Administration Of Chloroquine Research Articles

Possible Involvement of Nitric Oxide in the Antipruritic Effect of Metformin on Chloroquine-Induced Scratching in Mice

Background: Metformin ameliorates non-histamine-mediated itch. We have recently reported that the nitric oxide (NO) pathway is involved in chloroquine (CQ)-induced scratching behavior. Here we investigated the involvement of the NO pathway in the antipruritic effect of metformin on CQ-induced itch. Methods: Metformin (5–200 mg/kg, given intraperitoneally [i.p.]) was injected 4 h before CQ (400 µg/site, given intradermally [i.d.]) or compound 48/80 (100 µg/site, i.d.). A nonspecific nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 1 and 10 mg/kg, i.p.), or an NO precursor, L-arginine (10 and 100 mg/kg, i.p.) was administered 30 min before injection of CQ. A neural NOS (nNOS) inhibitor, 7-nitroindazole (7-NI; 1 and 10 nmol/site, i.d.) was concurrently administered with CQ. The scratching behavior was recorded for 30 min following the injection of CQ. We studied the changes in skin and spinal nitrite levels after treatments. Results: Our results showed that metformin (100 and 200 mg/kg) significantly reduced the CQ-induced scratching behavior but not the compound 48/80-induced scratching behavior. L-Arginine inhibited the antipruritic effect of metformin, while L-NAME and 7-NI significantly potentiated the inhibitory effects of a subeffective dose of metformin on the CQ-induced scratching behavior. The skin but not the spinal nitrite level was significantly increased after CQ administration. The elevated cutaneous nitrite level was reversed by effective doses of either metformin or 7-NI, but not by the subeffective doses of metformin + 7-NI. Conclusion: Acute injection of metformin significantly inhibits CQ-induced scratching behavior. This effect is mediated through inhibition of the NO pathway, especially by inhibiting the dermal nNOS enzyme.

Tongxinluo Ameliorates Myocardial Ischemia-Reperfusion Injury Mainly via Activating Parkin-Mediated Mitophagy and Downregulating Ubiquitin-Proteasome System.

To investigate the protective effects and mechanism of Chinese herbal compound Tongxinluo Capsule (, TXL) on the Parkin-mediated mitophagy and the ubiquitin-proteasome system in a rat model of myocardial ischemia-reperfusion injury (MIRI). Seventy adult male Sprague-Dawley rats were randomly divided into 7 groups: sham group, MIRI group, low- and high-dose TXL (0.5 and 1 g·kg-1·d-1, respectively) groups, atorvastatin (ATV) group (7.2 g·kg-1·d-1), chloroquine (CQ) group (10 g·kg-1·d-1), and highdose TXL + CQ group. After pharmacological administration for 7 days, rats underwent left anterior descending artery ligation surgery to establish the MIRI models with 50 min ischemia followed by 4 h reperfusion. Blood was taken for cardiac troponin I (cTnI) detection and hearts were harvested for infarct staining and apoptosis detection. The autophagy or mitophagy proteins and ubiquitinated proteins were detected by Western blotting. Compared with the sham group, the MIRI group exhibited a larger infarcted area (27.13%±0.01%, P<0.01), a higher apoptotic index (34.33%±2.03% vs.1.81%±0.03%, P<0.01), and higher cTnI expression (14.18±1.01 vs. 7.96±0.32, P<0.01). The mitochondrial integrity was damaged in the MIRI group, while TXL and ATV alleviated the damage of MIRI. More autophagosomes were observed in the high-dose TXL group than in the MIRI group (7.00±0.58 vs. 4.33±1.15, P<0.05). More amounts of PTEN-induced putative kinase protein 1 (PINK1) and Parkin translocated onto the mitochondria were detected in the high-dose TXL group than in the MIRI group (P<0.05). The ubiquitin response was signifificantly downregulated in the high-dose TXL group relative to the MIRI group (P<0.05). CQ administration abolished the activation of autophagy flux and the PINK1/ Parkin pathway induced by high-dose of TXL. TXL ameliorates MIRI via activating Parkin-mediated mitophagy in rats. The downregulation of the ubiquitin-proteasome system is also involved.

