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【Objective】 To observe the effect of puerarin on the concentration of Ca2+ and the expression of brain derived neurotrophic factor (BDNF) in hippocampal neurons of vascular dementia (VD) rats so as to explore the mechanism of puerarin in protecting nerve cells. 【Methods】 Male SD rats were randomly divided into sham operation group, model group, and puerarin intervention group. The vascular dementia model was established by ligating bilateral common carotid arteries at intervals of 3 days. Two weeks after the operation, the learning and memory abilities of the rats were evaluated by Morris water maze, and the expression of BDNF in the hippocampus of the rats was detected by immunohistochemistry and Western blotting. The mean fluorescence intensity was measured by flow cytometry to represent the intracellular free Ca2+ concentration. 【Results】 In the puerarin intervention group, the rats’ escape latency in Morris water maze was significantly shortened, the expression of BDNF in the hippocampus was significantly increased, and the concentration of Ca2+ in hippocampal neurons was decreased. Compared with the model group, the difference was statistically significant (all P<0.05). 【Conclusion】 Puerarin has neuroprotective effect on VD rats, and its mechanism may be related to the decrease of Ca2+ concentration in hippocampal neurons and the up-regulation of BDNF expression.
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Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an important intracellular channel for releasing Ca²⁺. In order to investigate the effects of the ITPR1 overexpression on Ca²⁺ concentration and lipid content in duck uterine epithelial cells and its effects on calcium transport-related genes, the structural domain of ITPR1 gene of duck was cloned into an eukaryotic expression vector and transfected into duck uterine epithelial cells. The overexpression of the ITPR1 gene, the concentration of Ca²⁺, the lipid content, and the expression of other 6 calcium transport-related genes was determined. The results showed that the concentration of Ca²⁺ in uterine epithelial cells was significantly reduced after transfection (P<0.05), the triglyceride content was significantly increased (P<0.01), and the high-density lipoprotein content was significantly decreased (P<0.01). The correlation analysis results showed that the overexpression of the C-terminal half of the ITPR1 gene was significantly positively correlated with the total cholesterol content (P<0.01), which was significantly positively correlated with the low-density lipoprotein content (P<0.05). The overexpression of the N-terminal half of the ITPR1 gene was significantly positively correlated with the triglyceride content (P<0.01), which was significantly negatively correlated with the concentration of Ca²⁺ (P<0.05). RT-qPCR results of 6 calcium transport-related genes showed that the overexpression of the C-terminal half of the ITPR1 gene significantly inhibited the expression of the IP3R2, VDAC2 and CAV1 genes, and the overexpression of the N-terminal half of the ITPR1 gene significantly promoted the expression of the IP3R3 and CACNA2D1 genes. In conclusion, the ITPR1 gene overexpression can promote Ca²⁺ release in duck uterus epithelial cells, promote the synthesis of triglyceride, low-density lipoprotein and cholesterol, and inhibit the production of high-density lipoprotein, and the ITPR1 gene overexpression affected the expression of all 6 calcium transport-related genes.
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Animales , Femenino , Calcio/metabolismo , Patos/genética , Células Epiteliales , Inositol , Receptores de Inositol 1,4,5-Trifosfato , Lípidos , ÚteroRESUMEN
Objective Idiopathic pulmonary fibrosis ( IPF) is a chronic inflammatory disease with unknown etiology and is lack of effective therapy. The aim of this study is to explore the function of Ca2+ and PI3K/AKT/mTOR pathway in the pathogenesis of IPF, and the impact of 1,25?( OH) 2 D3 on Ca2+ and PI3K/AKT/mTOR pathway in type Ⅱalveolar epithelial cells of rat with IPF. Methods 150 male SD rats were randomly divided into 2 groups: prevention group ( control groupⅠ, model groupⅠ, medication groupⅠ) and treatment group ( control groupⅡ, model groupⅡ, medication groupⅡ) . The tracheal exposure surgery was operated in control groupⅠ/Ⅱ, and then 200μL sterile physiological saline was administered by intraperitoneal injection of each rats 2 days and 14 days after surgery, separately. Bleomycin(BLM)(5 mg/kg) was in?jected into the trachea of model groupⅠ/Ⅱ, and then vitamin D3 solvent(0.1%ethanol and 99.9%glycol propylene, 1μL/g) was ad?ministered by intraperitoneal injection 2 days and 14 days after surger?y, separately. Bleomycin( BLM) ( 5 mg/kg) was injected into the tra?chea of medication groupⅠ/Ⅱ, and then 1,25?