倍半萜烯内酯类化合物对晚期氧化蛋白产物诱导足细胞炎症因子MCP-1表达的影响及其机制的研究

发布时间：2018-06-03 13:30

本文选题：倍半萜烯内酯类化合物 + 晚期氧化蛋白产物　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景与目的糖尿病肾病(DN)是糖尿病最常见的一种微血管并发症。DN的发病机制十分复杂,至今尚未完全明确。按传统的观点,DN并非是一种炎症性疾病,然而近年的研究表明炎症反应和DN关系密切,炎症反应在DN发生发展过程中起到了重要的促进作用。研究表明,单核细胞趋化蛋白(MCP)-1参与诱导巨噬细胞进入糖尿病肾脏,在DN动物模型中的表达进行性上升,并且其在肾小球和肾小管中的积累量分别与肾小球和肾小管的损伤成正相关。在DN中,MCP-1不仅发挥趋化作用,促进单核细胞和巨噬细胞游走和活化,同时也能通过上调黏附因子表达和促进其他炎症因子的表达直接引起肾脏的炎症反应和肾纤维化。晚期氧化蛋白产物(AOPPs)是一个新的致DN进展的炎症介质。DN患者体内AOPPs水平明显高于未出现肾脏损伤的糖尿病患者。在DN动物模型中,AOPPs的慢性积累显著增加了残余肾巨噬细胞的侵润和MCP-1的过度表达,促进了炎症反应。AOPPs可通过ROS依赖的p38MAPK通路的激活降低足细胞中的nephrin和podocin的表达,同时AOPPs也可诱导足细胞的凋亡,导致足细胞数目减少、密度降低和基底膜裸露,进而诱发了蛋白尿,加重肾病的进展。核因子κB (NF-κB)在调节炎症因子的过程中发挥着重要作用,AOPPs能激活NF-κB相关通路。我们之前的研究发现,DN大鼠中NF-κB表达显著增加；在体外培养的大鼠肾小球系膜细胞中,高糖可诱导系膜细胞增殖,激活NF-κB相关信号通路,诱导炎症因子MCP-1和转化生长因子-p1表达增加；AOPPs可以快速诱导系膜细胞内活性氧的产生,激活MAPKs/NF-κB信号转导通路,最终诱导系膜细胞中MCP-1mRNA和蛋白过度表达。倍半萜烯内酯(SLs)是一大类具有多样结构的天然生物活性化合物,最初是从菊科植物中分离所得,小白菊内酯(PTL)是SLs中最主要的生物活性成分。已有研究发现PTL和其他SLs能通过抑制IKK和IκBα磷酸化和/或影响NF-κB和DNA的结合能力发挥其抗炎作用。我们最新的研究结果表明,在体外培养的系膜细胞中,SLs,包括PTL, MCL和及小白菊内酯的衍生物-化合物2,以及其他相关的小白菊内酯衍生物,能有效地抑制高糖及AOPPs诱导的IκBα的降解和NF-κB的活化,进而抑制系膜细胞中炎症因子MCP-1mRNA和蛋白的表达。我们推钡AOPPs能通过激活NF-κB相关的信号通路来诱导足细胞中炎症因子MCP-1的过度表达；倍半萜烯内酯类化合物可通过抑制NF-κB相关的信号转导途径的激活来发挥其抗炎作用,具有保护DN肾脏的作用。本实验以体外培养的足细胞为对象,旨在验证上述推测,为将来应用SLs治疗DN及相关炎症性肾病的深化研究提供一定的理论依据。研究内容与方法 1.体外制备AOPPs 将20mg/ml MSA与40mmo/1HC10等体积混合,其摩尔比为1：140,室温放置30分钟后,于无内毒素PBS中透析24小时,以除去游离的HCLO用0.22μmm的微孔滤膜过滤除菌后4℃保存。20mg/ml MSA与PBS等体积混合,作为对照。AOPPs含量通过测定酸性条件下340nm的吸光度,以氯胺T为标准取得。内毒素含量用鲎试验法检测,该方法制备的AOPPs内毒素含量均低于0.25EU/ml。 2.足细胞的培养按Peter Mundel的方法。