七氟烷对大鼠肾缺血再灌注损伤的影响及其机制

doi:10.3760/cma.j.cn101202-20221209-00396

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摘要目的探讨七氟烷对大鼠肾缺血再灌注损伤的影响及其作用机制。方法选取6~8周龄、体质量（250±30） g雄性SD大鼠40只,按数字表法随机分成假手术组（Sham组）、肾缺血再灌注组（IR组）、七氟烷预处理组（Sev组）、七氟烷预处理+沉默信号调节因子1（SIRT1）抑制剂组（EX组）4组,每组10只。肾缺血再灌注损伤模型制备前2天和术前10 min,Sham组、IR组、Sev组大鼠腹腔注射1% 二甲基亚砜（DMSO）溶液（5 mL/kg）,EX组腹腔注射含抑制剂EX-527（1 mg/mL）的1% DMSO溶液（5 mL/kg）。Sham组：切除右侧肾脏,暴露左侧肾脏但不夹闭肾蒂;IR组：切除右侧肾脏,制备左侧肾脏缺血再灌注模型;Sev组、EX组：先吸入七氟烷45 min后,再按IR组方法制备缺血再灌注模型。于制备缺血再灌注模型术后3 h,收集各组大鼠肾脏组织与左心室血液,取材后以颈椎脱位法处死大鼠。观察项目：（1）检测各组大鼠左心室血液血清肌酐（Scr）、尿素氮（BUN）。（2）制备各组大鼠肾脏组织病理切片,光镜下观察肾组织病理变化,对肾小管按Paller评分标准进行评分,评估肾脏损伤情况。（3）采用原位末端脱氧核苷酸转移酶介导的dUTP切口末端标记技术（TUNEL）检测各组大鼠肾脏组织细胞凋亡情况。（4）取各组大鼠肾脏组织病理切片,在透射电子显微镜下观察肾脏组织自噬情况。（5）采用蛋白质印迹法（Western blotting）检测各组大鼠肾脏组织SIRT1、叉头盒转录因子O3（FoxO3）蛋白,以及凋亡蛋白（BAX/Bcl-2）、自噬相关蛋白（Beclin1、LC3A/B）的表达水平。结果（1）Sham组大鼠血清Scr和BUN分别为（50.74±5.91） μmol/L和（10.11±0.80） mmol/L,IR组分别为（90.18±11.22） μmol/L和（53.39±6.29） mmol/L,Sev组分别为（63.70±8.69） μmol/L和（27.68±3.41） mmol/L,EX组分别为（80.18±9.15） μmol/L和（33.20±3.57） mmol/L。与Sham组相比,IR组、Sev组、EX组血清Scr、BUN水平均升高;与IR组相比,Sev组、EX组血清Scr、BUN水平均下降;与Sev组相比,EX组血清Scr、BUN 水平均升高：差异均有统计学意义（P值均<0.05）。（2）Sham组肾小球和肾小管形态结构基本正常;IR组肾小管上皮组织肿胀明显,肾小管细胞排列紊乱,大量细胞核固缩,大量中性粒细胞浸润;Sev组：肾小管上皮细胞轻中度肿胀,少见核固缩坏死,中性粒细胞浸润明显减少;EX组：肾小管上皮组织肿胀明显,细胞核固缩和溶解增多,中性粒细胞浸润较多。Sham组、IR组、Sev组、EX组肾小管Paller评分分别为（0.61±0.15）、（5.05±0.55）、（2.68±0.33）、（4.51±0.49）分。与Sham组比较,IR组、Sev组、EX组Paller评分均升高;与IR组比较,Sev组、EX组Paller评分均下降;与Sev组相比,EX组Paller评分升高：差异均有统计学意义（P值均<0.05）。（3）Sham组、IR组、Sev组、EX组细胞凋亡率分别为8.71%±0.68%、38.15%±2.23%、14.51%±2.02%、32.55%±2.89%。与Sham组比较,IR组、Sev组、EX组细胞凋亡率均升高;与IR组比较,Sev组、EX组细胞凋亡率下降;与Sev组比较,EX组细胞凋亡率升高：差异均有统计学意义（P值均<0.05）。（4）与Sham比较,IR组、Sev组、EX组大鼠肾组织自噬小体数量均增加;与IR组比较,Sev组、EX组自噬小体数量增加且体积增大;与Sev组比较,EX组自噬小体数量明显减少：差异均有统计学意义（P值均<0.05）。（5）与Sham组比较,IR组和EX组大鼠肾脏组织FoxO3、Beclin1、LC3A/B蛋白、BAX/Bcl-2蛋白的相对表达量均升高,SIRT1的相对表达量均降低;Sev组SIRT1、FoxO3、Beclin1、LC3A/B、BAX/Bcl-2蛋白的相对表达量均升高：差异均有统计学意义（P值均<0.05）。与IR组比较,Sev组SIRT1、Beclin1、LC3A/B蛋白的相对表达量均升高,FoxO3、BAX/Bcl-2蛋白的相对表达量均降低;EX组SIRT1蛋白的相对表达量升高,FoxO3、BAX/Bcl-2蛋白的相对表达量均降低：差异均有统计学意义（P值均<0.05）。与Sev组比较,EX组SIRT1、Beclin1、LC3A/B蛋白的相对表达量均降低,FoxO3、BAX/Bcl-2蛋白的相对表达量均升高,差异均有统计学意义（P值均<0.05）。结论七氟烷可通过促进自噬,抑制细胞凋亡,减轻大鼠肾缺血再灌注损伤,其机制可能与激活SIRT1/FoxO3信号通路有关。

