra-oral tissues and disease states. Several natural bitter compounds from plants, such as bitter melon extract and noscapine, have displayed anti-cancer effects against various cancer types. In this study, we examined the prevalence of Tas2R subtype expression in several epithelial ovarian or prostate cancer cell lines, and the functionality of Tas2R14 was determined. qPCR analysis of five TAS2Rs demonstrated that mRNA expression often varies greatly in cancer cells in comparison to normal tissue. Using receptor-specific siRNAs, we also demonstrated that noscapine stimulation of ovarian cancer cells increased apoptosis in ovarian cancer cells in a receptor-dependent, but ROS-independent manner. This study furthers our understanding of the function of Tas2Rs in ovarian cancer by demonstrating that their activation has an impact on cell survival.

Proteomics analysis of Schwann cell-derived exosomes: a novel therapeutic strategy for central nervous system injury

Abstract: Exosomes are nanometer-sized vesicles involved in intercellular communication, and they are released by various cell types. To learn about exosomes produced by Schwann cells (SCs) and to explore their potential function in repairing the central nervous system (CNS), we isolated exosomes from supernatants of SCs by ultracentrifugation, characterized them by electron microscopy and immunoblotting and determined their protein profile using proteomic analysis. The results demonstrated that Schwann cell-derived exosomes (SCDEs) were, on average, 106.5 nm in diameter, round, and had cup-like concavity and expressed exosome markers CD9 and Alix but not tumor susceptibility gene (TSG) 101. We identified a total of 433 proteins, among which 398 proteins overlapped with the ExoCarta database. According to their specific functions, we identified 12 proteins that are closely related to CNS repair and classified them by different potential mechanisms, such as axon regeneration and inflammation inhibition. Gene Oncology analysis indicated that SCDEs are mainly involved in signal transduction and cell communication. Biological pathway analysis showed that pathways are mostly involved in exosome biogenesis, formation, uptake and axon regeneration. Among the pathways, the neurotrophin, PI3K-Akt and cAMP signaling pathways played important roles in CNS repair. Our study isolated SCDEs, unveiled their contents, presented potential neurorestorative proteins and pathways and provided a rich proteomics data resource that will be valuable for future studies of the functions of individual proteins in neurodegenerative diseases.

Chlorogenic acid isomers directly interact with Keap 1-Nrf2 signaling in Caco-2 cells

Abstract: Chlorogenic acid (CGA) exists as multiple isomers (e.g., 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) in foods such as coffee beverages, fruits and vegetables. This study aimed to investigate relative activities of these six different CGA isomers to modify redox biology in inflamed Caco-2 cells that involved Nrf2 signaling. Caco-2 cells were pre-treated with individual CGA isomers to assess the relative effectiveness to mitigate oxidative stress. Isomer-specific capacity of different CGA isomers for direct free radical scavenging activity and potential endogenous control of oxidative stress were determined using chemical assays and cell-based experiments, respectively. Molecular dynamics simulations of the CGA and Keap1-Nrf2 complex were performed to predict CGA structure-specific interactions. Results demonstrated that dicaffeoylquinic acid (diCQA including 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) isomers had greater (p < 0.05) affinity to ameliorate oxidative stress through direct free radical scavenging activity. This observation corresponded to greater (p < 0.05) capacity to activate Nrf2 signaling compared to caffeoylquinic acid (CQA including 3-CQA, 4-CQA, and 5-CQA) isomers in inflamed differentiated Caco-2 cells. Simulations revealed that differences between the ability of CQA and diCQA to interact with the Keap1-Nrf2 complex may be due to differences in relative orientation within this complex. The observed CGA isomer-specific affinity for CQA to activate Nrf2 signaling was confirmed by nuclear translocation of Nrf2 induced by CGA and greater (p < 0.05) upregulation of genes related to Nrf2 expression.

IL-17A/IL-17RA promotes invasion and activates MMP-2 and MMP-9 expression via p38 MAPK signaling pathway in non-small cell lung cancer.

