The mechanism of cell-damaging reactive oxygen generation by colloidal fullerenes

Because of the ability to induce cell death in certain conditions, the fullerenes (C₆₀) are potential anticancer and toxic agents. The colloidal suspension of crystalline C₆₀ (nano-C₆₀, nC₆₀) is extremely toxic, but the mechanisms of its cytotoxicity are not completely understood. By combining experimental analysis and mathematical modelling, we investigate the requirements for the reactive oxygen species (ROS)-mediated cytotoxicity of different nC₆₀ suspensions, prepared by solvent exchange method in tetrahydrofuran (THF/nC₆₀) and ethanol (EtOH/nC₆₀), or by extended mixing in water (aqu/nC₆₀). With regard to their capacity to generate ROS and cause mitochondrial depolarization followed by necrotic cell death, the nC₆₀ suspensions are ranked in the following order: THF/nC₆₀>EtOH/nC₆₀>aqu/nC₆₀. Mathematical modelling of singlet oxygen (¹O₂) generation indicates that the ¹O₂-quenching power (THF/nC₆₀nC₆₀nC₆₀) of the solvent intercalated in the fullerene crystals determines their ability to produce ROS and cause cell damage. These data could have important implications for toxicology and biomedical application of colloidal fullerenes.     

An Anticarsia gemmatalis multiple nucleopolyhedrovirus mutant, vApAg, induces hemocytes apoptosis in vivo and displays reduced infectivity in larvae of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae)

An Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) mutant, vApAg, induces apoptosis in a cell culture derived from Anticarsia gemmatalis (UFL-AG-286), reducing viral progeny. We have investigated apoptosis induction in vivo by vApAg in A. gemmatalis larvae and its correlation to infectivity reduction. LC₅₀, LD₅₀, LT₅₀ and the mean time to death of larvae were determined for vApAg and AgMNPV. Apoptosis was accessed for hemocytes of infected larvae using light and transmission electron microscopy. All types of hemocytes can be infected by vApAg. After 12 h post-infection (h p.i.), typical cellular modifications associated to nucleopolyhedrovirus infection were observed. Apoptosis becomes evident after 24 h p.i., and massive after 72 h p.i. Necrosis of infected cells was also observed. Despite cell death, hemocytes produced budded viruses and polyhedra. Pl and gh1-type hemocytes presented phagocytic activity. Agarose gel electrophoresis revealed fragmentation of hemocytes DNA at late times post-infection. The LC₅₀ and LD₅₀ were between five- and six-fold higher for vApAg. The LT₅₀ and the mean time to death were higher for vApAg in a same treatment or for a similar mortality induced by AgMNPV. These results show correlation of apoptosis and the reduced infectivity of vApAg in A. gemmatalis larvae.     

三氧化二砷逆转HL60/ADR细胞耐药的研究

背景：临床应用三氧化二砷(As₂O₃)治疗复发难治性急性早幼粒细胞白血病已获得较好效果。 目的：探讨As₂O₃逆转急性髓系白血病细胞株HL60/ADR耐药的作用及其机制。 方法：以HL60/ADR细胞为研究对象,分为空白对照组和As₂O₃组,空白对照组不加任何药物培养,As₂O₃组加入48 h IC50 As₂O₃进行培养。 结果与结论：As₂O₃能提高HL60/ADR细胞摄取阿霉素水平,降低HL60/ADR细胞多药耐药相关蛋白1表达率(P < 0.05),能够促进HL60/ADR细胞凋亡,细胞早期和晚期凋亡率均高于空白对照组(P < 0.01),并随时间增加而提高。可减低细胞IκBα、p65、抗凋亡蛋白Bcl-2表达,使促凋亡蛋白Bax,断裂的Caspase-3、Caspase-9、PARP表达升高,说明As₂O₃能够逆转HL60/ADR细胞耐药,与其上述变化有关。     

