余婷;张妮;陈思羽;王笑笑;左思洋;郭兵;刘丽荣;
YU Ting;ZHANG Ni;CHEN Siyu;WANG Xiaoxioa;ZUO Siyang;GUO Bing;LIU Lirong;Center for Clinical Laboratories,the Affiliated Hospital of Guizhou Medical University;School of Clinical Laboratory Science,Guizhou Medical University;Guizhou Provincial Key Laboratory of Common Chronic Disease Pathogenesis and Drug Research,Guizhou Medical University;

• 1:贵州医科大学附属医院临床检验中心
• 2:贵州医科大学医学检验学院
• 3:贵州医科大学
• 4:贵州医科大学贵州省常见慢性疾病发病机制及药物研究重点实验室

摘要(Abstract)：

目的探讨猫爪草(RTT)对缺血再灌注(IR)小鼠急性肾损伤(AKI)的作用及其可能机制。方法 15只雄性C57BL/6小鼠随机均分为假手术组(sham组)、IR组、RTT预处理组(IR+RTT组),IR+RTT组术前14 d每天按2 g/kg剂量予RTT预灌胃处理,IR组和sham组给予等体积双蒸水灌胃;sham组小鼠仅钝性剥离肾蒂,另两组小鼠夹闭双侧肾蒂45 min构建IR模型;术后24 h处死小鼠,取眼球血液及肾脏;生化法检测血肌酐(Scr)和尿素氮(BUN),HE染色观察肾组织改变,免疫组织化学、Western blot检测中性粒细胞明胶酶相关脂质运载蛋白(NGAL)、肿瘤坏死因子-α(TNF-α)、组蛋白H3赖氨酸36三甲基化(H3K36me3)、组蛋白H3赖氨酸4三甲基化(H3K4me3)的表达变化,Pearson相关性分析H3K36me3、H3K4me3、TNF-α与NGAL的相关性。结果 IR组小鼠血清Scr和BUN高于sham组(P<0.05),IR+RTT组小鼠血清Scr和BUN低于IR组(P<0.05);HE染色显示,sham组小鼠肾小管和肾小球形态结构清晰,IR组肾小管上皮细胞出现肿胀、脱落、坏死,IR+RTT组肾脏病变明显改善;免疫组织化学、Western blot结果显示,与sham组相比,IR组小鼠肾脏组织NGAL、TNF-α、H3K36me3、H3K4me3蛋白水平上调(P<0.05);与IR组相比,IR+RTT组小鼠肾脏组织NGAL、 TNF-α、H3K36me3、H3K4me3蛋白水平下调(P<0.05);Pearson相关性分析结果显示,IR-AKI小鼠肾组织H3K36me3、H3K4me3、TNF-α与NGAL表达水平均呈正相关关系(r=0.739、0.782、0.713,P<0.05)。结论 RTT对IR-AKI小鼠肾功能及肾组织损伤具有预防作用,其机制可能与下调H3K36me3、H3K4me3、TNF-α表达进而抑制炎症反应有关。
Objective To investigate the alleviating effect and possible mechanism of Ranunculus ternatus thunb(RTT) on acute kidney injury(AKI) induced by ischemia reperfusion(IR) in mice,and to provide new ideas and directions for clinical prevention and treatment of AKI.Methods Fifteen male C57 BL/6 mice were randomly divided into the sham operation group,IR group and IR+RTT group,with 5 mice in each group.IR+RTT group was preperfused with 2 g/kg RTT per day for14 days before operation,while IR group and sham group were given the same volume of double distilled water,and eyeball blood and kidneys were collected.The IR mouse model was established by clamping bilateral renal pedicle for 45 min,while the sham group was only performed with blunt stripping pedicle,and all mice were killed 24 hours after operation,then eyeball blood and kidneys were collected.Serum creatinine(Scr) and urea nitrogen(BUN),the indicators of renal function were detected by biochemical method.HE staining was used to observe the pathological changes of renal tissues.Immunohistochemical and Western blot were used to measure the expression of neutrophil gelatinase-associated lipocalin(NGAL),tumor necrosis factor-α(TNF-α),Tri-methylation of Histone H3 lysine 36(H3 K36 me3),and Tri-methylation of Histone H3 lysine 4(H3 K4 me3).Pearson Correlation Coefficient was used to analyze the correlations between H3 K36 me3,H3 K4 me3,TNF-α,and NGAL.Results Biochemical results showed that compared with the sham group,Scr and BUN levelswere significantly higher in IR group(P <0.05),and compared with IR group,the levels were significantly lower in IR+RTT group(P <0.05).HE staining showed significant morphological structure of renal tubules and glomeruli in sham group,and swelling,shedding,and necrosis in renal tubular epithelial cells in IR group,and there was obvious improvement of lesions in IR+RTT group.Immunohistochemical and Western blot showed that the protein levels of NGAL,TNF-α,H3 K36 me3,and H3 K4 me3 were significantly up-regulated in IR group compared with the sham group(P <0.05),and the protein levels of NGAL,TNF-α,H3 K36 me3,and H3 K4 me3 were significantly downregulated in IR+RTT group compared with IR group(P <0.05).Pearson correlation analysis showed that the expression of H3 K36 me3,H3 K4 me3,TNF-α were significantly positively correlated with the expression of NGAL(r=0.739、0.782、0.713,P <0.05).Conclusion RTT has a preventive effect on the injury of renal function and tissues in IR-AKI mice.It may be closely related to the inhibition of inflammatory response that is caused by down-regulating the expression of H3 K36 me3,H3 K4 me3,TNF-α.