Abstract: The present study is to investigate the effect and mechanism of action of interleukin (IL)-17A and its receptor IL-17RA on non-small cell lung cancer (NSCLC). A total of 139 NSCLC patients were included in the study. NSCLC tissues and tumor-adjacent tissues were collected from the patients. Human NSCLC cell lines H157, H1975, and A549 were used for in vitro studies. MTT assay was performed to determine cell proliferation. Wound healing assay was used to determine cell motility. Transwell assay was carried out to detect migration and invasion. Quantitative real-time polymerase chain reaction was conducted to measure mRNA expression, while Western blotting was used for determine protein expression. Immunohistochemistry was employed to evaluate IL-17RA expression in 139 primary human NSCLC tissues. Levels of IL-17RA in NSCLC tissues were higher than tumor-adjacent normal tissues, and associated with clinical outcomes. Kaplan-Meier survival analysis indicated that NSCLC patients with positive IL-17RA expression had a poor survival. In addition, IL-17A/IL-17RA affected NSCLC cell migration and invasion in vitro. Treatment with IL-17A/IL-17RA increased the expression of MMP-2 and MMP-9 in NSCLC cells. Furthermore, phosphorylation of p38 was enhanced in IL-17RA-overexpressing NSCLC cells. P38 MAPK-specific inhibitor SB203580 suppressed the migration and invasion of NSCLC cells. MMP-2 and MMP-9 were downstream effectors of IL-17RA and p38 signaling pathways. The present study demonstrates that P38 MAPK activity is crucial for IL-17A/IL-17RA to promote NSCLC metastasis. In addition, IL-17A/IL-17RA signaling may be a novel and promising cancer therapeutic target for the treatment of NSCLC.

Genistein protects against ox-LDL-induced senescence through enhancing SIRT1/LKB1/AMPK-mediated autophagy flux in HUVECs.

Abstract: The anti-senescence activity of genistein is associated with inducing autophagy; however, the underlying mechanisms are not fully understood. In this study, human umbilical vein endothelial cells (HUVECs) were pretreated with genistein (1000 nM) for 30 min and then exposed to ox-LDL (50 mg/L) for another 12 h. The study found that genistein inhibited the ox-LDL-induced senescence (reducing the levels of P16 and P21 protein, and the activity of SA-β-gal); meanwhile, the effect of genistein was bound up with enhancing autophagic flux (increasing LC3-II, and decreasing the level of P62, p-mTOR and p-P70S6K). Moreover, SIRT1/LKB1/AMPK pathway was involved in genistein accelerating autophagic flux and mitigating senescence in HUVECs. The present study illustrated that genistein was a promising therapeutic agent to delay aging process and extend longevity.

Chlamydia trachomatis plasmid-encoded protein Pgp3 inhibits apoptosis via the PI3K-AKT-mediated MDM2-p53 axis.

Abstract: Chlamydia trachomatis, the most common human pathogen that causes trachoma and sexually transmitted disease, has developed various strategies for inhibiting host cell apoptosis. Activation of the PI3K (phosphoinositide 3-kinase)/AKT-mediated MDM2 (murine double minute 2)-p53 pathway plays a prominent role in the apoptosis resistance arising from C. trachomatis infection. However, the precise upstream mechanisms by which C. trachomatis activates this pathway have not been adequately investigated. Here, we reveal that the secreted C. trachomatis plasmid-encoded protein Pgp3 inhibits apoptosis in HeLa cells. This process requires the activation of the PI3K/AKT signaling pathway, thereby leading to phosphorylation and nuclear entry of MDM2, and p53 degradation. PI3 K inhibitor LY294002 and MDM2 inhibitor Nutlin-3a block Pgp3-induced inhibition of HeLa cell apoptosis, suggesting a critical role for the PI3K/AKT pathway and its effect on the MDM2-p53 axis in Pgp3 anti-apoptotic activity.

Mangiferin attenuates cisplatin-induced acute kidney injury in rats mediating modulation of MAPK pathway

Abstract: Cisplatin has been confined due to the reported cases of nephrotoxicity. In the present study, an active xanthone, Mangiferin (from Mangifera indica) was investigated for its defensive role in cisplatin-induced nephrotoxicity. Male wistar albino rats were divided into six groups i.e., group 1 (normal); group 2 (cisplatin control); group 3, 4, and 5 (mangiferin 10, 20, and 40 mg/kg, i.p.); and per se (40 mg/kg; i.p.). The treatment was given for 10 days. On day 7, single dose of cisplatin 8 mg/kg i.p. was administered to induce nephrotoxicity in all groups except normal and per se. On day 11, animals were anesthetized, blood was taken from heart and serum was separated. Thereafter, rats were sacrificed and kidneys were isolated and preserved for histopathological, ultrastructural, immunohistochemical, and western blot analysis. Cisplatin control group showed significant impairment in renal function due to increased inflammation and oxidative stress which was also confirmed by histopathology and MAPK pathway proteins expression. However, pretreatment with mangiferin 20 and 40 mg/kg significantly reversed the renal function along with the structural changes and the levels of antioxidants. Mangiferin treatment attenuated DNA damage and apoptotic pathway.