原花青素对Aβ_(25-35)作用下PC12细胞的保护作用

目的:探讨原花青素对Aβ_(25-35)作用下PC12细胞的保护作用及机制。方法:25μmol/L的Aβ_(25-35)作用于PC12细胞48 h,预先加入25、50和100 mg/L的原花青素干预24 h。采用MTT法检测细胞存活率,DCFH-DA单染法检测细胞活性氧簇(ROS)的含量,JC-10单染法检测细胞线粒体膜电位,AnnexinⅤ/PI双染法检测细胞凋亡,Western blot检测凋亡蛋白caspase-3的蛋白水平。结果:原花青素可提高Aβ_(25-35)作用下PC12细胞的存活率,降低胞内ROS含量,阻止线粒体膜电位下降,抑制caspase-3的活化(P0.05或P0.01),从而抑制Aβ_(25-35)诱导的PC12细胞凋亡。结论:原花青素对Aβ_(25-35)作用下的PC12细胞具有明显的保护作用,其作用机制可能是通过清除Aβ_(25-35)诱导产生的ROS而减轻对线粒体膜的损伤作用,从而抑制细胞凋亡的发生。     

Biophysical and pharmacological properties of the voltage-gated potassium current of human pancreatic β-cells

Voltage-gated potassium (Kv) currents of human pancreatic islet cells were studied by whole-cell patch clamp recording. On average, 75% of the cells tested were identified as β-cells by single cell, post-recording RT-PCR for insulin mRNA. In most cells, the dominant Kv current was a delayed rectifier. The delayed rectifier activated at potentials above −20 mV and had a V _½ for activation of −5.3 mV. Onset of inactivation was slow for a major component (τ= 3.2 s at +20 mV) observed in all cells; a smaller component (τ= 0.30 s) with an amplitude of ∼25% was seen in some cells. Recovery from inactivation had a τ of 2.5 s at −80 mV and steady-state inactivation had a V _½ of −39 mV. In 12% of cells (21/182) a low-threshold, transient Kv current (A-current) was present. The A-current activated at membrane potentials above −40 mV, inactivated with a time constant of 18.5 ms at −20 mV, and had a V _½ for steady-state inactivation of −52 mV. TEA inhibited total Kv current with an  IC₅₀= 0.54 m m   and PAC, a disubstituted cyclohexyl Kv channel inhibitor, inhibited with an  IC₅₀= 0.57 μ m   . The total Kv current was insensitive to margatoxin (100 n m ), agitoxin-2 (50 n m ), kaliotoxin (50 n m ) and ShK (50 n m ). Hanatoxin (100 n m ) inhibited total Kv current by 65% at +20 mV. Taken together, these data provide evidence of at least two distinct types of Kv channels in human pancreatic β-cells and suggest that more than one type of Kv channel may be involved in the regulation of glucose-dependent insulin secretion.     

Src family kinases and lipid mediators in control of allergic inflammation

Summary:  The Src family kinases Fyn and Lyn are important modulators of the molecular events initiated by engagement of the high-affinity IgE receptor (FcɛRI). These kinases control many of the early signaling events and initiate the production of several lipid metabolites that have an important role in regulating mast cell responses. The intracellular level of phosphatidylinositol (3,4,5)-trisphosphate (PIP₃), which is produced by phosphatidylinositol 3-OH kinase, plays an important role in determining the extent of a mast cells response to a stimulus. Enhanced levels lead to a hyperdegranulating phenotype (as seen in SHIP-1^−/− and Lyn^−/− mast cells), whereas decreased levels cause hypodegranulation (as seen in Fyn^−/− mast cells). Downregulation of mast cell phosphatase and tensin homologue deleted on chromosone 10 expression, a phosphatase that reduces cellular levels of PIP₃, caused constitutive cytokine production, demonstrating that this response is particularly sensitive to PIP₃ levels. Lyn and Fyn are also intimately linked to other lipid kinases, like sphingosine kinases (SphK). By producing sphingosine-1-phosphate (S1P), SphKs contribute to mast cell chemotaxis and degranulation. In vivo studies now reveal that circulating S1P as well as that found within the mast cell is important in determining mast cell responsiveness. These studies demonstrate the connection between Src protein tyrosine kinases and lipid second messengers that control mast cell function and allergic responses.     

