【作者】 臧洪梅；

【导师】 李俊；

【作者基本信息】 安徽医科大学 ， 药剂学， 2019， 博士

【摘要】 急性肾损伤（Acute kidney injury,AKI）是一种常见的以肾功能迅速下降、组织损伤为特征的临床综合征,属于复杂的重症肾脏疾病。世界流行病学调查显示,住院患者中AKI的发病率和死亡率超过20%,严重到需要透析的AKI患者的死亡率高达50%,已经成为国内外研究的热点和焦点。AKI的防治目前以对症治疗为主,主要包括可逆性病因的及时纠正、干预治疗、保持肾脏的生理环境稳定、感染的有效控制、营养支持以及防治并发症为主。改善全球肾脏病预后组织并未推荐任何用于预防和治疗AKI的药物,因此除了肾脏透析之外,目前尚无明显抑制肾脏损伤的特异性治疗方法。近年来研究者致力于寻找新的药物治疗和预防AKI。绞股蓝皂苷XLIX（Gypenoside XLIX,Gyp XLIX）是从绞股蓝总皂苷中分离得到的达玛烷型四环三萜类皂苷,可降低糖尿病肾病模型大鼠的肾小球滤过率、尿蛋白含量以及血糖、血脂各项指标,通过上调Bcl2蛋白表达,下调Bax和VCAM-1蛋白表达,从而对糖尿病肾病大鼠肾脏功能具有保护作用。课题前期研究发现Gyp XLIX对顺铂诱导的急性肾损伤小鼠模型具有一定的保护作用。本课题将从体内外方面进一步研究Gyp XLIX对急性肾损伤、缺血再灌注损伤诱导的急性肾损伤的保护作用,并探讨其可能机制;从口服生物利用度和体内药动学方面探讨其成药性,为临床急性肾损伤的防治提供新思路。1.Gyp XLIX对顺铂刺激的HK2细胞的保护作用采用MTT法筛选Gyp XLIX对HK2细胞的无毒剂量,并考察无毒剂量下Gyp XLIX对HK2细胞活力的影响。将细胞分成对照组、模型组及模型加药组,模型组用Cis（20μM）刺激24 h,模型加药组用Gyp XLIX预处理12h,再用Cis（20μM）处理24 h。收集细胞,采用流式细胞术检测细胞凋亡情况,通过Western blot和Real-time PCR方法检测各组细胞肾小管损伤分子KIM-1蛋白及mRNA表达的水平,检测细胞凋亡蛋白Cle-caspase-3,程序性坏死调控蛋白RIPK1,RIPK3和p-MLKL的蛋白及mRNA表达;通过Real-time PCR方法检测炎性因子TNF-α,IL-6和单核细胞趋化蛋白MCP-1的mRNA表达。MTT结果显示,0.5至128μM范围内随着浓度的升高,HK2细胞的活力逐渐增强,并无抑制现象。顺铂刺激造成了HK2细胞超过50%的抑制率,而32、64、128μM浓度的Gyp XLIX均可显著提升HK2细胞受顺铂刺激后的生存率。Gyp XLIX处理组肾小管细胞损伤减轻;KIM-1的mRNA和蛋白的表达量降低;炎症相关因子MCP-1、IL-6、TNF-α的mRNA水平下降;程序性坏死相关蛋白RIPK1、RIPK3及凋亡相关蛋白Cle-caspase-3的蛋白表达均明显下调。以上结果表明,Gyp XLIX对顺铂刺激的HK2细胞具有一定的保护作用。2.Gyp XLIX对缺氧/复氧损伤HK2细胞的保护作用细胞在37℃,5%CO₂的培养箱,含5%FBS的DMEM-F12培养基中生长至60%,将细胞转至37℃的无血清、低糖培养基（94%N₂、5%CO₂和1.0%O₂）中培养12 h。随后,将细胞在含2%FBS的DMEM-F12培养基中培养6 h,进行复氧。模型加药组用Gyp XLIX预处理24 h。同上检测HK2细胞肾小管损伤分子KIM-1蛋白、炎症及细胞程序性死亡相关指标的变化,结果显示,Gyp XLIX处理组肾小管细胞损伤减轻;KIM-1的mRNA和蛋白的表达量降低;炎症相关因子MCP-1,IL-6,TNF-α的mRNA表达下降;程序性坏死相关蛋白RIPK1,RIPK3及凋亡相关蛋白Cle-caspase-3的蛋白表达均明显下调。以上结果表明,Gyp XLIX对缺氧/复氧刺激的HK2细胞具有一定的保护作用。3.Gyp XLIX对顺铂诱导的急性肾损伤小鼠模型的保护作用将40只6-8周龄,体重18-20 g的C57BL/6小鼠适应性饲养1周,随机分为正常组、模型组和模型给药组（低、中、高剂量组）。正常组（NC组）腹腔注射生理盐水作为对照;模型组腹腔注射20 mg/kg的顺铂诱导急性肾损伤模型。模型给药组先采用25,50,100 mg/mL的Gyp XLIX溶液腹腔注射预处理,12 h后腹腔注射20 mg/kg的顺铂,连续给Gyp XLIX 3天后麻醉状态下处理,取血和肾脏组织。结果表明,与模型组相比,Gyp XLIX处理组肾小管的细胞损伤减轻;肌酐和尿素氮含量明显降低;KIM-1的mRNA和蛋白的表达量降低;炎症相关因子MCP-1、IL-6、TNF-α的mRNA表达下降;程序性坏死相关蛋白RIPK1、RIPK3及凋亡相关蛋白Cle-caspase-3的表达均明显下调。4.Gyp XLIX对缺血再灌注诱导的急性肾损伤小鼠模型的保护作用将6-8周龄,体重18-20 g的C57BL/6小鼠适应性饲养1周,随机分为假手术组（Shame组）、缺血再灌注模型组（IRI组）、模型给药组（Gyp组）。在IRI手术前3天腹腔注射Gyp XLIX（50 mg/kg）,每日1次。建立IRI模型24 h后,取血和肾脏组织备用。结果表明,与模型组相比,Gyp XLIX治疗组肾小管的细胞损伤减轻;肌酐和尿素氮含量明显降低;KIM-1的mRNA和蛋白的表达量降低;炎症相关因子MCP-1,IL-6,TNF-α的mRNA表达下降;程序性坏死相关蛋白RIPK1,RIPK3及凋亡相关蛋白Cle-caspase-3的表达均明显下调。以上结果表明,在小鼠体内Gyp XLIX对缺血再灌注诱导的急性肾损伤具有一定的保护作用。5.Gyp XLIX对顺铂诱导的急性肾损伤小鼠和HK2细胞保护机制将细胞分为A（空白对照组）、B（Cis组）、C（Gyp XLIX组）和D（Gyp XLIX+Cis组）组,采用RNA-seq技术对Gyp XLIX处理后肾小管上皮细胞HK-2细胞差异表达的mRNA进行筛选,筛选条件为FDR<0.05且|log2FC|>1;并应用生物信息学分析方法进行基因富集情况的分析,探讨Gyp XLIX对肾脏保护作用的机制。筛选结果显示Gyp XLIX可以下调IGF通路相关蛋白中IGFBP-7的表达,从而可能对IGF通路及其下游通路产生一定影响,影响肾脏细胞的凋亡、增殖。进一步验证结果显示,Gyp XLIX可下调IGFBP-7表达,上调p-IGF1R表达。