Anti-neuroinflammatory effects of galangin in LPS-stimulated BV-2 microglia through regulation of IL-1β production and the NF-κB signaling pathways

Abstract: Neuroinflammation resulting from microglial activation is involved in the pathogenesis of neurodegenerative diseases, including Parkinson's diseases. Microglial activation plays an important role in neuroinflammation and contributes to several neurological disorders. Hence, inhibition of both microglial activation and the generation of pro-inflammatory cytokines may lead to an effective treatment for neurodegenerative diseases. In the present study, the anti-neuroinflammatory effects of galangin were investigated in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Galangin significantly decreased the generation of nitric oxide, interleukin-1β, and inducible nitric oxide synthase in LPS-stimulated BV-2 microglial cells. In addition, galangin inhibited the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase 1/2. Furthermore, it was observed that activation of both IκB-α and nuclear factor kappa B (NF-κB) was significantly increased following LPS stimulation, and this effect was suppressed by galangin treatment. In conclusion, galangin displayed an anti-neuroinflammatory activity in LPS-stimulated BV-2 microglial cells. Galangin inhibited LPS-induced neuroinflammation via the MAPK and NF-κB signaling pathways and might act as a natural therapeutic agent for the treatment of various neuroinflammatory conditions.

Empagliflozin restores the integrity of the endothelial glycocalyx in vitro.

Abstract: The antihyperglycemic agent empagliflozin not only improves glycemic control but has also been associated with clinically meaningful reductions in cardiovascular events. Studies have shown that empagliflozin significantly reduces cardiovascular death and heart failure-associated hospitalizations. Given that endothelial dysfunction is closely linked with the pathogenesis of atherosclerotic cardiovascular disease, we hypothesized that the cardiovascular benefits observed with empagliflozin may be a result of its positive impact on the health of the endothelial glycocalyx (GCX), a critical component for the endothelium homeostasis. Human abdominal aortic endothelial cells (HAAECs) were either statically cultured or subjected to a steady wall shear stress of 10 dyne/cm2. Empagliflozin (50 µM, 24 h) restored heparinase III-mediated GCX disruption and the normal mechanotransduction responses in GCX-compromised HAAECs while reducing the attachment of all-trans retinoic acid-transformed NB4 cells to HAAECs. The current body of work suggests that the cardioprotective properties previously reported for empagliflozin may in part be due to the ability of empagliflozin to preserve and restore the structural integrity of the GCX, which in turn helps to maintain vascular health by promoting an anti-inflammatory endothelium, in the presence of a pro-inflammatory environment. Further studies are needed to fully understand the mechanisms underlying the cardiovascular benefits of empagliflozin.

Hydrogen sulfide modulates SIRT1 and suppresses oxidative stress in diabetic nephropathy.

Abstract: DN is recognized as not only a leading cause of end stage renal disease (ESRD) but also an independent risk factor for cardiovascular disease (CVD). Novel therapeutic approaches to diabetic nephropathy (DN) are needed, or else, healthcare resources will be overwhelmed by the expected worldwide increase in associated cases of ESRD and CVD. Reactive oxygen species (ROS) and advanced glycation end product (AGE) are implicated in the development of DN. Hydrogen sulfide (H2S) is known for its antioxidant and antiapoptotic characteristics. Simultaneously diabetics have lower H2S levels. Thus, it is worth investigating the use of H2S in treatment of DN. To investigate the potential therapeutic role of H2S in DN. Sixty male rats were divided into four groups: control, DN, DN+NaHS30 µmol/kg/day and DN+NaHS100 µmol/kg/day. Fasting blood sugar (FBS), kidney function tests, SIRT1 activity, superoxide dismutase activity (SOD), malondialdehyde (MDA) and expression of caspase3 and p53 in renal tissues were assessed. Kidney was examined histopathologically. DN rats had higher FBS, renal dysfunction, decreased SIRT1 and SOD activity levels, increased caspase3 and p53 relative expression and increased MDA in renal tissues. NaHS increased SIRT1 and reversed biochemical, apoptotic, oxidant and pathologic parameters characteristic of DN, with better results using a dose of 100 µmol/kg/day. H2S has a protective role against DN through decreasing FBS, ROS, apoptosis and upregulating SIRT1, thus preserving renal cells from further damage caused by DM.