The scavenging of reactive oxygen species and the potential for cell protection by functionalized fullerene materials

We demonstrated that three different types of water-soluble fullerenes materials can intercept all of the major physiologically relevant ROS. C(60)(C(COOH)(2))(2), C(60)(OH)(22), and Gd@C(82)(OH)(22) can protect cells against H(2)O(2)-induced oxidative damage, stabilize the mitochondrial membrane potential and reduce intracellular ROS production with the following relative potencies: Gd@C(82)(OH)(22)> or =C(60)(OH)(22)>C(60)(C(COOH)(2))(2). Consistent with their cytoprotective abilities, these derivatives can scavenge the stable 2,2-diphenyl-1-picryhydrazyl radical (DPPH), and the reactive oxygen species (ROS) superoxide radical anion (O(2)(*-)), singlet oxygen, and hydroxyl radical (HO(*)), and can also efficiently inhibit lipid peroxidation in vitro. The observed differences in free radical-scavenging capabilities support the hypothesis that both chemical properties, such as surface chemistry induced differences in electron affinity, and physical properties, such as degree of aggregation, influence the biological and biomedical activities of functionalized fullerenes. This represents the first report that different types of fullerene derivatives can scavenge all physiologically relevant ROS. The role of oxidative stress and damage in the etiology and progression of many diseases suggests that these fullerene derivatives may be valuable in vivo cytoprotective and therapeutic agents.     

亚硒酸钠对UVB损伤人角质形成细胞的保护作用

目的:研究亚硒酸钠(Na2Se O3)对中波紫外线(UVB)损伤人角质形成细胞的保护作用。方法:培养永生化人角质形成细胞(Ha Ca T细胞),实验分为4组处理:(1)正常对照组;(2)Na2Se O3组:分别加入1 nmol/L、10 nmol/L、50 nmol/L、100 nmol/L、200 nmol/L和1μmol/L的Na2Se O3预孵育24 h;(3)UVB组:300、600和900 J/m2UVB照射;(4)Na2Se O3+UVB组:Na2Se O3预孵育24 h后进行UVB照射。采用MTT法检测细胞增殖活性,采用流式细胞仪检测300 J/m2UVB照射后细胞的凋亡率。结果:(1)UVB组与正常对照组比较,细胞增殖活性显著降低(P0.05),细胞活性与UVB照射剂量呈负相关;(2)Na2Se O3组与正常对照组比较,细胞增殖活性无明显差异;(3)不同浓度Na2Se O3+UVB组与UVB组比较,细胞增殖活性增加,差异显著(P0.05),其中100 nmol/L组促进细胞增殖作用最强;(4)300 J/m2UVB照射后,不同浓度Na2Se O3+UVB组与UVB组比较,凋亡率下降,差异显著(P0.05),其中100 nmol/L组抑制凋亡作用最强。结论:UVB对角质形成细胞有损伤作用,且与照射剂量呈正相关;Na2Se O3具有光保护性能,可减轻UVB辐射损伤人角质形成细胞。     

Effects of magnetic fields on the accumulation of thiobarbituric acid reactive substances induced by iron salt and H2O2 in mouse brain homogenates or phosphotidylcholine

