【作者】 陈燕；

【导师】 赵雪俭； 孟艳；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 研究背景：脓毒血症诱发的失控性全身性炎症反应综合征（SIRS）是导致多器官功能衰竭的重要原因，而急性肾损伤（AKI）是其中最严重的并发症，致死率极高。然而，外源性糖皮质激素能逆转这种危机状态，使心血管系统恢复稳态，各器官的功能恢复，从而可挽救患者生命[6]。糖皮质激素（GC）的生物学活性由糖皮质激素受体(Glucocorticoidreceptor，GR)介导，GR调节基因的表达，当GC与GR结合后可导致GR的活化，活化的GR入核后与正调节的糖皮质激素反应元件（Glucocorticoid response element，GRE）结合，能够诱导一些抗炎蛋白的表达，如IκB。目前认为，GR发挥抗炎作用的主要机制是通过蛋白与蛋白之间的相互作用来发挥转录抑制作用；而GC的大多数副作用主要是由其转录激活作用诱导的。实际上，糖皮质激素抢救危症和多种难治性疾病的功能很强大。尽管如此，糖皮质激素的副作用不容忽视,譬如，胃肠道应激性出血可成为各种危症患者的隐形杀手。此外，肥胖、骨质疏松症和胰岛素抵抗等副作用难以防治[7]。因此，临床急需有与糖皮质激素类似疗效而无糖皮质激素样副作用的药物。自古以来，人参被誉为“百草之王”。为用现代技术证实人参有“起死回生”的功效，本人就读的学科通过系列研究发现，人参二醇组皂苷（PDS）有与地塞米松相类似地改善失血性休克犬心肺微循环，进而逆转心肺功能等作用；在LPS诱导的急性肺损伤大鼠模型中，PDS和地塞米松都可上调IκB的表达，转位入核的NF-κB减少。使细胞因子的分泌明显减少，从而有效地阻遏了免疫系统的活化，实现对内毒素休克大鼠脏器损伤的逆转作用。特别是PDS没有地塞米松相类似的副作用。目的：拟在建立LPS诱导急性肾损伤（AKI）小鼠模型的基础上，对比研究PDS和地塞米松逆转LPS诱导AKI肾功能的作用与机制。揭示PDS和地塞米松相类似地改善AKI肾功能作用的分子机制，为人参二醇组皂苷开发成替代地塞米松等糖皮质激素的药物提供理论依据。方法与结果：1．成功地复制了LPS诱导的AKI早期动物模型，PDS与地塞米松有类似的保护AKI小鼠肾功能的作用1.1实验动物分组与处理C57BL/6小鼠随机分为4组（n=8）：盐水对照组（Control组）、LPS模型组（LPS组）、PDS+LPS组、Dexa+LPS组，对照组经腹腔注射0.9%氯化钠注射液0.5ml，其余三组分别经腹腔注射LPS10mg/kg。PDS+LPS组、Dexa+LPS组小鼠在LPS注射前1h分别经腹腔注射PDS（25.0mg/kg）或地塞米松（2.5mg/kg）。LPS处理12h后麻醉下，取肾脏、心脏、肝脏等组织冻存或固定，备蛋白表达与形态学观察；采集血液，全自动生化分析仪检测血清尿素氮（BUN）、肌酐（CREA）、谷丙转氨酶（ALT）、谷草转氨酶（AST）、乳酸脱氢酶（LDH）水平。1.2实验结果LPS组小鼠血尿素氮和肌酐水平与生理盐水对照组比较极显著增高（P<0.01）。LPS组肾脏组织切片HE染色见轻微改变。说明LPS（10mg/kg，12h）诱导的AKI模型成功，并属于AKI的早期阶段。LPS组血清谷丙转氨酶（ALT）、谷草转氨酶（AST）、乳酸脱氢酶（LDH）含量与对照组比较明显增高（P<0.01），显示LPS组已发生心脏和肝脏细胞的明显损伤，证明LPS诱导了全身炎症反应综合征。血尿素氮和肌酐含量，PDS+LPS组和Dexa+LPS组与LPS组比较均明显降低（P<0.05），说明PDS有与地塞米松相类似地保护肾功能的作用。2. PDS和地塞米松有类似的逆转LPS诱导AKI肾功能的机制2.1PDS和地塞米松影响LPS诱导的肾脏细胞凋亡相关蛋白表达应用免疫组化及免疫印迹法检测不同组凋亡相关蛋白的表达。结果显示LPS组凋亡蛋白Bax、Cleave-caspase3、Cyto C表达增多，而PDS+LPS组及Dexa+LPS组较LPS组表达减少；抗凋亡蛋白Bcl-2在LPS组表达减少，而PDS+LPS组及Dexa+LPS组较LPS组表达增多。提示注射LPS（10mg/kg）12小时后小鼠肾脏细胞发生凋亡，而PDS+LPS组及Dexa+LPS组凋亡程度较LPS组减轻。可见，PDS及地塞米松影响凋亡相关蛋白表达，减少LPS诱导的肾脏细胞凋亡。2.2PDS和地塞米松抑制NF-κB信号转导通路，减少AKI小鼠细胞因子的产生与释放2.2.1应用免疫印迹的方法检测IκB和NF-κB p65、p50的蛋白表达结果表明LPS组IκB的蛋白表达水平与对照组比较明显降低（P<0.05），PDS+LPS组IκB蛋白表达水平明显高于LPS组（P<0.05），Dexa+LPS组的IκB表达水平与LPS组比较也有增加趋势。LPS组的核NF-κB P65和P50的表达水平与对照组比较显著增高（P<0.05）。然而，与LPS组比较，PDS+LPS组和Dexa+LPS组的核NF-κB P50表达水平显著下调（P<0.05），NF-κB P65表达水平有下降趋势。可见，PDS和地塞米松抑制了NF-κB信号通路的活化。2.2.2应用酶联免疫法检测血清中TNF-α和IL-6含量结果显示，LPS组TNF-α和IL-6含量与对照组比较显著增高（P<0.01）。然而，PDS+LPS组的TNF-α和IL-6含量与LPS组比较均明显降低（P<0.05），Dexa+LPS组与LPS组比较TNF-α有下降趋势，IL-6显著降低（P<0.05）。这是PDS和地塞米松抑制了NF-κB信号通路的结果。2.3PDS和地塞米松有抑制肾脏组织氮氧自由基产生和上调SOD活性的作用LPS组iNOS蛋白表达水平和NO含量与对照组比较均显著增高（P<0.05）。然而，PDS+LPS组和Dexa+LPS组的iNOS蛋白表达水平和NO含量均显著地低于LPS组（P<0.05）。LPS组MDA含量与对照组比较明显增高（P<0.05），而清除氧自由基的酶SOD含量和Mn-SOD的蛋白表达水平明显下降（P<0.05）。LPS诱导的氧化应激加重AKI小鼠的肾脏功能障碍。然而，PDS和地塞米松则通过抗氧化应激作用保护LPS诱导的AKI肾脏功能。3．PDS与地塞米松有类似地增加细胞核GR表达的作用，可能是GR的功能配体3.1PDS明显增加了细胞核GR的表达应用免疫印迹的方法检测GR的表达。结果表明总的GR（t-GR）除对照组表达最低，其余3组t-GR表达水平均明显增高，组间没有差异。LPS组核GR（n-GR）的表达水平在各组中最低，以PDS+LPS组的n-GR表达水平为最高，Dexa+LPS组次之。故推测，对于GR来说，PDS有地塞米松样的GR配体作用。3.2PDS可能是糖皮质激素受体的功能配体，并呈剂量依赖性增加GR的转录活性在含有糖皮质激素反应元件的荧光素酶报告基因（GRE-Luciferase）与GR共转染293细胞中荧光强度很低。当加入地塞米松100μmol/L时，GR的转录活性增加36.5倍。在用不同剂量PDS取代地塞米松时，GR的转录活性如下：①加入25μg/mlPDS时，对GR的转录活性没有影响；②加入PDS为50μg/ml时GR的转录活性增加5.5倍；③当加入PDS为100μg/ml时，GR的转录活性增至12倍。可见，PDS有与地塞米松样和GR结合作用，并呈剂量依赖性增加GR的转录活性。因此，我们推测PDS可能是GR的功能配体。结论：1．人参二醇组皂苷有与地塞米松相类似地逆转LPS诱导的小鼠AKI作用。而且，这种逆转作用是通过相同的机制实现的。2．人参二醇组皂苷可能是GR功能性配体的依据：PDS和地塞米松有同样的增强细胞核糖皮质激素受体（GR）表达的作用；在含有糖皮质激素反应元件的荧光素酶报告基因（GRE-Luciferase）与GR共转染293细胞反应体系中，PDS有与地塞米松相似的与GR结合作用，并且，PDS呈剂量依赖性增加GR的转录活性，故推测PDS可能是GR的功能配体，尚需进一步研究证实。3.人参二醇组皂苷具有潜在临床应用前景。更多还原

