Injection Of Chloroquine Research Articles

Chloroquine enhances cisplatin-induced apoptosis of nasopharyngeal carcinoma cells by inhibiting autophagy via upregulating miR129

To investigate the role of miR129 in mediating the effect of chloroquine to enhance cisplatin- induced apoptosis in nasopharyngeal carcinoma cells (HNE1). MTT assay was used to detect the viability of HNE1 cells treated with different concentrations of cisplatin. Colony formation of HNE1 cells treated with cisplatin and chloroquine, alone or in combination, was observed using crystal violet staining. BALB/C unde mice were inoculated with HNE1 cells and randomly divided into 4 groups with 6 mice in each group. The mice received intraperitoneal injections of cisplatin and chloroquine, alone or in combination once every 3 days for 4 consecutive weeks, and the tumor growth was observed in each group. The expression of miR129 in HNE1 cells treated with chloroquine, cisplatin, or both was detected with qPCR. The effects of miR129 suppression with a miR129 inhibitor on the expressions of autophagy related proteins p62, LC3B, Beclin1 and the drug-resistant related protein P-glycoprotein (P-gp) were examined using Western blotting in HNE1 cells treated with chloroquine, cisplatin, or both; the changes in cell apoptosis were detected Annexin V/PI double staining. Chloroquine combined with cisplatin significantly inhibited HNE1 cell proliferation in vitro and the growth of HNE1 cell-derived tumor in nude mice as compared with cisplatin alone (P < 0.01). In cultured HNE1 cells, inhibition of the expression of miR129 significantly promoted autophagy and up-regulated P-gp expression (P < 0.01); Chloroquine obviously inhibited cisplatin-induced autophagy and up-regulated the expression of miR129 in HNE1 cells (P < 0.01). Transfection of the cells with the miR129 inhibitor abolished the inhibitory effect of chloroquine on cisplatin-induced autophagy, and significantly increased the cell survival rate (P < 0.05) and lower the cell apoptotic rate (P < 0.01) after combined treatment with chloroquine and cisplatin. Chloroquine enhances the pro-apoptotic effect of cisplatin by up-regulating miR129 to inhibit autophagy and drug resistance in HNE1 cells.

Bulleyaconitine A Inhibits Itch and Itch Sensitization Induced by Histamine and Chloroquine

Itch (pruritus), specifically chronic itch associated with disease conditions, significantly impairs the patient’s quality of life. At present, the mechanisms underlying this aversive experience are still unclear, and the effective treatment of itch is largely unmet. Here, we report that intragastrical administration of bulleyaconitine A (BLA), which has been used for treating chronic pain for 30 years in China, inhibited itch-like behaviors induced by intradermal injection of histamine and chloroquine in mice and rats, dose-dependently. We found that a single application of the pruritic agents at the skin region innervated by the sural nerve induced long-term potentiation (LTP) of C-fiber field potentials evoked by the stimulation of the same nerve in the spinal dorsal horn of rats. The spinal LTP was remarkably reversed by the spinal application of either BLA or gastrin-releasing peptide receptor (GRPR) antagonist (PD176252). The effect of PD176252 was completely occluded by BLA, while the effect of BLA was only partially occluded by PD176252. Repetitive injection (daily, for four days) of either histamine or chloroquine in the back of the neck enhanced scratching behaviors progressively, and the itch sensitization persisted for at least one week after the discontinuation of the injections. The behavioral change was accompanied with the potentiation of C-fiber synaptic transmission in the dorsal horn. Both the itch sensitization and synaptic potentiation were substantially attenuated by intragastrical BLA. Together, BLA was effective in inhibiting histamine-dependent and histamine-independent itches, and the mechanisms underlying these effects were involved but not limited to the inhibition of gastrin-releasing peptide (GRP)–GRPR signaling in the spinal dorsal horn.

