Microglial over-pruning of synapses during development in autism-associated SCN2A-deficient mice and human cerebral organoids.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus of understanding ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglia-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.

PDZD8-mediated endoplasmic reticulum-mitochondria associations regulate sympathetic drive and blood pressure through the intervention of neuronal mitochondrial homeostasis in stress-induced hypertension.

Neuronal hyperexcitation in the rostral ventrolateral medulla (RVLM) drives heightened sympathetic nerve activity and contributes to the etiology of stress-induced hypertension (SIH). Maintenance of mitochondrial functions is central to neuronal homeostasis. PDZD8, an endoplasmic reticulum (ER) transmembrane protein, tethers ER to mitochondria. However, the mechanisms of PDZD8-mediated ER-mitochondria associations regulating neuronal mitochondrial functions and thereby mediating blood pressure (BP) in the RVLM of SIH were largely unknown. SIH rats were subjected to intermittent electric foot shocks plus noise for 2 h twice daily for 15 consecutive days. The underlying mechanisms of PDZD8 were investigated through in vitro experiments by using small interfering RNA and through in vivo experiments, such as intra-RVLM microinjection and Western blot analysis. The function of PDZD8 on BP regulation in the RVLM was determined in vivo via the intra-RVLM microinjection of adeno-associated virus (AAV)2-r-Pdzd8. We found that the c-Fos-positive RVLM tyrosine hydroxylase (TH) neurons, renal sympathetic nerve activity (RSNA), plasma norepinephrine (NE) level, BP, and heart rate (HR) were elevated in SIH rats. ER-mitochondria associations in RVLM neurons were significantly reduced in SIH rats. PDZD8 was mainly expressed in RVLM neurons, and mRNA and protein levels were markedly decreased in SIH rats. In N2a cells, PDZD8 knockdown disrupted ER-mitochondria associations and mitochondrial structure, decreased mitochondrial membrane potential (MMP) and respiratory metabolism, enhanced ROS levels, and reduced catalase (CAT) activity. These effects suggested that PDZD8 dysregulation induced mitochondrial malfunction. By contrast, PDZD8 upregulation in the RVLM of SIH rats could rescue neuronal mitochondrial function, thereby suppressing c-Fos expression in TH neurons and decreasing RSNA, plasma NE, BP, and HR. Our results indicated that the dysregulation of PDZD8-mediated ER-mitochondria associations led to the loss of the activity homeostasis of RVLM neurons by disrupting mitochondrial functions, thereby participating in the regulation of SIH pathology.

Microglia-derived TNF-α contributes to RVLM neuronal mitochondrial dysfunction via blocking the AMPK-Sirt3 pathway in stress-induced hypertension.

BACKGROUND: Neuroinflammation in the rostral ventrolateral medulla (RVLM) has been associated with the pathogenesis of stress-induced hypertension (SIH). Neuronal mitochondrial dysfunction is involved in many pathological and physiological processes. However, the impact of neuroinflammation on neuronal mitochondrial homeostasis and the involved signaling pathway in the RVLM during SIH are largely unknown. METHODS: The morphology and phenotype of microglia and the neuronal mitochondrial injury in vivo were analyzed by immunofluorescence, Western blot, RT-qPCR, transmission electron microscopy, and kit detection. The underlying mechanisms of microglia-derived tumor necrosis factor-α (TNF-α) on neuronal mitochondrial function were investigated through in vitro and in vivo experiments such as immunofluorescence and Western blot. The effect of TNF-α on blood pressure (BP) regulation was determined in vivo via intra-RVLM microinjection of TNF-α receptor antagonist R7050. RESULTS: The results demonstrated that BP, heart rate (HR), renal sympathetic nerve activity (RSNA), plasma norepinephrine (NE), and electroencephalogram (EEG) power increased in SIH rats. Furthermore, the branching complexity of microglia in the RVLM of SIH rats decreased and polarized into M1 phenotype, accompanied by upregulation of TNF-α. Increased neuronal mitochondria injury was observed in the RVLM of SIH rats. Mechanistically, Sirtuin 3 (Sirt3) and p-AMPK expression were markedly downregulated in both SIH rats and TNF-α-treated N2a cells. AMPK activator A769662 upregulated AMPK-Sirt3 signaling pathway and consequently reversed TNF-α-induced mitochondrial dysfunction. Microinjection of TNF-α receptor antagonist R7050 into the RVLM of SIH rats significantly inhibited the biological activities of TNF-α, increased p-AMPK and Sirt3 levels, and alleviated neuronal mitochondrial injury, thereby reducing c-FOS expression, RSNA, plasma NE, and BP. CONCLUSIONS: This study revealed that microglia-derived TNF-α in the RVLM impairs neuronal mitochondrial function in SIH possibly through inhibiting the AMPK-Sirt3 pathway. Therefore, microglia-derived TNF-α in the RVLM may be a possible therapeutic target for the intervention of SIH.

