A Ca2+ chelator ameliorates chromium (VI)-induced hepatocyte L-02 injury via down-regulation of voltage-Dependent anion channel 1 (VDAC1) expression

Hexavalent chromium could result in cell malfunctions. Intracellular Ca²⁺ ([Ca²⁺]_i) content and VDAC1 expression are both important features related to cell survial. This study aimed to explore the mechanism of cell injury induced by Cr(VI) and tentatively offer clues to repairing this cell damage using [Ca²⁺]_i and VDAC1. L-02 hepatocytes were treated with Cr(VI)/BAPTA, and the levels of [Ca²⁺]_i and cell injury associated with Cr(VI) were determined in addition to the effect of BAPTA. The expression of VDAC1 in Cr(VI)-induced cells was evaluated. The results showed a dose-dependent elevation of the level of VDAC1 and the mRNA level of the VDAC1 biogenesis-related gene Sam50. BAPTA could ameliorate less severe damage induced by 4 μM Cr(VI) via reducing VDAC1 and Sam50. Additionally, cell injury caused by less than 4 μM Cr(VI) could be ameliorated by VDAC1 knockdown. Taken together, the findings of this study suggest that inhibition of intracellular Ca^2± overload could protect cells from damage and that VDAC1 plays a considerable role in Cr(VI)-induced liver injury.     

L-1416, a novel MDR reversing agent with possible reduced calcium antagonism

BackgroundMultidrug efflux transporter P-glycoprotein (P-gp) is highly expressed on membrane of tumor cells and supposed to be implicated in the resistance to tumor chemotherapy. However, currently none of P-gp inhibitors has been approved by Food and Drug Administration not only due to toxicity but also lack of efficacy in clinical trials.MethodsTo solve the problem, our lab synthesized a novel compound named 1416 [1-(2,6-dimethylphenoxy)-3,4-dimethoxyphenylethylamino) propane hydrochloride] with the hope of high P-gp inhibition and low side effects. Caco-2 cell monolayer and tumor bearing mice were used to evaluate the P-gp inhibition of 1416 in vitro and in vivo, respectively. One of its potential side effects, calcium antagonism was also evaluated.ResultsResults showed that 1416 showed a similar P-gp inhibition as verapamil in Caco-2 cell monolayer. No significant difference was observed in antitumor enhancement when the optical isomers of 1416 (D-1416 and L-1416) were co-administered with vinblastine. In calcium antagonism, L-1416 showed less calcium inhibition than both D-1416 and verapamil.ConclusionThe novel compound 1416 could significantly increase the antitumor effects of cytotoxic drugs and one of its optical isomers, L-1416, might be more promising due to its potential low calcium antagonism.     

HPLC法测定L-(+)-扁桃酸的光学纯度

目的采用HPLC法测定L-(+)-扁桃酸的光学纯度。方法采用Chirlcel OJ-H手性色谱柱,以正己烷-乙醇-三氟乙酸(96∶4∶0.3)为流动相,检测波长220 nm,流速1.0 m L·min-1,柱温30℃。结果 L-(+)-扁桃酸与D-(-)-扁桃酸的分离良好,L-(+)-扁桃酸0.2~1.2×103μg·m L-1与峰面积的线性关系良好(r=0.9998),检测限为0.4 ng(S/N=3),定量限为1.6 ng(S/N=10)。结论所用方法快速、准确、灵敏,适于L-(+)-扁桃酸光学纯度的测定。     

槲皮素抑制异烟肼诱导的L-02细胞线粒体氧化应激性DNA损伤

目的:探讨活性氧(ROS)介导的线粒体氧化损伤在异烟肼(INH)诱导L-02细胞DNA损伤中的作用及槲皮素对细胞的保护作用。方法:建立体外培养INH致肝细胞L-02损伤的模型,将细胞分为对照(control)组、INH组、槲皮素低剂量(Que low)及高剂量(Que high)组。利用彗星试验评价细胞DNA损伤;制备L-02细胞线粒体,应用荧光探针DCFH-DA和rhodamine 123检测细胞线粒体ROS水平及线粒体膜电位(ΔΨm);采用TBA法测定丙二醛(MDA)含量;应用黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)的活性;采用Western blotting法检测细胞中Bcl-2和Bax蛋白表达,计算Bax/Bcl-2值。结果:INH可诱导L-02细胞DNA损伤,使细胞线粒体ROS水平、细胞MDA含量及Bax/Bcl-2值明显增高,并使细胞ΔΨm值和SOD活性明显下降。而槲皮素能减轻细胞DNA损伤,减少细胞ROS水平,增加细胞ΔΨm值,降低细胞MDA含量,增加SOD活性,减少Bax/Bcl-2值。结论:INH可通过诱导细胞线粒体氧化应激导致L-02细胞DNA损伤。槲皮素能减轻INH诱导L-02细胞的DNA损伤,对L-02细胞具有保护作用,可能与其抑制ROS介导的线粒体氧化损伤有关。     

