Effects of Na+-H+ exchanger 1 knockdown on protein expression levels of ATP binding cassette transporter A1 and cholesterol efflux in hypoxic RAW264.7 cells / 中华老年医学杂志

Objective To explore the effects of Na+ H-exchanger 1(NHE1) knockdown on ATP binding cassette transporter A1 (ABCA1) protein expression levels and cholesterol efflux in the hypoxic RAW264.7 cells.Methods The RAW264.7 cells were infected with lentiviral vectors expressing shRNA specific for NHE1(siNHE1) or scramble RNA (siNC).The expression of NHE1 at mRNA or protein level was detected by qRT-PCR and Western blotting respectively in the infected cells after 24 h in a hypoxia condition.In the meantime,the methods of SNARF-1,Fluo-4 NW andSuc-LLVY-aminoluciferin were employed to determine NHE1 activity,intracellular Ca2+ ([Ca2+]i) and calpain activity,respectively.Furthermore,ABCA1 protein levels were detected by Western blotting in the 24 h hypoxic cells.In parallel,the intracellular cholesterol content and cholesterol efflux were analyzed by the methods of combined enzymatic HLPC and 3 H-cholesterol.Results The hypoxia condition versus the normoxia condition up-regulated NHE1 mRNA and protein expression level and activity by 2.48 folds,1.28 folds and 61.96％ (all P＜0.05),and increased[Ca2+]i and calpain activity by 4.51 folds and 2.41 folds(all P＜0.05).Whereas the NHE1 mRNA and protein expression and activity at the presence of hypoxia were inhibited by siNHE1 with the inhibition ratio of 84.95％,60.75％ and 66.44％,respectively (all P＜0.05)and[Ca2+]i and calpain activity were reduced by 59.23％ and 54.66％ (P＜0.05).Furthermore,the ABCA1 protein level was 61.67％ lower in the hypoxic cells than in the normoxic cells (P＜0.05),and siNHE1 was increased by 56.52％ after treatment of Hypoxia.Hypoxia elevated intracellular total cholesterol and cholesterol ester by 74.57 ％ and 101.81％ (all P＜0.05).Treatment with siNHE1 in the hypoxia condition can reduce total cholesterol and cholesterol ester by 34.24 ％ 及 49.66 ％ (all P＜0.05).Hypoxia reduced the cholesterol efflux by 34.79％(P＜0.05),which were partially reversed by siNHE1.Conclusions NHE1 might play an important role in hypoxia-induced ABCA1 protein attenuation and reverse cholesterol transport dysfunction through[Ca2+]i/calpain pathway.

Effects of Na+ /H+ exchanger 1 inhibitor on intestinal injury of rats with burn sepsis and the mechanism / 中华烧伤杂志

