【摘要】：糖尿病腎病(diabetic kidney disease, DKD)是糖尿病最主要微血管并發(fā)癥,患者一旦出現(xiàn)大量蛋白尿,腎功能將進行性下降。由于其發(fā)病機制復(fù)雜,目前的治療措施療效有限。中醫(yī)藥治療在控制DKD方面具有一定優(yōu)勢。前期研究結(jié)果顯示,導(dǎo)師經(jīng)驗方糖腎方可減少2型DKD患者尿蛋白排泄、提高估算的腎小球濾過率,升高血漿谷胱甘肽水平,具有一定的保護腎功能及抗氧化作用。普遍認(rèn)為,氧化應(yīng)激在DKD發(fā)生發(fā)展中起到重要作用,活性氧簇(reactive oxygen species, ROS)可激活多條信號通路,引起內(nèi)皮細(xì)胞、系膜細(xì)胞、足細(xì)胞、腎小管間質(zhì)損傷,從而加重DKD。近年來,主要表達(dá)于腎小管上皮細(xì)胞的肌醇加氧酶(myo-inositol oxygenase, MIOX)在DKD發(fā)生發(fā)展中的作用逐漸被發(fā)現(xiàn),認(rèn)為其參與調(diào)控細(xì)胞內(nèi)ROS生成,與氧化應(yīng)激密切相關(guān),可能成為DKD干預(yù)新靶點。為了進一步觀察、驗證糖腎方干預(yù)2型DKD的長期療效,探索其可能機制,本研究選用自發(fā)性2型DKD小鼠動物模型,觀察糖腎方對小鼠的干預(yù)作用。在此基礎(chǔ)上,體外實驗驗證MIOX與氧化應(yīng)激的關(guān)系。此外,課題組開展了糖腎方治療DKD有效性和安全性的隨機、雙盲、安慰劑平行對照、多中心臨床試驗,將氧化應(yīng)激指標(biāo)納為次要療效指標(biāo),以期為糖腎方保護DKD的抗氧化作用提供更全面證據(jù)。目的：1.研究糖腎方對白發(fā)性2型糖尿病db/db小鼠腎臟損傷的保護作用,探索其機制是否與抗氧化應(yīng)激、降低MIOX表達(dá)有關(guān)。2.探索MIOX在高糖培養(yǎng)的大鼠近端腎小管上皮細(xì)胞(NRK52E)中的表達(dá)變化及其在近端腎小管上皮細(xì)胞氧化應(yīng)激中的作用。3. 由于糖腎方治療DKD有效性和安全性的隨機、雙盲、安慰劑平行對照、多中心臨床試驗尚未揭盲,本研究歸納試驗實施過程中影響質(zhì)量控制的關(guān)鍵環(huán)節(jié),為提高研究質(zhì)量提供幫助。方法：1.糖腎方對db/db小鼠腎臟保護作用及機制研究采用8周齡自發(fā)性2型糖尿病模型db/db小鼠,隨機分為模型組(db/db組)、糖腎方治療組(db/db+TSF組),同周齡不發(fā)病db/m小鼠作為正常對照組(db/m組),db/db+TSF組小鼠采用糖腎方(2.4g/kg-d)連續(xù)灌胃給藥12周,db/m組和db/db組小鼠給予等量蒸餾水。所有小鼠均以常規(guī)普通飼料順應(yīng)性喂養(yǎng)2周后,開始干預(yù)。每日觀察小鼠一般狀態(tài),每周測量體重,每4周檢測血糖、24h尿白蛋白。實驗第12周進行動物取材,小鼠禁食12h后眼內(nèi)眥取血,全自動生化儀檢測肝、腎功能和血脂。之后迅速打開腹腔,摘取雙側(cè)腎臟并稱重,一部分腎組織用于組織病理分析,另一部分分離出腎皮質(zhì)用于分子生物學(xué)檢測。腎組織進行PAS染色觀察病理損傷,計算腎小球系膜基質(zhì)百分比,TBA法檢測血清、尿液中丙二醛含量,Wester blot.實時熒光定量PCR、免疫組化方法檢測小鼠腎組織中氧化應(yīng)激相關(guān)的NADPH氧化酶亞基、Nrf2通路、MIOX及足細(xì)胞標(biāo)志物表達(dá)。2. MIOX在高糖誘導(dǎo)腎小管上皮細(xì)胞氧化應(yīng)激中的作用采用5.5、10、20、30 mM濃度葡萄糖刺激大鼠近端腎小管上皮細(xì)胞(NRK52E) 48h,30 mM葡萄糖刺激NRK52E細(xì)胞12.24.36.48.60h。 Western blot方法檢測細(xì)胞內(nèi)MIOX、Nox4、 Nrf2表達(dá)變化,分別繪制葡萄糖對NRK52E氧化應(yīng)激損傷的劑量和時間依賴曲線,確定最佳刺激劑量和時間。設(shè)計MIOX基因siRNA Oligo干擾序列,篩選出敲降效率最高者轉(zhuǎn)染至NRK52E細(xì)胞,6h后更換終濃度5.5、30mM葡萄糖培養(yǎng)基,48h后收集細(xì)胞,DCFH-DA探針檢測細(xì)胞內(nèi)ROS水平,Western blot檢測細(xì)胞內(nèi)MIOX、Nox4、Nrf2、 NQO1蛋白表達(dá)。3. 歸納總結(jié)糖腎方治療DKD有效性和安全性的隨機、雙盲、安慰劑平行對照、多中心臨床試驗實施過程中的質(zhì)量控制要點。結(jié)果：1.糖腎方可改善自發(fā)性2型糖尿病模型db/db小鼠腎組織損傷,其機制與降脂、抗氧化應(yīng)激、保護足細(xì)胞有關(guān)(1)腎臟保護作用：給藥0至12周,db/db組體重明顯高于db/m組(P0.001),db/db+TSF組在第6周(小鼠16周齡)出現(xiàn)體重下降,并持續(xù)至實驗結(jié)束(P0.05)。糖腎方干預(yù)可降低db/db組升高的腎重(P0.01)。實驗期間,db/m組血糖保持穩(wěn)定,db/db和db/db+TSF組血糖均高于db/m組(P0.01),但兩組之間無差異。與db/m組比,db/db組血清AST、ALT升高(P0.01,P0.001),糖腎方干預(yù)可有效改善其異常的肝功能(P0.05,P0.05)。db/db組血清TC、FFA、LDL-C較db/m組顯著升高(P0.001,P0.05,P0.001),糖腎方可改善db/db小鼠血脂紊亂(P0.001,P0.05,P0.001)。實驗過程中,db/db組24h尿白蛋白較db/m組升高(P0.001),給藥第8周開始,db/db+TSF組24h尿白蛋白出現(xiàn)下降趨勢(P0.01),至給藥12周時與db/m組更接近(P0.05)。 PAS染色可見db/db組腎小球體積增大,系膜基質(zhì)增生,半定量結(jié)果表明腎小球系膜基質(zhì)百分比顯著升高(P0.01),糖腎方治療后腎小球系膜基質(zhì)百分比降低(P0.01)。(2)抗氧化應(yīng)激機制：TBA法檢測MDA水平發(fā)現(xiàn),db/db組小鼠血清、24h尿液MDA均較db/m組顯著升高(P0.01,P0.001),糖腎方干預(yù)可使其含量顯著下降(P0.05,P0.05)。實時熒光定量PCR結(jié)果顯示,db/db組小鼠腎組織Nox2、Nox4、 p22phox mRNA水平較db/m組小鼠顯著升高(P0.01,P0.05,P0.01),但p47phox無差異(P0.05),糖腎方干預(yù)可降低Nox2、Nox4、p22phox mRNA水平(P0.001,P0.05,P0.01)。