[Alleviating effects of hydrogen on hyperhomocysteinemia and fatty liver induced by high-methionine diet].

Objective: To investigate the alleviating effect of hydrogen (H2) on homocysteine (Hcy) levels and non alcoholic fatty liver in rats with hyperhomocysteinemia (HHcy). Methods: After one week of adaptive feeding, Wistar rats were randomly divided into three groups: the general diet group (CHOW), the high methionine group (HMD), and the high methionine plus hydrogen rich water group (HMD+HRW), with 8 rats in each group. The CHOW group was fed with AIN-93G feed, while the HMD and HMD+HRW groups were fed with AIN-93G+2% methionine feed to construct an HHcy model. The HMD+HRW group was also gavaged with hydrogen rich water (3 ml/animal, twice a day, with a hydrogen concentration of 0.8 mmol/L), and body weight data were recorded. After 6 weeks of feeding, the plasma and liver samples were processed and collected. The plasma homocysteine (Hcy) and lipid contents of each group were measured, and the histological morphology of the liver was observed. The activities of key enzymes in the Hcy metabolism pathway and mRNA expression were detected in the liver. Results: Compared with the CHOW group rats, the Hcy level in the blood of HMD rats was significantly increased significantly (P＜0.05). Pathological tissue sections showed liver enlargement, injury, and fatty liver in the rats; Compared with the HMD group rats, the HMD+HRW group rats showed a significant decrease in Hcy in the blood, reduced liver damage, and increased Hcy metabolism key enzyme activity and mRNA expression in the liver, with statistical differences (P＜0.05). Conclusion: Hydrogen has a significant improvement effect on liver injury induced by HMD diet in HHcy rats, possibly by enhancing the three metabolic pathways of Hcy to reduce excessive Hcy in the body, thereby improving liver metabolic function and symptoms of non-alcoholic fatty liver disease.

Edaravone Ameliorates Renal Warm Ischemia-Reperfusion Injury by Downregulating Endoplasmic Reticulum Stress in a Rat Resuscitation Model.

BACKGROUND: This study was conducted to explore whether the effect of edaravone (5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol3-one, EDR) can ameliorate renal warm ischemia-reperfusion injury (IRI) by modulating endoplasmic reticulum stress (ERS) and its downstream effector after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in a rat model. METHODS: The rats (n=10) experienced anaesthesia and intubation followed by no CA inducement were defined as the Sham group. Transoesophageal alternating current stimulation was employed to establish 8 min of CA followed by conventional CPR for a resuscitation model. The rats with successful restoration of spontaneous circulation (ROSC) randomly received EDR (3 mg/kg, EDR group, n=10) or equal volume normal saline solution (the NS group, n=10). At 24 hr after ROSC, serum creatinine (SCR), blood urea nitrogen (BUN) levels, and cystatin-C (Cys-C) levels were determined and the protein level of glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), extracellular signal-regulated kinase (ERK), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), Bax/Bcl-2, and caspase-3 were detected by Western blot method. RESULTS: At 24 hrs after ROSC, SCR, BUN and Cys-C were obviously increased and the proteins expression, including GRP78, CHOP and p-ERK1/2, cleaved-caspase 3 Bax/Bcl-2 ratio, were significantly upregulated in the NS group compared with the Sham group (p<0.05). The remarkable improvement of these adverse outcomes was observed in the EDR group (p<0.05). CONCLUSION: In conclusion, we found that EDR ameliorates renal warm IRI by downregulating ERS and its downstream effectors in a rat AKI model evoked by CA/CPR. These data may provide evidence for future therapeutic benefits of EDR against AKI induced by CA/CPR.

Liver ChREBP Protects Against Fructose-Induced Glycogenic Hepatotoxicity by Regulating L-Type Pyruvate Kinase.