Hydrogen peroxide-induced oxidative stress and its impact on innate immune responses in lung carcinoma A549 cells

Abstract: The immune responses, involved in recognition of cancer-specific antigens, are of particular interest as this may provide major leads towards developing new vaccines and antibody therapies against cancer. An effective treatment for cancer is still a challenge because there are many mechanisms through which the tumor cells can escape the host immune surveillance. Oxidative stress or respiratory burst which is host’s mechanism to kill the foreign particles is used as defense mechanism by the tumor cells. The tumor cells uses this oxidative stress to form neo-antigens which in turn makes them undetectable and can escape the host immune surveillance. The human lung carcinoma (A549) cells were treated using 100 µM H2O2 to induce oxidative stress, and the extent oxidative modifications were detected at the level of membrane and proteins in form of lipid peroxidation and protein carbonyls respectively. Nitric oxide and iNOS levels were estimated by Griess assay and immunostaining, respectively. The oxidized tumor proteins were visualized on one-dimensional SDS–PAGE. The H2O2-treated (15 min and 24 h post-treatment) A549 cells were co-cultured with THP-1 cells to subsequently visualize the phagocytic activity by Giemsa and CFSE staining to understand the role of neo (oxidized) tumor antigens in eliciting alteration in immune responses. A significant decline in the percent engulfed cells and decrease in the levels of reactive oxygen species was observed. Immunohistostaining for p47phox, which is an important indicator of the oxygen-dependent phagocytosis, showed a decrease in its levels when cells were treated for only 15 min with 100 µM H2O2, whereas at 24-h post-treatment there was no change in the p47phox levels. The study has established oxidative stress as a new pathogenic mechanism of carcinogenesis and will open new avenues for clinical intervention, adjunct therapies for cancer, and its control at the initial stage by targeting these neo-antigens.

The mitochondrial Nod-like receptor NLRX1 modifies apoptosis through SARM1.

Abstract: NLRX1, the mitochondrial NOD-like receptor (NLR), modulates apoptosis in response to both intrinsic and extrinsic cues Insights into the mechanism of how NLRX1 influences apoptosis remain to be determined Here, we demonstrate that NLRX1 associates with SARM1, a protein with a toll/interleukin-1 receptor (TIR)-containing domain also found in adaptor proteins downstream of toll-like receptors, such as MyD88 While a direct role of SARM1 in innate immunity is unclear, the protein plays essential roles in Wallerian degeneration (WD), a type of neuronal catabolism occurring following axonal severing or damage In non-neuronal cells, we found that endogenous SARM1 was equally distributed in the cytosol and the mitochondrial matrix, where association with NLRX1 occurred In these cells, the apoptotic role of NLRX1 was fully dependent on SARM1, indicating that SARM1 was downstream of NLRX1 in apoptosis regulation In primary murine neurons, however, Wallerian degeneration induced by vinblastine or NGF deprivation occurred in SARM1- yet NLRX1-independent manner, suggesting that WD requires the cytosolic pool of SARM1 or that NLRX1 levels in neurons are too low to contribute to WD regulation Together, these results shed new light into the mechanisms through which NLRX1 controls apoptosis and provides evidence of a new link between NLR and TIR-containing proteins

MicroRNA-32 targeting PTEN enhances M2 macrophage polarization in the glioma microenvironment and further promotes the progression of glioma