Western blot與免疫組化結(jié)果顯示,與db/m組相比,db/db組腎組織Nox2、Nox4蛋白表達(dá)升高(P0.05, P0.001; P0.05, P0.001),糖腎方可下調(diào)其表達(dá)(P0.05, P0.001; P0.05,P0.001)。實時熒光定量PCR發(fā)現(xiàn),與db/m組相比,db/db組小鼠腎組織Nrf2、QO1mRNA升高(P0.05,P0.01),db/db+TSF組上述指標(biāo)顯著下降(P0.05,P0.05)；db/db組HO-1 mRNA水平較db/m組下降(P0.05),糖腎方干預(yù)可升高其表達(dá)(P0.05)。Western blot與免疫組化顯示,糖腎方可下調(diào)db/db組腎組織內(nèi)升高的Nrf2、NQO1蛋白表達(dá)(P0.05, P 0.001;P0.05,P0.001)。此外,免疫組化還顯示,MIOX表達(dá)于近端腎小管,Western blot與免疫組化均證實db/db組小鼠腎小管內(nèi)MIOX顯著增多(P0.01,P0.001),而糖腎方可使其降低(P0.05,P0.001)。(3)保護足細(xì)胞機制：實時熒光定量PCR顯示,db/db組小鼠腎組織nephrin、podocin、 WT-1 mRNA水平均較db/m組顯著下降(P0.05,P0.05,P0.05),而糖腎方治療可使其表達(dá)升高(P0.01,P0.01,P0.05)。免疫組化結(jié)果同樣證實糖腎方具有升高db/db組小鼠nephrin podocin表達(dá)的作用(P0.01,P0.01)。2. MIOX介導(dǎo)高糖誘導(dǎo)的腎小管上皮細(xì)胞氫化應(yīng)激發(fā)生(1)高糖刺激對MIOX表達(dá)的影響：隨著葡萄糖濃度升高,NRK52E細(xì)胞MIOX、Nox4表達(dá)逐漸升高,Nrf2表達(dá)逐漸下降；30 mM葡萄糖刺激NRK52E細(xì)胞0、12、24、36、48、60h,細(xì)胞內(nèi)MIOX、Nox4、Nrf2表達(dá)均先升高后降低,Nrf2在刺激12h升至高峰,48h時下降顯著,MIOX、Nox4至48h時出現(xiàn)表達(dá)高峰。(2)敲降MIOX對細(xì)胞氧化應(yīng)激的影響：篩選得到敲降效率最高(72.1%)的MIOX基因siRNA Oligo干擾序列,轉(zhuǎn)染至NRK52E細(xì)胞,6h后更換終濃度5.5及30 mM葡萄糖培養(yǎng)基,48h后發(fā)現(xiàn),細(xì)胞內(nèi)ROS含量及MIOX、Nox4、NQO1蛋白表達(dá)升高,Nrf2表達(dá)下降,敲降MIOX可降低ROS水平及Nox4、NQOl表達(dá),Nrf2表達(dá)較高糖刺激進一步下降。3.受試者招募、數(shù)據(jù)管理、多級監(jiān)查、研究者和受試者依從性是試驗實施過程中質(zhì)量控制的重要環(huán)節(jié)。結(jié)論：1.糖腎方可降低自發(fā)性2型糖尿病模型db/db、鼠尿白蛋白排泄,改善腎組織病理損傷,具有腎臟保護作用,其機制可能與改善血脂紊亂、減輕氧化應(yīng)激、降低MIOX表達(dá)、保護足細(xì)胞損傷有關(guān)。2. MIOX可促進高糖引起的腎小管上皮細(xì)胞氧化應(yīng)激反應(yīng),其作用與促進Nox4表達(dá)有關(guān),這一結(jié)果為進一步探索中藥作用靶點提供了新思路。
[Abstract]:Diabetic nephropathy (diabetic kidney disease, DKD) is the most important microvascular complication of diabetes. Once a large number of albuminuria appears, the renal function will be reduced. Due to its complicated pathogenesis, the curative effect of the present treatment is limited. The treatment of traditional Chinese medicine has some advantages in the control of DKD. Fang sugar kidney can reduce the urinary protein excretion of type 2 DKD patients, improve the estimated glomerular filtration rate and increase the plasma glutathione level. It has a certain protection of renal function and antioxidant effect. It is generally believed that oxidative stress plays an important role in the development of DKD, and the active oxygen cluster (reactive oxygen species, ROS) can activate a number of signal pathways. It causes endothelial cells, mesangial cells, podocytes and tubulointerstitial damage, and thus aggravates DKD. in recent years. The role of myo-inositol oxygenase (MIOX), which is mainly expressed in renal tubular epithelial cells, is gradually discovered in the development of DKD, which is considered to be involved in the regulation of intracellular ROS formation, which is closely related to oxidative stress. To further observe the new target of DKD intervention. In order to further observe the long-term effect of sugar