【Abstract】 Background:Uncontrolled sepsis-induced systemic inflammatory responsesyndrome (SIRS) induced by sepsis is a main cause of multiple organfailure. And acute kidney injury (AKI) is one of the most seriouscomplications in SIRS with high mortality rate. However, exogenousglucocorticoids can reverse this state of crisis and save the lives of patientsthrough restore the cardiovascular system homeostasis and the organfunction. The biological activity of glucocorticoids (GC) is mediated by theglucocorticoid receptor (GR), which regulate gene expression. GR isactivated when the GC is combined with GR. Then activated GR enter intonucleus to combine with positive regulation GRE, which can induce theexpression of several inflammatory proteins such as IκB. It is believed thatthe transcriptional suppression which induced by the interaction of proteinand protein is the main mechanism of GR anti-inflammatory effects; whilemost of the main side effects of GC are induced by transcriptionalactivation. In fact, glucocorticoids are very powerful in rescuing riskdisease and a variety of refractory disease. However, the side effects ofglucocorticoids can not be ignored, for example, stress-inducedgastrointestinal bleeding can become dangerous in patients with a varietyof invisible killer. In addition, the side effects of obesity, osteoporosis,insulin resistance are difficult to control. Therefore, It is an urgent clinicalneed to find a drug with glucocorticoids similar curative effect without sideeffects of glucocorticoids. Since ancient times, ginseng is known as the "King of Herbs." The"revived" effect of Ginseng is confirmed by modern technology. Ourprevious results showed that the effect of panaxdiol saponins (PDS) issimilar to dexamethasone (Dexa) in improving cardiopulmonarymicrocirculation in hemorrhagic shock dogs. PDS and dexamethasone canupregulate the expression of IκB and reduce NF-κB of translocation intothe nucleus in acute lung injury induced by LPS in rat. So that thesecretion of cytokines was significantly reduced, the activation of theimmune system was surpressed, then the organ damage induced byendotoxin shock was reverse in rats. Particularly, PDS hadn’t side effects.Purpose:To establish acute kidney injury (AKI) induced by LPS in mouse, andexplore the effect and mechanism of PDS and dexamethasone on AKIinduced by LPS. It will provide a theoretical basis for PDS developed intoalternative glucocorticoid dexamethasone drugs.Methods and Results:1. LPS-induced AKI animal models were successfully established,PDS and dexamethasone had a similar effect in the protection of renalfunction in AKI mice1.1Group and TreatmentC57BL/6mice were randomly divided into four groups (n=8): salinegroup(Control group), LPS model group (LPS group), PDS+LPS group,Dexa+LPS group. The mice were injected intraperitoneally0.9%Nacl0.5ml in the control group, and the remaining mice were injectedintraperitoneally LPS10mg/kg. The mice of PDS+LPS group andDexa+LPS group were respectively injected intraperitoneally PDS(25.0mg/kg) or dexamethasone (2.5mg/kg) before1h of LPS injection.12h after LPS treatment, the kidney, heart and liver tissues of mice were taken under anesthesia and frozen or fixed them, prepared