本文選題：倍半萜烯內酯類化合物 + 晚期氧化蛋白產物��； 參考：《南方醫(yī)科大學》2014年碩士論文

【摘要】：研究背景與目的 糖尿病腎病(DN)是糖尿病最常見的一種微血管并發(fā)癥。DN的發(fā)病機制十分復雜,至今尚未完全明確。按傳統(tǒng)的觀點,DN并非是一種炎癥性疾病,然而近年的研究表明炎癥反應和DN關系密切,炎癥反應在DN發(fā)生發(fā)展過程中起到了重要的促進作用。研究表明,單核細胞趨化蛋白(MCP)-1參與誘導巨噬細胞進入糖尿病腎臟,在DN動物模型中的表達進行性上升,并且其在腎小球和腎小管中的積累量分別與腎小球和腎小管的損傷成正相關。在DN中,MCP-1不僅發(fā)揮趨化作用,促進單核細胞和巨噬細胞游走和活化,同時也能通過上調黏附因子表達和促進其他炎癥因子的表達直接引起腎臟的炎癥反應和腎纖維化。 晚期氧化蛋白產物(AOPPs)是一個新的致DN進展的炎癥介質。DN患者體內AOPPs水平明顯高于未出現(xiàn)腎臟損傷的糖尿病患者。在DN動物模型中,AOPPs的慢性積累顯著增加了殘余腎巨噬細胞的侵潤和MCP-1的過度表達,促進了炎癥反應。AOPPs可通過ROS依賴的p38MAPK通路的激活降低足細胞中的nephrin和podocin的表達,同時AOPPs也可誘導足細胞的凋亡,導致足細胞數(shù)目減少、密度降低和基底膜裸露,進而誘發(fā)了蛋白尿,加重腎病的進展。 核因子κB (NF-κB)在調節(jié)炎癥因子的過程中發(fā)揮著重要作用,AOPPs能激活NF-κB相關通路。我們之前的研究發(fā)現(xiàn),DN大鼠中NF-κB表達顯著增加；在體外培養(yǎng)的大鼠腎小球系膜細胞中,高糖可誘導系膜細胞增殖,激活NF-κB相關信號通路,誘導炎癥因子MCP-1和轉化生長因子-p1表達增加；AOPPs可以快速誘導系膜細胞內活性氧的產生,激活MAPKs/NF-κB信號轉導通路,最終誘導系膜細胞中MCP-1mRNA和蛋白過度表達。 倍半萜烯內酯(SLs)是一大類具有多樣結構的天然生物活性化合物,最初是從菊科植物中分離所得,小白菊內酯(PTL)是SLs中最主要的生物活性成分。已有研究發(fā)現(xiàn)PTL和其他SLs能通過抑制IKK和IκBα磷酸化和/或影響NF-κB和DNA的結合能力發(fā)揮其抗炎作用。我們最新的研究結果表明,在體外培養(yǎng)的系膜細胞中,SLs,包括PTL, MCL和及小白菊內酯的衍生物-化合物2,以及其他相關的小白菊內酯衍生物,能有效地抑制高糖及AOPPs誘導的IκBα的降解和NF-κB的活化,進而抑制系膜細胞中炎癥因子MCP-1mRNA和蛋白的表達。 我們推鋇AOPPs能通過激活NF-κB相關的信號通路來誘導足細胞中炎癥因子MCP-1的過度表達；倍半萜烯內酯類化合物可通過抑制NF-κB相關的信號轉導途徑的激活來發(fā)揮其抗炎作用,具有保護DN腎臟的作用。本實驗以體外培養(yǎng)的足細胞為對象,旨在驗證上述推測,為將來應用SLs治療DN及相關炎癥性腎病的深化研究提供一定的理論依據(jù)。 研究內容與方法 1.體外制備AOPPs 將20mg/ml MSA與40mmo/1HC10等體積混合,其摩爾比為1：140,室溫放置30分鐘后,于無內毒素PBS中透析24小時,以除去游離的HCLO用0.22μmm的微孔濾膜過濾除菌后4℃保存。20mg/ml MSA與PBS等體積混合,作為對照。AOPPs含量通過測定酸性條件下340nm的吸光度,以氯胺T為標準取得。內毒素含量用鱟試驗法檢測,該方法制備的AOPPs內毒素含量均低于0.25EU/ml。 2.足細胞的培養(yǎng) 按Peter Mundel的方法。足細胞于33℃含5%C02培養(yǎng)箱,,在10%FBS、50U/ml IFN-y的RPM11640培養(yǎng)液中增殖。為了得到分化表型,足細胞于37℃含5%C02培養(yǎng)箱,在10%FBS的RPM11640培養(yǎng)液中培養(yǎng)10-14天。待細胞分化后,使用無血清1640培養(yǎng)液培養(yǎng)24小時。根據(jù)不同的實驗目的分組,加用各種試劑和藥物刺激。本研究使用10-20代足細胞。 一、AOPPs對足細胞炎癥因子MCP-1表達的影響 1. qPCR法檢測足細胞MCP-1mRNA的表達。 