鲜枸杞子提取物通过p38 MAPK信号通路抑制人肝癌细胞HepG2诱导小鼠恶病质的作用及机制
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摘要
目的:探讨鲜枸杞子提取物(LBL)对人类肝癌细胞系HepG2诱导的小鼠恶病质模型的作用及其相关机制研究。方法:小鼠分为正常组,恶病质模型组,LBL低剂量组(LBL 5 mg·kg~(-1)),LBL高剂量组(LBL 25 mg·kg~(-1))。采用人肝癌细胞系HepG2成功建立小鼠恶病质模型,待小鼠的体质量明显下降时,连续灌喂生理盐水或不同剂量LBL 28 d后,记录各组小鼠的体质量,酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)检测血浆肌酸激酶(creatine kinase,CK),促炎因子白细胞介素-1β(interleukin-1β,IL-1β),白细胞介素-6(interleukin-6,IL-6),肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的水平,采用免疫组织化学检测小鼠肌肉降解水平及p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)的表达,蛋白免疫印迹法(Western blot)检测磷酸化p38 MAPK(phospho-p38 mitogen-activated protein kinase,p-p38 MAPK),炎症信号通路核转录因子-κB(nuclear factorκB,NF-κB)的表达。结果:与恶病质模型组比较,LBL低、高剂量组小鼠体质量明显下降(P<0.05,P<0.01),肌肉降解程度明显抑制(P<0.05),CK水平显著下降(P<0.01);LBL低、高剂量组小鼠血浆IL-1β,IL-6,TNF-α表达水平明显下调(P<0.05,P<0.01)。免疫组织化学结果显示,LBL低、高剂量组p38 MAPK蛋白表达降低(P<0.05),Western blot结果进一步证实p-p38 MAPK,NF-κB蛋白表达降低(P<0.01)。与LBL低剂量组比较,LBL高剂量组血浆肌酸激酶,p38 MAPK,p-p38 MAPK,NF-κB蛋白表达明显降低(P<0.05,P<0.01)。结论:鲜枸杞子提取物能够抑制人类肝癌细胞系HepG2诱导的小鼠恶病质模型的恶病质的进程,其作用机制可能与其下调促炎因子IL-1β,IL-6,TNF-α,从而抑制p38 MAPK,p-p38 MAPK,NF-κB的表达相关。
Objective: To investigate the inhibitory effect and mechanism of the extracts from fresh Lycii Fructus( LBL) on hepatoma HepG2 cell-induced cachexia in mouse. Method: The human hepatoma cell line HepG2 was injected into BALB/C mice to establish the cachexia model. Then the LBL was fed to the models respectively in low dose( 5 mg·kg~(-1)) or high dose( 25 mg·kg~(-1)). After 28 days of continuous feeding,the mice’s body weight was detected. The expression levels of creatine kinase( CK),interleukin-1β( IL-1β),interleukin-6( IL-6) and tumor necrosis factor-α( TNF-α) were evaluated by enzyme linked immunosorbent assay( ELISA).The muscle degradation and the expression of p38 mitogen-activated protein kinase( p38 MAPK) were detected by immunohistochemistry staining. The expression levels of phospho-p38 mitogen-activated protein kinase( p-p38 MAPK) and nuclear factor-κB( NF-κB) were determined by Western blot. Result: Compared with cachexia group,the loss of body weight and muscle decomposition were significantly inhibited both in the low dose LBL group and high dose LBL group( P < 0. 05,P < 0. 01). The level of plasma CK decreased significantly both in the low-dose LBL group and the high-dose LBL group( P < 0. 01). ELISA tests revealed lower expression levels of IL-1β,IL-6 and TNF-α in both the low-dose LBL group and the high-dose LBL group( P < 0. 05,P < 0. 01).Immunohistochemistry staining showed that the expression of p38 MAPK was inhibited both in the low-dose LBL group and the high-dose LBL group( P < 0. 05). Western blot indicated that the expressions of p-p38 MAPK and NF-κB were inhibited both in the low-dose LBL group and the high-dose LBL group( P < 0. 01). We found that the high-dose LBL group shows a higher inhibitory capability than the low-dose LBL group. Conclusion: LBL could inhibit the cachexia induced by hepatoma HepG2 cell line in mouse,suggesting LBL could reduce major cytokine and plasma inflammatory factors through p-p38 MAPK pathway.
