miR-424 protects PC-12 cells from OGD-induced injury by negatively regulating MKP-1.

OBJECTIVE: It's of great significance to investigate the novel targets of drugs for the treatment of stroke. In this study, we explored the neuroprotective role of miR-424 in oxygen glucose deprivation (OGD)-induced injuries in PC-12 cells. MATERIALS AND METHODS: PC-12 cells were subjected to OGD stimulation to mimic ischemic injury. The expressions of miR-424 and mitogen-activated protein kinase phosphatase-1 (MKP-1) were altered by transient transfection with miR-424 mimic, miR-424 inhibitor, pEX-MKP-1, or sh-MKP-1. Cell counting kit-8 (CCK-8) assay, flow cytometry, and quantitative reverse transcription polymerase chain reaction (qRT-PCR), were conducted to respectively detect cell viability, apoptotic cells, and the expression of miR-424 and MKP-1. The protein expressions of several factors were determined by Western blot. Meanwhile, relative luciferase activity assay was done to verify the predicted targets association. RESULTS: OGD induced injury in PC-12 cells by suppressing cell viability and inducing apoptosis. OGD also induced the expression of miR-424 in PC-12 cells. Overexpression of miR-424 protected PC-12 cells from OGD-induced injury by increasing cell viability and decreasing apoptosis. MKP-1 was a direct target of miR-424, and its expression was negatively regulated by miR-424. Up-regulation of expression of MKP-1 aggravated OGD-induced cell injury by inhibiting the expression of hypoxia-inducible factor 1α (HIF-1α), thus inhibiting the PI3K/AKT/mTOR pathways. CONCLUSIONS: miR-424 protected PC-12 cells from OGD-induced injury through direct suppression of MKP-1 expression, as MKP-1 promoted OGD-induced cell injury by inhibiting the expression of HIF-1α and PI3K/AKT/mTOR pathways.

Omega-3 fatty acids prevent early-life antibiotic exposure-induced gut microbiota dysbiosis and later-life obesity.

Early-life antibiotic exposure can disrupt the founding intestinal microbial community and lead to obesity later in life. Recent studies show that omega-3 fatty acids can reduce body weight gain and chronic inflammation through modulation of the gut microbiota. We hypothesize that increased tissue levels of omega-3 fatty acids may prevent antibiotic-induced alteration of gut microbiota and obesity later in life. Here, we utilize the fat-1 transgenic mouse model, which can endogenously produce omega-3 fatty acids and thereby eliminates confounding factors of diet, to show that elevated tissue levels of omega-3 fatty acids significantly reduce body weight gain and the severity of insulin resistance, fatty liver and dyslipidemia resulting from early-life exposure to azithromycin. These effects were associated with a reversal of antibiotic-induced dysbiosis of gut microbiota in fat-1 mice. These results demonstrate the beneficial effects of omega-3 fatty acids on antibiotic-induced gut dysbiosis and obesity, and suggest the potential utility of omega-3 supplementation as a safe and effective means for the prevention of obesity in children who are exposed to antibiotics.

Endogenous synthesis of n-3 PUFA modifies fatty acid composition of kidney phospholipids and eicosanoid levels in the fat-1 mouse.

The goal of the present study was to determine whether endogenous synthesis of n-3 polyunsaturated fatty acids (PUFA) in the fat-1 mouse is comparable to fish oil feeding with respect to kidney n-3 PUFA composition and eicosanoid levels. Wild-type and heterozygous fat-1 mice, capable of synthesizing n-3 PUFA endogenously, were given diets enriched in either n-3 or n-6 PUFA in a 2×2 factorial design and terminated after 12 weeks. Kidney phospholipid fatty acids were analysed by gas chromatography. Kidney eicosanoids were analysed by liquid chromatography tandem mass spectrometry. Relative to control mice fed n-6 PUFA, n-3 PUFA fed and fat-1 mice had higher levels of kidney phospholipid n-3 PUFA, and lower levels of n-6 PUFA and eicosanoids. However, mice fed n-3 PUFA mice had higher levels of n-3 PUFA and lower levels of eicosanoids as compared to fat-1 mice. In conclusion, diet feeding had a greater impact on kidney fatty acid composition and eicosanoid levels than the genetic effect of the fat-1 gene. However, the fat-1 mouse remains a close approximation that can be used as a complementary model to study the role of n-3 PUFA in the kidney.

