白芍总苷对角质形成细胞增殖及血管内皮生长因子和IL-23表达的影响
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摘要
背景
银屑病是一种常见的由多基因遗传决定的、多环境因素刺激诱导的慢性炎症性增殖性皮肤病,其病因和发病机制至今尚未完全阐明。目前银屑病发病的始动触发因素仍然不清楚,其发病机制涉及到免疫系统紊乱、血管新生和角质形成细胞增生等多个环节。其组织病理学最主要的特征是角质形成细胞(keratinocyte, KC)过度增生、炎症细胞浸润、新生血管形成。在银屑病的组织病理变化中真皮乳头血管的异常最先发生,而后出现炎症细胞的移行和表皮增生及分化异常。同时,大量的免疫活性细胞及其分泌的细胞因子渗出到血管外的组织中,诱导角质形成细胞活化、增殖并分泌大量细胞因子,由此形成细胞因子相互调控的恶性循环,促进疾病的进展。
在银屑病病理过程中,由表皮细胞分泌的很多血管源性细胞因子参与了血管增生,但以血管内皮生长因子(vascular endothelial growth factor, VEGF)最为重要。银屑病皮损中的KC是促进血管新生细胞因子的主要来源。研究表明,VEGF mRNA及蛋白表达水平在银屑病患者皮损中明显增加,患者非皮损处VEGFmRNA及蛋白水平表达亦增加,许多研究又表明严重类型的银屑病患者血清中VEGF水平也显著升高,且与疾病的活动有一定相关性。另有报道将编码VEGF的基因转入小鼠皮肤可致银屑病样改变,并出现特征性的Koebner现象,这进一步说明VEGF在银屑病发病过程中起到重要作用。同时,银屑病也是多种炎细胞共同参与的免疫性疾病。IL-23是新近发现的一种细胞因子,是由本身无生物活性的p19因子与IL-12的p40亚基通过二硫键相连组成的异二聚体。能够诱导记忆性T细胞产生IFN-γ,具有较强的抗感染免疫保护功能和抗肿瘤活性。研究表明银屑病病人皮损IL-23的表达明显增高。Piskin等发现UVB治疗银屑病后,临床症状得到改善,局部皮损IL-23的表达也下调。因此他们推测,角质形成细胞过表达IL-23可能对银屑病的炎性过程起一定作用。
白芍总苷胶囊(商品名帕夫林,total glucosides of paeony, TGP)是从中药白芍中提取的复合制剂,主要成分包括芍药苷、芍药内酯苷、羟基芍药苷、苯甲酰芍药苷等,其中芍药苷占总苷量的90%,是白芍的主要有效成分。其药理作用主要有抗炎、调节免疫功能、影响细胞增殖、止痛、保肝等作用。TGP对自身免疫过程中的多个环节都存在着调节作用。研究报道TGP可以抑制VEGF、金属基质蛋白酶(MMP)等活性物质的产生。目前国内对TGP治疗类风湿性关节炎、儿童特发性关节炎等自身免疫性疾病的研究较多,但对TGP治疗银屑病的机制尚不清楚。
促分裂原活化蛋白激酶(mitogen-activated protein kinases, MAPK)信号转导途径是哺乳动物细胞中重要的信号通路,能将多种细胞外刺激产生的信号通过级联反应从细胞膜传递到细胞核内,在细胞分化、增殖、凋亡、应激、炎症以及免疫反应等多种生理和病理过程中发挥着极其重要的作用。其任一环节的异常都可能导致细胞的异常增殖与分化。MAPK信号转导通路包括MAPK激酶激酶(MAPK kinase kinase, MAPKKK)、MAPK激酶(MAPK kinase, MAPKK). MAPK,此通路主要为三级酶联反应模式：激活因子作用于MAPKKK使其首先被激活,MAPKKK被激活后又能使MAPKK磷酸化而被激活,产生MAPK并激活一系列其它蛋白激酶,使细胞骨架成分磷酸化,亦可经核转位进入细胞核激活核内转录因子,调节转录因子的靶基因,完成对细胞刺激的反应。细胞因子、生长因子、激素以及各种应激刺激都可以作为激活因子激活这个三级酶联反应。MAPK是MAPK途径的核心,其底物绝大部分是核内转录因子。MAPKs主要包括3个经典的亚家族途径,即细胞外调节激酶1/2(extracellular signal-regulated kinase 1/2, ERK1/2), p38 MAPK (p38 mitogen-activated protein kinase, p38 MAPK)和c-Jun氨基末端激酶(c-Jun amino-terminal kinases, JNK)。p38是MAPK的亚类之一,主要被炎性细胞因子和环境应激刺激而激活。p38的激活和炎症因子TNF-α、IL-1、IL-6的产生关系密切,并在角质形成细胞的增生和分化中发挥重要作用。已有研究表明p38MAPK途径在银屑病的病理过程中发挥重要作用。有报道TGP可通过激活p38MAPK,调节前炎症介质TNF-α、IL-1的产生而发挥抗炎作用,表明TGP的治疗作用可能通过调节信号通路途径。因此,为了进一步探讨TGP对KC作用的机制,我们选择了p38MAPK信号途径来研究TGP作用的可能机制。
目前,TGP对KC的具体作用还不明确,因此本研究拟在体外培养的人表皮角质形成细胞系HaCaT细胞中,以主要由KC分泌的VEGF和IL-23为检测指标,观察TGP对体外培养的角质形成细胞的增殖、VEGF和IL-23表达和分泌的影响,及其信号通路的调控,探讨TGP治疗银屑病的可能机制。
目的
1.观察TGP对HaCaT细胞增殖的影响；
2.观察TGP对HaCaT细胞表达VEGF和IL-23的影响；
3.探讨P38MAPKs信号传导途径在TGP对HaCaT细胞表达VEGF和IL-23调控中的作用。
方法
1. HaCaT细胞的培养
永生化的角质形成细胞株HaCaT细胞用含10%FBS的1640置于37℃、5%CO2培养箱中进行培养。
2.实验设计
(1)根据参考文献选择不同浓度的TGP加入HaCaT细胞,测定HaCaT细胞增殖的改变；
(2)不同浓度TGP孵育HaCaT细胞48h后,测定VEGF和IL-23 mRNA及蛋白的表达；
(3) HaCaT细胞经p38 MAPK阻断剂SB203580预处理2h后,给予125mg/LTGP孵育,测定HaCaT细胞VEGF和IL-23 mRNA及蛋白的表达；
(4) 125mg/LTGP孵育HaCaT细胞0、5、15、30 min,检测p38MAPK磷酸化的改变；
(5) HaCaT细胞经p38 MAPK阻断剂SB203580预处理2 h后,给予TGP孵育,测定p38 MAPK磷酸化改变。
3.实验方法