Characterization of a chloroquine‐induced canine model of pruritus and skin inflammation

Chloroquine (CQ) is a prototypical systemic and intradermal pruritogen for histamine-independent (nonhistaminergic) itch in mice and humans. The predictive validity of this model is poorly documented in dogs. To determine pruritogenic and inflammatory effects of systemic and i.d. CQ injections in healthy dogs. Ten healthy purpose-bred laboratory beagles. All dogs were randomized to receive i.d. (200 and 400µg/site), intravenous (2mg/kg) and subcutaneous (3mg/kg) CQ injections. Dogs were video-recorded for 30min after i.d. injections and for 300min after i.v. and s.c. injections. Buffered saline injections served as controls for each route. Global wheal scores were evaluated at 30min post-i.d. injection by a blinded investigator. All dogs showed wheal and erythema at the CQ i.d. injection sites; global wheal scores of each CQ concentration were significantly increased compared to placebo (P≤0.05). Blinded evaluation revealed no significant increase in generalized pruritic behaviour (pruritic seconds) after i.v. or s.c. administration of CQ. Intradermal injections induced mild localized acute pruritic behaviours at the site of injections at 200µg (P=0.06) and 400µg (P=0.27) CQ in dogs. To the best of the authors' knowledge, this is the first report which shows that i.d. CQ injections may induce acute inflammation in healthy dogs. By contrast to the systemic CQ-induced pruritus reported previously in healthy mice and dogs, no significant pruritic behaviours were observed after CQ injection, regardless of the route of administration.

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.

Curcumin promotes functional recovery and inhibits neuronal apoptosis after spinal cord injury through the modulation of autophagy

Objective: The study was aimed to investigate whether the neuroprotective role of curcumin is associated with regulation of autophagy. Methods: Rat spinal cord injury (SCI) models were established according to Allen’s weight-drop trauma method. Curcumin was administered 30 min after the contusion and continued weekly. At 3, 7, 14, 21, and 28 days after SCI, functional recovery was evaluated using the Basso, Beattie and Bresnahan (BBB) scoring and the oblique plate test, following which, spinal cord tissues were obtained. Histological changes and apoptosis were then measured with H&E staining and TUNEL assay. Glia activation, inflammatory infiltration, inflammatory factor release, and myelination were observed through immunohistochemical (IHC) staining and ELISA. Autophagy and Akt activation were detected by western blotting. After autophagy was inhibited by injection of chloroquine, TUNEL, inflammatory factor release, myelin basic protein (MBP) IHC staining and functional recovery evaluation were performed again. Results: Curcumin treatment promoted functional recovery after SCI and reduced neuron apoptosis, improved spinal cord integrity, recovery, and re-myelination, and suppressed the inflammatory response. Autophagy was enhanced and Akt/mTOR pathway was inhibited by curcumin. Autophagy inhibition partially eliminated the protective effect of curcumin on SCI. Conclusion: Curcumin may exert its therapeutic effect on SCI through the enhancement of autophagy, in which, inhibition of the Akt/mTOR signaling pathway may be also involved.

The role of autophagy in the pathogenesis of exposure keratitis.

Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure‐induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3‐methyladenine (3‐MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy‐related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy‐related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress‐related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway‐related proteins. Real‐time quantitative PCR (qRT‐PCR) determined IL‐1β, IL‐6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure‐induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress‐induced autophagy promoted cell survival. Taken together, air exposure‐induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.

Serotoninergic projection from dorsal raphe nucleus to insular cortex is involved in acute itch sensation processing in mice

Previous studies have demonstrated that both dorsal raphe nucleus (DR) and insular cortex (IC) are critical for somatic sensory information transmission and regulation, especially for pain, and neurons in the DR send projection fibers to the IC. However, whether these ascending connections are involved in the processing of itch sensation remains unknown. In order to provide evidence for that, fluoro-gold (FG) retrograde tracing combined with immunofluorescent histochemical staining was performed for revealing the chemical nature of the projection neurons and the FOS expression induced by acute itch stimulation via intradermal histamine or chloroquine injection in the mouse. Both FOS- and p-ERK-containing neurons were increased in the DR and IC in the acute itch mice compared to those in the sham group. After FG was injected into the IC, FG-labeled retrograde neuronal cell bodies were observed in the whole extent of the brainstem, especially in the DR. About 81% of the total number of FG-labeled neurons in the DR showed serotonin (5-HT)-immunopositive staining. About 32% FG-labeled 5-HT-ergic neurons within DR expressed FOS in chroloquine-induced acute itch, whereas only 6% FG-labeled 5-HT-ergic neurons within DR expressed FOS in histamine-induced acute itch. These results provide morphological evidence for that there are 5-HT-ergic projections from the DR to IC which might be involved in the sensory information processing of acute itch. These results are helpful for understanding functional roles of 5-HT-ergic ascending projection under the condition of acute itch.