Docosahexaenoic acid (DHA) sensitizes brain tumor cells to etoposide-induced apoptosis.

In this study, we investigated whether DHA, a nutritionally important n-3 unsaturated fatty acid, modulated the sensitivity of brain tumor cells to the anticancer drug, etoposide (VP16). Medulloblastoma (MB) cell lines, Daoy and D283, and glioblastoma (GBM) cell lines, U138 and U87, were exposed to DHA or VP16 alone or in combination. The effects on cell proliferation and the induction of apoptosis were determined by using MTS and Hoechest 33342/PI double staining. U87 and U138 cells were found to be insensitive to the addition of DHA and VP16, whereas the two MB cell lines showed high sensitivity. DHA or VP16 alone showed little effect on cell proliferation or death in either the MB or GBM cell lines, but pretreatment with DHA enhanced the responsiveness to VP16 in the MB cell lines. To understand the mechanisms of combined DHA and VP16 on MB cells, pathway specific oligo array analyses were performed to dissect possible signaling pathways involved. The addition of DHA and VP16, in comparison to VP16 added alone, resulted in marked suppression in the expression of several genes involved in DNA damage repair, cell proliferation, survival, invasion, and angiogenesis, including PRKDC, Survivin, PIK3R1, MAPK14, NFκB1, NFκBIA, BCL2, CD44, and MAT1. These results suggest (1) that the effects of DHA and VP16 in brain tumor cells are mediated in part by the down regulation of events involved in DNA repair and the PI3K/MAPK signaling pathways and (2) that brain tumors genotypically mimicked by MB cells may benefit from therapies combining DHA with VP16.

Non-mammalian fat-1 gene prevents neoplasia when introduced to a mouse hepatocarcinogenesis model: Omega-3 fatty acids prevent liver neoplasia.

We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis model. Mice containing double mutations (DM) in c-myc and TGF-alpha (transforming growth factor-alpha), leading to liver neoplasia, were crossed with mice containing omega-3 desaturase. MRI analysis of triple mutant (TM) mice showed the absence of neoplasia at all time points for 92% of mice in the study. Pathological changes of TM (TGFalpha/c-myc/fat-1) mouse liver tissue was similar to control mouse liver tissue. Magnetic resonance spectroscopy (MRS) measurements of unsaturated fatty acids found a significant difference (p<0.005) between DM and TM transgenic (Tg) mice at 34 and 40 weeks of age. HPLC analysis of mouse liver tissue revealed markedly decreased levels of omega-6 fatty acids in TM mice when compared to DM (TGFalpha/c-myc) and control (CD1) mice. Mass spectrometry (MS) analysis indicated significantly decreased 16:0/20:4 and 18:1/20:4 and elevated 16:0/22:6 fatty acyl groups in both GPCho and GPEtn, and elevated 16:0/20:5, 18:0/18:2, 18:0/18:1 and 18:0/22:6 in GPCho, within TM mice compared to DM mice. Total fatty acid analysis indicated a significant decrease in 18:1n9 in TM mice compared to DM mice. Western blot analysis of liver tissue showed a significant (p<0.05) decrease in NF-kappaB (nuclear factor-kappaB) levels at 40 weeks of age in TM mice compared to DM mice. Microarray analysis of TM versus DM mice livers at 40 weeks revealed alterations in genes involved in cell cycle regulation, cell-to-cell signaling, p53 signaling, and arachidonic acid (20:4) metabolism. Endogenous omega-3 fatty acids were found to prevent HCC development in mice.

