Cytosolic Phospholipase A2 Expression Research Articles

Mitochondrial oxidative damage reprograms lipid metabolism of renal tubular epithelial cells in the diabetic kidney

The functional and structural changes in the proximal tubule play an important role in the occurrence and development of diabetic kidney disease (DKD). Diabetes-induced metabolic changes, including lipid metabolism reprogramming, are reported to lead to changes in the state of tubular epithelial cells (TECs), and among all the disturbances in metabolism, mitochondria serve as central regulators. Mitochondrial dysfunction, accompanied by increased production of mitochondrial reactive oxygen species (mtROS), is considered one of the primary factors causing diabetic tubular injury. Most studies have discussed how altered metabolic flux drives mitochondrial oxidative stress during DKD. In the present study, we focused on targeting mitochondrial damage as an upstream factor in metabolic abnormalities under diabetic conditions in TECs. Using SS31, a tetrapeptide that protects the mitochondrial cristae structure, we demonstrated that mitochondrial oxidative damage contributes to TEC injury and lipid peroxidation caused by lipid accumulation. Mitochondria protected using SS31 significantly reversed the decreased expression of key enzymes and regulators of fatty acid oxidation (FAO), but had no obvious effect on major glucose metabolic rate-limiting enzymes. Mitochondrial oxidative stress facilitated renal Sphingosine-1-phosphate (S1P) deposition and SS31 limited the elevated Acer1, S1pr1 and SPHK1 activity, and the decreased Spns2 expression. These data suggest a role of mitochondrial oxidative damage in unbalanced lipid metabolism, including lipid droplet (LD) formulation, lipid peroxidation, and impaired FAO and sphingolipid homeostasis in DKD. An in vitro study demonstrated that high glucose drove elevated expression of cytosolic phospholipase A2 (cPLA2), which, in turn, was responsible for the altered lipid metabolism, including LD generation and S1P accumulation, in HK-2 cells. A mitochondria-targeted antioxidant inhibited the activation of cPLA2f isoforms. Taken together, these findings identify mechanistic links between mitochondrial oxidative metabolism and reprogrammed lipid metabolism in diabetic TECs, and provide further evidence for the nephroprotective effects of SS31 via influencing metabolic pathways.

Electroacupuncture improves limb motor function by down-regulating cytosolic phospholi-pase A2 and reducing apoptosis of nerve cells in rats with spinal cord injury

To observe the effect of electroacupuncture（EA） on the expression of cytosolic phospholipase A2 （cPLA2） and apoptosis of nerve cells in rats with spinal cord injury （SCI）, so as to explore its mechanisms underlying improvement of SCI. Seventy-two female SD rats were randomly divided into model, EA, antagonist and EA+antagonist groups, with 18 rats in each group and other 18 rats were used as the sham operation （sham） group. The SCI model was established by referring to modified Allen's method with a weight impactor. The hindlimb motor function was assessed by using Basso-Beattie-Bresnahan （BBB） score. Rats of the EA group were subjected to EA stimulation at "Dazhui"（GV14）, "Yaoyangguan"（GV3）, bilateral "Ciliao"（BL32） and "Zusanli"（ST36） for 20 min, once a day for 14 days. Rats of the antagonist group received intravenous injection followed by intraperitoneal injection of arachidonyl trifluoromethyl ketone （AACOCF3, antagonist of cPLA2）, once every other day. Rats of the EA+antagonist group received EA treatment combined with antagonist injection. After the treatment, the rats were sacrificed and the spinal cord tissue was collected for detecting the protein expression of cPLA2, p-cPLA2, Bcl-2, Bax and Caspase-3 by Western blot, and the mRNA expression of cPLA2, Bcl-2, Bax and Caspase-3 using qRT-PCR. The morphological changes of the spinal cord were detected by Nissl staining. In comparison with the sham group, the BBB score, expression of Bcl-2 protein and mRNA were significantly down-regulated （P<0.01）, whereas the expression levels of Bax, Caspase-3 and p-cPLA2 proteins and mRNAs were considerably up-regulated in the model group （P<0.01）. Compared with the model group, the BBB score, expression levels of Bcl-2 protein and mRNA were significantly up-regulated （P<0.01, P<0.05）, while the expression levels of Bax, Caspase-3 and p-cPLA2 proteins in the EA, antagonist and EA+antagonist groups, Bax and cPLA2 mRNAs in both antagonist and EA+antagonist groups, and Caspase-3 mRNA in the EA+antagonist group were obviously down-regulated （P<0.01, P<0.05）. The effect of EA+antagonist was significantly superior to EA in increasing BBB score and in lowering expression of Bax and cPLA2 mRNAs （P<0.01, P<0.05）. Nissl staining showed reduced number of nerve cells and Nissl bodies, and striped dark blue cells in the model group, which was milder in the EA and antagonist groups, particularly in the EA+antagonist group. EA may improve the limb motor function of SCI rats, which may be related to its functions in down-regulating the expression of p-cPLA2, Bax and Caspase-3 and up-regulating Bcl-2 to reduce the apoptosis of nerve cells in the regional spinal cord.

