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中华肾病研究电子杂志

中华肾病研究电子杂志 ›› 2022, Vol. 11 ›› Issue (01) : 15 -21. doi: 10.3877/cma.j.issn.2095-3216.2022.01.003

论著

紫草素抑制缺血再灌注肾损伤后肾小管细胞的增殖和迁移
陈钰澜1, 陈健文2, 朱飞2, 王田田1, 张妍1, 刘娇娜2, 黄梦杰2, 吴玲玲2,†(), 陈香美2,†()   
  1. 1. 100853　北京，解放军总医院第一医学中心肾脏病医学部、解放军肾脏病研究所、肾脏疾病国家重点实验室、国家慢性肾病临床医学研究中心、肾脏疾病研究北京市重点实验室；100853　北京，解放军医学院
    2. 100853　北京，解放军总医院第一医学中心肾脏病医学部、解放军肾脏病研究所、肾脏疾病国家重点实验室、国家慢性肾病临床医学研究中心、肾脏疾病研究北京市重点实验室
  • 收稿日期:2021-12-06 出版日期:2022-02-28
  • 通信作者: 吴玲玲, 陈香美
  • 基金资助:
    国家自然科学基金重点项目(82030025)

Shikonin inhibited the proliferation and migration of the renal tubular cells after the renal ischemia-reperfusion injury

Yulan Chen1, Jianwen Chen2, Fei Zhu2, Tiantian Wang1, Yan Zhang1, Jiaona Liu2, Mengjie Huang2, Lingling Wu2,†(), Xiangmei Chen2,†()   

  1. 1. Department of Nephrology, First Medical Center of Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Diseases; Chinese PLA Medical School; Beijing 100853, China
    2. Department of Nephrology, First Medical Center of Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Diseases
  • Received:2021-12-06 Published:2022-02-28
  • Corresponding author: Lingling Wu, Xiangmei Chen
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引用本文：

陈钰澜, 陈健文, 朱飞, 王田田, 张妍, 刘娇娜, 黄梦杰, 吴玲玲, 陈香美. 紫草素抑制缺血再灌注肾损伤后肾小管细胞的增殖和迁移[J]. 中华肾病研究电子杂志, 2022, 11(01): 15-21.

Yulan Chen, Jianwen Chen, Fei Zhu, Tiantian Wang, Yan Zhang, Jiaona Liu, Mengjie Huang, Lingling Wu, Xiangmei Chen. Shikonin inhibited the proliferation and migration of the renal tubular cells after the renal ischemia-reperfusion injury[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2022, 11(01): 15-21.

目的

探讨丙酮酸激酶M2(PKM2)抑制剂紫草素对肾脏缺血再灌注损伤(IRI)后肾小管修复的影响。

方法

8周龄雄性C57BL/6J小鼠30只随机分为6组：假手术的Sham溶剂组和Sham紫草素组，双侧肾脏IRI模型的bIRI溶剂1 d组、bIRI紫草素1 d组、bIRI溶剂3 d组、bIRI紫草素3 d组。肾脏IRI模型建立，采用双侧肾蒂夹闭28 min处理，术后4 h腹腔注射3 mg/kg紫草素或溶剂，术后1 d或3 d处死小鼠留取样本。检测各组小鼠血清肌酐、尿素氮水平；通过PAS染色观察肾组织病理学改变；免疫荧光检测丙酮酸激酶M2、增殖细胞核抗原(PCNA)、翅荚百脉根凝集素(LTL)表达；Western印迹检测肾脏的PCNA表达。体外实验，人近端肾小管上皮HK2细胞分成8组：对照组(不做缺氧处理)、模型组(缺氧/复氧处理)、模型+紫草素剂量组(缺氧/复氧后给予0.5 μM、1.0 μM、1.5 μM、2.0 μM、3.0 μM、7.0 μM紫草素干预)。采用细胞计数试剂盒检测细胞存活率、细胞生长抑制率。细胞划痕实验检测细胞迁移力。

