P2X7-Receptor Pathway Involvement in the Anti-Inflammatory Activity of Medicinal Plants.

Medicinal plants used in European folk medicine attached to Lamiales, Gentianales or Asterales orders are used to treat inflammatory disorders. Many targets have been identified but to date, implication of purinergic receptor P2X7 activation has not yet been investigated. We managed to evaluate the protective effect on P2X7 activation by plant extracts used as anti-inflammatory in European folk medicine by the YO-PRO-1 uptake dye in vitro bioassay. Results revealed that among our selected plants, species from Scrophularia and Plantago genus were able to decrease significantly P2X7 activation (>50 % at 0.1 and 1 µg/mL). UPLC/MS, dereplication and metabolomic analysis of Scrophularia extracts, allowed us to identify the cinnamoyl-iridoid harpagoside as putative inhibitor of P2X7 activation. These results open a new research field regarding the anti-inflammatory mechanism of cinnamoyl-iridoids bearing plants, which may involve the P2X7 receptor.

Evaluation of Placental Toxicity of Five Essential Oils and Their Potential Endocrine-Disrupting Effects.

Pregnant women may use EOs in case of morning sickness, nausea, stress management, etc. Little is known about the potential danger that EOs represent for the placenta and therefore for the pregnancy. Our aim was to explore and compare the placental toxicity and potential endocrine disrupting effects of niaouli, orange, tea tree, wintergreen and ylang-ylang EOs, and their key compounds: 4-terpineol, 1,8-cineol, limonene, methyl salicylate and benzyl salicylate. We studied the release of four hormones and the activation of P2X7 receptor in JEG-Tox human placental cells as key biomarkers for endocrine toxicity. We observed that niaouli, orange, tea tree, wintergreen and ylang-ylang EOs and their key components disrupted at least one of the studied hormones but none of them activated the P2X7 cell death receptor. The tested EOs appear then to be more hormonal modulators rather than EDCs in human placental cells. The hormonal effects observed with the key components were very different from those observed with the EOs. EOs are very complex mixtures, and it is essential to study whole EOs rather than their components individually in safety assessment.

In Vitro Chemopreventive Potential of Phlorotannins-Rich Extract from Brown Algae by Inhibition of Benzo[a]pyrene-Induced P2X7 Activation and Toxic Effects.

Phlorotannins are polyphenols occurring exclusively in some species of brown algae, known for numerous biological activities, e.g., antioxidant, antiproliferative, antidiabetic, and antiallergic properties. Their effects on the response of human lung cells to benzo[a]pyrene (B[a]P) has not been characterized. Our objective was to in vitro evaluate the effects of a phlorotannin-rich extract obtained from the brown algae Ascophyllum nodosum and Fucus vesiculosus on B[a]P cytotoxic effects. The A549 cell line was incubated with B[a]P for 48 and 72 h in the presence or absence of the brown algae extract. Cytochrome P450 activity, activation of P2X7 receptor, F-actin disorganization, and loss of E-cadherin expression were assessed using microplate cytometry and fluorescence microscopy. Relative to control, incubation with the brown algae extract was associated with lower B[a]P-induced CYP1 activity, lower P2X7 receptor activation, and lower reactive oxygen species production. The brown algae extract inhibited the alterations of F-actin arrangement and the downregulation of E-cadherin expression. We identified a phlorotannins-rich extract that could be deeper investigated as a cancer chemopreventive agent to block B[a]P-mediated carcinogenesis.

Perception of pharmacy students toward opioid-related disorders and roles of community pharmacists: A French nationwide cross-sectional study.

Background: Community pharmacists are among the frontline health professionals who manage patients with an opioid-related disorder (ORD). Pharmacists frequently have a negative attitude toward these patients, which could have a negative impact on their management. However, education on ORD may improve the attitude of future healthcare professionals. This cross-sectional study aimed to assess French pharmacy students' perceptions of ORD. Methods: This online survey was performed by emails sent to French pharmacy schools (between January 14, 2019 and May 31, 2019). The primary outcome was the perception (visual analogic scale) of ORD as a disease, the roles of community pharmacies (delivery of opioid agonist therapy-OAT and harm reduction kits), and the efficacy of OAT. The secondary outcomes assessed professional experience, university experience of and education on ORD, and the individual characteristics of students. Results: Among the 1,994 students included, 76.3% perceived ORD as a disease and felt that it was normal for pharmacists to deliver OAT (78.9%) and harm reduction kits (74.6%). However, only 46.9% perceived OAT as being effective. Multivariable analyses showed that females had a more positive perception in recognizing ORD as a disease. The progression through university years increased the positive perception of ORD as a disease and the delivery of OAT and harm reduction kits by pharmacists. Education on substance-related disorders had no impact on any scores. Students who had already delivered OAT had a negative perception of their efficacy. The students who had already performed pharmacy jobs or traineeships had a negative perception of harm reduction kit delivery. Conclusion: Education on substance-related disorders had no impact on students' perceptions. It seemed that the maturity acquired through university years had a stronger impact on the students' perceptions of ORD. Efforts must be made to improve our teaching methods and reinforce the confidence of students in the roles of community pharmacists.

