Apigenin exhibits anti-inflammatory effects in LPS-stimulated BV2 microglia through activating GSK3β/Nrf2 signaling pathway

Abstract

Objective: Apigenin is a natural flavonoid compound extracted from Matricaria chamomilla. We evaluated the anti-inflammatory effects of apigenin in this study using the Lipopolysaccharide (LPS)-stimulated BV2 microglia.

Methods: BV2 cells were treated with apigenin for 1?h and then treated with LPS. The inflammatory cytokine productions were tested by qRT-PCR and ELISA. The expression of GSK3β, Nrf2, and NF-κB signaling pathways were measured by western blot analysis.

Results: Apigenin significantly attenuated LPS-induced TNF-α, IL-1β, and IL-6 production. Apigenin suppressed LPS-induced NF-κB activation. Furthermore, GSK3β, Nrf2, and HO-1 were concentration-dependently increased by apigenin. The suppression of apigenin on LPS-induced inflammatory response and NF-κB activation were prevented when Nrf2 was knocked out or by GSK3β inhibitor.

Conclusions: Collectively, apigenin suppressed LPS-induced microglia activation via activating GSK3β/Nrf2 signaling pathway.     

Regulation of IGF-I production and proliferation of human leiomyomal smooth muscle cells by Scutellaria barbata D. Don in vitro: isolation of flavonoids of apigenin and luteolin as acting compounds

Scutellaria barbata D. Don (Lamiaceae) (SB) is a perennial herb, which is natively distributed throughout Korea and southern China. This herb is known in traditional Chinese Medicine as Ban-Zhi-Lian and traditional Korean medicine as Banjiryun, respectively. SB has been used as an anti-inflammatory and antitumor agent. We aimed to determine the expression of growth factor molecules for growth inhibition after treatment of SB in two different human myometrial smooth muscle cell (SMC)s and leiomyomal SMCs. Water-soluble ingredients of SB, myometrial SMCs, and the leiomyomal cell lines were used in vitro. SB significantly reduced cell numbers in culture and arrested cell proliferation, and also induced apoptosis, indicating that the presence of an intact apoptotic pathway was demonstrated in these cells by SB. Uterine leiomyoma is the most common benign smooth muscle cell tumor of the myometrium. The expression of insulin-like growth factor-I (IGF-I) was measured at the mRNA and protein level in myometrium and leiomyomal cells with and without treatment with a water extract of SB for 3 days. IGF-I mRNA expression was significantly higher in leiomyomal cells than in myometrium cells. The IGF-I protein was more abundant in leiomyomal cells than in myometrium. When SB was treated to the cells, the IGF-I protein concentrations in myometrial and leiomyomal cells from the SB-treated cells were similar. The results indicated that IGF-I expression is probably associated with a proliferation of leiomyomal cells than myometrium. However, SB down-regulated the IGF-I expression where IGF-I contributes to the selective growth of the leiomyoma. Therefore, growth modulation of LMs by SB occurs via mechanisms dependent of apoptosis. The raw materials were extracted and subjected to functional isolation for the active molecules in the present assay systems. The five flavonoids were isolated and the chemical structures of resveratrol, baicalin, berberine, apigenin, and luteolin were determined. The effects of resveratrol, baicalin, and berberine on the above parameters have not been significantly evidenced, whereas apigenin and luteolin were effective. The anti-proliferative compounds apigenin and luteolin belong to the flavones, a class of flavonoids and are characterized as selectively inhibitors of the growth of LM cells. Our findings suggest that flavonoids of apigenin and luteolin are potentially useful for the development of therapeutic treatments of cancer. These data also suggest that SB reduces tumor volume with inducing a concomitant increase in the rate of apoptosis.     

Anti-rheumatoid arthritis effects of flavonoids from Daphne genkwa

ObjectiveThis study aims to select the most effective anti-Rheumatoid Arthritis (RA) component of flavonoids from Daphne genkwa Sieb. et Zucc. by anti-inflammatory and immunomodulatory effects in vitro, and to elucidate the mechanism.MethodsThe anti-inflammatory and immunomodulatory effects of total flavonoids (TF) and four flavonoid components (genkwanin, hydroxygenkwanin, luteolin and apigenin) were determined by pharmacological approach in LPS-induced RAW 264.7 macrophages and ConA-induced T lymphocytes. Principal component analysis (PCA) was used to obtain the optimal anti-RA component in vitro. Western blot and real-time quantitative PCR (q-PCR) were used to explore the mechanisms. Finally, the in vitro anti-RA effect was verified by human rheumatoid arthritis fibroblast-like synoviocytes (FLSs).ResultsTF and four flavonoids significantly reduced the expressions of NO, iNOS, TNF-α, IL-6, IFN-γ and IL-2. PCA showed that genkwanin was the most effective anti-RA component in vitro. Genkwanin inhibited nuclear factor-κB (NF-κB) pathway by decreasing the phosphorylation levels of IKK, IκB and NF-κB, and down-regulated the expressions of iNOS, COX-2 and IL-6 mRNA. Genkwanin also inhibited the abnormal proliferation of FLSs and down-regulated the secretions of NO and IL-6.ConclusionThe most effective anti-RA component was genkwanin. Genkwanin exerts anti-RA effect through down-regulating the activation of NF-κB pathway and mRNA expressions of inflammatory mediators, and also by inhibiting the abnormal proliferation of FLSs and its NO and IL-6 secretion levels.     