足细胞于33℃含5%C02培养箱,,在10%FBS、50U/ml IFN-y的RPM11640培养液中增殖。为了得到分化表型,足细胞于37℃含5%C02培养箱,在10%FBS的RPM11640培养液中培养10-14天。待细胞分化后,使用无血清1640培养液培养24小时。根据不同的实验目的分组,加用各种试剂和药物刺激。本研究使用10-20代足细胞。一、AOPPs对足细胞炎症因子MCP-1表达的影响 1. qPCR法检测足细胞MCP-1mRNA的表达。细胞同步于GO期后,0、50、100.200(μg/ml)的AOPPs和200(μg/ml)未经修饰的MSA刺激细胞24h；或者200μg/ml的AOPPs刺激细胞0、3、6、12、24、48h。提取细胞中总RNA。qPCR法检测足细胞MCP-1mRNA的表达。 2. ELISA法检测足细胞培养上清中MCP-1的表达。细胞同步于GO期后,0、50、100、200(μg/ml)的AOPPs和200(μg/ml)未经修饰的MSA刺激细胞24h；或者200的AOPPs刺激细胞0、3、6、12、24、48h。取细胞上清。ELISA法检测细胞上清中MCP-1的浓度。 3. Western blot方法检测足细胞的IKKβ, IκBα和NF-κB蛋白表达水平。足细胞分别与200μg/ml AOPPs或MSA共同孵育30min。提取细胞总蛋白。Western blot方法检测足细胞的IKKβ、IκBα和NF-κB蛋白表达水平。 4.抑制试验。 NF-κB特异性抑制剂PTL预孵育1h后,200μg/ml AOPPs刺激足细胞24h。提取细胞上清,ELISA法检测足细胞MCP-1的表达。二、SLsAOPPs诱导的足细胞炎症因子MCP-1表达的影响 1. qPCR法检测足细胞MCP-1mRNA的表达用不同浓度的SLs(MCL,化合物1,化合物2)预孵育1h后,200μg/ml AOPPs刺激足细胞24h, PTL(5μM)作为阳性对照。或者AOPPs刺激24h,而SLs,即MCL(5μM),化合物1(10μM),化合物2(2.5μM))作用预先设定的时间。提取细胞总RNA, qPC法检测足细胞MCP-1mRNA的表达。 2. ELISA法检测足细胞MCP-1的表达用不同浓度的SLs(MCL,化合物1,化合物2)预孵育1h后,200μg/ml AOPPs刺激足细胞24h, PTL(5μM)作为阳性对照。或者AOPPs刺激24h,而SLs,即MCL(5μM),化合物1(10μM),化合物2(2.5lμM))作用预先设定的时间。提取细胞上清,ELISA法检测足细胞MCP-1的表达。 3. Western blot法检测足细胞IKKβ、IkBα、NF-κB蛋白的表达。不同浓度的SLs (MCL,化合物1,化合物2)预孵育1h后,200μg/ml AOPPs刺激足细胞30min, PTL(10μM)作为阳性对照。或者AOPPs刺激30min,而SLs,即MCL (10μM),化合物1(20μM),化合物2(5μM))作用预先设定的时间。提取细胞总蛋白,Western blot法检测足细胞IKKβ、IkBα、NF-κB蛋白的表达。统计方法采用SPSS13.0统计软件包进行统计分析,所有数据结果均以均数加减标准差表示。多个样本均数的比较采用One-way ANOVA,两两比较采用LSD和SNK,p0.05为差异有统计学意义。所有数据均代表3次重复实验的结果。结果 AOPPs的鉴定 AOPPs-MSA中AOPPs含量为72.40±9.8nmol/mg蛋白质,未修饰的MSA中AOPPs含量为0.2±0.06nmol/mg蛋白质。