Ultrathin Niobium Carbide MXenzyme for Remedying Hypertension by Antioxidative and Neuroprotective Actions.

Hypertension, as a leading risk factor for cardiovascular diseases, is associated with oxidative stress and impairment of endogenous antioxidant mechanisms, but there is still a tremendous knowledge gap between hypertension treatment and nanomedicines. Herein, we report a specific nanozyme based on ultrathin two-dimensional (2D) niobium carbide (Nb2 C) MXene, termed Nb2 C MXenzyme, to fight against hypertension by achieving highly efficient reactive oxygen species elimination and inflammatory factors inhibition. The biocompatible Nb2 C MXenzyme displays multiple enzyme-mimicking activities, involving superoxide dismutase, catalase, glutathione peroxidase, and peroxidase, inducing cytoprotective effects by resisting oxidative stress, thereby alleviating inflammatory response and reducing blood pressure, which is systematically demonstrated in a stress-induced hypertension rat model. This strategy not only opens new opportunities for nanozymes to treat hypertension but also expands the potential biomedical applications of 2D MXene nanosystems.

Up-regulation of miR-335 and miR-674-3p in the rostral ventrolateral medulla contributes to stress-induced hypertension.

The rostral ventrolateral medulla (RVLM) is known as the vasomotor center that plays a crucial role in mediating the development of stress-induced hypertension (SIH). MicroRNAs (miRNAs) are involved in many different biological processes and diseases. However, studies that evaluated the roles of miRNAs in the RVLM during SIH do not exist. Here, we performed RNA sequencing to explore the genome-wide miRNA profiles in RVLM in an SIH rat model established by administering electric foot-shocks and noises. The function of miRNAs in blood pressure regulation was determined in vivo via the intra-RVLM microinjection of the agomir or antagomir. Furthermore, the underlying mechanisms of miRNAs on SIH were investigated through in vitro and in vivo experiments, like gain-of-function. We discovered 786 miRNA transcripts among which 4 were differentially expressed. The over-expression of miR-335 and miR-674-3p in RVLM dramatically increased the heart rate (HR), arterial blood pressure (ABP), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) levels of normotensive rats, whereas the knockdown of miR-335 and miR-674-3p in RVLM markedly reduced the HR, ABP, SBP, DBP, and MAP levels of SIH rats. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed that miR-335 and miR-674-3p participated in regulating the development of SIH from different aspects, like apoptosis-multiple species pathway. Sphk1, whose expression was markedly decreased in SIH, was identified as a novel target of miR-335. MiR-335 over-expression substantially reduced the expression of Sphk1 and promoted neural apoptosis, and its inhibition had opposite effects. Re-introduction of Sphk1 dramatically abrogated the apoptosis induced by miR-335. This study provides the first systematic dissection of the RVLM miRNA landscape in SIH. MiR-335 and miR-674-3p act as SIH promoters, and the identified miR-335/Sphk1/apoptosis axis represents one of the possible mechanisms. These miRNAs can be exploited as potential targets for the molecular-based therapy of SIH.

Overexpression of NaV1.6 in the rostral ventrolateral medulla in rats mediates stress-induced hypertension via glutamate regulation.

