【作者】 叶涛；

【导师】 叶章群；

【作者基本信息】 华中科技大学 ， 外科学（泌尿外科）， 2021， 博士

【摘要】 第一部分茶黄素通过调节氧化应激减少肾脏草酸钙晶体沉积及肾小管上皮细胞损伤的作用研究【目的】晶体诱导的细胞氧化损伤在草酸钙肾结石形成的早期阶段发挥着关键作用,小管上皮细胞损伤后会导致细胞表面黏附蛋白合成增多,进一步促进结晶的黏附聚集。本部分研究拟探讨茶黄素对晶体细胞损伤和肾脏草酸钙晶体沉积的的作用和影响。【方法】在动物实验中,将30只6-8周龄的雄性C57BL/6小鼠随机分为5组,利用腹腔注射乙醛酸构建结晶性肾损伤模型:正常对照组、结晶性肾损伤模型组、低剂量茶黄素治疗组（20 mg/kg）,中剂量茶黄素治疗组（50 mg/kg）和高剂量茶黄素治疗组（100 mg/kg）。偏光显微镜和Pizzolato染色观察小鼠肾组织中结晶沉积情况;PAS染色和原位TUNEL荧光染色评估肾小管损伤和细胞凋亡情况;DHE法检测肾组织中ROS生成水平;IHC染色检测小鼠肾脏中氧化应激相关蛋白NOX2、SOD2和成石相关蛋白CD44、OPN的表达情况;测定小鼠血清肌酐和尿素氮水平。在细胞实验中,CCK-8法检测HK-2细胞增殖活性确定茶黄素治疗浓度,然后根据处理方式分为4组:正常对照组、COM处理组、20μg/ml茶黄素治疗组、50μg/ml茶黄素治疗组。检测和评估各组细胞的细胞凋亡情况,ROS生成水平,H₂O₂、LDH、MDA和SOD表达情况,成石相关蛋白CD44、OPN的表达水平以及晶体对小管上皮细胞的黏附情况。【结果】我们成功建立了小鼠结晶性肾损伤模型,造模组小鼠肾脏草酸钙晶体沉积数量,小管损伤程度和细胞凋亡数量,ROS生成水平,CD44、OPN的表达水平,血清肌酐和尿素氮水平显著高于正常对照组。而在茶黄素治疗组,上述各指标水平均明显低于造模组,且降低效果与治疗剂量呈正相关。细胞实验中,≤50μg/ml的茶黄素浓度对HK-2细胞活性无明显影响。COM晶体处理后,细胞凋亡数量、ROS生成水平、H₂O₂、LDH、MDA和SOD表达水平、成石相关蛋白CD44、OPN的表达水平和晶体黏附量均明显高于正常对照组,而经茶黄素预处理后,这些指标水平均发生逆转,且50μg/ml茶黄素效果优于20μg/ml茶黄素。【结论】茶黄素能够抑制氧化应激,改善结晶性肾损伤,减少草酸钙晶体的黏附和沉积,进而可能发挥抑制草酸钙肾结石形成作用。第二部分茶黄素通过mi R-128-3p/SIRT1轴减轻草酸钙晶体诱导的肾脏氧化损伤的机制研究【目的】在第一部分研究中,我们证实了茶黄素具有减少肾脏草酸钙晶体沉积和肾小管上皮细胞损伤的功效。在该部分研究中我们将进一步探究茶黄素对草酸钙晶体诱导的肾脏氧化损伤发生保护的潜在分子机制机制,尤其是mi R-128-3p/SIRT1轴在其中扮演的角色。【方法】在小鼠实验中,首先通过IHC染色和q PCR检测造模和接受茶黄素预处理的小鼠肾脏中SIRT1的表达情况,通过FISH染色和q PCR检测小鼠肾脏中mi R-128-3p的表达情况,然后利用Pearson相关系数分析评估SIRT1和mi R-128-3p的表达关系。在细胞实验中,分别检测COM晶体和/或茶黄素预处理后SIRT1和mi R-128-3p的表达水平,通过双荧光素酶报告实验、q PCR和western blot明确mi R-128-3p对SIRT1的表达调控作用。最后利用mi R-128-3p mimic和Agomir-128分别在HK-2细胞和小鼠肾脏中激活上调mi R-128-3p的表达,通过一系列体内外实验观察茶黄素通过调节mi R-128-3p/SIRT1轴对草酸钙晶体诱导的氧化应激、结晶性肾损伤以及晶体对肾小管上皮细胞的黏附沉积的作用和影响。【结果】首先在小鼠实验中,我们发现乙醛酸诱导肾脏草酸钙结晶沉积模型时会降低SIRT1的表达同时升高mi R-128-3p的表达,而茶黄素治疗可使SIRT1表达升高而mi R-128-3p表达降低,通过Pearson相关系数分析也证实SIRT1与mi R-128-3p表达呈负相关。在细胞实验中我们同样发现COM晶体处理小管上皮细胞后会降低HK-2细胞中SIRT1的表达同时升高mi R-128-3p的表达,而茶黄素治疗可逆转它们的表达变化。通过双荧光素酶报告实验、q PCR和western blot的实验结果也证明了mi R-128-3p能够直接靶向SIRT1的3’-UTR抑制其表达。最后,进一步的细胞和动物实验结果也说明激活mi R-128-3p可加重草酸钙晶体诱导的氧化应激、细胞凋亡和结晶黏附沉积,同时还会部分逆转茶黄素对SIRT1的表达上调作用以及对结晶引起的肾脏氧化损伤的保护作用。【结论】在草酸钙晶体引起的肾脏损伤中,茶黄素可上调SIRT1表达和下调mi R-128-3p表达,降低肾脏氧化应激压力,减少晶体黏附沉积。调控mi R-128-3p/SIRT1轴可能是茶黄素抑制结晶性肾损伤发挥肾脏保护功能的重要途径。茶黄素有望为开展草酸钙肾结石防治工作提供新的思路和选择。更多还原

【Abstract】 Part I The effect of theaflavin on reducing calcium oxalate crystal deposition and renal tubular epithelial cell injury by regulating oxidative stressObjective:Crystal-induced cellular oxidative stress injury plays a key role in the early stage of calcium oxalate（Ca Ox）kidney stone formation,tubule epithelial cell injury could increase the expression of cell surface adhesive proteins,which further promoted the adhension and aggregation of Ca Ox crystals.This study aimed to explore the role and influence of theaflavin（TF）on crystal-cell injury and Ca Ox crystal deposition in kidney.Methods:The mice model of crystalline kidney injury was established