Cell viability score (CVS) as a good indicator of critical concentration of benzalkonium chloride for toxicity in cultured ocular surface cell lines

Cytotoxicity of benzalkonium chloride (BAK) is a major factor affecting drug cytotoxicity. This study aimed to determine the critical concentration of BAK for cultured ocular cells, using SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). Cell viability was determined following the exposure of cells to 11 concentrations of BAK for 10, 30, or 60 min using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and neutral red assays, and the cell viability score (CVS) was used to evaluate comprehensively the toxicity of BAK. The CVS system consists of two values. The CVS50 was determined by the number of measurements for viability ⩾50% of control. The CVS40/80 was calculated as follows: CVS40/80 = (number of measurements for viability values >80%) − (number of measurements for viability values <40%). Both %CVS50 and %CVS40/80 decreased with concentrations of BAK. When BAK concentrations were 0.01% or higher, %CVS50 and %CVS40/80 became 0 and less than −90, respectively. Meanwhile, when BAK concentrations were 0.001% or lower, %CVS50 became 100. In the case of %CVS40/80, when the BAK concentrations were 0.002% or lower, the values reached 75 or more, and when 0.0005% or lower, the %CVS40/80 value reached 100. Accordingly, BAK induced very low cytotoxicity in the cultured ocular cell lines at concentrations of 0.002% or lower. The concentration-dependency confirmed that the CVS score is useful for expressing drug cytotoxicity in a simple and comprehensive manner.     

Comparison of the ocular surface changes following the use of two different prostaglandin F2α analogues containing benzalkonium chloride or polyquad in rabbit eyes

Objective: The aim of this study is to compare the effect of prostaglandin analogues preserved with either 0.015% or 0.001% benzalkoium chloride (BAK); or 0.001% polyquad (PQ) on the ocular surface of rabbit eyes.

Methods: Forty white rabbits were randomized to receive four-times daily instillation of either 0.0015% tafluprost (TF) preserved with 0.001% BAK (TF-BAK); 0.004% travoprost (TR) with 0.015% BAK (TR-BAK) or 0.001% PQ (TR-PQ); or preservative-free artificial tears in one eye for a 4-week period. Tear samples collected from the 40 rabbits were analyzed by enzyme-linked immunosorbent assays (ELISA) to identify the presence of inflammatory cytokines: interleukin (IL)-1β and IL-6 on day 14. Subsequently, harvested cornea and bulbar conjunctiva were evaluated using light and transmission electron microscopy (TEM).

Results: IL-6 was significantly increased in TF-BAK and TR-BAK groups compared to controls and TR-PQ group (p?=?0.005); however, IL-1β level was not significantly different among four groups (p?=?0.360). Rabbits treated with TR-BAK showed decreased goblet cell density of bulbar conjunctiva and increased pyknotic change and vacuolization of corneal epithelial cells on light microscopy; similar change occurred but was less severe in TF-BAK group. The TR-PQ group showed similar results as the controls. The destruction of the microvillar architecture of bulbar conjunctiva and cornea was most prominent in the TR-BAK group.

Conclusions: Preservatives included in the anti-glaucoma eye-drops showed different ocular surface changes according to the concentration and type in the rabbits. Prostaglandin analogues preserved with higher level of BAK may cause more harmful effects on the ocular surface than PQ-preserved medications.     

Surfactant and UV-B-induced damage of the cultured bovine lens

Purpose: To evaluate in vitro methods for testing the toxicity of the surfactants, sodium dodecyl sulfate (SDS) and benzalkonium chloride (BAK), and Ultraviolet (UV)-B radiation to the bovine lens.

Methods: Lenses were dissected from bovine eyes––obtained from a local abattoir––and incubated in M199 culture medium at 37 °C, with 4% CO₂ and 96% air atmosphere. For the SDS and BAK experiments, the lenses (n=153) were exposed directly to 0.001%, 0.01%, 0.1%, and 1.0% solutions for 15 min. These lenses were then rinsed five times each with saline and medium. Another group of lenses (n=36) was irradiated with broadband UV-B at energy levels of 1.0 and 2.0 J/cm² (0.445 and 0.89 J/cm² in the biologically effective energy levels). For all of the above experiments, lens optical quality and cellular viability of lens epithelial cells were evaluated.