Endogenously produced n-3 fatty acids protect against ovariectomy induced bone loss in fat-1 transgenic mice.

Aging is associated with bone loss, leading to increased risk of fractures. Recently, there is growing interest in identifying nutritional supplements that can prevent bone loss with minimum side effects. There is increasing evidence for the beneficial effects of n-3 fatty acids in the prevention of bone loss. A transgenic mouse model (fat-1) that produces n-3 fatty acids endogenously and its wild type counterpart were used in this study to determine the effects of endogenously produced n-3 fatty acids on serum bone turnover markers, long bones, and lumbar vertebrae. Serum alkaline phosphatase and P1NP levels decreased significantly in wild type mice after ovariectomy. No significant changes were seen in osteocalcin. Cancellous and cortical bone mass were higher in the femur of fat-1 mice. In wild type mice, there was significant loss of bone after ovariectomy in the distal femur, femoral neck, proximal tibia, and fourth lumbar vertebra. However, in fat-1 mice, there was no, or significantly less, bone lost after ovariectomy in all the sites studied. We conclude that endogenously produced n-3 fatty acids can attenuate ovariectomy induced bone loss in the different bone sites studied, mainly as a consequence of decreased bone resorption at the endosteal surface.

A quasi-experimental study on a community-based behaviour change programme among injecting drug users in Sichuan, China.

The objective of this study is to explore an effective model of comprehensive intervention on HIV/AIDS among injecting drug users (IDUs) in communities located in urban areas and to evaluate its feasibility in Sichuan Province, China. A quasi-experimental study was designed so that various intervention measures were conducted in the intervention city but not in the control city. A Behaviour Surveillance Survey was introduced to evaluate intervention exposure and the effect of behaviour change. In the intervention city, services received by IDUs increased over time (P < 0.001). Awareness of HIV increased from 34.2% in 2003 to 58.3% in 2004, and to 67.4% in 2005 (P < 0.001). The proportion of IDUs surveyed who shared a needle the last time they injected drugs decreased from 17.1% in 2003 to 7.0% in 2005, and in terms of the past month from 42.4% in 2003 to 18.4% in 2005 (P < 0.001). Data from a multivariate logistic regression analysis showed that comprehensive intervention was a protective factor for behaviour change (odds ratio [OR] =0.561; 95% confidence interval [CI], 0.424-0.741). A well-designed and organized comprehensive intervention programme will effectively change the high-risk behaviour among IDUs in these communities. A more comprehensive, expanded and integrated response is needed when conducting an HIV/AIDS prevention programme.

Membrane effects of the n-3 fish oil fatty acids, which prevent fatal ventricular arrhythmias.

Fish oil fatty acids are known to exert beneficial effects on the heart and vascular systems. We have studied the membrane effects on ion channel conductance by the n-3 fish oil fatty acids that account for these beneficial effects. We have confirmed that these fatty acids prevent fatal cardiac arrhythmias in a reliable dog model of sudden cardiac death. This finding was followed by experiments indicating that the n-3 fatty acids electrically stabilize heart cells and do so largely through modulation of the fast voltage-dependent Na(+) currents and the L-type Ca(2+) channels in a manner, which makes the heart cells resistant to arrhythmias. Others and we have demonstrated that these membrane effects on the heart can prevent fatal cardiac arrhythmias in humans.

Interactions of n-3 fatty acids with ion channels in excitable tissues.

