Phospholipase A2 Activity Research Articles

Review on the Ethnobotany, Phytochemical and Pharmacological Profile of Senna occidentalis L. (Fabaceae): Potential Application as Remedy in the Treatment of Dysmenorrhea

Senna occidentalis L. has been used in several traditional medicines against various diseases and this is based on its botanical, ethnopharmacology, and phytochemistry profiles. This powerful herb is recognized for its antibacterial, antifungal, antidiabetic, anticancer, antimutagenic, protective, and inflammatory hepatic activity. Multiple chemical compounds, including achrosine, aloe-emodin, emodin, anthraquinones, etc., have been isolated from this plant. The results of this bibliographic research thus presented in this review have demonstrated the ability of certain extracts from S. occidentalis L. to lower the lipid peroxide content, the activity of gamma-glutamyl transpeptidase and phospholipase A2 in exudates of the granuloma of cotton pellets, thus resulting in a reduced availability of arachidonic acid, an important precursor in the biosynthesis of prostaglandins, which are the only likely source and/or cause of dysmenorrhea. Thus, based on its phytochemical profile and its pharmacological properties, it is therefore suggested that S. occidentalis would be a potential and effective remedy in the treatment of dysmenorrhea.

Analyzing the influence of age and sex in Bothrops pauloensis snake venom

Considerable heterogeneity and ontogenetic changes in venom composition have already been observed in different species of snakes within the Viperidae family. Since the venom of young and adult can cause distinct pathological effects and because the antivenom may be less effective in neutralizing envenoming by young snakes compared to adults, it is of paramount importance to understand the ontogenetic variation of snake venom. Thus, the present study aimed to analyze and compare the venom of Bothrops pauloensis snakes, searching for possible influences of ontogeny and sex in their biochemical and biological aspects. The venom of younger individuals was more complex in relation to high molecular mass proteins, with a greater abundance of metalloproteinases, while adults showed a greater abundance of medium and low molecular mass proteins, such as phospholipases A2 (PLA2), C-type lectins and serine proteases. The antivenom showed better immunorecognition towards the venom of adult snakes than younger ones, in addition to a deficiency in the recognition of medium molecular mass proteins, suggesting the need for an improvement in the antivenom. Younger snakes showed higher coagulant, caseinolytic, and hemorrhagic activity, while adult snakes showed higher L-amino acid oxidase (LAAO) activity and acted faster in lethality. Differences between males and females were observed mainly in the rate of loss of coagulant activity, change in PLA2 activity and lethality action time. Furthermore, considering only the adult groups, males showed a higher LAAO and thrombin-like activity, while females showed a higher caseinolytic and hyaluronidase activity. With the results obtained in this work, it was possible to conclude that there is an ontogenetic variation in the composition and some activities of the B. pauloensis snake venom, in addition to differences between the venom of males and females, reinforcing that there is an intraspecific variation that may result in different symptoms in their envenoming and, consequently, differences in the response to treatment with the antivenom.

Effect of the phospholipase A2 inhibitor Varespladib, and its synergism with crotalic antivenom, on the neuromuscular blockade induced by Crotalus durissus terrificus venom (with and without crotamine) in mouse neuromuscular preparations

The venom of the South American rattlesnake Crotalus durissus terrificus causes an irreversible neuromuscular blockade in isolated preparations due to action of the presynaptically-acting heterodimeric phospholipase A2 (PLA2) crotoxin. Some populations of this subspecies contain, in addition to crotoxin, the toxin crotamine, which acts directly on muscle fibers. In this study we used C. d. terrificus venoms with (crot+) or without (crot-) crotamine to test whether Varespladib, a PLA2 inhibitor, is able to abrogate the neuromuscular blockade induced by these venoms comparatively with crotalic antivenom. Mouse phrenic nerve-diaphragm preparations were exposed to venoms previously incubated with two different concentrations of Varepladib or antivenom, or with a mixture of these two agents, before addition to the bath. In another experimental setting, venoms were initially added to the system, followed by the addition of Varespladib or antivenom 10, 30, or 60 min after venom. At the highest concentrations tested, Varespladib and antivenom inhibited the action of the venom >80% and >70%, respectively. With lower concentrations the inhibition of neuromuscular blockade decreased, but when low doses of the two agents were incubated together with the venom, the inhibitory effect improved, underscoring a synergistic phenomenon. When added after venom, Varespladib was able to halt the progression of the neuromuscular blockade even when added at 60 min. Antivenom exhibited a lower ability to inhibit the toxic effect of the venoms in these conditions. In conclusion, the PLA2 inhibitor Varespladib is highly effective at abrogating the neuromuscular blocking activity of crotamine-positive and crotamine-negative C. d. terrificus venoms and seems to act synergistically with antivenom.

