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.

TRPV1 mediates astrocyte activation and interleukin-1ß release induced by hypoxic ischemia (HI).

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a serious birth complication with high incidence in both advanced and developing countries. Children surviving from HIE often have severe long-term sequela including cerebral palsy, epilepsy, and cognitive disabilities. The severity of HIE in infants is tightly associated with increased IL-1ß expression and astrocyte activation which was regulated by transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel in the TRP family. METHODS: Neonatal hypoxic ischemia (HI) and oxygen-glucose deprivation (OGD) were used to simulate HIE in vivo and in vitro. Primarily cultured astrocytes were used for investigating the expression of glial fibrillary acidic protein (GFAP), IL-1ß, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and activation of the nucleotide-binding, oligomerization domain (NOD)-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome by using Western blot, q-PCR, and immunofluorescence. Brain atrophy, infarct size, and neurobehavioral disorders were evaluated by Nissl staining, 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining and neurobehavioral tests (geotaxis reflex, cliff aversion reaction, and grip test) individually. RESULTS: Astrocytes were overactivated after neonatal HI and OGD challenge. The number of activated astrocytes, the expression level of IL-1ß, brain atrophy, and shrinking infarct size were all downregulated in TRPV1 KO mice. TRPV1 deficiency in astrocytes attenuated the expression of GFAP and IL-1ß by reducing phosphorylation of JAK2 and STAT3. Meanwhile, IL-1ß release was significantly reduced in TRPV1 deficiency astrocytes by inhibiting activation of NLRP3 inflammasome. Additionally, neonatal HI-induced neurobehavioral disorders were significantly improved in the TRPV1 KO mice. CONCLUSIONS: TRPV1 promotes activation of astrocytes and release of astrocyte-derived IL-1ß mainly via JAK2-STAT3 signaling and activation of the NLRP3 inflammasome. Our findings provide mechanistic insights into TRPV1-mediated brain damage and neurobehavioral disorders caused by neonatal HI and potentially identify astrocytic TRPV1 as a novel therapeutic target for treating HIE in the subacute stages (24 h).

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 Has a Job in the Brain.

Nuclear factor of activated T cells 5 (NFAT5) has recently been classified as a new member of the Rel family. In addition, there are 5 more well-defined members (NF-κB and NFAT1-4) in the Rel family, which participate in regulating the expression of immune and inflammatory response-related genes. NFAT5 was initially identified in renal medullary cells where it regulated the expression of osmoprotective-related genes during the osmotic response. Many studies have demonstrated that NFAT5 is highly expressed in the nuclei of neurons in fetal and adult brains. Additionally, its expression is approximately 10-fold higher in fetal brains. With the development of detection technologies (laser scanning confocal microscopy, transgene technology, etc.), recent studies suggest that NFAT5 is also expressed in glial cells and plays a more diverse functional role. This article aims to summarize the current knowledge regarding the expression of NFAT5, its regulation of activation, and varied biological functions in the brain.

Niche convergence and biogeographic history shape elevational tree community assembly in a subtropical mountain forest.

Niche convergence or conservatism have been proposed as essential mechanisms underlying elevational plant community assembly in tropical mountain ecosystems. Subtropical mountains, compared to tropical mountains, are likely to be shaped by a mixing of different geographic affinities of species and remain somehow unclear. Here, we used 31 0.1-ha permanent plots distributed in subtropical forests on the eastern and western aspects of the Gaoligong Mountains, southwest China between 1498 m and 3204 m a.sl. to evaluate how niche-based and biogeographic processes shape tree community assembly along elevational gradients. We analyzed the elevational patterns of taxonomic, phylogenetic and functional diversity, as well as of individual traits, and assessed the relative importance of environmental effects on these diversity measures. We then classified tree species as being either tropical affiliated or temperate affiliated and estimated their contribution to the composition of biogeographic affinities. Species richness decreased with elevation, and species composition showed apparent turnover across the aspects and elevations. Most traits exhibited convergent patterns across the entire elevational gradient. Phylogenetic and functional diversity showed opposing patterns, with phylogenetic diversity increasing and functional diversity decreasing with elevation. Soil nutrients, especially phosphorus and nitrogen, appeared to be the main abiotic variables driving the elevational diversity patterns. Communities at lower elevations were occupied by tropical genera, while highlands contained species of tropical and temperate biogeographic affinities. Moreover, the high phylogenetic diversity at high elevations were likely due to differences in evolutionary history between temperate and tropical species. Our results highlight the importance of niche convergence of tropical species and the legacy of biogeographic history on the composition and structure of subtropical mountain forests. Furthermore, limited soil phosphorus caused traits divergence and the partitioning for different forms of phosphorus may explain the high biodiversity found in phosphorus-limited subtropical forests.

[Function of bladder smooth muscle autophagy in cyclophosphamide-induced cystitis].