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	杨传铭
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关键词 ：再灌注损伤, 七氟烷, 肾损伤, 大鼠, 模型, 动物, 自噬, 沉默信号调节因子1, 叉头盒转录因子O3

Abstract：Objective This study aimed to investigate the effect of sevoflurane on renal ischemia-reperfusion injury in rats and its mechanism. Methods Forty male SD rats, aged 6-8 weeks old with a body weight of (250±30) g, were randomly divided into four groups: the sham surgery group (Sham group), renal ischemia-reperfusion group (IR group), sevoflurane pretreatment group (Sev group), and sevoflurane pretreatment+silencing signal regulator 1 (SIRT1) inhibitor group (EX group), with 10 rats in each group. Two days before the preparation of the renal ischemia-reperfusion injury model and 10 min before surgery, the rats in the Sham, IR, and Sev groups were intraperitoneally injected with 1% dimethyl sulfoxide (DMSO) solution (5 mL/kg), and the EX group was intraperitoneally injected with 1% DMSO solution (5 mL/kg) containing the EX-527 inhibitor (1 mg/mL). In the Sham group, the right kidney was removed, and the left kidney was exposed but not clamped. In the IR group, the right kidney was resected, and an left renal ischemia-reperfusion model was established. In the Sev group and EX group, sevoflurane was inhaled for 45 min, and then an ischemia-perfusion model was prepared in accordance with the method used in the IR group. Three hours after the preparation of the ischemia-reperfusion model, renal tissue and left ventricular blood of each group were collected, and the rats were sacrificed by cervical dislocation after the material was collected. Observation was performed as follows: (1) Serum creatinine (Scr) and urea nitrogen (BUN) were detected in the left ventricle of rats in each group. (2) Pathological sections of kidney histopathology of each group of rats were prepared; pathological changes in kidney tissue were observed under light microscopy, and renal tubules were scored in accordance with the Paller scoring standard to evaluate kidney damage. (3) In-situ terminal deoxynucleotidyl transferase-mediated dUTP incision-end labeling technology was used to detect apoptosis in kidney tissue cells of rats. (4) The pathological sections of kidney tissue in each group were taken, and the autophagy of kidney tissue was observed under transmission electron microscopy. (5) Western blotting was used to detect the expression level of SIRT1, forkhead box transcription factor O3 (FoxO3), and apoptotic (BAX/Bcl-2) and autophagy-related (Beclin1, LC3A/B) proteins in rat kidney tissue. Results (1) Scr and BUN detection results in each group were shown as follows: (50.74±5.91) μmol/L and (10.11±0.80) mmol/L in the Sham group, (90.18±11.22) μmol/L and (53.39±6.29) mmol/L in the IR group, (63.70±8.69) μmol/L and (27.68±3.41) mmol/L in the Sev group, and (80.18±9.15) μmol/L and (33.20±3.57) mmol/L in the EX group. Compared with the Sham group, serum Scr and BUN levels were increased in the IR group, Sev group, and EX group. Compared with the IR group, serum Scr and BUN levels decreased in the Sev group and EX group. Compared with the Sev group, serum Scr and BUN levels were increased in the EX group. The differences were statistically significant (all P values<0.05). (2) Renal histopathological changes in each group: the morphological structure of glomeruli and renal tubules in the Sham group was basically normal; in the IR group, the tubular epithelial tissue was swollen; the tubular cells were arranged disorderly; a large number of nuclei were solidified, and a large number of neutrophils were infiltrated; in the Sev group, tubular epithelial cells swell mildly, nuclear shrinkage necrosis occurred rarely, and neutrophil infiltration significantly