PO-123 Voluntary wheel training attenuate brain aging in SAMP8 mice

Objective Alzheimer's disease (AD), is a progressive neurodegenerative disease. The pathological hallmarks including the deposition of senile plaque and neurofibrillary tangles (NFTs) in AD. The underlying molecular mechanisms for AD are not fully understood. Studies have shown that exercise as an economical and popular fitness method, long-term exercise can reduce the risk of AD, delay the occurrence of AD or delay the progression of AD. This study was to investigate the protective effect of the Voluntary wheel on cerebral cortex senescence in SAMP8 mice.&#x0D; Methods Fifteen SPF-class 7-month-old SAMP8 mice were randomly divided into control group (CON), voluntary wheel group (V) and voluntary wheel with chloroquine group (VQ), with 5 mice in each group. Each group was under adaptive feeding for one week. After 3 days of adaptive voluntary wheel training for V and VQ groups, the experiment officially started. The V and VQ groups performed an 8-week voluntary wheel training and the VQ group received a daily intraperitoneal injection of chloroquine (40 mg/kg). After 8 weeks intervention, the Morris water maze test was used to determine the changes in spatial learning and memory ability of each group. After 24 hours of the end of this experiment, the mice were killed by breaking neck. The degree of hippocampal cell injury was detected by Nissl staining; the expression of apoptosis and senescence-associated protein in the cerebral cortex was detected by Western blot.&#x0D; Results Compared with the CON group, the escape latency of the V group mice in the Morris water maze test was significantly shorter (P&lt;0.01), the number of crossing target quadrants was significantly increased (P&lt;0.05), however, the VQ group exacerbate above response compared with CON group. Nissl staining showed that the cells of cerebral cortex in the CON group had obvious damage, while the V group showed significant improvement. The VQ group had a significant damage compared with the V group; Western blot results showed that the expression of AD -like pathological changes such as BACE1 protein in the cortex of group V was significantly decreased, and the expression of P-GSK-3β protein was increased (P&lt;0.01) compared with CON group. On the contrary, compared with V group, VQ alleviated the improvement effect of voluntary running. (P&lt;0.05); the expression of apoptosis-related protein increased, and thedecreased in CON group , while voluntary running intervention down-regulated BAX, Cleaved-caspase3 , increased the Bcl-2 protein level compared with the CON group (P&lt;0.05). Furthermore, compared with the CON group, the expression level of aging-related protein AC-P53 was decreased in the V group, and the expression level of Sirt1 was increased. &#x0D; Conclusions The 8-week voluntary wheel training can significantly improve the learning and memory ability of SAMP8. Mechanismly, voluntary wheel training can improve the senescence of cerebral cortex by inhibiting cell apoptosis, the expression of AD –like pathological protein and increasing Sirt1 protein level.

The behavioral study on the interactive aggravation between pruritus and depression.

BackgroundThe interactive aggravation of pruritus and depression is well‐known, but an appropriate experimental model that could mimic this behavioral phenomenon is still lacking. Thus, a systematic animal behavioral investigation was carried out in this study. This will promote the research and treatment of pruritus and depression.MethodsThe 2,4‐dinitrofluorobenzene (DNFB)‐induced chronic itch model was established to measure the depression index by forced swimming test (FST), tail suspension test (TST), and splash test (ST). The chronic unpredicted mild stress (CUMS)‐induced depression model was established to measure spontaneous itch and acute histamine or chloroquine‐induced itch behaviors. A depression and itch combining model was also established to measure the scratching and depression behaviors. The motor function of DNFB mice was analyzed by the rotarod test.ResultsThe scratching number, the immobility time in the FST and TST, and the grooming number in the ST test were all significantly increased in the chronic itch model. Mice receiving CUMS treatment showed significantly increased spontaneous scratching number, immobility time in the FST and TST tests, and grooming number in the ST. The combined model showed increased immobility time in FST and TST tests and increased grooming number in ST comparing to the depression model, and showed increased scratching number comparing to the chronic itch model. After histamine (His) or chloroquine (CQ) injection, the scratching numbers of CUMS mice were all significantly increased compared to those of His‐ and CQ‐control, respectively. Anti‐depression drug ketamine could significantly inhibit the depression‐like behaviors of CUMS mice, and simultaneously stopped the promoting effect on His‐induced acute itch.ConclusionsThis study established an appropriate cross aggravation experimental mode and demonstrated that there is cross aggravation between pruritus and depression. The illumination of related mechanisms underlying this cross aggravation effect will provide theoretical basis for the prevention and treatment of depression and pruritus.