BACKGROUND: The rostral ventrolateral medulla (RVLM) plays a key role in mediating the development of stress-induced hypertension (SIH). Furthermore, enhanced glutamate transport within glutamatergic neurons in the RVLM mediates pressor responses. Data from our previous studies suggest that the voltage-gated sodium channel NaV1.6 is overexpressed in neurons in the RVLM in SIH model rats and participates in the resulting elevation of blood pressure. However, previous studies have not investigated the relationship between NaV1.6 expression and glutamatergic neurons. METHODS: Here, we constructed an SIH rat model by knocking down NaV1.6 via microinjection of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA into the RVLM. Glutamate-related markers were quantified by Western blotting and immunofluorescence, and blood pressure was measured in the rats. RESULTS: Our findings showed that vesicular glutamate transporter 1 (VGluT1) protein expression in the RVLM was higher in SIH rats than in Control rats, and GAD67 protein expression in SIH rats was lower than that in Control rats. Therefore, the number of VGluT1-positive neurons increased, while the number of GAD67-labeled neurons decreased after stress. After knocking down NaV1.6 expression in the RVLM, VGluT1 expression and the number of VGluT1-positive neurons decreased relative to those in SIH rats, while GAD67 protein expression and the number of GAD67-labeled neurons increased relative to those in SIH rats. CONCLUSIONS: These results indicate that overexpression of NaV1.6 in the RVLM may mediate the transport and transformation of glutamate in neurons, and NaV1.6 may participate in SIH.

Deletion of Kv10.2 Causes Abnormal Dendritic Arborization and Epilepsy Susceptibility.

The abnormal function of the voltage-gated potassium channel Kv10.2 can induce epilepsy. However, the physiological function of Kv10.2 in the central nervous system remains unclear. In this study, we found that Kv10.2 knockout (KO) increased the complexity of neurons in the CA3 subarea of hippocampus. Kv10.2 KO led to enlarged somata, elongated dendritic length, and increased the number of dendritic tips in cultured rat hippocampus neurons. Kv10.2 KO also increased Synapsin I and PSD95 protein density in cultured rat hippocampal neurons. Whole cell patch-clamp recordings of brain slices in the CA3 subarea of hippocampus revealed that Kv10.2 KO increased the amplitude of spontaneous excitatory postsynaptic currents (sEPSC) and miniature excitatory postsynaptic currents (mEPSC), depolarized the resting membrane potential and increased the action potential firing, reduced the rheobase and increased the input resistance, which results in enhanced neuronal excitability. Furthermore, we made electroencephalogram (EEG) recordings of brain activity in freely moving rats before and after inducing seizures by pentylenetetrazole (PTZ) injection. Kv10.2 KO rats dramatically increased the EEG amplitude during epilepsy. Behavioral observation after seizure induction revealed that Kv10.2 KO rats demonstrated shortened onset latency, prolonged duration, and increased seizure severity when compared with wild type rats. Therefore, this study provides a new link between Kv10.2 and neuronal morphology and higher intrinsic excitability.

Rescuing Kv10.2 protein changes cognitive and emotional function in kainic acid-induced status epilepticus rats.

Voltage-gated potassium (Kv) channels are widely expressed in the central and peripheral nervous system and are crucial mediators of neuronal excitability. Importantly, these channels also actively participate in cellular and molecular signaling pathways that regulate the life and death processes of neurons. The current study used a kainic acid (KA)-induced temporal lobe epilepsy model to examine the role of the Kv10.2 gene in status epilepticus (SE). Lentiviral plasmids containing the coding sequence region of the KCNH5 gene (LV-KCNH5) were injected into the CA3 subarea of the right dorsal hippocampus within 24 h in post-SE rats to rescue Kv10.2 protein expression. Open-field and elevated plus maze test results indicated that anxiety-like behavior was ameliorated in the KA + LV-KCNH5 group rats compared with the SE group rats, and working memory was improved in the Y-maze test. However, the spatial reference memory of the LV-KCNH5 group rats did not improve in the Morris water maze test, and no difference was found in the light-dark transition box test. The results of this study indicate that Kv10.2 protein may play an important role in epilepsy, providing new potential therapeutic directions and drug targets for epilepsy treatment.

Infiltrating T helper 17 cells in the paraventricular nucleus are pathogenic for stress-induced hypertension.

Central neuroinflammation produced by both innate and adaptive immunities plays a major role in the development of stress-induced hypertension (SIH), but successful T cell immunoregulation for SIH requires that the T cells can access brain tissues. So far, both the effects of T helper 17 (Th17) cells on SIH and the pathway for T cells entry into the brain were unknown. Here we show that the blood pressure (BP), heart rate (HR) and the norepinephrine(NE) of the SIH rats were considerably higher, the numbers of Th17 cells and IL-17 were higher, relative to control. Anti-IL-17 attenuated the elevation of BP and HR of the SIH rats when microinjected into the paraventricular nucleus (PVN).Alb-FITC, after infusion into the carotid artery, were found in the brain parenchyma of the PVN in the SIH rats. We concluded that Th17 cells infiltrated the parenchyma of the paraventricular nucleus (PVN) via a compromised blood brain barrier (BBB) in response to stress and Th17 cells and IL-17 play an important role in the pathophysiology of SIH.