Abstract: This study was aimed to explore the molecular mechanism of macrophage polarization and its effect on glioma progression. THP1 cells were cocultured in conditioned medium from U87 human glioblastoma cells to simulate the glioma microenvironment. The expression of miR-32 and PTEN in THP1 cells was detected by real-time PCR. A luciferase reporter assay was conducted to confirm the target relation between miR-32 and PTEN. Western blot assays and ELISA were performed to detect PTEN, M2 macrophage-specific markers, PI3K/AKT signaling proteins, and apoptosis-related proteins. U87 cell proliferation was evaluated by CCK-8 and colony forming assays, and the migration ability of the cells was evaluated by Transwell and wound healing assays. The U87 culture supernatant promoted the M2 phenotype of THP1 cells. miR-32 was upregulated and PTEN was downregulated in THP1 cells with the M2 phenotype in the glioma microenvironment. Luciferase assays confirmed that PTEN expression was suppressed by miR-32 through interaction with the 3'UTR of PTEN. Overexpression of miR-32 suppressed PTEN expression in THP1 cells. Overexpression of miR-32 or downregulation of PTEN promoted the expression of M2 macrophage-specific markers, thereby enhancing M2 macrophage polarization. Additionally, miR-32 inhibited THP1 cell apoptosis via suppressing the PI3K/AKT signaling pathway. Most importantly, the proliferation and migration capacities of U87 cells treated with the THP1 culture supernatant after miR-32 overexpression were enhanced, and these effects could be reversed by cotransfection with pcDNA3.1-PTEN. miR-32 negatively modulates PTEN, thereby promoting M2 macrophage transformation through PI3K/AKT signaling, enhancing glioma proliferation and migration abilities.

CPT1 regulates the proliferation of pulmonary artery smooth muscle cells through the AMPK-p53-p21 pathway in pulmonary arterial hypertension.

Abstract: Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a dominant role in the development of pulmonary arterial hypertension (PAH). Some studies and our previous work found that disturbance of fatty acid metabolism existed in PAH. However, the mechanistic link between fatty acid catabolism and cell proliferation remains elusive. Here, we identified an essential role and signal pathway for the key rate-limiting enzyme of mitochondrial fatty acid β-oxidation, carnitine palmitoyltransferase (CPT) 1, in regulating PASMC proliferation in PAH. We found that CPT1 was highly expressed in rat lungs and pulmonary arteries in monocrotaline-induced PAH, accompanied by decreased adenosine triphosphate (ATP) production and downregulation of the AMPK-p53-p21 pathway. Platelet-derived growth factor (PDGF)-BB upregulated the expression of CPT1 in a dose- and time-dependent manner. PASMC proliferation and ATP production induced by PDGF-BB were partly reversed by the CPT1 inhibitor etomoxir (ETO). The overexpression of CPT1 in PASMCs also promoted proliferation and ATP production and subsequently inhibited the phosphorylation of AMPK, p53, as well as p21 in PASMCs. Furthermore, AMPK was activated by ETO, which increased the expression of p53 and p21, and the proportion of cells in the cell cycle G2/M phase in response to PDGF-BB stimulation in PASMCs. Our work reveals a novel mechanism of CPT1 regulating PASMC proliferation in PAH, and regulation of CPT1 may be a potential target for therapeutic intervention in PAH.

Inhibitions of anandamide transport and FAAH synthesis decrease apoptosis and oxidative stress through inhibition of TRPV1 channel in an in vitro seizure model.