关键词(KeyWords)： 缺血再灌注损伤;急性肾损伤;猫爪草;小鼠;组蛋白H3赖氨酸36三甲基化;组蛋白H3赖氨酸4三甲基化
ischemia reperfusion(IR);acute kidney injury(AKI);ranunculus ternatus thunb(RTT);mice;tri-methylation of histone H3 lysine 36(H3K36me3);tri-methylation of histone H3 lysine 4(H3K4me3)

Abstract:

Keywords:

基金项目(Foundation): 贵州省高层次留学人才创新创业择优资助项目[留学人才择优资助合同（2018）01];; 大学生创新创业训练计划立项项目（20195200148）

作者(Authors): 余婷;张妮;陈思羽;王笑笑;左思洋;郭兵;刘丽荣;
YU Ting;ZHANG Ni;CHEN Siyu;WANG Xiaoxioa;ZUO Siyang;GUO Bing;LIU Lirong;Center for Clinical Laboratories,the Affiliated Hospital of Guizhou Medical University;School of Clinical Laboratory Science,Guizhou Medical University;Guizhou Provincial Key Laboratory of Common Chronic Disease Pathogenesis and Drug Research,Guizhou Medical University;

DOI: 10.19367/j.cnki.2096-8388.2021.11.010

参考文献(References)：

• [1]马晓春，李旭．急性肾损伤定义诊断及其防治进展[J]．中国实用内科杂志，2012, 32(6):408-411.
• [2] SUSANTITAPHONG P, CRUZ D N, CERDA J, et al.World incidence of AKI:a meta-analysis[J]. Clinical Journal of the American Society of Nephrology, 2013, 8(9):1482-1493.
• [3]杨焕芳，何永成．急性肾损伤研究进展[J]．深圳中西医结合杂志，2016, 26(9):195-198.
• [4] OSTERMANN M, LIU K. Pathophysiology of AKI[J].Best Practice&Research Clinical Anaesthesiology, 2017,31(3):305-314.
• [5] SINGBARTL K, FORMECK C L, KELLUM J A. Kidneyimmune system crosstalk in AKI[J]. Seminars in Nephrology, 2019, 39(1):96-106.
• [6] RABB H, GRIFFIN M D, MCKAY D B, et al. Inflammation in AKI:current understanding, key questions,and knowledge gaps[J]. Journal of the American Society of Nephrology:JASN, 2016, 27(2):371-379.
• [7] MAS V R, LE T H, MALUF D G. Epigenetics in kidney transplantation[J]. Transplantation, 2016, 100(1):23-38.
• [8]苗耀东，李小江，贾英杰．猫爪草的化学成分及药理作用研究进展[J]．中草药，2014, 45(11):1651-1654.
• [9]吕小华，王会敏，韩红霞，等．猫爪草多糖免疫调节及抗氧化活性研究[J]．中国中药杂志，2010, 35(14):1862-1865.
• [10]胡春生，尹进，陈炜林，等．猫爪草对慢性咽炎人群的影响研究[J]．实用预防医学，2014, 21(12):1502-1505.
• [11]李爽，包宇实．缺血再灌注急性肾损伤机制研究进展[J]．医学综述，2020, 26(19):3848-3853.