Objective: To investigate the inhibitory effect and mechanism of the extracts from fresh Lycii Fructus( LBL) on hepatoma HepG2 cell-induced cachexia in mouse. Method: The human hepatoma cell line HepG2 was injected into BALB/C mice to establish the cachexia model. Then the LBL was fed to the models respectively in low dose( 5 mg·kg~(-1)) or high dose( 25 mg·kg~(-1)). After 28 days of continuous feeding,the mice’s body weight was detected. The expression levels of creatine kinase( CK),interleukin-1β( IL-1β),interleukin-6( IL-6) and tumor necrosis factor-α( TNF-α) were evaluated by enzyme linked immunosorbent assay( ELISA).The muscle degradation and the expression of p38 mitogen-activated protein kinase( p38 MAPK) were detected by immunohistochemistry staining. The expression levels of phospho-p38 mitogen-activated protein kinase( p-p38 MAPK) and nuclear factor-κB( NF-κB) were determined by Western blot. Result: Compared with cachexia group,the loss of body weight and muscle decomposition were significantly inhibited both in the low dose LBL group and high dose LBL group( P < 0. 05,P < 0. 01). The level of plasma CK decreased significantly both in the low-dose LBL group and the high-dose LBL group( P < 0. 01). ELISA tests revealed lower expression levels of IL-1β,IL-6 and TNF-α in both the low-dose LBL group and the high-dose LBL group( P < 0. 05,P < 0. 01).Immunohistochemistry staining showed that the expression of p38 MAPK was inhibited both in the low-dose LBL group and the high-dose LBL group( P < 0. 05). Western blot indicated that the expressions of p-p38 MAPK and NF-κB were inhibited both in the low-dose LBL group and the high-dose LBL group( P < 0. 01). We found that the high-dose LBL group shows a higher inhibitory capability than the low-dose LBL group. Conclusion: LBL could inhibit the cachexia induced by hepatoma HepG2 cell line in mouse,suggesting LBL could reduce major cytokine and plasma inflammatory factors through p-p38 MAPK pathway.
引文
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[17]朱彩平,张声华.枸杞多糖对H(22)肝癌小鼠的抑癌作用[J].中国公共卫生,2006,22(6):717-718.
[2]Lee D E,Brown J L,Rosa-Caldwell M E,et al.Cancer cachexia-induced muscle atrophy:evidence for alterations in microRNAs important for muscle size[J].Physiol Genomics,2017,49(5):253-260.
[3]GAO Y,WEI Y,WANG Y,et al.Lycium barbarum:a traditional chinese herb and a promising anti-aging agent[J].Aging Dis,2017,8(6):778-791.
[4]HU Q,JIA B L,GAO T S,et al.A study on the anticancer effect of ningxia wolfberry[J].J Tradit Chin Med,1989,9(2):117-124.
[5]张义军,陈丽.均匀设计法优选枸杞提取工艺[J].时珍国医国药,2008,19(10):2457-2458.
[6]郑亚兵,马胜林,周卫民,等.沙利度胺抗Lewis肺癌小鼠癌性恶病质的实验研究[J].北京医学,2010,32(7):559-561.
[7]龚梦鹃,谢媛媛,邹忠杰.枸杞对阴虚小鼠的抗疲劳作用[J].中国实验方剂学杂志,2012,18(14):171-174.
[8]任非非,刘敬霞,俞维,等.枸杞不同制剂对环磷酰胺致血虚大鼠全血细胞及骨髓造血功能的影响[J].中国实验方剂学杂志,2012,18(3):187-189.
[9]Suzuki H,Asakawa A,Amitani H,et al.Cancer cachexia-pathophysiology and management[J].J Gastroenterol,2013,48(5):574-594.
[10]Scheede-Bergdahl C,Watt H L,Trutschnigg B,et al.Is IL-6 the best pro-inflammatory biomarker of clinical outcomes of cancer cachexia[J].Clin Nutr,2012,31(1):85-88.
[11]Fortunati N,Manti R,Birocco N,et al.Proinflammatory cytokines and oxidative stress/antioxidant parameters characterize the bio-humoral profile of early cachexia in lung cancer patients[J].Oncol Rep,2007,18(6):1521-1527.
[12]洪梓德,莫志贤.中药抗肿瘤机制中的11种信号通路[J].中国实验方剂学杂志,2018,24(21):205-218.
[13]Barreto R,Waning D L,GAO H,et al.Chemotherapyrelated cachexia is associated with mitochondrial depletion and the activation of ERK1/2 and p38 MAPKs[J].Oncotarget,2016,7(28):43442-43460.
[14]Blackwell T S,Christman J W.The role of nuclear factor-kappa B in cytokine gene regulation[J].Am J Respir Cell Mol Biol,1997,17(1):3-9.
[15]Je J H,Lee J Y,Jung K J,et al.NF-kappa B activation mechanism of 4-hydroxyhexenal via NIK/IKK and p38 MAPK pathway[J].FEBS Lett,2004,566(1/3):183-189.
[16]章培军,邢雁霞,刘斌钰,等.枸杞多糖对实验性肝损伤保护作用的研究[J].中国药物与临床,2011,11(11):1286-1287.
[17]朱彩平,张声华.枸杞多糖对H(22)肝癌小鼠的抑癌作用[J].中国公共卫生,2006,22(6):717-718.