如源一号对脂多糖刺激Caco-2及其与THP-1共培养表达炎症趋化细胞因子的影响

Effects of RY-1 on Lipopolysaccharide stimulated Expression of pro-inflammatory cytokines and Chemokines in Human Colon Cancer Cells and Monocytes

目的探讨如源一号(RY-1)治疗HIV感染致慢性腹泻的作用机制。方法采用人结肠癌细胞系Caco-2体外培养、和共培养的Caco-2与人单核细胞系THP-1,以脂多糖(LPS)刺激模拟肠菌移位诱导炎症模型。以CCK8法检测RY-1的细胞毒性;以定量PCR法和ELISA法定量检测共培养的Caco-2/THP-1上清中炎症细胞因子(TNF-α、IL-1β、IL-6、IL-8、TGF-β、COX-2、iNOS、IL-23)和趋化因子(CCL2和CCL20)的水平,Griess法检测一氧化氮(NO)含量;以WB法检测Caco-2 NF-κB通路的改变。结果RY-1在本研究的最大浓度1 mg/mL无细胞毒性。与模型组相比较,RY-1可显著抑制LPS刺激增高的IL-6 mRNA(最高抑制59.5%)和上清蛋白浓度(最高抑制7.9%);此作用不依赖于抑制NF-κB。对LPS刺激Caco-2表达IL-8、CCL2 mRNA的增高也有抑制。此外,RY-1可抑制共培养的Caco-2/THP-1上清中的IL-6(最高抑制13.2%)、CCL2(最高抑制29.4%)、CCL20(最高抑制29.4%)和TNF-α(最高抑制8.6%)。RY-1还可显著促进共培养的Caco-2/THP-1表达IL-23蛋白(高浓度时增加39.0%)。结论RY-1可能通过抑制IL-6、CCL2,直接抑制肠黏膜炎症、保护肠黏膜屏障功能;还可上调IL-23,可能通过多机制对肠黏膜屏障起到保护作用。

Objective To explore the possible therapeutic mechanism of RY-1 to treat chronic diarrhea among people living with HIV(PLWH).Methods Cultured human colorectal adenocarcinoma Caco-2,as well as co-culture of Caco-2 and THP-1(a human acute monocytic leukemia cell line),were subjected to lipopolysaccharide(LPS)stimulation to recapitulate the features of inflammation in the gastrointestinal tract caused by microbial translocation during HIV-1 infection.The cytotoxicity of RY-1 on Caco-2 cells was evaluated with CCK8.The expression profiles of proinflammatory cytokines(such as TNF-α,IL-1β,IL-6,IL-8,TGF-β,COX-2,iNOS,IL-23),and chemokines(CCL2 and CCL20)were determined by quantitative PCR and ELISA assay.The concentrations of nitric oxidant in the supernatant were also quantified by the Griess reagent.The phosphorylation of NF-κB p65 and IκBαwere also quantified by Western blot.Results RY-1 in all concentrations tested in this study(maximum 1 mg/mL)showed no cytotoxicity on Caco-2 cells.Compared to the normal control group.RY-1 treatments significantly decreased LPS-stimulated upregulation of IL-6 with a maximum inhibition of 59.5%on mRNA and 7.9%on supernatant protein concentration.This effect is irrelevant to NF-κB pathway activation.Moreover,RY-1 also suppressed LPS-stimulated transcription of IL-8 and CCL2 significantly.In LPS stimulated co-culture of Caco-2/THP-1,RY-1 decreased the supernatant concentration of IL-6(13.2%),CCL2(29.4%),CCL20(29.4%),and TNF-α(8.6%).RY-1 also augmented the IL-23 production in LPS-stimulated co-cultured Caco-2/THP-1 significantly with a maximum increase of 39.0%.Conclusion RY-1 might exert its protective effect against chronic diarrhea in HIV-1 infection by a synergetic mechanism probably through inhibiting the intestinal inflammation mediated by IL-6 and CCL2 and maintaining the intestinal barrier function by augmented IL-23 production.