Results: The analysis of optical quality, using a scanning laser in vitro assay system, of exposed lenses treated with SDS and BAK at concentrations of 0.01%, 0.1%, and 1.0%, and with UV-B at energy levels of 0.445 and 0.89 J/cm² showed a dose- and time-dependent increase in back vertex distance (BVD) variability, indicating loss of sharp focus in comparison with control lenses. Both 0.001% SDS and 0.001% BAK-treated lenses did not show any optical damage until 8-days after exposure. Lenses treated with 0.01% SDS showed recovery from optical damage 6-days later after exposure. Optical damage was not shown immediately for UV-B-exposed lenses. The Alamar Blue assay data for SDS, BAK and UV-B-exposed lenses, except the 0.001% SDS treated lens group, showed also dose- and time-dependent decreases in cellular viability in comparison with the control lenses, and there was no cellular recovery during the entire culture period. Lenses treated with 0.001% SDS did not show biological damage until 8-days after exposure. It appears that cellular changes appeared earlier than optical changes.

Conclusions: The findings suggest that cultured bovine lenses can be evaluated by assays that probe optical properties and cellular function after exposure to surfactants and UV-B irradiation, and that the optical and biological assay methods are valuable for in vitro mild ocular toxicity research.     

Interaction Between Polyalkylcyanoacrylate Nanoparticles and Peritoneal Macrophages: MTT Metabolism,NET Reduction,and NO Production

Purpose. The nature of interactions between macrophages and drug carriers is of primordial importance either in the design of more effective therapeutic strategies for macrophage-associated pathogenesis or in establishing new approaches for pharmacological action avoiding macrophages. Methods. Polyalkylcyanoacrylate nanoparticles (PMCA, PECA, PBCA and PIBCA nanoparticles) were assayed for their toxicity on peritoneal resident and thioglycolate-elicited macrophages. Cellular viability was assessed by MTT tetrazolium salt assay, oxidative burst by NBT reduction and NO production by nitrite evaluation. Results. The nanoparticles tested led to cellular morphological modifications and induced toxicity in both types of macrophages in culture. The polyalkylcyanoacrylate nanoparticles uptake by peritoneal macrophages caused an increase in respiratory burst, as assessed by the NBT reduction assay, and induced the release of soluble toxic factors to the culture medium. The association of LPS with the PMCA nanoparticles significantly stimulated the production of nitric oxide (NO) by resident macrophages. In contrast, the association of PBCA nanoparticles with LPS does not increase the nitrite production as compared with LPS alone, which may be due to a different physico-chemical interaction between LPS and the two types of polymers. Conclusions. In cultured mice peritoneal macrophages, nanoparticles of PACA induce the production of oxygen reactive products, which cause changes in the cell metabolism of both resident and elicited macrophages. PMCA nanoparticles in association with LPS significantly increase the expression of the inducible isoform of nitric oxide synthase, leading to the release of large amount of NO, which may be highly cytotoxic to the cultured cells in the presence of peroxide generated from the oxidative burst.     

Local and Systemic Toxicity in Mice Following Subcutaneous Implantation of Latex Penrose Drains

Penrose drains are widely used in surgical procedures as an aid in wound healing. The studies presented here investigated the potential toxicity associated with the implantation of latex Penrose drains in BALB/c and B6C3F1 mice. Animals were implanted subcutaneously in the dorsal surface of the neck with 100, 150, or 200 mg of Perry latex drain or 200 mg of Bard (comparative control) latex drain for up to 36 hours. High‐dose (200 mg) exposure to the Perry drain induced severe local and systemic toxicity, resulting in mortality within 24 hours. Time‐ and dose‐responsive effects included decreased response to stimulus, inflammation at the implantation site, epaxial myositis, lesions consistent with hepatic glycogen depletion, apoptotic necrosis of the adrenal “X zone,” and massive thymic apoptosis and atrophy. Negligible levels of endotoxin were quantified from Perry drain samples using the Limulus Amebocyte Lysate Assay. Extraction studies revealed the presence of zinc diethyldithiocarbamate (ZDEC) in the Perry drains but not in the control drains. No other differences were noted from gas chromatography mass spectrometry (GCMS) analyses. Quantitation studies measured ZDEC levels at 2.22 ± 0.04 µg/mg in Perry samples. When ZDEC was eluted from Perry drains prior to implantation, animals exhibited no signs of toxicity. Although FDA regulations limit accelerators to 1.5% of rubber medical products, these studies indicate that the presence of ZDEC in concentrations lower than 0.25% of the drain weight may induce local toxicity and delayed wound healing.     