Abstract: The expression level of TRPV1 is high in hippocampus which is a main epileptic area in the brain. In addition to the actions of capsaicin (CAP) and reactive oxygen species (ROS), the TRPV1 channel is activated in neurons by endogenous cannabinoid, anandamide (AEA). In the current study, we investigated the role of inhibitors of TRPV1 (capsazepine, CPZ), AEA transport (AM404), and FAAH (URB597) on the modulation of Ca2+ entry, apoptosis, and oxidative stress in in vitro seizure-induced rat hippocampus and human glioblastoma (DBTRG) cell line. The seizure was induced in the hippocampal and DBTRG neurons using in vitro 4-aminopyridine (4-AP) to trigger a seizure-like activity model. CPZ and AM404 were fully effective in reversing 4-AP-induced intracellular free Ca2+ concentration of the hippocampus and TRPV1 current density of DBTRG. However, AEA and CAP did not activate TRPV1 in the URB597-treated neurons. Hence, we observed TRPV1 blocker effects of URB597 in the DBTRG neurons. In addition, the AM404 and CPZ treatments decreased intracellular ROS production, mitochondrial membrane depolarization, apoptosis, caspases 3 and 9 values in the hippocampus. In conclusion, the results indicate that inhibition of AEA transport, FAAH synthesis, and TRPV1 activity can result in remarkable neuroprotective effects in the epileptic neurons. Possible molecular pathways of involvement of capsazepine (CPZ) and AM4040 in anandamide and capsaicin (CAP)-induced apoptosis, oxidative stress, and Ca2+ accumulation through TRPV1 channel in the seizure-induced rat hippocampus and human glioblastoma neurons. The TRPV1 channel is activated by different stimuli including reactive oxygen species (ROS), anandamide (AEA), and CAP and it is blocked by capsazepine (CPZ). Cannabinoid receptor type 1 (CB1) is also activated by AEA. The AEA levels in cytosol are decreased by fatty acid amide hydrolase (FAAH) enzyme. Inhibition of FAAH through URB597 induces stimulation of CB1 receptor through accumulation AEA. URB597 acts antiepileptic effects through inhibition of TRPV1. Overloaded Ca2+ concentration of mitochondria can induce an apoptotic program by stimulating the release of apoptosis-promoting factors such as caspases 3 and caspase 9 by generating ROS due to respiratory chain damage. AM404 and CPZ reduce TRPV1 channel activation and Ca2+ entry in the in vitro 4-AP seizure model-induced hippocampal and glioblastoma neurons.

MiR-19b-3p attenuates IL-1β induced extracellular matrix degradation and inflammatory injury in chondrocytes by targeting GRK6

Abstract: Osteoarthritis (OA) is characterized by degradation of articular cartilage. MiRNAs are involved in the regulation of chondrogenesis and OA. We aimed to investigate effects and mechanisms of miR-19b-3p in regulating chondrocytes viability, cartilage degradation and inflammatory response. Primary chondrocytes were isolated from cartilages in control subjects and patients with OA. Murine ATDC5 cells were pre-conditioned with IL-1β in vitro. Expressions and interaction of miR-19b-3p with G protein-coupled receptor kinase 6 (GRK6), and their effects on inflammation, chondrocytes viability and cartilage degradation were determined after miR-19b-3p mimic or GRK6 siRNA transfection. MiR-19b-3p was significantly decreased in OA chondrocytes and IL-1β-stimulated ATDC5 cells, in paralleled with the elevated type-II-collagen, aggrecan, MMP13 and GRK6 expression. MiR-19b-3p mimic dramatically increased the viability of chondrocytes and suppressed cell apoptosis. It also increased type-II-collagen, aggrecan expression and glycosaminoglycan (sGAG) content, and decreased the expression of MMP-1 and MMP-13 that controlled by IL-1β. Overexpression of miR-19b-3p inhibited the production of IL-6 and IL-8 in ATDC5 cells. However, the protective effects of miR-19b-3p mimic on IL-1β induced cell death; IL-8 production and sGAG decrease were greatly discounted by GRK6 lentiviral vectors. Luciferase reporter assay confirmed that GRK6 gene was a direct target ofmiR-19b-3p. GRK6 siRNA transfection antagonized the IL-1β-induced chondrocytes injury, extracellular matrix degradation and inflammatory response. MiR-19b-3p mimic and GRK6 siRNA showed comparable inhibitory effect on IL-1β-provoked NF-κB as reflected by the expression of p-p65. NF-κB translocation inhibition with PS1154 reversed the effects of IL-1β on IL-8 and sGAG. Collectively, miR-19b-3p attenuated OA by targeting GRK6-NF-κB pathway.

Silencing circular RNA circ_0010729 protects human cardiomyocytes from oxygen–glucose deprivation-induced injury by up-regulating microRNA-145-5p