Electroacupuncture improves limb motor function by down-regulating cytosolic phospholi-pase A2 and reducing apoptosis of nerve cells in rats with spinal cord injury

To observe the effect of electroacupuncture（EA） on the expression of cytosolic phospholipase A2 （cPLA2） and apoptosis of nerve cells in rats with spinal cord injury （SCI）, so as to explore its mechanisms underlying improvement of SCI. Seventy-two female SD rats were randomly divided into model, EA, antagonist and EA+antagonist groups, with 18 rats in each group and other 18 rats were used as the sham operation （sham） group. The SCI model was established by referring to modified Allen's method with a weight impactor. The hindlimb motor function was assessed by using Basso-Beattie-Bresnahan （BBB） score. Rats of the EA group were subjected to EA stimulation at "Dazhui"（GV14）, "Yaoyangguan"（GV3）, bilateral "Ciliao"（BL32） and "Zusanli"（ST36） for 20 min, once a day for 14 days. Rats of the antagonist group received intravenous injection followed by intraperitoneal injection of arachidonyl trifluoromethyl ketone （AACOCF3, antagonist of cPLA2）, once every other day. Rats of the EA+antagonist group received EA treatment combined with antagonist injection. After the treatment, the rats were sacrificed and the spinal cord tissue was collected for detecting the protein expression of cPLA2, p-cPLA2, Bcl-2, Bax and Caspase-3 by Western blot, and the mRNA expression of cPLA2, Bcl-2, Bax and Caspase-3 using qRT-PCR. The morphological changes of the spinal cord were detected by Nissl staining. In comparison with the sham group, the BBB score, expression of Bcl-2 protein and mRNA were significantly down-regulated （P<0.01）, whereas the expression levels of Bax, Caspase-3 and p-cPLA2 proteins and mRNAs were considerably up-regulated in the model group （P<0.01）. Compared with the model group, the BBB score, expression levels of Bcl-2 protein and mRNA were significantly up-regulated （P<0.01, P<0.05）, while the expression levels of Bax, Caspase-3 and p-cPLA2 proteins in the EA, antagonist and EA+antagonist groups, Bax and cPLA2 mRNAs in both antagonist and EA+antagonist groups, and Caspase-3 mRNA in the EA+antagonist group were obviously down-regulated （P<0.01, P<0.05）. The effect of EA+antagonist was significantly superior to EA in increasing BBB score and in lowering expression of Bax and cPLA2 mRNAs （P<0.01, P<0.05）. Nissl staining showed reduced number of nerve cells and Nissl bodies, and striped dark blue cells in the model group, which was milder in the EA and antagonist groups, particularly in the EA+antagonist group. EA may improve the limb motor function of SCI rats, which may be related to its functions in down-regulating the expression of p-cPLA2, Bax and Caspase-3 and up-regulating Bcl-2 to reduce the apoptosis of nerve cells in the regional spinal cord.