by intraperitoneal injection of glyoxylic acid,30 male C57BL/6 mice（6-8 week old）were randomly divided into 5 groups:normal control group,crystallized renal injury model group,low-dose TF treatment group（20 mg/kg）,medium-dose TF treatment group（50 mg/kg）,and high-dose TF treatment group（100 mg/kg）.Polarized light microscope and Pizzolato staining were used to observe Ca Ox crystal deposition in renal tissues of mice.Tubular injury and cell apoptosis were assessed by PAS staining and TUNEL fluorescence in situ staining.ROS production level in renal tissues was evaluated using DHE method.IHC staining was used to detect the protein expression of NOX2,SOD2,CD44,and OPN in the mice kidneys.Serum creatinine and urea nitrogen of mice were measured using commercial kits.In cell experiments,the proliferation activity of HK-2 cells was detected by CCK-8 method to determine the concentration of TF treatment,and then the cells were divided into four groups based on treatment methods:normal control group,COM treatment group,20μg/ml TF treatment group,and 50μg/ml TF treatment group.Cell apoptosis,ROS production,H₂O₂,LDH,MDA,and SOD levels,stone-related protein CD44 and OPN expression,and crystal adhesion to tubular epithelial cells were detected and evaluated in each group.Results:We successfully established the mouse model of crystalline kidney injury.The amount of Ca Ox crystal deposition,the degree of tubular injury,the number of cell apoptosis,the level of ROS production,the expression levels of CD44 and OPN,and the levels of serum creatinine and urea nitrogen in the model group mice were significantly increased compared with those in the normal control group.However,in TF treatment groups,the levels of all above indexes were obviously lower than those in the model group,and the reduction effect was positively correlated with the treatment dose.In cell experiments,the concentration of TF≤50μg/ml had no significant effect on the activity of HK-2 cells.After COM crystal treatment,the number of cell apoptosis,ROS production,H₂O₂,LDH,MDA,and SOD levels,the expression of CD44 and OPN,and crystal adhesion amounts significantly increased compared to those of normal control group;but after TF pretreatment,the levels of these indexes were reversed.The effect of 50μg/ml TF was better than that of 20μg/ml TF.Conclusions:TF could inhibit oxidative stress and improve crystalline kidney injury,reducing the adhesion and deposition of Ca Ox crystals,and thus may contribute to the inhibition of kidney stone formation.Part II The mechanism of theaflavin alleviating oxidative injury of kidney induced by calcium oxalate crystals through mi R-128-3p/SIRT1 axisObjective: According to the results of Part I,we confirmed the effect of theaflavin（TF）in reducing calcium oxalate（Ca Ox）crystal