Chloroquine Diphosphate Research Articles

Anti-dengue viral activity of Glycyrrhiza glabra roots in Vero cells

Despite being a global public health problem, there are no antiviral agents for dengue. Plants are the sources of most approved drugs and many phytochemicals have exhibited in vitro antiviral activity. We explored the antiviral potential of the aqueous extract of Glycyrrhiza glabra roots (GGaq) on dengue viruses. Plaque reduction antiviral assay for half maximal inhibitory concentrations (IC50) was done in Vero cells infected with dengue serotypes 1–4 and exposed to varying concentrations of GGaq. Half-maximal cytotoxic concentration (CC50) of the GGaq was 651.9 µg/mL. The IC50 of the four dengue serotypes (10–50 µg/mL) indicated moderate inhibition by GGaq. 98–100% inhibition of all dengue serotypes was seen with GGaq at the maximum nontoxic dose. Preparative thin layer chromatography of GGaq, isolated sub fractions E and F which had low to moderate antiviral activity (IC50 12.65–85.95 and IC50 13.14–69.27 respectively). They exhibited good therapeutic potential only for dengue serotype-4. Virus adsorption to cells was significantly inhibited by GG (50%) and sub fraction E (24.9%). Chloroquine diphosphate was used as the positive control in all assays. The aqueous extract of GG was non-toxic and had better antiviral activity than sub fractions E and F.

N-alkylation of amines for the synthesis of potential antiviral agents: A structural modification approach

The threat of emerging viral outbreaks has increased the need for fast and effective development of therapeutics against emerging pathogens. One approach is to modify the structure of existing therapeutic agents to achieve the desired antiviral properties. Here, we attempted to synthesize a new antiviral compound by modifying the structure of chloroquine using the N-alkylation of the primary amine (N1,N1-diethylpentane-1,4-diamine) that is used in chloroquine synthesis. Chloroquine is commonly used to treat malaria. Like chloroquine, chloroquine is used for treating conditions such as rheumatoid arthritis, lupus, and malaria. For instance, in malaria treatment, it targets and inhibits the growth of the malaria parasite, aiding in its elimination from the body. The synthesized compounds MP1, C1, and TT1 were further tested in vitro against the B.1 lineage of SARS-CoV-2. One of the compounds, MP1, demonstrated minor effectiveness, with an IC50 of XX at only a high concentration (at a concentration of 60 μM) and decreased both the number of SARS-CoV-2 copies and the amount of infectious virus. Although the synthesized compounds failed to markedly inhibit SARS-CoV-2, this could be a pontial mechanism for manipulating the drug structure against other pathogens. MP1, TT1, C1, and chloroquine diphosphate were used as ligands for molecular docking to determine the principal interactions between these compounds and the active site of the protein downloaded from the Protein Data Bank (PDB ID: 6lzg). Finally, ADMET assays were performed on the synthesized compounds to determine their pharmacokinetics and bioavailability.

In vitro antimalarial therapeutic interventions using various combinations of antibiotics and standard antimalarials against multi drug resistant Indian field isolates of Plasmodium falciparum

Resistance to antimalarials is considered to be major cause of increased malaria morbidity and mortality. Present study was undertaken to explore an effective drug combination against selected mutant parasite various combinations using standard antimalarials {Chloroquine diphosphate (CQ), Quinine (QUIN), Mefloquine (MQ), Piperaquine (PPQ), Artemether (ARTM), Arteether (ARTE), Dihydro-artemisinin (DHA), Lumefantrine (LUME)and Atovaquone (ATQ)} belonging to three different classes and two antibiotics, Azithromycin (AZI) and Doxycycline (DOXY) have been tried against K1 (chloroquine-resistant) and 3D7 (chloroquine-sensitive) strains as well as selected arteether torelant parasite (MZRI-R) and field isolates (MZRI & MZRII) of P. falciparum. SYBR Green I fluorescence (MSF) assay was used to determine IC50 values. Results of present studies highlighted that all the combinations used exhibited additive effect with varying intensity depending upon the drug ratios used. We also observed that combination of ARTM plus LUME was more advantageous than DOXY plus DHA/CQ and AZI plus CQ/ATQ for treatment of multidrug resistant parasites whereas combinations of LUME plus PPQ, ATQ plus DOXY/AZI can be used for treatment of CQ resistant parasite.