Atg7‐Dependent Autophagy Is Essential for Cardiac Function with High Fat Diet.

Heart is a high energy‐demanding tissue. Since 60%–90% energy is from fatty acid oxidation in mitochondria, the regulation of lipid metabolism in the heart is critical. Autophagy, a self‐degradative process, has been reported to be involved in lipid metabolism. Autophagy includes macroautophagy, microautophagy, and chaperone‐mediated autophagy. It has been known that macroautophagy is essential not only for maintaining physiological cardiac function, but also in pathological stressors such as ischemia, and cardiac hypertrophy. However, the molecular pathway through which macroautophagy regulates cardiac lipid metabolism remains unclear. Therefore, it is critical to determine whether macroautophagy is essential in cardiac lipid metabolism.In this study, we evaluated the function of autophagy in cardiac lipid metabolism. First, in order to evaluate the effects of high fat diet (HFD) on cardiac autophagy, cardiac‐specific expression of Tandem fluorescent‐tagged LC3 (mRFP‐EGFP‐LC3)(TF‐LC3) mice were fed either normal diet (ND) or 60% HFD. Through the injection of Chloroquine, an inhibitor of the fusion between autophagosome and lysosome, we evaluated autophagy flux with ND and HFD. In TF‐LC3 mice, autophagic flux was increased with 6 weeks' HFD, but inhibited with 3 months' HFD. Then, we detected mitophagy with HFD. Mitophagy was activated with HFD, as assessed in transgenic mice expressing the mitochondrial‐targeted form of the fluorescent biosensor Keima(Mito‐Keima). Mito‐Keima fluorescence shows pH‐dependent excitation characteristics, shifting excitation maxima to a higher wavelength after mitochondria come into contact with the acidic milieu of lysosomes in the content of mitophagy.Next, to elucidate the role of autophagy in cardiac lipid metabolism, we used genetically altered mouse lines for Atg7, which is responsible for macroautophagy. We generated atg7‐cardiac‐specific knock out (atg7cko) mice. Atg7cko mice were fed HFD for two months. Then through measuring cardiac function by PV‐Loop, atg7cko mice developed severe cardiac systolic (ESPVR=25±2.2 in WT with HF, ESPVR=15±1.3 in atg7cko with HF, p&lt;0.05) and diastolic (EDPVR=0.11±0.02 in WT with HF, EDPVR=0.15±0.01 in atg7cko with HF, p&lt;0.05) dysfunction. Electronic microscope (EM) analysis showed that dysfunctional mitochondria accumulated in atg7cko mice with HFD. Through Tg‐mito‐keima mice crossing with WT or atg7cko mice with ND or HFD, mitophagy was impaired in atg7cko mice with ND and HFD. Mitochondrial membrane potential, mitochondrial oxygen consumption rate (OCR) and fatty acid oxidation (FAO) were decreased in atg7cko mice with HFD. EM analysis and oil red o staining showed lipid droplets were accumulated and Fibrosis and cell death were increased in atg7cko mice during HFD. These results suggested that inhibition of mitophagy and accumulation of dysfunctional mitochondria developed in atg7cko mice with HFD.Taken together, these data indicated that atg7‐dependent autophagy is essential for cardiac function with HFD.Support or Funding InformationNational Institute of Health RO1(1R01HL138720‐01)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pharmacological evidence of involvement of nitric oxide pathway in anti-pruritic effects of sumatriptan in chloroquine-induced scratching in mice.