Excessive fructose consumption is closely linked to the pathogenesis of metabolic disease. Carbohydrate response element-binding protein (ChREBP) is a transcription factor essential for fructose tolerance in mice. However, the functional significance of liver ChREBP in fructose metabolism remains unclear. Here, we show that liver ChREBP protects mice against fructose-induced hepatotoxicity by regulating liver glycogen metabolism and ATP homeostasis. Liver-specific ablation of ChREBP did not compromise fructose tolerance, but rather caused severe transaminitis and hepatomegaly with massive glycogen overload in mice fed a high-fructose diet, while no obvious inflammation, cell death, or fibrosis was detected in the liver. In addition, liver ATP contents were significantly decreased by ChREBP deficiency in the fed state, which was rendered more pronounced by fructose feeding. Mechanistically, liver contents of glucose-6-phosphate (G6P), an allosteric activator of glycogen synthase, were markedly increased in the absence of liver ChREBP, while fasting-induced glycogen breakdown was not compromised. Furthermore, hepatic overexpression of LPK, a ChREBP target gene in glycolysis, could effectively rescue glycogen overload and ATP reduction, as well as mitigate fructose-induced hepatotoxicity in ChREBP-deficient mice. Taken together, our findings establish a critical role of liver ChREBP in coping with hepatic fructose stress and protecting from hepatotoxicity by regulating LPK.

Association between miR-499 rs3746444 polymorphism and coronary heart disease susceptibility: An evidence-based meta-analysis of 5063 cases and 4603 controls.

MicroRNA-499 (miR-499) rs3746444 polymorphism has been associated with the risk of coronary heart disease (CHD). However, results from several studies are inconsistent. This meta-analysis aimed to further investigate the possible association between miR-499 rs3746444 polymorphism and CHD risk. A total of 9 case-control studies included 5063 CHD cases and 4603 healthy subjects. The A allele at rs374644 was associated with significantly decreased CHD risk in the total population according to the allelic model (OR = 0.80, 95% CI = 0.68-0.93, P = 0.005), homozygous model (OR = 0.52, 95% CI = 0.39-0.71, P < 0.001) and heterozygous model (OR = 0.57, 95% CI = 0.43-0.77, P < 0.001). A similar trend was found specifically in Asian and Chinese populations. In contrast, the wild-type GG genotype at rs374644 was associated with significantly increased CHD risk in the total population, according to the dominant model (OR = 1.83, 95% CI = 1.39-2.42, P < 0.001), and a similar trend was found in Asian and Chinese populations. These results indicate that in the total population, as well as in Asian and Chinese populations, the wild-type GG genotype at rs374644 may be related to increased susceptibility to CHD, while the A allele may be protective against CHD.

ZBTB20 regulates EGFR expression and hepatocyte proliferation in mouse liver regeneration.

Liver has a unique regenerative capacity, however, its regulatory mechanism is not fully defined. We have established the zinc-finger protein ZBTB20 as a key transcriptional repressor for alpha-fetoprotein (AFP) gene in liver. As a marker of hepatic differentiation, AFP expression is closely associated with hepatocyte proliferation. Unexpectedly, here we showed that ZBTB20 acts as a positive regulator of hepatic replication and is required for efficient liver regeneration. The mice specifically lacking ZBTB20 in hepatocytes exhibited a remarkable defect in liver regeneration after partial hepatectomy, which was characterized by impaired hepatocyte proliferation along with delayed cyclin D1 induction and diminished AKT activation. Furthermore, we found that epithelial growth factor receptor (EGFR) expression was dramatically reduced in the liver in the absence of ZBTB20, thereby substantially attenuating the activation of EGFR signaling pathway in regenerating liver. Adenovirus-mediated EGFR overexpression in ZBTB20-deficient hepatocytes could largely restore AKT activation in response to EGFR ligands in vitro, as well as hepatocyte replication in liver regeneration. Furthermore, ZBTB20 overexpression could significantly restore hepatic EGFR expression and cell proliferation after hepatectomy in ZBTB20-deficient liver. Taken together, our data point to ZBTB20 as a critical regulator of EGFR expression and hepatocyte proliferation in mouse liver regeneration, and may serve as a potential therapeutic target in clinical settings of liver regeneration.

