This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.
Osteogenesis ; Core Binding Factor Alpha 1 Subunit/pharmacology* ; Large-Conductance Calcium-Activated Potassium Channels/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Calcium/metabolism* ; Cell Differentiation ; RNA, Messenger/metabolism* ; Cell Proliferation ; Osteoblasts

OBJECTIVE: To explore the mechanisms of large-conductance calcium-activated potassium channel (BKCa) involved in inflammatory response in sepsis. METHODS: The serum levels of BKCa were measured by enzyme-linked immunosorbent assay (ELISA) in patients with sepsis (28 cases), patients with common infection (25 cases) and healthy people (25 cases). The relationship between levels of BKCa and acute physiology and chronic health evaluation II (APACHE II) were analyzed. Cultured RAW 264.7 cells were stimulated by lipopolysaccharide (LPS). In some experiments, a cell model of sepsis was constructed using Nigericin as the second stimulus signal. The mRNA and protein expressions of BKCa in RAW 264.7 cells stimulated with LPS (0, 50, 100, 1 000 μg/L) were measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RAW 264.7 cells were transfected with small interfering RNA of BKCa (siRNA-BKCa), and the levels of caspase-1 precursor (pro-caspase-1), interleukin-1β precursor (pro-IL-1β) in cell, and the levels of caspase-1 p20, IL-1β p17 of cell culture medium, and NOD-like receptor protein 3 (NLRP3), nuclear factor-κB (NF-κB) were measured by Western blotting. The apoptosis were detected by staining with propidium iodide (PI), the release rate of lactate dehydrogenase (LDH) were measured, and the expression of apoptotic protein Gasdermin D (GSDMD) was measured by Western blotting to evaluate the effect of silencing BKCa on cell pyrosis. RESULTS: The level of serum BKCa in patients with sepsis was significantly higher than that in patients with common infection and health peoples (ng/L: 165.2±25.9 vs. 102.5±25.9, 98.8±20.0, both P < 0.05). In addition, the level of serum BKCa in patients with sepsis was significantly positively correlated with APACHE II score (r = 0.453, P = 0.013). LPS could construct a sepsis cell model by which LPS could promote BKCa expression in mRNA and protein with a concentration-dependent manner. The mRNA and protein expressions of BKCa in the cells stimulated by 1 000 μg/L LPS were significantly higher than that in the blank group (0 μg/L) [BKCa mRNA (2^-ΔΔCt): 3.00±0.36 vs. 1.00±0.16, BKCa/β-actin: 1.30±0.16 vs. 0.37±0.09, both P < 0.05]. Compared with the control group, the ratios of caspase-1 p20/pro-caspase-1 and IL-1β p17/pro-IL-1β in the model group were significantly increased (caspase-1 p20/pro-caspase-1: 0.83±0.12 vs. 0.27±0.05, IL-1β p17/pro-IL-1β: 0.77±0.12 vs. 0.23±0.12, both P < 0.05), however, transfection of siRNA-BKCa induced the decrease both of them (caspase-1 p20/pro-capase-1: 0.23±0.12 vs. 0.83±0.12, IL-1β p17/pro-IL-1β: 0.13±0.05 vs. 0.77±0.12, both P < 0.05). Compared with the control group, the number of apoptotic cells, LDH release rate and GSDMD expression in the model group were significantly increased [LDH release rate: (30.60±8.40)% vs. (15.20±7.10)%, GSDMD-N/GSDMD-FL: 2.10±0.16 vs. 1.00±0.16, both P < 0.05], however, transfection of siRNA-BKCa induced the decrease both of them [LDH release rate: (15.60±7.30)% vs. (30.60±8.40)%, GSDMD-N/GSDMD-FL: 1.13±0.17 vs. 2.10±0.16, both P < 0.05]. The mRNA and protein expressions of NLRP3 in sepsis cells were significantly higher than those in the control group [NLRP3 mRNA (2^-ΔΔCt): 2.06±0.17 vs. 1.00±0.24, NLRP3/GAPDH: 0.46±0.05 vs. 0.15±0.04, both P < 0.05]. However, the expression of NLRP3 after siRNA-BKCa transfection was significantly lower than that in model group [NLRP3 mRNA (2^-ΔΔCt): 1.57±0.09 vs. 2.06±0.17, NLRP3/GAPDH: 0.19±0.02 vs. 0.46±0.05, both P < 0.05]. Compared with the control group, the NF-κB p65 nuclear transfer of sepsis cell were significantly increased (NF-κB p65/Histone: 0.73±0.12 vs. 0.23±0.09, P < 0.05). However, the NF-κB p65 expression in the nucleus were decreased after siRNA-BKCa transfection (NF-κB p65/Histone: 0.20±0.03 vs. 0.73±0.12, P < 0.05). CONCLUSIONS BKCa is involved in the pathogenesis of sepsis, and its possible mechanism is to activate NF-κB/NLRP3/caspase-1 signaling pathway to induce inflammatory factor production and cell death.
Humans ; Histones ; Caspase 1 ; Large-Conductance Calcium-Activated Potassium Channels ; Lipopolysaccharides ; NF-kappa B ; NLR Family, Pyrin Domain-Containing 3 Protein ; L-Lactate Dehydrogenase ; Sepsis ; RNA, Small Interfering ; Caspases