In this study, we examined the effects of magnetic fields (MFs) on the generation of thiobarbituric acid reactive substances (TBARS) in the mouse brain homogenates or phosphotidylcholine (PC) solution, incubated with FeCl₃ and/or H₂O₂. Active oxygen species were generated and lipid peroxidation was induced in mouse brain homogenates by incubation with iron ions, resulting in the accumulation of TBARS. Lipid peroxidation was induced in PC by incubation with iron ions and H₂O₂. Exposure to sinusoidal MFs (60 Hz, 0.2–1.2 mT), symmetric sawtooth-waveform MFs (50 Hz, 25–600 mT/s), rectangular MFs (1/0.4–1/16 Hz, 3.3 mT) and static MFs (1, 5–300 mT) had no effect on the accumulation of TBARS in brain homogenates induced by FeCl₃. In contrast, when the homogenates were incubated with FeCl₃ in static MFs (2–4 mT), the accumulation of TBARS was decreased. However, this inhibitory effect disappeared when EDTA was added to the homogenate and incubated with H₂O₂. The accumulation of TBARS in PC solution incubated with FeCl₃ and H₂O₂ was also inhibited by the static MF. These results indicate that only static MFs had an inhibitory effect on iron-induced lipid peroxidation and the effectiveness of this magnetic field on iron ion-induced active oxygen species generation is restricted to a so called ‘window’ of field intensity of 2–4 mT.     

4-Hydroxynonenal modulates the long-term potentiation induced by L-type Ca2+ channel activation in the rat dentate gyrus in vitro

Increased oxyradical production and membrane lipid peroxidation (MLP) occur under physiological and degenerative conditions in neurons. We investigated whether 4-hydroxynonenal (4HN), one of the membrane lipid peroxidation products, affects long-term potentiation (LTP) in the rat dentate gyrus in vitro. Treatment of hippocampal slices with 4HN (10 μM) enhanced LTP without affecting basal evoked potentials. The enhancement was completely inhibited by 2 μM nifedipine, a blocker of L-type Ca²⁺ channels. In cultured dentate gyrus neurons, treatment of the cells with 4HN for 24 h resulted in a significant amount of cell death that was detoxified by glutathione, whereas short-term treatment with 4HN (6 h) had no effect. Nifedipine partially but significantly suppressed the 4HN-induced cell death. These results suggest that 4HN modulates LTP and induces delayed cell death through L-type Ca²⁺ channel activation in the dentate gyrus. 4HN thereby plays an important role in both physiological and pathophysiological events in the hippocampus.     

Optical biodetection of cadmium and lead ions in water

A three layer waveguiding silicon dioxide (SiO₂)/silicon nitride (Si₃N₄)/SiO₂ structure on silicon substrate was proposed as an optically efficient biosensor for calibration of heavy metal ions in drinking water. The catalytic activities of urease and acetylcholine esterase (AchE) were inhibited by the presence of cadmium (Cd²⁺) and lead (Pb²⁺) ions. The detection limit as low as 1 ppb was achieved by employing the technique of total reflection at the interface between the Si₃N₄ core and composite polyelectrolyte self-assembled (PESA) membranes containing cyclotetrachromotropylene (CTCT) as an indicator.     

Lymphocyte-Associated Complement: Role of C8 in Certain Cell-mediated Lytic Reactions

⁵¹Cr- labelled chicken erythrocytes (E) were treated with human     and C7 to form     , susceptible to lysis by the terminal complement components C8 and C9 ('reactive lysis') Addition of purified and extensively washed human blood lymphocytes, but not of erythrocytes, to     resulted in a similar but cell-mediated reactive lysis. Contamination of the effector cell preparations with plasma was excluded. The reaction does not require living effector cells. Its dependency on C8 was proved by inhibition with rabbit anti-human C8 as well as with its F(ab')₂ fragments. Although terminal complement components may be of importance for cell-mediated lysis of complement-carrying target cell, no effector role could be assigned to them in antibody-dependent lymphocyte mediated lysis Thus, lysis of antibody-coated chicken erythrocytes in the absence of added complement by purified human lymphocytes was not inhibited by the F(ab')₂ fragment of anti-C8.     