Estrogen enhances mismatch repair by induction of MLH1 expression via estrogen receptor-ß.

Epidemiological data demonstrated that hormone replace treatment has protective effect against colorectal cancer (CRC). Our previous studies showed that this effect may be associated with DNA mismatch repair. This study aims to investigate the mechanism of estrogen induction of MLH1, and whether colorectal tumor proliferation can be inhibited through induction of MLH1 by estrogen signal pathway. Human CRC cell lines were used to examine the regulation of MLH1 expression by over-expression and depletion of estrogen receptor-α (ERα) and estrogen receptor-ß (ERß), under the treatment with 17ß-estradiol or ß-Estradiol 6-(O-carboxy-methyl)oxime:BSA, followed by a real-time Q-PCR and Western blotting analysis. Luciferase reporter and chromatin immunoprecipitation assays were used to identify the estrogen response elements in the proximal promoter of MLH1 gene. Then, the influence of estrogen-induced MLH1 on CRC tumor growth were determined in vitro and in vivo. We found that mismatch repair ability and microsatellite stability of cells were enhanced by estrogen via induction of MLH1 expression, which was mediated by ERß, through a transcriptional activation process. Furthermore, we identified that ERß exerted an inhibitory effect on CRC tumor proliferation in vitro and in vivo, combined with 5-FU, through up-regulation of MLH1 expression. Finally, we concluded that estrogen enhances mismatch repair ability and tumor inhibition effect in vitro and in vivo, via induction of MLH1 expression mediated by ERß.

Regulation of hepatic lipogenesis by the zinc finger protein Zbtb20.

Hepatic de novo lipogenesis (DNL) converts carbohydrates into triglycerides and is known to influence systemic lipid homoeostasis. Here, we demonstrate that the zinc finger protein Zbtb20 is required for DNL. Mice lacking Zbtb20 in the liver exhibit hypolipidemia and reduced levels of liver triglycerides, along with impaired hepatic lipogenesis. The expression of genes involved in glycolysis and DNL, including that of two ChREBP isoforms, is decreased in livers of knockout mice. Zbtb20 binds to and enhances the activity of the ChREBP-α promoter, suggesting that altered metabolic gene expression is mainly driven by ChREBP. In addition, ChREBP-ß overexpression largely restores hepatic expression of genes involved in glucose and lipid metabolism, and increases plasma and liver triglyceride levels in knockout mice. Finally, we show that Zbtb20 ablation protects from diet-induced liver steatosis and improves hepatic insulin resistance. We suggest ZBTB20 is an essential regulator of hepatic lipogenesis and may be a therapeutic target for the treatment of fatty liver disease.

Advances in the study of Lynch syndrome in China.

Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer, is an autosomal dominant genetic condition that has a high risk of colon cancer as well as other cancers due to inherited mutations in mismatch repair (MMR) genes. During the last decades, there have been great advances in research on Chinese Lynch syndrome. This review mainly focuses on the genetic basis, clinicopathologic features, diagnosis, intervention, chemoprevention, and surveillance of Lynch syndrome in China. In addition to frequently altered MMR genes, such as MLH1, MSH2, MSH6, and MLH3, other MMR-associated genes, such as those encoding human exonuclease 1, transforming growth factor ß receptor 2, and alanine aminopeptidase, metastasis-associated protein 2, adenomatosis polyposis coli down-regulated 1, and hepatic and glial cell adhesion molecule have also been implicated in Chinese Lynch syndrome. Most Chinese researchers focused on the clinicopathologic features of Lynch syndrome, and it is noticeable that the most frequent extracolonic tumor in northeast China is lung cancer, which is different from other areas in China. The Chinese diagnostic criteria for Lynch syndrome have been established to identify gene mutation or methylation. With regard to chemoprevention, celecoxib may be effective to prevent polyps relapse in Lynch syndrome carriers. Additionally, a colonoscopy-based surveillance strategy for the prevention and early detection of neoplasms in Lynch-syndrome carriers has been proposed.