Objective@#To observe the effects of Na+ /H+ exchanger 1 (NHE1) inhibitor on intestinal injury of rats with burn sepsis, and to explore the possible mechanism preliminarily.@*Methods@#Ninety SD rats were divided into control group, pure sepsis group, and NHE1 inhibitor group according to the random number table, with 30 rats in each group. Full-thickness scald (hereinafter referred to as burn) model with 20% total body surface area were reproduced on the back of rats in pure sepsis and NHE1 inhibitor groups, and then 50 μL liquid of Pseudomonas aeruginosa ATCC 27853 (2×105 colony forming unit/mL) were injected into the center of wounds on the back. Rats in NHE1 inhibitor group were intraperitoneally injected with 0.1 mmol/L NHE1 inhibitor cariporide (0.4 mg/kg) rapidly after the successful establishment of burn sepsis model, while rats in pure sepsis group were injected with the same volume of normal saline. Except for not being made burn wounds nor receiving bacterination, rats in control group were treated the same as those in pure sepsis group. Rats with burn sepsis in each group were laparotomized and injected with 200 mL fluorescein isothiocyanate (FITC)-dextran in the concentration of 0.1 mol/L in terminal ileum at 12 hours post injury, and their left ventricular blood and terminal ileum were collected 30 minutes later. The serum content of FITC-dextran was detected with fluorescence spectrophotometer (n=10); the morphology of intestinal tissue was observed with HE staining (n=10); the content of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in serum and intestinal tissue was determined with enzyme-linked immunosorbent assay (n=20); the activity of myeloperoxidase (MPO) in serum and intestinal tissue was detected with colorimetric method (n=20); the protein expression of nuclear factor-kappa B-p65 (NF-κB-p65) and phosphorylation levels of mitogen-activated protein kinase (MAPK) signal pathway related proteins p38MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinase 1/2 (JNK1/2) were determined by Western blotting (n=4). The same samples of rats in control group were collected for related detection at the same time point as above. Data were processed with one-way analysis of variance and SNK test.@*Results@#(1) The serum content of FITC-dextran of rats in pure sepsis group was significantly higher than that in control group (P<0.01), while the serum content of FITC-dextran of rats in NHE1 inhibitor group was significantly lower than that in pure sepsis group (P<0.01). Compared with that in control group, infiltration of a large number of inflammatory cells, ulcer and necrosis of intestinal mucosa of rats in pure sepsis group were observed. The injury condition of intestine of rats in NHE1 inhibitor group was better than that in pure sepsis group. (2) The serum content of IL-6, TNF-α, and MPO of rats in pure sepsis group was (387±42) and (164.7±10.1) ng/mL, and (7.5±1.5) U/mL, respectively, significantly higher than that in control group [(75±17) and (13.1±6.5) ng/mL, and (2.3±0.7) U/mL, respectively, with P values below 0.01]. The serum content of IL-6, TNF-α, and MPO of rats in NHE1 inhibitor group was (176±37) and (64.9±9.3) ng/mL, and (5.9±0.8) U/mL, respectively, which was significantly lower than that in pure sepsis group (with P values below 0.01). (3) The content of IL-6, TNF-α, and MPO in intestinal tissue of rats in pure sepsis group was (190±13) and (172.8±29.7) ng/mL, and (8.7±1.5) U/mL, respectively, significantly higher than that in control group [respectively (20±3) and (11.9±2.3) ng/mL, and (2.9±0.3) U/mL, with P values below 0.01]. The content of IL-6, TNF-α, and MPO of intestinal tissue of rats in NHE1 inhibitor group was (35±6) and (45.2±6.1) ng/mL, and (5.3±0.6) U/mL, respectively, significantly lower than that in pure sepsis group (with P values below 0.01). (4) The protein expression of NF-κB-p65 and phosphorylation levels of p38MAPK and ERK1/2 in intestinal tissue of rats in pure sepsis group were significantly higher than those in control group (with P values below 0.01); the protein expression of NF-κB-p65 and the phosphorylation level of p38MAPK in intestinal tissue of rats in NHE1 inhibitor group were significantly lower than those in pure sepsis group (with P values below 0.01); phosphorylation levels of JNK1/2 in intestinal tissue of rats in the three groups were similar (with P values above 0.05).@*Conclusions@#The inhibition of NHE1 can significantly alleviate the intestinal injury, and the mechanisms may be attributed to the regulation of NF-κB and p38MAPK signal pathway, resulting in inhibition of the inflammatory response of intestinal tract.

Sustained Intracellular Acidosis Triggers the Na⁺/H⁺ Exchager-1 Activation in Glutamate Excitotoxicity

The Na⁺/H⁺ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular H⁺ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-β (PKC-β) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to 100 μM glutamate or 20 mM NH₄Cl. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-β, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to NH₄Cl. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-β, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-β/ERK1/2/p90RSK pathway in neuronal cells.

The effects of NHE -1 on the proliferative and invasive abilities of esophageal carcinoma cell line KYSE-70 in vitro / 肿瘤

Objective: To observe the effects of NHE -1 (Na+/H+ exchanger 1) gene on the proliferative and invasive abilities of esophageal cancer cell line KYSE-70. Methods: The expressions of NHE-1 mRNA and protein in esophageal cancer EC-9706, KYSE-450, EC-1 and KYSE-70 cells were detected by realtime fluorescence quantitative-PCR and Western blotting, respectively. Chemically synthesized siRNA (small interference RNA) targeting NHE -1 was transfected into KYSE-70 cells. The expression levels of NHE-1 mRNA and protein in esophageal cancer KYSE-70 cells 24, 48 and 72 h after transfection with NHE-1 siRNA were detected by real-time fluorescence quantitative-PCR and Western blotting, respectively. The proliferative and invasive abilities of KYSE-70 cells were detected by MTT method and Boyden chamber assay, respectively. Results: NHE-1 mRNA and protein were highly expressed in KYSE-70 cells. As compared with the negative control (transfected with nonsense siRNA) and blank control (KYSE-70 cells without transfection), the expression levels of NHE-1 mRNA and protein in KYSE-70 were significantly decreased after transfection with siRNA targeting NHE -1 (all P < 0.05). As compared with the negative control, blank control and the KYSE-70 cells transfected with NHE-1 siRNA for 24 h, the proliferative and invasive abilities of KYSE-70 were inhibited after transfection with NHE-1 siRNA for 48 and 72 h (all P < 0.05). Conclusion: Silencing the expression of NHE -1 gene can inhibit the proliferative and invasive abilities of esophageal cancer KYSE-70 cells in vitro . NHE -1 gene is expected to become an important target for gene therapy of esophageal cancer. Copyright © 2013 by TUMOR.