3.1.甲基噻唑基四唑(Methyl thiazolyl tetrazolium, MTT)法测定HaCaT细胞增殖将HaCaT细胞分组给予不同浓度TGP干预,加入5g/L的MTT液20μl继续培养4h,再加入二甲基亚砜150μl,使结晶物充分溶解,在酶标仪490nm处测量各孔吸光值。
3.2.实时荧光定量聚合酶链式反应(Real-Time RT-PCR)检测VEGF和IL-23 mRNA的表达
将所收集的细胞提取总RNA,经逆转录反应得到cDNA,以管家基因GAPDH作为参照,通过real-time RT-PCR技术检测VEGF和IL-23 mRNA的表达。
3.3.双抗体夹心ELISA法检测VEGF和IL-23蛋白表达
将所收集的细胞培养上清进行孵育、酶反应、显色等步骤,酶标仪450nm处测量各孔吸光值,根据标准曲线计算出相应浓度。
3. 4. Western blot检测p38 MAPK蛋白表达
将所收集的细胞提取总蛋白,经过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、蛋白印迹、显色、凝胶成像分析系统成像、应用Qwin图像分析软件进行半定量分析,检测p38 MAPK蛋白的表达。
结果
1.TGP对HaCaT细胞增殖的影响
根据预实验结果和细胞生长曲线,统计分析显示：TGP在低浓度0.5mg/L、2.5 mg/L时对HaCaT细胞的增殖有促进作用(P<0.01)；浓度增高为12.5mg/L～125 mg/L时反而对细胞的增殖有抑制作用(P<0.01),且随TGP浓度的增加抑制作用愈明显,与对照组相比,差异有统计学意义。TGP 125 mg/L时抑制作用最明显。
2.TGP对HaCaT细胞表达和分泌VEGF的影响
(1) 0.5mg/LTGP、2.5 mg/LTGP作用于HaCaT细胞48h, VEGF的分泌量和VEGFmRNA的表达量均高于对照组(P<0.05)。12.5 mg/LTGP、62.5mg／LTGP.125 mg／LTGP作用于HaCaT细胞48h,随药物浓度的增加,VEGF的分泌量和VEGFmRNA的表达量均低于对照组(P<0.05)。其中TGP浓度为125 mg/L时VEGF分泌量和VEGFmRNA的表达量最低。
(2)p38 MAPK阻断剂SB203580对TGP诱导的HaCaT细胞VEGF mRNA表达和VEGF蛋白分泌的影响p38MAPK抑制剂SB203580预处理2 h后再给予125 mg／LTGP作用48h,HaCaT细胞分泌VEGF的量和VEGFmRNA的表达量均较单独125 mg/LTGP作用时明显增高(P<0.01)。
3.TGP对HaCaT细胞表达和分泌IL-23的影响
(1)0.5mg/LTGP.2.5mg/LTGP作用于HaCaT细胞48h,IL-23的分泌量和IL一23mRNA的表达量均高于对照组(P<0.05).62.5mg/LTGP. 125mg/LTGP作用于HaCaT细胞48h,随药物浓度的增加,IL-23的分泌量和IL-23mRNA的表达量均低于对照组(P<0.05)。其中TGP浓度为125mg/L时IL-23分泌量和IL-23mRNA的表达量最低。TGP浓度为12.5mg/L时IL-23分泌量和IL-23mRNA的表达量与对照组相比没有统计学意义(P>0.05)。
(2)p38 MAPK阻断剂SB203580对TGP诱导的HaCaT细胞IL-23mRNA的表达和IL-23蛋白分泌的影响p38MAPK抑制剂SB203580预处理2 h后再给予125mg/LTGP作用48h,HaCaT细胞分泌IL-23的量和IL-23mRNA的表达量均较单独125mg/LTGP作用时明显增高(P<0.01)。4.TGP对HaCaT细胞p38磷酸化的影响4.1 TGP对HaCaT细胞p38 MAPK磷酸化的影响125mg/LTGP作用于孵育的HaCaT细胞,在不同时间段(5min.10min. 30min)收集细胞,p-p38蛋白表达于5min达到高峰,10min后p-p38表达水平逐渐减弱,但与对照组表达水平比较差别仍有统计学意义(P<0.01)；T-p38蛋白表达与对照组比较差异无统计学意义(P>0.05)。4.2 p38 MAPK阻断剂SB203580对TGP诱导HaCaT细胞p38 MAPK磷酸化的影响
HaCaT细胞经p38MAPK阻断剂SB203580(10μM)预处理2h后,再给予125mg/LTGP孵育5min,结果经SB203580预处理组p-p38表达水平明显低于单独TGP给药组,两组相比有统计学意义(P<0.01)。
结论
1.TGP能够抑制HaCaT细胞的增殖；
2.TGP能够抑制HaCaT细胞VEGF和IL-23 mRNA和蛋白的表达；
3.TGP能够活化HaCaT细胞p38 MAPK信号途径；
4. p38 MAPK信号途径参与了TGP对HaCaT细胞VEGF、IL-23 mRNA和蛋白表达的抑制作用。
Background
Psoriasis is a common, chronic, inflammatory disease. The cause and pathogenesis of psoriasis is still unknown. The disease is characterized by keratinocyte hyperproliferation, vascular hyperplasia and infiltration of T lymphocytes, neutrophils, and other types of leucocytes in affected skin. It is generally believed to be a complex autoimmune inflammatory disease with a genetic basis. Cytokine production by keratinocytes has multiple consequences for the migration of inflammatory cells,may have systemic effects on the immune system,influences keratinocyte proliferation and differentiation processes,and finally affects the production of other cytokines by keratinocytes.