for proteinexpression and morphological observation. The blood were collected to testblood urea nitrogen (BUN), serum creatinine (CREA), alanineaminotransferase (ALT), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) level by automatic biochemical analyzer.1.2ResultsThe blood urea nitrogen and creatinine levels in LPS group mice weresignificantly higher (P<0.01) than that of saline control group. The kidneyin LPS group had minor morphological changes in HE staining. Thissuggested that LPS-induced AKI animal models were successfullyestabalished, and is in the early stages of AKI. Serum alanineaminotransferase (ALT), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) levels in LPS group were significantly higher thanthat of control group (P <0.01). It suggested that the heart and liver cells inthe LPS group was significantly damage. It proved that LPS can induce asystemic inflammatory response syndrome. Blood urea nitrogen andcreatinine levels in PDS+LPS group and Dexa+LPS group weresignificantly lower than that of LPS group (P<0.05). This suggested thatthe PDS and dexamethasone had similarly protection of renal function.2. PDS and dexamethasone have a similar mechanism in the reversalof LPS-induced AKI renal function2.1The effect of PDS and dexamethasone on apoptosis-relatedproteins in LPS-induced renal cellWe detected the expression of apoptosis-related proteins in differentgroups by immunohistochemistry and Western blot. The results showedthat the expression of Bax, Cyto C, cleaved caspase3increased in LPSgroup, while the expression in PDS+LPS group and Dexa+LPS group werelower than LPS group; the expression of anti-apoptotic protein Bcl-2in LPS group decreased and the expression in PDS+LPS group andDexa+LPS group increased compared with LPS group. This suggested thatapoptosis occurred in renal cell after12h of LPS (10mg/kg) injection, andthe degree of apoptosis reduced in PDS+LPS group and Dexa+LPS groupcompared with LPS group. it is thus clear that PDS and dexamethasoneaffect the expression of apoptosis-related proteins and reduce LPS-inducedrenal cell apoptosis.2.2PDS and dexamethasone can reduce the production and release ofcytokines through inhibiting NF-κB signaling pathway in mice AKI2.2.1The expression of IκB and NF-κB p65, p50protein was detectedby Western blotThe results showed that IκB protein expression levels in LPS groupwere significantly lower than that of control group (P<0.05). IκB proteinexpression levels in PDS+LPS group were significantly higher than that ofthe LPS group (P<0.05). Compaired with LPS group, IκB expression levelsin Dexa+LPS group also had an increasing trend. Nuclear NF-κB P65andP50expression levels in LPS group were significantly higher than that ofcontrol group (P<0.05). However, compared with the LPS group, nuclearNF-κB P50expression levels in PDS+LPS group and Dexa+LPS groupwere significantly reduced (P<0.05)，nuclear NF-κB P65expression levelshad a downward trend. So PDS and dexamethasone inhibited the activationof NF-κB signaling pathway.2.2.2The serum TNF-α and IL-6levels were tested by enzyme-linkedimmunosorbent assayThe results showed that TNF-α and IL-6levels in LPS group wassignificantly higher than that of control group (P <0.01). However, TNF-αand IL-6levels in PDS+LPS group were significantly lower than