( OH) 2 D3( 2μg/kg) was administered by intraperitoneal injection 2 days and 14 days after surgery, separately. IPF model was built by injecting Bleomycin into the trachea of rats, 1,25?(OH)2D3(2μg/kg) was used to prevent and treat IPF by intraperitoneal injection in medication group. The hydroxyproline content of lung tissue in each group was measured, type Ⅱalveolar epithelial cells were separated from lung tissue and labeled with Fluo?3AM, then concentration of Ca2+ was detected by Laser scanning confocal microscope. The mRNA levels of PI3K, AKT and mTOR in the typeⅡalveolar epithelial cells were tested by RT?PCR. Results Compared with control groupⅠ/Ⅱ at each time point, hydroxyproline content of lung tissue, Ca2+ concentration and expression of PI3K, AKT and mTOR in typeⅡalveolar epithelial cells in model groupⅠ/Ⅱand medication groupⅠ/Ⅱwere sig?nificantly raised( P<0.05 or P<0.01) , but these were significantly reduced in medication groupⅠ/Ⅱcompared with model groupⅠ/Ⅱ( P<0.05 or P<0.01) . Correlation analysis showed that there is significant positive correlation between Ca2+ concentration and mRNA expression levels of PI3K, AKT and mTOR in model groupⅠ/Ⅱ(r=0.5988, r=0.6230, r=0.6603,P<0.01)and medication groupⅠ/Ⅱ( r=0.701 2, r=0.632 3,r=0.740 3,P<0.01) . Conclusion The PI3K/AKT/mTOR pathway plays an important role in devel?opment of IPF. 1,25?( OH) 2 D3 is able to reduce Ca2+concentration in typeⅡalveolar epithelial cells and inhibit the PI3K/AKT/mTOR pathway, and then inhibit the development of IPF.
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Objective To evaluate the effect of silencing leptin by small interfering RNA(siRNA)on the expression of lep‐tin ,and apoptosis ,proliferation and intracellular Ca2+ concentration([Ca2+ ]i )of hepatic stellate cells(HSCs)and to provide evi‐dence for liver fibrosis gene therapy.Methods HSCs were divided into normal control group ,blank vector group ,siRNA nega‐tive control group and leptin‐siRNA group.After transfection of the leptin‐siRNAs into HSCs ,cell proliferation was measured by MTT assay.Cell cycle and apoptosis were measured by flow cytometry.Expression of leptin was detected by immunocyto‐chemistry and Western blot. [Ca2+ ]i was measured by Fura‐2/AM loading.Results Compared with the normal control group , the blank vector group and the siRNA control group ,the protein expression of leptin and the cell growth were significantly in‐hibited in the leptin‐siRNA group(P<0.05). The proliferation rate of HSCs was significantly different at different time points (24 ,48 and 72 h)(P<0.05).The cell apoptosis rate was increased significantly in the leptin‐siRNA group(P<0.01).At the same time ,Leptin‐siRNA‐induced [Ca2+ ]i was also significantly reduced(P<0.05).Conclusion The leptin gene may play an important role in liver fibrosis progression and is potentially a novel predictive and prognostic marker for liver fibrosis.
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Objective To investigate the function of phosphatidylinositol phospholipase C encoded by LB361 gene of L.interrogans ( L-PI-PLC) and its mechanism in inducing macrophage apoptosis .Meth-ods The PI-PLC domains in the sequence of LB 361 gene of L.interrogans serovar Lai strain were analyzed by bioinformatics method .Prokaryotic expression system was established to express the recombinant L -PI-PLC ( rL-PI -PLC).The enzymatic activity of rL-PI-PLC in hydrolyzing phosphatidylinositol -4,5-bisphos -phate (PIP2) substrate into inositol-1,4,5-trisphosphate (IP3) was determined by IP3 fluorescence polariza-tion assay.LB361gene expressions at mRNA and protein levels as well as the secretion of LB 361gene prod -ucts were detected by real-time fluorescent quantitative RT -PCR and Western blot assay after infection of hu-man THP-1 macrophages with L.interrogans serovar Lai strain.A LB361 gene-transfected THP -1cell line was generated for evaluation of the mechanism of LB 361 gene products in elevating intracellular free Ca 2+( [Ca 2+] i) concentration and inducing the apoptosis of transfected THP -1 cells with the use of laser confocal microscopy and flow cytometry.Re sul ts The rL-PI-PLC hydrolyzed PIP2 into IP3 with a Km of 199 μmol/L and a Kcat of 8.566×10-5 S-1 .The expressions of LB361gene at mRNA and protein levels were both signifi -cantly up-regulated after infection of THP-1 cells with L.interrogans serovar Lai strain .Moreover , the exter-nal secretion of L-PI-PLC was also found during infection .The concentrations of IP 3 and [ Ca2+] i in the LB361 gene-transfected THP-1 cells were significantly increased compared to those in the non-transfected THP-1 cells, resulting in a high [Ca2+]i-dependent apoptosis of partial THP-1 cells.Conclusion PI-PLC is encoded by the LB361 gene of L.interrogans, which could induce the apoptosis of macrophages through el-evating [ Ca2+] i concentration during infection of microphages with L.interrogans.