基于Cole-Cole模型评价纳米二氧化钛的细胞毒性作用

利用Cole-Cole模型评价不同剂量纳米二氧化钛(Nano-TiO₂)的细胞毒性作用,并探讨电生理机制。150和300 mg/L的Nano-TiO₂悬液作用人胃癌MGC803细胞24 h后,制备成细胞悬浮液。在1 kHz～100 MHz范围,采用Agilent 4294A精密阻抗分析仪测量了人胃癌MGC803细胞悬浮液电阻抗的幅值和相位角,经电阻抗频谱和Nyquist图的曲线拟合的残差分析,建立Cole-Cole模型参数,评估Nano-TiO₂对MGC803细胞导电性能的影响。结果表明,150和300 mg/L的Nano-TiO₂引起第一电阻抗增量(ΔZ₁)分别减少18.18%(P<0.001)和39.39%(P<0.001),第二电阻抗增量(ΔZ₂)分别减少6.56%(P<0.001)、8.2%(P<0.001),降低了MGC803细胞膜及其核膜的电阻,增加了其导电性能;第一特征频率(f_C1)分别增加19.74%(P<0.001)和29.67%(P<0.001),第二特征频率(f_C2)分别增加6.28%(P<0.001)、23.43%(P<0.001);第一散射角(β₁)分别降低1.35%(P>0.05)和2.70%(P<0.05)。Cole-Cole模型可评价Nano-TiO₂的细胞毒性作用并解释其电生理机制,为纳米颗粒的细胞毒理研究提供一种电特性方法。     

A prostaglandin D2 receptor antagonist modifies experimental asthma in sheep

Background Prostaglandin (PG) D₂ is the major cylooxygenase metabolite released by mast cells upon allergen stimulation, and elicits responses through either the prostanoid DP1 receptor and/or the chemoattractant receptor homologous molecule expressed on T-helper type 2 (Th2) cells (CRTH2/DP2). Experimental evidence suggests that stimulation of one or both these receptors contributes to asthma pathophysiology.
Objective The aim of this study was to test the hypothesis that the prostanoid DP1 receptor contributes to asthma pathophysiology by determining the efficacy of an orally active antagonist for this receptor, S-5751, on allergen-induced bronchoconstriction, airway hyperresponsiveness (AHR) and cellular inflammation in the sheep model of asthma.
Methods PGD₂-induced cyclic adenosine monophosphate (cAMP) production in platelet-rich plasma was used to establish the in vitro efficacy of S-5751. In vivo , sheep naturally allergic to Ascaris suum were challenged with an aerosolized antigen with and without S-5751 treatment (given 4 days before and for 6 days after the challenge).
Results S-5751 inhibited PGD₂-induced cAMP production in platelet-rich plasma with an IC₅₀ value of 0.12 μ m . S-5751 at 30 mg/kg, but not at 3 mg/kg, reduced the early bronchoconstriction and inhibited the late bronchoconstriction. AHR and inflammatory cell infiltration in bronchoalveolar lavage fluid at days 1 and 7 were also inhibited with the 30 mg/kg dose. The responses observed with S-5751 at 30 mg/kg were comparable with those with montelukast treatment (0.15 mg/kg, twice a day, intravenous); however, S-5751 did not block inhaled leukotrieneD₄-induced broncoconstriction.
Conclusion Prostanoid DP1 receptor inhibition may represent an alternative target for asthma therapy.     

T Cell Subsets and T Cell Function in Cartilage-Hair Hypoplasia

Cartilage hair hypoplasia is a rare autosomal recessive form of short-limbed dwarfism associated with a cellular immunodeficiency. In eight patients, the authors studied the presence of T cell subsets and in vitro T cell function in order to address the basis for the immunological disorder. Both the proliferative response to phytohaemagglutinin (PHA) and the PHA-induced IL2 production were 60% lower compared with controls ( P   =  0.007 and 0.005, respectively). The impaired proliferative response could not be restored by addition of IL-2. This result is in accordance with a decrease in the percentage of activated T cells expressing the p 55 subunit of the IL-2 receptor complex (CD25). The results define more precisely that T cells from cartilage hair hypoplasia patients are defective in the transition from the G₀ to the G₁ phase of the cell cycle. Furthermore, the data demonstrate that several CHH patients show a reduced proportion of CD45RA⁺'naive' T cells. However, the in vitro impairment of T cell function cannot solely be explained by imbalance between 'naive' and 'memory' T cells. Although CHH patients with a history of recurrent respiratory tract infections showed the most aberrant in vitro immune parameters, a clear relationship between clinical data and in vitro parameters could not be established for the whole patient group.     