reduced; in the EX group, renal tubular epithelial tissue swelling was evident, nucleus contraction and lysis were increased, and neutrophil infiltration is high. The tubular Paller scores of the Sham group, IR group, Sev group, and EX group accounted for (0.61±0.15),(5.05±0.55),(2.68±0.33),(4.51±0.49). Compared with the Sham group, the Paller scores were higher in the IR group, Sev group, and EX group. Compared with the IR group, the Paller scores of the Sev group and EX group decreased. Compared with the Sev group, the Paller score was increased in the EX group, and the difference was statistically significant (all P value<0.05). (3) Apoptosis of renal histocytes in each group: the apoptotic rates in the Sham group, IR group, Sev group, and EX group accounted for 8.71%±0.68%, 38.15%±2.23%, 14.51%±2.02%, and 32.55%±2.89%, respectively. Compared with the Sham group, the apoptotic rate of cells in the IR group, Sev group, and EX group was increased. Compared with the IR group, the apoptotic rate of cells in the Sev group and EX group decreased. Compared with the Sev group, the apoptotic rate of cells in the EX group was increased, and the difference was statistically significant (all P value<0.05). (4)Autophagy in renal tissues of each rat group was compared under electron microscopy. Compared with the Sham group, the number of autophagies in the IR group, Sev group, and EX group increased. Compared with the IR group, the number and volume of autophagies increased in the Sev group and EX group. Compared with the Sev group, the number of autophagies in the EX group was significantly reduced. The difference was statistically significant (all P value<0.05). (5) The expression level of SIRT1, FoxO3, and apoptotic (BAX/Bcl-2) and autophagy-related (Beclin1 and LC3A/B) proteins was compared in renal tissues of rats. Compared with the Sham group, the relative expression level of FoxO3, Beclin1, LC3A/B protein, and BAX/Bcl-2 protein in the IR and EX groups was increased, whereas that of of SIRT1 was decreased. In addition, the relative expression level of SIRT1, FoxO3, Beclin1, LC3A/B, and BAX/Bcl-2 proteins in the Sev group was all increased, with statistical significance (all P values<0.05). Compared with the IR group, the relative expression level of SIRT1, Beclin1, and LC3A/B proteins was increased in the Sev group, whereas the relative expression level of FoxO3 and BAX/Bcl-2 proteins was decreased. The relative expression level of SIRT1 protein was increased in the EX group, whereas the relative expression level of FoxO3 and BAX/Bcl-2 proteins was decreased. The differences were statistically significant (all P values<0.05). Compared with the Sev group, the relative expression level of SIRT1, Beclin1, and LC3A/B proteins in the EX group was decreased, whereas the relative expression level of FoxO3 and BAX/Bcl-2 proteins was increased: the differences were statistically significant (all P values<0.05). Conclusion Sevoflurane treatment can protect rats from renal ischemia-reperfusion injury by promoting autophagy and inhibiting apoptosis by an underlying mechanism related to the activation of the SIRT1-FoxO3 signaling pathway.

Key words： Reperfusion injury Sevoflurane Kidney injury Rats Models animal Autophagy Silent information regulator sirtuin 1 Forkhead transcription factor O3

收稿日期: 2022-12-09

基金资助:安徽省高校省级自然科学研究重点项目（KJ2017A246、KJ2020A0581）; 蚌埠医学院研究生科研创新计划项目（Byycxz20019）

通讯作者: 李晓红,Email：Lxh552@hotmail.com

引用本文:

杨传铭, 易铭, 韩冰, 张一粟, 李晓红. 七氟烷对大鼠肾缺血再灌注损伤的影响及其机制[J]. 中华解剖与临床杂志, 2023, 28(11): 749-757.
Yang Chuanming, Yi Ming, Han Bing, Zhang Yisu, Li Xiaohong. Protective effect and mechanism of sevoflurane in renal ischemia-reperfusion injury in rats. Chinese Journal of Anatomy and Clinics, 2023, 28(11): 749-757.