Lipidomic Profiling of Bronchoalveolar Lavage Fluid Extracellular Vesicles Indicates Their Involvement in Lipopolysaccharide-Induced Acute Lung Injury

Emerging data support the pivotal role of extracellular vesicles (EVs) in normal cellular physiology and disease conditions. However, despite their abundance, there is much less information about the lipid mediators carried in EVs, especially in the context of acute lung injury (ALI). Our data demonstrate that C57BL/6 mice subjected to intranasal Escherichia coli lipopolysaccharide (LPS)-induced ALI release, a higher number of EVs into the alveolar space, compared to saline-treated controls. EVs released during ALI originated from alveolar epithelial cells, macrophages, and neutrophils and carry a diverse array of lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The eicosanoids in EVs correlated with cellular levels of arachidonic acid, expression of cytosolic phospholipase A2, cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome epoxygenase p450 proteins in pulmonary macrophages. Furthermore, EVs from LPS-toll-like receptor 4 knockout (TLR4^-/-) mice contained significantly lower amounts of COX and LOX catalyzed eicosanoids and ω-3 PUFA metabolites. More importantly, EVs from LPS-treated wild-type mice increased TNF-α release by macrophages and reduced alveolar epithelial monolayer barrier integrity compared to EVs from LPS-treated TLR4^−/− mice. In summary, our study demonstrates for the first time that the EV carried PUFA metabolite profile in part depends on the inflammatory status of the lung macrophages and modulates pulmonary macrophage and alveolar epithelial cell function during LPS-induced ALI.

Anti-inflammatory and Cytoprotective Effect of Clinacanthus nutans Leaf But Not Stem Extracts on 7-Ketocholesterol Induced Brain Endothelial Cell Injury.

Clinacanthus nutans (Lindau) (C. nutans) has diverse uses in traditional herbal medicine for treating skin rashes, insect and snake bites, lesions caused by herpes simplex virus, diabetes mellitus and gout in Singapore, Malaysia, Indonesia, Thailand and China. We previously showed that C. nutans has the ability to modulate the induction of cytosolic phospholipase A2 (cPLA2) expression in SH-SY5Y cells through the inhibition of histone deacetylases (HDACs). In the current study, we elucidated the effect of C. nutans on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induced a dose-dependent loss of hCMEC/D3 cell viability, and such damage was significantly inhibited by C. nutans leaf extracts but not stem extracts. 7KC also induced a marked increase in mRNA expression of pro-inflammatory cytokines, IL-1β IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX-2) in brain endothelial cells, and these increases were significantly inhibited by C. nutans leaf but not stem extracts. HPLC analyses showed that leaf extracts have a markedly different chemical profile compared to stem extracts, which might explain their different effects in counteracting 7KC-induced inflammation. Further study is necessary to identify the putative phytochemicals in C. nutans leaves that have anti-inflammatory properties.

Cellular Microenvironment Stiffness Regulates Eicosanoid Production and Signaling Pathways.

Pathological changes in the biomechanical environment are implicated in the progression of idiopathic pulmonary fibrosis (IPF). Stiffened matrix augments fibroblast proliferation and differentiation and activates TGF-β1 (transforming growth factor-β1). Stiffened matrix impairs the synthesis of the antifibrogenic lipid mediator prostaglandin E2 (PGE2) and reduces the expression of the rate-limiting prostanoid biosynthetic enzyme cyclooxygenase-2 (COX-2). We now show that prostaglandin E synthase (PTGES), the final enzyme in the PGE2 biosynthetic pathway, is expressed at lower levels in the lungs of patients with IPF. We also show substantial induction of COX-2, PTGES, prostaglandin E receptor 4 (EP4), and cytosolic phospholipase A2 (cPLA2) expression in human lung fibroblasts cultured in soft collagen hydrogels or in spheroids compared with conventional culture on stiff plastic culture plates. Induction of COX-2, cPLA2, and PTGES expression in spheroid cultures was moderately inhibited by the p38 mitogen-activated protein kinase inhibitor SB203580. The induction of prostanoid biosynthetic enzyme expression was accompanied by an increase in PGE2 levels only in non-IPF-derived fibroblast spheroids. Our study reveals an extensive dysregulation of prostanoid biosynthesis and signaling pathways in IPF-derived fibroblasts, which are only partially abrogated by culture in soft microenvironments.

Organ function, sphingolipid levels and inflammation in tunicamycin induced endoplasmic reticulum stress in male rats.