The release and activity of HMGB1 in ferroptosis

Damage-associated molecular pattern molecules (DAMPs) are endogenous danger signals that alert the innate immune system and shape the inflammation response to cell death. However, the release and activity of DAMPs in ferroptosis, a recently identified form of regulated necrosis characterized by iron overload and lipid peroxidation, still remain poorly understood. Here, we demonstrate that HMGB1 is a DAMP released by ferroptotic cells in an autophagy-dependent manner. Both type I and II ferroptosis activators, including erastin, sorafenib, RSL3, and FIN56, induce HMGB1 release in cancer and noncancer cells. In contrast, genetic ablation (using ATG5−/− or ATG7−/− cells) or pharmacologic inhibition (the administration of bafilomycin A1 or chloroquine) of autophagy was found to block ferroptosis activator-induced HMGB1 release. Mechanically, autophagy-mediated HDAC inhibition promotes HMGB1 acetylation, resulting in HMGB1 release in ferroptosis. Moreover, AGER, but not TLR4, is required for HMGB1-mediated inflammation in macrophages in response to ferroptotic cells. These studies suggest that HMGB1 inhibition might have some potential therapeutic effects in ferroptosis-associated human disease.

Electroretinogram as an early detection of chloroquine retinal toxicity in pigmented rabbits

Objective Chloroquine (CQ) is a useful drug. It has less systemic hazards than many of the other medications used for immune diseases, but may cause retinopathy. The ophthalmologists do not discover the retinopathy till the appearance of bull’s eye. The aim of the work is to try to use electroretinogram (ERG) and its b/a ratio in early detection of CQ retinopathy for pigmented rabbits chronically treated with CQ. Materials and methods Twenty adult pigmented rabbits (1.5–2 kg) were classified into four groups (five each): the first group served as control and the other three groups of rabbits were intramuscularly injected with CQ diphosphate (14 mg/kg body weight) three times/week for 50, 70, and 90 days, respectively. Each group is subjected to ERG recording, and then decapitation of animal and measurement of oxidant, antioxidant parameters, and histopathological examination in the retinal tissue were done. Results All results show no change by the 50th day after CQ administration. All the changes appear by the 70th day. The data indicated there is a deformation in ERG waves and significant decrease (P Conclusion Our data showed the possibility of using b/a ratio as an early indicator to CQ retinal toxicity in pigmented rabbits. It is also suggested that CQ increases the oxidative stress levels, so supplementation by antioxidants should be a part of the treatment regime.

Cadmium results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells

Environmental exposure to cadmium (Cd) links to neurodegenerative disorders. Autophagy plays an important role in controlling cell survival/death. However, how autophagy contributes to Cd's neurotoxicity remains enigmatic. Here, we show that Cd induced significant increases in autophagosomes with a concomitant elevation of LC3-II and p62 in PC12 cells and primary neurons. Using autophagy inhibitor 3-MA, we demonstrated that Cd-increased autophagosomes contributed to neuronal apoptosis. Impairment of Cd on autophagic flux was evidenced by co-localization of mCherry and GFP tandem-tagged LC3 puncta in the cells. This is further supported by the findings that administration of chloroquine (CQ) potentiated the basic and Cd-elevated LC3-II and p62 levels, autophagosome accumulation and cell apoptosis, whereas rapamycin relieved the effects in the cells in response to Cd. Subsequently, we noticed that Cd evoked the phosphorylation of Akt and BECN1. Silencing BECN1 and especially expression of mutant BECN1 (Ser295A) attenuated Cd-increased autophagosomes and cell death. Of note, inhibition of Akt with Akt inhibitor X, or ectopic expression of dominant negative Akt (dn-Akt), in the presence or absence of 3-MA, significantly alleviated Cd-triggered phosphorylation of Akt and BECN1, autophagosomes, and apoptosis. Importantly, we found that Cd activation of Akt functioned in impairing autophagic flux. Collectively, these results indicate that Cd results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells. Our findings underscore that inhibition of Akt to improve autophagic flux is a promising strategy against Cd-induced neurotoxicity and neurodegeneration.

Chloroquine Restores Ganglioside Homeostasis and Improves Pathological and Behavioral Outcomes Post-stroke in the Rat.