Modified lipid metabolism and cytosolic phospholipase A2 activation in mesangial cells under pro-inflammatory conditions.

Diabetic kidney disease is a consequence of hyperglycemia and other complex events driven by early glomerular hemodynamic changes and a progressive expansion of the mesangium. The molecular mechanisms behind the pathophysiological alterations of the mesangium are yet to be elucidated. This study aimed at investigating whether lipid signaling might be the missing link. Stimulation of human mesangial cells with high glucose primed the inflammasome-driven interleukin 1 beta (IL-1β) secretion, which in turn stimulated platelet-derived growth factor (PDGF-BB) release. Finally, PDGF-BB increased IL-1β secretion synergistically. Both IL-1β and PDGF-BB stimulation triggered the formation of phosphorylated sphingoid bases, as shown by lipidomics, and activated cytosolic phospholipase cPLA2, sphingosine kinase 1, cyclooxygenase 2, and autotaxin. This led to the release of arachidonic acid and lysophosphatidylcholine, activating the secretion of vasodilatory prostaglandins and proliferative lysophosphatidic acids. Blocking cPLA2 release of arachidonic acid reduced mesangial cells proliferation and prostaglandin secretion. Validation was performed in silico using the Nephroseq database and a glomerular transcriptomic database. In conclusion, hyperglycemia primes glomerular inflammatory and proliferative stimuli triggering lipid metabolism modifications in human mesangial cells. The upregulation of cPLA2 was critical in this setting. Its inhibition reduced mesangial secretion of prostaglandins and proliferation, making it a potential therapeutical target.

MO614: Modified Lipid Metabolism and Cytosolic Phospholipase A2 Activation in Mesangial Cells Under Pro-Inflammatory Conditions

Abstract BACKGROUND AND AIMS Diabetic kidney disease (DKD) is thought to be a consequence of hyperglycemia and driven by early glomerular hemodynamic changes and progressive expansion of the mesangium. The molecular mechanism behind the pathophysiological alterations of the mesangium is yet to be elucidated. This study aimed at investigating whether lipid signaling might be the missing link. METHOD Human mesangial cells (HMCs) were stimulated with high glucose, IL-1β or PDGF-BB. Production of IL-1β and PDGF-BB, COX-2 induction and cPLA2 activation and prostaglandin secretion were investigated using a combination of ELISA, western blot, lipidomics, qPCR and activity assays. Inhibition of cPLA2 was evaluated using ELISA, migration and proliferation assays. Validation of data was performed in silico using the Nephroseq database [1] and a recently published glomerular transcriptomic DKD database. [2] RESULTS Stimulation of HMCs with high glucose primed IL-1β secretion through inflammasome NLRP3 signaling. In turn, IL-1β was able to stimulate PDGF-BB release. PDGF-BB increased IL-1β secretion synergistically. Both IL-1β and PDGF-BB stimuli caused lipid metabolism variations and in particular triggered the formation of phosphorylated sphingoid bases (PSBs) and the activation of the enzymes cPLA2 and COX-2. This led to the formation of prostanoids from arachidonic acid produced by cPLA2. As a result, prostaglandins were secreted. Another product of cPLA2 activation was lysophosphatidic acid (LPA), a proliferation effector. Blocking cPLA2-mediated release of arachidonic acid could in fact reduce HMCs’ proliferation and secretion of prostaglandins. CONCLUSION Hyperglycemia primes glomerular inflammatory and proliferative stimuli triggering lipid metabolism modifications in HMCs. The upregulation of cPLA2 was found to be critical in this setting. cPLA2 inhibition with AACOCF3 reduced mesangial secretion of prostaglandins and proliferation, making it a potential target for DKD therapy.

