Ying Zong

NLRP3 inflammasome activation mediates radiation-induced pyroptosis in bone marrow-derived macrophages.

A limit to the clinical benefit of radiotherapy is not an incapacity to eliminate tumor cells but rather a limit on its capacity to do so without destroying normal tissue and inducing inflammation. Recent evidence reveals that the inflammasome is essential for mediating radiation-induced cell and tissue damage. In this study, using primary cultured bone marrow-derived macrophages (BMDM) and a mouse radiation model, we explored the role of NLRP3 inflammasome activation and the secondary pyroptosis underlying radiation-induced immune cell death. We observed an increasing proportion of pyroptosis and elevating Caspase-1 activation in 10 and 20 Gy radiation groups. Nlrp3 knock out significantly diminished the quantity of cleaved-Caspase-1 (p10) and IL-1β as well as the proportion of pyroptosis. Additionally, in vivo research shows that 9.5 Gy of radiation promotes Caspase-1 activation in marginal zone cells and induces death in mice, both of which can be significantly inhibited by knocking out Nlrp3. Thus, based on these findings, we conclude that the NLRP3 inflammasome activation mediates radiation-induced pyroptosis in BMDMs. Targeting NLRP3 inflammasome and pyroptosis may serve as effective strategies to diminish injury caused by radiation.

Up-Regulated ATF4 Expression Increases Cell Sensitivity to Apoptosis in Response to Radiation

Background/Aims: Activating transcription factor 4 (ATF4) is a member of the activating transcription factor family which regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses. ATF4 is also over-expressed in human solid tumors, although its effect on responsiveness to radiation is largely unexplored. Methods: Real-time PCR was used to detect ATF4 mRNA levels in cells treated with different doses of 60Coγ radiation. Cell viability was assayed using a cell counting kit. The cell cycle was analyzed using flow cytometry, and cell apoptosis was assayed using Annexin V-PI double labeling. Small interfering RNA (siRNA) against ATF4 was transfected into ECV304 cells using Lipofectamine 2000. An ATF4 over-expression plasmid (p-ATF4-CGN) was transfected into HEK293 cells that endogenously expressed low levels of ATF4. The levels of intracellular reactive oxygen species (ROS) were measured using CM-H2DCFDA as a probe. Results: ATF4 mRNA and protein expression levels were higher after radiation and increased in a dose- and time-dependent manner in AHH1 lymphoblast cells (P < 0.05). An increase in ATF4 levels was also observed after radiation in primary murine spleen cells, human endothelial ECV304 cells, human liver LO2 cells, breast cancer MCF7 cells, and human hepatocellular carcinoma HEPG2 cells. No change was observed in human embryonic kidney 293 (HEK293) cells. Over-expressing ATF4 in HEK293 cells inhibited cell proliferation, increased cell apoptosis and significantly increased the proportion of cells in G1 phase. Conversely, when ATF4 expression was knocked down using siRNA in ECV304 cells, it protected the cells from radiation-induced apoptosis. These findings suggest that ATF4 may play a role in radiation-induced cell killing by inhibiting cell proliferation and promoting cell apoptosis. Conclusions: In this study, we found that radiation up-regulated the expression of ATF4. We used ATF4 knockdown and over-expression systems to show that ATF4 may play a role in radiation-induced cellular apoptosis.

Is the CARD8 rs2043211 polymorphism associated with susceptibility to Crohn’s disease? A meta-analysis

Abstract Many studies have reported the association between the CARD8 gene polymorphism rs2043211 and the susceptibility to Crohn’s disease (CD), but the results have remained quite contradictory. Therefore, the aim of the meta-analysis was to explore whether the CARD8 rs2043211 polymorphism has an effect on CD risk. We performed a systematic literature search for related articles published up to July 2014 in multiple databases. Six eligible articles containing eight studies were selected. Odds ratios (ORs) as well as their corresponding 95% confidence intervals (CIs) were used to estimate the association between the CARD8 polymorphism and CD risk in different genotypic models. Heterogeneity analysis was also performed and publication bias was taken into account. Subgroup analyses were conducted according to different ethnicities, as well as different types of CD. In the pooled analyses, no statistical significant association was found between the CARD8 polymorphism and CD risk in the overall population or Caucasian subgroup in the additive model (overall population: OR = 0.93, 95% CI = 0.87–1.01; Caucasian: OR = 0.93, 95% CI = 0.83–1.05). However, subgroup analysis based on different CD types showed a significant association between the CARD8 polymorphism and CD risk in the additive model (ileal CD: OR = 0.83, 95% CI = 0.70–0.98; stenotic or fistulizing CD: OR = 0.81, 95% CI = 0.72–0.92). Our results indicated that CD may involve different types of pathogenesis and have variable clinical manifestations. In patients with ileal, stenotic or fistulizing CD, the mutant-type rs2043211 polymorphism may generate a potentially protective effect.