Perturbations of ganglioside homeostasis have been observed following stroke whereby toxic simple gangliosides GM2 and GM3 accumulate, while protective complex species GM1 and GD1 are reduced. Thus, there is a need for therapeutic interventions which can prevent ganglioside dysregulation after stroke. A pharmacological intervention using chloroquine was selected for its transient lysosomotropic properties which disrupt the activity of catabolic ganglioside enzymes. Chloroquine was administered both in vitro (0.1μM), to primary cortical neurons exposed to GM3 toxicity, and in vivo (45mg/kgi.p.), to 3-month-old male Wistar rats that underwent a severe stroke injury. Chloroquine was administered for seven consecutive days beginning 3days prior to the stroke injury. Gangliosides were examined using MALDI imaging mass spectrometry at 3 and 21days after the injury, and motor deficits were examined using the ladder task. Chloroquine treatment prevented ganglioside dysregulation 3days post-stroke and partially prevented complex ganglioside depletion 21days post-stroke. Exogenous GM3 was found to be toxic to primary cortical neurons which was protected by chloroquine treatment. Motor deficits were prevented in the forelimbs of stroke-injured rats with chloroquine treatment and was associated with decreased inflammation, neurodegeneration, and an increase in cell survival at the site of injury. Chloroquine administration prevents ganglioside dysregulation acutely, protects against GM3 toxicity in neurons, and is associated with long-term functional and pathological improvements after stroke in the rat. Therefore, targeting lipid dysregulation using lysosomotropic agents such as chloroquine may represent a novel therapeutic avenue for stroke injuries.

Potential Chronotherapeutic Optimization of Antimalarials in Systemic Lupus Erythematosus: Is Toll-Like Receptor 9 Expression Dependent on the Circadian Cycle in Humans?

Toll-like receptor 9 (TLR9) belongs to the group of endosomal receptors of the innate immune system with the ability to recognize hypomethylated CpG sequences from DNA. There is scarce information about TLR9 expression and its association with the circadian cycle (CC). Different patterns of TLR9 expression are regulated by the CC in mice, with an elevated expression at Zeitgeber time 19 (1:00 a.m.); nevertheless, we still need to corroborate this in humans. In systemic lupus erythematosus (SLE), the inhibitory effect of chloroquine (CQ) on TLR9 is limited. TLR9 activation has been associated with the presence of some autoantibodies: anti-Sm/RNP, anti-histone, anti-Ro, anti-La, and anti-double-stranded DNA. Treatment with CQ for SLE has been proven to be useful, in part by interfering with HLA-antigen coupling and with TLR9 ligand recognition. Studies have shown that TLR9 inhibitors such as antimalarial drugs are able to mask TLR9-binding sites on nucleic acids. The data presented here provide the basic information that could be useful for other clinical researchers to design studies that will have an impact in achieving a chronotherapeutic effect by defining the ideal time for CQ administration in SLE patients, consequently reducing the pathological effects that follow the activation of TLR9.

Therapeutic potential of CPI-613 for targeting tumorous mitochondrial energy metabolism and inhibiting autophagy in clear cell sarcoma.

Clear cell sarcoma (CCS) is an aggressive type of soft tissue tumor that is associated with high rates of metastasis. In the present study, we found that CPI-613, which targets tumorous mitochondrial energy metabolism, induced autophagosome formation followed by lysosome fusion in HS-MM CCS cells in vitro. Interestingly, CPI-613 along with chloroquine, which inhibits the fusion of autophagosomes with lysosomes, significantly induced necrosis of HS-MM CCS cell growth in vitro. Subsequently, we established a murine orthotropic metastatic model of CCS and evaluated the putative suppressive effect of a combination of CPI-613 and chloroquine on CCS progression. Injection of HS-MM into the aponeuroses of the thigh, the most frequently affected site in CCS, resulted in massive metastasis in SCID-beige mice. By contrast, intraperitoneal administration of CPI-613 (25 mg/kg) and chloroquine (50 mg/kg), two days a week for two weeks, significantly decreased tumor growth at the injection site and abolished metastasis. The present results imply the inhibitory effects of a combination of CPI-613 and chloroquine on the progression of CCS.

Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria.