Atrial Ca²⁺ handling abnormalities, mainly involving the dysfunction of ryanodine receptor (RyR) and sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), play a role in the pathogenesis of atrial fibrillation (AF). Previously, we found that the expression and function of transient receptor potential vanilloid subtype 4 (TRPV4) are upregulated in a sterile pericarditis (SP) rat model of AF, and oral administration of TRPV4 inhibitor GSK2193874 alleviates AF in this animal model. The aim of this study was to investigate whether oral administration of GSK2193874 could alleviate atrial Ca²⁺ handling abnormalities in SP rats. A SP rat model of AF was established by daubing sterile talcum powder on both atria of Sprague-Dawley (SD) rats after a pericardiotomy, to simulate the pathogenesis of postoperative atrial fibrillation (POAF). On the 3rd postoperative day, Ca²⁺ signals of atria were collected in isolated perfused hearts by optical mapping. Ca²⁺ transient duration (CaD), alternan, and the recovery properties of Ca²⁺ transient (CaT) were quantified and analyzed. GSK2193874 treatment reversed the abnormal prolongation of time to peak (determined mainly by RyR activity) and CaD (determined mainly by SERCA activity), as well as the regional heterogeneity of CaD in SP rats. Furthermore, GSK2193874 treatment relieved alternan in SP rats, and reduced its incidence of discordant alternan (DIS-ALT). More importantly, GSK2193874 treatment prevented the reduction of the S2/S1 CaT ratio (determined mainly by RyR refractoriness) in SP rats, and decreased its regional heterogeneity. Taken together, oral administration of TRPV4 inhibitor alleviates Ca²⁺ handling abnormalities in SP rats primarily by blocking the TRPV4-Ca²⁺-RyR pathway, and thus exerts therapeutic effect on POAF.
Administration, Oral ; Animals ; Atrial Fibrillation/etiology* ; Calcium/metabolism* ; Myocytes, Cardiac/metabolism* ; Pericarditis/pathology* ; Rats ; Rats, Sprague-Dawley ; Ryanodine Receptor Calcium Release Channel/pharmacology* ; Sarcoplasmic Reticulum/pathology* ; TRPV Cation Channels