OBJECTIVE: To explore the existence of bladder smooth muscle autophagy and its function in cyclophosphamide (CYP) -induced cystitis. METHODS: A total of 30 Sprague-Dawley rats were randomized into control (n = 6) and experimental groups (4 h, 48 h, 72 h, 96 h, n = 24) . The experimental group received a single intraperitoneal injection of 150 mg/kg CYP in 0.6 ml saline while the control group had the same volume of saline injection. The changes of microtubule-associated protein light chain 3 (LC3), interleukin-6 (IL-6), IL-1ß and tumor necrosis factor-alpha (TNF-α) proteins in bladder muscular layer were measured with Western blot. Co-location of LC3 and alpha-small muscle action (α-SMA) was detected with double-labeled immunofluorescence. Autophagic vacuoles were observed with electron microscopy. The changes in bladder inflammation were measured by hematoxylin and eosin staining. RESULTS: Autophagy was confirmed in detrusor myocytes with electron microscope, LC3 Western blot and double-labeled immunofluorescence. As compared with the control group, the expression level of LC3 in experimental groups decreased (0.08 ± 0.01 vs 0.13 ± 0.01, P < 0.05) at 4 h and increased 1.19, 0.75 and 0.20 fold (0.29 ± 0.04 vs 0.13 ± 0.01, P < 0.05; 0.23 ± 0.03 vs 0.13 ± 0.01, P < 0.05;0.16 ± 0.03 vs 0.13 ± 0.01, P > 0.05) at 48, 72 and 96 h respectively.Electron microscopy and double-labeled immunofluorescent also supported the above change of LC3 by Western blot.In addition, as compared with the control groups, IL-6, IL-1ß and TNF-α and bladder histological scores of bladder muscular layer significantly increased in experimental groups (all P < 0.05) . CONCLUSIONS: Autophagy exists in bladder smooth muscle. In cyclophosphamide-induced cystitis, bladder smooth muscle autophagy activation may not be sufficient to protect bladder muscle layer inflammation in rats.

Botany, traditional uses, phytochemistry and pharmacological activity of Crataegus pinnatifida (Chinese hawthorn): a review.

OBJECTIVES: Crataegus pinnatifida (C. pinnatifida), including C. pinnatifida Bge. and its variant C. pinnatifida Bge. var. major N, E. Br., has traditionally been used as a homologous plant for traditional medicine and food in ethnic medical systems in China. Crataegus pinnatifida, especially its fruit, has been used for more than 2000 years to treat indigestion, stagnation of meat, hyperlipidemia, blood stasis, heart tingling, sores, etc. This review aimed to provide a systematic summary on the botany, traditional uses, phytochemistry, pharmacology and clinical applications of C. pinnatifida. KEY FINDINGS: This plant contains flavonoids, phenylpropanoids, terpenoids, organic acids, saccharides and essential oils. Experimental studies showed that it has hypolipidemic, antimyocardial, anti-ischemia, antithrombotic, anti-atherosclerotic, anti-inflammatory, antineoplastic neuroprotective activity, etc. Importantly, it has good effects in treating diseases of the digestive system and cardiovascular and cerebrovascular systems. SUMMARY: There is convincing evidence from both in vitro and in vivo studies supporting the traditional uses of C. pinnatifida. However, multitarget network pharmacology and molecular docking technology should be used to study the interaction between the active ingredients and targets of C. pinnatifida. Furthermore, exploring the synergy of C. pinnatifida with other Chinese medicines to provide new understanding of complex diseases may be a promising strategy.

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.

Optimal pressure in penile rehabilitation with a vacuum erection device: evidence based on a rat model.

Vacuum erection device (VED), used to treat radical prostatectomy (RP)-associated erectile dysfunction, has attracted considerable attention. However, the optimal negative pressure remains to be determined. This investigation explored the optimal pressure for VED therapy in penile rehabilitation. Thirty-six 9-week-old male rats were randomly divided into six groups: control groups (sham group, bilateral cavernous nerve crush [BCNC] group) and VED therapy groups (-200 mmHg group, -300 mmHg group, -400 mmHg group, -500 mmHg group). BCNC group and VED therapy groups underwent BCNC surgery. Intracavernosal pressure (ICP)/mean arterial pressure (MAP) ratio was calculated to assess erectile function. Masson's trichrome (MT) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR) were performed to explore cellular and molecular changes of the penis. Compared to the BCNC group, ICP/MAP ratios in all VED treatment groups were improved significantly (all P < 0.05), but there were no statistically significant differences among VED therapy groups. With increased pressure, complications gradually emerged and increased in frequency. Expression of molecular indicators, such as endothelial nitric oxide synthase (eNOS) and alpha-smooth muscle actin (α-SMA), increased after VED therapy, and hypoxia-inducible factor 1α (HIF-1α) and transforming growth factor beta (TGF-ß) decreased. In addition, VED therapy improved the outcomes of MT and TUNEL assay. This investigation demonstrated a pressure of -200 mmHg in a rat model is optimal for VED therapy for penile rehabilitation after RP. No further benefits were observed with increased pressure, despite an increase in complications.

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.

Bumetanide reduce the seizure susceptibility induced by pentylenetetrazol via inhibition of aberrant hippocampal neurogenesis in neonatal rats after hypoxia-ischemia.

Hypoxia-ischemia brain damage (HIBD) is one of prevalent causes of neonatal mortality and morbidity. Our data demonstrated that hypoxia-ischemia (HI) induced Na+-K+-Cl--co-transporter 1 (NKCC1) increasing in hippocampus. Previous studies demonstrated that NKCC1 regulates various stages of neurogenesis. In this study, we studied the role of increased NKCC1 in regulating of HI-induced neurogenesis. HIBD model was established in 7days old Sprague-Dawley rat pup, and the expression of NKCC1 was detected by western blot and qPCR. Brain electrical activity in freely rats was monitored by electroencephalography (EEG) recordings. HI-induced neurogenesis was detected by immunofluorescence staining. Neurobehavioral test was to investigate the neuro-protective role of bumetanide, an inhibitor of NKCC1, on neonatal rats after HI. The results showed that bumetanide treatment significantly reduced brain electrical activity and the seizure stage of epilepsy induced by pentylenetetrazol (PTZ) in vivo after HI. In addition, bumetanide restored aberrant hippocampal neurogenesis and associated cognitive function. Our data demonstrated that bumetanide reduces the susceptibility of epilepsy induced by PTZ in rats suffering from HI injury during neonatal period via restoring the ectopic newborn neurons in dentate gyrus (DG) and cognitive function.