Oxidized Lipids Control Lipid Order and Phospholipase A2 Activity in Model Membranes

Oxidative stress is an important etiologic factor in the pathogenesis of various diseases. The formation of oxidized phospholipid species in vivo induces membrane remodelling with direct pathological implications with a prominent inflammatory component. Secretory phospholipases A2 (sPLA2) are involved in the regulation of inflammation and immune response. Their activity is highly dependent on the lipid membrane composition, structure and organization. In this work, we studied the impact of oxidized phosphatidylcholines (OxPCs) on the membrane lipid order and the sPLA2 activity. The effects of two of the most physiologically active OxPCs, 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero- 3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3- phosphocholine (PGPC) were compared using 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC) vesicles. Both OxPCs reduce the membrane lipid order and sPLA2 activity at physiological temperature. Moreover, these changes depend on the chemical nature of the oxidized chains.

Homologues Phospholipases A2 isolated from Naja naja atra snake venom exhibit different activity against intact human erythrocytes

Phospholipases A2 (PLA2) catalyze the hydrolysis of fatty acid esters in the 2‐position of 3‐snphospholipids and are found in intracellular and secreted forms. Secretory PLA2s (sPLA2s) are small proteins (13‐15 kDa) classified into five groups based on primary structure differences. Despite their structural similarity, sPLA2s show a wide range of enzymatic and pharmacological activities. Most sPLA2s have high activity toward anionic membranes due to cationic residue presence on their binding recognition site (BRS). Within the same snake venom, several PLA2s can be found, which present differences in their ability to hydrolyze phospholipids in biological bilayers.In this study, Chinese cobra, Naja naja atra (N. n. atra) venom was purified by Size Exclusion Chromatography using a Superdex 75 and cation exchange using a column packed with Carboxymethyl Cellulose (CM‐Cellulose) to pre‐separate protein components from the venom. Subsequently, fractions displaying PLA2 activity were pooled, concentrated, and applied to a GE mini‐CM‐Cellulose column coupled to the AKTA Fast Protein Liquid Chromatography (FPLC) system. The final purification presents four peaks but only two present PLA2 activity (Band D and E). Peak purity was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE).We have shown that hydrophobic residue presence on the PLA2 BRS is crucial for binding and hydrolytic activity toward human erythrocyte membranes. PLA2 activity was assayed against human erythrocytes and monomeric substrate to determine differences in hydrolytic activity. Primary sequence alignment shows 94% homology, however Band E presented 40% less activity against human erythrocytes, despite having 100% active site homology. Molecular modeling of both sPLA2s revealed differences at the Cys‐19 and Trp‐19 position and spatial orientation differences at the Trp‐61, Tyr‐63 Phe‐64, and Lys‐65 positions. Due to amino acid differences at the BRS, differences in hydrolytic activity may be linked to the anchorage of sPLA2 to the erythrocyte surface.

Petasites for Migraine Prevention: New Data on Mode of Action, Pharmacology and Safety. A Narrative Review.

Petasins are the pharmacologically active ingredients of butterbur and of therapeutic benefit in the treatment of migraine and tension headaches. Here, we summarize the pharmacology, safety and clinical efficacy of butterbur in the prevention of migraine attacks and present new data on its mode of action. We review published literature and study reports on the safety and clinical efficacy of the butterbur root extract Petadolex® and report new findings on petasins in dampening nociception by desensitizing calcium-conducting TRP ion channels of primary sensory neurons. Importantly, butterbur diminishes the production of inflammatory mediators by inhibiting activities of cyclooxygenases, lipoxygenases and phospholipase A2 and desensitizes nociception by acting on TRPA1 and TRPPV1 ion channels. It inhibits the release of calcitonin-gene related peptide (CGRP) of meningeal afferents during migraine attacks. We also evaluated the safety of a butterbur root extract in repeated dose studies for up to 6 months. A no-observable-adverse-effect-level at 15-fold of the maximal clinical dose (3 mg/kg/day MCD) was established for rats. At supratherapeutic doses, i.e., 45–90-fold MCD, we observed bile duct hyperplasia, and mechanistic studies revealed regulations of solute carriers to likely account for bile duct proliferations. Additionally, liver function tests were performed in cultures of primary human hepatocytes and did not evidence hepatotoxicity at therapeutic butterbur level and with migraine co-medications. Lastly, in randomized, double-blinded and placebo-controlled trials with Petadolex® migraine attack frequency was reduced significantly at 150 mg/day, and no relevant abnormal liver function was reported. Together, butterbur is effective in the prevention of migraine attacks by blocking CGRP signaling.

