虫草多糖对肿瘤坏死因子α诱导肝细胞凋亡的影响

赵佳男; 彭景华; 刘平; 胡义扬

doi:10.3969/j.issn.1001-5256.2021.06.029

虫草多糖对肿瘤坏死因子α诱导肝细胞凋亡的影响

DOI: 10.3969/j.issn.1001-5256.2021.06.029

赵佳男¹,
彭景华¹,
刘平^{1, 2, 3},
胡义扬^{1, 2, 3, 4, ,}

1.
上海中医药大学肝病研究所，上海 201203
2.
上海市中医临床重点实验室，上海 201203
3.
肝肾疾病病证教育部重点实验室，上海 201203
4.
上海中医药大学附属曙光医院临床药理研究所，上海 201203

基金项目:

国家自然科学基金重点项目 (81530101)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明: 赵佳男负责撰写论文；彭景华、刘平负责审校；胡义扬负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

作者简介:
赵佳男(1996—)，男，主要从事中医药防治慢性肝病的研究

通信作者:
胡义扬，yyhuliver@163.com

中图分类号: R575.2
计量
- 文章访问数: 417
- HTML全文浏览量: 110
- PDF下载量: 31
- 被引次数: 0
出版历程
- 收稿日期: 2021-02-02
- 录用日期: 2021-03-10
- 出版日期: 2021-06-20

Effect of Cordyceps polysaccharides on hepatocyte apoptosis induced by tumor necrosis factor-α

Jianan ZHAO¹,
Jinghua PENG¹,
Ping LIU^{1, 2, 3},
Yiyang HU^{1, 2, 3, 4
, ,}

1.
Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2.
Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203, China
3.
Key Laboratory of Liver and Kidney Diseases, Ministry of Education, Shanghai 201203, China
4.
Institute of Clinical Pharmacology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