Serum and urinary lipoproteins in the human nephrotic syndrome: evidence for renal catabolism of lipoproteins

The urinary excretion of lipoproteins and the possibility of catabolic alterations on glomerular filtration were investigated in four nephrotic subjects differing in etiology, serum lipoprotein profile, and 24 hr urinary output of protein and lipids. The apolipoproteins and lipoproteins of urine were compared with those of serum with respect to distribution profile, physical properties, and composition. Lipoprotein particles resembling the serum very low, intermediate, low, and high density lipoproteins (VLDL, IDL, LDL, and HDL, respectively) in density, particle size, and morphology were isolated from the urine. As expected from molecular sieving effects during glomerular filtration, the urinary HDL were more abundant than the lower density lipoproteins even when the plasma LDL was elevated markedly. However, little sieving effect was seen within the urinary HDL, which comprised a broad spectrum of particle sizes including the larger HDL₂, whose average diameter was similar to that of the plasma HDL. A sieving effect was not seen in the urinary LDL, except for a greatly increased proportion, about 20% of total particles, of HDL-like species. Intact apolipoproteins were not found in the concentrated urinary fraction isolated by ultrafiltration between the limits of 10⁴ and 5 × 10⁴ daltons. On the basis of immunoreactivity, gel electrophoresis, and amino acid composition, apolipoproteins B and AI are the major and minor proteins, respectively, of urinary LDL, and apo B is the major protein of the urinary IDL and VLDL. Apolipoproteins AI, AII, CI, CIII, and possibly AIV were isolated from the urinary HDL. As much as 20% of the protein moiety of the urinary HDL appeared to be large apolipoprotein fragments with molecular weights and isoelectric points similar to those of apo CII and apo CIII. The fragments were derived in part from apo AI, the least acidic form of which was lost preferentially. The lower density classes of urinary lipoproteins also appeared to have lost apo E and apo C's and to have undergone partial proteolysis. Apparently, the surface-exposed, readily exchangeable apolipoproteins are subject to proteolytic degradation upon glomerular filtration.     

c-jun末端激酶在亚砷酸钠致人胚胎肝细胞损伤中的表达

目的探讨亚砷酸钠染毒对人胚胎肝(L-02)细胞中c-jun末端激酶(JNK)的变化。方法将处于对数生长期的L-02细胞分别暴露于终浓度为0(对照)、50、100、150μmol/L的亚砷酸钠溶液中培养24 h,采用四甲基偶氮唑蓝(MTT)法检测细胞生长情况,采用流式细胞术检测染毒24 h时的细胞周期及细胞凋亡率;采用蛋白杂交(Western-blot)法检测JNK及p-JNK的蛋白表达水平。结果与对照组相比,各浓度亚砷酸钠染毒组L-02细胞的存活率及G0-G1期构成比均较低,JNK、p-JNK的蛋白表达水平和S期构成比及凋亡率均较高;而G2-M期构成比在50μmol/L亚砷酸钠染毒组较低,在100、150μmol/L亚砷酸钠染毒组均较高,差异均有统计学意义(P0.05)。且随着亚砷酸钠染毒浓度的升高,L-02细胞凋亡率及JNK和p-JNK蛋白的表达水平均呈上升趋势,细胞存活率呈下降趋势。结论亚砷酸钠诱导L-02细胞凋亡可能与JNK及p-JNK表达的增加有关。     

Protective effect of blueberry anthocyanins in a CCl4-induced injury model in human embryonic liver cells

This study investigates the possible mechanism of the protective effects of blueberry anthocyanins on human embryonic liver L-02 cells. Results of the WST-8 method showed that different concentrations of blueberry anthocyanins protected the human embryonic liver L-02 cells against CCl₄-induced injury in a dose-dependent manner with IC₅₀ at 6.2×g/L. Cell clone formation inhibition assay demonstrated that cell clones increased with increased concentration of anthocyanins. Propidium iodide (PI) staining analysis showed that anthocyanins can decrease cell cycle in the G1 phase. DNA ploidy analysis showed a weakened percentage of hypodiploid cells in a dose-dependent manner. Moreover, Annexin V-FITC/PI staining analysis showed a dose-dependent effect of anthocyanins on late apoptotic and necrotic cells. Western blot assay showed gradually decreased caspase-3 protein expression levels with increased anthocyanin concentration. In summary, these findings provided pharmacological evidence supporting the clinical application and protective effect of blueberry anthocyanins against acute liver injury.     

