Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4) Mitigates Seizures.

Astrocytes are critical regulators of the immune/inflammatory response in several human central nervous system (CNS) diseases. Emerging evidence suggests that dysfunctional astrocytes are crucial players in seizures. The objective of this study was to investigate the role of transient receptor potential vanilloid 4 (TRPV4) in 4-aminopyridine (4-AP)-induced seizures and the underlying mechanism. We also provide evidence for the role of Yes-associated protein (YAP) in seizures. 4-AP was administered to mice or primary cultured astrocytes. YAP-specific small interfering RNA (siRNA) was administered to primary cultured astrocytes. Mouse brain tissue and surgical specimens from epileptic patient brains were examined, and the results showed that TRPV4 was upregulated, while astrocytes were activated and polarized to the A1 phenotype. The levels of glial fibrillary acidic protein (GFAP), cytokine production, YAP, signal transducer activator of transcription 3 (STAT3), intracellular Ca2+([Ca2+]i) and the third component of complement (C3) were increased in 4-AP-induced mice and astrocytes. Perturbations in the immune microenvironment in the brain were balanced by TRPV4 inhibition or the manipulation of [Ca2+]i in astrocytes. Knocking down YAP with siRNA significantly inhibited 4-AP-induced pathological changes in astrocytes. Our study demonstrated that astrocytic TRPV4 activation promoted neuroinflammation through the TRPV4/Ca2+/YAP/STAT3 signaling pathway in mice with seizures. Astrocyte TRPV4 inhibition attenuated neuroinflammation, reduced neuronal injury, and improved neurobehavioral function. Targeting astrocytic TRPV4 activation may provide a promising therapeutic approach for managing epilepsy.

Complement C3 Aggravates Post-epileptic Neuronal Injury Via Activation of TRPV1.

Epilepsy is a brain condition characterized by the recurrence of unprovoked seizures. Recent studies have shown that complement component 3 (C3) aggravate the neuronal injury in epilepsy. And our previous studies revealed that TRPV1 (transient receptor potential vanilloid type 1) is involved in epilepsy. Whether complement C3 regulation of neuronal injury is related to the activation of TRPV1 during epilepsy is not fully understood. We found that in a mouse model of status epilepticus (SE), complement C3 derived from astrocytes was increased and aggravated neuronal injury, and that TRPV1-knockout rescued neurons from the injury induced by complement C3. Circular RNAs are abundant in the brain, and the reduction of circRad52 caused by complement C3 promoted the expression of TRPV1 and exacerbated neuronal injury. Mechanistically, disorders of neuron-glia interaction mediated by the C3-TRPV1 signaling pathway may be important for the induction of neuronal injury. This study provides support for the hypothesis that the C3-TRPV1 pathway is involved in the prevention and treatment of neuronal injury and cognitive disorders.

Silencing of circIgf1r plays a protective role in neuronal injury via regulating astrocyte polarization during epilepsy.

Epilepsy is a common brain disorder, repeated seizures of epilepsy may lead to a series of brain pathological changes such as neuronal or glial damage. However, whether circular RNAs are involved in neuronal injury during epilepsy is not fully understood. Here, we screened circIgf1r in the status epilepticus model through circRNA sequencing, and found that it was upregulated after the status epilepticus model through QPCR analysis. Astrocytes polarizing toward neurotoxic A1 phenotype and neurons loss were observed after status epilepticus. Through injecting circIgf1r siRNA into the lateral ventricle, it was found that knocking down circIgf1r in vivo would induce the polarization of astrocytes to phenotype A2 and reduce neuronal loss. The results in vitro further confirmed that inhibiting the expression of circIgf1r in astrocytes could protect neurons by converting reactive astrocytes from A1 to the protective A2. In addition, knocking down circIgf1r in astrocytes could functionally promote astrocyte autophagy and relieve the destruction of 4-AP-induced autophagy flux. In terms of mechanism, circIgf1r promoted the polarization of astrocytes to phenotype A1 by inhibiting autophagy. Taken together, our results reveal circIgf1r may serve as a potential target for the prevention and treatment of neuron damage after epilepsy.

Clinical characteristics and plasma antibody titer of patients with COVID-19 in Zhejiang, China.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first affected humans in China on December 31, 2019 (Shi et al., 2020). Coronaviruses generally cause mild, self-limiting upper respiratory tract infections in humans, such as the common cold, pneumonia, and gastroenteritis (To et al., 2013; Berry et al., 2015; Chan et al., 2015). According to the Report of the World Health Organization (WHO)-China Joint Mission on COVID-19 (WHO, 2020), the case fatality rate of COVID-19 increases with age, while the rate among males is higher than that among females (4.7% and 2.8%, respectively). Since an effective vaccine and specific anti-viral drugs are still under development, passive immunization using the convalescent plasma (CP) of recovered COVID-19 donors may offer a suitable therapeutic strategy for severely ill patients in the meantime. So far, several studies have shown therapeutic efficacy of CP transfusion in treating COVID-19 cases. A pilot study first reported that transfusion of CP with neutralizing antibody titers above 1:640 was well tolerated and could potentially improve clinical outcomes through neutralizing viremia in severe COVID-19 cases (Chen et al., 2020). Immunoglobulin G (IgG) and IgM are the most abundant and important antibodies in protecting the human body from viral attack (Arabi et al., 2015; Marano et al., 2016). Our study aimed to understand the aspects of plasma antibody titer levels in convalescent patients, as well as assessing the clinical characteristics of normal, severely ill, and critically ill patients, and thus provide a basis for guiding CP therapy. We also hoped to find indicators which could serve as a reference in predicting the progression of the disease.

