出 处:《中国药房》2020年第22期2701-2705,共5页China Pharmacy
基 金:国家自然科学基金资助项目(No.81774004,No.81973479)。
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、10 mg/mL生草乌和诃子制草乌作用4、8、12、24 h对细胞抑制率的影响,采用Hoechst 33258染色法观察2、4、6 mg/mL生草乌和诃子制草乌作用24 h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、2 mg/mL生草乌和诃子制草乌作用24 h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、1 mg/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2 mg/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24 h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4~10 mg/mL时,诃子制草乌在各时间点(除8、10 mg/mL作用24 h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、6 mg/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。0.05~0.5 mg/mL的生草乌和诃子制草乌对RAW264.7细胞均无毒性,0.25、0.5 mg/mL的生草乌和0.1、0.25、0.5 mg/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、0.5 mg/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。OBJECTIVE:To compare cytotoxicity and anti-inflammatory effects of raw Aconitium kusnezoffii and A.kusnezoffii processed with Terminalia chebula.METHODS:Using H9 c2 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/m L 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/m L 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/m L 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/m L raw A.kusnezoffii and A.kusnezoffii processed with T.chebula on the release of NO,TNF-αand IL-6 in RAW264.7 inflammation cells induced by LPS.RESULTS:When the mass concentration was 0.5,1 mg/m L,neither raw A.kusnezoffii and A.kusnezoffii processed with T.chebula had no inhibitory effect on H9 c2 cells.When the mass concentration was 2 mg/m L,the inhibitory effects of A.kusnezoffii processed with T.chebula on H9 c2 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/m L,the inhibitory rate of A.kusnezoffii processed with T.chebula on H9 c2 cells at different time points(except for 24 h culture of 8,10 mg/m L)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/m L 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/m L raw A.kusnez
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