结果

肾小管损伤面积评分：bIRI紫草素1 d组高于bIRI溶剂1 d组(P<0.01)，bIRI紫草素3 d组高于bIRI溶剂3 d组(P＝0.0337)，而假手术的Sham溶剂组和Sham紫草素组之间没有明显差别。免疫荧光(P＝0.0331)和免疫印迹(P＝0.0228)结果均显示，bIRI紫草素3 d组小鼠肾组织的PCNA表达较bIRI溶剂3 d组明显减少。在体外实验中，与模型组比较，模型+紫草素剂量组(1.5 μM、2.0 μM、3.0 μM、7.0 μM紫草素干预)的HK2细胞存活率显著降低(P<0.001)，细胞生长抑制率升高(P<0.001)。细胞划痕实验显示，1.5 μM紫草素能够显著抑制复氧后肾小管细胞的迁移(P<0.001)。

结论

肾脏IRI损伤后，紫草素可能通过减少肾小管上皮细胞的再生和迁移来抑制肾小管修复，从而加重肾损伤。

关键词: 急性肾损伤, 肾小管上皮细胞, 紫草素, 增殖, 迁移
Objective

To investigate the effect of pyruvate kinase M2 (PKM2) inhibitor shikonin on renal tubular repair after renal ischemia-reperfusion injury (IRI).

Methods

Thirty 8-week-old male C57BL/6J mice were randomly divided into 6 groups: sham solvent group (sham-operation plus solvent), sham shikonin group (sham-operation plus shikonin); bIRI solvent 1 d group (bilateral IRI plus solvent 1 d) and bIRI shikonin 1 d group (bilateral IRI plus shikonin 1 d); bIRI solvent 3 d group (bilateral IRI plus solvent 3 d), bIRI shikonin 3 d group (bilateral IRI plus shikonin 3 d). To establish the renal IRI model, bilateral renal pedicles were clipped for 28 min, and 3 mg/kg shikonin or solvent was intraperitoneally injected 4 h after the operation. The mice were sacrificed 1 or 3 days after the operation for samples collection. The serum creatinine and blood urea nitrogen levels of mice in each group were measured. The renal histopathological changes were observed by PAS staining. The expressions of PKM2, proliferating cell nuclear antigen (PCNA), and Lotus tetragonolobus lectin (LTL) were detected by immunofluorescence. Western blotting was used to detect renal PCNA expression. For the in vitro experiments, the human proximal tubular epithelial HK2 cells were divided into 8 groups: control group (without hypoxia treatment), model group (with hypoxia/reoxygenation treatment), shikonin doses groups (hypoxia/reoxygenation plus shikonin of 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 3.0 μM, and 7.0 μM). The cell survival rate and cell growth inhibition rate were detected by the cell counting kit 8 (CCK-8). The scratch wound healing assay was applied to detect cell migration ability.

Results

The renal tubular injury area scores in the bIRI shikonin 1 d group and the bIRI shikonin 3 d group, were higher than those in the bIRI solvent 1 d group and bIRI solvent 3 d group, respectively (P=0.0337), while the renal tubular injury area scores in the sham solvent group and the sham shikonin group did not differ from each other significantly. Both immunofluorescence (P=0.0331) and western blotting (P=0.0228) results showed that the expression of PCNA in the kidneys of mice was significantly lower in the bIRI shikonin 3 d group than in the bIRI solvent 3 d group. During the in vitro experiments, compared with the model group, the shikonin doses groups (1.5 μM, 2.0 μM, 3.0 μM, and 7.0 μM) showed significantly lower survival rates of HK2 cells (P<0.001), as well as higher rates of cell growth inhibition (P<0.001). The scratch wound healing assay displayed that shikonin of 1.5 μM could obviously inhibit the migration of renal tubular HK2 cells after reoxygenation (P<0.001).

