OBJECTIVE: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout. METHODS: The Sidt2 knockout (Sidt2^-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed. RESULTS: Sidt2^-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2^-/- HL7702 cells. CONCLUSION Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
Humans ; Lysosome-Associated Membrane Glycoproteins/metabolism* ; Autophagy ; Apoptosis ; Hepatocytes ; Lysosomes/metabolism* ; Chloroquine/pharmacology* ; Nucleotide Transport Proteins/metabolism*

Animals ; Chloroquine/pharmacology* ; Longevity ; Rats

OBJECTIVE: To explore the combined pro-apoptosis effect of HSP90 inhibitor BIIB021 and chloroquine (CQ) in chronic myeloid leukemia (CML) cells bearing T315I mutation and its mechanism. METHODS: The p210-T315I cells were divided into 4 groups by different treatment: control, BIIB021, CQ, and BIIB021 + CQ. After treated with BIIB021 or/and CQ for 24 hours, Annexin V/PI binding assay was used to detect apoptosis rates of CML cells. DAPI staining was used to observe nuclear fragmentation, and Western blot was used to detect the expression of caspase 3, PARP (apoptosis related proteins) and p62, LC3-I/II (autophagy related proteins). P210-T315I cells were inoculated subcutaneously into mice and CML mouse models were established. The mice in treatment groups were injected with BIIB021 and/or CQ while mice in control group were treated with PBS and normal saline. The tumor volume of mice was measured every 4 days, and protein level of cleaved-caspase 3 and LC3-II in tumor tissue were detected by immunohistochemistry. RESULTS: The results showed that BIIB021 induced apoptosis of CML cells in a dose-dependent manner ( r=0.91). CQ could enhance the apoptosis-inducing effect of BIIB021. Flow cytometry analysis results showed that the apoptosis rate of p210-T315I cells in combination group was higher than that in BIIB021 or CQ only group (P<0.05). DAPI staining showed nuclear fragmentation in combination group could be observed more obviously. Western blot analysis showed that BIIB021 could induce LC3-I to convert to LC3-II and decrease p62 protein levels (P<0.05). Moreover, the combination group had higher expression of LC3-II, p62 (P<0.05), activated PARP and activated caspase 3 than BIIB021 only group (P<0.05). Besides, experiment in vivo showed the mean tumor volume in co-treatment group was lower than that in single drug group (P<0.01). Immunohistochemistry of tumor tissue also showed the protein level of cleaved-caspase 3 and LC3-II in combined group was higher than that in BIIB021 only group. CONCLUSION HSP90 inhibitor BIIB021 induced significant apoptosis of CML cells bearing T315I both in vivo and in vitro. CQ can enhance this effect probably by autophagy inhibition.
Adenine/analogs & derivatives* ; Animals ; Apoptosis ; Autophagy ; Caspase 3/metabolism* ; Cell Line, Tumor ; Chloroquine/therapeutic use* ; Fusion Proteins, bcr-abl/pharmacology* ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy* ; Mice ; Mutation ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Pyridines

Chloroquines are the long-established prescription drug, which are often used clinically to treat malaria and connective tissue diseases. Since December 2019, corona virus disease 2019 （COVID-19） outbreaks caused by 2019 novel coronavirus （2019-nCoV） has occurred in China and many countries around the world. Due to the lack of drugs against COVID-19, the disease spreads rapidly and the mortality rate is relatively high. Therefore, specific drugs against 2019-nCoV need to be quickly screened. The antimalarial drug chloroquine phosphate which has already been approved is confirmed to have an anti-2019-nCoV effect and has been included in diagnostic and therapeutic guidelines. However, awareness of the risk of chloroquine phosphate causing acute poisoning or even death should be strengthened. The current dosage recommended in clinical treatment is larger than that in previous treatment of malaria and the period of treatment is longer. Many provinces have required close clinical monitoring of adverse reactions. This paper reviews the pharmacological effects, poisoning and toxicological mechanisms, in vivo metabolism and distribution, and forensic issues of chloroquine drugs, in order to provide help to forensic practice and clinical work.
Betacoronavirus ; COVID-19 ; China ; Chloroquine/therapeutic use* ; Coronavirus Infections/drug therapy* ; Forensic Toxicology ; Humans ; Pandemics ; Pneumonia, Viral/drug therapy* ; SARS-CoV-2 ; COVID-19 Drug Treatment