DNA damage accumulates with age (Lombard et al., 2005). However, whether and how robust DNA repair machinery promotes longevity is elusive. Here, we demonstrate that ATM-centered DNA damage response (DDR) progressively declines with senescence and age, while low dose of chloroquine (CQ) activates ATM, promotes DNA damage clearance, rescues age-related metabolic shift, and prolongs replicative lifespan. Molecularly, ATM phosphorylates SIRT6 deacetylase and thus prevents MDM2-mediated ubiquitination and proteasomal degradation. Extra copies of Sirt6 extend lifespan in Atm-/- mice, with restored metabolic homeostasis. Moreover, the treatment with CQ remarkably extends lifespan of Caenorhabditis elegans, but not the ATM-1 mutants. In a progeria mouse model with low DNA repair capacity, long-term administration of CQ ameliorates premature aging features and extends lifespan. Thus, our data highlights a pro-longevity role of ATM, for the first time establishing direct causal links between robust DNA repair machinery and longevity, and providing therapeutic strategy for progeria and age-related metabolic diseases.

Trehalose-Induced Activation of Autophagy Improves Cardiac Remodeling After Myocardial Infarction

BackgroundTrehalose (TRE) is a natural, nonreducing disaccharide synthesized by lower organisms. TRE exhibits an extraordinary ability to protect cells against different kinds of stresses through activation of autophagy. However, the effect of TRE on the heart during stress has never been tested. ObjectivesThis study evaluated the effects of TRE administration in a mouse model of chronic ischemic remodeling. MethodsWild-type (WT) or beclin1+/− mice were subjected to permanent ligation of the left anterior descending artery (LAD) and then treated with either placebo or trehalose (1 mg/g/day intraperitoneally for 48 h, then 2% in the drinking water). After 4 weeks, echocardiographic, hemodynamic, gravimetric, histological, and biochemical analyses were conducted. ResultsTRE reduced left ventricular (LV) dilation and increased ventricular function in mice with LAD ligation compared with placebo. Sucrose, another nonreducing disaccharide, did not exert protective effects during post-infarction LV remodeling. Trehalose administration to mice overexpressing GFP-tagged LC3 significantly increased the number of GFP-LC3 dots, both in the presence and absence of chloroquine administration. TRE also increased cardiac LC3-II levels after 4 weeks following myocardial infarction (MI), indicating that it induced autophagy in the heart in vivo. To evaluate whether TRE exerted beneficial effects through activation of autophagy, trehalose was administered to beclin 1+/− mice. The improvement of LV function, lung congestion, cardiac remodeling, apoptosis, and fibrosis following TRE treatment observed in WT mice were all significantly blunted in beclin 1+/− mice. ConclusionsTRE reduced MI-induced cardiac remodeling and dysfunction through activation of autophagy.

Interactive effects of alcohol and chloroquine on hematologic profile of Wistar rats.

Abstract Background There is paucity of information on the adverse effects of alcohol – chloroquine interaction on hematological parameters. To investigate the effects of concurrent administration of chloroquine and ethanol on hematologic parameters of adult Wistar rats and the ameliorative role of vitamin B12 and folic acid supplementation on any adverse effects. Methods Some 30 adult Wistar rats weighing 120–200 g were assigned to six major groups of five rats each according to their weights. The control group A was fed with normal rat chow and water. The experimental groups B–F were administered with drugs for a period of 7 days as follows: B (chloroquine only); C (ethanol only); D (chloroquine+ethanol); E (chloroquine+ethanol+vitamin B12); and F (chloroquine+ethanol+vitamin B12+folic acid). Blood samples were collected from each animal by cardiac puncture to determine red blood cell (RBC) count, white blood cell (WBC) count, packed cell volume (PCV), hemoglobin (Hb), mean corpuscular hemoglobin concentration, mean cell volume (MCV), and mean cell hemoglobin (MCH). Liver enzymes, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were analyzed to test for liver function. Results Group D indicated lower PCV, Hb, MCV, MCH, and greater WBC compared with control. Group E indicated greater (p&lt;0.05) PCV, Hb, and RBC compared with Group D. Greater (p&lt;0.05) MCV and MCH, and lower WBC and RBC were observed in Group F compared with Group D. Furthermore, Group D rats indicated greater (p&lt;0.05) mean AST and ALT compared with Groups A, E, and F. Conclusions Concurrent administration of chloroquine and alcohol indicated adverse effects on hematologic parameters of Wistar rats. The administration of vitamin B12 and folic acid appeared to ameliorate these adverse effects.