kidney prescription intervention on the long-term effect of type 2 DKD and explore its possible mechanism, this study selected spontaneous 2 DKD mice model and observed the intervention effect of sugar kidney prescription on mice. On this basis, the relationship between MIOX and oxygen stress was verified in vitro. In addition, the subject group carried out the sugar kidney prescription. A randomized, double-blind, placebo-controlled, multicenter clinical trial with a randomized, double-blind, placebo-controlled, multicenter clinical trial to treat DKD effectiveness and safety, and to provide more comprehensive evidence for the antioxidant effect of DKD in the sugar kidney prescription. Objective: 1. to study the protective effect of sugar kidney prescription on renal injury in db/db mice with type 2 diabetic type white hair. Whether the mechanism is associated with antioxidant stress, reducing the expression of MIOX and reducing the expression of.2. to explore the expression of MIOX in the proximal renal tubular epithelial cells (NRK52E) in high glucose cultured rats and its role in the oxidative stress of proximal renal tubular epithelial cells..3. is randomized, double blind, placebo parallel control, due to the efficacy and safety of sugar kidney prescription in the treatment of DKD. The multicenter clinical trial has not been blinded. This study summarizes the key links affecting the quality control during the implementation of the test, and provides help to improve the quality of the study. Method: 1. the protective effect and mechanism of the 1. sugar kidney prescription on the renal protection and mechanism of 8 weeks old spontaneous 2 diabetes model db/db mice were randomly divided into model group (group db/db), sugar kidney prescription In the treatment group (group db/db+TSF), the db/m mice of the same week age were used as the normal control group (group db/m), and the group db/db+TSF mice were given the sugar kidney recipe (2.4g/kg-d) for 12 weeks, and the db/m and db/db mice were given the same amount of distilled water. All the mice were fed with the routine normal feed CIS feeding for 2 weeks, and the general state of the mice was observed daily. The body weight was measured every week, blood glucose was measured every 4 weeks, 24h urine albumin was tested for Twelfth weeks, and the mice were harvested for Twelfth weeks. After fasting the mice, the blood was taken in the eye canthus. The liver, kidney function and blood lipid were detected by the automatic biochemical analyzer. Then the abdominal cavity was opened and the kidneys were extracted and weighed. Some kidney groups were used for histopathological analysis, and the other part of the renal cortex was separated. In molecular biology test, renal tissue was stained by PAS staining, calculated the percentage of glomerular mesangial matrix, TBA method to detect serum, MDA content in urine, Wester blot. real-time fluorescence quantitative