Conclusion

After renal IRI injury, shikonin may inhibit the renal tubular repair process through reducing the regeneration and migration of renal tubular epithelial cells, thereby aggravating the renal injury.

Key words: Acute kidney injury, Tubular epithelial cell, Shikonin, Proliferation, Migration
图1 小鼠肾皮髓质交界病理切片结果注：A：PAS×100；B：PAS×400; 1：Sham溶剂组；2：Sham紫草素组；3：bIRI溶剂1d组；4：bIRI紫草素1 d组；5：bIRI溶剂3 d组；6：bIRI紫草素3 d组
图2 各组肾小管损伤评分、血清肌酐、尿素氮水平注：A：各组肾小管病理损伤评分结果；B：各组血清肌酐检测结果；C：各组血清尿素氮检测结果；1: Sham溶剂组；2：Sham紫草素组；3：bIRI溶剂1 d组；4：bIRI紫草素1 d组；5：bIRI溶剂3 d组；6：bIRI紫草素3 d组；与bIRI溶剂1 d组比较，aP<0.01；与bIRI溶剂3 d组比较，bP<0.05
图3 术后3 d肾组织PCNA、LTL与PKM2免疫荧光染色及PCNA表达量注：A：PCNA、LTL、PKM2免疫荧光染色共定位，箭头所指为损伤后增殖的近端小管上皮细胞表达PKM2(免疫荧光×400，比例尺＝50 μm)；B：PCNA、DAPI免疫荧光染色(免疫荧光×400，比例尺＝50 μm)；C：免疫荧光染色图片统计细胞核PCNA阳性率；D：肾组织蛋白质Western印迹检测PCNA表达水平；PCNA：增殖细胞核抗原；LTL：翅荚百脉根凝集素；PKM2：丙酮酸激酶M2；β-actin：β肌动蛋白；与bIRI溶剂3 d组比较，aP<0.05
图4 紫草素对缺氧/复氧HK2细胞存活率和生长抑制率的影响注：A：HK2细胞存活率；B：HK2细胞生长抑制率；与模型组比较，aP<0.001；与对照组比较，bP<0.05
图5 紫草素对缺氧/复氧HK2细胞迁移的影响(40×)注：与模型组比较，aP<0.001
[11]
Uckett DL, Alquraishi M, Chowanadisai W, et al. The role of PKM2 in metabolic reprogramming: insights into the regulatory roles of non-coding RNAs [J]. Int J Mol Sci, 2021, 22(3): 1171.
[12]
Mehta RL, Cerdá J, Burdmann EA, et al. International Society of Nephrology′s 0by25 initiative for acute kidney injury (zero preventable deaths by 2025): a human rights case for nephrology [J]. Lancet, 2015, 385(9987): 2616-2643.
[13]
Sharfuddin AA, Molitoris BA. Pathophysiology of ischemic acute kidney injury [J]. Nat Rev Nephrol, 2011, 7(4): 189-200.
[14]
Ash SR, Cuppage FE. Shift toward anaerobic glycolysis in the regenerating rat kidney [J]. Am J Pathol, 1970, 60(3): 385-402.
[15]
Lee YB, Min JK, Kim JG, et al. Multiple functions of pyruvate kinase M2 in various cell types [J]. J Cell Physiol, 2022, 237(1): 128-148.
[16]
Lan R, Geng H, Singha PK, et al. Mitochondrial pathology and glycolytic shift during proximal tubule atrophy after ischemic AKI [J]. J Am Soc Nephrol, 2016, 27(11): 3356-3367.
[17]
Blum JE, Gheller BJ, Benvie A, et al. Pyruvate kinase m2 supports muscle progenitor cell proliferation but is dispensable for skeletal muscle regeneration after injury [J]. J Nutr, 2021, 151(11): 3313-3328.
[18]
Zhang X, Zheng C, Gao Z, et al. PKM2 promotes angiotensin-Ⅱ-induced cardiac remodelling by activating TGF-β/Smad2/3 and Jak2/Stat3 pathways through oxidative stress [J]. J Cell Mol Med, 2021, 25(22): 10711-10723.
[19]
Jiang H, Zou Y, Zhao J, et al. Pyruvate kinase M2 mediates glycolysis in the lymphatic endothelial cells and promotes the progression of lymphatic malformations [J]. Am J Pathol, 2021, 191(1): 204-215.