#2928 Fabry disease may imbalance integrin α3 related to endothelial dysfunction

Abstract Background and Aims Fabry Disease (FD) is a rare disease characterized by a mutation in the GLA gene and consequently by the functional anomaly of the α-Galactosidase A (α-Gal) enzyme. The lack of this functional enzyme leads to lysosomal accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different cell types, causing several symptoms throughout the body, which aggravate during the patient's longevity. We already demonstrated that exposure of endothelial cells to chloroquine, to mimic FD effects, leads to lysosomal accumulation in human endothelial cells. In the present study, we analyzed the endothelial changes in ultrastructural morphology and cell adhesion under evaluation of Integrin α3 expression, an important integrin that stabilizes cell adhesion and is involved in several signaling pathways in endothelial cells. Method Human endothelial cells (EA.hy926, ATCC CRL-2922) were exposed to chloroquine diphosphate (0.5 µg/mL) for 72 hours. The ultrastructural morphology was analyzed through scanning electron microscopy (SEM) and the Integrin α3 expression was analyzed through Western Blot and RT-qPCR. Results Through SEM (Fig. 1C-F) it was observed that after the exposure to chloroquine diphosphate, there was a decrease in cell expansion and adhesion and an increase in vesicular content. Curiously, the analysis of the Integrin α3 gene (ITGA3) and protein expression (Fig. 1A-B) demonstrated a significant increase (P < 0.05) in cells that were exposed to chloroquine diphosphate compared to non-exposed cells (control cells). Conclusion The in vitro condition of the FD in human endothelial cells demonstrated an increase in Integrin α3; however, there was a decrease in cell adhesion and expansion. The evaluation of the Integrin α3 expression could be important for monitoring the endothelial dysfunction in FD, as it is intrinsically involved in cell adhesion, remodeling, and fibrosis. These important changes can lead to renal impairment, proteinuria, and decreased glomerular filtration rate.

Dissimilar effect of organometallic ruthenium complexes on the viability of MDR and non-MDR experimental models

Ruthenium complexes containing triphenylphosphine diamide ligands were prepared, characterized, and tested for their biological activity against various cancer cell lines and the malaria parasite, Plasmodium falciparum. The effect of M (mono-substituted) and B (bis-substituted) complexes on the human cervical carcinoma (HeLa) cell line was investigated using the MTT assay. Five (B2, B3, B5, B6, and B13) of the 24 synthesized ruthenium complexes showed significant effects with IC50 values ranging between 0.3 and 2.3 μM. Evaluation of the potential biomolecular targets of B2 and B13 by fluorescence spectroscopy revealed relevant interactions with BSA and only a weak affinity for ctDNA. Complexes M2, B2, M13 and B13 were selected for further biological characterization. Their effect on the viability of two ovarian cancer cell lines was compared to normal cell lines, denoting their selectivity. Upon treatment of four different drug-resistant gynaecological cancer cell lines, differing in their multidrug-resistant phenotypes, the efficacy of the bis-substituted complexes was shown to be greater than their mono-substituted counterparts. The non-MDR cells are sensitive to all the tested complexes, compared to MDR cells which are less sensitive. Upon investigation of complexes M2, M13, B2, and B13 against sensitive and multidrug-resistant parasite strains of P. falciparum, the bis-substituted complexes were again shown to be the most potent, with submicromolar activity against both strains. Furthermore, the resistance indexes for the complexes were approximately equal to 1, which is at least 5-fold lower than chloroquine diphosphate, suggesting the ability of these complexes to retain their activity in resistant forms of the parasite.