Chloroquine (CQ) induces histamine-independent itch in human and mice. We recently reported the role of intradermal nitric oxide (NO)/cyclic guanosine monophosphate pathway in CQ-evoked scratching in mice. Chloroquine stimulates neuronal nitric oxide synthase (nNOS) activity to over-producing NO in the skin. Sumatriptan, a 5-hydroxytryptamine 1b/1d receptors (5-HTR1b/1d) agonist, is involved in pain and used to treat migraine and cluster headaches. According to previous studies, sumatriptan inhibits NOS activity. Thus, we aimed to investigate the effect of sumatriptan on CQ-induced scratching. We used the rostral back model of itch. Chloroquine was injected intradermally into the rostral back of NMRI mice, and the scratching behavior was evaluated by measuring the number of bouts over 30 min. We evaluated the effect of sumatriptan and combination of sumatriptan and a non-selective NO synthase inhibitor, L-N-nitro arginine methyl ester (L-NAME), on the scratching behavior. Additionally, the changes of skin, hippocampus, and cortical nitrite level after different treatments were studied. Intraperitoneal and intradermal sumatriptan attenuates CQ-induced itch which reversed by GR-127935, the selective 5-HTR1b and 5-HTR1d antagonist. Co-administration of subeffective doses of sumatriptan and L-NAME significantly decreases the scratching behavior. Intradermal injection of CQ significantly increases the intradermal nitrite levels while it does not have any significant effects on hippocampal or cortical nitrite concentrations. Likewise, the effective doses of intraperitoneal and intradermal sumatriptan significantly reduce intradermal nitrite levels. We concluded that sumatriptan suppresses CQ-induced itch most likely by activating 5-HT1b/1d receptors. This effect probably mediates through NO pathway.

Neuronal aromatase expression in pain processing regions of the medullary and spinal cord dorsal horn.

In both acute and chronic pain conditions, women tend to be more sensitive than men. This sex difference may be regulated by estrogens, such as estradiol, that are synthesized in the spinal cord and brainstem and act locally to influence pain processing. To identify a potential cellular source of local estrogen, here we examined the expression of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Our studies focused on primary afferent neurons and on their central targets in the spinal cord and medulla as well as in the nucleus of the solitary tract, the target of nodose ganglion-derived visceral afferents. Immunohistochemical staining in an aromatase reporter mouse revealed that many neurons in laminae I and V of the spinal cord dorsal horn and caudal spinal trigeminal nucleus and in the nucleus of the solitary tract express aromatase. The great majority of these cells also express inhibitory interneuron markers. We did not find sex differences in aromatase expression and neither the pattern nor the number of neurons changed in a sciatic nerve transection model of neuropathic pain or in the Complete Freund's adjuvant model of inflammatory pain. A few aromatase neurons express Fos after cheek injection of capsaicin, formalin, or chloroquine. In total, given their location, these aromatase neurons are poised to engage nociceptive circuits, whether it is through local estrogen synthesis or inhibitory neurotransmitter release.

Pharmacological evidence for the involvement of adenosine triphosphate sensitive potassium channels in chloroquine-induced itch in mice.

Chloroquine (CQ) evokes itch in human and scratching behavior in rodents through a histamine-independent pathway. Chloroquine directly excites peripheral sensory neurons which convey itch signals to the central nervous system. It has been revealed that ATP-sensitive potassium channels (KATP channels) are important in regulating neuronal excitability. Thus, we aimed to investigate the involvement of KATP channels in CQ-induced itch which may also reveal a linkage between metabolic state of cells and itch. Intradermal (id) injection of CQ at dose of 400μg/site induces the scratching behavior. KATP channel openers, diazoxide (DZX) and minoxidil (MIN), and a KATP channel blocker, glibenclamide (GLI), were administered intraperitoneally (ip) before CQ. Then the behavior was recorded for 30min, in an unmanned condition, and the scratching bouts were counted by an expert observer who was blinded to the experiments. Furthermore, quantitative reverse transcription-PCR (qRT-PCR) was used to investigate the possible changes in dermal expression of Kcnj8 and Kcnj11, the genes encoding the KATP channels. Our results show that either DZX (10mg/kg, ip) or MIN (10mg/kg, ip) significantly attenuated CQ-induced scratching behavior in mice. Moreover, pretreatment with GLI (3mg/kg, ip) significantly reversed the anti-pruritic effects of DZX and MIN. Our finding of qRT-PCR analysis also show that the expression of Kcnj8 is decreased after CQ injection. We suggest that KATP channels are possibly involved in CQ-induced itch. While, further studies will be significant to better elucidate the association of metabolic state of cells and itch.