AOPPs和未经修饰的MSA经鲎试验法检测样本内毒素含量结果为内毒素含量低于0.025EU/ml。一、AOPPs对足细胞炎症因子MCP-1表达的影响 (1)不同浓度干预的结果：足细胞分别与0、50、100、200、400μg/mlAOPPs或MSA(200μg/ml)共同孵育24h,随着AOPPs浓度的增高,细胞上清中MCP-1mRNA和MCP-1蛋白的表达水平亦逐渐增加,AOPPs(200μg/ml)时达最高(p0.001)；未经修饰的MSA对足细胞MCP-1mRNA和MCP-1蛋白的表达水平无明显影响(P＞0.05)。 (2)干预不同时间的结果：AOPPs(200μg/ml)或MSA(200μg/ml)与足细胞共同孵育0、3、6、12、24、48h、MCP-1mRNA和MCP-1蛋白的表达水平随刺激时间的延长而升高,24h达最高(p0.001)。未经修饰的MSA对足细胞MCP-1mRNA和MCP-1蛋白的表达水平无明显影响(P＞0.05)。 (3)足细胞与200μg/ml AOPPs或MSA共同孵育30min,与正常对照组相比,AOPPs刺激组足细胞IκBα蛋白表达逐渐降低(p0.01); p-IKKα/β和P-NF-κBp65蛋白表达显著增加(p0.05),而总IKKβ和NF-κB p65蛋白表达未见明显差异。未经修饰MSA刺激组对足细胞IκBα IKKp和NF-κB蛋白的表达水平无显著差异(p0.05) (4)抑制试验：用NF-κB特异性抑制剂PTL(10μM)预孵育足细胞30min,再用AOPPs刺激24h后测定足细胞MCP-1的表达。与AOPPs干预组相比,用PTL‘预孵育组细胞上清中的MCP-1分泌减少,差异具有统计学意义(P0.001)。二、SLs对AOPPs诱导的足细胞炎症因子MCP-1表达的影响 (1)不同浓度的SLs (MCL,化合物1,化合物2)预孵育1h后,200μg/ml AOPPs刺激足细胞24h, PTL(5μM)作为阳性对照。或者AOPPs刺激24h,而SLs,即MCL (5μM),化合物1(10μM),化合物2(2.5μM))作用预先设定的时间。结果显示MCL,化合物1,化合物2呈剂量和时间依赖型抑制AOPPs诱导的足细胞MCP-1mRNA和蛋白的表达,其中5μMMCL,10μM化合物1,2.5μM化合物2抑制作用最强,和阳性对照组5μM PTL作用相当。 (2)不同浓度的SLs (MCL,化合物1,化合物2)预孵育1h后,200μg/ml AOPPs刺激足细胞30min,PTL(10μM)作为阳性对照。或者AOPPs刺激30min,而SLs,即MCL (10μM),化合物1(20μM),化合物2(5μM))作用预先设定的时间。结果显示MCL,化合物1,化合物2呈剂量和时间依赖型抑制AOPPs诱导的足细胞IKKβ蛋白的磷酸化、IκBα蛋白的降解和NF-κB蛋白的活化。其中10μM MCL,20μM化合物1,5μM化合物2抑制作用最强,和阳性对照组10μMPTL作用相当。结论一、AOPPs可通过IKK/NF-κB通路诱导体外培养的足细胞过度表达炎症因子MCP-1。二、SLs(MCL,化合物1和化合物2)能通过抑制IKK/NF-κB通路的激活来降低AOPPs诱导的足细胞MCP-1的过度表达而发挥抗炎作用,说明SLs可能具有防治DN及相关免疫炎症性肾病的作用,但其作用机制尚有待进一步研究。同时,与PTL相比,自主合成的SLs在制备工艺、稳定性、生物利用度、毒副作用等方面均具有显著的优势,具有更为广阔的开发与临床应用前景。
[Abstract]:Background and purpose of study