Cytotoxic and Anti-Inflammatory Effects of Ent-Kaurane Derivatives Isolated from the Alpine Plant Sideritis hyssopifolia.

This paper reports the isolation and structural characterization of four new ent-kaurane derivatives from the Lamiaceae plant Sideritis hyssopifolia. Planar structures and relative configurations were determined using both mass spectrometry and nuclear magnetic resonance (1D and 2D). Absolute configurations were determined by comparing experimental and theoretical electronic circular dichroism spectra. The cytotoxic and microbial activities of all new compounds were tested. Compounds that were non-cytotoxic were further evaluated for anti-inflammatory activity.

The Algoplus Score to Assess Acute Postoperative pain in Elderly patients-A Pilot Observational Study.

Standard verbal or analogue scales may not be accurate to assess acute postoperative pain in elderly patients. This study was designed to field test the Algoplus tool, developed specifically for this population and based on observation of patient behavior. Prospective, observational cohort. Single center, French University hospital. Forty-eight patients, aged over 65, scheduled for surgery under general anesthesia, and observed on admission to the postanesthesia care unit, immediately after extubation, during the different steps of analgesic intervention (demand, relief with intravenous opioid titration, plus intermediate measures when relevant), and either at discharge or 3 hours after admission. A numerical rating scale (NRS) was used to guide analgesia. The Algoplus score and the state of alertness or sedation were noted. NRS scores and Algoplus scores were significantly related, and both scores significantly decreased under the effect of analgesia, but the correlation was low. In early observations, the Algoplus score was higher than that predicted by the NRS score, in relation to residual sedation. Female gender tended to lower the Algoplus score compared to the NRS score. When the NRS score exceeded 3/10, indicating the need for analgesic intervention, the Algoplus score was generally lower than the recommended trigger for analgesia (2/5). These results are promising, but further evidence of a clinical benefit to the use of Algoplus for acute postoperative pain is needed. In future studies, scoring should be adjusted to take into account the time from extubation, the state of sedation, and the patient's gender in order to interpret results.

Advancing Endocrine Disruptors via In Vitro Evaluation: Recognizing the Significance of the Organization for Economic Co-Operation and Development and United States Environmental Protection Agency Guidelines, Embracing New Assessment Methods, and the Urgent Need for a Comprehensive Battery of Tests.

Efforts are being made globally to improve the evaluation and understanding of endocrine-disrupting chemicals. Recognition of their impact on human health and the environment has stimulated attention and research in this field. Various stakeholders, including scientists, regulatory agencies, policymakers, and industry representatives, are collaborating to develop robust methodologies and guidelines for assessing these disruptors. A key aspect of these efforts is the development of standardized testing protocols and guidelines that aim to provide consistent and reliable methods for identifying and characterizing endocrine disruptors. When evaluating the potential endocrine-disrupting activity of chemicals, no single test is capable of detecting all relevant endocrine-disrupting agents. The test battery approach is designed to reduce the risk of false negative results for compounds with toxic potential. A weight-of-evidence approach is therefore necessary for endocrine disruptor evaluation. This approach considers various types of data from multiple sources, assessing the overall strength, consistency, and reliability of the evidence. OECD guidelines are highly regarded for their scientific rigor, transparency, and consensus-based development process. It is crucial to explore and develop new methodologies that can effectively evaluate the risks associated with potential endocrine disruptors. Integrating these methods into a comprehensive weight-of-evidence framework will enhance risk assessments and facilitate informed decisions regarding the regulation and management of these substances, ensuring the protection of human health and the environment from their adverse effects.

Acute pain evaluation with Algoplus scale in Cambodian patients.