細胞同步于GO期后,0、50、100.200(μg/ml)的AOPPs和200(μg/ml)未經(jīng)修飾的MSA刺激細胞24h；或者200μg/ml的AOPPs刺激細胞0、3、6、12、24、48h。提取細胞中總RNA。qPCR法檢測足細胞MCP-1mRNA的表達。 2. ELISA法檢測足細胞培養(yǎng)上清中MCP-1的表達。 細胞同步于GO期后,0、50、100、200(μg/ml)的AOPPs和200(μg/ml)未經(jīng)修飾的MSA刺激細胞24h；或者200的AOPPs刺激細胞0、3、6、12、24、48h。取細胞上清。ELISA法檢測細胞上清中MCP-1的濃度。 3. Western blot方法檢測足細胞的IKKβ, IκBα和NF-κB蛋白表達水平。 足細胞分別與200μg/ml AOPPs或MSA共同孵育30min。提取細胞總蛋白。Western blot方法檢測足細胞的IKKβ、IκBα和NF-κB蛋白表達水平。 4.抑制試驗。 NF-κB特異性抑制劑PTL預孵育1h后,200μg/ml AOPPs刺激足細胞24h。提取細胞上清,ELISA法檢測足細胞MCP-1的表達。 二、SLsAOPPs誘導的足細胞炎癥因子MCP-1表達的影響 1. qPCR法檢測足細胞MCP-1mRNA的表達 用不同濃度的SLs(MCL,化合物1,化合物2)預孵育1h后,200μg/ml AOPPs刺激足細胞24h, PTL(5μM)作為陽性對照�；蛘逜OPPs刺激24h,而SLs,即MCL(5μM),化合物1(10μM),化合物2(2.5μM))作用預先設定的時間。提取細胞總RNA, qPC法檢測足細胞MCP-1mRNA的表達。 2. ELISA法檢測足細胞MCP-1的表達 用不同濃度的SLs(MCL,化合物1,化合物2)預孵育1h后,200μg/ml AOPPs刺激足細胞24h, PTL(5μM)作為陽性對照�；蛘逜OPPs刺激24h,而SLs,即MCL(5μM),化合物1(10μM),化合物2(2.5lμM))作用預先設定的時間。提取細胞上清,ELISA法檢測足細胞MCP-1的表達。 3. Western blot法檢測足細胞IKKβ、IkBα、NF-κB蛋白的表達。 不同濃度的SLs (MCL,化合物1,化合物2)預孵育1h后,200μg/ml AOPPs刺激足細胞30min, PTL(10μM)作為陽性對照�；蛘逜OPPs刺激30min,而SLs,即MCL (10μM),化合物1(20μM),化合物2(5μM))作用預先設定的時間。提取細胞總蛋白,Western blot法檢測足細胞IKKβ、IkBα、NF-κB蛋白的表達。 統(tǒng)計方法 采用SPSS13.0統(tǒng)計軟件包進行統(tǒng)計分析,所有數(shù)據(jù)結果均以均數(shù)加減標準差表示。多個樣本均數(shù)的比較采用One-way ANOVA,兩兩比較采用LSD和SNK,p0.05為差異有統(tǒng)計學意義。所有數(shù)據(jù)均代表3次重復實驗的結果。結果 AOPPs的鑒定 AOPPs-MSA中AOPPs含量為72.40±9.8nmol/mg蛋白質,未修飾的MSA中AOPPs含量為0.2±0.06nmol/mg蛋白質。AOPPs和未經(jīng)修飾的MSA經(jīng)鱟試驗法檢測樣本內毒素含量結果為內毒素含量低于0.025EU/ml。 一、AOPPs對足細胞炎癥因子MCP-1表達的影響 (1)不同濃度干預的結果：足細胞分別與0、50、100、200、400μg/mlAOPPs或MSA(200μg/ml)共同孵育24h,隨著AOPPs濃度的增高,細胞上清中MCP-1mRNA和MCP-1蛋白的表達水平亦逐漸增加,AOPPs(200μg/ml)時達最高(p0.001)；未經(jīng)修飾的MSA對足細胞MCP-1mRNA和MCP-1蛋白的表達水平無明顯影響(P＞0.05)。 (2)干預不同時間的結果：AOPPs(200μg/ml)或MSA(200μg/ml)與足細胞共同孵育0、3、6、12、24、48h、MCP-1mRNA和MCP-1蛋白的表達水平隨刺激時間的延長而升高,24h達最高(p0.001)。未經(jīng)修飾的MSA對足細胞MCP-1mRNA和MCP-1蛋白的表達水平無明顯影響(P＞0.05)。 (3)足細胞與200μg/ml AOPPs或MSA共同孵育30min,與正常對照組相比,AOPPs刺激組足細胞IκBα蛋白表達逐漸降低(p0.01); p-IKKα/β和P-NF-κBp65蛋白表達顯著增加(p0.05),而總IKKβ和NF-κB p65蛋白表達未見明顯差異。