摘要
目的:考察石榴皮的有效成分尿石素A(UA)对肿瘤恶病质模型小鼠的恶病质心肌损伤以及模拟肿瘤恶病质损伤刺激下的体外培养心肌细胞损伤的作用,并探索其可能的作用机制。方法:通过在BALB/c小鼠皮下接种鼠源C26结直肠癌细胞建立肿瘤恶病质小鼠模型,后灌胃给予UA(25、50 mg/kg)18 d,采用苏木精-伊红(H-E)染色心肌切片观察组化特征,用蛋白质印迹(Western blot)法分析心肌中凋亡及自噬相关蛋白的表达水平;体外培养大鼠H9c2心肌细胞,通过给予C26肿瘤细胞的条件培养基模拟肿瘤恶病质损伤刺激,用磺酰罗丹明B(SRB)比色法检测细胞存活率,用膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙啶(PI)法流式细胞术检测细胞凋亡率,考察给予不同剂量UA(5、10μmol/L)对心肌细胞存活率及凋亡率的影响;利用mRFP-GFP-微管相关蛋白1轻链3(LC3B)自噬指示双荧光标记H9c2心肌细胞,考察UA对心肌细胞在模拟肿瘤恶病质损伤刺激下自噬水平及自噬相关蛋白的影响。结果:与健康对照组小鼠相比,模型组小鼠的心脏质量显著下降(P<0.001),心脏切片H-E染色结果显示心肌细胞的横截面积变小;50 mg/kg UA给药可以显著抑制肿瘤恶病质小鼠心脏的萎缩(P<0.01),增加心肌细胞横截面积。与对照组小鼠相比,模型组小鼠心肌组织中剪切型半胱氨酸天冬氨酸蛋白酶-3(Cleaved Caspase-3)/Caspase-3的比值显著升高(P<0.05),自噬蛋白LC3BⅡ的表达显著升高(P<0.001);25、50 mg/kg UA给药均可显著抑制肿瘤恶病质小鼠心肌的凋亡(P<0.05,P<0.01),50 mg/kg UA给药可以进一步促进肿瘤恶病质小鼠心肌的自噬(P<0.05)。与对照组相比,C26细胞条件培养基刺激下的H9c2心肌细胞的凋亡率明显增加(P<0.001);10μmol/L UA能够显著改善C26条件培养基诱导的心肌细胞凋亡(P<0.001)。C26条件培养基诱导H9c2心肌细胞中Cleaved Caspase-3/Caspase-3比值升高(P<0.001);10μmol/L UA显著抑制Cleaved Caspase-3/Caspase-3的比值升高(P<0.05)。C26条件培养基可使mRFP-GFP-LC3B双荧光标记H9c2心肌细胞的绿色和红色荧光信号强度显著增强(P<0.05),即自噬体及自噬溶酶体均增加。10μmol/L UA可以显著增强C26条件培养基刺激下的H9c2心肌细胞的绿色和红色荧光信号强度(P<0.05)。C26条件培养基可以显著升高H9c2心肌细胞中自噬蛋白LCBⅡ的表达(P<0.05),10μmol/L UA可以显著增加C26条件培养基刺激下的自噬蛋白LCBⅡ的表达(P<0.05)。结论:UA可以改善肿瘤恶病质心肌损伤,其作用机制可能是通过促进自噬、减少凋亡,从而发挥保护心肌细胞对抗肿瘤恶病质损伤的作用。
Objective:To investigate the effect of active ingredient of Pomegranate peel urolithin A(UA)on cachexia myocardial injury in cancer cachexia model mice and cardiomyocytes injury culture in vitro stimulated by simulated cancer cachexia injury,and to explore its possible mechanism.Methods:The mouse model of cancer cachexia was established by subcutaneous inoculation of murine C26 colon cancer cells into BALB/c mice,after which UA(25 or 50 mg/kg)was administared intragastrically for 18 days.Hematoxylin-eosin(H-E)staining was used to observe the histochemical characteristics of myocardial sections.Western blot assay was used to analyze the expression levels of apoptosis-related and autophagy-related proteins in the myocardium.Rat H9c2 cardiomyocytes were cultured in vitro.The conditioned medium of C26 tumor cells was given to simulate the stimulation of cancer cachexia injury.The cell survival rate was detected by sulforhodamine B(SRB)assay and the apoptosis rate was detected by flow cytometry with AnnexinV-FITC/PI staining.The influence of UA at different doses(5μmol/L or 10μmol/L)on the survival rate and apoptosis rate of cardiomyocytes was investigated.The monomeric red fluorescew protein mRFP-green fluorescent protein GFP-microtubule associated protein 1 light chain 3(LC3B)dual⁃fluorescence-labeled autophagy-indicating H9c2 cardiomyocytes were used to investigate the effect of UA on autophagy levels and autophagy-related proteins of