Physico-chemical and spectroscopic study of some biologically potent molecules in aqueous solution of an anti-malarial drug molecule with reference to diverse molecular interactions simultaneously optimized by DFT

The various molecular interactions of amino acids, l-Asparagine (L-Asn) and l-Glutamine (L-Gln) in the aqueous solutions of Chloroquine diphosphate (CDP) had been explored comprehensively by various physicochemical as well as spectroscopic methodologies. Here we exposed the chemistry of l-Asparagine and l-Glutamine in aqueous solution of CDP by means of the apparent molar volume (ϕv), limiting apparent molar volume (ϕv0), viscosity B coefficients, molar refraction (RM), limiting molar refraction (RM0), limiting slope of surface tension and the studies revealed dominancy of solute-solvent interaction, over the solvent-solvent and solute-solute interactions in solution. A significant evidence on the nature of solute – solvent interaction obtained from 1H as well as 13C NMR spectroscopic measurement. CDP – amino acids binding constants were calculated from UV–visible spectroscopic measurement, showing significant binding ability. This work has a theoretical background after the practical outcomes, which was executed by the DFT calculations.

Comprehensive Analysis of MICALL2 Reveals Its Potential Roles in EGFR Stabilization and Ovarian Cancer Cell Invasion.

Molecules interacting with CasL (MICALs) are critical mediators of cell motility that act by cytoskeleton rearrangement. However, the molecular mechanisms underlying the regulation of cancer cell invasion remain elusive. The aim of this study was to investigate the potential role of one member of MICALs, i.e., MICALL2, in the invasion and function of ovarian cancer cells. We showed by bioinformatics analysis that MICALL2 expression was significantly higher in tissues of advanced-stage ovarian cancer and associated with poor overall survival of patients. MICALL2 was strongly correlated with the infiltration of multiple types of immune cells and T-cell exhaustion markers. Moreover, enrichment analyses showed that MICALL2 was involved in the tumor-related matrix degradation pathway. Mechanistically, MMP9 was identified as the target gene of MICALL2 for the regulation of invadopodium formation and SKOV3, HO-8910PM cell invasion. In addition, EGFR-AKT-mTOR signaling was identified as the downstream pathway of MICALL2 in the regulation of MMP9 expression. Furthermore, MICALL2 silencing promoted EGFR degradation; however, this effect was abrogated by treatment with the autophagy inhibitors acadesine and chloroquine diphosphate. Silencing of MICALL2 resulted in a suppressive activity of Rac1 while suppressing Rac1 activation attenuated the pro-EGFR, pro-MMP9, and proinvasive effects induced by the overexpression of MICALL2. Collectively, our results indicated that MICALL2 participated in the process of immune infiltration and invasion by ovarian cancer cells. Moreover, MICALL2 prevented EGFR degradation in a Rac1-dependent manner, consequently leading to EGFR-AKT-mTOR-MMP9 signaling activation and invadopodia-mediated matrix degradation.

Effect of Tempuyung (Sonchus arvensis L.) Leaf Extract on Kidney Histology of Mice Exposed to Plasmodium berghei

Plasmodium berghei is a parasite that causes complications or damage to the kidneys with elevated values of blood urea nitrogen (BUN) and creatinine in the blood. Tempuyung (Sonchus arvensis L.) leaf extract contains antioxidant compounds in the form of flavonoids which has the potential to reverse kidney damage. This study aimed to determine the effect of various doses of tempuyung leaf extract on kidney histology in mice exposed to Plasmodium berghei. This study used a completely randomized design method with 28 male BALB/c mice aged 6-8 weeks. They were divided into 7 treatment groups, namely KN (Normal Control), K+ (received 10mg/kg BW (bodyweight) of chloroquine diphosphate solution), K- (infected with P. berghei only), P1 (received 1 mg/kg BW of S. arvensis), P2 (10mg/kg BW of S. arvensis), P3 (received S. arvensis 100mg/kg BW), P4 (received S. arvensis 200mg/kg BW). Except in KN treatment group, all other treatment groups were infected with P. berghei .prior to oral administration of S. arvensis. The S. arvensis treatment was carried out for 7 days after which the blood serum was taken intracardially, and the kidneys were removed for histological studies. The results showed that tempuyung leaf extract had a significant effect on the repair of glomerular cells that experienced hydropic degeneration and necrosis but has no significant effect on glomerular atrophy. Thus, tempuyung leaves have the potential to reverse kidney complications caused by malaria.

