| 杨小春,吕建,孙景毅,等.水飞蓟宾对肢体缺血再灌注大鼠肝损伤及线粒体凋亡途径的影响[J].安徽医药,2025,29(3):494-501. |
| 水飞蓟宾对肢体缺血再灌注大鼠肝损伤及线粒体凋亡途径的影响 |
| Effects of silybin on liver injury and mitochondrial apoptosis pathway in limb ischemia-reperfusion rats |
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| DOI：10.3969/j.issn.1009-6469.2025.03.013 |
| 中文关键词: 肝损伤 再灌注损伤 线粒体凋亡 水飞蓟宾 丙氨酸转氨酶 超氧化物歧化酶 胱天蛋白酶 -3 胱天蛋白酶 -9 |
| 英文关键词: Liver injury Reperfusion Injury Mitochondrial apoptosis Silybin Glutamic-pyruvic transaminase Superoxide dismutase Caspase-3 Caspase-9 |
| 基金项目:秦皇岛市科学技术研究与发展计划项目( 202101A104) |
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| 中文摘要: |
| 目的探讨水飞蓟宾对肢体缺血再灌注大鼠肝损伤的保护作用及其具体机制。方法 2022年 1月至 2023年 1月将 50只大鼠按照随机数字表法分为正常组、模型组、水飞蓟宾低浓度组、水飞蓟宾中浓度组和水飞蓟宾高浓度组，每组 10只。水飞蓟宾低、中和高浓度组大鼠分别给予 100、200和 400 mg·kg.1 ·d.1水飞蓟宾灌胃，连续 7d。于末次给药 2h后，双后肢根部环扎橡皮筋阻断血流 4h，再灌注 4h，制备肢体缺血再灌注肝损伤模型。计算肝指数和脾指数，检测肝脏损伤指标［丙氨酸转氨酶(ALT)、天冬氨酸转氨酶( AST)以及乳酸脱氢酶( LDH)］、氧化应激指标［超氧化物歧化酶( SOD)、谷胱甘肽过氧化物酶( GSH-Px)和丙二醛］以及血清中炎症反应指标［白细胞介素 -1β(IL-1β)和肿瘤坏死因子 α(TNF-α)］线粒体通透性转运孔( mPTP)孔开放程度，肝组织进行苏木精 -伊红( HE)染色和脱氧核糖核苷酸末端转移酶介导的缺口末端标，记法( TUNEL)染色，逆转录实时荧光定量聚合酶链式反应( RT-qPCR)法检测 B淋巴细胞瘤 -2(Bcl-2)和 Bcl-2相关 X蛋白( Bax)mRNA表达水平，蛋白质印迹法检测肝组织中活化胱天蛋白酶( cleaved caspase)-3和 cleaved caspase-9蛋白表达水平以及 Bax/Bcl-2，并分别检测肝细胞胞质和线粒体中细胞色素 C(Cyt-c)蛋白表达水平。结果模型组肝细胞排列紊乱，肝细胞肿胀，部分出现空泡变性，大量炎症细胞浸润；水飞蓟宾低、中和高浓度组大鼠肝组织病理损伤减轻，出现空泡化、坏死的细胞数减少，炎症细胞浸润减少。与正常组比较，模型组肝指数、脾指数、 ALT、AST和 LDH、TNF-α和 IL-1β以及丙二醛含量、细胞凋亡率均升高， SOD和 GSH-Px活性降低， mPTP孔开放程度增强， BaxmRNA表达水平以及 cleaved caspase-3、cleaved caspase-9蛋白表达水平和 Bax/Bcl-2升高，胞质中 Cyt-c蛋白表达水平升高， Bcl-2mRNA表达水平以及线粒体中 Cyt-c蛋白表达水平降低(P<0.05)；与模型组比较，水飞蓟宾低、中和高浓度组肝指数［(7.41±1.05)mg/g、(6.33±1.02)mg/g、(5.45±0.75)mg/g比( 9.37±1.34)mg/g］和脾指数、 ALT［(194.22±36.82)IU/ L、(111.06±33.97)IU/L、(77.36±9.37)IU/L比( 275.27±44.75)IU/L］、 AST［(221.73±47.55)IU/L、(105.90±15.41)IU/L、(83.95±7.90) IU/L比( 384.94±47.90)IU/L］和 LDH［( 459.47±48.05)U/L、(371.86±40.60)U/L、(316.88±23.56)U/L比( 548.90±53.68)U/L］含量、 TNF-α和 IL-1β以及丙二醛含量、细胞凋亡率降低， SOD和 GSH-Px活性升高， mPTP孔( 1.44±0.23、1.24±0.2、1.05±0.15比 1.63± |
| 英文摘要: |
| Objective To investigate the protective effect and mechanism of silybin on liver injury in limb ischemia-reperfusion rats. Methods From January 2022 to January 2023, 50 rats were randomly divided into normal group, model group, silybin low concentra-tion group, silybin medium concentration group and silybin high concentration group according to the random number table method,with 10 rats in each group. Rats in low, medium, and high concentration groups were given 100, 200 and 400 mg·kg.1·d.1 silybin intra-gastric administration, for consecutive 7 days, respectively. 2 h after the last administration, the blood flow was blocked for 4 h by tyingrubber bands around the root of the double hind limbs, and the limb ischemia-reperfusion liver injury model was prepared for 4 h. Theliver index and spleen index were calculated, liver injury indexes [alanine aminotransferase (ALT), aspartate transaminase (AST), andlactate dehydrogenase (LDH)], oxidative stress indexes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondial-dehyde (MDA)], serum inflammatory response indexes [interleukin-1β (IL-1β) and tumor Necrosis factor α (TNF-α)], mitochondrial per-meability transport pore (mPTP) openness were detected, liver tissue was stained by hematoxylin-eosin (HE) and Terminal deoxynucleo-tidyl transferase dUTP nick end labeling (TUNEL), and the mRNA expression levels of B lymphoblastoma-2 (Bcl-2) and Bcl-2 associat-ed X protein (Bax) were detected by Reverse Transcription quantitative PCR (RT-qPCR). The expression levels of spliced cysteine as-partate proteolytic enzyme 3 (cleaved caspase 3) and cleaved caspase 9 and Bax/Bcl-2 were detected by Western blotting, and the cyto-chrome C (Cyt-c) protein expression levels were detected in the cytoplasm and mitochondria of hepatocytes, respectively.Results In the model group, the arrangement of hepatocytes was disordered, the hepatocytes were swollen, some of them showed vacuolar degenera-tion, and a large number of inflammatory cells infiltrated. The pathological changes of liver tissue in the low, medium and high concen-tration silibinin groups were alleviated, the number of cells with vacuolization and necrosis was reduced, and the infiltration of inflam-matory cells was reduced. Compared with normal group, liver index and spleen index, contents of ALT, AST and LDH, contents of TNF-α, IL-1β and malondialdehyde, cell apoptosis rate were increased in model group, SOD and GSH-Px activities were decreased, and the openness of mPTP pore was enhanced. Bax mRNA expression level, cleaved caspase-3, cleaved caspase-9 protein expression levels and Bax/Bcl-2 were increased, and Cyt-c protein expression level in cytoplasm was increased. The expression levels of Bcl-2 mRNA and Cyt-c protein in mitochondria were decreased (P<0.05). Compared with model group, liver index [(7.41±1.05) mg/g, (6.33±1.02) mg/ g, (5.45±0.75) mg/g vs. (9.37±1.34) mg/g] and spleen index, contents of ALT [(194.22±36.82) IU/L, (111.06±33.97) IU/L, (77.36±9.37) IU/L vs. (275.27±44.75) IU/L], AST [(221.73±47.55) IU/L,(105.90±15.41) IU/L, (83.95±7.90) IU/L vs. (384.94±47.90) IU/L] and LDH [(459.47±48.05) U/L, (371.86±40.60) U/L, (316.88±23.56) U/L vs. (548.90±53.68) U/L], contents of TNF-α, IL-1β and malondialde-hyde, apoptosis rate were decreased, SOD and GSH-Px activities were increased, and mPTP hole (1.44±0.23, 1.24±0.2, 1.05±0.15 vs. 1.63±0.25) opening degrees were weakened in silybin low and high concentration groups. Bax mRNA expression levels were decreased,cleaved caspase-3 and cleaved caspase-9 protein expression levels and Bax/Bcl-2 , and cytoplasmic Cyt-c (0.96±0.06, 0.44±0.05, 0.16±0.05 vs. 1.13±0.14) protein expression levels were decreased. The expression levels of Bcl-2 mRNA and Cyt-c (0.73±0.07, 0.82±0.06, 1.05±0.08 vs. 0.18±0.05) protein in mitochondria were increased (P<0.05).Conclusion Silybin can reduce the degree of liver injury and inhibit the apoptosis of liver cells in limb ischemia-reperfusion rats, which may play a role by reducing oxidative stress responseand inflammatory response and blocking mitochondrial apoptosis pathway. |
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