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Objective To investigate the influence of three kinds of different Ca~(2+) concentration of dialysate on serum calcium,calcium-phosphorus product,serum PTH and CRP in maintenance hemodialysis patients.Methods 45 patients of maintenance hemodialysis with ages ≥18 years were selected.They were divided randomly three groups,and be given three different kinds of Ca~(2+) concentration of dialysate to carry out hemodialysis with 3 months.Then the patients' variation of blood pressure and whether there were low calcium spasm or ostealgia were abserved during hemodialysis whole range.In addition,changes of their serum calcium、phosphorus、iPTH and CRP at pretherapy and three months after reatment were also detected.Results After three months of hemodialysis,serum iPTH in patients with low Ca~(2+) concentration dialysate increased obviously,and the serum CRP decreased significantly(P0.05),and serum CRP increase notably(P
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Usage of the fruit and bark of a Melia-family plant as a digestive tract-parasiticide and agricultural insecticide was recorded about two thousant years ago in ancient China. Toosendanin (TSN), a triterpenoid, is an effectual ingredient extracted from the plant. Studies have demonstrated that TSN selectively affects neurotransmitter release, effectively antagonizes botulism, induces cell differentiation and apoptosis and inhibits proliferation of various human cancer cells, inhibits feeding and dovelopment in insects and modifies K+- and Ca2+-channel activity. The research data to demonstrate that TSN inhibits K+-channel and facilitates L-type Ca2+-channel are summarized, and the mechanism of action of TSN is discussed.
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To investigate the relationship between intracellular free Ca2+ concentration ([Ca2+]i)and calcium-activated chloride (Clca) channels of pulmonary artery smooth muscle cells (PASMCs) in rats under acute and chronic hypoxic conditions, acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca2+indicator Fura-2/AM was used to observe [Ca2+]i of rat PASMCs under normal and chronic hypoxic condition. The effect of Clca channels on PASMCs proliferation was assessed by MTT assay.The Clca channel blockersniflumic acid (NFA) and indaryloxyacetic acid (IAA-94) exerted inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. Under chronic hypoxic condition, [Ca2+ ]i was increased. Under normoxic condition, [Ca2+]i was (123.63±18.98) nmol/L, and in hypoxic condition, [Ca2+]i was (281. 75±16.48) nmol/L (P＜0.01). Under normoxic condition, [Ca2+]i showed no significant change and no effect on Clca channels was observed (P＞0. 05). Chronic hypoxia increased [Ca2+]i which opened Clca channels. The NFA and IAA-94blocked the channels and decreased [Ca2+]i from (281. 75±16.48) nmol/L to (117.66±15.36)nmol/L (P＜0.01). MTT assay showed that under chronic hypoxic condition NFA and IAA-94 decreased the value of absorbency (A value) from 0. 459±0. 058 to 0. 224±0. 025 (P＜0.01).Hypoxia increased [Ca2+]i which opened Clca channels and had a positive-feedback in [Ca2+]i. Thismay play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition,Clca channel may play a part in the regulation of proliferation of PASMCs.
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To prove the buffering contribution of mitochondria to the increase of intracellular Ca2+ level ([Ca2+]i) via background nonselective cation channel (background NSCC), we examined whether inhibition of mitochondria by protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) affects endothelial Ca2+ entry and Ca2+ buffering in freshly isolated rabbit aortic endothelial cells (RAECs). The ratio of fluorescence by fura-2 AM (R340/380) was measured in RAECs. Biological state was checked by application of acetylcholine (ACh) and ACh (10microM) increased R340/380 by 1.1+/-0.15 (mean+/-S.E., n=6). When the external Na+ was totally replaced by NMDG+, R340/380 was increased by 1.19+/-0.17 in a reversible manner (n=27). NMDG+-induced [Ca2+]i increase was followed by oscillatory decay after [Ca2+]i reached the peak level. The increase of [Ca2+]i by NMDG+ was completely suppressed by replacement with Cs+. When 1microM CCCP was applied to bath solution, the ratio of [Ca2+]i was increased by 0.4+/-0.06 (n=31). When 1microM CCCP was used for pretreatment before application of NMDG+, oscillatory decay of [Ca2+]i by NMDG+ was significantly inhibited compared to the control (p < 0.05). In addition, NMDG+-induced increase of [Ca2+]i was highly enhanced by pretreatment with 2microM CCCP by 320+/-93.7%, compared to the control (mean+/-S.E., n=12). From these results, it is concluded that mitochondria might have buffering contribution to the [Ca2+]i increase through regulation of the background NSCC in RAECs.