Atherogenicity of low-density lipoproteins after switching from a protease inhibitor to dolutegravir: a substudy of the NEAT022 study.

The aim of this study was to investigate whether switching from a ritonavir-boosted PI-based regimen to a dolutegravir-based regimen improved the atherogenic properties of LDL particles in patients with HIV. This was a substudy of the NEAT022 study (ClinicalTrials.gov NCT02098837). Adults with HIV with a Framingham score >10% or aged >50 years and being treated with a stable boosted PI-based regimen were randomized to either switch to dolutegravir or continue with boosted PI. At baseline and Week 48, we assessed atherogenic LDL properties: LDL particle size and phenotype (A, intermediate, B), oxidized LDL (ox-LDL) and lipoprotein-associated phospholipase A2 (Lp-PLA2) activity. Eighty-six participants (dolutegravir 44; PI 42) were included. Participants had a median (IQR) age of 54 (51-57) years and 79.1% were male. In the dolutegravir arm, after 48 weeks, we observed: (1) an increase in LDL size [median 1.65 Å (IQR -0.60 to 4.20); P = 0.007], correlated with the decrease in triglyceride concentration [Spearman correlation = -0.352 (P = 0.001)], with a corresponding decrease of subjects with atherogenic LDL phenotype B (36.4% to 20.5%; P = 0.039); (2) a decrease in Lp-PLA2 activity [median 1.39 μmol/min/mL (IQR -2.3 to 0.54); P = 0.002]; and (3) a decrease in ox-LDL [median 14 U/L (IQR -102 to 13); P = 0.006]. In the PI arm, none of these favourable lipid modifications was observed. Forty-eight weeks after switching from a PI-based to a dolutegravir-based regimen, patients with Framingham score >10% or aged >50 years showed improvement of several atherogenic lipid features, including LDL particle phenotype, ox-LDL and Lp-PLA2.

Current Knowledge on Mammalian Phospholipase A1, Brief History, Structures, Biochemical and Pathophysiological Roles.

Phospholipase A1 (PLA1) is an enzyme that cleaves an ester bond at the sn-1 position of glycerophospholipids, producing a free fatty acid and a lysophospholipid. PLA1 activities have been detected both extracellularly and intracellularly, which are well conserved in higher eukaryotes, including fish and mammals. All extracellular PLA1s belong to the lipase family. In addition to PLA1 activity, most mammalian extracellular PLA1s exhibit lipase activity to hydrolyze triacylglycerol, cleaving the fatty acid and contributing to its absorption into the intestinal tract and tissues. Some extracellular PLA1s exhibit PLA1 activities specific to phosphatidic acid (PA) or phosphatidylserine (PS) and serve to produce lysophospholipid mediators such as lysophosphatidic acid (LPA) and lysophosphatidylserine (LysoPS). A high level of PLA1 activity has been detected in the cytosol fractions, where PA-PLA1/DDHD1/iPLA1 was responsible for the activity. Many homologs of PA-PLA1 and PLA2 have been shown to exhibit PLA1 activity. Although much has been learned about the pathophysiological roles of PLA1 molecules through studies of knockout mice and human genetic diseases, many questions regarding their biochemical properties, including their genuine in vivo substrate, remain elusive.

Efficient immobilized phospholipase A1 on Mo-basing nanomaterials for enzymatic degumming.

Six kinds of Mo-basing nanomaterials (MoO3 , MoO3 @Ru, Mo-PDA, MoPC , MoP, CNT@MoS2 ) were successfully synthesized, which were employed as carriers to immobilize phospholipase A1 (PLA1) for the hydrolysis of phospholipids (PLs). PLA1 was immobilized by a simple adsorption-precipitation-cross-linking to form an "enzyme net" covering on nanoparticles. The greatest advantage of these nanoparticles was their strong hydrophilic surface. It not only permitted their dispersion in the aqueous phase, but also showed the strong affinity for PLs in the organic phase, because amphiphilic PLs had the polar head group and higher hydrophilicity than other oils components. Michaelis-Menten analysis revealed that higher catalytic activity and enzyme-substrate affinity were observed in several immobilized PLA1 than its free form. MoO3 was confirmed to be the best candidate for carrier. The highest specific activity of MoO3 -immobilized PLA1 reached 43.1U/mg, which was about 1.8 times higher than that of free PLA1 (24.4U/mg). In addition, the stability and recycling were also enhanced. The robust immobilized PLA1 was prepared in this work, showing great potential for the enzymatic degumming.