[20]
Zheng D, Jiang Y, Qu C, et al. Pyruvate kinase M2 tetramerization protects against hepatic stellate cell activation and liver fibrosis [J]. Am J Pathol, 2020, 190(11): 2267-2281.
[21]
叶寅寅，江蕾，丁昊，等. M2型丙酮酸激酶参与成纤维细胞激活及增殖介导的肾间质纤维化的研究进展[J]. 中华肾脏病杂志，2019, 35(10): 795-800.
[22]
Wei Q, Su J, Dong G, et al. Glycolysis inhibitors suppress renal interstitial fibrosis via divergent effects on fibroblasts and tubular cells [J]. Am J Physiol Renal Physiol, 2019, 316(6): F1162-F1172.
[23]
Ding H, Jiang L, Xu J, et al. Inhibiting aerobic glycolysis suppresses renal interstitial fibroblast activation and renal fibrosis [J]. Am J Physiol Renal Physiol, 2017, 313(3): F561-F575.
[24]
Alquraishi M, Puckett DL, Alani DS, et al. Pyruvate kinase M2: a simple molecule with complex functions [J]. Free Radic Biol Med, 2019, 143: 176-192.
[25]
Liu M, Wang Y, Ruan Y, et al. PKM2 promotes reductive glutamine metabolism [J]. Cancer Biol Med, 2018, 15(4): 389-399.
[26]
Chen X, Chen S, Yu D. Protein kinase function of pyruvate kinase M2 and cancer [J]. Cancer Cell Int, 2020, 20(1): 523.
[1]
Zuk A, Bonventre JV. Acute kidney injury [J]. Annu Rev Med, 2016, 67: 293-307.
[2]
Sawhney S, Marks A, Fluck N, et al. Post-discharge kidney function is associated with subsequent ten-year renal progression risk among survivors of acute kidney injury [J]. Kidney Int, 2017, 92(2): 440-452.
[3]
Warburg O. On the origin of cancer cells [J]. Science, 1956, 123(3191): 309-314.
[4]
Zhou HL, Zhang R, Anand P, et al. Metabolic reprogramming by the S-nitroso-CoA reductase system protects against kidney injury [J]. Nature, 2019, 565(7737): 96-100.
[5]
李佳颖，胡雨婷，陈丽萌. 代谢重编程与近端肾小管损伤的研究进展[J]. 中华肾脏病杂志，2020, 36(11): 892-896.
[6]
Zhang Q, Liu Q, Zheng S, et al. Shikonin inhibits tumor growth of ESCC by suppressing PKM2 mediated aerobic glycolysis and STAT3 phosphorylation [J]. J Cancer, 2021, 12(16): 4830-4840.
[7]
Ye Y, Xu L, Ding H, et al. Pyruvate kinase M2 mediates fibroblast proliferation to promote tubular epithelial cell survival in acute kidney injury [J]. FASEB J, 2021, 35(7): e21706.
[8]
Patel S, Das A, Meshram P, et al. Pyruvate kinase M2 in chronic inflammations: a potpourri of crucial protein-protein interactions [J]. Cell Biol Toxicol, 2021, 37(5): 653-678.
[9]
Cheon JH, Kim SY, Son JY, et al. Pyruvate kinase M2: a novel biomarker for the early detection of acute kidney injury [J]. Toxicol Res, 2016, 32(1): 47-56.
[10]
Lin HY, Wang X, He C, et al. Progress on biosynthesis and function of the natural products of Zi Cao as a traditional Chinese medicinal herb [J]. Yi Chuan, 2021, 43(5): 459-472.
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