芹菜素抗肿瘤作用及其机制研究进展

肿瘤的发生是一个多因素参与的复杂的过程。当今世界各地的研究学者们试图从不同的角度探讨其发生发展机制及抑制途径,除了遗传学、基因学的研究,人们也找到了一些能够抑制肿瘤生长的药物,如植物源性药物芹菜素。芹菜素具有较强的抗炎、抗氧化、抗肿瘤等药理作用和生物学特性。近期研究发现它具有低毒、无诱变等特点,也因此受到肿瘤领域的专家学者们的广泛关注。研究就抗癌药物芹菜素的生物学作用方面做…简单阐述。     

芹黄素对人眼Tenon囊成纤维细胞增生的影响

背景 青光眼滤过手术后人Tenon囊成纤维细胞(HTFs)的增生是滤过手术失败的丰要原因,寻找抑制术后HTFs增生的药物可提高滤过手术的成功率. 目的 观察芹黄素对HTFs体外生长的抑制效应并探讨其作用机制.方法 斜视手术中获取的人眼Tenon囊组织剪成1 mm×1 mm×1 mm的组织块,进行原代培养,免疫荧光法进行细胞波形蛋白检测以鉴定培养的HTFs.取第3～5代HTFs接种于96孔板中,加入0、20、40、80、160 μmol/L芹黄素后继续培养,于24、48、72 h后分别取一块96孔板,在酶联免疫检测仪上检测560 nm波长处各组细胞的吸光度(A560)值,磺基罗丹明B(SRB)法测定细胞的增生能力.分别在培养基中加入40、80、160 μmol/L芹黄素,同时在不同浓度芹黄素组培养基中均加入10 μg/L BrdU,继续培养48 h,在各组同一个视野下分别拍摄荧光显微镜和光学显微镜照片,计算BrdU的标记率,以未添加任何芹黄素(0μmol/L)组为对照组.用Hoechst 33258染色后在荧光显微镜下观察培养细胞的形态学改变,采用流式细胞技术检测细胞的凋亡情况和细胞周期的变化. 结果 培养的细胞生长状态良好,免疫荧光法检测可见细胞对波形蛋白呈阳性反应,为细胞质中均匀的绿色荧光,确定培养的细胞为HTFs.SRB法检测结果显示,HTFs的增生值(A560)随着芹黄素浓度的增加而逐渐下降,同浓度芹黄素组HTFs A560值随着作用时间的延长逐渐下降,差异均有统计学意义(F组别=480.306,P=0.000;F时间 =555.144,P=0.000).0、40、80 μmol/L芹黄素作用HTFs 48 h后BrdU的标记率分别为(87.860±0.632)％、(61.520±4.306)％、(23.480±4.472)％,各组之间的总体比较差异有统计学意义(F=299.347,P=0.000),其中40 μmol/L、80 μ mol/L芹黄素组HTFs BrdU的标记率均明显低于0 μmol/L芹黄素组,差异均有统计学意义(P＜0.05).Hoechse 33258染色发现,不同浓度芹黄素组的细胞数目减少,随着芹黄素浓度的增加,细胞核固缩和变形的细胞数目增加.流式细胞术检测发现,随着芹黄素浓度的增加,G0/G1期细胞的百分数逐渐增加,而S期和G2/M期细胞的百分数逐渐下降,差异均有统计学意义(FG0/G1 =58.621,P=0.000;Fs=32.357,P=0.001;Fc2/M =83.998,P=0.000).0、40、80、160 μmol/L芹黄素作用72 h后,细胞的凋亡率分别为(4.77±0.21)％、(13.24-±1.35)％、(18.33±1.86)％、(31.58±2.77)％,4个组的总体差异有统计学意义(F=204.791,P＜0.05). 结论 芹黄素通过诱导细胞凋亡,并将细胞阻滞于G0/G1期而抑制HTFs的增生,其作用呈剂量和时间依赖性.     