Lipoprotein-associated Phospholipase A2 Activity Contributes to the Coronary Artery Disease with Metabolic Syndrome

Background: Coronary artery disease (CAD) is an inflammatory process characterized by atherosclerosis in coronary arteries and it is a major cause of death and disability in developed countries. Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been consistently associated with CAD risk factors and predictive of CVD outcomes; additionally, it is consistently higher among type 2 diabetics than nondiabetics. However, the relationships of circulating Lp-PLA2 activity with incident CAD among patients with metabolic syndrome (MetSynd) have not been examined sufficiently.
 Objective: The aim is to determine contribution of Lp-PLA2 to coronary artery disease (CAD) in patients with Metabolic Syndrome (MetSynd).
 Subjects and methods: This is a cohort prospective study based on 412 patients male and female were eligible and aged 25-75 years old patients and gave consent to participate in study. The study included socio-demographics, clinical biochemistry and the presence of co-morbid diseases. The data were analyzed using descriptive and multivariate analyses.
 Results: There was a significant difference between MetSynd Positive and normal subjects with respect to age groups, gender, BMI, smoking, nargile use, thyroid, COPD, CAD, hypertension, diabetic and stroke. Also, there was a significant difference between MetSynd versus normal subjects with respect to BMI, Waist Circumference, hemoglobin, HbA1c, vitamin B12, fasting blood glucose, vitamin D, calcium, creatinine, triglyceride, uric acid, ferritin, systolic BP (mm Hg) and diastolic BP (mm Hg), creatine kinase-myocardial band (CK-MB) (p=0.001); Lp-PLA2 activity, (p=0.001); HOMA-IR index,(p=0.004), insulin (p=0.001); C-reactive protein (p=0.004);White blood cell (WBC) (p=0.008); Platelet p= 0.018) Mean Plate Volume (p= 0.032); red cell distribution width (p=0.001); and vitamin D levels (p=0.018), respectively. The multivariate stepwise regression analysis indicated that Lp-PLA2 (p<0.001), BMI (kg/m2) (p<0.001), systolic BP (p<0.001), MetSynd (p=0.002), CK-MB (p=0.019), Calcium) (p= 0.023), Triglyceride (p= 0.029), Total-cholesterol (p= 0.046) were considered as risk predictors of the CAD patients after adjusting for age and gender.
 Conclusion: Lp-LPA2 contributes to CAD in the presence of MetSynd, as well as Lp-PLA2 could be utilized as a useful predictor in cases of CAD with MetSynd.

Dengue NS1 induces phospholipase A2 enzyme activity, prostaglandins, and inflammatory cytokines in monocytes

IntroductionDengue virus (DENV) NS1 is a non-structural secretory protein associated with severe disease and known to cause vascular leak leading to dengue haemorrhagic fever (DHF). As phospholipases A2 (PLA2) enzymes, platelet activating factor, and leukotrienes are elevated in dengue, we sought to investigate whether NS1 potentially contributes to disease pathogenesis by inducing PLA2s. MethodsTHP-1 cells and primary human monocytes of healthy adults (n = 6) were co-cultured with DENV1 NS1, LPS and media alone. The latter two were used as positive and negative controls. The cell culture supernatants and lysates were harvested at 12 and 24 h and the activity of secretory and cytoplasmic PLA2, prostaglandins (PGE2 and PGD2) were measured by ELISA and cytokines levels were measured using a magnetic Luminex assay. Expression of PLA2G4A, PLA2G2A, PLA2G5, PLA2G10, PLA2G7, GAPDH, NLRP3 and DDX58 genes were assessed using quantitative RT-PCR. ResultscPLA2 (p = 0.005), sPLA2 (p = 0.04), PGE2 metabolite (p = 0.02) and PGD2 metabolite (p = 0.04) levels were significantly higher at 12 h in monocytes co-cultured with NS1. Levels of IP-10 (p = 0.005) and IL-10 (p = 0.009) was significantly higher at 24 h, whereas IFNα level was significantly higher (p = 0.013) only at 12 h. IL-1β (p = 0.028 and p = 0.031) and TNFα (p = 0.007 and p = 0.011) showed significantly higher levels at both time points. At 12 h significant upregulation of PLA2G4A (p < 0.0001) was seen, whereas PLA2G7 (p = <0.0001), NLRP3 (p = 0.0009) and DDX58 (p = 0.0056) were significantly downregulated. This pattern changed at 24 h with PLA2G4A (p = 0.0069) showing a marked downregulation and PLA2G7, DDX58 and NLRP3 showing an upregulation, although not significant. ConclusionDengue NS1 induces the production of PLA2 enzymes, prostaglandins and inflammatory cytokines from primary human monocytes, which could play a role in vascular leak in dengue.