通过siRNA沉默IGFBP-7基因发现,沉默IGFBP-7后不仅可以抑制顺铂诱导的KIM-1表达的升高、炎症反应以及细胞程序性坏死的发生,同时可以激活IGF通路,引起其下游PI3K-AKT-mTOR通路的激活,促进细胞增殖和修复,因此对顺铂诱导的肾脏损伤具有保护作用。6.Gyp XLIX体内药动学性质预测及大鼠体内药动学、组织分布研究药动学研究表明,Gyp XLIX口服吸收较为迅速,体内滞留时间较长,可快速分布到大鼠全身器官,在肾脏中浓度较高,为其临床用于治疗急性肾损伤提供了较好的基础,但是存在口服生物利用度较低的情况,可进一步研究其体内代谢过程及寻求提高其生物利用度的方法,为其临床应用奠定理论基础。综上所述,本课题研究发现在顺铂诱导的AKI和缺血再灌注损伤引起的AKI小鼠模型中、顺铂刺激的HK2细胞以及缺氧\复氧HK2细胞中,Gyp XLIX不仅可以抑制的KIM-1的表达,抑制炎症、凋亡和程序性坏死,还可通过下调IGFBP-7表达,上调p-IGF1R表达,激活IGF通路,进而活化下游PI3K-AKT-mTOR通路,促进细胞增殖,对急性肾损伤模型产生保护作用。Gyp XLIX水溶性小,分子量大,在体内快速分布,在肾脏中浓度较高,但是生物利用度低,后期将进行体内药物代谢和制剂学研究,为急性肾损伤的防治提供新的思路。更多还原

【Abstract】 Acute kidney injury（AKI）is a common,complex,and severe clinical syndrome characterized by rapid decline in renal function,combined with tissue damage.According to the world epidemiological survey,the incidence of AKI in hospitalized patients are21%,and the mortality rate of AKI patients requiring dialysis treatment is 50%.At present,the prevention and treatment of AKI are mainly focused on symptomatic treatment,which involves timely correction of reversible causes,interventional treatment,maintaining the stability of the renal physiological environment of the kidney,effectively controlling infection,providing nutritional support,and preventing/treating complications.No drugs for the prevention and treatment of AKI have been recommended by Kidney disease improving global outcomes（KDIGO）.Therefore,except for renal dialysis,there is no specific treatment for AKI.Recently,researchers have devoted themselves to finding new drugs for preventing and treating AKI.At present,the drugs used for the treatment and prevention of AKI are mainly divided into three types based on their general mechanism of action:promoting the anti-inflammatory response,inhibiting cellular apoptosis,and promoting cellular proliferation.Gypenoside XLIX（Gyp XLIX）is a tetracyclic,triterpene saponin of the Damarane type that is isolated from gypenoside.It has been reported that Gyp XLIX reduces the glomerular filtration rate,urinary protein content,blood sugar levels,and blood lipid levels in a rat model of diabetic nephropathy.It preserves renal function in this rat model of diabetic nephropathy by upregulating the protein expression of Bcl2 while downregulating the expression of Bax and VCAM-1 protein.Previous studies conducted by our group have shown that Gyp XLIX has a protective effect against cisplatin-induced AKI in mice.This study will further examine the protective effects and mechanism of Gyp XLIX on cisplatin-induced and ischemia reperfusion-induced AKI in vitro and in vivo.This study will also identify its pharmacokinetic properties of Gyp XLIX after oral administration in vivo.This study will explore the druggability of Gyp XLIX and provide new ideas for the prevention and treatment of AKI.1.Protective effects of Gyp XLIX on cisplatin-treated