VEGF is a crucial regulator of angiogenesis and vascular permeability in both physiological and pathological conditions such as tumor growth and chronic inflammation. VEGF is expressed and secreted by epidermal keratinocytes in normal human skin. Keratinocytes overexpress VEGF in clinically involved and uninvolved skin of patients with chronic plaque psoriasis. In transgenic mice with epidermis-specific overexpression of VEGF and enhanced skin vascularity and vascular permeability, chronic transgenic delivery of VEGF to the skin induced inflammation and all characteristics of psoriasis spontaneously, and the VEGF antagonist reversed the phenotype. These findings indicate VEGF might play an important role in the pathogenesis of psoriasis. IL-23 is produced by dendritic cells, other antigen-presenting cells and keracinocytes. IL-23 is clearly elevated in psoriasis lesions.Importantly, IL-23 levels decrease with clinical improvement of psoriasis following effective treatment, providing a direct correlation between overproduction of IL-23 and active psoriasis.In other mouse studies, recombinant IL-23 injected into normal-appearing skin produced erythematous,thick, scaly skin, with histologic features reminiscent of psoriasis.
Paeonia lactiflora Pall is a kind of Chinese traditional herbal medicine. Total glucosides of paeony (TGP) consists of more than 90% paeoniflorin and other components such as hydroxypaeoniflorin, paeonin, albiflorin,benzoylpaeoniflorin,etc. Previous studies have demonstrated that TGP exerts anti-inflammatory,analgesic effects in the rat model of AA and carrageenan-induced arthritis. However, the mechanism of TGP on psoriasis remains unclear and need further investigation.
The mitogen-activated protein kinase (MAPK) pathways are the best characteriz-ed of intracellular protein kinase cascades. MAPK plays a role in signal transduction associated with cell proliferation, differentiation and the production of cytokines. There are three well-characterized MAPK subfamilies in mammalian cells:extra cellular-signal-regulated protein kinase 1/2 (ERK1/2), the p38 mitogen-activated protein kinases (p38 MAPK) and the c-Jun N-terminal kinase (JNK). A strong link has been established between the p38 pathway and inflammation.The activation of the p38 pathway plays essential roles in the production of proinflammatory cytokines TNF-aand IL-1.However, little is known about the exact role of TGP in HaCaT cells. We hypothesized that TGP may modulate VEGF and IL-23 expression. We examined whether this effect functioned via the mitogen-activated protein kinases (MAPKs) signal transduction pathway, particularly p38 mitogen-activated protein kinase (p38 MAPK).
In light of this, we performed a study to investigate the effect of total glucosides of paeony(TGP) on cell-proliferation and secretion of inflammatory cytokines VEGF and IL-23 in human HaCaT keratinocytes and whether p38 pathway is involved in this progress.
Objective
1. Investigate the effect of TGP on the proliferation of HaCaT cells;
2.Investigate the effect of CRH on the expression of VEGF and IL-23 on HaCaT cells;
3. Examine whether the effect of TGP on the expression of VEGF and IL-23 functioned via MAPKs signal transduction pathway.
Materials and methods
1. Cell culture
The immortalized human HaCaT keratinocytes were maintained at 37℃and 5% carbon dioxide (CO2) in 1640 supplemented with 10% heat-inactivated fetal bovine serum,100 U/ml penicillin and 100μg/ml streptomycin.
2. Cell pretreatment
HaCaT cells were seeded at density 4×104 cells/ml, grown for 48 h until 70% confluence. The cells were pretreated with 10μM SB203580 and incubated for 2 h before application of TGP.
3. Methods
3.1. MTT assay for cell proliferation
HaCaT cells were incubated with TGPof different concentrations respectively, and then followed by MTT assay.
3.2 Real-time RT-PCR
After the experimental treatment, the total RNA was extracted from HaCaT cells. The reverse transcription of RNA to cDNA was performed. cDNA were amplified with real-time RT-PCR. VEGF mRNA and IL-23 mRNA expression were normalized to the expressed housekeeping gene human GAPDH. Samples were tested in triplicate and the average values were used for quantification.
3.3. ELISA
After stimulation for 48 h, culture supernatants of cells were collected, centrifuged (15 000 rpm,5 min) and stored at -80℃until analysis. The concentrations of VEGF and IL-23 in the culture supernatant were measured by commercially available enzyme linked immunosorbent assay (ELISA) kits according to manufacturer's instructions. Each supernatant was analyzed in triplicate.
3.4. Western blot
The phosphorylation of p38 MAPK was detected by western blot analysis, SB203580 was used to block p38MAPKs pathway. Densitometric analysis of the band intensity was carried out using Qwin software.
Results
1. TGP inhibited HaCaT proliferation
TGP increased HaCaT proliferation in a lower concentration and TGP 12.5mg/L～125mg/L inhibited keratinocytes proliferation distinctly.
2. Effect of TGP on VEGF production in HaCaT cells
TGP increased the expression of VEGF mRNA and protein in a lower concentration and TGP inhibited the expression of VEGF mRNA and protein in a higher concentration.
3. Effect of TGP on IL-23 production in HaCaT cells
TGP increased the expression of IL-23 mRNA and protein in a lower concentration and TGP inhibited the expression of IL-23 mRNA and protein in a higher concentration
4. The role of MAPKs pathway in the effect of TGP in HaCaT cells
4.1 TGP activated p38 MAPK phosphorylation in HaCaT cells
TGP induced a rapid phosphorylation of p38 MAPK in a time dependent manner and with a peak at 5 min.
4.2. The effect of p38 MAPK inhibitor SB203580 on phosphorylation of p38 MAPK in HaCaT cells
Pretreating HaCaT cells with the p38 MAPK inhibitor SB203580 inhibited the TGP-induced phosphorylation of p38 MAPK. These data indicated that TGP activated p38 MAPK phosphorylation in HaCaT cells.