Antioxidant role of autophagy in maintaining the integrity of glomerular capillaries

ABSTRACT Autophagy is a lysosomal degradation system by which cytosolic materials and damaged organelles are broken down into basic components. To explore the physiological role of autophagy in glomerular endothelial cells (GEnCs), we compared the autophagic flux among cells in the kidney under starvation. Inhibition of autophagy by chloroquine administration significantly increased the number of autophagosomes or autolysosomes in GEnCs and proximal tubular cells, but not in podocytes, suggesting that the GEnCs exhibit substantial autophagic activity. Next, we analyzed endothelial and hematopoietic cell-specific atg5-deficient mice (atg5-conditional KO [cKO] mice). Glomeruli of 4-wk-old atg5-cKO mice exhibited slightly distended capillary loops accompanied by an accumulation of reactive oxygen species (ROS). Glomeruli of 8-wk-old atg5-cKO mice showed a lobular pattern with thickening of the capillary loops and mesangial matrix expansion; however, the vasculature of other organs was preserved. The atg5-cKO mice died by 12 wk of age, presumably due to pancytopenia resulting from the defect in their hematopoietic lineages. Therefore, we subjected 4-wk atg5-cKO mice to irradiation followed by bone marrow transplantation from normal littermates. Transplanted mice recapitulated the glomerular phenotypes of the atg5-cKO mice with no obvious histological changes in other organs. Twelve-mo-old transplanted mice developed mesangiolysis and glomerulosclerosis with significant deterioration of kidney function. Administration of N-acetyl-l-cysteine, a ROS scavenger, to atg5-cKO mice rescued the glomerular phenotypes. These data suggest that endothelial autophagy protects glomeruli from oxidative stress and maintains the integrity of glomerular capillaries. Enhancing endothelial autophagy may provide a novel therapeutic approach to minimizing glomerular diseases.

Effects of concurrent chloroquine and ethanol administration on the rat kidney morphology.

IntroductionThe use of antimalarial chloroquine in malaria-endemic regions of Africa is rampant and it is not uncommon to find individuals taken the drug concurrent with alcohol. Effects of anti-malarial drug chloroquine (Chq) and ethanol (Et) combination on kidney volume and function using rat model was investigated.Methods32 adult male rats were randomly distributed into four groups of 8 rats each. Group C serve as control and received vehicle only, while Q is Chq treated only, E is Et treated and QE is Et and Chq treated. Chq was administered intraperitoneally at 1mg/100g body weight weekly and 6% Et in drinking water provided ad libitum. Urine volume was collected before the treatment began and after the treatment. After eight weeks, all animals were euthanized; kidneys were harvested and fixed in 10% neutral formalin. The fixed left kidneys were scanned with computed tomography and the scan slices were used to estimate 3-dimensional kidney volume on ImageJ.ResultsTotal kidney volume was none significantly increased in Q, E and QE treated compared to control groups (p = 0.5150). Also, microscopic analysis showed increased proximal tubule diameter (p = 0.1426) and epithelial hypertrophy (p = 0.2530) and significant urinary space shrinkage (p = 0.00001). The initial urine volume was not significantly different between the control and treated groups (p = 0.9864) however, following treatment urine volume was significantly reduced in QE rats group (p = 0.0029).ConclusionThe results suggest chloroquine and ethanol combination as potential cause of kidney injury through structural damage and function derangement.

Comparative approaches between quantitative-MSI and quantitative-WBA: Application to chloroquine administration in a Long-Evans male rat model

Comparative approaches between quantitative-MSI and quantitative-WBA: Application to chloroquine administration in a Long-Evans male rat model

Autophagy regulates TGF-β2-induced epithelial-mesenchymal transition in human retinal pigment epithelium cells.