Increased Expression of Kv10.2 in the Hippocampus Attenuates Valproic Acid-Induced Autism-Like Behaviors in Rats.

The role of potassium channels provides suggestive evidence for the etiology of autism. The voltage-gated potassium channel Kv10.2 (KCNH5) is widely expressed in the brain. However, the inherent relationship between Kv10.2 and autism is still unclear. Herein, a rat valproic acid (VPA)-induced autism spectrum disorder model was established. The expression level of Kv10.2 was obviously decreased in the hippocampus of VPA rats. Kv10.2 was mainly localized in neurons. Subsequently, a recombinant lentivirus expressing Kv10.2 was used to upregulate the expression of Kv10.2 in the hippocampus of VPA-exposed rats. The results were promising as injection of the Kv10.2 lentivirus in the hippocampus relieved anxiety and stereotypical behavior, and improved the social and exploratory abilities of rats that were prenatally exposed to VPA. In addition, spectral analysis of electroencephalogram data revealed that animals exposed to VPA exhibited increased high-frequency activity compared with the control rats, and this activity recovered to a certain extent after upregulation of Kv10.2 expression by lentivirus injection. These results suggest that changes in Kv10.2 may play an important role in the etiology of autism, thus providing a promising direction for further research on autism.

Berberine activates bitter taste responses of enteroendocrine STC-1 cells.

Glucagon-like peptide-1 (GLP-1) is involved in the regulation of insulin secretion and glucose homeostasis. GLP-1 release is stimulated when berberine interacts with a novel G protein family (TAS2Rs) in enteroendocrine cells. In this study, we used STC-1 cells and examined a marked increase in Ca2+ in response to various bitter compounds. Ca2+ responses to traditional Chinese medicine extracts, including berberine, phellodendrine and coptisine, in STC-1 cells were suppressed by the phospholipase C (PLC) inhibitor U-73122, suggesting the involvement of bitter taste receptors in changing the physiological status of enteroendocrine cells in a PLC-dependent manner. STC-1 cells showed berberine-up-regulated preproglucagon (GLP-1 precursor) mRNA and GLP-1 secretion. A QPCR analysis demonstrated that TAS2R38, a subtype of the bitter taste receptor, was associated with GLP-1 secretion. Berberine-mediated GLP-1 secretion was attenuated in response to small interfering RNA silencing of TAS2R38. The current studies demonstrated that Gα-gustducin co-localized with GLP-1 and Tas2r106 in the STC-1 cells. We further utilized inhibitors of PLC and TRPM5, which are known to participate in taste signal transduction, to investigate the underlying pathways mediated in berberine-induced GLP-1 secretion. Berberine-induced GLP-1 release from enteroendocrine cells is modulated in a PLC-dependent manner through a process involving the activation of bitter taste receptors. Together, our data demonstrated a berberine-mediated GLP-1 secretion pathway in mouse enteroendocrine cells that could be of therapeutic relevance to hyperglycemia and the role of bitter taste receptors in the function of the small intestine.

Neuroinflammation contributes to autophagy flux blockage in the neurons of rostral ventrolateral medulla in stress-induced hypertension rats.