Conclusions
1. TGP inhibited keratinocytes proliferation;
2. VEGF and IL-23 mRNA expression and protein production were downregulated by TGP;
3. TGP induced a rapid phosphorylation of p38 MAPK
4. TGP downregulated VEGF、IL-23 expression in HaCaT cells by p38 MAPK pathways.
银屑病是一种常见的由多基因遗传决定的、多环境因素刺激诱导的慢性炎症性增殖性皮肤病,其病因和发病机制至今尚未完全阐明。目前银屑病发病的始动触发因素仍然不清楚,其发病机制涉及到免疫系统紊乱、血管新生和角质形成细胞增生等多个环节。其组织病理学最主要的特征是角质形成细胞(keratinocyte, KC)过度增生、炎症细胞浸润、新生血管形成。在银屑病的组织病理变化中真皮乳头血管的异常最先发生,而后出现炎症细胞的移行和表皮增生及分化异常。同时,大量的免疫活性细胞及其分泌的细胞因子渗出到血管外的组织中,诱导角质形成细胞活化、增殖并分泌大量细胞因子,由此形成细胞因子相互调控的恶性循环,促进疾病的进展。
在银屑病病理过程中,由表皮细胞分泌的很多血管源性细胞因子参与了血管增生,但以血管内皮生长因子(vascular endothelial growth factor, VEGF)最为重要。银屑病皮损中的KC是促进血管新生细胞因子的主要来源。研究表明,VEGF mRNA及蛋白表达水平在银屑病患者皮损中明显增加,患者非皮损处VEGFmRNA及蛋白水平表达亦增加,许多研究又表明严重类型的银屑病患者血清中VEGF水平也显著升高,且与疾病的活动有一定相关性。另有报道将编码VEGF的基因转入小鼠皮肤可致银屑病样改变,并出现特征性的Koebner现象,这进一步说明VEGF在银屑病发病过程中起到重要作用。同时,银屑病也是多种炎细胞共同参与的免疫性疾病。IL-23是新近发现的一种细胞因子,是由本身无生物活性的p19因子与IL-12的p40亚基通过二硫键相连组成的异二聚体。能够诱导记忆性T细胞产生IFN-γ,具有较强的抗感染免疫保护功能和抗肿瘤活性。研究表明银屑病病人皮损IL-23的表达明显增高。Piskin等发现UVB治疗银屑病后,临床症状得到改善,局部皮损IL-23的表达也下调。因此他们推测,角质形成细胞过表达IL-23可能对银屑病的炎性过程起一定作用。
白芍总苷胶囊(商品名帕夫林,total glucosides of paeony, TGP)是从中药白芍中提取的复合制剂,主要成分包括芍药苷、芍药内酯苷、羟基芍药苷、苯甲酰芍药苷等,其中芍药苷占总苷量的90%,是白芍的主要有效成分。其药理作用主要有抗炎、调节免疫功能、影响细胞增殖、止痛、保肝等作用。TGP对自身免疫过程中的多个环节都存在着调节作用。研究报道TGP可以抑制VEGF、金属基质蛋白酶(MMP)等活性物质的产生。目前国内对TGP治疗类风湿性关节炎、儿童特发性关节炎等自身免疫性疾病的研究较多,但对TGP治疗银屑病的机制尚不清楚。
促分裂原活化蛋白激酶(mitogen-activated protein kinases, MAPK)信号转导途径是哺乳动物细胞中重要的信号通路,能将多种细胞外刺激产生的信号通过级联反应从细胞膜传递到细胞核内,在细胞分化、增殖、凋亡、应激、炎症以及免疫反应等多种生理和病理过程中发挥着极其重要的作用。其任一环节的异常都可能导致细胞的异常增殖与分化。MAPK信号转导通路包括MAPK激酶激酶(MAPK kinase kinase, MAPKKK)、MAPK激酶(MAPK kinase, MAPKK). MAPK,此通路主要为三级酶联反应模式：激活因子作用于MAPKKK使其首先被激活,MAPKKK被激活后又能使MAPKK磷酸化而被激活,产生MAPK并激活一系列其它蛋白激酶,使细胞骨架成分磷酸化,亦可经核转位进入细胞核激活核内转录因子,调节转录因子的靶基因,完成对细胞刺激的反应。细胞因子、生长因子、激素以及各种应激刺激都可以作为激活因子激活这个三级酶联反应。MAPK是MAPK途径的核心,其底物绝大部分是核内转录因子。MAPKs主要包括3个经典的亚家族途径,即细胞外调节激酶1/2(extracellular signal-regulated kinase 1/2, ERK1/2), p38 MAPK (p38 mitogen-activated protein kinase, p38 MAPK)和c-Jun氨基末端激酶(c-Jun amino-terminal kinases, JNK)。p38是MAPK的亚类之一,主要被炎性细胞因子和环境应激刺激而激活。p38的激活和炎症因子TNF-α、IL-1、IL-6的产生关系密切,并在角质形成细胞的增生和分化中发挥重要作用。已有研究表明p38MAPK途径在银屑病的病理过程中发挥重要作用。有报道TGP可通过激活p38MAPK,调节前炎症介质TNF-α、IL-1的产生而发挥抗炎作用,表明TGP的治疗作用可能通过调节信号通路途径。因此,为了进一步探讨TGP对KC作用的机制,我们选择了p38MAPK信号途径来研究TGP作用的可能机制。
目前,TGP对KC的具体作用还不明确,因此本研究拟在体外培养的人表皮角质形成细胞系HaCaT细胞中,以主要由KC分泌的VEGF和IL-23为检测指标,观察TGP对体外培养的角质形成细胞的增殖、VEGF和IL-23表达和分泌的影响,及其信号通路的调控,探讨TGP治疗银屑病的可能机制。
目的
1.观察TGP对HaCaT细胞增殖的影响；
2.观察TGP对HaCaT细胞表达VEGF和IL-23的影响；
3.探讨P38MAPKs信号传导途径在TGP对HaCaT细胞表达VEGF和IL-23调控中的作用。
方法
1. HaCaT细胞的培养
永生化的角质形成细胞株HaCaT细胞用含10%FBS的1640置于37℃、5%CO2培养箱中进行培养。
2.实验设计
(1)根据参考文献选择不同浓度的TGP加入HaCaT细胞,测定HaCaT细胞增殖的改变；
(2)不同浓度TGP孵育HaCaT细胞48h后,测定VEGF和IL-23 mRNA及蛋白的表达；
(3) HaCaT细胞经p38 MAPK阻断剂SB203580预处理2h后,给予125mg/LTGP孵育,测定HaCaT细胞VEGF和IL-23 mRNA及蛋白的表达；
(4) 125mg/LTGP孵育HaCaT细胞0、5、15、30 min,检测p38MAPK磷酸化的改变；