Protective mechanism of NHE-1-siRNA on human renal tubular epithelial cell from ischemic reperfusion injury induced by antimycin A / 中华肾脏病杂志

Objective To explore the mechanism of protecting cells from ischemic reperfusion injury by constructing specific small interference RNA (siRNA) to inhibit Na+-H+exchanger-1 (NHE-1) expression in human renal tubular epithelial cell (HKC). Methods The siRNA was designed and synthesized based on human NHE-1 complete sequence,and was transfected into HKC.The irrespective siRNA transfected group was used as control.The cells were treated with 10 μmol/L antimyein A to induce ischemia and anoxyaemia environment.NHE-1expression was examined by RT-PCR and Western blot.The intraeellular pH (pHi),Ca2+ or Na+ concentrations were detected by BCECF/AM,Fluo-3/AM and SBFI-AM,respectively,combining with laser eonfocal assay system.Nucleic morphology was determined by Hoechst 33342.Cellular apoptosis was examined by Annexin V/PI staining and flow eytometry.Fluorescent probe JC-1 was used to detect the change of mitechondrial transmembrane potential. Results The specific siRNA could efficiently inhibit NHE-1 expression in HKC.Compared with the irrespective siRNA transfected group,the mRNA and protein expression of NHE-1 was significantly down-regulated in NHE-1 siRNA transfeeted group (all P＜0.05).After treatment with antimyein A,the mRNA and protein expression of NHE-1 was significantly up-regulated in both groups,however,it was less than that in irrespective siBNA transfected group.At the same time,the ratio of apoptosis decreased (8.9% +2.9% vs 18.8%±3.2% , 17.4%±3.6% ,P＜0.05) and mitochondrial transmembrane potential rose significantly in NHE-1 siRNA transfected group as compared to irrespective siRNA transfected group and antimycin A group.The intracellular Na+,H+ and Ca2+concentrations increased in NHE-1 siRNA transfected group treated with antimyein A,but their levels were lower than those in irrespective siRNA transfected group with the same treatment(P＜0.05). Conclusions The synthesized siBNA can inhibit the expression of NHE-1 and can protect HKC from isehemia reperfasion injury induced by antimyein A.The mechanism might be via suppressing the expression of NHE-1 to delay intracelluar Na+ accumulation,attenuate intracellular Ca2+ overloading,and inhibit the decrease of mitechondrion transmembrane potential and reduce cellular apoptosis.

Role of Na~+/H~+ Exchanger 1 on Injury of Human Renal Tubular Cell Induced by Postasphyxial-Serum of Neonates / 实用儿科临床杂志

Objective To explore the role of Na~+/H~+ exchanger 1(NHE1) in injury of human renal tubular cells(HK-2) induced by postasphyxial-serum of neonates.Methods HK-2 was used as the target cell.The attacking concentration of postasphyxial-serum of neonates was 200 mL/L.First,the experiment was designed as control group and asphyxia group,the expression of NHE1 in the HK-2 was detected by immunohisto chemical method in the cells.Second,the experiment group was designed as control group,asphyxia group,and pretreatment with 5-N-Ethyl-N-isopropylamiloride(EIPA) group,then the change of morphology was observed under inverted microscope,and the cell viability was measured by methyl thiazolyl tetrazolium(MTT) method,and the leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.Results Compared with blank control group,the expression of NHE1 in the HK-2 was increased signi-ficantly in asphyxia group,the changes of morphology of HK-2 was most serious and obvious,the cell viability decreased,and the leakage rate of LDH increased significantly in asphyxia group.But compared with asphyxia group,the change of morphology of HK-2 was greatly improved,the cell injury was decreased obviously,the leakage rate of LDH was increased and viability was decreased in pretreatment group in a dose 2 ?mol/L.Conclusions The postasphyxial-serum may induce the expression of NHE1,which plays an important role in injury of renal tubular cell induced by postasphyxial-serum in neonates,and inhibiting activity of NHE1 may relieve the injury of renal tubular cells induced by postasphyxial-serum in neonates.