The mycotoxin Deoxynivalenol inhibits the cell surface expression of activation markers in human macrophages

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. It exhibits several toxic effects including impaired growth and immune dysregulation. Macrophages play pivotal role in the host defense; upon activation, they express several specific cell surface receptors that are important in adhesion and cell signaling. Several studies have demonstrated that DON can affect macrophages, however, very few data are available concerning the effect of DON on human macrophages, and the effect on macrophage cell surface receptors is unknown. In the present study, human blood monocytes, differentiated in vitro into macrophages, were activated with IFN-γ, in the presence or absence of low concentrations of DON. The expression of CD11c, CD13, CD14, CD18, CD33, CD35, CD54, CD119 and HLA-DP/DQ/DR was analyzed by flow cytometry. As expected, macrophage activation by IFN-γ upregulated the expression of CD54, CD14, CD119 and HLA-DP/DQ/DR. Incubation with DON decrease the cell surface expression of these activation markers in a dose-dependent manner. When cells were treated with 5 μM DON, the mean fluorescence intensity measured for the expression of these receptors was the same as that observed in non-activated macrophages. This inhibitory effect of DON was only observed when the mycotoxin was applied before the activation signal. Taken together, our results suggest that low concentration of DON alter macrophage activation as measured by the expression of cell surface markers. This may have implications for human health when consuming DON contaminated feed.     

Cryptotanshinone inhibits LPS-induced proinflammatory mediators via TLR4 and TAK1 signaling pathway

Cryptotanshinone (CTN), one of the major constituents of tanshinones, was investigated for anti-inflammatory activity in the murine macrophage cell line RAW 264.7. CTN inhibited the production of nitric oxide (NO) production, as well as expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated macrophages. Since CTN was considered as inhibiting LPS-triggered phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation, we consequently evaluated the expression of toll-like receptor 4 (TLR4) and CD14, as well as phosphorylation of TGF-β-activated kinase 1 (TAK1). CTN reduced the expression of CD14 and TLR4, and suppressed LPS-induced phosphorylation of TAK1. Furthermore, CTN significantly increased the survival rate against LPS challenge in D-galactosamine-sensitized mice, which was in line with in vitro results. These results suggested that CD14/TLR4 and TAK1 might be the potential molecular targets for addressing the protective effects of CTN on LPS-induced inflammatory effects in macrophages.     

Direct inhibition,but indirect sensitization of pacemaker activity to sympathetic tone by the interaction of endotoxin with HCN‐channels

In critically ill patients regulation of heart‐rate is often severely disturbed. Interaction of bacterial endotoxin (lipopolysaccharide, LPS) with hyperpolarization‐activated cyclic nucleotide‐gated cation‐(HCN)‐channels may interfere with heart‐rate regulation. This study analyzes the effect of LPS, the HCN‐channel blocker ivabradine or Ca²⁺‐channel blockers (nifedipine, verapamil) on pacemaking in spontaneously beating neonatal rat cardiomyocytes (CM) in vitro. In vivo, the effect of LPS on the heart‐rate of adult CD1‐mice with and without autonomic blockade is analyzed telemetrically. LPS (100 ng/mL) and ivabradine (5 μg/mL) reduced the beating‐rate of CM by 20.1% and 24.6%, respectively. Coincubation of CM with both, LPS and ivabradine, did not further reduce the beating‐rate, indicating interaction of both compounds with HCN‐channels, while coincubation with Ca²⁺‐channel blockers and LPS caused additive beating‐rate reduction. In CD1‐mice (containing an active autonomic‐nervous‐system), injection of LPS (0.4 mg/kg) expectedly resulted in increased heart‐rate. However, if the autonomic nervous system was blocked by propranolol and atropine, in line with the in vitro data, LPS induced a significant reduction of heart‐rate, which was not additive to ivabradine. The in vivo and in vitro results indicate that LPS interacts with HCN‐channels of cardiomyocytes. Thus, LPS indirectly sensitizes HCN‐channels for sympathetic activation (tachycardic‐effect), and in parallel directly inhibits channel activity (bradycardic‐effect). Both effects may contribute to the detrimental effects of septic cardiomyopathy and septic autonomic dysfunction.     