Crosstalk between autophagy inhibitor and salidroside-induced apoptosis: A novel strategy for autophagy-based treatment of hepatocellular cancer

Autophagy regulates many cell function related to cancer, including cell proliferation, invasion and apoptosis. Therefore, we investigated the potential value of crosstalk between autophagy and apoptosis. The present study demonstrated that seven autophagy related genes were screened from the biological network of salidroside (Sal) acting on liver cancer. The GO analysis showed that these genes were mainly involved in apoptosis and autophagy. The KEGG analysis showed that these genes regulated the process of liver cancer through Th17 cell differentiation, PI3K-Akt signaling pathway and other pathways. Moreover, seven genes were positively correlated with tumor purity, number of B cells, number of CD4+ T cells, number of CD8+ T cells, number of macrophages, number of dendritic cells and number of neutrophils. The overall survival time of liver cancer patients in the high expression group of BIRC5, HSP90AB1 and MTOR was lower than that in the low expression group (P < 0.05), while the overall survival time of the liver cancer patients in the high expression group of DLC1 and FOXO1 was higher than that in the low expression group (P < 0.05). In the pan-cancer analysis, we also found that BIRC5, HSP90AB1, MTOR, and ITGA6 were highly expressed in various cancers, while DLC1, FOXO1, and FOS were low expressed in various cancers. In the molecule docking analysis, we found that FOS, HSP90AB1, and MTOR had the best binding ability. Notably, in the vitro validation experiments, Sal was confirmed to induce autophagy and apoptosis, inhibite invasion and metastasis of liver cancer cells through the PI3K/Akt/mTOR signaling pathway. Meanwhile, inhibition of autophagy by chloroquine diphosphate (CQ) promoted Sal-induced mitochondrial apoptosis via corresponding cell and animal experiments. We speculated that Sal-induced autophagy might be a protective mechanism, inhibition of autophagy could further promote the progression of liver cancer. It may provide important insight into the molecular mechanism of crosstalk between autophagy and apoptosis, and provide a new theoretical basis of Sal combined with autophagy inhibitors as a adjuvant chemotherapeutic strategy for human liver cancer.

Response mechanisms to acid stress promote LF82 replication in macrophages.

Adherent-invasive E. coli (AIEC) LF82 is capable of adhering to and invading intestinal epithelial cells, as well as replicating within macrophages without inducing host cell death. We compared the transcriptomics of LF82 at pH=7.5 and pH=5.8 by RNA-sequencing, and qRT-PCR verified differentially expressed genes (DEGs). The deletion mutants of DEGs in the treatment group (pH=5.8) compared to the control group (pH=7.5) were constructed by λ recombinant. The replication differences between the mutants and WT infected Raw 264.7 at 24 h.p.i were analyzed by combining LB solid plate count and confocal observation. NH4Cl and chloroquine diphosphate (CQ) were used for acid neutralization to study the effect of pH on the replication of LF82 in macrophages. Na2NO3 was added to RPMI 1640 to study the effect of nitrate on the replication of LF82 in macrophages. 0.3% solid LB was used for flagellar motility assay and Hela was used to study flagellar gene deletion mutants and WT adhesion and invasion ability. In this study, we found that infection with LF82 results in acidification of macrophages. Subsequent experiments demonstrated that an intracellular acidic environment is necessary for LF82 replication. Transcriptome and phenotypic analysis showed that high expression of acid shock genes and acid fitness genes promotes LF82 replication in macrophages. Further, we found that the replication of LF82 in macrophages was increased under nitrate treatment, and nitrogen metabolism genes of LF82 were upregulated in acid treatment. The replication in macrophages of ΔnarK, ΔnarXL, ΔnarP, and Δhmp were decreased. In addition, we found that the expression of flagellar genes was downregulated in acidic pH and after LF82 invading macrophages. Motility assay shows that the movement of LF82 on an acidic semisolid agar plate was limited. Further results showed that ΔfliC and ΔfliD decreased in motility, adhesion ability, and invasion of host cells, but no significant effect on replication in macrophages was observed. In this study, we simulated the acidic environment in macrophages, combined with transcriptome technology, and explained from the genetic level that LF82 promotes replication by activating its acid shock and fitness system, enhancing nitrate utilization, and inhibiting flagellar function.