PCR, and immunohistochemical method to detect the oxidative stress related NADPH oxidase subunit in mouse kidney tissue, Nrf2 pathway, MIOX and podocyte standard The effect of.2. MIOX on oxidative stress induced by high glucose in renal tubular epithelial cells was stimulated by 5.5,10,20,30 mM concentration of glucose stimulated proximal renal tubular epithelial cells (NRK52E) 48h, and 30 mM glucose stimulated NRK52E cells 12.24.36.48.60h. Western blot method to detect intracellular MIOX. The dose and time dependence curve of NRK52E oxidative stress damage were used to determine the optimal dose and time. The MIOX gene siRNA Oligo interference sequence was designed, the highest knockdown efficiency was transfected into NRK52E cells, 6h was replaced by the final concentration 5.5,30mM glucose medium, 48h was collected after the collection of fine cell, and DCFH-DA probe was used to detect the intracellular ROS level, Western blo T detection of intracellular MIOX, Nox4, Nrf2, NQO1 protein expression.3. induction and summary of the efficacy and safety of sugar kidney prescription in the treatment of DKD efficacy and safety of random, double blind, placebo parallel control, the implementation of multicenter clinical trials of quality control points. Results: 1. sugar kidney can improve the spontaneous 2 diabetes model db/db mice renal tissue damage, its mechanism and reduction Lipid, antioxidant stress, protection of podocytes (1) renal protection: 0 to 12 weeks, group db/db was significantly higher than group db/m (P0.001), group db/db+TSF had weight decline in sixth weeks (16 weeks of age of mice) and continued to the end of the experiment (P0.05). Sugar kidney intervention could reduce the renal weight of db/db group (P0.01). During the experiment, the blood sugar of db/m group was maintained. The blood glucose of group db/db and db/db+TSF was higher than that of group db/m (P0.01), but there was no difference between the two groups. Compared with the db/m group, the serum AST and ALT increased (P0.01, P0.001) in the db/db group. Rat blood lipid disorder (P0.001, P0.05, P0.001). The 24h urine albumin in the group db/db was higher than that in the db/m group (P0.001). At the beginning of the eighth week, the 24h urine albumin in the group db/db+TSF decreased (P0.01), which was closer to the db/m group (P0.05) at the 12 week of administration. The percentage of glomerular mesangial matrix increased significantly (P0.01), the percentage of glomerular mesangial matrix decreased (P0.01) after the treatment of sugar kidney. (2) the antioxidant stress mechanism: the level of MDA in the serum of db/db mice was significantly higher than that of the db/m group (P0.01, P0.001) in the serum of db/db mice (P0.01, P0.001), and the glucose and kidney intervention could significantly decrease its content (P0.05, P0.0). 