Suppression of tumour necrosis factor‐α by Schizonepeta tenuifolia water extract via inhibition of IκBα degradation and Jun N‐terminal kinase/stress‐activated protein kinase activation

Objectives The anti‐inflammatory effects of an aqueous extract of Schizonepeta tenuifolia on lipopolysaccharide (LPS)‐induced tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) in vivo and in vitro have been investigated. Methods C57BL/6 mice were orally administered phosphate‐buffered saline (control) or S. tenuifolia water extract (50, 200, 500 or 1000 mg/kg) for 10 days before intraperitoneal administration of LPS (1.3 mg/kg). Blood samples were obtained 1 h after LPS challenge, followed by determination of TNF‐α and IL‐6 levels. Peritoneal macrophages from thioglycollate‐injected mice were obtained and stimulated with LPS and S. tenuifolia water extract for viability assay, cytokine analysis, real‐time RT PCR and Western blotting. Key findings Oral administration of S. tenuifolia water extract to mice significantly reduced LPS‐induced serum levels of TNF‐α, but not IL‐6. When peritoneal macrophages were treated in vitro with S. tenuifolia water extract, the inhibition of LPS‐induced TNF‐α was more pronounced than that of IL‐6 at the level of secreted protein and mRNA. S. tenuifolia water extract reduced the degradation of IκBα and the nuclear relocation of p65 NF‐κB, but the phosphorylation of IκBα was not affected. Inhibition of c‐Jun N‐terminal kinase/stress‐activated protein kinase (JNK/SAPK) by S. tenuifolia water extract led secondarily to the inhibition of phospho‐c‐Jun and phospho‐ATF‐2. Conclusions These results indicated that the downregulation of TNF‐α by S. tenuifolia water extract may have involved the inhibition of both IκBα degradation and activation of c‐Jun and ATF‐2 involving suppression of JNK/SAPK.     

A comparison of the fourth-generation fluoroquinolones gatifloxacin 0.3% and moxifloxacin 0.5% in terms of ocular tolerability

SUMMARY

Purpose: To compare the ocular tolerability of the commercially available ophthalmic solutions of the fourth-generation fluoroquinolones, gatifloxacin 0.3% (Zymart, Allergan, Inc., Irvine, CA) with benzalkonium chloride (BAK) and moxifloxacin 0.5% (Vigamoxt) without BAK.

Methods: A baseline evaluation was conducted on 30 healthy volunteers for conjunctival hyperemia, conjunctival vascularity, pupil size, and anterior chamber (AC) cell and flare. Pupils were measured under scotopic conditions with a Colvard pupillometer. Conjunctival hyperemia and vascularity, and AC reaction were measured on a Likert-like scale of 0-3. Subjects then received drops in both eyes from masked bottles of gatifloxacin ophthalmic solution 0.3% with BAK (in one eye determined randomly) and moxifloxacin ophthalmic solution 0.5% without BAK (in the contralateral eye) in a double-masked fashion. Subjects graded pain and ocular irritation in each eye on a scale of 1-10 after 5min with their eyes closed. The examination was then repeated.

Results: The average age of this study population was 34.4years. The groups of eyes receiving moxifloxacin 0.5% demonstrated an increase in mean conjunctival hyperemia (0.21 [range: 0-1] at baseline to 1.52 [range: 0-3] at 5min.)

that was significantly greater (p?=?0.0005) compared with that of the group receiving gatifloxacin 0.3% (0.22 [range: 0-1] at baseline to 0.45 [range: 0-2] at 5min). The group receiving moxifloxacin 0.5% showed an increase in conjunctival vascularity (0.55 [range: 0-1] at baseline to 1.61 [range: 0.5-3] at 5?min.) that was significantly greater (p?=?0.0005) compared with that of the group receiving gatifloxacin 0.3% (0.52 [range: 0-1] at baseline to 0.68 [range: 0-2] at 5?min.). Significantly less pain (1.2 vs. 3.2, p?=?0.001) and irritation (0.64 vs. 3.42, p?=?0.001) occurred with gatifloxacin 0.3% than with moxifloxacin 0.5%. Pupil size was significantly reduced (5.65mm-5.05mm) in eyes receiving moxifloxacin 0.5% (p?=?0.004) and no significant change occurred in pupil size (5.60mm-5.65mm) in eyes that received gatifloxacin 0.3% (p?=?0.878). No AC reaction was noted with either medication.