Neurokinin-1 receptor desensitization to consecutive microdialysis infusions of substance P in human skin

Unloaded shortening velocity ( V ₀) of human triceps surae muscle was measured in vivo by applying the 'slack test', originally developed for determining V ₀ of single muscle fibres, to voluntary contractions at varied activation levels (ALs). V ₀ was measured from 10 subjects at five different ALs defined as a fraction (5, 10, 20, 40 and 60%) of the maximum voluntary contraction (MVC) torque. Although individual variability was apparent, V ₀ tended to increase with AL  ( R ²= 0.089; P = 0.035)  up to 60%MVC (8.6 ± 2.6 rad s⁻¹). This value of V ₀ at 60%MVC was comparable to the maximum shortening velocity of plantar flexors reported in the previous studies. Electromyographic analysis showed that the activities of soleus, medial gastrocnemius and lateral gastrocnemius muscles increased with AL during isometric contraction and after the application of quick release in a similar manner. Also, it showed that the activity of an antagonist, tibialis anterior muscle, was negligible, even though a slight increase took place after the quick release of agonist. Correlation analysis showed that there were no significant correlations between V ₀ and MVC torque normalized with respect to body mass, although the correlation coefficient was relatively high at low ALs. The results suggest that in human muscle, V ₀ represents the unloaded velocity of the fastest muscle fibres recruited, and increases with AL possibly because of progressive recruitment of faster fibres. Individual variability may be explained, at least partially, by the difference in fibre-type composition.     

Fe3O4纳米粒子的细胞相容性

背景：无论是作为化疗药物载体还是作为肿瘤热疗介质,Fe₃O₄纳米粒子与肿瘤细胞微观结构的作用都有待于深入研究。 目的：分析Fe₃O₄纳米粒子的细胞相容性,探讨其在骨肉瘤化疗中作为药物载体的应用和存在的问题。 方法：参照GB/T16886.5-2003 (医疗器械生物学评价 第5部分：体外细胞毒性试验)的评价标准和要求,偶联十六烷基三甲基溴化铵、聚乙二醇、油酸钠Fe₃O₄纳米粒子胶体溶液,分别与大鼠原发骨肉瘤细胞和人成纤维细胞共培养的方式对其进行细胞毒性测试。 结果及结论：偶联油酸钠Fe₃O₄纳米粒子细胞相容性良好,参照GB/T16886.5-2003标准属于安全范围。偶联十六烷基三甲基溴化铵Fe₃O₄纳米粒子细胞相容性差,不适宜作为化疗药物载体进入人体。偶联聚乙二醇Fe₃O₄纳米粒子有一定的细胞相容性,作为磁靶向热化疗载体还需研究。     

Natural and artificial substrates for retinal pigment epithelial monolayer transplantation

The aim of this study was to culture retinal pigment epithelial (RPE) cells on natural and synthetic substrates for future use in RPE monolayer transplantation in the eye. The extracellular capsules surrounding the human lens and a hydrogel biomaterial were used as substrates for monolayer culture. All materials were seeded with either pig or human retinal pigment epithelial cells and were maintained in tissue culture conditions. Upon confluency, the cell density was calculated and cell viability determined. All monolayers were stained with phalloidin–rhodamine for F-actin and antibodies to tight junction-associated protein, ZO₁. The final cell density of human RPE monolayers on the hydrogel and lens capsule was 3200±187 and 3350±120 cells/mm² respectively. Pig RPE cells had a final cell density of 3740±205 cells/mm² on the lens capsule and 3025± cells/mm² on the hydrogel. F-actin staining revealed a circumferential ring of actin filaments in all the cells grown on substrates. ZO₁ immunohistochemisty demonstrated staining along the lateral cell borders of all cell types. The successful culture of RPE cells on these substrates may have the potential for transplanting cell monolayers in the eye to improve outcomes for degenerative diseases in the retina.     