Episodic ataxia (EA) is a group of disorders characterized by recurrent spells of vertigo, truncal ataxia, and dysarthria. Episodic ataxia type 2 (EA2), the most common subtype of EA, is an autosomal dominant disease caused by mutation of the CACNA1A gene. EA2 has been rarely reported in the Chinese population. Here we present an EA2 family admitted to Xiangya Hospital in October 2018. The proband was a 22-year-old male who complained of recurrent spells of vertigo, slurred speech, and incoordination for 4 years. Brain magnetic resonance imaging (MRI) showed cerebellar atrophy. He had neuropsychological development disorder in childhood, and cognitive assessment in adulthood showed cognitive impairment. The proband's mother and grandmother had a similar history. Peripheral blood samples from the proband and family members were collected, and genomic DNA was isolated. Whole exome sequencing of the proband detected a heterozygous frameshift mutation c.2042_2043del (p.Q681Rfs*100) of CACNA1A gene. This mutation was verified in the proband and 2 family members using Sanger sequencing. One family member carrying this mutation was free of symptoms and signs, suggesting an incomplete penetrance of the mutation. We reported a variant c.2042_2043del of CACNA1A gene as the pathogenic mutation in a Chinese EA2 family for the first time. This case enriched the clinical spectrum of CACNA1A related EA2, and contributed to the understanding of clinical and genetic characteristics of EA2 to reduce misdiagnosis.
Adult ; Ataxia ; Calcium Channels/genetics* ; Humans ; Male ; Mutation ; Nystagmus, Pathologic ; Pedigree ; Vertigo ; Young Adult

OBJECTIVE: To investigate the effects of CACNA1H gene knockout (KO) on autistic-like behaviors and the morphology of hippocampal neurons in mice. METHODS: In the study, 25 CACNA1H KO mice of 3-4 weeks old and C57BL/6 background were recruited as the experimental group, and 26 wild type (WT) mice of the same age and background were recruited as the control group. Three-chamber test and open field test were used to observe the social interaction, anxiety, and repetitive behaviors in mice. After that, their brain weight and size were measured, and the number of hippocampal neurons were observed by Nissl staining. Furthermore, the CACNA1H heterozygote mice were interbred with Thy1-GFP-O mice to generate CACNA1H^-/--Thy1⁺(KO-GFP) and CACNA1H^+/+-Thy1⁺ (WT-GFP) mice. The density and maturity of dendritic spines of hippocampal neurons were observed. RESULTS: In the sociability test session of the three-chamber test, the KO mice spent more time in the chamber of the stranger mice than in the object one (F_{1, 14}=95.086, P < 0.05; Post-Hoc: P < 0.05), without any significant difference for the explored preference index between the two groups (t=1.044, P>0.05). However, in the social novelty recognition test session, no difference was observed between the time of the KO mice spend in the chamber of new stranger mice and the stranger one (F_{1, 14}=18.062, P < 0.05; Post-Hoc: P>0.05), and the explored preference index of the KO mice was less than that of the control group (t=2.390, P < 0.05). In the open field test, the KO mice spent less time in the center of the open field apparatus than the control group (t=2.503, P < 0.05), but the self-grooming time was significantly increased compared with the control group (t=-2.299, P < 0.05). Morphological results showed that the brain weight/body weight ratio (t=0.356, P>0.05) and brain size (t=-0.660, P>0.05) of the KO mice were not significantly different from those of the control group, but the number of neurons were significantly reduced in hippocampal dentate gyrus compared with the control group (t=2.323, P < 0.05). Moreover, the density of dendritic spine of dentate gyrus neurons in the KO-GFP mice was significantly increased compared with the control group (t=-2.374, P < 0.05), without any significant difference in spine maturity (t=-1.935, P>0.05). CONCLUSION CACNA1H KO mice represent autistic-like behavior, which may be related to the decrease in the number of neurons and the increase in the density of dendritic spine in the dentate gyrus.
Animals ; Autistic Disorder/genetics* ; Calcium Channels, T-Type/genetics* ; Gene Knockout Techniques ; Hippocampus ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Neurons

Autoantibodies ; Calcium Channels ; Humans ; Myasthenia Gravis ; Receptors, Cholinergic/metabolism*