Chloroquine attenuates diet-induced obesity and glucose intolerance through a mechanism that might involve FGF-21, but not UCP-1-mediated thermogenesis and inhibition of adipocyte autophagy

Chloroquine diphosphate (CQ), a weak base used to inhibit autophagic flux and treat malaria and rheumatoid diseases, has been shown, through unknown mechanisms, to improve glucose and lipid homeostasis in patients and rodents. We investigate herein the molecular mechanisms underlying these CQ beneficial metabolic actions in diet-induced obese mice. For this, C57BL6/J mice fed with either a chow or a high-fat diet (HFD) and uncoupling protein 1 (UCP-1) KO and adipocyte Atg7-deficient mice fed with a HFD were treated or not with CQ (60 mg/kg of body weight/day) during 8 weeks and evaluated for body weight, adiposity, glucose homeostasis and brown and white adipose tissues (BAT and WAT) UCP-1 content. CQ reduced body weight gain and adipose tissue and liver masses in mice fed with a HFD, without altering food intake, oxygen consumption, respiratory exchange ratio, spontaneous motor activity and feces caloric content. CQ attenuated the insulin intolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia and hypercholesterolemia induced by HFD intake, such effects that were associated with increases in serum and liver fibroblast growth factor 21 (FGF-21) and BAT and WAT UCP-1 content. Interestingly, CQ beneficial metabolic actions of reducing body weight and adiposity and improving glucose homeostasis were preserved in HFD-fed UCP-1 KO and adipocyte Atg7 deficient mice. CQ reduces body weight gain and adiposity and improves glucose homeostasis in diet-induced obese mice through mechanisms that might involve FGF-21, but not UCP1-mediated nonshivering thermogenesis or inhibition of adipocyte autophagy.

Synthesis of Calix[4]resorcinarene Derivatives as Antimalarial Agents through Heme Polymerization Inhibition Assay

Malaria is an endemic disease in tropical countries, including Indonesia, with a high annual mortality rate. Because of that, serious attention shall be given to find new antimalarial agents that are highly active for medical treatment. In this work, we designed and synthesized three calix[4]resorcinarene derivatives and evaluated them as antimalarial agents through in vitro heme polymerization inhibitory assay. The calix[4]resorcinarenes were prepared from resorcinol and corresponding aldehyde derivatives in ethanol media through a cyclo-condensation reaction. The calix[4]resorcinarene products were obtained in 31.1–85.1% yield. The synthesized compounds were subjected to structure elucidation using spectroscopy techniques. The antimalarial activity of calix[4]resorcinarene with aromatic substituent (IC50 = 0.198 mg/mL) was higher than the aliphatic ones (IC50 = 0.282–0.814 mg/mL). It was found that all calix[4]resorcinarenes in this work exhibited stronger antimalarial activity than chloroquine diphosphate as the positive control (IC50 = 1.157 mg/mL). The calix[4]resorcinarenes could interact with hydrogen bonding, thus inhibiting the heme polymerization process. These findings demonstrate that calix[4]resorcinarene derivatives are potential antimalarial agents to be developed for effective medical treatment in the near future.

Toxicological effects of hydroxychloroquine sulfate and chloroquine diphosphate substances on the early-life stages of fish in the COVID-19 pandemic context.

Hydroxychloroquine sulfate (HCQ) and chloroquine diphosphate (CQ) have been used at increased rates to treat COVID-19 but can constitute a potential environmental risk. The objective was to evaluate the toxicity of sublethal concentrations of HCQ and CQ in zebrafish embryos/larvae. The 50% lethal concentrations (LC50) of HCQ and CQ at 96 h post-fertilization (hpf) were calculated by testing various concentrations on 2,160 embryos. The LC50 obtained were 560 and 800 µM for HCQ and CQ, respectively. Next, the embryotoxicity assay was performed, where 1,200 embryos were subjected to sublethal concentrations of HCQ and CQ. The hatching and heart rates were recorded. After euthanasia, photomicrographs of all larvae were taken to measure the total length, pericardial and yolk sac areas. The embryos exposed to sublethal concentrations of HCQ and CQ showed delayed hatching at 72 hpf, as well as an increase in the heart rate, larger pericardial and yolk sac areas, and body malformations at 96 hpf. The findings show that HCQ and CQ are toxic to fish in the early development phases. Understanding the mechanisms of toxicity will help extrapolate the effects of 4-aminoquinoline derivatives when they reach the aquatic environment in the context of the COVID-19 pandemic.