TRPV1 translocated to astrocytic membrane to promote migration and inflammatory infiltration thus promotes epilepsy after hypoxic ischemia in immature brain.

BACKGROUND: Neonatal hypoxic-ischemic brain damage (HIBD), a leading cause of neonatal mortality, has intractable sequela such as epilepsy that seriously affected the life quality of HIBD survivors. We have previously shown that ion channel dysfunction in the central nervous system played an important role in the process of HIBD-induced epilepsy. Therefore, we continued to validate the underlying mechanisms of TRPV1 as a potential target for epilepsy. METHODS: Neonatal hypoxic ischemia and oxygen-glucose deprivation (OGD) were used to simulate HIBD in vivo and in vitro. Primarily cultured astrocytes were used to assess the expression of TRPV1, glial fibrillary acidic protein (GFAP), cytoskeletal rearrangement, and inflammatory cytokines by using Western blot, q-PCR, and immunofluorescence. Furthermore, brain electrical activity in freely moving mice was recorded by electroencephalography (EEG). TRPV1 current and neuronal excitability were detected by whole-cell patch clamp. RESULTS: Astrocytic TRPV1 translocated to the membrane after OGD. Mechanistically, astrocytic TRPV1 activation increased the inflow of Ca2+, which promoted G-actin polymerized to F-actin, thus promoted astrocyte migration after OGD. Moreover, astrocytic TRPV1 deficiency decreased the production and release of pro-inflammatory cytokines (TNF, IL-6, IL-1ß, and iNOS) after OGD. It could also dramatically attenuate neuronal excitability after OGD and brain electrical activity in HIBD mice. Behavioral testing for seizures after HIBD revealed that TRPV1 knockout mice demonstrated prolonged onset latency, shortened duration, and decreased seizure severity when compared with wild-type mice. CONCLUSIONS: Collectively, TRPV1 promoted astrocyte migration thus helped the infiltration of pro-inflammatory cytokines (TNF, IL-1ß, IL-6, and iNOS) from astrocytes into the vicinity of neurons to promote epilepsy. Our study provides a strong rationale for astrocytic TRPV1 to be a therapeutic target for anti-epileptogenesis after HIBD.

NFAT5 and HIF-1α Coordinate to Regulate NKCC1 Expression in Hippocampal Neurons After Hypoxia-Ischemia.

Hypoxic-ischemic encephalopathy (HIE) is a serious birth complication with severe long-term sequelae such as cerebral palsy, epilepsy and cognitive disabilities. Na+-K+-2Cl- cotransporters 1 (NKCC1) is dramatically upregulated after hypoxia-ischemia (HI), which aggravates brain edema and brain damage. Clinically, an NKCC1-specific inhibitor, bumetanide, is used to treat diseases related to aberrant NKCC1 expression, but the underlying mechanism of aberrant NKCC1 expression has rarely been studied in HIE. In this study, the cooperative effect of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor of activated T cells 5 (NFAT5) on NKCC1 expression was explored in hippocampal neurons under hypoxic conditions. HI increased HIF-1α nuclear localization and transcriptional activity, and pharmacological inhibition of the HIF-1α transcription activity or mutation of hypoxia responsive element (HRE) motifs recovered the hypoxia-induced aberrant expression and promoter activity of NKCC1. In contrast, oxygen-glucose deprivation (OGD)-induced downregulation of NFAT5 expression was reversed by treating with hypertonic saline, which ameliorated aberrant NKCC1 expression. More importantly, knocking down NFAT5 or mutation of the tonicity enhancer element (TonE) stimulated NKCC1 expression and promoter activity under normal physiological conditions. The positive regulation of NKCC1 by HIF-1α and the negative regulation of NKCC1 by NFAT5 may serve to maintain NKCC1 expression levels, which may shed light on the transcription regulation of NKCC1 in hippocampal neurons after hypoxia.

Ablative Hypofractionated Radiation Therapy Enhances Non-Small Cell Lung Cancer Cell Killing via Preferential Stimulation of Necroptosis In Vitro and In Vivo.