(5) HaCaT细胞经p38 MAPK阻断剂SB203580预处理2 h后,给予TGP孵育,测定p38 MAPK磷酸化改变。
3.实验方法
3.1.甲基噻唑基四唑(Methyl thiazolyl tetrazolium, MTT)法测定HaCaT细胞增殖将HaCaT细胞分组给予不同浓度TGP干预,加入5g/L的MTT液20μl继续培养4h,再加入二甲基亚砜150μl,使结晶物充分溶解,在酶标仪490nm处测量各孔吸光值。
3.2.实时荧光定量聚合酶链式反应(Real-Time RT-PCR)检测VEGF和IL-23 mRNA的表达
将所收集的细胞提取总RNA,经逆转录反应得到cDNA,以管家基因GAPDH作为参照,通过real-time RT-PCR技术检测VEGF和IL-23 mRNA的表达。
3.3.双抗体夹心ELISA法检测VEGF和IL-23蛋白表达
将所收集的细胞培养上清进行孵育、酶反应、显色等步骤,酶标仪450nm处测量各孔吸光值,根据标准曲线计算出相应浓度。
3. 4. Western blot检测p38 MAPK蛋白表达
将所收集的细胞提取总蛋白,经过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、蛋白印迹、显色、凝胶成像分析系统成像、应用Qwin图像分析软件进行半定量分析,检测p38 MAPK蛋白的表达。
结果
1.TGP对HaCaT细胞增殖的影响
根据预实验结果和细胞生长曲线,统计分析显示：TGP在低浓度0.5mg/L、2.5 mg/L时对HaCaT细胞的增殖有促进作用(P<0.01)；浓度增高为12.5mg/L～125 mg/L时反而对细胞的增殖有抑制作用(P<0.01),且随TGP浓度的增加抑制作用愈明显,与对照组相比,差异有统计学意义。TGP 125 mg/L时抑制作用最明显。
2.TGP对HaCaT细胞表达和分泌VEGF的影响
(1) 0.5mg/LTGP、2.5 mg/LTGP作用于HaCaT细胞48h, VEGF的分泌量和VEGFmRNA的表达量均高于对照组(P<0.05)。12.5 mg/LTGP、62.5mg／LTGP.125 mg／LTGP作用于HaCaT细胞48h,随药物浓度的增加,VEGF的分泌量和VEGFmRNA的表达量均低于对照组(P<0.05)。其中TGP浓度为125 mg/L时VEGF分泌量和VEGFmRNA的表达量最低。
(2)p38 MAPK阻断剂SB203580对TGP诱导的HaCaT细胞VEGF mRNA表达和VEGF蛋白分泌的影响p38MAPK抑制剂SB203580预处理2 h后再给予125 mg／LTGP作用48h,HaCaT细胞分泌VEGF的量和VEGFmRNA的表达量均较单独125 mg/LTGP作用时明显增高(P<0.01)。
3.TGP对HaCaT细胞表达和分泌IL-23的影响
(1)0.5mg/LTGP.2.5mg/LTGP作用于HaCaT细胞48h,IL-23的分泌量和IL一23mRNA的表达量均高于对照组(P<0.05).62.5mg/LTGP. 125mg/LTGP作用于HaCaT细胞48h,随药物浓度的增加,IL-23的分泌量和IL-23mRNA的表达量均低于对照组(P<0.05)。其中TGP浓度为125mg/L时IL-23分泌量和IL-23mRNA的表达量最低。TGP浓度为12.5mg/L时IL-23分泌量和IL-23mRNA的表达量与对照组相比没有统计学意义(P>0.05)。
(2)p38 MAPK阻断剂SB203580对TGP诱导的HaCaT细胞IL-23mRNA的表达和IL-23蛋白分泌的影响p38MAPK抑制剂SB203580预处理2 h后再给予125mg/LTGP作用48h,HaCaT细胞分泌IL-23的量和IL-23mRNA的表达量均较单独125mg/LTGP作用时明显增高(P<0.01)。4.TGP对HaCaT细胞p38磷酸化的影响4.1 TGP对HaCaT细胞p38 MAPK磷酸化的影响125mg/LTGP作用于孵育的HaCaT细胞,在不同时间段(5min.10min. 30min)收集细胞,p-p38蛋白表达于5min达到高峰,10min后p-p38表达水平逐渐减弱,但与对照组表达水平比较差别仍有统计学意义(P<0.01)；T-p38蛋白表达与对照组比较差异无统计学意义(P>0.05)。4.2 p38 MAPK阻断剂SB203580对TGP诱导HaCaT细胞p38 MAPK磷酸化的影响
HaCaT细胞经p38MAPK阻断剂SB203580(10μM)预处理2h后,再给予125mg/LTGP孵育5min,结果经SB203580预处理组p-p38表达水平明显低于单独TGP给药组,两组相比有统计学意义(P<0.01)。
结论
1.TGP能够抑制HaCaT细胞的增殖；
2.TGP能够抑制HaCaT细胞VEGF和IL-23 mRNA和蛋白的表达；
3.TGP能够活化HaCaT细胞p38 MAPK信号途径；
4. p38 MAPK信号途径参与了TGP对HaCaT细胞VEGF、IL-23 mRNA和蛋白表达的抑制作用。
Background
Psoriasis is a common, chronic, inflammatory disease. The cause and pathogenesis of psoriasis is still unknown. The disease is characterized by keratinocyte hyperproliferation, vascular hyperplasia and infiltration of T lymphocytes, neutrophils, and other types of leucocytes in affected skin. It is generally believed to be a complex autoimmune inflammatory disease with a genetic basis. Cytokine production by keratinocytes has multiple consequences for the migration of inflammatory cells,may have systemic effects on the immune system,influences keratinocyte proliferation and differentiation processes,and finally affects the production of other cytokines by keratinocytes.