Steroids from Toona sureni-derived Endophytic Fungi Stemphylium sp. MAFF 241962 and Their Heme Polymerization Inhibition Activity

Abstract Antimalarial drug resistance is a major cause of the increasing incidence of malaria worldwide, necessitating an urgent demand for the development of new antimalarial drugs. However, the availability of bioactive natural compounds derived from plants is often limited. To address this issue, continuous exploration of bioactive secondary metabolites from endophytic fungi derived from medicinal plants has been recognized as a viable alternative. Therefore, this research aimed to isolate and characterize three ergosteroids, namely isocyathisterol (1), ergosterol-5,8-peroxide (3), cerevisterol (4), and a phytosterol, β-sitosterol (2), from the rice cultures of endophytic fungus Stemphylium sp. MAFF 241962, derived from Toona sureni. Endophytic fungi species were determined using molecular analysis of the internal transcribed region (ITS) of the ribosomal DNA. After comparing the sequence data to the NCBI database using BLAST, endophytic fungi were identified as Stemphylium sp. 241962 with 100% similarity. The chemical structures were elucidated using spectroscopic methods, including 1D and 2D NMR. Antimalarial activities of compounds 1-4 were evaluated using heme polymerization inhibition activity (HPIA) method. The results showed moderate inhibition activities with IC50 values of 7.70 ± 0.11, 9.48 ± 0.09, 7.88 ± 0.10, and 8.36 ± 0.56 mg/mL, respectively, compared to positive control chloroquine diphosphate with IC50 values of 1.59 ± 0.03 mg/mL. Keywords: Steroid, Stemphylium sp., Toona sureni, Antimalarial activity, Heme polymerization inhibition

Vaspin alleviates pathological cardiac hypertrophy by regulating autophagy-dependent myocardial senescence

Abstract Background Visceral adipose tissue–derived serine protease inhibitor (vaspin), a secretory adipokine, protects against insulin resistance. Recent studies have demonstrated that serum vaspin levels are decreased in patients with coronary artery disease and that vaspin protects against myocardial ischemia-reperfusion injury and atherosclerosis. However, it remains unclear whether vaspin exerts specific effects on pathological cardiac hypertrophy. Methods An in vivo study was conducted using a cardiac hypertrophy model established by subcutaneous injection of isoproterenol (ISO) in C57BL/6 and vaspin-ko mice. Rapamycin was administered intraperitoneally to mice, for further study. H9c2 cells and neonatal rat ventricular myocytes (NRVMs) were treated with ISO to induce hypertrophy. Human vaspin fusion protein, the proteasome inhibitor MG132, and chloroquine diphosphate were used for further mechanistic studies. Results Here, we provide the first evidence that vaspin knockdown results in markedly exaggerated cardiac hypertrophy, fibrosis, and cardiomyocyte senescence in mice treated with ISO. Conversely, the administration of exogenous recombinant human vaspin protected NRVMs in vitro against ISO-induced hypertrophy and senescence. Furthermore, vaspin significantly potentiated the ISO-induced decrease in autophagy. Both rapamycin and chloroquine diphosphate regulated autophagy in vivo and in vitro, respectively, and participated in vaspin-mediated cardioprotection. Moreover, the PI3K-AKT-mTOR pathway plays a critical role in vaspin-mediated autophagy in cardiac tissues and NRVMs. Our data showed that vaspin downregulated the p85 and p110 subunits of PI3K by linking p85 and p110 to NEDD4L-mediated ubiquitination degradation. Conclusion Our results show, for the first time, that vaspin functions as a critical regulator that alleviates pathological cardiac hypertrophy by regulating autophagy-dependent myocardial senescence, providing potential preventive and therapeutic targets for pathological cardiac hypertrophy.