Beneficial effect and potential molecular mechanism of chloroquine on sperm motility and fertilizing ability in yellow catfish

Sperm motility is a critical determinant of male fertility in vertebrates including fish species. Chloroquine, an autophagy inhibitor, has been reported to own debated influence on sperm quality of mammalians. The objectives of this study were to determine the effect of chloroquine on sperm motility and fertilizing efficiency in yellow catfish, an important freshwater fish species in China. Gradient doses of chloroquine were intraperitoneally injected into yellow catfish that resulted in an increasing expression level of LC3-II and inhibition of autophagy. Subsequently, various sperm parameters were assessed with the computer-assisted sperm analysis (CASA) system. Chloroquine injection significantly increased straight line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP) of sperm compared with the control. Consequently, 1μM chloroquine/g body weight was revealed to be an optimal dose for in vivo treatment, which led to elevation of fertility rate, whereas no effect on the hatching rate. The comparative transcriptome analysis between chloroquine-treated and control testes were conducted in order to figure out the possible molecular mechanism of chloroquine to regulate sperm motility. Interestingly, Gene Ontology (GO) analysis indicated that chloroquine treatment not only inhibited autophagy pathway but also significantly reduced toll-like receptor signaling pathway, suggesting a possible trade-off between male reproduction and immunity. KEGG analysis showed up-regulation of many pathways including PI3K-AKT signaling pathway which has been reported to be correlated with spermatogenesis and sperm maturation in yellow catfish. Our data reveals a potential trade-off between reproductive traits and immune function, and suggests that the application of chloroquine could improve sperm quality and fertilization efficiency in broodstock fish. Statement of relevanceOur research demonstrated that an optimal dose of chloroquine could efficiently inhibit autophagy and improve sperm motility and fertilization efficiency. The comparative transcriptome analysis between chloroquine-treated and control testes were conducted in order to figure out the possible molecular mechanism of chloroquine to regulate sperm mobility.

Analysis of spinal neurotransmitters in histaminergic and non-histaminergic itch

Background: Chronic intractable itch is a profound problem that decreases quality of life and frequently accompanies a variety of inflammatory skin conditions and systemic diseases. The development of antipruritic treatments therefore requires an understanding of the fundamental mechanisms of itch. Objectives: We investigated the roles of substance P (SP), gastrin-releasing peptide (GRP), and glutamate in the spinal neurotransmission of histamine-dependent and -independent itch by using in vivo electrophysiology. Results: In anesthetized mice, responses of single neurons in the superficial dorsal horn to intradermal (i.d.) injection of chloroquine (CQ) were partially reduced by spinal application of the AMPA/kainite antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Co-application of CNQX plus a neurokinin-1 receptor (NK-1R) antagonist produced stronger inhibition, while co-application of CNQX, NK-1R, and GRP receptor (GRPR) antagonists completely inhibited neuronal firing. Neuronal responses elicited by i.d. histamine were abolished by CNQX alone. In behavioral studies, individual intrathecal administration of a GRPR, NK-1, or AMPA antagonist each significantly attenuated CQ-evoked scratching behavior. Co-administration of the NK-1 and AMPA antagonists was more effective, and administration of all 3 antagonists abolished scratching. Intrathecal CNQX alone prevented histamine-evoked scratching behavior. Conclusion: These results suggest that SP, GRP, and glutamate each partially contribute to histamine-independent itch. Meanwhile, histamine-evoked itch is mediated primarily by glutamate. Co-application of NK-1, GRP, and AMPA receptor antagonists may prove beneficial in treating chronic and intractable itch such as atopic dermatitis.