[Study of reason of "the older, the better" of Pericarpium Citri Reticulatae (PCR) which contributed increase of flavonoids].

This study is to explore the reason of "the older, the better" of PCR and itsincrease of flavonoids. We identified the fun- gus isolated from the PCR using microscopic and molecular identification. HPLC method was used to determine the content of 4 fla- vonoids and to clarifythe regularity of them; UV spectrophotometry method was used to determine the total content of flavonoids; reverse thinking was applied to screen the fungus that have close relation to the change of flavonoids. Finally, we have isolated and identified 25 fungusfrom the PCR, which belong to 2 genus and 4 species, including pencillium commune, P. minioluteeum, P. citrinum, Aspergillus flavus and A. niger. The content of flavonoids was increased in the mildew PCR due to A. niger and other fungus. Therefore, "the ol- der, the better" of PCR had its scientific reason that the increase of flavonoids had a close relation of the metabolic activity of A. niger and other fungus.

[Adenovirus-mediated protein-kinase-GIalpha suppresses the hypoxia-induced proliferation and phenotype-switching of pulmonary arterial smooth muscle cell].

OBJECTIVE: To observe the proliferation and phenotype-switching of pulmonary arterial smooth muscle cell (PASMC) induced by hypoxia and interfered by Ad-PKGIα. And to investigate the potential regulative role of PKGIα gene in the molecule mechanism of hypoxia pulmonary vessel remodeling (HPVR). METHODS: To establish the pure PASMC cultured by tissue-sticking methods. Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) and Western blot were used to examine the PKGIα mRNA and protein expression after PASMC were transfected by Ad-PKG. The mRNA and protein expressive change of smooth muscle alpha actin (SM-alpha-actin) determined the degree of cell phenotype-switching. The changes of PASMC proliferation were determined by flow cytometry and ³H-TdR incorporated way. RESULTS: Ad-PKGIα could transfect into PASMC and highly express. Hypoxia down-regulated the expression of SM-alpha-actin protein (44.25 ± 5.34 in normoxia, 32.18 ± 4.19 in 12 h hypoxia condition, 21.90 ± 2.44 in 24 h hypoxia condition, P < 0.05), that could be blocked by the transfection of Ad-PKGIα. Hypoxia could push PASMC mitosis and proliferating (³H-TdR incorporated way: 7570 ± 371 in normoxia, 12,020 ± 831 in 12 h hypoxia condition, 14,924 ± 1491 in 24 h hypoxia condition, P < 0.05), that could be blocked by the transfection of Ad-PKGIα, too. CONCLUSIONS: The results suggested that PKGIα signaling pathway might play an important role in the molecule mechanism of HPVR. And PKGIα gene might be a target point of gene therapy.

What is the optimal dose of epinephrine during cardiopulmonary resuscitation in a rat model?

OBJECTIVE: Because different species may require different doses of drug to produce the same physiologic response, we were provoked to evaluate the dose-response of epinephrine during cardiopulmonary resuscitation (CPR) and identify what is the optimal dose of epinephrine in a rat cardiac arrest model. METHODS: Rat cardiac arrest was induced via asphyxia, and then the effects of different doses of epinephrine (0.04, 0.2, and 0.4 mg/kg IV, respectively) and saline on the outcome of CPR were compared (n = 10/each group). The primary outcome measure was restoration of spontaneous circulation (ROSC), and the secondary was the change of spontaneous respiration and hemodynamics after ROSC. RESULTS: Rates of ROSC were 9 of 10, 8 of 10, 7 of 10, and 1 of 10 in the low-dose, medium-dose, and high-dose epinephrine groups and saline group, respectively. The rates of withdrawal from the ventilator within 60 minutes in the low-dose (7 of 9) and medium-dose epinephrine groups (7 of 8) were higher than in the high-dose epinephrine group (1 of 7, P < .05). Mean arterial pressures were comparable, but the heart rate in the high-dose epinephrine group was the lowest among epinephrine groups after ROSC. These differences in part of time points reached statistical significance (P < .05). CONCLUSION: Different doses of epinephrine produced the similar rate of ROSC, but high-dose epinephrine inhibited the recovery of spontaneous ventilation and caused relative bradycardia after CPR in an asphyxial rat model. Therefore, low and medium doses of epinephrine were more optimal for CPR in a rat asphyxial cardiac arrest model.