Diabetic nephropathy ( DN ) is one of the most common microvascular complications in diabetic nephropathy . The pathogenesis of DN is very complicated and has not been completely defined . In recent years , the expression of MCP - 1 plays an important role in the development of DN . In DN , MCP - 1 plays an important role in the development of DN .

In the DN animal model , the expression of nephrin and podocin in the foot cells can be reduced by the activation of the p38MAPK via the ROS - dependent p38 MAPK pathway , which leads to the reduction of the number of cells , the reduction of the density and the exposure of the basement membrane , which in turn induces proteinuria and increases the progression of the nephropathy .

NF - 魏B ( NF - 魏B ) plays an important role in the regulation of inflammatory factors , and AOPP can activate NF - 魏B - related pathways . Previous studies have shown that NF - 魏B expression in DN rats is significantly increased ;
In rat mesangial cells cultured in vitro , high glucose can induce mesangial cell proliferation , activate NF - 魏B related signal pathway , induce inflammatory factor MCP - 1 , and increase the expression of transforming growth factor - p1 ;
AOPP can rapidly induce the production of active oxygen in mesangial cells , activate MAPKs / NF - 魏B signal transduction pathways , and eventually induce MCP - 1 mRNA and protein overexpression in mesangial cells .

Polyterpene lactones are a large variety of natural bioactive compounds , which are originally isolated from the plants of Compositae . It has been found that in vitro cultured mesangial cells can exert their anti - inflammatory effects by inhibiting IKK and I魏B 伪 phosphorylation and / or affecting the binding ability of NF - 魏B and DNA . Our latest research results show that in the mesangial cells cultured in vitro , the degradation of I魏B 伪 and activation of NF - 魏B in mesangial cells can be effectively inhibited , and the expression of MCP - 1mRNA and protein in mesangial cells can be inhibited .

We can induce the overexpression of MCP - 1 in the cells by activating the signal pathway associated with NF - 魏B .
The sesquiterpenes lactone compound can exert its anti - inflammatory effect by inhibiting the activation of the signal transduction pathway related to NF - 魏B , and has the function of protecting DN ' s kidney .

Study contents and methods

1 . In vitro preparation of AOPP

20 mg / ml MSA was mixed with 40 mmo / 1HC10 in a molar ratio of 1 : 140 , room temperature for 30 minutes , and then dialysed for 24 hours in an endotoxin - free PBS to remove the free HCLO . After filtration of the bacteria with a 0.22 . mu.m microporous filter membrane , 20 mg / ml MSA was obtained by measuring the absorbance at 340 nm in the acid condition . The content of the endotoxin was detected by a limulus test method . The endotoxin content was lower than 0.25 EU / ml .

2 . Culture of foot cells

According to the method of Peter Mundel , sufficient cells were cultured in a 5 % CO2 incubator at 33 & deg ; C to proliferate in a 10 % FBS , 50 U / ml IFN - y RPM11640 culture broth . To obtain a differentiated phenotype , sufficient cells were cultured in a 5 % CO2 incubator at 37.degree . C. , cultured for 10 - 14 days in a 10 % FBS - free RPM11640 culture broth . After differentiation of cells , a serum - free 1640 medium was used for 24 hours . Various reagents and drug stimuli were added according to different experimental purposes . 10 - 20 generations of cells were used in this study .

Effect of One - and AODV on the Expression of MCP - 1 in Foot - cell Inflammatory Factors

1 . The expression of MCP - 1 mRNA was detected by qPCR .

At 0 , 50 , and 100 . 200 ( 渭g / ml ) AOpods and 200 ( 渭g / ml ) of unmodified MSA stimulated the cells for 24 h after the cells were synchronized to GO phase ;
At 0 , 3 , 6 , 12 , 24 , 48 h , the expression of MCP - 1 mRNA was detected by qPCR .

2 . The expression of MCP - 1 was detected by ELISA .

0 , 50 , 100 , 200 ( 渭g / ml ) AOpods and 200 ( 渭g / ml ) of unmodified MSA stimulated cells for 24 h after cell synchronization .
The concentration of MCP - 1 in the supernatant of cells was determined by ELISA .

3 . Western blot was used to detect IKK尾 , I魏B 伪 and NF - 魏B protein expression levels .

The total protein of IKK尾 , I魏B 伪 and NF - 魏B were detected by Western blot .

4 . Inhibition test .

After 1 hour incubation of NF - 魏B specific inhibitor , the cells were stimulated with 200 渭g / ml AOAC for 24 h . The supernatant of cells was extracted and the expression of MCP - 1 was detected by ELISA .

Expression of MCP - 1 in Human Foot Cells Induced by Two , SLsAODV

1 . Expression of MCP - 1 mRNA in Foot Cells by qPCR

The expression of MCP - 1 mRNA was detected by the total RNA and qPC method . The total RNA and qPC method were used to detect MCP - 1 mRNA expression .