The TRPC family consists of multiple important cationic channels in mammals that participate in a variety of physiological and pathological processes. Our previous studies have shown that transforming growth factor-β1 (TGF-β1) increases the expression of TRPC6 in podocytes, but the roles of other members of the TRPC family in podocytes require further investigation. In this study, we investigated the effect of TGF-β1 on the expression of the TRPC family and the role of the TRPC family in the changes of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in podocytes induced by TGF-β1. The model of podocyte injury was established by treatment with TGF-β1 in immortalized glomerular podocytes (MPC5) in vitro. qRT-PCR and Western blot were used to detect the effect of TGF-β1 on the mRNA and protein expression of each TRPC family member. After the expression of each TRPC family member was knocked down by a siRNA-based approach and blocked by SKF96365, respectively, free cytosolic Ca²⁺ was measured using the fluorescent Ca²⁺ indicator Fluo-3/AM, and the dynamic change of [Ca²⁺]_i in podocytes was detected by a dynamic high-speed calcium imaging system. The results showed that TGF-β1 increased the protein expression of TRPC1/3/6 in podocytes, but had no effects on the protein expression of TRPC4. The protein expression levels of TRPC5/7 were only affected by 4 ng/mL and 8 ng/mL TGF-β1, respectively. TGF-β1 increased TRPC1/3/6 mRNA levels in podocytes, however had no effects on TRPC4/5/7 mRNA. TGF-β1 significantly increased [Ca²⁺]_i in podocytes. Knockdown of TRPC1/4/5/7 in podocytes had no significant effect on the [Ca²⁺]_i induced by TGF-β1, but TRPC3/6 knockdown significantly decreased the [Ca²⁺]_i. There was no significant difference in the [Ca²⁺]_i between the TRPC6 siRNA-treated group and SKF96365-treated group, but the [Ca²⁺]_i of the TRPC3 siRNA-treated group was significantly higher than that of SKF96365-treated group. These results demonstrate that TGF-β1 increases the expression of the TRPC1/3/6 in podocytes. TGF-β1 increases [Ca²⁺]_i in podocytes, which is dependent on the TRPC3/6 expression. Our results also suggest that the effect of TRPC6 on [Ca²⁺]_i in podocytes may be greater than that of TRPC3.
Animals ; TRPC6 Cation Channel/metabolism* ; Calcium/metabolism* ; TRPC Cation Channels/metabolism* ; Podocytes/metabolism* ; Transforming Growth Factor beta1/metabolism* ; RNA, Small Interfering/metabolism* ; RNA, Messenger/metabolism* ; Mammals/metabolism*

OBJECTIVE: To analyze the clinical phenotype and genetic characterization of a child with early infantile epileptic encephalopathy. METHODS: The proband was subjected to history taking and was diagnosed based on his clinical manifestation, magnetic resonance imaging (MRI) and whole exome sequencing (WES). Sanger sequencing was carried out to determine the origin of pathogenic variant. RESULTS: The proband unconsciously tilts his head to one side with squint, which revealed an abnormal discharge. MRI indicated suspicious abnormal signal shadow in the left posterior frontal cortex in addition with inflammation signs in the right maxillary sinus and ethmoid sinus. WES revealed that the proband has carried a heterozygous c.5789G>A variant in the CACNAIA gene. The result of Sanger sequencing was in keeping with that of WES. Neither of his parents has carried the same variant. CONCLUSION The heterozygous c.5789G>A variant of the CACNAIA gene probably underlay the early infantile epileptic encephalopathy 42 in the proband, which has a de novo origin.
Calcium Channels/genetics* ; Genetic Testing ; Heterozygote ; Humans ; Infant ; Mutation ; Spasms, Infantile/genetics* ; Whole Exome Sequencing