that ofLPS group (P<0.05). Compared with LPS group, TNF-α level in Dexa+LPS group had a downward trend. IL-6of Dexa+LPS group weresignificantly lower (P<0.05). It is due to NF-κB signaling pathway wassuppressed by PDS and dexamethasone.2.3PDS and dexamethasone can inhibit nitrogen-oxygen free radicalsproduction and increase SOD activity in kidney tissueiNOS protein expression and NO levels in LPS group weresignificantly higher than that of control group (P<0.05). However, iNOSprotein expression and NO levels in PDS+LPS group and Dexa+LPS groupwere significantly lower than that of LPS group (P<0.05).MDA levels in LPS group were significantly higher than that ofcontrol group (P<0.05), while the SOD levels and Mn-SOD enzymeprotein expression which scavenging oxygen free radicals decreasedobviously (P<0.05). Oxidative stress induced by LPS aggravated kidneydysfunction in AKI mice. However, PDS and dexamethasone protectedkidney function of LPS-induced AKI through resistance to oxidative stress.3. PDS and dexamethasone have a similar effect of increasing nuclearGR expression. PDS may be a functional ligand of GR3.1PDS significantly increased the expression of GR in the nucleusThe expression of glucocorticoid receptor was detected by Westernblot. The results showed that total GR（t-GR） in control group was thelowest and the t-GR expression of remaining three groups weresignificantly increased, no difference between the groups. The nuclear GR(n-GR) expression levels in LPS group was the lowest in each group, then-GR expression levels in PDS+LPS group was highest, Dexa+LPS groupfollowed. It is speculated that for GR, PDS has dexamethasone-like GRligand effects.3.2PDS may be a functional ligand of the glucocorticoid receptor andincreases transcriptional activity of GR in a dose-dependent The low fluorescence intensity can be found in containingglucocorticoid response element luciferase reporter gene (GRE-Luciferase)and GR co-transfected293cells. When adding dexamethasone(100μmol/L), the transcription activity of GR increased by36.5times.When replacing dexamethasone with different doses of PDS, GRtranscription activity was as follows:①When adding PDS (25μg/ml), thetranscriptional activity of the GR was not affected;②W henadding PDS(50μg/ml), the transcriptional activity of GR increased by5.5times;③When adding PDS(100μg/ml), the transcriptional activity of GRincreased by12times. So, PDS can bind with GR just like dexamethasoneand increase GR transcriptional activity in a dose-dependent. Thus, wethink that PDS may be a functional ligand of GR.Conclusion:1. Panaxdiol saponins and dexamethasone had similarly reversedeffect on LPS-induced AKI mice. Moreover, this reversed effect wasachieved by the same mechanisms.2. PDS may be the functional ligand of GR: PDS and dexamethasonehave the same effect of enhancing the nuclear glucocorticoid receptor (GR)expression. In the reaction system of containing glucocorticoid responseelement luciferase reporter gene (GRE-Luciferase) and GR co-transfected293cells, PDS and dexamethasone have similar effect of binding with GRand PDS increases transcriptional activity of GR in a dose-dependent. Thuswe think that PDS may be a functional ligand of GR. Further research isneeded to confirm the finding.3. PDS has potential clinical application prospect.更多还原