• [12]邱立强，夏豪，江洪，等．斑蝥素阻断核转录因子κB信号通路抑制血管平滑肌细胞增殖和迁移[J]．中国循环杂志，2019, 34(5):503-510.
• [13]刘佳琨．猫爪草的药理药用研究进展[J]．医学信息，2014, 27(10):491-492.
• [14]刘莉，王凤云，韩亮．中药猫爪草的研究进展[J]．广东药科大学学报，2020, 36(1):140-144.
• [15]杨金伟，张莹．猫爪草提取部位及有效成分抗肿瘤作用的研究进展[J]．药物评价研究，2021, 44(2):446-451.
• [16]韩红霞，吕世静．猫爪草多糖对小鼠急性化学性肝损伤保护作用的研究[J]．检验医学与临床，2010, 7(9):769-770,773.
• [17]何潇，陈彦旭，李曼，等．猫爪草多糖的抗肝损伤活性研究[J]．国际药学研究杂志，2018, 45(5):360-366.
• [18]安淑娴．中性粒细胞明胶酶相关脂质运载蛋白作为急性肾损伤新的生物学标志物的研究进展[J]．国际泌尿系统杂志，2012, 32(1):107-110.
• [19]徐卫卫，伍聪聪，王笑笑，等．组蛋白H3K36me3与缺血再灌注诱导小鼠急性肾损伤的相关性研究[J]．中国病理生理杂志，2020, 36(3):525-531.
• [20]丁勇，许超，吴季辉，等．表观遗传学研究进展[J]．中国科学：生命科学，2017, 47(1):3-15.
• [21] HENIKOFF S, GREALLY J M. Epigenetics, cellular memory and gene regulation[J]. Current Biology, 2016,26(14):644-648.
• [22]RACEDO S E, WRENZYCKI C, LEPIKHOV K, et al.Epigenetic modifications and related mRNA expression during bovine oocyte in vitro maturation[J]. Reproduction, Fertility, and Development, 2009, 21(6):738-748.
• [23]LIU B, CHEN Z Y, SONG X W, et al. Oryza sativa dicer-like4 reveals a key role for small interfering RNA silencing in plant development[J]. The Plant Cell, 2007,19(9):2705-2718.
• [24]LI L X, FAN L X, ZHOU J X, et al. Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease[J]. The Journal of Clinical Investigation, 2017, 127(7):2751-2764.
• [25]SHAKESPEAR M R, HALILI M A, IRVINE K M, et al. Histone deacetylases as regulators of inflammation and immunity[J]. Trends in Immunology, 2011, 32(7):335-343.
• [26]BLACK J C, VAN RECHEM C, WHETSTINE J R. Histone lysine methylation dynamics:establishment, regulation, and biological impact[J]. Molecular Cell, 2012,48(4):491-507.
• [27]ZHAO S Q, ZHONG Y Y, FU X X, et al. H3K4 methylation regulates LPS-induced proinflammatory cytokine expression and release in macrophages[J]. Shock(Augusta, Ga), 2019, 51(3):401-406.
• [28]金冉阳，杨永健．白黎芦醇对舒尼替尼诱导肾损伤的作用及机制研究[J]．中国循环杂志，2021, 36(2):192-198.

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