OBJECTIVE: Evaluation of acute pain in patients with language barriers is often difficult during humanitarian missions. Algoplus, a behavioral scale validated for acute pain evaluation in Caucasians with verbal communication difficulties, was tested during a clinical mission in Cambodia in patients admitted to hospital for acute pain. METHODS: Patients (N = 33, 19 men and 14 women [38 ± 3 years old]) suffering from acute pain were admitted to Calmette Hospital, Phnom Penh, Cambodia during June 2010. Patients spoke Khmer only; a medical trainee who did no speak Khmer performed the Algoplus scale, and a bilingual Khmer pain specialist doctor asked the patient to score pain intensity on a numerical scale (0-10). The relevance of the scale and of each item (facial expression, complaints, look, body position, and atypical behavior yes/no) was studied. Internal consistency was assessed by Cronbach alpha analysis and convergent validity by correlation coefficient. The relationship between Algoplus items and pain intensity was analysed. RESULTS: Pain intensity (6.3 ± 2.2) and Algoplus scores (2.1 ± 1.2) are correlated (r = 0.61, P < 0.001): increases in Algoplus are correlated to increases in pain intensity. Internal consistency is 0.51, and scales comparison per item is significant for facial expression (P = 0.028) and complaints (P = 0.005), but not for the other items. CONCLUSION: This feasibility study shows that despite a correlation with pain intensity, Algoplus may underestimate acute pain in this population. It is, however, an interesting tool for future studies to explore facial expression and complaints as proxies of pain in non-communicative patients.

Effects of vegetable oils on biochemical and biophysical properties of membrane retinal pigment epithelium cells.

The aim of this study was to investigate the effect of vegetable oil enrichment of retinal pigment epithelial (RPE) cells on their biochemical and biophysical properties. For this, RPE cells were incubated with 4 different vegetables oils (olive oil, corn oil, argan oil, and camelina oil). The cytotoxicity of these vegetable oils was assessed in vivo on 8-week-old mice and in vitro by using the neutral red and YO-PRO-1 tests. Membrane fluidity was evaluated by fluorescence anisotropy using the fluorescent probe diphenylhexatriene, and membrane fatty acid composition was assessed by gas chromatography. None of the oils tested displayed cytotoxic effects. In vitro, omega-3 rich oils improved membrane fluidity by 47% compared with the control cells. The omega-3 PUFA content within membranes decreased by 38% to 55% when cells were incubated separately with olive oil, corn oil, or argan oil, and increased when cells were incubated with a mixture of those oils, or with camelina oil alone (50% and 103% increase, respectively). Our results show that the fatty acids in vegetable oil incorporate into retinal cells and increase the plasma membrane fluidity.

Forskolin Induces Endocrine Disturbance in Human JEG-3 Placental Cells.

Forskolin, used in folk medicine since ancient times, is now available as a dietary supplement, with an indication as a fat burner and appetite suppressant. However, the safety of forskolin is poorly documented especially for pregnant women. The question that we raised is what about the safety of forskolin in pregnant women? As the placenta, an endocrine organ, is the key organ of pregnancy, we evaluated the in vitro placental toxicity of forskolin. We focused first on the activation of a P2X7 degenerative receptor as a key biomarker for placental toxicity, and second on steroid and peptide hormonal secretion. We observed that forskolin activated P2X7 receptors and disturbed estradiol, progesterone, hPL and hyperglycosylated hCG secretion in human placental JEG-Tox cells. To the best of our knowledge, we highlighted, for the first time, that forskolin induced endocrine disturbance in placental cells. Forskolin does not appear to be a safe product for pregnant women and restrictions should be taken.

Pregnant Women and Endocrine Disruptors: Role of P2X7 Receptor and Mitochondrial Alterations in Placental Cell Disorders.

In pregnant women, the lungs, skin and placenta are exposed daily to endocrine-disrupting chemicals (EDCs). EDCs induce multiple adverse effects, not only on endocrine organs, but also on non-endocrine organs, with the P2X7 cell death receptor being potentially the common key element. Our objective was first to investigate mechanisms of EDCs toxicity in both endocrine and non-endocrine cells through P2X7 receptor activation, and second, to compare the level of activation in lung, skin and placental cells. In addition, apoptosis in placental cells was studied because the placenta is the most exposed organ to EDCs and has essential endocrine functions. A total of nine EDCs were evaluated on three human cell models. We observed that the P2X7 receptor was not activated by EDCs in lung non-endocrine cells but was activated in skin and placenta cells, with the highest activation in placenta cells. P2X7 receptor activation and apoptosis are pathways shared by all tested EDCs in endocrine placental cells. P2X7 receptor activation along with apoptosis induction could be key elements in understanding endocrine placental and skin disorders induced by EDCs.