Abstract: Circular RNAs (circRNAs) are effector molecules that exert functions in cardiovascular diseases. Nevertheless, the effects of circRNAs on myocardial ischemia remain uninvestigated. This paper aimed to explore the functions of circ_0010729 in oxygen–glucose–deprivation (OGD)-caused injury of human cardiomyocytes (HCM). HCM were exposed to OGD environment for 4 h. Then the expression of circ_0010729 was evaluated by RT-qPCR. After transfection, cell viability, apoptosis, and migration were examined to evaluate the impact of overexpression and knockdown of circ_0010729 on OGD-induced cell injury. The regulation between circ_0010729 and microRNA-145-5p (miR-145-5p) was verified. After miR-145-5p inhibitor transfection, whether aberrant miR-145-5p expression affected the modulation of circ_0010729 in OGD-induced cell injury was measured. Western blot was utilized to analyze mTOR and MEK/ERK pathway-related proteins. OGD treatment enhanced circ_0010729 expression and evoked cell injury in HCM. Moreover, OGD-induced injury was aggrandized by circ_0010729 overexpression via suppressing cell growth and migration in HCM. Knockdown of circ_0010729 attenuated OGD-induced injury. In addition, circ_0010729 negatively regulated miR-145-5p expression. MiR-145-5p inhibition reversed the effects of silencing circ_0010729 on OGD-induced injury and mTOR and MEK/ERK pathways. We demonstrated that silencing circ_0010729 activated mTOR and MEK/ERK pathways by up-regulating miR-145-5p, thereby protecting HCM from OGD-induced injury.

Mesenchymal stem cells attenuate sepsis-induced liver injury via inhibiting M1 polarization of Kupffer cells

Abstract: Sepsis is a leading cause of death in intensive care units that can result in acute hepatic damage Animal experiments and clinical trials have shown that mesenchymal stem cell (MSC) therapy has some beneficial in several liver diseases However, the protective effects of MSC therapy on sepsis-induced hepatic damage and associated mechanisms are not completely understood The aim of the present study was to investigate the effects of MSCs on sepsis-induced liver injury and underlying mechanisms A rat model of sepsis-induced liver injury was established by cecal ligation and puncture, and serum alanine aminotransferase and aspartate transaminase activities as well as liver histological changes were measured Inflammatory cytokines, Kupffer cell M1 phenotype markers, and associated signal molecules were also determined in septic rats and in lipopolysaccharide (LPS)-treated Kupffer cells Our results showed that injection of MSCs attenuated sepsis-induced liver injury Treatment with MSCs inhibited activation of Kupffer cells towards M1 phenotype, attenuated TNF-α and IL-6 expression, and promoted IL-4 and IL-10 expression in septic rats and LPS-treated Kupffer cells Furthermore, MSCs also inhibited the nuclear translocation of nuclear factor-kappa B in LPS-challenged Kupffer cells and the liver of septic rats These results indicated that MSCs attenuated sepsis-induced liver injury through suppressing M1 polarization of Kupffer cells

Anti-inflammatory and anticancer effects of p-methoxycinnamic acid, an active phenylpropanoid, against 1,2-dimethylhydrazine-induced rat colon carcinogenesis.

Abstract: p-methoxycinnamic acid (p-MCA) is an active phenolic acid found in rice bran, turmeric, brown rice, Kaempferia galanga, buckwheat inflorescence, etc. Earlier, we have reported that p-methoxycinnamic acid possesses antioxidant and antilipidperoxidative effects on 1,2-dimethylhyrdrazine (DMH)-induced colon carcinogenesis. The purpose of this study is to unravel the anti-inflammatory and anticancer properties of p-MCA against DMH-induced colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control, group 2 rats received 40 mg/kg b.wt. of p-MCA in 0.1% carboxymethylcellulose (CMC) every day, and colon cancer was induced in groups 3–6 using DMH at the dose of (20 mg/kg b.wt. subcutaneously) once a week for 15 weeks. In addition, along with DMH, groups 4 (initiation), 5 (post initiation) and 6 (entire period) rats received p-MCA (40 mg/kg b.wt.) p.o. every day during different time periods for the total experimental period of 30 weeks. Colon of animals treated with DMH showed an increased number of aberrant crypt foci (ACFs), increased nuclear translocation of transcription factor NF-κB p65 subunit, increased expression of inflammatory markers (iNOS, COX-2), cytokines (tumour necrosis factor-α, interleukin-6), cyclin D1, antiapoptotic protein (Bcl-2), metastasis marker (matrix metalloproteinase-2 (MMP-2)) and angiogenic marker (vascular endothelial growth factor VEGF) and decreased expression of pro-apoptotic proteins (Bax, caspases 3 and 9). On supplementing with p-MCA (40 mg/kg b.wt.) throughout the entire experimental period, DMH-induced pathological alterations reversed significantly to normal.