#6827 DI(2-ETHYLHEXYL) PHTHALATE INDUCES RENAL PROXIMAL TUBULAR CELLS TO UNDERGO EPITHELIAL-MESENCHYMAL TRANSITION VIA AHR SIGNALING

Abstract Background and Aims Environmental factors account for the majority of identified risks associated with CKD of uncertain etiology (CKDu). Among the various environmental factors, plasticizer di(2-ethylhexyl) phthalate (DEHP) is widely used in modern life. The exposure to DEHP is extensive but mostly underestimated in the general population. The link between DEHP exposure and renal injuries has been scarcely reported but the underlying mechanisms are largely unknown. This study aimed to examine the mechanisms by which DEHP causes renal tubular injuries. Method Human proximal tubular cells (PTC), HK2 cells, were used as a cellular model to test the central hypothesis of this study. Cells were treated with various concentrations of DEHP at different time points. WST-1 assay was used to examine the impacts of DEHP on cell viability. Bright field microscopy and immunofluorescence were used to analyze the morphological evidence of epithelial-mesenchymal transition (EMT). Western blots were used to investigate the changes in the expression of EMT-related proteins and arylhydrocarbon receptor (AhR) signaling. Inhibitors of AhR signaling were employed to investigate the crucial roles of AhR signaling in DEHP-induced EMT in HK2 cells. Results By using WST-1 assay, we found that DEHP did not affect cell viability when the examined concentration was below 50 μM, which resembled the low-level environmental exposures. However, when the concentration was higher than 100 μM, DEHP reduced the cell viability in a dose-dependent manner. In response to DEHP, HK-2 cells changed from cuboid to spindle shape when cultured with DEHP (25 μM). This morphological evidence became evident when the treatment time was longer than 48 hours. At the protein levels, DEHP resulted in down-regulation of e-cadherin and upregulation of vimentin and α-SMA. DEHP also lead to upregulation of AhR. The inhibitor of AhR signaling, CQDP (chloroquine diphosphate) reversed the DEHP-induced EMT, which were evidenced by morphology and by the expression patterns of EMT-related proteins. Conclusion At cellular level, our results suggested that low-level environmental exposures of DEHP led to EMT in renal PTC via AhR signaling.

Ranking the risks of eighty pharmaceuticals in surface water of a megacity: A multilevel optimization strategy

Pharmaceuticals in freshwater posed ecological risks to aquatic ecosystem, however, most risk assessments of pharmaceuticals were conducted at screening level, which were limited by the availability of the toxicity data. In this study, risks of 80 pharmaceuticals including 35 antibiotics, 13 antiviral drugs, 13 illicit drugs, and 19 antidepressants in surface water of Beijing were assessed with a proposed multilevel environmental risk optimization strategy. Target pharmaceuticals were detected in surface water samples with the detection frequency from 1.7 % to 100 % and the total concentrations from 31.1 ng/L to 2708 ng/L. Antiviral drugs were the dominant pharmaceuticals. Preliminary screening-level risk assessment indicated that 20 pharmaceuticals posed low to high risks with risk quotient from 0.14 (chloroquine diphosphate) to 27.8 (clarithromycin). Thirteen pharmaceuticals were recognized with low to high risks by an optimized risk assessment method. Of them, the refined probabilistic risk assessment of joint probability curves coupling with a quantitative structure activity relationship-interspecies correlation estimation (QSAR-ICE) model was applied. Clarithromycin, erythromycin and ofloxacin were identified to pose low risks with maximum risk products (RP) of 1.23 %, 0.41 % and 0.35 %, respectively, while 10 pharmaceuticals posed de minimis risks. Structural equation modeling disclosed that human land use and climate conditions influenced the risks of pharmaceuticals by indirectly influencing the concentrations of pharmaceuticals. The results indicated that the multilevel strategy coupling with QSAR–ICE model was appropriate and effective for screening priority pollutants, and the strategy can be used to prioritize pharmaceuticals and other emerging contaminants in the aquatic environment.