Cocktail Effect of Endocrine Disrupting Chemicals: Application to Chlorpyrifos in Lavender Essential Oils.

Chlorpyrifos is a pesticide that is toxic to human health and has been banned for the past decade. Due to its persistent and bioaccumulative properties, chlorpyrifos is still present in soil. Pregnant women can be exposed to chlorpyrifos through drinking water and herbal products, such as essential oils (EOs), resulting in adverse effects to the mother and fetus. Our objective was to evaluate and compare the potential endocrine disrupting effects of chlorpyrifos "free" or in contaminated lavender EO. We studied the release of four hormones and the activation of the P2X7 cell death receptor in human placental JEG-Tox cells as key biomarkers of endocrine toxicity for pregnant women (hPlacentox assay). We observed that "free" chlorpyrifos disrupted placental hormones and activated the P2X7 receptor, whereas chlorpyrifos in lavender EO disrupted only the placental hormones. We confirm that chlorpyrifos can be classified as an endocrine disrupting chemical (EDC) for pregnant women and point out that its endocrine disrupting effect may not be apparent when present in lavender EOs. Our results reveal the existence of specific reverse cocktail effects that may have protective properties against EDCs.

JEG-3 placental cells in toxicology studies: a promising tool to reveal pregnancy disorders.

Placental alterations are responsible for adverse pregnancy outcomes like preeclampsia and intrauterine growth restriction. And yet, placenta toxicology has not become a fully-fledged toxicology field. Because placenta is very often seen only as a barrier between the mother and the fetus, there is a lack and therefore a need for an experimental human model with technical recommendations to study placenta toxicology. In vitro approaches are recommended in experimental toxicology as they focus on a specific biological process and yield high-throughput screening methods. In the present study, we first established incubation conditions to preserve signatures of the human JEG-3 cell line identity while enabling toxicity detection. JEG-3 cells prepared in our incubation conditions were renamed JEG-Tox cells. As placental alterations are mainly triggered by uncontrolled apoptosis, we second used known apoptotic agents pregnant women are exposed to, to check that JEG-Tox cells can trigger apoptosis. Ethanol, bisphenol F, quinalphos, 4,4'-DDT, benzalkonium chloride, phenoxyethanol, propylparaben, and perfluorooctanic acid all induced chromatin condensation in JEG-Tox cells. Our incubation conditions allow JEG-Tox cells to keep placental cell identity and to respond to toxic chemicals. JEG-Tox cells are a pertinent model for placenta toxicology and could be used to better understand pregnancy alterations.

Bisphenol A, Bisphenol F, and Bisphenol S: The Bad and the Ugly. Where Is the Good?

BACKGROUND: Bisphenol A (BPA), a reprotoxic and endocrine-disrupting chemical, has been substituted by alternative bisphenols such as bisphenol F (BPF) and bisphenol S (BPS) in the plastic industry. Despite their detection in placenta and amniotic fluids, the effects of bisphenols on human placental cells have not been characterized. Our objective was to explore in vitro and to compare the toxicity of BPA to its substitutes BPF and BPS to highlight their potential risks for placenta and then pregnancy. METHODS: Human placenta cells (JEG-Tox cells) were incubated with BPA, BPF, and BPS for 72 h. Cell viability, cell death, and degenerative P2X7 receptor and caspases activation, and chromatin condensation were assessed using microplate cytometry and fluorescence microscopy. RESULTS: Incubation with BPA, BPF, or BPS was associated with P2X7 receptor activation and chromatin condensation. BPA and BPF induced more caspase-1, caspase-9, and caspase-3 activation than BPS. Only BPF enhanced caspase-8 activity. CONCLUSIONS: BPA, BPF, and BPS are all toxic to human placental cells, with the P2X7 receptor being a common key element. BPA substitution by BPF and BPS does not appear to be a safe alternative for human health, particularly for pregnant women and their fetuses.

Lipidomic analysis of human corneal epithelial cells exposed to ocular irritants highlights the role of phospholipid and sphingolipid metabolisms in detergent toxicity mechanisms.