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http://www.cjac.com.cn/CN/10.3760/cma.j.cn101202-20221209-00396 或 http://www.cjac.com.cn/CN/Y2023/V28/I11/749

[1]	Lugo-Baruqui JA, Ayyathurai R, Sriram A, et al.Use of mannitol for ischemia reperfusion injury in kidney transplant and partial nephrectomies-review of literature[J]. Curr Urol Rep, 2019,20(1):6. DOI: 10.1007/s11934-019-0868-6
[2]	Steichen C, Giraud S, Bon D, et al.Barriers and advances in kidney preservation[J]. Biomed Res Int, 2018,2018:9206257. DOI: 10.1155/2018/9206257
[3]	Hou J, Rao M, Zheng W, et al.Advances on cell autophagy and its potential regulatory factors in renal ischemia-reperfusion injury[J]. DNA Cell Biol, 2019,38(9):895-904. DOI: 10.1089/dna.2019.4767
[4]	Ding C, Han F, Xiang H, et al.Role of prostaglandin E2 receptor 4 in the modulation of apoptosis and mitophagy during ischemia/reperfusion injury in the kidney[J]. Mol Med Rep, 2019,20(4):3337-3346. DOI: 10.3892/mmr.2019.10576
[5]	Guo X, Li Z, Zhu X, et al.A coherent FOXO3-SNAI2 feed-forward loop in autophagy[J]. Proc Natl Acad Sci U S A, 2022,119(11):e2118285119. DOI: 10.1073/pnas.2118285119
[6]	Murtaza G, Khan AK, Rashid R, et al.FOXO transcriptional factors and long-term living[J]. Oxid Med Cell Longev,2017, 2017: 3494289. DOI: 10.1155/2017/3494289
[7]	Zhou F, Wang YK, Zhang CG, et al.MiR-19a/b-3p promotes inflammation during cerebral ischemia/reperfusion injury via SIRT1/FoxO3/SPHK1 pathway[J]. J Neuroinflammation, 2021,18(1):122. DOI: 10.1186/s12974-021-02172-5
[8]	Ji Z, Wu W, Zhou F, et al.Effects of sevoflurane exposure on apoptosis and cell cycle of peripheral blood lymphocytes, and immunologic function[J]. BMC Anesthesiol, 2021,21(1):87. DOI: 10.1186/s12871-021-01305-w
[9]	Xu X, Deng R, Zou L, et al.Sevoflurane participates in the protection of rat renal ischemia-reperfusion injury by down-regulating the expression of TRPM7[J]. Immun Inflamm Dis, 2023,11(1): e753. DOI: 10.1002/iid3.753
[10]	Yamamoto M, Morita T, Ishikawa M, et al.Specific microRNAs are involved in the reno‑protective effects of sevoflurane preconditioning and ischemic preconditioning against ischemia reperfusion injury in rats[J]. Int J Mol Med, 2020,45(4):1141-1149. DOI: 10.3892/ijmm.2020.4477
[11]	胡彦,王锁刚,翟琼瑶,等.两种术式大鼠急性肾缺血再灌注损伤模型建立的对比研究[J].医学研究生学报,2021,34(3): 238-243. DOI: 10.16571/j.cnki.1008-8199.2021.03.003.Hu Y, Wang SG, Zhai QY, et al.A comparative study on the establishment of two surgical models of acute renal ischemia-reperfusion injury in rats[J]. Journal of Medical Postgraduates,2021,34(3): 238-243. DOI: 10.16571/j.cnki.1008-8199.2021.03.003
[12]	Lee HT, Ota-Setlik A, Fu Y, et al.Differential protective effects of volatile anesthetics against renal ischemia-reperfusion injury in vivo[J]. Anesthesiology, 2004,101(6):1313-1324. DOI: 10.1097/00000542-200412000-00011