Conclusions: The group of eyes receiving gatifloxacin 0.3% with BAK demonstrated greater ocular tolerability in comparison to the group receiving moxifloxacin 0.5% without BAK. Moxifloxacin-induced pupillary miosis may be due to prostaglandin release in the anterior chamber. A limitation of this study is the relatively young age of the study population.     

Effects of schisandrin on transcriptional factors in lipopolysaccharide-pretreated macrophages

Schisandrin is the main active ingredient isolated from Schisandra chinensis Baill. Recent studies have demonstrated that schisandrin exhibits anti-inflammatory effects in vivo and in vitro. In this study, we examined whether the order of lipopolysaccharide (LPS) treatment affects the mechanism of schisandrin anti-inflammatory activity. We found that the antiinflammatory mechanisms are not the same depending on whether macrophages were treated with schisandrin before or after LPS. The main difference is that inhibitor kappaBα (IκBα) degradation was not inhibited when macrophages were pretreated by LPS before schisandrin and was weakly inhibited when macrophages were pretreated by schisandrin before LPS.     

Inhibitory action of daunorubicin on lipopolysaccharide-stimulated inducible-type nitric oxide synthase induction in alveolar macrophages

The purpose of this study was to investigate the effects of daunorubicin on lipopolysaccharide (LPS)-stimulated inducible-type nitric oxide synthase (iNOS) expression in macrophages. LPS-stimulated iNOS expression and NO production were significantly inhibited in alveolar macrophages from rats administrated daunorubicin (4 mg/kg body weight per day) for 5 consecutive days. Incubation of macrophages with daunorubicin at 1 muM but not at 0.1 and 0.5 muM significantly inhibited LPS-stimulated NO production and iNOS induction. Activation of extracellular regulated kinase (ERK) by LPS was markedly attenuated in both macrophages isolated from in vivo daunorubicin-treated rats and those incubated in vitro with daunorubicin at 1 microM. ERK activation, iNOS induction, and NO production following LPS stimulation were all markedly inhibited in the presence of U0126, an ERK inhibitor. The viability of macrophages was decreased by incubation with daunorubicin at 0.5 and 1 microM, while treatment of rats with daunorubicin did not affect viability of macrophages isolated from the rats. These results suggest that in vivo treatment of rats with daunorubicin attenuates LPS-induced iNOS expression of macrophages through inhibition of ERK activation, while inhibition of iNOS induction by in vitro incubation with daunorubicin may be mainly due to its cytotoxicity.     

Endotoxin and muramyl dipeptide modulate surface receptor expression on human mononuclear cells

Endotoxin (lipopolysaccharide (LPS), 100 ng/ml) and muramyl dipeptide (MDP 100 ng/ml), two immunomodulatory bacterial cell wall products, were incubated with human whole blood, and the expression of receptors involved in antigen presentation, costimulation, and cell activation was investigated by use of flow cytometry. On monocytes, LPS and MDP increased surface expression of human leukocyte antigen-DR (HLA-DR), CD18, CD54 (intercellular adhesion molecule-1, ICAM-1), and CD86 (B7-2). On lymphocytes, LPS but not MDP increased HLA-DR expression after 18 h. The expression of CD28, CD49d/CD29, and CD106 (vascular cell adhesion molecule-1, VCAM-1) remained unchanged on both monocytes and lymphocytes. The early increase (1-6 h) of CD18 and ICAM-1 expression led us to hypothesize that CD18-dependent costimulatory signals were involved in the later (6 h) increase of monocyte HLA-DR expression. However, blocking studies using monoclonal antibodies against CD18 (IB4, 15 microg/ml) demonstrated that the LPS- and MDP-induced increase of HLA-DR and ICAM-1 expression on monocytes was not mediated through CD18. LPS induced the expression of the early activation marker CD69 by a CD14-dependent but CD18-independent mechanism, whereas MDP did not induce CD69 expression. Analysis of leukocyte subsets demonstrated that CD4(+) T-cells, CD8(+) T-cell, CD19(+) B-cells, CD56(+) natural killer (NK)-cells, and CD14(+) monocytes increased the expression of CD69 after stimulation with LPS. Collectively, these data demonstrate a stronger immunomodulatory effect of LPS compared with MDP which may, in part, explain the established difference of toxicity between these two bacterial cell wall products.     