Rapamycin (Rap) is an immunosuppressant, which is mainly used in the anti-rejection of organ transplantation. Meanwhile, it also shows great potential in the fields of anticancer, neuroprotection and anti-aging. Rap can inhibit the activity of mammalian target of Rap (mTOR). It activates the transcription factor EB (TFEB) to up-regulate lysosomal function and eliminates the inhibitory effect of mTOR on ULK1 (unc-51 like autophagy activating kinase 1) to promote autophagy. Recent research showed that Rap can directly activate the lysosomal cation channel TRPML1 in an mTOR-independent manner. TRPML1 activation releases lysosomal calcium. Calcineurin functions as the sensor of the lysosomal calcium signal and activates TFEB, thus promoting lysosome function and autophagy. This finding has greatly broadened and deepened our understanding of the pharmacological roles of Rap. In this review, we briefly introduce the canonical Rap-mTOR-ULK1/TFEB signaling pathway, and then discuss the discovery of TRPML1 as a new target of Rap and the pharmacological potential of this novel Rap-TRPML1-Calcineurin-TFEB pathway.
Autophagy ; Calcium/metabolism* ; Calcium Channels ; Lysosomes/metabolism* ; Signal Transduction ; Sirolimus

Objective: To investigate whether large conductance calcium-activated potassium channel (BK(Ca)) was involved in the migration of pericytes (PC) in the mice of senile cochlear stria vascularis capillaries PC. Methods: C57BL/6J mice were divided into 3-month (n=10) and 12-month groups (n=10). Auditory brainstem response (ABR) was used to test the hearing threshold of each group. The immunofluorescence was used to detect the expression changes of osteopontin (OPN) and β-BK(Ca) channels on cochlear stria vascularis PC. The morphological changes of perivascular cells in cochlea were observed by transmission electron microscope (TEM). Cell experiment: The PC, which were in the stria vascularis of the cochlea were primary cultured and identified. A cell senile model was made with D-gal. The appropriate intervention concentration of low galactose (D-gal) was determined by CCK8. β-galactosidase (SA-β-gal) staining was used to evaluate the cell decrept level. The change of BK(Ca) channels current on PC were recorded by whole cell patch clamp technique. The expression of BK(Ca) channels on PC was detected by immunofluorescence. The migration and invasion ability of two groups were detected by using Scratch test and Transwell. The levels of OPN and β-BK(Ca) channels were detected by Western blot. SPSS 22.0 software was used to analyze the data. Results: The ABR threshold in the 12-month group was higher than 3-month group (t=12.66, P<0.01). In the 12-month group, the expression of β-BK(Ca) channel was lower and the expression of OPN was increased (t=14.64, P<0.01; t=20.73, P<0.01). In TEM, cochlear stria vascularis PC were tightly connected to endothelial cells in 3-month group, while PC were loosely connected to endothelial cells or PC soma were separated from the capillary in 12-month group. Cell experiment: The positive rate of PC in the primary cultured cochlear stria vascularis is above 95%. Compared with the SA-β-gal stained cells in the control group, the positive rate of 15 mg/ml D-gal intervention PC was 85% (t=36.90, P<0.01). Whole cell patch clamp BK(Ca) channels current decreased in the D-gal group compared with the young group PC (t=12.18, P<0.05). The OPN expression in the senile group was higher than control group (t=16.30, P<0.01), while the β-BK(Ca) channels expression was decreased (t=11.98, P<0.01; t=15.72, P<0.05), and migration ability raised (t=7.91, P<0.01;t=7.59, P<0.01). After intervened of BK(Ca) channels specific blocker IBTX in the D-gal group, the expression of OPN and migration were increased (t=4.26, P<0.05; t=5.88, P<0.01; t=21.97, P<0.01). Conclusion: PC migration capacity were increased during the senile period, and the expression of β-BK(Ca) channel was decreased. The administration of IBTX, a specific blocker of BK(Ca) channel, at the cell level could increase the migration capacity, suggesting that BK(Ca) might be involved in the migration of PC in the stria vascularis of the aging cochlea.
Aging ; Animals ; Cochlea ; Endothelial Cells ; Large-Conductance Calcium-Activated Potassium Channels ; Mice ; Mice, Inbred C57BL ; Pericytes ; Stria Vascularis