Transforming growth factor (TGF)-β2-induced epithelial-mesenchymal transition (EMT) in human retinal pigment epithelium (RPE) cells has an important role in the pathophysiology of intraocular fibrotic disorders, which may cause vision impairment and blindness. Autophagy, an intracellular homeostatic pathway, contributes to the physiological and pathological processes of RPE. Furthermore, autophagy has previously been reported to function in the EMT process in numerous tissue and cell types. However, the association between autophagy and the EMT process in RPE cells has not yet been fully determined. The present study demonstrated that TGF-β2-treated human RPE cells (ARPE-19 cell line) exhibited a significantly increased autophagic flux compared with control cells, as determined by western blot analysis of the protein levels of microtubule-associated protein 1 light chain 3-II and p62 (also termed sequestosome 1). Furthermore, it was demonstrated that autophagy activation enhanced the TGF-β2-induced EMT process in ARPE-19 cells, and inhibition of autophagy by chloroquine administration attenuated TGF-β2-induced EMT, which was determined by analyzing the expression of mesenchymal and epithelial markers by reverse transcription-quantitative polymerase chain reaction and/or western blotting. A transwell migration and invasion assays was also performed that demonstrated that autophagy activation by rapamycin enhanced TGF-β2-stimulated RPE cell migration and invasion, and inhibition of autophagy reduced TGF-β2-stimulated RPE cell migration and invasion. These results also demonstrated that autophagy activation enhanced the TGF-β2-induced EMT process in ARPE-19 cells, and inhibition of autophagy attenuated TGF-β2-induced EMT. Overall, the results of the present study demonstrated that TGF-β2-induced EMT may be regulated by autophagy, thus indicating that autophagy may serve as a potential therapeutic target for the attenuation of EMT in intraocular fibrotic disorders.

Impaired Protein Quality Control During Left Ventricular Remodeling in Mice With Cardiac Restricted Overexpression of Tumor Necrosis Factor.

Sustained inflammation in the heart is sufficient to provoke left ventricular dysfunction and left ventricular remodeling. Although inflammation has been linked to many of the biological changes responsible for adverse left ventricular remodeling, the relationship between inflammation and protein quality control in the heart is not well understood. To study the relationship between chronic inflammation and protein quality control, we used a mouse model of dilated cardiomyopathy driven by cardiac restricted overexpression of TNF (tumor necrosis factor; Myh6-sTNF). Myh6-sTNF mice develop protein aggregates containing ubiquitin-tagged proteins within cardiac myocytes related to proteasome dysfunction and impaired autophagy. The 26S proteasome was dysfunctional despite normal function of the core 20S subunit. We found an accumulation of autophagy substrates in Myh6-sTNF mice, which were also seen in tissue from patients with end-stage heart failure. Moreover, there was evidence of impaired autophagosome clearance after chloroquine administration in these mice indicative of impaired autophagic flux. Finally, there was increased mammalian target of rapamycin complex 1 (mTORC1) activation, which has been linked to inhibition of both the proteasome and autophagy. Myh6-sTNF mice with sustained inflammatory signaling develop proteasome dysfunction and impaired autophagic flux that is associated with enhanced mTORC1 activation.

Autophagy regulates proliferation and biliary differentiation of hepatic oval cells via the MAPK/ERK signaling pathway.

Hepatic oval cells (HOCs) are thought to possess self‑renewal ability and a bipotential capacity for differentiation, which allows them to differentiate into hepatocytes and cholangiocytes. Autophagy serves an important role in self‑renewal and differentiation of stem cells; however, how autophagy contributes to proliferation and differentiation of hepatic progenitor cells has yet to be elucidated. In the present study, autophagy was regulated by rapamycin (Rapa) and chloroquine (Chlo) administration. The results demonstrated that Chlo‑treated HOCs exhibited decreased autophagic activity alongside a decreased tendency to proliferate, as determined by Cell Counting Kit‑8. In addition, activation of autophagy by Rapa enhanced the biliary differentiation of HOCs. Furthermore, increased phosphorylated (p)‑extracellular signal‑regulated kinase (ERK)/p‑p38 expression was observed following the induction of autophagy, thus indicating that the mitogen‑activated protein kinase (MAPK)/ERK signaling pathway was activated by autophagy to exert effects on the stimulation of HOC proliferation and differentiation. In conclusion, the present study demonstrated that autophagy regulates proliferation and biliary differentiation of HOCs via the MAPK/ERK signaling pathway. These results suggest a role for autophagy in stimulating the proliferation and differentiation of HOCs.