BACKGROUND: Neuroinflammation plays hypertensive roles in the uninjured autonomic nuclei of the central nervous system, while its mechanisms remain unclear. The present study is to investigate the effect of neuroinflammation on autophagy in the neurons of the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone reside. METHODS: Stress-induced hypertension (SIH) was induced by electric foot-shock stressors with noise interventions in rats. Systolic blood pressure (SBP) and the power density of the low frequency (LF) component of the SAP spectrum were measured to reflect sympathetic vasomotor activity. Microglia activation and pro-inflammatory cytokines (PICs (IL-1ß, TNF-α)) expression in the RVLM were measured by immunoblotting and immunostaining. Autophagy and autophagic vacuoles (AVs) were examined by autophagic marker (LC3 and p62) expression and transmission electron microscopy (TEM) image, respectively. Autophagy flux was evaluated by RFP-GFP-tandem fluorescent LC3 (tf-LC3) vectors transfected into the RVLM. Tissue levels of glutamate, gamma aminobutyric acid (GABA), and plasma levels of norepinephrine (NE) were measured by using high-performance liquid chromatography (HPLC) with electrochemical detection. The effects of the cisterna magna infused minocycline, a microglia activation inhibitor, on the abovementioned parameters were analyzed. RESULTS: SIH rats showed increased SBP, plasma NE accompanied by an increase in LF component of the SBP spectrum. Microglia activation and PICs expression was increased in SIH rats. TEM demonstrated that stress led to the accumulation of AVs in the RVLM of SIH rats. In addition to the Tf-LC3 assay, the concurrent increased level of LC3-II and p62 suggested the impairment of autophagic flux in SIH rats. To the contrary, minocycline facilitated autophagic flux and induced a hypotensive effect with attenuated microglia activation and decreased PICs in the RVLM of SIH rats. Furthermore, SIH rats showed higher levels of glutamate and lower level of GABA in the RVLM, while minocycline attenuated the decrease in GABA and the increase in glutamate of SIH rats. CONCLUSIONS: Collectively, we concluded that the neuroinflammation might impair autophagic flux and induced neural excitotoxicity in the RVLM neurons following SIH, which is involved in the development of SIH.

CREB1 regulates glucose transport of glioma cell line U87 by targeting GLUT1.

Glioma is stemmed from the glial cells in the brain, which is accounted for about 45% of all intracranial tumors. The characteristic of glioma is invasive growth, as well as there is no obvious boundary between normal brain tissue and glioma tissue, so it is difficult to resect completely with worst prognosis. The metabolism of glioma is following the Warburg effect. Previous researches have shown that GLUT1, as a glucose transporter carrier, affected the Warburg effect, but the molecular mechanism is not very clear. CREB1 (cAMP responsive element-binding protein1) is involved in various biological processes, and relevant studies confirmed that CREB1 protein regulated the expression of GLUT1, thus mediating glucose transport in cells. Our experiments mainly reveal that the CREB1 could affect glucose transport in glioma cells by regulating the expression of GLUT1, which controlled the metabolism of glioma and affected the progression of glioma.

Expression and functional activity of bitter taste receptors in primary renal tubular epithelial cells and M-1 cells.

The kidney is essential in the maintenance of in vivo homeostasis by body fluid and electrolyte conservation and metabolic waste removal. Previously, we reported the expression of a novel G protein family (Tas2rs), which includes bitter taste receptors, in the kidney tubule system, including the nephrons and the collecting duct system. Bitter taste receptors could affect kidney function via Ca2+ intake. Alkaloids such as phenylthiocarbamide stimulate these receptors and cause an increase in Ca2+ intake. In this study, we determined the expression of bitter taste receptors in the immature kidney and small intestine and in primary renal epithelial cells and M-1 (collecting tubule cell line) cells, by using QPCR and immunostaining. We found no expression of bitter taste receptors in the immature kidney and small intestine several days after birth; the relative abundance of Tas2rs transcripts varied depending on the developmental stage. Tas2rs were expressed in primary renal epithelial cells and M-1 cells. The traditional Chinese medicinal plant extracts phellodendrine and coptisine caused a rapid rise in intracellular Ca2+ concentration, which was inhibited by the phospholipase C (PLC) inhibitor U-73122. Thus, phellodendrine and coptisine could change the physiological status of renal cells in vitro by mediation of bitter taste receptors in a PLC-dependent manner. Our results provide new insights on the expression and role of bitter taste receptors in renal development and function.

2D-SAR and 3D-QSAR analyses for acetylcholinesterase inhibitors.

Alzheimer's disease (AD) accounts for almost three quarters of dementia patients and interferes people's normal life. Great progress has been made recently in the study of Acetylcholinesterase (AChE), known as one of AD's biomarkers. In this study, acetylcholinesterase inhibitors (AChEI) were collected to build a two-dimensional structure-activity relationship (2D-SAR) model and three-dimensional quantitative structure-activity relationship (3D-QSAR) model based on feature selection method combined with random forest. After calculation, the prediction accuracy of the 2D-SAR model was 89.63% by using the tenfold cross-validation test and 87.27% for the independent test set. Three cutting ways were employed to build 3D-QSAR models. A model with the highest [Formula: see text] (cross-validated correlation coefficient) and [Formula: see text](non-cross-validated correlation coefficient) was obtained to predict AChEI activity. The mean absolute error (MAE) of the training set and the test set was 0.0689 and 0.5273, respectively. In addition, molecular docking was also employed to reveal that the ionization state of the compounds had an impact upon their interaction with AChE. Molecular docking results indicate that Ser124 might be one of the active site residues.