Disorders of the endoplasmic reticulum (ER) lead to cellular damage but can cause cell death if ER dysfunction is prolonged. We aimed to examine liver/kidney functions, neutral sphingomyelinase (N-SMase) activity, sphingolipid levels, cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2) protein expression in rats under ER stress. ER stress was induced by tunicamycin (TM) and the ER stress inhibitor taurodeoxycholic acid (TUDCA) was injected before induction of ER stress. ER stress was confirmed by increased tissue levels of GRP78. Hematological and biochemical profiles were measured by autoanalyzers while hepatic and renal injury was evaluated via microscopy and histopathological scoring. Tissue levels of C16-C24 sphingomyelins (SM), C16-C24 ceramides (CERs) and sphingosine-1-phosphate (S1P) were determined by LC-MS/MS. Tissue cPLA2 and COX-2 were measured by western blot and activity assays. Tunicamycin treatment caused kidney and liver function test abnormalities, increased hematocrit and hemoglobin levels but decreased white blood cell counts. Histopathological findings showed hepatic necroinflammation and renal tubular damage in rats treated with TM. TUDCA administration attenuated WBC abnormalities and TM- induced hepatic/renal functional impairment in ER stress, as evident by significantly restored serum ALT, AST, creatinine, and total bilirubin levels. A significant increase was observed in N-SMase activity, tissue levels of C16-C24 CERs, cPLA2 and COX-2 expression in liver and kidney tissue under ER stress. TUDCA administration decreased tissue CER levels, cPLA2 and COX-2 expression as well as prostaglandin E2 (PGE2) formation. These results signify that ER stress causes hepatic and renal toxicity as well as CER-induced PGE2 formation in liver and kidney.

Expression of Cytosolic Phospholipase A2 (cPLA2)-Arachidonic Acid (AA)-Cyclooxygenase-2 (COX-2) Pathway Factors in Lung Cancer Patients and Its Implication in Lung Cancer Early Detection and Prognosis.

BackgroundThe aim of this study was to elucidate the involvement of cPLA2-AA-COX-2 pathway factors and their potential role in lung cancer early diagnosis and prognosis.Material/MethodsWe selected 80 lung cancer patients as the cancer group, and 30 normal patients were selected as the normal group. Serum contents of COX-2, cPLA2, COX-1, mPGES, PGE2, and PGI2 were measured, and mRNA levels of COX-2, cPLA2, COX-1, and mPGES in serum were determined. Spearman’s P-test was used to analyze the correlation between expression of PGI2 and mPGES in serum and the clinical characteristics of these lung cancer patients. The factors affecting the prognosis lung cancer were analyzed by COX regression model.ResultsThe serum contents of COX-2, cPLA2, COX-1, mPGES, PGE2, and PGI2 in the cancer patient group were significantly higher (p<0.05) than in the normal group; after treatment, the serum contents of these factors were significantly decreased (p<0.05). However, distant metastasis had a significant effect on serum contents of mPGES and PGI2 (p<0.05), but not on the other factors. The mRNA levels of COX-2, cPLA2, COX-1, and mPGES in cancer patients were significantly higher than in normal patients. In addition, the 5-year survival rate of patients with high expression of mPGES and/or PGI2 was lower than that of the low expression group. Cox regression analysis showed that the expression of mPGES and PGI2 had statistical significance in predicting the prognosis of lung cancer.ConclusionsThe cPLA2-AA-COX-2 pathway is closely associated with lung cancer. These findings are important for clinical diagnosis of lung cancer.

Effect of Kaempferol on Cyclooxygenase 2 (Cox2) and Cytosolic Phospholipase A2 (cPLA2) Protein Expression in BALB/c Mice