Repurposing of the anti-malaria drug chloroquine for Zika Virus treatment and prophylaxis

One of the major challenges of the current Zika virus (ZIKV) epidemic is to prevent congenital foetal abnormalities, including microcephaly, following ZIKV infection of pregnant women. Given the urgent need for ZIKV prophylaxis and treatment, repurposing of approved drugs appears to be a viable and immediate solution. We demonstrate that the common anti-malaria drug chloroquine (CQ) extends the lifespan of ZIKV-infected interferon signalling-deficient AG129 mice. However, the severity of ZIKV infection in these mice precludes the study of foetal (vertical) viral transmission. Here, we show that interferon signalling-competent SJL mice support chronic ZIKV infection. Infected dams and sires are both able to transmit ZIKV to the offspring, making this an ideal model for in vivo validation of compounds shown to suppress ZIKV in cell culture. Administration of CQ to ZIKV-infected pregnant SJL mice during mid-late gestation significantly attenuated vertical transmission, reducing the ZIKV load in the foetal brain more than 20-fold. Given the limited side effects of CQ, its lack of contraindications in pregnant women, and its worldwide availability and low cost, we suggest that CQ could be considered for the treatment and prophylaxis of ZIKV.

Macrophage roles in the clearance of apoptotic cells and control of inflammation in the prostate gland after castration.

Androgen deprivation results in massive apoptosis in the prostate gland. Macrophages are actively engaged in phagocytosing epithelial cell corpses. However, it is unknown whether microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis (LAP) is involved and contribute to prevent inflammation. Flow cytometry, RT-PCR and immunohistochemistry were used to characterize the macrophage subpopulation residing in the epithelial layer of the rat ventral prostate (VP) after castration. Stereology was employed to determine variations in the number of ED1 and ED2. Mice were treated with either chloroquine or L-asparagine to block autophagy. M1 (iNOS-positive) and M2 macrophages (MRC1+ and ARG1+) were not found in the epithelium at day 5 after castration. The percentage of CD68+ (ED1) and CD163+ (ED2) phenotypes increased after castration but only CD68+ cells were present in the epithelium. RT-PCR showed increased content of the autophagy markers Bcl1 and LC3 after castration. In addition, immunohistochemistry showed the presence of LC3+ and ATG5+ cells in the epithelium. Double immunohistochemistry showed these cells to be CD68+ /LC3+ , compatible with the LAP phenotype. LC3+ cells accumulate significantly after castration. Chloroquine and L-asparagine administration caused inflammation of the glands at day 5 after castration. CD68+ macrophages phagocytose apoptotic cell corpses and activate the LAP pathway, thereby contributing to the preservation of a non-inflammed microenvironment. Marked inflammation was detected when autophagy blockers were administered to castrated animals.

Lipopolysaccharide mediates hepatic stellate cell activation by regulating autophagy and retinoic acid signaling

ABSTRACTBacterial translocation and lipopolysaccharide (LPS) leakage occur at a very early stage of liver fibrosis in animal models. We studied the role of LPS in hepatic stellate cell (HSC) activation and the underlying mechanisms in vitro and in vivo. Herein, we demonstrated that LPS treatment led to a dramatic increase in autophagosome formation and autophagic flux in LX-2 cells and HSCs, which was mediated through the AKT-MTOR and AMPK-ULK1 pathway. LPS significantly decreased the lipid content, including the lipid droplet (LD) number and lipid staining area in HSCs; pretreatment with macroautophagy/autophagy inhibitors or silencing ATG5 attenuated this decrease. Furthermore, lipophagy was induced by LPS through the autophagy-lysosomal pathway in LX-2 cells and HSCs. Additionally, LPS-induced autophagy further reduced retinoic acid (RA) signaling, as demonstrated by a decrease in the intracellular RA level and Rar target genes, resulting in the downregulation of Bambi and promoting the sensitization of the HSC's fibrosis response to TGFB. Compared with CCl4 injection alone, CCl4 plus LPS injection exaggerated liver fibrosis in mice, as demonstrated by increased Col1a1 (collagen, type I, α 1), Acta2, Tgfb and Timp1 mRNA expression, ACTA2/α-SMA and COL1A1 protein expression, and Sirius Red staining area, which could be attenuated by injection of an autophagy inhibitor. LPS also reduced lipid content in HSCs in vivo, with this change being attenuated by chloroquine (CQ) administration. In conclusion, LPS-induced autophagy resulted in LD loss, RA signaling dysfunction, and downregulation of the TGFB pseudoreceptor Bambi, thus sensitizing HSCs to TGFB signaling.