cardiomyocytes under stimulation of simulated tumor cachexia injury.Results:Compared with the healthy control group,the heart weight of C26 tumor cachexia mice model group was significantly decreased(P<0.001),and the cross-sectional area of cardiomyocytes was also decreased showed by H-E staining.The administration of 50 mg/kg UA significantly inhibited cardiac atrophy in mice with cancer cachexia(P<0.01),and enhanced the cross-section area of cardiomyocytes.Compared with the healthy control group,the ratio of apoptosis-related protein Cleaved Caspase-3/Caspase-3 in myocardial tissue of the C26 tumor cachexia mice group was significantly increased(P<0.05),and the expression level of autophagy protein LC3BⅡwas significantly increased(P<0.001).Administration of 25 and 50 mg/kg UA significantly inhibited myocardial apoptosis in cancer cachexia mice(P<0.05,P<0.01),and administration of 50 mg/kg UA further promoted myocardial autophagy in cancer cachexia mice(P<0.05).Compared with the control group,the apoptosis rate of H9c2 cardiomyocytes stimulated by C26 cell conditioned medium was significantly increased(P<0.001).10μmol/L UA significantly improved cardiomyocyte apoptosis induced by C26 conditioned medium(P<0.001).The ratio of Cleaved Caspase-3/Caspase-3 in H9c2 cardiomyocytes induced by C26 conditioned medium was increased(P<0.001).10μmol/L UA significantly inhibited the ratio increase of Cleaved Caspase-3/Caspase-3(P<0.05).The intensity of green and red fluorescence signal of mRFP⁃GFP⁃LC3B dual-fluorescence-labeled H9c2 cardiomyocytes was significantly increased by C26 conditioned medium(P<0.05),which suggested an increase in autophagosome and autophagolysosome.10μmol/L UA significantly enhanced the green and red fluorescence signal intensity of H9c2 cardiomyocytes stimulated by C26 conditioned medium(P<0.05).C26 conditioned medium significantly increased the expression level of autophagy protein LCBⅡin H9c2 cardiomyocytes(P<0.05),and 10μmol/L UA significantly increased the expression level of autophagy protein LCBⅡstimulated by C26 conditioned medium(P<0.05).Conclusion:UA can ameliorate the myocardial injury in cancer cachexia,and its mechanism of action may be to protect cardiomyocytes against the cancer cachexia injury by promoting autophagy and reducing apoptosis.
作者
邓雪
王琼森
刘璇
DENG Xue;WANG Qiongsen;LIU Xuan(Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《上海中医药大学学报》
2025年第4期56-65,共10页
Academic Journal of Shanghai University of Traditional Chinese Medicine
基金
国家自然科学基金资助项目(82374085)
上海市自然科学基金资助项目(23ZR1460500)。
关键词
尿石素A
石榴皮
肿瘤恶病质
心肌损伤
凋亡
自噬
urolithin A
Pomegranate peel
cancer cachexia
myocardial injury
apoptosis
autophagy
作者简介
邓雪,女,硕士,主要从事中药药理学研究;通信作者:刘璇,研究员,博士生导师,E-mail:xuanliu@shutcm.edu.cn。