In summary, we have shown that the conventional explanation for the site of action of a ligand which alters the conductance of a membrane ion channel is that the ligand interacts or binds with the ion channel protein, changing its conductance, is inadequate to explain the primary site of action of the antiarrhythmic n-3 PUFAs. We have shown that when a neutral asparagine is replaced by a positively charged lysine in the N406 amino acid site in the alpha-subunit of the human cardiac sodium channel, the n-3 fatty acids lose their inhibitory action on the sodium current. The inadequacy of this finding to explain the primary site of action of the n-3 PUFAs is demonstrated by the inhibitory effect on all other cardiac ion channels, so far tested. We show that ion channels, which share no amino acid homology with the PUFAs, have their conductance also reduced in the presence of the PUFAs, Thus a more general conceptual framework or paradigm is needed to account for the broad action of the PUFAs on diverse different ion channels lacking amino acid homology. We have been testing the membrane tension hypothesis of Andersen and associates. According to this hypothesis, the fatty acids are not acting directly on the ion channel protein but accumulating in the phospholipid membrane in immediate juxtaposition to the site in the membrane where the ion channel protein penetrates the membrane phospholipid bilayer. This alters membrane tensions exerted by the phospholipid membrane on the ion channel, which in turn causes conformational changes in the ion channel, altering the conductance of the ion channel. Our preliminary data seem to support this membrane tension hypothesis.

Evidence of a lysosomal pathway for apoptosis induced by the synthetic retinoid CD437 in human leukemia HL-60 cells.

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene carboxylic acid (AHPN/CD437) has been proven to be a potent inducer of apoptosis in a variety of tumor cell types. However, the mechanism of its action remains to be elucidated. Recent studies suggest that the lysosomal protease cathepsin D, when released from lysosomes to the cytosol, can initiate apoptosis. In this study, we examined whether cathepsin D and free radicals are involved in the CD437-induced apoptosis. Exposure of human leukemia HL-60 cells to CD437 resulted in rapid induction of apoptosis as indicated by caspase activation, phosphatidylserine exposure, mitochondrial alterations and morphological changes. Addition of the antioxidants alpha-tocopherol acetate effectively inhibited the CD437-induced apoptosis. Measurement of the intracellular free radicals indicated a rise in oxidative stress in CD437-treated cells, which could be attenuated by alpha-tocopherol acetate. Interestingly, pretreatment of cells with the cathepsin D inhibitor pepstatin A blocked the CD437-induced free radical formation and apoptotic effects, suggesting the involvement of cathepsin D. However, Western blotting revealed no difference in cellular quantity of any forms of cathepsin D between control cells and CD437-treated cells, whereas immunofluorescence analysis of the intracellular distribution of cathepsin D showed release of the enzyme from lysosomes to the cytosol. Labeling of lysosomes with lysosomotropic probes confirmed that CD437 could induce lysosomal leakage. The CD437-induced relocation of cathepsin D could not be prevented by alpha-tocopherol acetate, suggesting that the lysosomal leakage precedes free radical formation. Furthermore, a retinoic acid nuclear receptor (RAR) antagonist failed to block these effects of CD437, suggesting that the action of CD437 is RAR-independent. Taken together, these data suggest a novel lysosomal pathway for CD437-induced apoptosis, in which lysosomes are the primary target and cathepsin D and free radicals act as death mediators.

Partitioning of polyunsaturated fatty acids, which prevent cardiac arrhythmias, into phospholipid cell membranes.

It has been demonstrated in animal studies that polyunsaturated fatty acids (PUFA) prevent ischemia-induced malignant ventricular arrhythmias, a major cause of sudden cardiac death in humans. To learn how these PUFA, at low micromolar concentrations, exert their antiarrhythmic activity, we studied their effects in vitro on the contractions of isolated cardiac myocytes and the conductances of their sarcolemmal ion channels. These fatty acids directly stabilize electrically every cardiac myocyte by modulating the conductances of specific ion channels in their sarcolemma. In this study, we determined the molar ratio of PUFA to the moles of phospholipid (PL) in cell membranes to learn if the ratio is so low as to preclude the possibility that the primary site of action of PUFA is on the packing of the membrane PL. [(3)H]-arachidonic acid (AA) was used to measure the incorporation of PUFA, and the inorganic phosphorous of the PL was determined as a measure of the moles of PL in the cell membrane. Our results indicate that the mole percent of AA to moles of phospolipid is very low (< or =1.0) at the concentrations that affect myocyte contraction and the conductance of voltage-dependent Na(+) and L-type Ca(2)+ channels in rat cardiomyocytes and in alpha-subunits of human myocardial Na(+) channels. In conclusion, it seems highly unlikely that these fatty acids are affecting the packing of PL within cell membranes as their way of modulating changes in cell membrane ion currents and in preventing arrhythmias in our contractility studies. -- Pound, E. M., J. X. Kang, and A. Leaf. Partitioning of polyunsaturated fatty acids, which prevent cardiac arrhythmias, into phospholipid cell membranes. J. Lipid Res. 2001. 42: 346--351.