HK2 CellsThe MTT assay was used to determine a non-toxic concentration of Gyp XLIX on HK2cells to investigate the effect of Gyp XLIX on the viability of HK2 cells.Cells were divided into three groups;the model group was stimulated with cisplatin（Cis 20μM）for24 h,while the drug group was pretreated with varying concentrations of Gyp XLIX and treated with cisplatin（20μM）for 24 h.Cells were collected and apoptosis was detected using flow cytometry.The expression of KIM-1,cle-caspase-3,RIPK1,RIPK3,and p-MLKL proteins and their mRNA were detected using western blot and real-time PCR.Real-time PCR was used to detect the difference in the expression of several inflammatory factors:TNF-a,IL-6,and MCP-1.The results of the MTT assay showed that the viability of HK2 cells increased gradually with the increase in concentration from0.5 to 128μM,and no inhibition was observed.Cisplatin-stimulated HK2 cells showed a rate of growth that was over 50%lower than control group.Gyp XLIX（32,64,and 128μM）significantly improved the survival rate of HK2 cells exposed to cisplatin.In the Gyp XLIX-treatment group,the cellular damage was alleviated,and the expression of KIM-1 was decreased.The expression of MCP-1,IL-6,and TNF-αwas decreased after treatment with Gyp XLIX.Furthermore,the expression of programmed necrosis-related proteins（RIPK1,RIPK3）and apoptosis-related proteins（cle-caspase-3）were significantly lower after treatment with Gyp XLIX.These results suggest that Gyp XLIX has a protective effect on HK2 cells exposed to cisplatin.2.Protective effect of Gyp XLIX on hypoxia/reoxygenation injured HK2 cellsCells in the Hypoxia-reoxygenation injury group were cultured in DMEM-F12 medium containing 5%FBS（37℃,5%CO₂）and grew up to 60%.Then,the cells were cultured in serum-free and low-sugar medium under hypoxic conditions（94%N₂,5%CO₂ and1.0%O₂）at 37℃for 12 hours.Subsequently,the cells were cultured in DMEM-F12medium containing 2%FBS for 6 hours under normal conditions to promote reoxygenation.The cells in the drug group were treated with Gyp XLIX for 24 hours.The results showed that hypoxia-reoxygenation injury caused HK2 cellular damage,increased the expression of KIM-1,and upregulated expression of MCP-1,IL-6,and TNF-α,upregulated the expression of RIPK1 and RIPK3 as well as cle-caspase-3.Gyp XLIX treatment reduced the damage to HK2 cells and decreased the expression of KIM-1,MCP-1,IL-6,TNF-α,RIPK1,RIPK3,and cle-caspase-3.These results suggest that Gyp XLIX has a protective effect on HK2 cells subjected to ischemia-reperfusion injury.3.Protective effect of Gyp XLIX on cisplatin-induced AKI in miceForty C57BL/6 mice（aged 6-8 weeks and weighing 18-20 g）were fed in a specific pathogen-free facility for 1 week.They were randomly divided into five groups:the normal