摘要

摘要: 目的探讨虫草多糖对肝细胞凋亡的影响和作用机制。方法以人正常肝细胞L02作为研究对象，将不经任何药物处理的人正常肝细胞L02作为正常组；利用不同浓度的TNFα(5、10、20、40 ng/ml)进行干预24 h后筛选肝细胞凋亡模型的造模条件并作为模型组；根据实验设计，使用3种不同浓度的虫草多糖(50、100、200 μg/ml)预作用12 h后给予或者不给予筛选后的TNFα模型浓度24 h作为实验组，收取样本进行检测，采用CCK8法检测细胞增殖活力；采用Annexin V/PI双染法检测肝细胞凋亡数；采用RT-PCR法检测细胞凋亡(Bax、caspase3、caspase8、caspase9、死亡受体Fas)的mRNA表达；采用蛋白免疫印迹法检测cleaved-caspase3、cleaved-caspase8蛋白表达。计量资料多组间比较采用单因素方差分析或Kruskal-Wallis H检验，进一步两两比较采用LSD-t检验或Dunnett-t检验。结果 CCK8法检测结果显示，40 ng/ml的TNFα诱导L02肝细胞24 h后，L02肝细胞增殖较正常组显著降低(73.54%±14.19% vs 100.00%±23.61%，P＜0.01)，3种不同浓度的虫草多糖在对肝细胞无明显细胞毒性的前提下，较模型组(93.02%±7.21%)，细胞增殖均显著升高(P值均＜0.01)，分别为108.10%±9.05%、114.30%±8.79%、117.70%±9.66%。Annexin V/PI双染法检测结果显示，与正常组细胞凋亡数量比较，模型组细胞凋亡数量显著升高(7.71%±1.20% vs 11.57%±1.41%，P＜0.05)，3种不同浓度的虫草多糖较模型组(18.91%±0.80%)均明显降低细胞凋亡数量，分别为15.16%±0.16%、13.28%±1.57%、16.91%±0.21%(P值均＜0.05)。RT-PCR法和蛋白免疫印迹检测结果显示，40 ng/ml TNFα造模后细胞凋亡相关的Bax、死亡受体Fas的mRNA表达较正常组均增强，差异均有统计学意义(P值均＜0.05)，并且激活形式的cleaved-caspase3、cleaved-caspase8的蛋白表达显著增强(P值均＜0.05)，与模型组比较，50 μg/ml的虫草多糖可显著降低Bax、caspase3、caspase9的mRNA表达和cleaved-caspase8蛋白表达，100 μg/ml的虫草多糖可显著降低Bax、caspase3、caspase8、Fas、caspase9 mRNA和cleaved-caspase8蛋白表达，200 μg/ml的虫草多糖可显著降低caspase3、caspase8、Fas、caspase9 mRNA和cleaved-caspase3、cleaved-caspase8蛋白表达，3种虫草多糖浓度作用具有一定量效趋势。结论虫草多糖可以有效抑制TNFα诱导的L02正常肝细胞凋亡。
- 肝硬化 /
- 虫草多糖 /
- 细胞凋亡
Abstract: Objective To investigate the effect of Cordyceps polysaccharides on hepatocyte apoptosis and its mechanism of action. Methods Normal human L02 hepatocytes without any drug treatment was established as normal group. The L02 hepatocytes were treated with different concentrations of tumor necrosis factor-α (TNF-α) (5, 10, 20, and 40 ng/ml) for 24 hours to screen out the modeling conditions for the model of hepatocyte apoptosis, which were established as model group. According to the experimental design, L02 hepatocytes pretreated with three different concentrations of Cordyceps polysaccharides (50, 100, and 200 μg/ml) for 12 hours, with or without TNF-α treatment for 24 hours at the selected concentration, were established as experimental group. Related samples were collected for analysis. CCK8 assay was used to measure cell proliferation; Annexin V/PI double staining was used to measure the number of apoptotic hepatocytes; RT-PCR was used to measure the mRNA expression of cell apoptosis genes (Bax, caspase-3, caspase-8, caspase-9, and death receptor Fas); Western blotting was used to measure the protein expression of cleaved-caspase-3 and cleaved-caspase-8. A one-way analysis of variance or the Kruskal-Wallis H test was used for comparison of continuous data between multiple groups, and the least significant difference t-test or the Dunnett-t test was used for further comparison between two groups. Results CCK8 assay showed that compared with the normal group, the L02 hepatocytes induced by 40 ng/ml TNF-α for 24 hours had a significant reduction in proliferation (73.54%±14.19% vs 100.00%±23.61%, P < 0.01), and compared with the model group under the premise that Cordyceps polysaccharides had no obvious cytotoxicity to hepatocytes at the three concentrations, the 50, 100, and 200 μg/ml experimental groups had a significant increase in cell proliferation (108.10%±9.05%/114.30%±8.79%/117.70%±9.66% vs 93.02%±7.21%, all P < 0.01). Annexin V/PI double staining showed that compared with the normal group, the model group had a significant increase in the number of apoptotic cells (11.57%±1.41% vs 7.71%±1.20%, P < 0.05), and compared with the model group, the 50, 100, and 200 μg/ml experimental groups had a significant reduction in the number of apoptotic cells (15.16%±0.16%/13.28%±1.57%/16.91%±0.21% vs 18.91%±0.80%, all P < 0.05). RT-PCR and Western blotting showed that compared with the normal group, the L02 hepatocytes induced by 40 ng/ml TNF-α had significant increases in the mRNA expression of the apoptosis-related genes Bax, caspase-9, and death receptor Fas (all P < 0.05), as well as significant increases in the protein expression of the activated forms of cleaved-caspase-3 and cleaved-caspase-8 (P < 0.05). Compared with the model group, the 50 μg/ml experimental group had significant reductions in the mRNA expression of Bax, caspase-3, and caspase-9 and the protein expression of cleaved-caspase-8; the 100 μg/ml experimental group had significant reductions in the mRNA expression of Bax, caspase-3, caspase-8, Fas, and caspase-9 and the protein expression of cleaved-caspase-8; the 200 μg/ml experimental group had significant reductions in the mRNA expression of caspase-3, caspase-8, Fas, and caspase-9 and the protein expression of cleaved-caspase-3 and cleaved-caspase-8. The three concentrations of Cordyceps polysaccharides had a certain dose-effect trend. Conclusion Cordyceps polysaccharides can effectively inhibit the apoptosis of normal L02 hepatocytes induced by TNF-α.
- Liver Cirrhosis /
- Cordyceps Sinensis Mycelia Polysaccharide /
- Apoptosis