Since the outbreak of coronavirus disease 2019 (COVID-19) in the late 2019, a variety of antiviral drugs have been used in the first-line clinical trial. The Diagnostic and Treatment Protocol for COVID-19 (Trial Version 6) in China recommends chloroquine phosphate for the first time as an anti-coronavirus trial drug. As a classic drug for treatment of malaria and rheumatism, chloroquine phosphate has been used clinically for more than 80 years, and has also shown good results in the treatment of various viral infections. As the plasma drug concentration varies greatly among different races and individuals and due to its narrow treatment window, chloroquine in likely to accumulate in the body to cause toxicity. Among the treatment regimens recommended for COVID-19, reports concerning the safety of a short-term high-dose chloroquine regimen remain scarce. In this review, the authors summarize the current research findings of chloroquine phosphate in the treatment of COVID-19, and examine the pharmacokinetic characteristics, antiviral therapy, the therapeutic mechanism and safety of chloroquine.
Antiviral Agents ; Betacoronavirus ; drug effects ; China ; Chloroquine ; analogs & derivatives ; therapeutic use ; Coronavirus Infections ; drug therapy ; Humans ; Pandemics ; Pneumonia, Viral ; drug therapy

Key Findings There is insufficient evidence to support the routine use of HCQ or CQ for the treatment of COVID-19. Results from interim analyses of 2 large RCTs, the Recovery and the Solidarity trials, reportedly showed no clinical benefit from HCQ for hospitalized patients with COVID-19. There are 3 randomized controlled trials that investigated the efficacy and safety of HCQ compared to standard therapy. Overall quality of evidence was very low. Meta-analyses from the “COVID-19 Living Data” project suggests that the use of HCQ may increase the incidence of adverse events at day 14 to day 28 (RR 2.49, 95% confidence interval: 1.04 to 5.98, moderate quality of evidence); the most common adverse event across the two trials is diarrhea (n=8). In a statement dated June 5, 2020, the investigators of the Recovery trial announced their decision to halt further enrollment to the HCQ arm of the trial because an interim analysis showed no clinical benefit from the use of HCQ in hospitalized patients with COVID. On June 15, 2020, the US FDA revoked the emergency use authorization for HCQ and CQ as treatment for COVID-19. On June 18, 2020, the WHO announced that recruitment to the HCQ arm of the Solidarity trial has been halted.
Chloroquine ; Hydroxychloroquine ; COVID-19

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has recently been noted as a repositioning candidate for angiogenesis inhibition and cancer therapy. However, the definite anti-angiogenic mechanism of MBZ remains unclear. In this study, we explored the inhibitory mechanism of MBZ in endothelial cells (ECs) and developed a novel strategy to improve its anti-angiogenic therapy. Treatment of ECs with MBZ led to inhibition of EC proliferation in a dose-dependent manner in several culture conditions in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or FBS, without selectivity of growth factors, although MBZ is known to inhibit VEGF receptor 2 kinase. Furthermore, MBZ inhibited EC migration and tube formation induced by either VEGF or bFGF. However, unexpectedly, treatment of MBZ did not affect FAK and ERK1/2 phosphorylation induced by these factors. Treatment with MBZ induced shrinking of ECs and caused G2-M arrest and apoptosis with an increased Sub-G1 fraction. In addition, increased levels of nuclear fragmentation, p53 expression, and active form of caspase 3 were observed. The marked induction of autophagy by MBZ was also noted. Interestingly, inhibition of autophagy through knocking down of Beclin1 or ATG5/7, or treatment with autophagy inhibitors such as 3-methyladenine and chloroquine resulted in marked enhancement of anti-proliferative and pro-apoptotic effects of MBZ in ECs. Consequently, we suggest that MBZ induces autophagy in ECs and that protective autophagy can be a novel target for enhancing the anti-angiogenic efficacy of MBZ in cancer treatment.
Apoptosis ; Autophagy* ; Caspase 3 ; Chloroquine ; Endothelial Cells* ; Fibroblast Growth Factor 2 ; Helminths ; Intercellular Signaling Peptides and Proteins ; Mebendazole* ; Microtubules ; Phosphorylation ; Phosphotransferases ; Receptors, Vascular Endothelial Growth Factor ; Vascular Endothelial Growth Factor A

OBJECTIVE: The aim of this study was to investigate the effects of 2 anti-malarial drugs, chloroquine (CQ) and hydroxychloroquine (HCQ), on inhibition of vascular smooth muscle cell (VSMC) proliferation both in vivo and in vitro via Adenosine monophosphate-activated protein kinase (AMPK) activation. METHODS: Protein and mRNA levels were determined by western blot analysis and real-time reverse transcription-polymerase chain reaction in primary rat VSMCs treated with CQ and HCQ, respectively. Cell proliferation was measured by flow cytometry and cell counting. Mice carotid arteries were ligated and treated with CQ or HCQ every other day for 3 weeks. Pathological changes of carotid arteries were visualized by both microscopy and fluorescence microscopy. RESULTS: CQ and HCQ increase AMPK phosphorylation in VSMCs. Both CQ and HCQ decrease platelet-derived growth factor-induced VSMC proliferation and cell cycle progression in an AMPK-dependent manner. In addition, CQ and HCQ inhibit Smad3 phosphorylation and VSMC proliferation induced by transforming growth factor-β1. Moreover, CQ and HCQ diminished neointimal proliferation in a mouse model of carotid artery ligation-induced neointima formation. CONCLUSION: The results demonstrated that CQ and HCQ inhibit cell proliferation and cell cycle progression in VSMCs via the AMPK-dependent signaling pathway. Carotid artery ligation-induced intima thickness was reduced in mouse arteries treated with CQ or HCQ, suggesting a role for antimalarial drugs in treating atherosclerosis and restenosis.
Adenosine ; AMP-Activated Protein Kinases ; Animals ; Antimalarials ; Arteries ; Atherosclerosis ; Blotting, Western ; Carotid Arteries ; Cell Count ; Cell Cycle ; Cell Proliferation ; Chloroquine ; Flow Cytometry ; Hydroxychloroquine ; In Vitro Techniques ; Mice ; Microscopy ; Microscopy, Fluorescence ; Muscle, Smooth, Vascular ; Neointima ; Phosphorylation ; Protein Kinases ; Rats ; RNA, Messenger