Dose-response of vasopressin in a rat model of asphyxial cardiac arrest.

The advantage of vasopressin over epinephrine in the treatment of cardiac arrest (CA) is still being debated, and it is not clear whether a high dose of vasopressin is beneficial or detrimental during or after cardiopulmonary resuscitation (CPR) in a rat model of CA. In this study, asphyxial CA was induced in 40 male Sprague-Dawley rats. After 10 minutes of asphyxia, CPR was initiated; and the effects of different doses of vasopressin (low dose, 0.4 U/kg; medium dose, 0.8 U/kg; and high dose, 2.4 U/kg; intravenous; n = 10 in each group) and a saline control (isotonic sodium chloride solution, 1 mL, intravenous) were compared. Outcome measures included the rate of restoration of spontaneous circulation (ROSC) and changes of hemodynamic and respiratory variables after ROSC. The rates of ROSC were 1 of 10 in the saline group and 8 of 10 in each of the 3 vasopressin groups. There were no differences in mean aortic pressure or changes of respiratory function after CPR among the vasopressin groups. However, the heart rate was lower in the high-dose vasopressin group than in the low- and medium-dose groups. These findings indicate that different doses of vasopressin result in a similar outcome of CPR, with no additional benefits afforded by a high dose of vasopressin during or after CPR, in a rat model of asphyxial CA. The mechanism and physiologic significance of the relative bradycardia that occurred in the high-dose vasopressin group are currently unknown and require further investigation.

[Effects of all-trans retinoic acid on phenotype switch and migration of human pulmonary artery smooth muscle cells in vitro and the possible mechanisms].

OBJECTIVE: To investigate the effects of all-trans retinoic acid (atRA) on phenotype switch and migration of human pulmonary artery smooth muscle cells (PASMC) in vitro and the possible mechanisms. METHODS: Cultured human PASMC were divided into 8 groups randomly: group A (control group), group B (vehicle group), group C (treated with 10% fetal cattle serum), group D, group E, group F, group G, and group H (treated with atRA at the concentrations of 0.001 micromol/L, 0.01 micromol/L, 0.1 micromol/L, 1 micromol/L, 10 micromol/L respectively as well as 10% fetal cattle serum). Then, expression of JWA mRNA in human PASMC was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), cell migration was measured using modified Boyden's chamber, and expression of SM-alpha-actin mRNA and protein was detected by RT-PCR and immunohistochemistry respectively. The difference between the groups was analyzed. RESULTS: There was no significant difference in the JWA mRNA expression of human PASMC between group A (0.125 +/- 0.014), group B (0.164 +/- 0.018) and group C (0.164 +/- 0.006), but the JWA mRNA expression in group D (0.326 +/- 0.018), group E (0.440 +/- 0.033), group F (0.460 +/- 0.040), group G (0.589 +/- 0.024) and group H (0.821 +/- 0.050) were significantly higher than those of group A, group B and group C (all P < 0.01). The number of migrating cells in group C (30.4 +/- 7.4) was higher than those in group A (7.2 +/- 1.9) and group B (6.8 +/- 2.3, all P < 0.01), but the number of migrating cells in group D (21.8 +/- 2.9), group E (17.2 +/- 2.3), group F (14.4 +/- 3.5), group G (12.6 +/- 3.4) and group H (8.8 +/- 2.4) was significantly lower than that in group C (all P < 0.01), and there was no significant difference between group G, group H and group A, group B. The SM-alpha-actin protein expression in group C (0.219 +/- 0.018) was significantly lower than those in group A (0.319 +/- 0.011) and group B (0.325 +/- 0.005, all P < 0.01), but the protein expression in group E (0.328 +/- 0.016), group F (0.386 +/- 0.025), group G (0.442 +/- 0.017) and group H (0.501 +/- 0.018) was significantly higher than that in group C (all P < 0.01), while that in group F, group G and group H was significantly higher than those in group A and group B (all P < 0.01). The SM-alpha-actin mRNA expression in group C (0.144 +/- 0.009) was significantly lower than those in group A (0.299 +/- 0.023) and group B (0.296 +/- 0.041, all P < 0.01), but the mRNA expression in group D (0.487 +/- 0.014), group E (0.501 +/- 0.020), group F (0.611 +/- 0.018), group G (0.774 +/- 0.013) and group H (0.851 +/- 0.026) was significantly higher than those in group A, group B and group C (all P < 0.01). CONCLUSION: atRA increased JWA gene expression in human pulmonary artery smooth muscle cells, and meanwhile, the cells differentiated into a contractive phenotype and the cell migration decreased.