Inflammation and Nitro-oxidative Stress as Drivers of Endocannabinoid System Aberrations in Mood Disorders and Schizophrenia.

The endocannabinoid system (ECS) is composed of the endocannabinoid ligands anandamide (AEA) and 2-arachidonoylgycerol (2-AG), their target cannabinoid receptors (CB1 and CB2) and the enzymes involved in their synthesis and metabolism (N-acyltransferase and fatty acid amide hydrolase (FAAH) in the case of AEA and diacylglycerol lipase (DAGL) and monoacylglycerol lipase (MAGL) in the case of 2-AG). The origins of ECS dysfunction in major neuropsychiatric disorders remain to be determined, and this paper explores the possibility that they may be associated with chronically increased nitro-oxidative stress and activated immune-inflammatory pathways, and it examines the mechanisms which might be involved. Inflammation and nitro-oxidative stress are associated with both increased CB1 expression, via increased activity of the NADPH oxidases NOX4 and NOX1, and increased CNR1 expression and DNA methylation; and CB2 upregulation via increased pro-inflammatory cytokine levels, binding of the transcription factor Nrf2 to an antioxidant response element in the CNR2 promoter region and the action of miR-139. CB1 and CB2 have antagonistic effects on redox signalling, which may result from a miRNA-enabled negative feedback loop. The effects of inflammation and oxidative stress are detailed in respect of AEA and 2-AG levels, via effects on calcium homeostasis and phospholipase A2 activity; on FAAH activity, via nitrosylation/nitration of functional cysteine and/or tyrosine residues; and on 2-AG activity via effects on MGLL expression and MAGL. Finally, based on these detailed molecular neurobiological mechanisms, it is suggested that cannabidiol and dimethyl fumarate may have therapeutic potential for major depressive disorder, bipolar disorder and schizophrenia.

Effects of Peroxiredoxin 6 and Its Mutants on the Isoproterenol Induced Myocardial Injury in H9C2 Cells and Rats.

Background Peroxiredoxin 6 (PRDX6) is an important antioxidant enzyme, with a potential application value in the treatment of diseases caused by oxidative damage. Methods PRDX6 and a mutant (mPRDX6) were heterologously expressed by using an E.coli expression system and purified by Ni-affinity chromatography. Isoproterenol (ISO) was used to induce a myocardial cell injury model and an animal myocardial injury model. After the treatment with PRDX6 and mPRDX6, the proliferation activity of H9C2 cells was detected by Cell Counting Kit-8 (CCK8) method; the apoptosis was evaluated by flow cytometry, and the histological changes of myocardial cells were observed by hematoxylin and eosin (H&E) staining, the levels of catalase (CAT), glutathione peroxidase (GPX), malondialdehyde (MDA), and superoxide dismutase (SOD) in ISO-treated H9C2 cells as well as in the heart tissue and serum of rats treated with ISO were detected, and the expression levels of Bax, Bcl-2 and peroxisome proliferators-activated receptors-γ (PPAR-γ) proteins were detected by Western blot. Results PRDX6 and mPRDX6 were successfully expressed and purified. The results of efficacy study showed that the mutant mPRDX6, in which the phospholipaseA2 (PLA2) activity of PRDX6 was deleted by site directed mutation, had a better protective effect against the myocardial injury than PRDX6. CCK8 results showed that compared with that in ISO group, the proliferation activity of H9C2 cells was significantly enhanced (P < 0.01), the apoptosis rate was significantly decreased (P < 0.01), and the fluorescence intensity of reactive oxygen species (ROS) was significantly decreased (P < 0.01) in mPRDX6 group. The results of H&E staining showed that the myocardial injury was alleviated to a certain extent in mPRDX6 group. Compared with those in ISO group, the activities of CAT, GPX, and SOD in H9C2 cells and the heart tissue and serum of rats were significantly increased (P < 0.05), while the contents of MDA were significantly decreased (P < 0.05). Western blot analysis showed that the expression level of Bcl-2 in H9C2 cells was significantly decreased (P < 0.01), and that of Bax and PPAR-γ was significantly increased (P < 0.05). Conclusion mPRDX6 has a protective effect against the myocardial injury induced by ISO, and the mechanism may be related to its antioxidation. This study may provide a theoretical basis for the research and development of drugs used for the treatment of myocardial injury.