VEGF is a crucial regulator of angiogenesis and vascular permeability in both physiological and pathological conditions such as tumor growth and chronic inflammation. VEGF is expressed and secreted by epidermal keratinocytes in normal human skin. Keratinocytes overexpress VEGF in clinically involved and uninvolved skin of patients with chronic plaque psoriasis. In transgenic mice with epidermis-specific overexpression of VEGF and enhanced skin vascularity and vascular permeability, chronic transgenic delivery of VEGF to the skin induced inflammation and all characteristics of psoriasis spontaneously, and the VEGF antagonist reversed the phenotype. These findings indicate VEGF might play an important role in the pathogenesis of psoriasis. IL-23 is produced by dendritic cells, other antigen-presenting cells and keracinocytes. IL-23 is clearly elevated in psoriasis lesions.Importantly, IL-23 levels decrease with clinical improvement of psoriasis following effective treatment, providing a direct correlation between overproduction of IL-23 and active psoriasis.In other mouse studies, recombinant IL-23 injected into normal-appearing skin produced erythematous,thick, scaly skin, with histologic features reminiscent of psoriasis.
Paeonia lactiflora Pall is a kind of Chinese traditional herbal medicine. Total glucosides of paeony (TGP) consists of more than 90% paeoniflorin and other components such as hydroxypaeoniflorin, paeonin, albiflorin,benzoylpaeoniflorin,etc. Previous studies have demonstrated that TGP exerts anti-inflammatory,analgesic effects in the rat model of AA and carrageenan-induced arthritis. However, the mechanism of TGP on psoriasis remains unclear and need further investigation.
The mitogen-activated protein kinase (MAPK) pathways are the best characteriz-ed of intracellular protein kinase cascades. MAPK plays a role in signal transduction associated with cell proliferation, differentiation and the production of cytokines. There are three well-characterized MAPK subfamilies in mammalian cells:extra cellular-signal-regulated protein kinase 1/2 (ERK1/2), the p38 mitogen-activated protein kinases (p38 MAPK) and the c-Jun N-terminal kinase (JNK). A strong link has been established between the p38 pathway and inflammation.The activation of the p38 pathway plays essential roles in the production of proinflammatory cytokines TNF-aand IL-1.However, little is known about the exact role of TGP in HaCaT cells. We hypothesized that TGP may modulate VEGF and IL-23 expression. We examined whether this effect functioned via the mitogen-activated protein kinases (MAPKs) signal transduction pathway, particularly p38 mitogen-activated protein kinase (p38 MAPK).
In light of this, we performed a study to investigate the effect of total glucosides of paeony(TGP) on cell-proliferation and secretion of inflammatory cytokines VEGF and IL-23 in human HaCaT keratinocytes and whether p38 pathway is involved in this progress.
Objective
1. Investigate the effect of TGP on the proliferation of HaCaT cells;
2.Investigate the effect of CRH on the expression of VEGF and IL-23 on HaCaT cells;
3. Examine whether the effect of TGP on the expression of VEGF and IL-23 functioned via MAPKs signal transduction pathway.
Materials and methods
1. Cell culture
The immortalized human HaCaT keratinocytes were maintained at 37℃and 5% carbon dioxide (CO2) in 1640 supplemented with 10% heat-inactivated fetal bovine serum,100 U/ml penicillin and 100μg/ml streptomycin.
2. Cell pretreatment
HaCaT cells were seeded at density 4×104 cells/ml, grown for 48 h until 70% confluence. The cells were pretreated with 10μM SB203580 and incubated for 2 h before application of TGP.
3. Methods
3.1. MTT assay for cell proliferation
HaCaT cells were incubated with TGPof different concentrations respectively, and then followed by MTT assay.
3.2 Real-time RT-PCR
After the experimental treatment, the total RNA was extracted from HaCaT cells. The reverse transcription of RNA to cDNA was performed. cDNA were amplified with real-time RT-PCR. VEGF mRNA and IL-23 mRNA expression were normalized to the expressed housekeeping gene human GAPDH. Samples were tested in triplicate and the average values were used for quantification.
3.3. ELISA
After stimulation for 48 h, culture supernatants of cells were collected, centrifuged (15 000 rpm,5 min) and stored at -80℃until analysis. The concentrations of VEGF and IL-23 in the culture supernatant were measured by commercially available enzyme linked immunosorbent assay (ELISA) kits according to manufacturer's instructions. Each supernatant was analyzed in triplicate.
3.4. Western blot
The phosphorylation of p38 MAPK was detected by western blot analysis, SB203580 was used to block p38MAPKs pathway. Densitometric analysis of the band intensity was carried out using Qwin software.
Results
1. TGP inhibited HaCaT proliferation
TGP increased HaCaT proliferation in a lower concentration and TGP 12.5mg/L～125mg/L inhibited keratinocytes proliferation distinctly.
2. Effect of TGP on VEGF production in HaCaT cells
TGP increased the expression of VEGF mRNA and protein in a lower concentration and TGP inhibited the expression of VEGF mRNA and protein in a higher concentration.
3. Effect of TGP on IL-23 production in HaCaT cells
TGP increased the expression of IL-23 mRNA and protein in a lower concentration and TGP inhibited the expression of IL-23 mRNA and protein in a higher concentration
4. The role of MAPKs pathway in the effect of TGP in HaCaT cells
4.1 TGP activated p38 MAPK phosphorylation in HaCaT cells
TGP induced a rapid phosphorylation of p38 MAPK in a time dependent manner and with a peak at 5 min.
4.2. The effect of p38 MAPK inhibitor SB203580 on phosphorylation of p38 MAPK in HaCaT cells
Pretreating HaCaT cells with the p38 MAPK inhibitor SB203580 inhibited the TGP-induced phosphorylation of p38 MAPK. These data indicated that TGP activated p38 MAPK phosphorylation in HaCaT cells.
Conclusions
1. TGP inhibited keratinocytes proliferation;
2. VEGF and IL-23 mRNA expression and protein production were downregulated by TGP;
3. TGP induced a rapid phosphorylation of p38 MAPK
4. TGP downregulated VEGF、IL-23 expression in HaCaT cells by p38 MAPK pathways.
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31.陈刚,邓小红,郭莉霞等.白芍总苷对巨噬细胞核转录因子KB活化的影响及其机制研究.中国中药杂志.2008；3(33)：669-671.