Subchronic safety evaluation of EPO-018B, a pegylated peptidic erythropoiesis stimulating agent, after 5-week subcutaneous injection in Cynomolgus monkeys and Sprague-Dawley rats.

EPO-018B, a synthetic peptide-based erythropoiesis stimulating agent (ESA), is coupled to polyethylene glycol (PEG) and designed to specifically bind and activate the erythropoietin (EPO) receptor to result in production of red blood cells. This study was designed to evaluate the potential subchronic toxicity of EPO-018B for Cynomolgus monkeys and Sprague-Dawley rats both at 0, 0.5, 5 and 50 mg/kg every week for 5 weeks, followed by 6-week recovery for rats and 12-week recovery for monkeys. The No Observed Adverse Effect Level (NOAEL) for rats and monkeys were both considered to be at least 0.5 mg/kg/day, the minimum toxic dose to be 5.0 mg/kg/day and the severe toxic dose to be more than 50.0 mg/kg/day. The toxicological effects included the exaggerated pharmacology and secondary sequelae that resulted from an erythropoiesis-stimulating agent treatment to healthy animals. Most treatment induced effects were reversible or showed ongoing recovery upon discontinuation of treatment. The anticipated patient population for EPO-018B treatment is targeted to be the anemia patients caused by chronic renal failure or chemotherapy against to cancer and is expected to have an ideal clinical application prospect.

Evaluation of subchronic toxicity of SIM010603, a potent inhibitor of receptor tyrosine kinase, after 28-day repeated oral administration in SD rats and beagle dogs.

SIM010603, a promising multi-targeted receptor tyrosine kinase (RTK) inhibitor, is now being considered for evaluation in phase clinical trial. In this work, the subchronic toxicity of SIM010603 in SD rats and beagle dogs have been characterized. Rats and dogs received SIM010603 orally (0-20 and 0-10mg/kg/day, respectively) on a consecutive daily dosing schedule for 28 days following a 14 days recovery period. Sunitinib was used as a positive control. The No Observed Adverse Effect Level (NOAEL) of SIM010603 was 5mg/kg/day for rats, and undefined for dogs. The treatment resulted in unscheduled mortality in dogs receiving 10mg/kg of SIM010603 or Sunitinib. The adverse effects of SIM010603 on rats and dogs mainly included gastrointestinal toxicity, skeletal toxicity, myelosuppression, thymus atrophy, bronchopneumonia, cardiovascular dysfunction, and pancreatic toxicity. Similar observations have also been noted with this class of RTK signaling inhibitors and are consistent with pharmacologic perturbations of physiologic/angiogenic processes associated with the intended molecular targets. Most treatment-induced effects were reversible or showed ongoing recovery upon discontinuation of treatment. SIM010603 has shown comparable toxicity effect on beagle dogs, while better tolerability on SD rats when compared to Sunitinib.

Toxicity of Smokeless Tobacco Extract after 184-Day Repeated Oral Administration in Rats

The use of smokeless tobacco (ST) is growing rapidly and globally. The consumption of ST is associated with an increased risk for developing chronic diseases, such as diabetes, hypercholesterolemia, and myocardial infarction, and has led to many public health problems. It is very important to access the toxicity of ST. This experiment presents data from 184-day toxicology studies in Sprague-Dawley (SD) rats designed to characterize the chronic effects of a smokeless tobacco extract (STE). The control group and treatment groups were matched for a range of nicotine levels. Animals were given STE by oral gavage with doses of 3.75 (low-dose), 7.50 (mid-dose) and 15.00 (high-dose) mg·nicotine/kg body weight/day for 184 days, followed by 30 days for recovery. Variables evaluated included body weights, feed consumption, clinical observations, clinical and anatomic pathology (including organ weights), and histopathology. Decreased body weights and organ weights (heart, liver and kidney) were found in animals in the mid-dose and high-dose groups. STE also showed moderate and reversible toxicity in esophagus, stomach, liver, kidney and lung.