Circulating Biomarkers Predict Symptomatic but Not Asymptomatic Carotid Artery Stenosis

Background: Subjects exposed to risk factors such as age, gender, hypertension, diabetes mellitus, and smoking are prone to atherosclerotic events. Aims: The main aim of this longitudinal cohort study was to determine whether the role of novel plasma biomarkers for atherosclerotic carotid artery disease is different in subjects developing symptomatic carotid artery stenosis (CAS), as opposed to those with incident asymptomatic CAS. Methods: The following biomarkers were measured in 5,550 middle-aged subjects in a population-based cohort study: C-reactive protein (CRP), lipoprotein-associated phospholipase A2 mass and activity, proneurotensin, midregional proadrenomedullin (MR-proADM), midregional proatrial natriuretic peptide (MR-proANP), N-terminal pro-B-type natriuretic peptide (NT pro-BNP), copeptin, and cystatin C. After exclusion of those with prevalent CAS, subjects were thereafter followed in national patient registers for 23.4 (interquartile range 19.5–24.3) years regarding incident symptomatic and asymptomatic CAS. Results: Among 110 patients with confirmed incident CAS, 56 were symptomatic and 54 were asymptomatic. When including conventional risk markers in a Cox regression analysis, NT pro-BNP (hazard ratio [HR] 1.59; 95% confidence interval [CI]: 1.20–2.11), MR-proADM (HR 1.40; CI: 1.13–1.73), cystatin C (HR 1.21; CI: 1.02–1.43), and CRP (HR 1.53; CI: 1.13–1.73) were independently associated with incident symptomatic CAS, whereas no plasma biomarker was associated with incident asymptomatic CAS. Conclusion: Plasma biomarkers NT pro-BNP, MR-proADM, cystatin C, and CRP were independently associated with incident symptomatic CAS, whereas no such association could be demonstrated with incident asymptomatic CAS. As these biomarkers indicate future development of clinically relevant atherosclerotic CAS, their potential utility in relation to intensified preventive measures and selection of potential candidates for carotid surgery should be further evaluated.

The IRE1α Inhibitor KIRA6 Blocks Leukotriene Biosynthesis in Human Phagocytes

The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) has been linked to inflammation and lipid mediator production. Here we report that the potent IRE1α inhibitor, KIRA6, blocks leukotriene biosynthesis in human phagocytes activated with lipopolysaccharide (LPS) plus N-formyl-methionyl-leucyl-phenylalanine (fMLP) or thapsigargin (Tg). The inhibition affects both leukotriene B4 (LTB4) and cysteinyl leukotriene (cys-LTs) production at submicromolar concentration. Macrophages made deficient of IRE1α were still sensitive to KIRA6 thus demonstrating that the compound’s effect on leukotriene production is IRE1α-independent. KIRA6 did not exhibit any direct inhibitory effect on key enzymes in the leukotriene pathway, as assessed by phospholipase A2 (PLA2), 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S) enzyme activity measurements in cell lysates. However, we find that KIRA6 dose-dependently blocks phosphorylation of p38 and ERK, mitogen-activated protein kinases (MAPKs) that have established roles in activating cytosolic PLA2α (cPLA2α) and 5-LOX. The reduction of p38 and ERK phosphorylation is associated with a decrease in cPLA2α phosphorylation and attenuated leukotriene production. Furthermore, KIRA6 inhibits p38 activity, and molecular modelling indicates that it can directly interact with the ATP-binding pocket of p38. This potent and unexpected, non-canonical effect of KIRA6 on p38 and ERK MAPKs and leukotriene biosynthesis may account for some of the immune-modulating properties of this widely used IRE1α inhibitor.