32.王玉英,熊小利,龙剑文.白芍总苷对静止期银屑病患者血清TNF-a及IL-8的影响.现代中西医结合杂志.2009；18(18)：2111-2112.
33. Fusenig NE, Boukamp P. Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes. Mol Carcinog. Nov 1998;23(3):144-158.
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35. Kwon YW, Kwon KS, Moon HE, et al. Insulin-like growth factor-II regulates the expression of vascular endothelial growth factor by the human keratinocyte cell line HaCaT. J Invest Dermatol. Jul 2004;123(1):152-158.
36. Mildner M, Weninger W, Trautinger F, Ban J, Tschachler E. UVA and UVB radiation differentially regulate vascular endothelial growth factor expression in keratinocyte-derived cell lines and in human keratinocytes. Photochem Photobiol. Oct 1999;70(4):674-679.
37. Bonnekoh B, Wevers A, Geisel J, Rasokat H, Mahrle G. Antiproliferative potential of zidovudine in human keratinocyte cultures. J Am Acad Dermatol. Sep 1991;25(3):483-490.
38. Stein M, Bernd A, Ramirez-Bosca A, Kippenberger S, Holzmann H. Measurement of anti-inflammatory effects of glucocorticoids on human keratinocytes in vitro. Comparison of normal human keratinocytes with the keratinocyte cell line HaCaT. Arzneimittelforschung. Nov 1997; 47(11):1266-1270.
39. Muller K, Prinz H. Antipsoriatic anthrones with modulated redox properties.4. Synthesis and biological activity of novel 9,10-dihydro-1, 8-dihydroxy-9-oxo-2-anthracenecarboxylic and hydroxamic acids.J Med Chem.1997;40(17):2780-2787.
40.王兴旺,陈敏珠,徐叔云.白芍总甙对T淋巴细胞亚群的作用.中国药理学通报.1992；8(5)：340.
41.王兴旺,陈敏珠,徐叔云.白芍总甙对免疫系统的影响.中国病理生理杂志.1991；7(6)：609-611.
42. Wu H,Li W,Wang T,et al.Paeoniflorin suppress NFkappaB activation through modulation of IkappaBalphaandenhances5 fluorouracil induced apoptosis inhuman gastric carcinoma cells. BiomedPharmacother. 2008;62(9):659-666.
43. Mao QQ,Ip SP,Ko KM,etal.Paeony glycosides protect against cortico sterone induced neurotoxicity in PC12cells.Cell Mol Neurobiol.2009; 29(5):643-647.
44. Barker JN.The pathophysiology of psoriasis.Lancet. Jul 27 1991;338(8761):227-230.
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46. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW. The vascular endothelial growth factor family:identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol. Dec 1991;5(12):1806-1814.
47. Dvorak AM, FengD. The vesicuacuolar organelle (VVO).A new ndothelial cell permeability organelle J Histochem Cyt ochem.2001; 49:419-432.
48. Carmeliet P, Collen D. Molecular basis of angiogenesis. Role of VEGF and VE-cadherin. Ann N YAcad Sci. May 2000;902:249-262;discussion 262-244.
49. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW. The vascular endothelial growth factor family:identification of a fourth molecular species and characterization of alternative splicing of KHA.Mol Endocrinol.Dec 1991;5(12):1806-1814.
50. Man XY, Yang XH, Cai SQ, Yao YG, Zheng M. Immunolocalization and expression of vascular endothelial growth factor receptors (VEGFRs) and neuropilins (NRPs) on keratinocytes in human epidermis.Mol Med.Jul-Aug 2006; 12(7-8):127-136.
51. Ettehadi P, Greaves MW, Wallach D, Aderka D, Camp RD. Elevated tumour necrosis factor-alpha (TNF-alpha) biological activity in psoriatic skin lesions. Clin Exp Immunol.Apr 1994;96(1):146-151.
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56. Levy AP.A cellular paradigm for the failure to increase vascular endothelial growth factor in chronically hypoxic ststes.Coron Artery Dis.Sep 1999;10(6):427-430.
57. Lee S,French MA,Price P.IL-23 and IFN-gamma deficiency in immunodeficient HIV patients who achieved a long-term increase in CD-4 T-cell counts on highly active antiretroviral therapy. AIDS. 2004;18:1337-1340.
58. Oniki S,Nagai H,Horikawa T,et al. Interleukin-23 and interleukin-27 exert quite differente antitumor and vaccine effects on poorly immunogenic melanoma.Cancer Res.s.2006;66:6395-6404.
59. Chan JR,Blumenschein W,Murphy E,et al.IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2-dependent mechanisms with implications for psoriasis pathogenesis.J Exp Med.2006; 203 (12):2577-2587.
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29.朱蕾,魏伟,郑咏秋.白芍总苷对胶原性关节炎大鼠滑膜细胞的作用及机制.药学学报.2006,41(2)：166-170.
30.王斌,陈敏珠,徐叔云.白芍总甙对佐剂性关节炎大鼠滑膜细胞功能和脾细胞增殖反应的影响.中国药理学与毒理学杂志.1994；8(2)：129-131.
31.陈刚,邓小红,郭莉霞等.白芍总苷对巨噬细胞核转录因子KB活化的影响及其机制研究.中国中药杂志.2008；3(33)：669-671.
32.王玉英,熊小利,龙剑文.白芍总苷对静止期银屑病患者血清TNF-a及IL-8的影响.现代中西医结合杂志.2009；18(18)：2111-2112.
33. Fusenig NE, Boukamp P. Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes. Mol Carcinog. Nov 1998;23(3):144-158.
34. Park HJ, Kim HJ, Lee JH, et al. Corticotropin-releasing hormone (CRH) downregulates interleukin-18 expression in human HaCaT keratinocytes by activation of p38 mitogen-activated protein kinase (MAPK) pathway. J Invest Dermatol. Apr 2005;124(4):751-755.
35. Kwon YW, Kwon KS, Moon HE, et al. Insulin-like growth factor-II regulates the expression of vascular endothelial growth factor by the human keratinocyte cell line HaCaT. J Invest Dermatol. Jul 2004;123(1):152-158.