NLRP3 inflammasome activation mediates fatigue-like behaviors in mice via neuroinflammation

Numerous experimental and clinical studies have suggested that the interaction between the immune system and the brain plays an important role in the pathophysiology of chronic fatigue syndrome (CFS). The NLRP3 inflammasome is an important part of the innate immune system. This complex regulates proinflammatory cytokine interleukin-1β (IL-1β) maturation, which triggers different kinds of immune-inflammatory reactions. We employed repeated forced swims to establish a model of CFS in mice. NLRP3 knockout (KO) mice were also used to explore NLRP3 inflammasome activation in the mechanisms of CFS, using the same treatment. After completing repeated swim tests, the mice displayed fatigue-like behaviors, including locomotor activity and reduced fall-off time on the rota-rod test, which was accompanied by significantly higher mature IL-1β level in the prefrontal cortex (PFC) and malondialdehyde (MDA) level in serum. We also found increased NLRP3 protein expression, NLRP3 inflammasome formation and increased mature IL-1β production in the PFC, relative to untreated mice. The NLRP3 KO mice displayed significantly moderated fatigue behaviors along with decreased PFC and serum IL-1β levels under the same treatment. These findings demonstrated the involvement of NLRP3 inflammasome activation in the mechanism of swimming-induced fatigue. Future therapies targeting the NLRP3/IL-1β pathway may have significant potential for fatigue prevention and treatment.

Teriravone Induces Neurotoxicity in Beagle Dogs

Stroke is the medical emergency and can result in permanent neurological damage, complications, and death [1]. For last three decades, stroke remains the second most common cause of mortality and recently has become the third leading cause of global disease burden estimated using disability-adjusted life years [2,3]. Oxidative stress has been implicated as a pathogenetic mechanism for neurodegeneration in stroke. Edaravone, a potent radical scavenger, has high liposolubility and good permeability through the blood–brain barrier and has been used for acute ischemic stroke in Japan since June 2001 [4]. Its neuroprotective effect has been confirmed by a large number of studies [5,6]. However, edaravone was also reported to induce adverse effects, such as acute renal failure, hepatitis, and disseminated intravascular coagulation [7]. It is necessary to develop a new generation of more effective neuroprotective drugs with lower toxicity to meet the increasing clinical needs. Teriravone, a new compound based on the structure of edaravone, is provided by Tianjing Zhongrui Pharmaceutical Company. The structures of teriravone and edaravone are shown in Figure 1A. In this study, the preclinical chronic toxicity and neurotoxic mechanisms of teriravone were evaluated in Beagle dogs to provide data to support its further development. Twenty-six Beagle dogs (13 male, 13 female), aged 6-8 months and weighted 7.8 1.2 kg, were provided by Shanghai Experimental Animal Co. Ltd. Twenty-six Beagle dogs were randomly divided into four groups: control group and three groups treated with teriravone (5, 15, 45 mg/kg/day). There were eight dogs in high-dose group and six dogs in the other groups, with equal numbers of each sex. Dogs received intravenous teriravone or its pharmaceutical adjuvant once daily for 90 consecutive days followed by a recovery period of 30 days. Clinical symptoms were observed daily throughout the study. Dynamic detections of hematology, serum chemistry, urinanalysis, and electrocardiogram were conducted. At the end of the treatment (d 90) and the end of the recovery period (d 120), half of the animals of each group were killed, respectively, for histopathological examination. Additionally, mesencephalon of dogs in control and teriravone 45 mg/kg/day groups was collected for the examination of differentially expressed genes by using microarray chip. Real-time PCR was used to validate differentially expressed genes IL2, LAMC2, and NRXN1. At last, protein levels of laminin gamma 2 (LAMC2), IL2, and neurexin (NRXN1) in mesencephalon were determined by Western blot. Neurotoxic symptoms such as limbs stiff, standing instability, side lying, and opisthotonus appeared during the administration of teriravone at doses of 15 and 45 mg/kg/day. Some animals in (A)

Evaluation of subchronic toxicity of GRD081, a dual PI3K/mTOR inhibitor, after 28-day repeated oral administration in Sprague-Dawley rats and beagle dogs.

GRD081, a newly developed dual PI3K/mTOR inhibitor, is now being considered for evaluation in phase I clinical trial. In this work, the subchronic toxicity of GRD081 in Sprague-Dawley (SD) rats and beagle dogs has been characterized. Rats and dogs received GRD081 orally (2, 5, 10 and 1, 2, 4 mg/kg/day, respectively) on a consecutive daily dosing schedule for 28 days following a 14 days of recovery period. The treatment resulted in unscheduled mortality in rats receiving 5 mg/kg/day and 10 mg/kg/day. The adverse effects of GRD081 on rats and dogs mainly included myelosuppression, immunosuppression, hematological toxicity, and moderate liver, pancreas and kidney toxicity. These observations are consistent with pharmacologic perturbations of physiologic processes associated with the intended molecular targets for this class of PI3K/mTOR signaling inhibitors. Most of the treatment-induced effects were reversible upon discontinuation of treatment. The no-observed-adverse-effect level (NOAEL) of GRD081 was 1mg/kg/day for beagle dogs and less than 2 mg/kg/day for SD rats.