Biochemical Characterization of the Pharmacological Effects of Yagari in Experimentally Induced Benign Prostatic Hyperplasia

AbstractBenign prostatic hyperplasia is a progressive pathologic condition common in ageing men, constituting a health burden. Benign prostatic hyperplasia is characterized by the proliferation of prostatic tissues, prostate enlargement, and lower urinary tract symptoms. The use of herbal medicine in the management and treatment of benign prostatic hyperplasia has shown some promise. The efficacy and potency of some polyherbal extracts have been scientifically proven in the management and treatment of diseases, while many others are yet to be investigated. The aim of this study was to determine the effect of the herbal mixture Yagari on experimentally induced benign prostatic hyperplasia in rats and to identify its pharmacologically active agents. The effect of the herbal mixture on experimentally induced benign prostatic hyperplasia in rats was determined using 36 male Wistar rats grouped randomly into 6 groups of 6 rats each. The test rats were treated subcutaneously using a combination of dihydrotestosterone and estradiol valerate in a 10:1 ratio for 28 days according to the standard method. The test rats were thereafter treated with the herbal mixture for 21 days. Prostate-specific antigen, inflammatory cytokines, assay of prostate function hormonal and prostate function enzyme activities, and kidney function test were evaluated in the blood samples collected by ocular puncture applying standard methods. Prostates were harvested and examined for histopathological changes. Biological activity-guided fractionation of Yagari in a silica gel column was carried out and using phospholipase A2 activity as a biomarker. The identity of the bioactive compounds was determined using mass spectroscopy and nuclear magnetic resonance. The herbal mixture showed a positive effect on prostatic hyperplasia by decreasing urinary obstruction through the inhibition of 5-alpha reductase, anti-inflammatory activity, and decreased level of sex hormones. Characterization by spectral studies revealed apigenin (4´,5,7-trihydroxyflavone, molecular weight).

Traces of Bothrops snake venoms in necrotic muscle preclude myotube formation in vitro

Deficient skeletal muscle regeneration, which often leads to permanent sequelae, is a common clinical finding in envenomations caused by snakes of the family Viperidae, such as those of Bothrops alternatus and B. diporus in South America. The causes of such poor muscle regenerative outcome are still incompletely understood. Using a murine experimental model of envenomation by the venoms of these two species, we assessed whether traces of venom components that remain in muscle tissue days after envenomation affect myoblasts and myotube formation in culture. The kinetics of drop in venom concentration in the tissue was assessed by ELISA and Western blot, and by the quantification of venom phospholipase A2 activity. A rapid drop of venom components was observed in muscle, although a band of 58–63 kDa remained even 168 h after venom injection, and venom phospholipase A2 activity was detected in muscle tissue days after envenomation. Muscle homogenates from envenomated animals were cytotoxic to myoblasts in culture and inhibited the formation of myotubes even in conditions where homogenates were devoid of cytotoxicity. These deleterious effects were abrogated when homogenates were incubated with antivenom. Our findings agree with previous observations with the venom of Bothrops asper and provide further evidence that one of the causes of the poor skeletal muscle regeneration after Bothrops sp venom-induced myonecrosis is the deleterious action on myogenic cells of traces of venom components remaining in the tissue.

Single-molecule phospholipase A2 becomes processive on melittin-induced membrane deformations

While it is established that the topology of lipid membranes plays an important role in biochemical processes, few direct observations exist regarding how the membranes are actively restructured and its consequences on subsequent reactions. In this work, we investigated how the two major components of bee venom, melittin and phospholipase A2 (PLA2), achieve activation by such membrane remodeling. Their membrane-disrupting functions have been reported to increase when both are present, but the mechanism of this synergism had not been established. Using membrane reconstitution, we found that melittin can form large-scale membrane deformities upon which PLA2 activity is 25-fold higher. Tracking of single-molecule PLA2 revealed that its processive behavior on these deformities underlies the enhanced activity. These results show how melittin and PLA2 work synergistically to enhance the lytic effects of the bee venom. More broadly, they also demonstrate how the membrane topology may be actively altered to modulate cellular membrane-bound reactions.