36. Mildner M, Weninger W, Trautinger F, Ban J, Tschachler E. UVA and UVB radiation differentially regulate vascular endothelial growth factor expression in keratinocyte-derived cell lines and in human keratinocytes. Photochem Photobiol. Oct 1999;70(4):674-679.
37. Bonnekoh B, Wevers A, Geisel J, Rasokat H, Mahrle G. Antiproliferative potential of zidovudine in human keratinocyte cultures. J Am Acad Dermatol. Sep 1991;25(3):483-490.
38. Stein M, Bernd A, Ramirez-Bosca A, Kippenberger S, Holzmann H. Measurement of anti-inflammatory effects of glucocorticoids on human keratinocytes in vitro. Comparison of normal human keratinocytes with the keratinocyte cell line HaCaT. Arzneimittelforschung. Nov 1997; 47(11):1266-1270.
39. Muller K, Prinz H. Antipsoriatic anthrones with modulated redox properties.4. Synthesis and biological activity of novel 9,10-dihydro-1, 8-dihydroxy-9-oxo-2-anthracenecarboxylic and hydroxamic acids.J Med Chem.1997;40(17):2780-2787.
40.王兴旺,陈敏珠,徐叔云.白芍总甙对T淋巴细胞亚群的作用.中国药理学通报.1992；8(5)：340.
41.王兴旺,陈敏珠,徐叔云.白芍总甙对免疫系统的影响.中国病理生理杂志.1991；7(6)：609-611.
42. Wu H,Li W,Wang T,et al.Paeoniflorin suppress NFkappaB activation through modulation of IkappaBalphaandenhances5 fluorouracil induced apoptosis inhuman gastric carcinoma cells. BiomedPharmacother. 2008;62(9):659-666.
43. Mao QQ,Ip SP,Ko KM,etal.Paeony glycosides protect against cortico sterone induced neurotoxicity in PC12cells.Cell Mol Neurobiol.2009; 29(5):643-647.
44. Barker JN.The pathophysiology of psoriasis.Lancet. Jul 27 1991;338(8761):227-230.
45. Diaz BV, Lenoir MC, Ladoux A, Frelin C, Demarchez M, Michel S. Regulation of vascular endothelial growth factor expression in human keratinocytes by retinoids. J Biol Chem. Jan 7 2000;275(1):642-650.
46. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW. The vascular endothelial growth factor family:identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol. Dec 1991;5(12):1806-1814.
47. Dvorak AM, FengD. The vesicuacuolar organelle (VVO).A new ndothelial cell permeability organelle J Histochem Cyt ochem.2001; 49:419-432.
48. Carmeliet P, Collen D. Molecular basis of angiogenesis. Role of VEGF and VE-cadherin. Ann N YAcad Sci. May 2000;902:249-262;discussion 262-244.
49. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW. The vascular endothelial growth factor family:identification of a fourth molecular species and characterization of alternative splicing of KHA.Mol Endocrinol.Dec 1991;5(12):1806-1814.
50. Man XY, Yang XH, Cai SQ, Yao YG, Zheng M. Immunolocalization and expression of vascular endothelial growth factor receptors (VEGFRs) and neuropilins (NRPs) on keratinocytes in human epidermis.Mol Med.Jul-Aug 2006; 12(7-8):127-136.
51. Ettehadi P, Greaves MW, Wallach D, Aderka D, Camp RD. Elevated tumour necrosis factor-alpha (TNF-alpha) biological activity in psoriatic skin lesions. Clin Exp Immunol.Apr 1994;96(1):146-151.
52. Bhushan M, McLaughlin B, Weiss JB, Griffiths CE. Levels of endothelial cell stimulating angiogenesis factor and vascular endothelial growth factor are elevated in psoriasis. Br J Dermatol. Dec 1999;141(6):1054-1060.
53. Creamer D, Allen MH, Groves RW, et al. Circulating vascular permeability factor/vascular endothelial growth factor in erythroderma. Lancet.Oct 19 1996;348 (9034):1101.
54. Creamer D, Allen M, Jaggar R, et al. Mediation of systemic vascular hyperpermeability in severe psoriasis by circulating vascular endothelial growth factor. Arch Dermatol.Jun 2002;138(6):791-796.
55. Nielsen HJ, Christensen IJ, Svendsen MN, et al. Elevated plasma levels of vascular endothelial growth factor and plasminogen activator inhibitor-1 decrease during improvement of psoriasis.Inflamm Res.Nov 2002;51(11):563-567.
56. Levy AP.A cellular paradigm for the failure to increase vascular endothelial growth factor in chronically hypoxic ststes.Coron Artery Dis.Sep 1999;10(6):427-430.
57. Lee S,French MA,Price P.IL-23 and IFN-gamma deficiency in immunodeficient HIV patients who achieved a long-term increase in CD-4 T-cell counts on highly active antiretroviral therapy. AIDS. 2004;18:1337-1340.
58. Oniki S,Nagai H,Horikawa T,et al. Interleukin-23 and interleukin-27 exert quite differente antitumor and vaccine effects on poorly immunogenic melanoma.Cancer Res.s.2006;66:6395-6404.
59. Chan JR,Blumenschein W,Murphy E,et al.IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2-dependent mechanisms with implications for psoriasis pathogenesis.J Exp Med.2006; 203 (12):2577-2587.
60. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK,JNK,and p38 protein kinases. Science. Dec 6 2002;298(5600):1911-1912.
61. Cano E, Mahadevan LC. Parallel signal processing among mammalian MAPKs. Trends Biochem Sci. Mar 1995;20(3):117-122.
62. Zhang X, Yang D, Ma S, Liu H. Up-regulation of activities of mitogen-activated protein kinase in psoriatic lesions.J Dermatol Sci.Feb 2005;37(2):118-119.
63. Johansen C, Kragballe K, Westergaard M, Henningsen J, Kristiansen K, Iversen L. The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin. Br J Dermatol. Jan 2005; 152(1):37-42.
64. Yu XJ, Li CY, Dai HY, et al. Expression and localization of the activated mitogen-activated protein kinase in lesional psoriatic skin. Exp Mol Pathol. Dec 2007;83(3):413-418.