[13]	Paller MS, Hoidal JR, Ferris TF.Oxygen free radicals in ischemic acute renal failure in the rat[J]. J Clin Invest, 1984,74(4):1156-1164. DOI: 10.1172/JCI111524
[14]	万子琳. 七氟烷预处理及后处理对心肌缺血再灌注损伤的保护作用及其机制的研究进展[J].云南医药, 2021, 42(6): 581-584
[15]	Zhao Z, Li Y, Chi F, et al.Sevoflurane postconditioning ameliorates cerebral ischemia-reperfusion injury in rats via TLR4/MyD88/TRAF6 signaling pathway[J]. Aging (Albany NY), 2022,14(24):10153-10170. DOI: 10.18632/aging.204461
[16]	He B, Yang F, Ning Y, et al.Sevoflurane alleviates hepatic ischaemia/reperfusion injury by up-regulating miR-96 and down-regulating FOXO4[J]. J Cell Mol Med, 2021,25(13):5899-5911. DOI: 10.1111/jcmm.16063
[17]	邱云, 高素敏, 吴一晨, 等. 七氟烷后处理对小鼠肾脏缺血再灌注损伤的保护作用及机制[J]. 南京医科大学学报(自然科学版), 2018, 38(8): 1077-1080,1130. DOI:10.7655/NYDXBNS20180812.Qiu Y, Gao SM, Wu YC, et al.The protective effect and mechanism of sevoflurane postconditioning on renal ischemia/ reperfusion injury in mice[J]. Journal of Nanjing Medical University(Natural Sciences),2018,38(8):1077-1080,1130. DOI: 10.7655/NYDXBNS20180812
[18]	Aras B, Akçilar R, Koçak FE, et al.Effect of ukrain on ischemia/reperfusion-induced kidney injury in rats[J]. J Surg Res, 2016,202(2): 267-275. DOI: 10.1016/j.jss.2015.12.037
[19]	李红宇, 陈如平, 吴建民, 等.BCL-2/Bax的比值决定着细胞凋亡[J].中国医药实践杂志, 2012 (1): 40-41, 27
[20]	Darwish SF, Mahmoud A, Abdel Mageed SS, et al.Dapagliflozin improves early acute kidney injury induced by vancomycin in rats: insights on activin A/miRNA-21 signaling and FOXO3a expression[J]. Eur J Pharmacol, 2023,955:175908. DOI: 10.1016/j.ejphar.2023.175908
[21]	Xu X, Deng R, Zou L, et al.Sevoflurane participates in the protection of rat renal ischemia-reperfusion injury by down-regulating the expression of TRPM7[J]. Immun Inflamm Dis, 2023,11(1): e753. DOI: 10.1002/iid3.753
[22]	Hu CY, Guo YQ, Hao YH, et al.Research on mechanism of sevoflurane in alleviating cerebral ischemia-reperfusion injury in rats through JNK signaling pathway[J]. Eur Rev Med Pharmacol Sci, 2020,24(7):3907-3914. DOI: 10.26355/eurrev_202004_20857
[23]	Liu Y, Levine B.Autosis and autophagic cell death: the dark side of autophagy[J]. Cell Death Differ, 2015,22(3):367-376. DOI: 10.1038/cdd.2014.143
[24]	Jiang M, Wei Q, Dong G, et al.Autophagy in proximal tubules protects against acute kidney injury[J]. Kidney Int, 2012,82(12):1271-1283. DOI: 10.1038/ki.2012.261
[25]	Zhen Y, Stenmark H.Autophagosome biogenesis[J]. Cells, 2023,12(4):668. DOI: 10.3390/cells12040668
[26]	胡彦, 王锁刚, 翟琼瑶, 等. 积雪草苷调控SIRT1-FOXO3-PINK1-Parkin通路介导的线粒体自噬保护肾缺血再灌注损伤的机制研究[J]. 天津医药, 2021, 49(11):1148-1153. DOI:10.11958/20210354.Hu Y, Wang SG, Zhai QY, et al.The study of asiaticoside regulating SIRT1-FOXO3-PINK1-Parkin pathway-mediated protective mechanism of mitochondrial autophagy on renal ischemia-reperfusion injury[J]. Tianjin Medical Journal, 2021, 49(11):1148-1153. DOI: 10.11958/20210354