纳米Ag—SiO2颗粒对小鼠成纤维细胞（L-929）的毒性研究

目的：探讨纳米Ag—SiO2颗粒对小鼠成纤维细胞（L-929）的体外毒性作用。方法：使用透射电镜（TEM）对纳米颗粒进行表征;CCK一8法观察在24h、48h、72h不同浓度纳米颗粒对L-929细胞活性的影响;倒置显微镜观察染毒24h后细胞形态的改变,并检测细胞上清液中乳酸脱氢酶（LDH）漏出率。结果：L-929细胞的生长活性随着纳米Ag—SiO2颗粒浓度的升高和染毒时间的延长而下降,低浓度组（6．25,12．5μg／mL）与对照组无明显差异,倒置显微镜下细胞形态正常,生长良好。而高浓度组（50,100μg／mL）与对照组有明显差异,透射电镜下观察细胞有明显的空泡变性,培养液上清中LDH活性明显升高。结论：纳米Ag—SiO2颗粒的体外细胞毒性呈剂量一时间依赖关系,低浓度组具有良好的生物相容性,高浓度组具有一定的细胞毒性,其毒性可能与细胞膜的完整性遭到破坏有关．     

Histone demethylase JHDM2A regulates H3K9 dimethylation in response to arsenic-induced DNA damage and repair in normal human liver cells

Long-term arsenic exposure is a worldwide public health problem that causes serious harm to human health. The liver is the main target organ of arsenic toxicity; arsenic induces disruption of the DNA damage repair pathway, but its mechanisms remain unclear. In recent years, studies have found that epigenetic mechanisms play an important role in arsenic-induced lesions. In this study, we conducted experiments in vitro using normal human liver cells (L-02) to explore the mechanism by which the histone demethylase JHDM2A regulates H3K9 dimethylation (me2) in response to arsenic-induced DNA damage. Our results indicated that arsenic exposure upregulated the expression of JHDM2A, downregulated global H3K9me2 modification levels, increased the H3K9me2 levels at the promoters of base excision repair (BER) genes (N-methylpurine-DNA glycosylase [MPG], XRCC1 and poly(ADP-ribose)polymerase 1) and inhibited their expression levels, causing DNA damage in cells. In addition, we studied the effects of overexpression and inhibition of JHDM2A and found that JHDM2A can participate in the molecular mechanism of arsenic-induced DNA damage via the BER pathway, which may not be involved in the BER process because H3K9me2 levels at the promoter region of the BER genes were unchanged following JHDM2A interference. These results suggest a potential mechanism by which JHDM2A can regulate the MPG and XRCC1 genes in the process of responding to DNA damage induced by arsenic exposure and can participate in the process of DNA damage repair, which provides a scientific basis for understanding the epigenetic mechanisms and treatments for endemic arsenic poisoning.     

Licorice root extract and magnesium isoglycyrrhizinate protect against triptolide-induced hepatotoxicity via up-regulation of the Nrf2 pathway

Triptolide, the predominant biologically active component of the Chinese herb Tripterygium wilfordii Hook f., possesses numerous pharmacological activities, including anti-inflammatory, anti-fertility, anti-neoplastic, and immunosuppressive effects. However, toxicity and severe adverse effects, particularly hepatotoxicity, limit the clinical application of triptolide. Licorice root extract contains various bioactive compounds and is potent hepatoprotective. Magnesium isoglycyrrhizinate, a magnesium salt of the 18α-glycyrrhizic acid stereoisomer of glycyrrhizic acid, is used clinically in China to treat chronic viral hepatitis and acute drug-induced liver injury. The aim of this study was to investigate the role of the factor erythroid 2-related factor 2 pathway in the protective effects of LE and MIG against triptolide-induced hepatotoxicity. Hepatotoxicity models were established in L-02 cells and rats using triptolide, and the protective effects of LE and MIG were investigated in vitro and in vivo, respectively. LE and MIG significantly protected against triptolide-induced cytotoxicity. Additionally, triptolide decreased the mRNA and protein levels of Nrf2 and down-regulated Nrf2 target genes, including UGT1A, BSEP, and MRP2, while pretreatment with LE and MIG reversed these effects. Finally, Nrf2-involved antioxidant responses were activated in the presence of LE and MIG.