Kaempferol, a phytochemical found in many edible plants, is known to alleviate diseases such as cancer, allergy, and inflammation. The objective of this study was to investigate whether kaempferol could reduce omega-6 and ovalbumin-mediated allergic reactions at lung and trachea in BALB/c mice. Mice were allocated into five groups: 1) control group (CON); 2) positive control group with orally administration of omega-6 (POS); 3) bovine serum albumin (BSA) sensitization group (with BSA injection and ovalbumin inhalation); 4) BSA+K10 group: BSA injection, 10 μg/g of kaempferol administration and ovalbumin inhalation; and 5) BSA+K20 group: BSA injection, 20 μg/g of kaempferol administration and ovalbumin inhalation. Results revealed that serum histamine level was the highest (p<0.01) in BSA group. In lung tissue and trachea, cyclooxygenase 2 (Cox2) expression was significantly (p<0.05) higher in the BSA group compared to that in other groups. However, phosphorylated cytosolic phospholipase A2 (p-cPLA2) expression in the trachea was not significantly different among groups. Taken together, results of this study suggest that kaempferol might be useful for alleviating inflammation reaction associated with Cox2 expression. However, the exact mechanism of action involved in the effect of kaempferol on inflammatory response remains unclear.

Sevoflurane attenuates ventilator‑induced lung injury by regulating c‑PLA2 expression.

The aim of the present study was to investigate the potential role of club cell secretory protein (CCSP), an endogenous modulator, in reducing pulmonary inflammation induced by sevoflurane following one‑lung ventilation (OLV). Healthy Japanese white rabbits were randomly assigned to six groups: Sham‑operated group (group S); respiratory management of OLV group (group O); OLV+sevoflurane treated group (group OF), club cells exfoliated+sham‑operated group (group NA), club cells exfoliated+OLV group (group NAO); and club cells exfoliated+OLV+sevoflurane treated group (group NAOF). At the end of the experimental observation, all animals in the different groups were sacrificed and lung injury was evaluated according to the lung wet/dry weight ratio and histological scoring system. Lung homogenates were harvested to detect the mRNA and protein expression of cytosolic phospholipase A2 (c‑PLA2) and CCSP. The content of arachidonic acid was measured using an ELISA. Following OLV treatment, c‑PLA2 expression was increased, CCSP expression was decreased and lung injury scores were significantly increased. Sevoflurane inhalation in the OLV‑treated group induced an upregulation of CCSP and a downregulation of c‑PLA2 expression. In the group NAO, in which the club cells were simultaneously exfoliated, OLV caused more severe lung damage and induced higher expression of c‑PLA2 compared with that in group O. However, sevoflurane inhalation reduced the extent of lung injury and the expression of c‑PLA2, even when the endogenous modulator of lung inflammation, CCSP, was exfoliated (group NAOF). These results indicated that OLV promoted lung inflammation through the CCSP and c‑PLA2 pathway. However, the results from the club cells exfoliated group indicated that the CCSP may not be involved in the protective effect exerted by sevoflurane inhalation.

Decreased Expression of Cytosolic Phospholipase A2 in Normal Conjunctiva May be Contributing to the Formation of Pterygium

Decreased Expression of Cytosolic Phospholipase A2 in Normal Conjunctiva May be Contributing to the Formation of Pterygium

Casein glycomacropeptide hydrolysates inhibit PGE2 production and COX2 expression in LPS-stimulated RAW 264.7 macrophage cells via Akt mediated NF-κB and MAPK pathways.

A casein glycomacropeptide hydrolysate (GMPH) was found to possess inhibitory activity against lipopolysaccharide (LPS)-induced inflammatory response in our previous study. In the current study, the inhibitory effect and the underlying molecular mechanism of GMPH on inflammatory response in LPS-stimulated RAW264.7 macrophages were further investigated. Results showed that GMPH significantly suppressed LPS-induced intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) production. GMPH reduced the production of prostaglandin E2 (PEG2) and the expression of cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) in LPS-stimulated macrophages. GMPH also attenuated LPS-induced phosphorylation of MAPK (c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38) and protein kinase B (Akt). Furthermore, GMPH inhibited nuclear transcription factor kappa-B (NF-κB) activation by suppressing the nuclear translocation of NF-κB p65, which was markedly reversed by LY294002, an Akt inhibitor. These results demonstrated that GMPH exerts anti-inflammatory functions through the inactivation of MAPK and Akt in LPS-stimulated RAW264.7 macrophages, therefore may hold potential to ameliorate inflammation-related metabolic disorders.

Effects of shinbuto and ninjinto on prostaglandin E2 production in lipopolysaccharide-treated human gingival fibroblasts.