[27]	Prerna K, Dubey VK.Beclin1-mediated interplay between autophagy and apoptosis: new understanding[J]. Int J Biol Macromol, 2022, 204: 258-273. DOI: 10.1016/j.ijbiomac.2022.02.005
[28]	李轶男,裴汉军,余学文. 远隔缺血预处理上调线粒体自噬减轻小鼠急性肾缺血再灌注损伤[J].智慧健康,2022,8(23):44-48. DOI: 10.19335/j.cnki.2096-1219.2022.23.010.Li YN, Pei HJ, Yu XW.Remote ischemic preconditioning up-regulates mitophagy and alleviates acute renal ischemia-reperfusion injury in mice[J]. Smart Healthcare, 2022,8(23):44-48. DOI: 10.19335/j.cnki.2096-1219.2022.23.010
[29]	Wu Y, Shi H, Xu Y, et al.Ebselen ameliorates renal ischemia-reperfusion injury via enhancing autophagy in rats[J]. Mol Cell Biochem, 2022,477(6):1873-1885. DOI: 10.1007/s11010-022-04413-4
[30]	吴凡, 李泽庚, 董昌武, 等. 芪白平肺胶囊通过调节SIRT1/FoxO3a通路改善COPD气虚痰瘀证大鼠炎症及氧化应激状态[J].细胞与分子免疫学杂志,2019, 35(2): 115-120. DOI: 10.13423/j.cnki.cjcmi.008771
[31]	蔡翔, 王美君, 徐芬, 等.卡格列净通过SIRT1-FOXO3α信号通路改善小鼠肾小球系膜细胞自噬功能[J].中国病理生理杂志,2023,39(1):131-141. DOI:10. 3969/j. issn. 1000-4718. 2023. 01. 016.Cai X, Wang MJ,Xu F, et al.Canagliflozin improves autophagy in mouse renal mesangial cells through SIRT1-FOXO3α signaling pathway[J]. Chinese Journal of Pathophysiology,2023,39(1):131-141. DOI:10. 3969/j.issn.1000-4718.2023.01.016
[32]	赵方, 高飞, 李绍慧, 等. 降脂通络软胶囊调控SIRT1/FoxO3通路对膜性肾病大鼠细胞凋亡的影响[J].中国实验方剂学杂志, 2022, 28(7): 113-120. DOI: 10.13422/j.cnki.syfjx.20220739.Zhao F, Gao F,Li SH, et al.Jiangzhi tongluo soft capsules regulate SIRT1/FoxO3 pathway to affect cell apoptosis in rats with membranous nephropathy[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2022,28(7): 113-120. DOI: 10.13422/j.cnki.syfjx.20220739
[33]	Kim JY, Mondaca-Ruff D, Singh S, et al.SIRT1 and autophagy: implications in endocrine disorders[J]. Front Endocrinol (Lausanne), 2022,13:930919. DOI: 10.3389/fendo.2022.930919
[34]	Morevati M, Egstrand S, Nordholm A, et al.Effect of NAD+ boosting on kidney ischemia-reperfusion injury[J]. PLoS One, 2021,16(6):e0252554. DOI: 10.1371/journal.pone.0252554
[35]	Zhao X, Liu Y, Zhu G, et al.SIRT1 downregulation mediated manganese-induced neuronal apoptosis through activation of FOXO3a-Bim/PUMA axis[J]. Sci Total Environ, 2019,646:1047-1055. DOI: 10.1016/j.scitotenv.2018.07.363
[36]	林琴琴, 王湘怡, 耿元文, 等. 间歇运动调控miR-155/SIRT1/FoxO3a通路抑制心梗大鼠肾脏细胞凋亡[J].天津体育学院学报,2021,36(3):360-365. DOI: 10.13297/j.cnki.issn1005-0000.2021.03.016.Lin QQ, Wang XY, Geng YW, et al.Aerobic interval training activates the miR-155/SIRT1/FoxO3a signalling pathway to inhibit renal apopto-sis in rats model with myocardial infarction[J]. Journal of Tianjin University of Sport, 2021, 36(3): 360-365. DOI: 10.13297/j.cnki.issn1005-0000.2021.03.016.