deposition and alleviating tubular epithelial cell damage.In this part,we aimed to explore the possible mechanism of TF’s protective effect on Ca Ox crystal induced renal oxidative damage,and especially to discuss the role of mi R-128-3p/SIRT1 axis.Methods: Firstly,the expression of SIRT1 in the kidneys of mice was detected by IHC and q PCR,and the expression of mi R-128-3p was detected by FISH and q PCR.Then,the expression relationship between SIRT1 and mi R-128-3p was evaluated by Pearson correlation coefficient analysis.In cell experiments,the expression levels of SIRT1 and mi R-128-3p after COM crystals with or without TF pretreatment were detected respectively,and the regulatory effect of mi R-128-3p on SIRT1 was determined using dual luciferase reporting assay,q PCR,and western blot.Finally,mi R-128-3p mimic and Agomir-128 were used to increase the expression of mi R-128-3p in HK-2 cells and the kidneys of mice,respectively.A series of experiments in vivo and in vitro were conducted to observe the effects of TF on Ca Ox crystal induced oxidative stress,crystalline kidney injury,and crystal adhesion and deposition on renal tubular epithelial cells by regulating mi R-128-3p/SIRT1 axis.Results: Firstly,we observed decreased expression of SIRT1 and increased expression of mi R-128-3p in the kidneys of glyoxylic acid-induced crystal group mice compared to those in normal control group,while TF treatment could upregulate SIRT1 expression and downregulate mi R-128-3p expression.Pearson correlation coefficient analysis confirmed that the expression of SIRT1 was negatively correlated with mi R-128-3p expression.In cell experiments,we also found that COM crystal treatment could decrease the expression of SIRT1 and increase the expression of mi R-128-3p,while TF treatment could reverse their expression changes.Subsequently,dual luciferase report assay,q PCR,and western blot results indicated that mi R-128-3p could directly target the 3’-UTR of SIRT1 to inhibit its expression.Finally,further results in cell and animal experiments also confirmed that mi R-128-3p activation could aggravate Ca Ox crystal induced oxidative stress,cell apoptosis,and crystal adhesion,and also partially reverse the upregulation of SIRT1 expression and the protective effect on crystalline kidney oxidative injury by TF supplement.Conclusions: TF could increase the expression of SIRT1 but decrease the expression of mi R-128-3p,to inhibite renal oxidative stress and reduce the adhesion and deposition of Ca Ox crystal.The regulation of mi R-128-3p/SIRT1 axis might be an important pathway for TF to alleviate renal injury caused by Ca Ox crystal and exert renal protection function.TF is expected to provide a new idea and choice for the prevention and treatment of Ca Ox kidney stones.更多还原