Detergent chemicals, widely used in household products, in pharmaceutical, medical, cosmetic and industrial fields, have been linked to side effects and involved in several eye diseases. On the ocular surface, detergents can interfere with the corneal epithelium, the most superficial layer of the cornea, representing a line of defence against external aggression. Despite its major role in numerous biological functions, there is still little data regarding disruption of lipid homeostasis induced by ocular irritants. To this purpose, a lipidomic analysis using UPLC-HRMS/MS-ESI ± was performed on human corneal epithelial (HCE) cells incubated with three widely known ocular irritants: benzalkonium chloride (BAK), sodium lauryl sulfate (SLS) and Triton X-100 (TXT). We found that these ocular irritants lead to a profound modification of the HCE cell lipidome. Indeed, the cell content of ceramide species increased widely while plasmalogens containing polyunsaturated fatty acid species, especially docosahexaenoic acids, decreased. Furthermore, these irritants upregulated the activity of phospholipase A2. The present study demonstrates that BAK, SLS and TXT induced disruption of the cell lipid homeostasis, highlighting that lipids mediate inflammatory and cell death processes induced by detergents in the cornea. Lipidomics may thus be regarded as a valuable tool to investigate new markers of corneal damage.

In Situ Gelling Ophthalmic Drug Delivery System for the Optimization of Diagnostic and Preoperative Mydriasis: In Vitro Drug Release, Cytotoxicity and Mydriasis Pharmacodynamics.

Mydriasis is required prior to many eye examinations and ophthalmic surgeries. Nowadays, phenylephrine hydrochloride (PHE) and tropicamide (TPC) are extensively used to induce mydriasis. Several pharmaceutic dosage forms of these two active ingredients have been described. However, no optimal therapeutic strategy has reached the market. The present work focuses on the formulation and evaluation of a mucoadhesive ion-activated in situ gelling delivery system based on gellan gum and hydroxyethylcellulose (HEC) for the delivery of phenylephrine and tropicamide. First, in vitro drug release was studied to assess appropriate sustained drug delivery on the ocular surface region. Drug release mechanisms were explored and explained using mathematical modeling. Then, in situ gelling delivery systems were visualized using scanning electron microscopy illustrating the drug release phenomena involved. Afterward, cytotoxicity of the developed formulations was studied and compared with those of commercially available eye drops. Human epithelial corneal cells were used. Finally, mydriasis intensity and kinetic was investigated in vivo. Mydriasis pharmacodynamics was studied by non-invasive optical imaging on vigilant rabbits, allowing eye blinking and nasolacrimal drainage to occur physiologically. In situ gelling delivery systems mydriasis profiles exhibited a significant increase of intensity and duration compared with those of conventional eye drops. Efficient mydriasis was achieved following the administration of a single drop of in situ gel reducing the required amount of administered active ingredients by four- to eight-fold compared with classic eye drop regimen.

High molecular weight hyaluronan decreases UVB-induced apoptosis and inflammation in human epithelial corneal cells.

PURPOSE: The aim of this study was to investigate high molecular weight hyaluronan (HMW-HA) protection on human corneal epithelial (HCE) cells against ultraviolet B (UVB) radiation-induced toxic effects. METHODS: The HCE cell line was incubated with HMW-HA or phosphate-buffered salt solution (PBS), rinsed, and exposed to UVB radiation. Cell viability, reactive oxygen species (ROS) and glutathione (GSH) levels, 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) release, p53 phosphorylation, caspase-3, -8, -9 activation, and interleukin (IL)-6 and -8 production were assessed to evaluate and to compare UVB-induced toxicity between cells treated with HMW-HA and cells treated with PBS. RESULTS: Data indicate that HMW-HA had significant protective effects against UVB radiation. HMW-HA increased HCE cell viability, decreased IL-6 and -8 production, and decreased caspase-3 and -8 activation. However, HMW-HA had no significant effect on ROS and GSH levels, 8-oxo-dG release, and p53 phosphorylation. CONCLUSIONS: To our knowledge, we report for the first time the ability of HMW-HA to protect cells against UV irradiation. According to our results, HMW-HA provides anti-inflammatory and anti-apoptotic signals to cells exposed to UVB.

Ocular burn: rinsing and healing with ionic marine solutions and vegetable oils.