Previously, we revealed that several kampo medicines used for patients with excess and/or medium patterns (kakkonto (TJ-1), shosaikoto (TJ-9), hangeshashinto (TJ-14), and orento (TJ-120)) reduced prostaglandin (PG)E2 levels using LPS-treated human gingival fibroblasts (HGFs). Recently, we examined other kampo medicines used for patients with the deficiency pattern [bakumondoto (TJ-29), shinbuto (TJ-30), ninjinto (TJ-32), and hochuekkito (TJ-41)] and the herbs comprising shinbuto and ninjinto using the same experimental model. Shinbuto and ninjinto concentration-dependently reduced LPS-induced PGE2 production by HGFs, whereas hochuekkito weakly reduced and bakumondoto did not reduce PGE2 production. Shinbuto and ninjinto did not alter cyclooxygenase (COX) activity or the expression of molecules involved in the arachidonic acid cascade. Therefore, we next examined which herbs compromising shinbuto and ninjinto reduce LPS-induced PGE2 production. Among these herbs, shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) strongly and concentration-dependently decreased LPS-induced PGE2 production. However, both shokyo and kankyo increased the expression of cytosolic phospholipase (cPL)A2 but did not affect annexin1 or COX-2 expression. These results suggest that shokyo and kankyo suppress cPLA2 activity. We demonstrated that kampo medicines suppress inflammatory responses in patients with the deficiency pattern, and in those with excess or medium patterns. Moreover, kampo medicines that contain shokyo or kankyo are considered to be effective for the treatment of inflammatory diseases.

Retinoic acid attenuates oxidative injury in bovine mammary epithelial cells induced by hydrogen peroxide

The objective of this study was to explore how retinoic acid (RA) attenuates oxidative injury induced by hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;) in bovine mammary epithelial cells (BMEC). Subconfluence BMEC were randomly divided into four groups with six replicates: the control group (incubated in serum-free medium without RA or H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2 &lt;/sub&gt;for 30 h), H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; group (pre-incubated for 24 h without RA, then for another 6 h with 600 μM H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;), RA group (incubated with 1 mg/ml RA for 30 h without H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;), and RA + H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; group (RA prevention group, pre-incubated with 1 mg/ml RA for 24 h and then for another 6 h with 600 mM H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;). The results showed that the H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; treatment significantly decreased several measured traits, including the cell viability, glutathione peroxidase (GPX) and thioredoxin reductase (TRXR) activities, selenoprotein P (SELP) content, catalase and superoxide dismutase activities, total antioxidant capacity, and GPX1, TRXR1, and SELP gene expression, as well as GPX1 and TRXR1 protein expression. H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; treatment also increased the malondialdehyde and reactive oxygen species contents and induced a marked increase of several measured traits, including the arachidonic acid (ARA) concentration, cytosolic phospholipase A2 and 5-lipoxygenase gene expression and activity, and 15-hydroxy twenty-four arachidonic acid and hydroxy peroxide tetracosenic arachidonic acid contents. RA pre-treatment prevented corresponding increases in parameters related to ARA metabolism and increased the activity of TRXR. Moreover, RA pre-treatment attenuated the phosphorylation levels of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase and effectively decreased the ARA content. These results suggest that RA protected BMEC from oxidative stress by elevating TRXR activity, which inhibited the MAPK signaling pathway and led to a decreased concentration of ARA.

The Protective Effect of Selenium on Bovine Mammary Epithelial Cell Injury Caused by Depression of Thioredoxin Reductase.

To elucidate the effect of selenium (Se) on antioxidant function of mammary glands in dairy cows and the underlying mechanism, an experiment was conducted using a single-factor completely randomized design study. Bovine mammary epithelial cells (BMECs) were randomly divided into four groups: control, Se treatment, 2,4-dinitrochlorobenzene (DNCB) inhibition, and Se prevention. Treatment of BMECs with Se was found to significantly reverse decreased cell proliferation and the expression of thioredoxin reductase (TrxR) after DNCB exposure. DNCB-induced activation of apoptosis signaling kinase-1 (ASK-1), which activates the mitogen-activated protein kinase (MAPK) pathway, was reduced in BMECs treated with Se. Additionally, our results indicated that Se treatment resulted in lower intracellular accumulation of arachidonic acid (ARA) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) due to suppressed expression of cytosolic phospholipase A2 (cPLA2) regulated by p38MAPK and c-Jun N-terminal kinase (JNK) in DNCB-stimulated BMECs. Taken together, these findings suggest that Se treatment improved the antioxidant function of dairy cow mammary glands and protected cells from oxidative damage primarily by increasing the activity of TrxR, inhibiting the activation of the MAPK signaling pathway, and thus decreasing the content of ARA and its related metabolites.

