生草乌和诃子制草乌细胞毒性及抗炎作用的比较研究
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篇名: 生草乌和诃子制草乌细胞毒性及抗炎作用的比较研究
TITLE: Comparative Study of Cytotoxicity and Anti-inflammatory Effects between Raw Aconitium kusnezoffii and Aconitium kusnezoffii Processed with Terminalia chebula
摘要: 目的:比较生草乌和诃子制草乌的细胞毒性及抗炎作用。方法:以大鼠心肌细胞H9c2为对象,采用CCK-8法检测0.5、1、2、4、6、8、10mg/mL生草乌和诃子制草乌作用4、8、12、24h对细胞抑制率的影响,采用Hoechst33258染色法观察2、4、6mg/mL生草乌和诃子制草乌作用24h对细胞形态学特征的影响。以小鼠巨噬细胞RAW264.7为对象,采用CCK-8法检测0.05、0.1、0.25、0.5、0.75、1、1.5、2mg/mL生草乌和诃子制草乌作用24h对细胞存活率的影响,采用酶联免疫吸附试验法检测0.05、0.1、0.25、0.5mg/mL生草乌和诃子制草乌对LPS致RAW264.7炎症细胞中一氧化氮(NO)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)释放的影响。结果:当质量浓度为0.5、1mg/mL时,生草乌和诃子制草乌对H9c2细胞几乎均无抑制作用;当质量浓度为2mg/mL时,诃子制草乌在各时间点对H9c2细胞的抑制率均显著高于生草乌(P<0.05或P<0.01),且作用24h的细胞荧光强度虽强于生草乌,但其细胞核完整;当质量浓度为4~10mg/mL时,诃子制草乌在各时间点(除8、10mg/mL作用24h外)对H9c2细胞的抑制率均显著低于生草乌(P<0.05或P<0.01),且4、6mg/mL生草乌组细胞的荧光强度强于诃子制草乌组,同时生草乌组的细胞核破碎现象更为严重。0.05~0.5mg/mL的生草乌和诃子制草乌对RAW264.7细胞均无毒性,0.25、0.5mg/mL的生草乌和0.1、0.25、0.5mg/mL的诃子制草乌对RAW264.7炎症细胞中NO的释放以及0.05、0.1、0.25、0.5mg/mL的生草乌和诃子制草乌对RAW264.7炎症细胞中TNF-α、IL-6的释放均有显著的抑制作用(P<0.05或P<0.01),其中相同质量浓度下诃子制草乌对NO释放的抑制作用优于生草乌,对TNF-α、IL-6释放的抑制作用弱于生草乌。结论:草乌经诃子汤炮制后毒性有所降低,尤其是以中或高浓度、短期内使用的毒性低于生草乌。同时,诃子制草乌的抗炎作用与同浓度生草乌相当。
ABSTRACT: OBJECTIVE:To comp are cytotoxicity and anti-inflammatory effects of raw Aconitium kusnezoffii and A. kusnezoffii processed with Terminalia chebula . METHODS :Using H 9c2 cardiomyocytes isolated from rat as subjects ,CCK-8 assay was used to detect the effects of 0.5,1,2,4,6,8,10 mg/mL raw A. kusnezoffii and A. kusnezoffii processed with T. chebula on cell inhibition rate after cultured for 4,8,12,24 h. Hoechst 33258 staining was used to observe the effects on cell morphology characteristics after treated with 2,4,6 mg/mL raw A. kusnezoffii and A. kusnezoffii processed with T. chebula . Using macrophages RAW264.7 cells as subjects ,CCK-8 assay was used to detect the effects of 0.05,0.1,0.25,0.5,0.75,1,1.5,2 mg/mL raw A. kusnezoffii and A. kusnezoffii processed with T. chebula on cell survival rate after cultured for 24 h. ELISA assay was used to detect the effects of 0.05,0.1,0.25,0.5 mg/mL raw A. kusnezoffii and A. kusnezoffii processed with T. chebula on the release of NO , TNF-α and IL-6 in RAW 264.7 inflammation cells induced by LPS. RESULTS :When the mass concentration was 0.5,1 mg/mL, neither raw A. kusnezoffii and A. kusnezoffii processed with T. chebula had no inhibitory effect on H 9c2 cells. When the mass concentration was 2 mg/mL,the inhibitory effects of A. kusnezoffii processed with T. chebula on H 9c2 cells was higher than that of raw A. kusnezoffii (P<0.05 or P<0.01);fluorescence intensity of cells treated for 24 h was stronger than that of raw A. kusnezoffii,but its nucleus was intact. When the mass concentration was 4-10 mg/mL,the inhibitory rate of A. kusnezoffii processed with T. chebula on H 9c2 cells at different time points (except for 24 h culture of 8,10 mg/mL)was significantly lower than raw A. kusnezoffii (P<0.05 or P<0.01). The characteristics of cell morphology also showed that the fluorescence intensity of raw A. kusnezoffii group at 4,6 mg/mL was stronger than that of A. kusnezoffii processed with T. chebula group,and the cell nucleus fragmentation was more serious in the raw A. kusnezoffii group. 0.05-0.5 mg/mL raw A. kusnezoffii and A. kusnezoffii processed with T. chebula had no toxicity to RAW264.7 cells. 0.25,0.5 mg/mL raw A. kusnezoffii and 0.1,0.25,0.5 mg/mL A. kusnezoffii processed with T. chebula showed significant inhibitory effect on the release of NO ,0.05,0.1,0.25,0.5 mg/mL raw A. kusnezoffii and A. kusnezoffii processed with T. chebula showed significant inhibitory effect on the release of TNF-α and IL-6 in RAW 264.7 cell(P<0.05 or P< 0.01). The inhibitory effects of A. kusnezoffii processed with T. chebula at the same concentration on the release of NO was better than that of raw A. kusnezoffii ,but poorer than raw A. kusnezoffii in the inhibitory effects on the release of TNF-α and IL-6. CONCLUSIONS:The toxicity of A. kusnezoffii can be reduced after processed with T. chebula ,especially the toxicity of it in medium or high concentration and short-term use is lower than that of raw A. kusnezoffii . At the same time ,the anti-inflammatory effect of A. kusnezoffii processed with T. chebula is comparable to that of raw A. kusnezoffii at the same concentration.
期刊: 2020年第31卷第22期
作者: 支美汝,韩舒,刘凯洋,韩喜桃,唐雅楠,刘子琴,王宏月,李飞,杜红
AUTHORS: ZHI Meiru,HAN Shu,LIU Kaiyang ,HAN Xitao,TANG Yanan ,LIU Ziqin,WANG Hongyue ,LI Fei,DU Hong
关键字: 生草乌;诃子制草乌;毒性;抗炎;H9c2细胞;RAW264.7细胞
KEYWORDS: Raw Aconitium kusnezoffii;Aconitium kusnezoffii processed with Terminalia chebula ; Cytotoxicity;Anti-
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