未經(jīng)修飾MSA刺激組對足細胞IκBα IKKp和NF-κB蛋白的表達水平無顯著差異(p0.05) (4)抑制試驗：用NF-κB特異性抑制劑PTL(10μM)預孵育足細胞30min,再用AOPPs刺激24h后測定足細胞MCP-1的表達。與AOPPs干預組相比,用PTL‘預孵育組細胞上清中的MCP-1分泌減少,差異具有統(tǒng)計學意義(P0.001)。 二、SLs對AOPPs誘導的足細胞炎癥因子MCP-1表達的影響 (1)不同濃度的SLs (MCL,化合物1,化合物2)預孵育1h后,200μg/ml AOPPs刺激足細胞24h, PTL(5μM)作為陽性對照。或者AOPPs刺激24h,而SLs,即MCL (5μM),化合物1(10μM),化合物2(2.5μM))作用預先設定的時間。結果顯示MCL,化合物1,化合物2呈劑量和時間依賴型抑制AOPPs誘導的足細胞MCP-1mRNA和蛋白的表達,其中5μMMCL,10μM化合物1,2.5μM化合物2抑制作用最強,和陽性對照組5μM PTL作用相當。 (2)不同濃度的SLs (MCL,化合物1,化合物2)預孵育1h后,200μg/ml AOPPs刺激足細胞30min,PTL(10μM)作為陽性對照�；蛘逜OPPs刺激30min,而SLs,即MCL (10μM),化合物1(20μM),化合物2(5μM))作用預先設定的時間。結果顯示MCL,化合物1,化合物2呈劑量和時間依賴型抑制AOPPs誘導的足細胞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抑制作用最強,和陽性對照組10μMPTL作用相當。 結論 一、AOPPs可通過IKK/NF-κB通路誘導體外培養(yǎng)的足細胞過度表達炎癥因子MCP-1。 二、SLs(MCL,化合物1和化合物2)能通過抑制IKK/NF-κB通路的激活來降低AOPPs誘導的足細胞MCP-1的過度表達而發(fā)揮抗炎作用,說明SLs可能具有防治DN及相關免疫炎癥性腎病的作用,但其作用機制尚有待進一步研究。同時,與PTL相比,自主合成的SLs在制備工藝、穩(wěn)定性、生物利用度、毒副作用等方面均具有顯著的優(yōu)勢,具有更為廣闊的開發(fā)與臨床應用前景。
[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 偽 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 偽 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 偽 and NF - 魏B protein expression levels .

The total protein of IKK尾 , I魏B 偽 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 偽 , NF - 魏B was detected by Western blot .

The expression of IKK尾 , IkB 偽 , 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 鹵 9.8 nmol / mg protein , and the unmodified MSA was 0.2 鹵 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 偽 protein was gradually decreased ( p < 0.01 ) , and the expression of total IKK偽 / 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 偽 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 .
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R587.2;R692

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