Perception of injections in semi-urban communities in Sokoto, northwest Nigeria

Objective: The hazards associated with injections constitute a serious public health challenge worldwide, especially in the developing countries such as Nigeria. To adequately address this menace, there is a need for a survey to assess the perception of health-care seekers and providers on injections generally to provide a platform upon which targeted intervention can be made. This study aimed at evaluating the semi-urban community perception on injections and awareness of dangers associated with it. Materials and Methods: This was a cross-sectional using validated interviewer administered questionnaires. A total of 102 respondents were recruited using a multi-stage sampling technique from semi-urban area. The questionnaire sought their demographic profiles, awareness of hazards associated with injections use, and perceptions. Results: Out all the respondents, 96.1% had injections in the last 3 months. Only 47.1% seek medical help in government health facilities. Majority considered injections to be more effective than oral drugs [80.4%, odds 4.10 and 95% confidence interval (CI) 2.53-6.65]. Most of the respondents preferred injections (74.5%, odds 2.92 and 95% CI 1.88-4.55). Chloroquine injection was the most cited by injections the respondents (70.6%). And only, 29.4% (odds 0.42 and 95% CI 0.27-0.64) had adverse events following injections. Awareness of hazards associated with injections was not too good as (27.5%, odds 0.38 and 95% CI 0.25-0.58) admitted that injection carries no risks. Conclusion: Awareness on dangers associated with injections was poor with erroneous perception that injections generally were more effective than oral drugs. Chloroquine is still widely used as an anti-malaria drug in private settings despite its ban. Incidence of injection is high in semi-urban communities.

Spinal neurons that contain gastrin-releasing peptide seldom express Fos or phosphorylate extracellular signal-regulated kinases in response to intradermal chloroquine.

BackgroundGastrin-releasing peptide (GRP) is thought to play a role in the itch evoked by intradermal injection of chloroquine. Although some early studies suggested that GRP was expressed in pruriceptive primary afferents, it is now thought that GRP in the spinal cord is derived mainly from a population of excitatory interneurons in lamina II, and it has been suggested that these are involved in the itch pathway. To test this hypothesis, we used the transcription factor Fos and phosphorylation of extracellular signal-regulated kinases (ERK) to look for evidence that interneurons expressing GRP were activated following intradermal injection of chloroquine into the calf, in mice that express enhanced green fluorescent protein (EGFP) in these cells.ResultsInjection of chloroquine resulted in numerous Fos- or phospho-ERK (pERK) positive cells in the somatotopically appropriate part of the superficial dorsal horn. The proportion of all neurons in this region that showed Fos or pERK was 18% and 21%, respectively. However, among the GRP–EGFP, only 7% were Fos-positive and 3% were pERK-positive. As such, GRP–EGFP cells were significantly less likely than other neurons to express Fos or to phosphorylate ERK.ConclusionsBoth expression of Fos and phosphorylation of ERK can be used to identify dorsal horn neurons activated by chloroquine injection. However, these results do not support the hypothesis that interneurons expressing GRP are critical components in the itch pathway.

Single Adult-human Equivalent Dose of Intramuscular Chloroquine did not Lower Blood Glucose Level in Fasted Wistar Rats

Objectives: The 4-Aminoquinoline, Chloroquine is still indicated for treatment of malaria fever due to sensitive Plasmodium species. The hypoglycemic effects of Chloroquine have been sparsely reported in human and animal models of type 2 diabetes and a widely held perception among health caregivers in this part of the world is that administering intramuscularly Chloroquine to fasting malaria patients could be complicated by syncope directly caused by hypoglycemia. This study investigated the effects of a single adult human intramuscular bolus of Chloroquine on the blood glucose level in fasting normoglycemic Wistar rats. Methods: Fifteen acclimatized adult male rats were randomly divided into three groups of 5 per group. Group I and II rats were fasted overnight and received 4.17 mg/kg of intramuscular Chloroquine in the morning. Group III rats were also fasted but received 1ml of sterile water injection. Fasting blood sugar level was determined immediately after injection and at 2 hour and 4 hour intervals using Accucheck glucometer. Results: The results were recorded as Mean ± SEM. The mean sugar levels of the groups were compared with each other within a given time point as well between the different time intervals within a given group using Student’s t test of significance. There were no observable statistical significant differences between the Mean blood glucose levels of the groups. Conclusion: The results of this study suggest that intramuscular Chloroquine injection in fasted normoglycemic rats did not lower significantly lower their blood glucose level and syncope in fasting adult humans immediately following treatment by this route was likely the result of a cardiovascular complication rather than from a Chloroquine-induced hypoglycemic effect.