group,the model group,and the treated group（low,medium,and high-dose group）.Animals in the normal group（NC group）were injected intraperitoneally with saline,while animals in the model and treated groups were injected intraperitoneally with20 mg/kg of cisplatin to induce AKI.The treated group was injected intraperitoneally Gyp XLIX（25,50,or 100 mg/mL）daily for 3 days.Under anesthesia,mice were euthanized and blood and kidney tissues were collected.Compared with the model group,the cellular damage was much less severe in the renal tubules of mice in the Gyp XLIX-treated group.Serum levels of creatinine and urea nitrogen were significantly reduced after treatment with Gyp XLIX.The expression of KIM-1,MCP-1,IL-6,TNF-α,RIPK1,RIPK3,and cle-caspase-3 were significantly reduced.These results suggest that Gyp XLIX has an protective effect on cisplatin-induced AKI in mice in vivo.4.Protective effect of Gyp XLIX against ischemia reperfusion-induced acute renal injury in miceC57BL/6 mice（aged 6-8 weeks and weighing 18-20 g）were fed in a specific pathogen-free facility for 1 week.They were randomly divided into three groups:the sham-operated group（Sham group）,the model group（IRI group）,and the administration group（Gyp group）.Gyp XLIX（50 mg/kg）was injected intraperitoneally once per day for three days before IRI.After 24 hours of establishing the IRI model,blood and kidney tissues were collected.These results showed that the damage to the renal tubules in the Gyp XLIX-treated mice was much less severe than levels seen in the model group.Furthermore,the serum levels of creatinine and urea nitrogen were significantly decreased after Gyp XLIX treatment.The expression of KIM-1,MCP-1,IL-6,TNF-α,RIPK1,RIPK3,and cle-caspase-3 were decreased after Gyp XLIX treatment.These results suggest that Gyp XLIX has a protective effect against in vivo AKI induced by ischemia-reperfusion in mice.5.Protective mechanism of Gyp XLIX against cisplatin-induced damage in HK2 cellsCells were divided into the following four groups:A（control）,B（Cis group）,C（Gyp XLIX group）and D（Gyp XLIX+Cis group）.RNA-seq was used to screen differentially expressed genes among HK-2 cells treated with Gyp XLIX.The screening conditions were as follows:FDR<0.05 and|log2FC|>1.Bioinformatics analyses were used to analyze the difference of genes further explore the protective mechanisms of Gyp XLIX treatment.Gyp XLIX downregulated the expression of mRNA for inflammatory cytokines such as TNF-α,IL-6,and IL-8;alleviate the inflammatory response in cisplatin-exposed HK2 cells,lowered the expression of KIM-1 mRNA,and reduced renal injury.To verify these results,we found that Gyp XLIX downregulated the expression of TNF-α,IL-6,and MCP-1,as well as protein levels of p-P65,which reduced inflammation.At the same