Generation of induced pluripotent stem cells from neonatal mouse cochlear cells.

The sensory epithelium (SE) within the mammalian cochleae has a limited capacity for regeneration, and the loss of mammalian cochlear hair cells always lead to permanent hearing loss. Previous reports show that early postnatal cochlea harbors stem/progenitor-like cells nominated otospheres which have a limited regenerative/repair capacity, while these cell populations are progressively lost during the postnatal development. Induced pluripotent stem cells (iPS cells) directly reprogrammed from non-embryonic cells have captured great attentions in the scientific community. In the present study, we determine whether Yamanaka׳s factors can induce the reprogramming of cochlear cells into iPS cells. We introduce defined factors Oct3/4, Sox2 and Klf4 into otospheres derived from postnatal day-1 (P1) mouse SE, and analyze characteristics alterations in cochlear cells. After transduction, otospheres generated colonies exhibiting a normal karyotype and morphology similar to that of mouse embryonic stem cells (ESCs). Moreover, these cochlear iPS cells also express ESC-like markers. Importantly, the cochlear iPS cells show pluripotency in vitro and in vivo, as evidenced by differentiation into three germ layers by embryoid body formation, as well as high efficient formation of teratomas containing three germ layers in immunodeficient mice. Thus, pluripotent cochlear iPS cells can be generated from cochlear cells by using three Yamanaka׳s transcription factors. These attempts represent the first step toward generating fully pluripotent iPS cells from mammalian cochleae with defined exogenous genes.

The effects of the CCR6/CCL20 biological axis on the invasion and metastasis of hepatocellular carcinoma.

Chemokines and their receptors have recently been shown to play major roles in cancer metastasis. Chemokine receptor 6 (CCR6) and its ligand, CCL20, were highly expressed in a variety of human cancers. In our present study, we aimed to clarify whether CCR6/CCL20 was correlated with the migration of hepatocellular carcinoma (HCC). RT-PCR and Western blot results showed that CCR6 was overexpressed in different invasive potential HCC cell lines (p<0.05), while the expression of CCL20 had no obvious difference (p>0.05). CCR6 was suppressed by siRNA in HCCLM6, and then the biological behaviors of HCCLM6 cells were observed. The results showed that the CCR6/CCL20 biological axis increased the capacity of proliferation and adhesion, as well as the chemotactic migration and the level of cytokines related to degraded extracellular matrix. In conclusion, these findings indicate that CCR6 indeed participates in regulating the migration and invasion of HCC, and it might become a prognostic factor of HCC.

miR-193b-3p and miR-346 Exert Antihypertensive Effects in the Rostral Ventrolateral Medulla.

BACKGROUND: Rostral ventrolateral medulla (RVLM) neuron hyperactivity raises sympathetic outflow, causing hypertension. MicroRNAs (miRNAs) contribute to diverse biological processes, but their influence on RVLM neuronal excitability and blood pressure (BP) remains widely unexplored. METHODS AND RESULTS: The RVLM miRNA profiles in spontaneously hypertensive rats were unveiled using RNA sequencing. Potential effects of these miRNAs in reducing neuronal excitability and BP and underlying mechanisms were investigated through various experiments. Six hundred thirty-seven miRNAs were identified, and reduced levels of miR-193b-3p and miR-346 were observed in the RVLM of spontaneously hypertensive rats. Increased miR-193b-3p and miR-346 expression in RVLM lowered neuronal excitability, sympathetic outflow, and BP in spontaneously hypertensive rats. In contrast, suppressing miR-193b-3p and miR-346 expression in RVLM increased neuronal excitability, sympathetic outflow, and BP in Wistar Kyoto and Sprague-Dawley rats. Cdc42 guanine nucleotide exchange factor (Arhgef9) was recognized as a target of miR-193b-3p. Overexpressing miR-193b-3p caused an evident decrease in Arhgef9 expression, resulting in the inhibition of neuronal apoptosis. By contrast, its downregulation produced the opposite effects. Importantly, the decrease in neuronal excitability, sympathetic outflow, and BP observed in spontaneously hypertensive rats due to miR-193b-3p overexpression was greatly counteracted by Arhgef9 upregulation. CONCLUSIONS: miR-193b-3p and miR-346 are newly identified factors in RVLM that hinder hypertension progression, and the miR-193b-3p/Arhgef9/apoptosis pathway presents a potential mechanism, highlighting the potential of targeting miRNAs for hypertension prevention.