Anatomical evidence of pruriceptive trigeminothalamic and trigeminoparabrachial projection neurons in mice.

Itch is relayed to higher centers by projection neurons in the spinal and medullary dorsal horn. We employed a double-label method to map the ascending projections of pruriceptive and nociceptive trigeminal and spinal neurons. The retrograde tracer fluorogold (FG) was stereotaxically injected into the right thalamus or lateral parabrachial area (LPb) in mice. Seven days later, mice received intradermal (id) microinjection of histamine, chloroquine, capsaicin, or vehicle into the left cheek. Histamine, chloroquine, and capsaicin intradermally elicited similar distributions of Fos-positive neurons in the medial aspect of the superficial medullary and spinal dorsal horn from the trigeminal subnucleus caudalis to C2. Among neurons retrogradely labeled from the thalamus, 43%, 8%, and 22% were Fos-positive following id histamine, chloroquine, or capsaicin. Among the Fos-positive neurons following pruritic or capsaicin stimuli, ∼1-2% were retrogradely labeled with FG. Trigeminoparabrachial projection neurons exhibited a higher incidence of double labeling in the superficial dorsal horn. Among the neurons retrogradely labeled from LPb, 36%, 29%, and 33% were Fos positive following id injection of histamine, chloroquine, and capsaicin, respectively. Among Fos-positive neurons elicited by id histamine, chloroquine, and capsaicin, respectively, 3.7%, 4.3%, and 4.1% were retrogradely labeled from LPb. The present results indicate that, overall, relatively small subpopulations of pruriceptive and/or nociceptive neurons innervating the cheek project to thalamus or LPb. These results imply that the vast majority of pruritogen- and algogen-responsive spinal neurons are likely to function as interneurons relaying information to projection neurons and/or participating in segmental nocifensive circuits.

High-fat diet increases autophagic flux in pancreatic beta cells in vivo and ex vivo in mice.

Defective beta cell function during lipid oversupply and type 2 diabetes is associated with dysregulation of lysosomal function and autophagy. Whether this dysregulation represents augmentation or inhibition is unclear because of technical limitations in assaying autophagy. The current aim was to determine the effects of high-fat feeding on true autophagic flux in beta cells in vivo in mice, and to establish the relationship between autophagy, endoplasmic reticulum (ER) stress and apoptosis. Green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) mice were fed chow or high-fat diets for 8-10 weeks and injected with 100 mg kg(-1) day(-1) chloroquine for 5 days, prior to being killed, to block clearance of autophagic markers. Pancreases and livers were fixed and GFP-LC3 aggregates or autophagosomes were detected by fluorescence or electron microscopy, respectively. Independently, islets isolated from chow or high-fat-fed mice were treated for 2 h with chloroquine ex vivo, and immunoblotting was performed for markers of autophagy (LC3 lipidation - LC3II and p62/SQSTM1), ER stress (C/EBP homology protein [CHOP], phosphorylated eukaryotic initiation factor 2α [p-eIFα] and inositol requiring enzyme 1α [p-IRE1α]) and apoptosis (cleaved caspase-3). Numbers of autophagosomes and GFP puncta were increased in beta cells by combined high-fat feeding and chloroquine injection, indicative of enhanced autophagic flux. By contrast, GFP puncta were attenuated in liver under the same conditions. Relative to chow-fed controls, islets isolated from fat-fed mice exhibited higher LC3II levels when treated ex vivo with chloroquine. The combination of high-fat feeding and acute chloroquine treatment induced CHOP, p-eIF2α and caspase-3, but not either treatment alone. We provide the first in vivo demonstrations that high-fat feeding increases autophagic flux in pancreatic beta cells, and that this serves to protect against induction of terminal ER stress. We also highlight an approach for monitoring dietary alterations in autophagic flux using ex vivo manipulation of isolated islets.