time,Gyp XLIX could downregulated the apoptosis-related protein cle-caspase-3 and programmed necrosis-related proteins RIPK1,RIPK3,and MLKL.Furthermore,reducing the expression of KIM-1 gene and protein protected against AKI.The results of gene sequencing analyses also showed that Gyp XLIX may activate the IGF pathway by downregulating IGFBP-7.This action would thereby activate the downstream PI3K-AKT pathway,inhibit apoptosis,and prevent the programmed necrosis of renal cells.Further verification showed that Gyp XLIX downregulated IGFBP-7 and upregulated the expression of p-IGF1R.Silencing the IGFBP-7 gene by siRNA prevents the upregulation of KIM-1,reduces inflammation,and prevents cisplatin-induced programmed necrosis.Furthermore,it also activates the IGF pathway and the downstream PI3K-AKT pathway,and promotes cellular proliferation and repair.Therefore,Gyp XLIX has protective effect against cisplatin-induced renal injury.6.Study of the pharmacokinetics of Gyp XLIX in vivoThe LC/MS/MS method was developed for the determination of Gypenoside in rat plasma and tissue homogenates.Gyp A was used as an internal standard and MRM was used as the monitoring mode.This method was applied to the in vivo pharmacokinetic properties and tissue distribution of Gyp XLIX.This method is simple to operate,has a high level of precision,high accuracy,high recovery,high sensitivity,and produces no obvious matrix effects.Pharmacokinetic studies showed that Gyp XLIX is rapidly absorbed following oral administration and has a long in vivo retention time.Gyp XLIX is rapidly distributed to all organs,especially the kidneys.These data provide a strong basis for the clinical application of Gyp XLIX for the treatment of AKI.However,in view of its low bioavailability following oral administration,we should further study the in vivo metabolism of Gyp XLIX and seek ways to improve its bioavailability.This will further lay a theoretical foundation for its clinical use.In summary,the present study found that Gyp XLIX protects against cisplatin and ischemia reperfusion-induced AKI in mice and alleviated the damage resulting from cisplatin exposure and hypoxia-reoxygenation in HK2 cells.Gyp XLIX inhibits the expression of KIM-1,reduces inflammation,prevents apoptosis and programmed necrosis.Furthermore,it also activates the IGF pathway and the downstream PI3K-AKT-mTOR pathway by downregulating IGFBP-7 expression and increasing levels of p-IGF1R.Gyp XLIX has poor solubility in water,a high molecular weight,rapid distribution in vivo,and high concentrations in the kidney.Unfortunately,its bioavailability is low after oral administration.Future in vivo metabolism and pharmacokinetic studies will be carried out to provide new ideas for the prevention and treatment of AKI.更多还原