5) real time fluorescence quantitative PCR results showed that the levels of Nox2, Nox4 and p22phox mRNA in the kidney tissues of db/db mice were significantly higher than those in the db/m group (P0.01, P0.05, P0.01), but there was no difference in p47phox (P0.05). The expression of Nox2, Nox4 protein increased (P0.05, P0.001; P0.05, P0.001) in the renal tissue, and the expression of P0.05, P0.001, P0.05, P0.001 was lowered in the sugar kidney. Compared with group db/m (P0.05), glucose and kidney intervention could increase its expression (P0.05).Western blot and immunohistochemistry, and sugar kidney can down regulate the Nrf2, NQO1 protein expression (P0.05, P 0.001, P0.05, P0.001) in the renal tissue of db/db group. Besides, immunohistochemistry also showed that the expression was expressed in proximal renal tubules. The MIOX in the renal tubules increased significantly (P0.01, P0.001) in the group of mice (P0.05, P0.001). (3) the protection of the podocyte mechanism: real-time fluorescent quantitative PCR showed that nephrin, podocin and WT-1 mRNA in the db/db group were significantly lower than those of the db/m group. 1, P0.05). The results of immunohistochemistry also confirmed the effect of sugar kidney on the increase of nephrin podocin expression in group db/db mice (P0.01, P0.01).2. MIOX mediated high glucose induced renal tubular epithelial cell hydrogen stress (1) the effect of high glucose stimulation on the expression of MIOX: with the increase of glucose concentration, NRK52E cell MIOX, Nox4 expression gradually increased. The expression decreased gradually; 30 mM glucose stimulated NRK52E cell 0,12,24,36,48,60h, and the expression of MIOX, Nox4 and Nrf2 in the cells all increased first and then decreased, Nrf2 in the stimulation of 12h to peak, 48h decreased significantly, MIOX, Nox4 to 48h. (2) the effect of knock down on cell oxidative stress: screening to get the highest knock efficiency (72.1%) Gene siRNA Oligo interference sequence, transfected to NRK52E cells, after 6h to replace the final concentration of 5.5 and 30 mM glucose medium. After 48h, ROS content and MIOX, Nox4, NQO1 protein expression increased, Nrf2 expression decreased. Management, multilevel monitoring, the compliance of the researchers and subjects is an important link in the quality control of the experiment. Conclusion: 1. sugar kidney can reduce the model db/db, urinary albumin excretion, improve the pathological damage of renal tissue, and have the role of renal protection. The mechanism may improve the disorder of blood lipid and reduce oxidative stress. Low MIOX expression and protective foot cell injury related to.2. MIOX can promote the oxidative stress response of renal tubular epithelial cells induced by high glucose, and their role is related to the promotion of Nox4 expression. This result provides a new idea for further exploration of the target target of traditional Chinese medicine.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R259
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本文編號：2154259