Ozone inhalation induces exacerbation of eosinophilic airway inflammation and hyperresponsiveness in allergen-sensitized mice

Background:  Ozone (O₃) exposure evokes asthma exacerbations by mechanisms that are poorly understood. We used a murine model to characterize the effects of O₃ on allergic airway inflammation and hyperresponsiveness and to identify factors that might contribute to the O₃-induced exacerbation of asthma.
Methods:  BALB/c mice were sensitized and challenged with Aspergillus fumigatus ( Af ). A group of sensitized and challenged mice was exposed to 3.0 ppm of O₃ for 2 h and studied 12 h later (96 h after Af challenge). Naive mice and mice exposed to O₃ alone were used as controls. Bronchoalveolar lavage (BAL) cellular and cytokine content, lung function [enhanced pause (P_enh)], isometric force generation by tracheal rings and gene and protein expression of Fas and FasL were assessed. Apoptosis of eosinophils was quantified by FACS.
Results:  In sensitized mice allergen challenge induced a significant increase of P_enh and contractile force in tracheal rings that peaked 24 h after challenge and resolved by 96 h. O₃ inhalation induced an exacerbation of airway hyperresponsiveness accompanied by recurrence of neutrophils and enhancement of eosinophils 96 h after allergen challenge. The combination of allergen and O₃ exposure inhibited Fas and FasL gene and protein expression and eosinophil apoptosis and increased interleukin-5 (IL-5), granulocyte-macrophage-colony stimulating factor (GM-CSF) and G-CSF protein levels.
Conclusions:  O₃ affects airway responsiveness of allergen-primed airways indirectly by increasing viability of eosinophils and eosinophil-mediated pathological changes.     

四硫代钼酸铵作为新型硫化氢供体的鉴定及其对皮肤细胞的保护作用

 目的: 本研究旨在检测金属络合物四硫代钼酸铵(ATTM)的硫化氢(H₂S)释放能力并进一步观察其减轻氧化应激诱导的皮肤细胞损伤的作用。方法:反应瓶法检测ATTM在细胞培养基中释放的H₂S,二氯二氢荧光素二乙酸酯或罗丹明123染色结合荧光显微镜照相术分别检测细胞内活性氧簇(ROS)的含量和线粒体膜电位(ΔΨ_m),用试剂盒检测细胞存活率和乳酸脱氢酶(LDH)的释放。结果:与H₂S供体GYY4137类似,ATTM在细胞培养基中可剂量依赖性地释放H₂S。在25~400 μmol/L 的浓度范围内,ATTM处理对人皮肤细胞(HaCaT细胞)的存活率无明显影响(P>0.05)。紫外线照射或外源性给予ROS供体(过氧化氢,H₂O₂)处理均可增加HaCaT细胞内ROS的含量。400 μmol/L H₂O₂处理可明显降低HaCaT细胞的存活率(P<0.01),而在H₂O₂处理前给予ATTM预处理,细胞存活率明显提高,其中在100和200 μmol/L浓度时ATTM具有最好的细胞保护作用(P<0.01)。400 μmol/L H₂O₂处理还可损害细胞的ΔΨ_m和细胞膜并使LDH释放增加(P<0.01)。而在细胞遭受损伤前给予200 μmol/L ATTM预处理可明显改善ΔΨ_m的水平(P<0.05)并抑制LDH从细胞内释放(P<0.01)。结论:ATTM具有释放H₂S的能力并可保护人皮肤细胞对抗氧化应激诱导的损伤效应。