PURPOSE: To investigate how necroptosis (ie, programmed necrosis) is involved in killing of non-small cell lung cancer (NSCLC) after ablative hypofractionated radiation therapy (HFRT). METHODS AND MATERIALS: Deoxyribonucleic acid damage, DNA repair, and the death form of NSCLC cells were assessed after radiation therapy. The overexpression and silencing of receptor-interacting protein kinases 3 (RIP3, a key protein involved activation of necroptosis)-stable NSCLC cell lines were successfully constructed. The form of cell death, the number and area of colonies, and the regulatory proteins of necroptosis were characterized after radiation therapy in vitro. Finally, NSCLC xenografts and patient specimens were used to examine involvement of necroptosis after ablative HFRT in vivo. RESULTS: Radiation therapy induced expected DNA damage and repair of NSCLC cell lines, but ablative HFRT at ≥10 Gy per fraction preferentially stimulated necroptosis in NSCLC cells and xenografts with high RIP3 expression, as characterized by induction and activation of RIP3 and mixed-lineage kinase domain-like protein and release of immune-activating chemokine high-mobility group box 1. In contrast, RNA interference of RIP3 attenuated ablative HFRT-induced necroptosis and activation of its regulatory proteins. Among central early-stage NSCLC patients receiving stereotactic body radiation therapy, high expression of RIP3 was associated with improved local control and progression-free survival (all P < .05). CONCLUSIONS: Ablative HFRT at ≥10 Gy per fraction enhances killing of NSCLC with high RIP3 expression via preferential stimulation of necroptosis. RIP3 may serve as a useful biomarker to predict favorable response to stereotactic body radiation therapy.

Ethical Inspection about laboratory animals.

Laboratory animals and animal experiments are foundations and important support conditions for life sciences, especially for medical research. The animal experiments have drawn extensive attention from the society because of the ethical issue. This paper takes Wenzhou Medical University as an example to give a brief introduction to the ethical review about laboratory animals in the university so as to further draw attention and concerns from the public about the ethical issue of laboratory animals. We successively introduce its scientific projects, nurturing environment and ethical review of laboratory animals.

Effects of zedoary turmeric oil on P450 activities in rats with liver cirrhosis induced by thioacetamide.

The aim of this study was to elucidate the effects of zedoary turmeric oil (ZTO) on P450 activities (CYP1A2, CYP2C9, CYP2C19, CYP2B6, CYP2D6 and CYP3A4) in rats with liver cirrhosis induced by thioacetamide (TAA). For the induction of liver cirrhosis, rats were given TAA in their drinking water at a concentration of 0.03% for consecutive 5 weeks and then 0.04% for the next consecutive 5 weeks throughout the establishment of cirrhosis. Then the cirrhotic rats were ip given saline, ZTO 100, 200 and 400 mg/kg, respectively, once daily for 2 weeks. When cirrhosis model was established at week 10, all rats of five groups were administered intragastrically with 15 mg/kg phenacetin, 0.6 mg/kg tolbutamide, 15 mg/kg omeprazole, 15 mg/kg bupropion, 15 mg/kg metoprolol, and 10 mg/kg midazolam. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The degree of liver cirrhosis was assessed by HE staining. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) from the model group increased by approximately 4-fold, and a decreased level of albumin (Alb) was also observed, as compared to the control group (P < 0.05). However, ZTO was found to reverse those changes of serum levels observed in the model group, and the 200 mg/kg ZTO treatment group showed the most obvious reverse tendency with significantly decreased ALT, AST and increased Alb levels (P < 0.05). The results indicated that ZTO with the dose of 100 mg/kg could inhibit the activities of CYP450 isoforms CYP2C9 and CYP2D6 in vivo in cirrhotic rats induced by TAA, while ZTO with the dose of 400 mg/kg could induce the activity of CYP2C19 in vivo in cirrhotic rats induced by TAA. However, ZTO showed no influence on cirrhotic rat hepatic CYP1A2, CYP2B6 and CYP3A4 activity in vivo. This has certain guiding significance to clinical treatment.

Comparison of different methods of intestinal obstruction in a rat model.

AIM: To investigate different methods of creating incomplete intestinal obstruction in a rat model and to compare their electrophysiologic, morphologic and histologic characteristics. METHODS: Rat ileum was partially obstructed by the respective application of: braided silk (penetrated the mesentery and surrounded intestine); half ligation (penetrated directly and ligated 1/2 cross-section of the intestine); wide pipe (6 mm in width, surrounded the intestine); narrow pipe (2 mm in width, surrounded the intestine). A control was also included (no obstruction). Various behavioral and electrophysiologic variables, as well as morphologic and immunohistochemical observations were recorded by blinded investigators at different time points (12, 24, 48, 72 h), including daily general condition, ileal wet weight and circumference, macromorphous and micromorphous intestine, bowel movement capability in vivo and in vitro, slow wave and neural electrical activity, and the number of c-Kit positive interstitial cells of Cajal (ICC). RESULTS: Despite being of a similar general condition, these methods resulted in different levels of obstruction in each group compared with the control at different time points (12, 24, 48, 72 h). However, these fields of the wide pipe rat showed significantly differences when compared with the other three obstructed groups at 12 to 72 h, including macroscopic and histological presentation, intestinal transit ratio and contractility, circumference and wet weight, amplitude and frequency of nerve electrical discharge and slow wave, and ICC numbers (all P < 0.01). CONCLUSION: The wide pipe rat method is significantly more reliable and stable than the other methods of obstruction, demonstrating that use of the wide pipe method can be a useful model of incomplete intestinal obstruction.