PURPOSE: We investigated the effects of various rinsing and healing protocols on corneal wound repair and inflammation following alkali burn in rabbits. METHODS: We conducted in vitro, in vivo and ex vivo studies. First, different rinse solutions were tested in vitro after incubation of ocular cells with methanol or NaOH. Cell viability was then assessed using the neutral red test (cytofluorometry). Second, NaOH was applied to rabbit corneas and associations of rinse solutions (NaCl 0.9% or controlled ionization marine solutions) with N-acetylcysteine or vegetable oils (from Calophyllum inophyllum and Aleurites moluccana) were tested in vivo. The regeneration of the corneal epithelium and the infiltration of inflammatory cells were evaluated using in vivo confocal microscopy and ex vivo histological cuts. RESULTS: The association of a controlled ionization marine solution with 10% C. inophyllum oil and 90% A. moluccana oil induced regeneration of the corneal epithelium and a decrease in inflammatory cells. CONCLUSIONS: Irrigation with marine solution followed by treatment with a mixture of C. inophyllum and A. moluccana oils is a promising treatment for ocular burns.

Effects of toxic cellular stresses and divalent cations on the human P2X7 cell death receptor.

PURPOSE: The purpose of this study was to investigate responses to toxic cellular stresses in different human ocular epithelia. METHODS: Reactivity with a specific anti-P2X7 antibody was studied using confocal fluorescence microscopy on conjunctival, corneal, lens, and retinal cell lines as well as using impression cytology on human ocular cells. Activation of the P2X7 receptor by selective agonists (ATP and benzoylbenzoyl-ATP) and inhibition by antagonists (oATP, KN-62, and PPADS) were evaluated using the quinolinium,4-[(3-methyl-2-(3H)-benzoxazolylidene) methyl]-1-[3-(triethylammonio)propyl]di-iodide (YO-PRO-1) test in cytofluorometry. Different specific stresses were then induced by a chemical toxin (benzalkonium chloride) and a chemical oxidant (tert-butyl hydroperoxide) to assess the role of the P2X7 receptor. Modulation of P2X7 receptor activation was performed with several ionic solutions. RESULTS: Our data show that four cell lines express the P2X7 cell death purinergic receptor as judged by reactivity with a specific anti-P2X7 antibody, activation by the selective P2X7 agonist benzoylbenzoyl-ATP and to a lesser extent by ATP (YO-PRO-1 dye uptake), and inhibition by three antagonists (oATP, KN-62, and PPADS). Benzalkonium chloride, a widely used preservative, induced dramatic membrane permeabilization through P2X7 pore opening on conjunctival and corneal epithelia. Reactive oxygen species, induced by tert-butyl hydroperoxide, lead to P2X7 receptor activation on retinal pigment epithelium. Modulation of P2X7 receptor activation was obtained with extracellular Ca(2+) and Mg(2+) and with a controlled ionization marine solution rich in different divalent cations. This marine solution could be proposed as a new ophthalmic solution. CONCLUSIONS: Our observations reveal a novel pathway for epithelial cells apoptosis/cytolysis by inducing different toxic stresses and their modulation by using ionic solutions.

In vitro modulation of preservative toxicity: high molecular weight hyaluronan decreases apoptosis and oxidative stress induced by benzalkonium chloride.

OBJECTIVE: Benzalkonium chloride (BAK) is one of the most often used preservative in pharmaceutical products and it is known to induce toxic effects. Hyaluronan (HA), a linear biopolymer, is involved in several biological processes. The aim of this work is to in vitro investigate if HA is able to decrease BAK toxicity. METHODS: Two human epithelial cell lines were treated with different incubation time protocol with BAK and three different molecular weights HA (HA 20k Da, HA 100 kDa and HA 1000 kDa, 0.2%, w/v). Flow cytometry, fluorescence microscopy, microplate cytofluorometry and confocal microscopy were performed to evaluate expression of CD44 receptor, cell viability, oxidative stress, mitochondrial mass, chromatin condensation, plasma-membrane permeability, DNA fragmentation and cytoskeleton morphology. RESULTS: The three HAs studied induce neither oxidative stress nor apoptosis. HA 1000 kDa significantly decreases oxidative stress, apoptosis and necrosis induced by BAK. Experiments with HA 20 kDa or HA 100 kDa did not show the same effects. For instance, the more molecular weight decreases, the more protection decreases. Moreover, we suggest that HA interacts with cell plasma-membrane and inhibits cell death receptors. CONCLUSION: High molecular weight HA (1000 kDa, 0.2%) is an effective protective agent against BAK.