HTML全文

图 1 不同浓度TNFα对L02肝细胞增殖的影响

与正常组比较，△△P＜0.01。

下载: 全尺寸图片幻灯片

图 2 不同浓度TNFα对L02肝细胞凋亡数的影响

下载: 全尺寸图片幻灯片

图 3 不同浓度虫草多糖的肝细胞毒性试验

注：与正常组比较，△P＜0.05，△△P＜0.01。

下载: 全尺寸图片幻灯片

图 4 不同浓度的虫草多糖对TNFα诱导L02肝细胞增殖的影响

注：与正常组比较，△P＜0.05，△△P＜0.01；与模型组M比较，**P＜0.01。

下载: 全尺寸图片幻灯片

图 5 不同浓度虫草多糖对TNFα诱导L02肝细胞凋亡数的影响

注：与正常组比较，△△P＜0.01；与模型组比较，*P＜0.05，**P＜0.01；与100 μg/ml组比较，&&P＜0.01。

下载: 全尺寸图片幻灯片

图 6 不同浓度虫草多糖对TNFα诱导L02肝细胞凋亡的相关蛋白表达影响

注：与正常组比较，△P＜0.05，△△P＜0.01；与模型组比较，**P＜0.01。

下载: 全尺寸图片幻灯片

表 1 引物序列及片段长度

引物名称	引物序列	产物长度(bp)
Bax	5′TCG CCC TTT TCT ACT TTG CC 3′	97
caspase3	5′CTG GTA TGT GTG GAT GAT GCT 3′	100
caspase8	5′CTG GGA GAA GGA AAG TTG GAC 3′	68.5
caspase9	5′TGC CTC AAT GCC AGT AAC 3′	129
Fas	5′ATT TTT GCC TTG GTG CTC A 3′	94
GAPDH	5′GGG AAG GTG AAG GTC GGA GT 3′	105
ACTB	5′AAG GTG ACA GCA GTC GGT T 3′	195

下载: 导出CSV

表 3 不同浓度虫草多糖对TNFα诱导L02肝细胞凋亡的Bax、caspase 9、caspase 8、Fas、caspase 3的mRNA表达影响

组别	Bax	caspase 9	Fas	caspase 8	caspase 3
正常组	0.92(0.63~1.90)	1.00±0.05	0.97(0.85~1.14)	1.01(0.83~1.15)	0.94(0.87~1.20)
模型组	2.38(1.46~4.14)¹⁾	1.11±0.32	1.66(1.20~3.87)¹⁾	1.33(0.92~4.44)	1.41(1.03~2.80)
50 μg/ml	0.81(0.78~1.11)³⁾	0.83±0.14²⁾	1.06(0.86~3.18)	0.90(0.83~1.40)	0.90(0.66~1.16)²⁾
100 μg/ml	1.09(0.65~1.64)²⁾	0.75±0.07³⁾	0.83(0.77~1.19)²⁾	0.85(0.76~1.02)	0.83(0.68~0.95)³⁾
200 μg/ml	1.88(0.90~3.69)	0.77±0.10³⁾	0.77(0.72~0.88)³⁾	0.82(0.75~0.84)²⁾	0.79(0.65~0.95)³⁾
统计值	H=14.17	F=5.298	H=15.88	H=10.45	H=14.63
P值	0.006	＜0.001	0.003	0.003	0.005
注：与正常组比较，1)P＜0.05；与模型组比较，2)P＜0.05，3)P＜0.01。