Adenoviral gene transfer of Caenorhabditis elegans n--3 fatty acid desaturase optimizes fatty acid composition in mammalian cells.

Omega--3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n--3 PUFAs is insufficient in most Western people, whereas the level of n--6 PUFAs is relatively too high, with an n--6/n--3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n--3 PUFAs in mammals relies on chronic dietary intake of fat rich in n--3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n--3 PUFAs or to convert n--6 to n--3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n--3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n--3 PUFA contents and dramatically balance the ratio of n--6/n--3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n--6 PUFAs to the corresponding n--3 PUFAs, and changed the n--6/n--3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n--6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

Prevention of fatal cardiac arrhythmias by polyunsaturated fatty acids.

In animal feeding studies, and probably in humans, n-3 polyunsaturated fatty acids (PUFAs) prevent fatal ischemia-induced cardiac arrhythmias. We showed that n-3 PUFAs also prevented such arrhythmias in surgically prepared, conscious, exercising dogs. The mechanism of the antiarrhythmic action of n-3 PUFAs has been studied in spontaneously contracting cultured cardiac myocytes of neonatal rats. Adding arrhythmogenic toxins (eg, ouabain, high Ca(2+), lysophosphatidylcholine, beta-adrenergic agonist, acylcarnitine, and the Ca(2+) ionophore) to the myocyte perfusate caused tachycardia, contracture, and fibrillation of the cultured myocytes. Adding eicosapentaenoic acid (EPA: 5-15 micromol/L) to the superfusate before adding the toxins prevented the expected tachyarrhythmias. If the arrhythmias were first induced, adding the EPA to the superfusate terminated the arrhythmias. This antiarrhythmic action occurred with dietary n-3 and n-6 PUFAs; saturated fatty acids and the monounsaturated oleic acid induced no such action. Arachidonic acid (AA; 20:4n-6) is anomalous because in one-third of the tests it provoked severe arrhythmias, which were found to result from cyclooxygenase metabolites of AA. When cyclooxygenase inhibitors were added with the AA, the antiarrhythmic effect was like those of EPA and DHA. The action of the n-3 and n-6 PUFAs is to stabilize electrically every myocyte in the heart by increasing the electrical stimulus required to elicit an action potential by approximately 50% and prolonging the relative refractory time by approximately 150%. These electrophysiologic effects result from an action of the free PUFAs to modulate sodium and calcium currents in the myocytes. The PUFAs also modulate sodium and calcium channels and have anticonvulsant activity in brain cells.

[Determination of 4 antiseptics and 6 sunscreen agents in cosmetic products by reversed-phase high performance liquid chromatography].

A method for the quantitative determination of 4 antiseptics (methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, n-propyl p-hydroxybenzoate, n-butyl p-hydroxybenzoate) and 6 sunscreen agents (2-hydroxy 4-methoxybenzophenone-5-sulfonic acid, 2-hydroxy 4-methoxybenzophenone, phenyl salicylate, 2-ethylhexyl 4-dimethyl aminobenzoate, 2-ethylhexyl 4-methoxycinnamate, 2-ethylhexyl salicylate) in cosmetic products was developed successfully. It was based on a reversed-phase high performance liquid chromatographic separation under the conditions of isocratic elution with a mixture of methanol-THF-water (30:100:100, V/V) by using a column packed with 10 microns YWG-C18, and the UV detection at 254 nm and 310 nm. The recoveries(n = 6) ranged from 87.2% to 106.5%, and the relative standard deviations(n = 6) were from 1.2% to 3.3%.