Oxymatrine attenuated isoproterenol-induced heart failure in rats via regulation of COX-2/PGI2 pathway.

Oxymatrine (OMT) is an active constituent of traditional Chinese herb Sophora japonica Ait which has been shown to exert potent anti-inflammatory,anti-oxidant and anti-fibrosis properties. Our previous studies have demonstrated that OMT has protective effects on isoproterenol-induced heart failure in rats through regulation of DDAH/ADMA metabolism pathway.In this study,we further investigated whether OMT could attenuate isoproterenol-induced heart failure through the regulation of COX-2/PGI2 pathway. Heart failure was induced in Sprague-Dawley rats by 5mg/kg isoproterenol subcutaneous injection for 7days. The rats were maintained on normal diet and randomly divided into five groups: control, isoproterenol, isoproterenol with OMT (50, 100mg/kg), and OMT alone groups (n=12 in each group). Serum brain natruretic peptide (BNP, a heart failure biomarker), histopathological variables, expression of Cytosolic phospholipase A2 (cPLA2), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and Prostacyclin synthase (PGIS) were analysed. Administration of OMT significantly reduced the increased BNP in plasm of isoproterenol-induced rats, attenuated cardiac fibrosis,suppressed overexpression of myocardial COX-1 expression, up-regulated COX-2 and PGIS expression, but had no effects on isoproterenol-induced elevated protein cPLA2. And compared with control group, any indexes in sham rats treated with OMT (100mg/kg) alone were unaltered. These results demonstrated that OMT has cardioprotective effects on isoproterenol-induced heart failure in rats by regulating COX-2/PGI2 pathway.

Studies on Shokyo, Kanzo, and Keihi in Kakkonto Medicine on Prostaglandin E2 Production in Lipopolysaccharide-Treated Human Gingival Fibroblasts.

We previously demonstrated that a kampo medicine, kakkonto, decreases lipopolysaccharide- (LPS-) induced prostaglandin E2 (PGE2) production by human gingival fibroblasts. In this study, we examined the herbs constituting kakkonto that exhibit this effect. Shokyo strongly and concentration dependently and kanzo and keihi moderately decreased LPS-induced PGE2 production. Shokyo did not alter cyclooxygenase-2 (COX-2) activity, cytosolic phospholipase A2 (cPLA2), annexin 1 and COX-2 expression, and LPS-induced extracellular signal-regulated kinase (ERK) phosphorylation. Kanzo inhibited COX-2 activity but increased annexin 1 and COX-2 expression and did not alter LPS-induced ERK phosphorylation. Keihi inhibited COX-2 activity and LPS-induced ERK phosphorylation but slightly increased COX-2 expression and did not alter cPLA2 and annexin 1 expression. These results suggest that the mechanism of shokyo is through the inhibition of cPLA2 activity, and that of kanzo and keihi is through the inhibition of COX-2 activity and indirect inhibition of cPLA2 activity. Therefore, it is possible that shokyo and kakkonto are clinically useful for the improvement of inflammatory responses.

Clinacanthus nutans Extracts Modulate Epigenetic Link to Cytosolic Phospholipase A2 Expression in SH-SY5Y Cells and Primary Cortical Neurons.