下载: 导出CSV

参考文献(10)

[1]	Chinese Society of Hepatology, Chinese Medical Association; Chinese Society of Gastroenterology, Chinese Medical Association; Chinese Society of Infectious Diseases, Chinese Medical Association. Consensus on the diagnosis and therapy of hepatic fibrosis (2019)[J]. J Clin Hepatol, 2019, 35(10): 2163-2172. DOI: 10.3969/j.issn.1001-5256.2019.10.007. 中华医学会肝病学分会, 中华医学会消化病学分会, 中华医学会感染病学分会. 肝纤维化诊断及治疗共识(2019年)[J]. 临床肝胆病杂志, 2019, 35(10): 2163-2172. DOI: 10.3969/j.issn.1001-5256.2019.10.007.
[2]	YAN X, ZHOU T, TAO Y, et al. Salvianolic acid B attenuates hepatocyte apoptosis by regulating mediators in death receptor and mitochondrial pathways[J]. Exp Biol Med (Maywood), 2010, 235(5): 623-632. DOI: 10.1258/ebm.2009.009293.
[3]	GUICCIARDI ME, GORES GJ. Apoptosis as a mechanism for liver disease progression[J]. Semin Liver Dis, 2010, 30(4): 402-410. DOI: 10.1055/s-0030-1267540.
[4]	PENG JH, LI XM, HU YY, et al. Effect of Cordyceps polysaccharide on lipid peroxidation of rats with dimethylnitrosamine-induced liver fibrosis[J]. China J Chin Mater Med, 2013, 38(3): 391-396. DOI: 10.4268/cjcmm20130320. 彭景华, 李雪梅, 胡义扬, 等. 虫草多糖对二甲基亚硝胺诱导的肝纤维化大鼠脂质过氧化及肝细胞再生的影响[J]. 中国中药杂志, 2013, 38(3): 391-396. DOI: 10.4268/cjcmm20130320.
[5]	WANG XB, LIU P, TANG ZP. Effect of Cordyceps mycelia extract on injury and phenotype shift of liver sinusoidal endothelial cells in rats with dimethylnitrosamine-induced liver cirrhosis[J]. J Beijing Univ Chin Med, 2008, 31(7): 448-451, 505. DOI: 10.3321/j.issn:1006-2157.2008.07.005. 王宪波, 刘平, 唐志鹏. 虫草菌丝提取物对二甲基亚硝胺大鼠肝窦内皮细胞损伤和表型改变的影响[J]. 北京中医药大学学报, 2008, 31(7): 448-451, 505. DOI: 10.3321/j.issn:1006-2157.2008.07.005.
[6]	HU YY. Pay attention to the study on active antiliver fibrosis components of Chinese herbal medicine[J]. Chin J Integr Med, 2012, 18(8): 563-564. DOI: 10.1007/s11655-012-1029-7.
[7]	PENG J, LI X, FENG Q, et al. Anti-fibrotic effect of Cordyceps sinensis polysaccharide: Inhibiting HSC activation, TGF-β1/Smad signalling, MMPs and TIMPs[J]. Exp Biol Med (Maywood), 2013, 238(6): 668-677. DOI: 10.1177/1535370213480741.
[8]	TIAN H, LIU L, LI Z, et al. Chinese medicine CGA formula ameliorates liver fibrosis induced by carbon tetrachloride involving inhibition of hepatic apoptosis in rats[J]. J Ethnopharmacol, 2019, 232: 227-235. DOI: 10.1016/j.jep.2018.11.027.
[9]	CROW MT, MANI K, NAM YJ, et al. The mitochondrial death pathway and cardiac myocyte apoptosis[J]. Circ Res, 2004, 95(10): 957-970. DOI: 10.1161/01.RES.0000148632.35500.d9.
[10]	PATEL T. Apoptosis in hepatic pathophysiology[J]. Clin Liver Dis, 2000, 4(2): 295-317. DOI: 10.1016/s1089-3261(05)70112-4.