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Acetilcolina , Baños , Carbonil Cianuro m-Clorofenil Hidrazona , Células Endoteliales , Fluorescencia , Fura-2 , MitocondriasRESUMEN
Objective To investigate the stimulative effects of CollagenⅠon the increased adhesion of rabbit bone marrow stromal cells (BMSCs), cytoskeleton actin organization and intracellular free Ca~(2+) concentration. Methods The third generation BMSCs isolated from mature rabbits were cultured at different initial concentrations on cover-slice coated by collagenⅠin RPMI1640 containing 10% fetal calf serum, and cultured on the same kind of cover-slice untreated with collagenⅠas control. The cells adhesive behavior at different times was assessed. Cellular actin organization was described as either typeⅠor typeⅡcells. In general, typeⅠcells are round and represent a preliminary stage of actin assembly, while typeⅡcells are elongated with organized actin fiber network. At the same time intracellular free Ca~(2+) concentration was measured by using calcium fluorescent probe Fluo-3/AM and laser confocal microscope. Results We found more typeⅡcells in BMSCs cultured with collagen typeⅠsix hours after culture than in the control group. At 12 hours 89% of the BMSCs were typeⅡcells, while only 55% were typeⅡcells in the control group. This indicated active cellular actin organization after being modified by collagen typeⅠ. We also found that the BMSCs cultured with collagen typeⅠincreased intracellular free Ca~(2+) concentration in monolayer culture. Conclusions CollagenⅠis effective in promoting the cellular adhesion, which suggests that a kind of internal relationship or cross-talk may exist between cellular actin organization, intracellular free Ca~(2+) concentration and cell adhesion. Further study, however, is needed.
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Aim To evaluate the inhibitory effect of panaxynol(PNN) on the proliferation of rat aortic smooth muscle cell(RASMC) and its mechanisms.Methods Cell proliferation was determined using cell count and -TdR incorporation test.Fura-3/AM and confocal were used to measure intracelluar free Ca~(2+) concentration.Expression of mitochondrial transcription factor 1(mtTF1) mRNA was tested by using RT-PR.Results PNN inhibited the RASMC proliferation and DNA synthesis induced by serum and PDGF-BB in a dose-dependent manner.9 ?mol?L~(-1) of PNN inhibited the increase of intracelluar free Ca~(2+) concentration induced by PDGF-BB.PDGF-BB upregulated the expression of mtTF1 mRNA,which could be suppressed by 3,9 ?mol?L~(-1) of PNN significantly.Conclusions PNN can inhibit RASMC proliferation significantly,which might be related to the decrease of intracellular free Ca~(2+) concentration and mtTF1 mRNA expression.
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In a myocyte freshly isolated from rabbit cerebral artery, the characteristics of Ca2+ release by histamine or caffeine were studied by microspectrofluorimetry using a Ca2+-binding fluorescent dye, fura-2. Histamine (5 micrometer) or caffeine (10 mM) induced a phasic rise of cytoplasmic free Ca2+ concentration ((Ca2+)c) which could occur repetitively with extracellular Ca2+ but only once or twice in Ca2+-free bathing solution. Also, the treatment with inhibitor of sarcoplasmic reticulum Ca2+-ATPase suppressed the rise of (Ca2+)c by histamine or caffeine. In Ca2+-free bathing solution, short application of caffeine in advance markedly attenuated the effect of histamine, and vice versa. In normal Ca2+-containing solution with ryanodine (2 micrometer), the caffeine-induced rise of (Ca2+)c occurred only once and in this condition, the response to histamine was also suppressed. On the other hand, in the presence of ryanodine, histamine could induce repetitive rise of (Ca2+)c while the amplitude of peak rise became stepwisely decreased and eventually disappeared. These results suggest that two different Ca2+-release mechanisms (caffeine-sensitive and histamine-sensitive) are present in rabbit cerebral artery myocyte and the corresponding pools overlap each other functionally. Increase of (Ca2+)c by histamine seems to partially activate ryanodine receptors present in caffeine-sensitive pool.