Reduction of Streptolysin O (SLO) Pore-Forming Activity Enhances Inflammasome Activation

Pore-forming toxins are utilized by bacterial and mammalian cells to exert pathogenic effects and induce cell lysis. In addition to rapid plasma membrane repair, macrophages respond to pore-forming toxins through activation of the NLRP3 inflammasome, leading to IL-1β secretion and pyroptosis. The structural determinants of pore-forming toxins required for NLRP3 activation remain unknown. Here, we demonstrate using streptolysin O (SLO) that pore-formation controls IL-1β secretion and direct toxicity. An SLO mutant incapable of pore-formation did not promote direct killing, pyroptosis or IL-1β production. This indicated that pore formation is necessary for inflammasome activation. However, a partially active mutant (SLO N402C) that was less toxic to macrophages than wild-type SLO, even at concentrations that directly lysed an equivalent number of red blood cells, enhanced IL-1β production but did not alter pyroptosis. This suggests that direct lysis may attenuate immune responses by preventing macrophages from successfully repairing their plasma membrane and elaborating more robust cytokine production. We suggest that mutagenesis of pore-forming toxins represents a strategy to enhance adjuvant activity.     

Methanol Extract of Biophytum sensitivum Alters the Cytokine Profile and Inhibits iNOS and COX-2 Expression in LPS/Con A Stimulated Macrophages

Biophytum sensitivum has been used in traditional folk medicine to treat numerous diseases. The molecular mechanism of B. sensitivum pharmacological and biochemical actions of macrophages in inflammation has not been clearly elucidated. We examined how the methanol extract of B. sensitivum regulates the production of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and nitric oxide (NO) in vitro and in vivo. The extract inhibits the production of NO and proinflammatory cytokines in lipopolysaccharide (LPS) or Concanavalin (Con) A-stimulated primary macrophages. In vitro L929 bioassay revealed the inhibition of TNF-α production by B. sensitivum treatment. Moreover, the extract could suppress the inducible nitric oxide synthase and cyclo-oxygenase-2 mRNA expression in LPS or Con A-stimulated macrophages. These findings provide evidence that B. sensitivum possesses potential anti-inflammatory activity and may be beneficial for the treatment of endotoxin shock or sepsis.     

益母草碱抑制NLRP3炎症小体过度激活调控巨噬细胞M1/M2表型分化

目的研究益母草碱对脂多糖(LPS)诱导小鼠腹腔巨噬细胞免疫应答影响及相关机制。方法分离小鼠腹腔巨噬细胞,用脂多糖和益母草碱预处理24 h,MMT法检测巨噬细胞活性;Griess法检测NO释放量;ELISA法检测IL-1β、IL-18、IL-6、TNF-α的释放量;RT-PCR法检测NLRP3、ASC、caspase-1、TNF-α、iNOS、Arg-1和CD206的mRNA表达量;Western blot检测NLRP3、ASC、caspase-1蛋白表达量。结果益母草碱能显著抑制脂多糖引起的巨噬细胞上清液中NO、IL-1β、IL-18、IL-6、TNF-α的释放。RT-PCR及Western blot实验结果显示,益母草碱可以抑制脂多糖引起的巨噬细胞中NLRP3、ASC、caspase-1的mRNA及蛋白表达;益母草碱还能明显抑制脂多糖所诱导的巨噬细胞向M1型分化,并促进巨噬细胞向M2型分化。结论益母草碱能通过抑制NLRP3炎症小体,促进脂多糖诱导的巨噬细胞由M1表型向M2表型分化。     

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.