芹菜素对结肠癌SW480细胞MMP及CD44v6作用的实验研究

目的观测不同浓度芹菜素处理后基质金属蛋白酶-2及基质金属蛋白酶-9的分泌情况;检测CD44v6跨膜糖蛋白黏附分子的表达。方法酶谱分析法和酶联免疫吸附法测定不同浓度芹菜素处理后基质金属蛋白酶-2及基质金属蛋白酶-9的分泌情况;免疫组织化学法检测不同浓度芹菜素处理后细胞CD44v6的表达。结果酶谱分析法和酶联免疫吸附法测定均显示不同浓度芹菜素处理后基质金属蛋白酶-2及基质金属蛋白酶-9的分泌量随着药物浓度的升高而减少,呈时间和剂量。SW480细胞表达的CD44v6与芹菜素浓度呈负相关。结论芹菜素抑制SW480细胞分泌基质金属蛋白酶基质金属蛋白酶-2和基质金属蛋白酶-9,芹菜素是一种良好的基质金属蛋白酶抑制剂。芹菜素抑制SW480细胞粘附分子CD44v6的表达,这可能是它抑制肿瘤侵袭和转移的机制之一。     

转基因鼠C6细胞内在缝隙连接通讯功能测定及其化学调控

目的研究Q细胞转染单纯疱疹胸苷激酶基因（herpes simplex virus—thymidine kinase,HSV-TK）前后缝隙连接通讯（GJIC）功能及其化学调节变化。方法用划痕标记染料示踪技术检测不同组别（C6组,C6-TK^-组,C6-TK^＋）C6细胞的GJIC功能以及加用缝隙连接蛋白上调剂apigenin后的功能变化。结果用药前C6组,C6-TK^-组及C6-TK^＋组荧光自伤沿列细胞传递未超过邻近的1列细胞内;用药后3组细胞在荧光显微镜下均可见划痕后的荧光染料可传递至比邻3—4列细胞。结论C6细胞间通讯微弱,基因转染对其无明显影响;Apigenin则可显著增强C6细胞间连接通讯从而提高其GJIC功能及“旁观者效应”。     

芹菜素对胶质瘤细胞增殖抑制作用的研究

目的 探讨芹菜素对人胶质瘤细胞U251的增殖抑制作用.方法 用四甲基偶氮唑盐(MTT)比色法,检测不同浓度(20μmol/L、40μmol/L、80μmol/L)的芹菜素作用不同时间(24h、48h、72h),对人胶质瘤细胞株U251生长的影响.结果 低剂量的芹菜素(20μmol/L)无明显抑制胶质瘤U251细胞增殖的作用,而中剂量(40μmol/L)及高剂量的芹菜素(80μmol/L)能抑制胶质瘤U251细胞增殖,且抑制作用呈剂量和时间依赖性.结论 芹菜素有抑制人胶质瘤细胞U251增殖的作用.     

芹菜素对同型半胱氨酸诱导的人脐静脉内皮细胞氧化损伤的保护作用

目的:探讨芹菜素(Apigenin)对同型半胱氨酸氧化损伤的人脐静脉内皮细胞的保护作用。方法:体外培养内皮细胞,将细胞分为7组,即空白对照组(control)、溶剂对照组(DMSO)、Hcy氧化损伤组(Hcy)、氧化损伤加入槲皮素对照组(quercetin+Hcy)、氧化损伤加入芹菜素低、中、高浓度组(Apigenin-L+Hcy、Apigenin-M+Hcy、Apigenin-H+Hcy)。将5mmol/L Hcy作用于加入槲皮素及不同浓度芹菜素预培养6h的内皮细胞,继续培养24h,以细胞毒四唑盐(MTT)比色试验,乳酸脱氢酶(LDH)、谷胱甘肽过氧化酶(GSH-px)、丙二醛(MDA)、活性氧,NADPH酶活性、流式细胞凋亡率为检测指标。结果:芹菜素呈剂量依赖性降低同型半胱氨酸对内皮细胞活力的影响,降低MDA、LDH量,增加培养液中GSH-px活性,并显著减少活性氧、NADPH酶活性和细胞凋亡数量,各指标差异有显著性(P0.01)。结论:芹菜素可保护和修复同型半胱氨酸诱导的内皮细胞的损伤,其作用可能与抗氧化、抑制内皮细胞凋亡有关。     

HPLC测定香青兰药材中的黄酮类成分

目的 建立测定香青兰药材含量的方法.方法 采用HPLC测定.结果 所测香青兰药材中山奈素、洋芹素、异鼠李素,各成分的线性关系良好,其线性范围分别为6.06～60.60 μg·ml-1(r=0.9996)、3.04～30.40 μg·ml-1(r=0.9997)、2.64～26.40μg·ml-1(r=0.9996).其回收率分别为96.2%(RSD=1.1%)、96.9%(RSD=1.1%)、97.2%(RSD=1.2%)(n=3).结论 所建方法准确、简便,为科学地评价香青兰药材的质量提供了依据.