[Role of Fas and FasL expression in apoptosis of pulmonary artery smooth muscle cells in hypoxia induced by inhibition of Na+/H+ exchanger isoform-1 in rat].

OBJECTIVE: To investigate the role of Fas and FasL expression in apoptosis of pulmonary artery smooth muscle cells (PASMCs) in hypoxia induced by inhibition of Na(+)/H(+) exchanger isoform-1 (NHE-1) in rat. METHODS: The cells were cultured in hypoxia (<1% O(2)) for 2, 6, 12, 24 and 48 hours respectively. Then cell apoptosis was observed with terminal deoxynudeotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The changes in fas and fasl mRNA expression in PASMCs were detected by semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR) in vitro. The expression of Fas and FasL protein in cells was determined immunohistochemically. RESULTS: No significant changes in the expression of fas/fasL mRNA and protein between cells transfected with NHE-1 ribozyme gene and cells transfected with pLXSN, or non-transfected control in hypoxia. CONCLUSION: Apoptosis of PASMCs induced by NHE-1 inhibition may occur independently of Fas/FasL death pathway.

[Role of bcl-2 and bax expression in apoptosis of hypoxic pulmonary artery smooth muscle cells of rats induced by Na+/H+ exchanger isoform-1 inhibition in vitro].

OBJECTIVE: To investigate the role of bcl-2 and bax expression in apoptosis of hypoxic pulmonary artery smooth muscle cells (PASMCs) of rats induced by Na+/H+ exchanger isoform-1 (NHE-1) inhibition in vitro. METHODS: Cultured rat PASMCs transfected with NHE-1 ribozyme gene (PRZ cell), cultured rat PASMCs transfected only with pLXSN vector (PX cell) and non-transfected rat PASMCs (PA cell) were obtained in our previous studies. The cells were cultured under low oxygen tension (<1% O2) for 0, 2, 6, 12, 24 and 48 hours respectively. Then cell apoptosis was examined with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Intracellular Ca2+([Ca2+]i) of cells was measured using fluorescence dye Fura-2/AM. The changes in mRNA expression of bcl-2 and bax in cells were assessed by semi quantitative reverse transcription-polymerase chain reaction (sqRT-PCR). The expression of Bcl-2 protein and Bax protein of cells was detected immunohistochemically. RESULTS: In contrast to normal PASMCs and cells transfected with pLXSN, the apoptosis rates in recombinant vectors transfected cells increased significantly and continuously. [Ca2+]i values were also increased in these cells, but the increase extent was remarkably lower than in the normal PASMCs (all P<0.01). The expression of bax mRNA and protein of cells transfected with recombinant vectors was increased significantly compared with cells of other two groups. Meanwhile, the expression of bcl-2 mRNA and protein of these cells were decreased significantly. CONCLUSION: The NHE-1 specific hammerhead ribozymes induce apoptosis of PASMCs under low oxygen tension by inhibiting the increase in intracellular Ca2+ concentration, increasing bax expression and decreasing bcl-2 expression.