Autophagy regulates the release of exercise factors and their beneficial effects on spatial memory recall

Exercise promotes learning and memory recall as well as rescues cognitive decline associated with aging. The positive effects of exercise are mediated by circulatory factors that predominantly increase Brain Derived Neurotrophic Factor (BDNF) signaling in the hippocampus. Identifying the pathways that regulate the release of the circulatory factors by various tissues during exercise and that mediate hippocampal Mus musculus Bdnf expression will allow us to harness the therapeutic potential of exercise. Here, we report that two weeks of voluntary exercise in male mice activates autophagy in the hippocampus by increasing LC3B protein levels (p = 0.0425) and that autophagy is necessary for exercise-induced spatial learning and memory retention (p < 0.001; exercise + autophagy inhibitor chloroquine CQ versus exercise). We place autophagy downstream of hippocampal BDNF signaling and identify a positive feedback activation between the pathways. We also assess whether the modulation of autophagy outside the nervous system is involved in mediating exercise's effect on learning and memory recall. Indeed, plasma collected from young exercise mice promote spatial learning (p = 0.0446; exercise versus sedentary plasma) and memory retention in aged inactive mice (p = 0.0303; exercise versus sedentary plasma), whereas plasma collected from young exercise mice that received the autophagy inhibitor chloroquine diphosphate failed to do so. We show that the release of exercise factors that reverse the symptoms of aging into the circulation is dependent on the activation of autophagy in young animals. Indeed, we show that the release of the exercise factor, beta-hydroxybutyrate (DBHB), into the circulation, is autophagy-dependent and that DBHB promotes spatial learning and memory formation (p = 0.0005) by inducing hippocampal autophagy (p = 0.0479). These results implicate autophagy in peripheral tissues and in the hippocampus in mediating the effects of exercise on learning and memory recall and identify DBHB as a candidate endogenous exercise factor whose release and positive effects are autophagy-dependent.

Sensitive imaging of Endoplasmic reticulum (ER) autophagy with an acidity-reporting ER-Tracker

ABSTRACT Macroautophagic/autophagic turnover of endoplasmic reticulum (reticulophagy) is critical for cell health. Herein we reported a sensitive fluorescence-on imaging of reticulophagy using a small molecule probe (ER-proRed) comprised of green-emissive fluorinated rhodol for ER targeting and nonfluorescent rhodamine-lactam prone to lysosome-triggered red fluorescence. Partitioned in ER to exhibit green fluorescence, ER-proRed gives intense red fluorescence upon co-delivery with ER into acidic lysosomes. Serving as the signal of reticulophagy, the turning on of red fluorescence enables discernment of reticulophagy induced by starvation, varied levels of reticulophagic receptors, and chemical agents such as etoposide and sodium butyrate. These results show ER probes optically activatable in lysosomes, such as ER-proRed, offer a sensitive and simplified tool for studying reticulophagy in biology and diseases. Abbreviations: Baf-A1, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CQ, chloroquine diphosphate; ER, endoplasmic reticulum; FHR, fluorinated hydrophobic rhodol; GFP, green fluorescent protein; Reticulophagy, selective autophagy of ER; RFP, red fluorescent protein; ROX, X-rhodamine; UPR, unfolded protein response

Combination of chloroquine diphosphate and salidroside induces human liver cell apoptosis via regulation of mitochondrial dysfunction and autophagy.

Hepatocellular carcinoma (HCC) is the leading cause of cancer‑associated death in the world. Chemotherapy remains the primary treatment method for HCC. Despite advances in chemotherapy and modalities, recurrence and resistance limit therapeutic success. Salidroside (Sal), a bioactive component extracted from the rhizome of Rhodiolarosea L, exhibits a spectrum of biological activities including antitumor effects. In the present study, it was demonstrated that Sal could induce apoptosis and autophagy of 97H cells by using CCK‑8 assay, transmission electron microscopy (TEM), Hoechst33342 staining, MDC staining, western blotting. Pretreatment with Sal enhanced apoptosis and autophagy via upregulation of expression levels of Bax, Caspase‑3, Caspase‑9, light chain (LC)3‑II and Beclin‑1 proteins and downregulation of expression levels of Bcl‑2, LC3‑I and p62 protein in 97H cells. Furthermore, Sal was demonstrated to inhibit activation of the PI3K/Akt/mTOR signaling pathway and, when combined with autophagy inhibitor chloroquine diphosphate (CQ), increased phosphorylation of PI3K, Akt and mTOR proteins. The combined treatment with Sal and CQ not only decreased Sal‑induced autophagy, but also accelerated Sal‑induced apoptosis. Therefore, Sal‑induced autophagy might serve a role as a defense mechanism in human liver cancer cells and its inhibition may be a promising strategy for the adjuvant chemotherapy of liver cancer.