IFN-γ deficiency in the rostral ventrolateral medulla contributes to stress-induced hypertension by impairing microglial synaptic engulfment.

BACKGROUND: Dysfunctional neurons and microglia in the rostral ventrolateral medulla (RVLM) have been implicated in the pathogenesis of stress-induced hypertension (SIH). Functional perturbation of microglial synaptic engulfment can induce aberrant brain circuit activity. IFN-γ is a pleiotropic cytokine that plays a role in regulating neuronal activity. However, existing research on the exploration of the effects of microglia on synapses in the RVLM is lacking, particularly on the function of IFN-γ in microglial synaptic engulfment involved in SIH. METHODS: A SIH rat model was established by electric foot shocks combined with noise stimulation. The underlying mechanism of IFN-γ on synaptic density and microglial synaptic engulfment was investigated through in-vivo and in-vitro experiments involving gain of function, immunofluorescence, quantitative real-time PCR, western blot, and morphometric analysis. Furthermore, the function of IFN-γ in neuronal activity, renal sympathetic nerve activity (RSNA), and blood pressure (BP) regulation was determined through in-vivo and in-vitro experiments involving Ca 2+ imaging, immunofluorescence, platinum-iridium electrode recording, ELISA, the femoral artery cannulation test, and the tail-cuff method. RESULTS: The BP, heart rate, RSNA, plasma norepinephrine, and the number of c-Fos-positive neurons in SIH rats increased compared with those in control rats. Pre and postsynaptic densities in the RVLM also increased in SIH rats. IFN-γ and CCL2 expression levels were significantly reduced in the RVLM of the SIH group, whose microglia also exhibited an impaired capacity for synapse engulfment. IFN-γ elevation increased CCL2 expression and microglial synaptic engulfment and decreased synaptic density in vivo and in vitro . However, CCL2 inhibition reversed these effects. Moreover, the reduction of neuronal excitability, RSNA, plasma norepinephrine, and BP by IFN-γ was abrogated through CCL2 expression. CONCLUSION: IFN-γ deficiency in the RVLM impaired the microglial engulfment of synapses by inhibiting CCL2 expression and increasing synaptic density and neuronal excitability, thereby contributing to SIH progression. Targeting IFN-γ may be considered a potential strategy to combat SIH.

LncRNA INPP5F ameliorates stress-induced hypertension via the miR-335/Cttn axis in rostral ventrolateral medulla.

AIMS: The rostral ventrolateral medulla (RVLM) is an essential vasomotor center responsible for regulating the development of stress-induced hypertension (SIH). Long non-coding RNAs (lncRNAs) play critical roles in various physiopathology processes, but existing research on the functions of RVLM lncRNAs on SIH has been lacking. In this study, we investigated the roles of RVLM lncRNAs in SIH. METHODS: Genome-wide lncRNA profiles in RVLM were determined by RNA sequencing in a SIH rat model established using electric foot shocks plus noises. The hypotensive effect of lncRNA INPP5F and the underlying mechanisms of lncRNA INPP5F on SIH were explored through in vivo and in vitro experiments, such as intra-RVLM microinjection and immunofluorescence. RESULTS: We discovered 10,179 lncRNA transcripts, among which the lncRNA INPP5F expression level was significantly decreased in SIH rats. Overexpression of lncRNA INPP5F in RVLM dramatically reduced the blood pressure, sympathetic nerve activity, and neuronal excitability of SIH rats. LncRNA INPP5F overexpression markedly increased Cttn expression and reduced neural apoptosis by activating the PI3K-AKT pathway, and its inhibition had opposite effects. Mechanistically, lncRNA INPP5F acted as a sponge of miR-335, which further regulated the Cttn expression. CONCLUSION: LncRNA INPP5F was a key factor that inhibited SIH progression, and the identified lncRNA INPP5F/miR-335/Cttn/PI3K-AKT/apoptosis axis represented one of the possible mechanisms. LncRNA INPP5F could serve as a therapeutic target for SIH.