Clinacanthus nutans Lindau (C. nutans), commonly known as Sabah Snake Grass in southeast Asia, is widely used in folk medicine due to its analgesic, antiviral, and anti-inflammatory properties. Our recent study provided evidence for the regulation of cytosolic phospholipase A2 (cPLA2) mRNA expression by epigenetic factors (Tan et al. in Mol Neurobiol. doi: 10.1007/s12035-015-9314-z , 2015). This enzyme catalyzes the release of arachidonic acid from glycerophospholipids, and formation of pro-inflammatory eicosanoids or toxic lipid peroxidation products such as 4-hydroxynonenal. In this study, we examined the effects of C. nutans ethanol leaf extracts on epigenetic regulation of cPLA2 mRNA expression in SH-SY5Y human neuroblastoma cells and mouse primary cortical neurons. C. nutans modulated induction of cPLA2 expression in SH-SY5Y cells by histone deacetylase (HDAC) inhibitors, MS-275, MC-1568, and TSA. C. nutans extracts also inhibited histone acetylase (HAT) activity. Levels of cPLA2 mRNA expression were increased in primary cortical neurons subjected to 0.5-h oxygen-glucose deprivation injury (OGD). This increase was significantly inhibited by C. nutans treatment. Treatment of primary neurons with the HDAC inhibitor MS-275 augmented OGD-induced cPLA2 mRNA expression, and this increase was modulated by C. nutans extracts. OGD-stimulated increase in cPLA2 mRNA expression was also reduced by a Tip60 HAT inhibitor, NU9056. In view of a key role of cPLA2 in the production of pro-inflammatory eicosanoids and free radical damage, and the fact that epigenetic effects on genes are often long-lasting, results suggest a role for C. nutans and phytochemicals to inhibit the production of arachidonic acid-derived pro-inflammatory eicosanoids and chronic inflammation, through epigenetic regulation of cPLA2 expression.

The Novel cPLA2 Inhibitor AK106-001616 Has a Protective Effect on SOD1&amp;lt;sup&amp;gt;G93A&amp;lt;/sup&amp;gt;-Induced Cell Death in NSC34 Murine Motor Neuron-Like Cell

The expression of cytosolic phospholipase A2 (cPLA2) expression is up-regulated in animal model of ALS and in patients with familial amyotrophic lateral sclerosis (fALS). Inhibition of cyclooxygenase 2 (COX2), which is a downstream enzyme of cPLA2, ameliorates the impairment of motor function in the ALS model mice. Therefore, the arachidonic acid cascade, including the cPLA2-COX2 pathway, is an important therapeutic target of ALS. The current study was designed to investigate the potential of AK106-001616, an inhibitor of cPLA2, in protection of motor neuron cell death induced by mutant superoxide dismutase (SOD1G93A). AK106-001616 (1 - 10 μM) protected NSC34 cells (mouse motor neuron like cells) against SOD1G93A-induced motor neuron cell death. Furthermore, aspirin, an inhibitor of COX1/2, reduced the SOD1G93A-induced motor neuron cell death at a concentration that inhibited COX2. Celecoxib, a selective COX2 inhibitor, also reduced the SOD1G93A-induced motor neuron cell death. These results suggest that the arachidonic acid cascade is important for SOD1G93A-induced motor neuron cell death and AK106-001616 has a potent neuroprotective effect against it. AK106-001616 may be a useful therapeutic agent against SOD1G93A-induced ALS.

Astrocytes Release Polyunsaturated Fatty Acids by Lipopolysaccharide Stimuli.

We previously reported that levels of long-chain fatty acids (FAs) including docosahexaenoic acids (DHA) increase in the hypothalamus of inflammatory pain model mice. However, the precise mechanisms underlying the increment of free fatty acids (FFAs) in the brain during inflammation remains unknown. In this study, we characterized FFAs released by inflammatory stimulation in rat primary cultured astrocytes, and tested the involvement of phospholipase A2 (PLA2) on these mechanisms. Lipopolysaccharide (LPS) stimulation significantly increased the levels of several FAs in the astrocytes. Under these conditions, mRNA expression of cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2) in LPS-treated group increased compared with the control group. Furthermore, in the culture media, the levels of DHA and arachidonic acid (ARA) significantly increased by LPS stimuli compared with those of a vehicle-treated control group whereas the levels of saturated FAs (SFAs), namely palmitic acid (PAM) and stearic acid (STA), did not change. In summary, our findings suggest that astrocytes specifically release DHA and ARA by inflammatory conditions. Therefore astrocytes might function as a regulatory factor of DHA and ARA in the brain.