[The effect of Na(+)/H(+) exchanger-1 (NHE-1) antisense expression vector on NHE-1 gene expression in human lung adenocarcinoma cells and its biological significance].

OBJECTIVE: To explore the inhibitory effect of Na(+)/H(+) exchanger-1 (NHE-1) antisense expression vector on NHE-1 gene in human lung neoplasm cells and to observe its biological effect. METHODS: NHE-1 antisense vector (pNHE-1) was introduced into human lung adenocarcinoma cell line A549 cells with cationic liposome transfection methods. Semi-quantification RT-PCR was performed to analyze the expression level of NHE-1 mRNA in A549 cells and transfected A549 cells. Intracellular pH (pHi) values were measured with fluorescence spectrophotometer. The growth characteristics of the cells was observed. Tumor cell apoptosis was observed with in situ apoptosis assessment. RESULTS: PCR verified the integration of antisense vector with the genomic DNA of transfected cells in positive clones. Semi-quantification RT-PCR showed that the NHE-1 gene expression level was significantly lower in transfected A549 cells (0.42 +/- 0.06) than in those untransfected (0.71 +/- 0.08, P < 0.01) and those transfected with pLXSN (0.69 +/- 0.16, P < 0.01). Compared with A549 cells, pHi values decreased significantly in transfected A549 cells at 12 h, 24 h and 48 h (6.990 +/- 0.005, 6.840 +/- 0.005 and 6.750 +/- 0.005 respectively vs 7.150 +/- 0.004, 7.140 +/- 0.007 and 7.120 +/- 0.008 respectively, P < 0.001). The growth of transfected A549 cells was significantly slower (P < 0.01), the double time was longer (3.20 vs 4.97) and the apoptosis rate was markedly higher [(24.50 +/- 3.62)% vs (1.50 +/- 0.55)%, P < 0.01]. CONCLUSION: The antisense vector was successfully transfected to A549 cells with liposome and integrated with transfected cell genome, and the expression of NHE-1 mRNA of A549 cells was inhibited, thereby resulted in a decrease of the pHi value by means of reduction of Na(+)/H(+) exchanging activity, with the inhibition of proliferation and growth of A549 cells and increased of A549 cell apoptosis.

[Effects of sodium hydrogen exchanger-1 hammerhead ribozyme on proliferation of pulmonary artery smooth muscle cells in rats in vitro].

AIM: To explore the effects of sodium hydrogen exchanger (NHE-1) specific hammerhead ribozyme on the expression and activity of NHE-1 and pHi in pulmonary artery smooth muscle cells (PASMCs) in rats, and its role in PASMCs proliferation. METHODS: According to the secondary structure of NHE-1 mRNA in rats, NHE-1 specific hammerhead ribozyme was designed with the assistance of computer. The recombinant vector of retroviral plasmid pLXSN and NHE-1 hammerhead ribozyme, PRZ, was transfected into the cultured PASMCs. G418 resistant cell clones were screened with 60 microg/ml G418. Then, the expression of NHE-1 mRNA was detected by RT-PCR, intracellular pH was measured with fluorescent probe-BCECF, 22Na and 3H-TdR incorporation were determined respectively. RESULTS: Compared with the cells transfected with pLXSN and non-transfected cells, NHE-1 mRNA, pHi value, 3H-TdR and 22Na incorporation decreased significantly in cells transfected with recombinant vector PRZ. No significance was found between the pLXSN transfected group and non-transfected group. CONCLUSION: NHE-1 hammerhead ribozyme can cleave the target RNA specifically, reduce the expression of NHE-1 mRNA, induce intracellular acidosis and consequently prohibit the proliferation of PASMCs.