2 . Expression of MCP - 1 in Foot Cells by ELISA

The expression of MCP - 1 was detected by ELISA .

3 . The expression of IKK尾 , IkB 伪 , NF - 魏B was detected by Western blot .

The expression of IKK尾 , IkB 伪 , NF - 魏B was detected by Western blot .

statistical method

Statistical analysis was carried out with SPSS 13.0 statistical software package . All data results were expressed by mean addition and subtraction standard deviation . One - way ANOVA was used to compare the mean values of multiple samples , and LSD and SNK were used in both comparisons . All the data represent the results of 3 replicate experiments .

Identification of AOLINGS

AOPP - MSA was 72.40 卤 9.8 nmol / mg protein , and the unmodified MSA was 0.2 卤 0.06 nmol / mg protein . The results showed that the endotoxin content was lower than 0.025 EU / ml .

Effect of One - and AODV on the Expression of MCP - 1 in Foot - cell Inflammatory Factors

( 1 ) The results of different concentration intervention : The expression level of MCP - 1 mRNA and MCP - 1 protein in the supernatant of the supernatant of the cells increased gradually with the increase of AOPP concentration , while the expression level of MCP - 1 mRNA and MCP - 1 protein in the supernatant of the cells increased gradually with the increase of AOPP concentration ( p0.001 ) .
The expression level of MCP - 1 mRNA and MCP - 1 protein was not significantly affected by unmodified MSA ( P > 0.05 ) .

( 2 ) The expression level of MCP - 1 mRNA and MCP - 1 protein was increased with the increase of stimulation time at 0 , 3 , 6 , 12 , 24 , 48 h , and the expression level of MCP - 1 mRNA and MCP - 1 protein increased with the increase of stimulation time ( P > 0.05 ) .

( 3 ) Compared with the normal control group , the expression of I魏B 伪 protein was gradually decreased ( p < 0.01 ) , and the expression of total IKK伪 / 尾 and P - NF - 魏B P65 protein was significantly increased ( p < 0.05 ) .

( 4 ) Inhibition test : The expression of MCP - 1 was determined by pre - incubation with NF - 魏B specific inhibitor ( 10 渭M ) for 30 min , then the expression of MCP - 1 was measured after 24 h of AOAC stimulation . Effect of two or two weeks on the expression of MCP - 1 induced by AOI The expression of MCP - 1mRNA and protein in human foot cells induced by AOI was inhibited by dose - dependent and time - dependent manner . The results showed that the inhibitory effect of the compound 1 and compound 2 on MCP - 1 mRNA and protein expression was inhibited by dose - dependent and time - dependent inhibition of AOI . The results showed that the inhibitory effect of the compound 1 and compound 2 on MCP - 1 mRNA and protein was strongest in 5 渭M CL , 10 渭M Compound 1 , 2.5 渭M Compound 2 and 5 渭M in the positive control group . ( 2 ) After pre - incubation for 1 h at different concentrations , 30 minutes after pre - incubation with 200 渭g / ml AOI , the phosphorylation of IKK尾 protein , the degradation of I魏B 伪 protein and activation of NF - 魏B protein were observed . The results showed that the inhibition of phosphorylation of IKK尾 protein and the activation of NF - 魏B were inhibited by the dose and time dependent manner . The results showed that the inhibitory effect of the compound 1 , compound 2 on the phosphorylation of IKK尾 protein and the activation of NF - 魏B protein were observed in 10 渭M , 20 渭M , 5 渭M , and 10 渭MPTL in the positive control group . Conclusion One , AOPP can induce the in vitro cultured human foot cells to overexpress the inflammatory factor MCP - 1 via IKK / NF - 魏B pathway . Two , two , two , compound 1 and compound 2 can reduce the excessive expression of IKK / NF - 魏B pathway to reduce the over - expression of MCP - 1 induced by AOI , but the mechanism of action is still to be further studied . At the same time , the self - synthesized microorganism has obvious advantages in the aspects of preparation process , stability , bioavailability , toxic side effect and the like , and has wider development and clinical application prospect .
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692

【参考文献】