参苓白术散通过自噬调节肠隐窝上皮细胞损伤的作用机制  

Shen Ling Baizhu Powder Regulates Intestinal Crypt Epithelial Cell Damage by Autophagy

作　　者：刘玉晖 徐伟[2] 曾兰芳 游宇[1] LIU Yuhui;XU Wei;ZENG Lanfang;YOU Yu(College of Pharmacy,Jiangxi University of TraditionalChinese Medicine,Nanchang 330004 Jiangxi,China;Department of Gastroenterology,the First AffiliatedHospital of Nanchang University,Nanchang 330006 Jiangxi,China)

机构地区：[1]南昌大学第一附属医院消化内科,江西南昌330006 [2]江西中医药大学药学院,江西南昌330004

出　　处：《中药新药与临床药理》2019年第10期1165-1171,共7页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目（81460627）

摘　　要：目的探究参苓白术散通过细胞自噬调节脂多糖(LPS)诱导的肠隐窝上皮细胞(IEC-6)损伤的作用与机制。方法 IEC-6细胞分组:空白组、LPS模型组、LPS+参苓白术散含药血清低、中、高(5%、10%、20%)剂量组、自噬抑制剂3-甲基腺嘌呤组(3-MA)、自噬诱导剂雷帕霉素靶蛋白(mTOR)阻断剂雷帕霉素组(Rapamycin)。采用MTT方法检测LPS的细胞毒性、检测参苓白术散含药血清浓度梯度活性;通过ELISA试剂盒测定IEC-6细胞上清液中白介素1β(IL-1β)、白介素8(IL-8)、白介素10(IL-10)和肿瘤坏死因子α(TNF-α)的浓度;通过RT-PCR法检测ATG5、ATG12、ATG13、ATG16的mRNA水平;使用RFP-GFP-LC3腺病毒转染IEC-6细胞,测定IEC-6细胞自噬体的数目。结果 LPS浓度为100μg·mL-1时,IEC-6细胞抑制率为29.3%,以该浓度进行后续实验;不同浓度参苓白术散含药血清对细胞活力均无明显作用;模型组与空白组比较,促炎因子IL-1β、IL-8和TNF-α浓度上升,抑炎因子IL-10浓度下降,ATG5、ATG13、ATG16L2 mRNA水平上升(均P <0.05),模型组中没有明显的自噬体斑点形成;含药血清组与模型组比较,促炎因子IL-1β、IL-8和TNF-α浓度下降,抑炎因子IL-10浓度上升,ATG5、ATG13、ATG16L2 mRNA水平下降(均P <0.05),ATG12和ATG16L1 mRNA水平无显著性差异(P> 0.05),含药血清组和自噬诱导剂Rapamycin组自噬斑点的形成明显增多;自噬抑制剂3-MA组与模型组比较,自噬体斑点没有差别。结论参苓白术散含药血清能抑制促炎症因子IL-1β、IL-8和TNF-α的释放,增加抑炎症因子IL-10的释放,减轻IEC-6细胞的炎症损伤,这一过程可能与调控自噬相关通路Beclin1有关。Objective To explore the effect and mechanism of Shen Ling Baizhu powder on regulating intestinal crypt epithelial cells(IEC-6)injury induced by lipopolysaccharide(LPS)through autophagy.Methods IEC-6 cells were divided into groups as follows:blank group,LPS model group,LPS+Shen Ling Baizhu powder serum low,medium and high(5%,10%,20%)dose groups,autophagy inhibitor 3-methyladenine group(3-MA),autophagy inducer(rapamycin target protein mTOR blocker)rapamycin group(Rapamycin).The cytotoxicity of LPS was detected by MTT method,and the concentration gradient activity of serum containing Shen Ling Baizhu powder was detected.Concentrations of interleukin-1β(IL-1β),interleukin 8(IL-8),interleukin-10(IL-10)and tumor necrosis factor alpha(TNF-α)in IEC-6 cells supernatant were determined by ELISA kit.RT-PCR was conducted to determine the mRNA levels of ATG5,ATG12,ATG13,and ATG16.IEC-6 cells were transfected with RFP-GFPLC3 adenovirus to determine the number of autophagosomes in IEC-6 cells.Results When the concentration of LPS was 100μg·mL-1,the inhibition rate of IEC-6 cells was 29.3%,and the subsequent experiments were carried out at this concentration.The serum containing different concentrations of Shen Ling Baizhu powder had no significant effect on cell viability.Compared with the blank group,the concentration of pro-inflammatory factors IL-1β,IL-8 and TNF-αincreased,the concentration of anti-inflammatory factor IL-10 decreased,and the levels of ATG5,ATG13 and ATG16 L2 mRNA increased with significant difference(P<0.05).There was no obvious autophagosome spot formation in the model group.Compared with the model group,the concentration of pro-inflammatory factors IL-1β,IL-8 and TNF-αdecreased,and the concentration of anti-inflammatory factor IL-10 increased with significant difference(P<0.05).The mRNA levels of ATG5,ATG13 and ATG16 L2 decreased(P<0.05),and there was no significant difference in ATG12 and ATG16 L1 mRNA levels(P>0.05).The formation of autophagic spots were significantly increased in the drug-cont

关 键 词：参苓白术散 脂多糖 炎症因子 细胞自噬 细胞自噬体 肠隐窝上皮细胞 

分 类 号：R285[医药卫生—中药学]