The antiarrhythmic and anticonvulsant effects of dietary N-3 fatty acids.

It has been shown in animals and probably in humans, that n-3 polyunsaturated fatty acids (PUFAs) are antiarrhythmic. We report recent studies on the antiarrhythmic actions of PUFAs. The PUFAs stabilize the electrical activity of isolated cardiac myocytes by modulating sarcolemmal ion channels, so that a stronger electrical stimulus is required to elicit an action potential and the refractory period is markedly prolonged. Inhibition of voltage-dependent sodium currents, which initiate action potentials in excitable tissues, and of the L-type calcium currents, which initiate release of sarcoplasmic calcium stores that increase cytosolic free calcium concentrations and activate the contractile proteins in myocytes, appear at present to be the probable major antiarrhythmic mechanism of the PUFAs.

Mannose 6-phosphate/insulin-like growth factor II receptor mediates the growth-inhibitory effects of retinoids.

Both retinoids and the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) have been shown to play an important role in controlling cell growth during embryonic development and oncogenesis. Our recent work (Kang et al., Proc. Natl. Acad. Sci. USA, 94: 13671-13676, 1997; Kang et al., Proc. Natl. Acad. Sci. USA, 95: 13687-13691, 1998) revealed a direct biochemical interaction between retinoic acid (RA) and the M6P/IGF2R, thereby leading us to hypothesize that the M6P/IGF2R may mediate a growth-inhibiting effect of RA. To test this hypothesis, cell growth and apoptosis in response to RA and various receptor-selective retinoids were examined in cells that lack or overexpress the M6P/IGF2R. RA and those retinoids capable of binding to the M6P/IGF2R induced a remarkable morphological change with characteristics of round shape and reduced spreading, apoptosis, and growth inhibition in stably transfected mouse P388D1 cells overexpressing the M6P/IGF2R but not in the M6P/IGF2R-deficient P388D1 cells. These effects of RA were neither blocked by a potent RA nuclear receptor (RAR) antagonist (AGN193109), nor mimicked by a selective RAR agonist (TTNPB), suggesting that the observed effects of RA are independent of RARs. Similar effects of the retinoids were observed in cultured neonatal rat cardiac myocytes that have high levels of the M6P/IGF2R. Furthermore, overexpression of the M6P/IGF2R in a RA-resistant cancer cell line (HL-60R) that lacked functional RARs gave the cells a susceptibility to RA-induced apoptosis. These data suggest that the M6P/ IGF2R may play an important role in mediating retinoid-induced apoptosis/growth-inhibition and provide insight into the similar biological effects of RA and the M6P/IGF2R on fetal development and carcinogenesis.

Functional and electrophysiologic effects of polyunsaturated fatty acids on exictable tissues: heart and brain.

It has been shown in animals and probably in humans, that n-3 polyunsaturated fatty acids (PUFAs) are antiarrhythmic. The free PUFAs stabilize the electrical activity of isolated cardiac myocytes by inhibiting sarcolemmal ion channels, so that a stronger electrical stimulus is required to elicit an action potential and the relative refractory period is markedly prolonged. This appears at present to be the probable major antiarrhythmic mechanism of the PUFAs. They similarly inhibit the Na+ and Ca2+ currents in rat hippocampal neurons which results in an increase in the electrical threshold for generalized seizures using the cortical stimulation model in rats.

n-3 fatty acids in the prevention of cardiac arrhythmias.

In animals and probably in humans n-3 polyunsaturated fatty acids (PUFA) are antiarrhythmic. A report follows on the recent studies of the antiarrhythmic actions of PUFA. The PUFA stabilize the electrical activity of isolated cardiac myocytes by inhibiting sarcolemmal ion channels, so that a stronger electrical stimulus is required to elicit an action potential and the relative refractory period is markedly prolonged. This appears at present to be the probable major antiarrhythmic mechanism of PUFA.