OBJECTIVES: To investigate the role of autophagy inhibitor chloroquine (CQ) in acute ethanol-induced liver injury and its mechenism. METHODS: Twenty-one C57BL/6 male mice were randomly divided into three groups:control group, ethanol group, CQ + ethanol group (=7). Mice in ethanol group were administered 33% (v/v) ethanol at a dose of 4.5 g/kg body weight. Ethanol-induced liver steatosis in each group was detected by hematoxylin and eosin staining. Hepatic lipid accumulation was detected by staining with Oil red O. Hepatic tissue triglyceride (TG) levels, serum aspartate aminotransferase(AST) and alanine aminotransferase(ALT) were determined by biochemical assays. Protein expression of microtubule-associated protein 1 light chain 3(LC3) and nuclear factorκB p65(NF-κB p65) were measured by Western blot and immunofluorescence. Pro-inflammatory factors tumor necrosis factor-α(TNF-α)、interleukin 6(IL-6) were detected by ELISA. RESULTS: Compared with control group, ethanol induced liver injury proved by accumulation of hepatic lipids, TG levels, AST and ALT activities were significantly increased by ethanol, protein expression of LC3-Ⅱ was also markedly increased by ethanol. Compared with ethanol group, addition of CQ increased furtherthe level of LC3-Ⅱexpression, and TG amount, serum AST and ALT activities, and the expression of NF-κB p65, TNF-αand IL-6. CONCLUSIONS Acute ethanol-intake could induce liver steatosis and inflammation, and autophagy inhibitor CQ exacerbatedethanol-induced liver injury, suggested that autophagy might be protective effect in acute ethanol-induced liver disease.
Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Autophagy ; drug effects ; Chloroquine ; pharmacology ; Interleukin-6 ; analysis ; Liver ; drug effects ; Liver Diseases, Alcoholic ; drug therapy ; Male ; Mice ; Mice, Inbred C57BL ; Microtubule-Associated Proteins ; metabolism ; Random Allocation ; Transcription Factor RelA ; metabolism ; Triglycerides ; analysis ; Tumor Necrosis Factor-alpha ; analysis

Recent studies have shown that the chemokine receptor CXCR3 and its ligand CXCL10 in the dorsal root ganglion mediate itch in experimental allergic contact dermatitis (ACD). CXCR3 in the spinal cord also contributes to the maintenance of neuropathic pain. However, whether spinal CXCR3 is involved in acute or chronic itch remains unclear. Here, we report that Cxcr3 mice showed normal scratching in acute itch models but reduced scratching in chronic itch models of dry skin and ACD. In contrast, both formalin-induced acute pain and complete Freund's adjuvant-induced chronic inflammatory pain were reduced in Cxcr3 mice. In addition, the expression of CXCR3 and CXCL10 was increased in the spinal cord in the dry skin model induced by acetone and diethyl ether followed by water (AEW). Intrathecal injection of a CXCR3 antagonist alleviated AEW-induced itch. Furthermore, touch-elicited itch (alloknesis) after compound 48/80 or AEW treatment was suppressed in Cxcr3 mice. Finally, AEW-induced astrocyte activation was inhibited in Cxcr3 mice. Taken together, these data suggest that spinal CXCR3 mediates chronic itch and alloknesis, and targeting CXCR3 may provide effective treatment for chronic pruritus.
Acetamides ; therapeutic use ; Animals ; Chemokine CXCL10 ; metabolism ; Chloroquine ; toxicity ; Chronic Disease ; Cyclopropanes ; adverse effects ; Dehydration ; complications ; Dinitrofluorobenzene ; adverse effects ; Disease Models, Animal ; Formaldehyde ; toxicity ; Freund's Adjuvant ; toxicity ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Motor Activity ; drug effects ; Pain ; chemically induced ; Pruritus ; chemically induced ; pathology ; Pyrimidines ; therapeutic use ; Receptors, CXCR3 ; antagonists & inhibitors ; genetics ; metabolism ; Skin ; pathology ; Spinal Cord ; drug effects ; metabolism ; pathology ; Time Factors ; p-Methoxy-N-methylphenethylamine ; toxicity