人参皂苷Rg_(1)、Rb_(1)对脂多糖体外诱导肠上皮屏障损伤的保护作用

Protective Effect of Ginsenosides Rg_(1) and Rb_(1) Against Intestinal Epithelial Barrier Injury Induced by Lipopolysaccharide in Vitro

目的:基于人髓系白血病单核细胞(THP-1)与肠上皮细胞(Caco-2)共培养体系,研究人参皂苷Rg_(1)和人参皂苷Rb_(1)对脂多糖(LPS)诱导的THP-1细胞炎症因子释放的影响,及其对THP-1细胞活化致Caco-2细胞炎性损伤的保护作用。方法:首先制备THP-1与Caco-2细胞共培养微流控芯片,实验分为空白组、LPS组和给药组。空白组细胞正常培养;LPS组在上层Caco-2细胞形成单层屏障后,在下层THP-1细胞中加入LPS(1 mg·L^(-1));给药组在LPS组的基础上在THP-1细胞中分别加入33 mg·L^(-1)的人参皂苷Rg_(1)和人参皂苷Rb_(1)。THP-1细胞与Caco-2细胞共培养24 h后采用异硫氰酸荧光素-葡聚糖(FITC-Dextran)示踪法检测下层芯片通道中的FITC-Dextran荧光值。THP-1细胞实验分为空白组、LPS组、给药组。空白组THP-1细胞正常培养;LPS组在THP-1细胞中加入LPS(1 mg·L^(-1));给药组在LPS组的基础上分别加入相应剂量的人参皂苷Rg_(1)和人参皂苷Rb_(1)(11、33、100 mg·L^(-1))。细胞培养24 h后细胞增殖与活性检测(CCK-8)检测THP-1细胞活性,实时荧光定量聚合酶链式反应(Real-time PCR)检测THP-1细胞炎性细胞因子白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)表达。Caco-2细胞实验分为空白组、LPS组、给药组。空白组Caco-2细胞正常培养;其他组将第二部分THP-1细胞实验中对应组的细胞上清置换于Caco-2细胞中,继续培养24 h后CCK-8检测Caco-2细胞活性,Real-time PCR检测Caco-2细胞炎性细胞因子IL-6、IL-8、TNF-α及紧密连接蛋白封闭蛋白(Occludin)表达,蛋白免疫印迹法(Western blot)检测Caco-2细胞紧密连接蛋白Occludin的表达。结果:在THP-1与Caco-2细胞共培养体系中,与LPS组比较,人参皂苷Rg_(1)和Rb_(1)均能有效保护LPS诱导肠上皮屏障通透性升高(P<0.01)。Rg_(1)和Rb_(1)拮抗LPS诱导的THP-1细胞IL-6、IL-1β、TNF-α炎性细胞因子表达升高(P<0.05)。经Rg_(1)和Rb_(1)处理的THP-1细胞上清与Caco-2细胞共培养后,与LPS组比较,显著降低Caco-2细胞IL-6、IL-8、TNF-α炎性细胞因子表达(P<0.01),上调紧密连接蛋白Occludin表达。结论:在THP-1与Caco-2细胞共培养体外模拟肠道上皮屏障功能模型中,人参皂苷Rg_(1)和Rb_(1)通过调节THP-1细胞释放炎性细胞因子,进而调控Caco-2细胞的炎性反应和细胞屏障完整性,在LPS诱导的体外肠上皮屏障损伤中发挥保护作用。

Objective:To investigate the effects of ginsenoside Rg_(1) and ginsenoside Rb_(1) on the release of inflammatory factors of human myeloid leukemia monocytes(THP-1)induced by lipopolysaccharide(LPS)and their protective effects on the inflammatory injury of intestinal epithelial cells(Caco-2)induced by THP-1cell activation based on the co-culture system of THP-1and Caco-2.Method:Firstly,the microfluidic chip of co-culture of THP-1and Caco-2 cells was prepared.In the experiment,a blank group,an LPS group,and drug intervention groups were set up.The cells in the blank group were cultured conventionally.In the LPS group,LPS(1 mg·L^(-1))was added to the lower THP-1cells after the upper Caco-2 cells formed a monolayer barrier.On the basis of the LPS group,33 mg·L^(-1)ginsenoside Rg_(1) and 33 mg·L^(-1) ginsenoside Rb_(1) were added to THP-1cells respectively.After the co-culture of THP-1cells and Caco-2 cells for 24 hours,the fluorescein isothiocyanate(FITC)-Dextran fluorescence value in the lower chip channel was detected by FITC-Dextran tracer method.A blank group,an LPS group,and drug intervention groups were set up in the THP-1cell experiment.THP-1cells in the blank group were cultured conventionally.In the LPS group,LPS(1 mg·L^(-1))was added to THP-1cells.Ginsenoside Rg_(1)and ginsenoside Rb_(1) of the corresponding doses(11,33,100 mg·L^(-1))were added to the drug intervention groups respectively on the basis of the LSP group.After 24 hours of cell culture,the activity of THP-1cells was detected by cell counting kit-8(CCK-8).Real-time quantitative polymerase chain reaction(Realtime PCR)was used to detect the expression of inflammatory cytokines such as interleukin-6(IL-6),interleukin-1β(IL-1β),and tumor necrosis factor(TNF)-αof THP-1cells.A blank group,an LPS group,and drug intervention groups were set up in the Caco-2 cell experiment.Caco-2 cells in the blank group were cultured conventionally,and in other groups,the corresponding cell supernatant in the second part of the THP-1cell experiment was employed in Caco-2 cells.After 24 hours of cell culture,the activity of Caco-2 cells was detected by CCK-8.Real-time PCR was used to detect the expression of IL-6,interleukin-8(IL-8),TNF-α,and Occludin of Caco-2 cells.The expression of tight junction protein Occludin in Caco-2 cells was detected by Western blot.Result:Both ginsenoside Rg_(1) and ginsenoside Rb_(1) could effectively protect LPS-induced intestinal epithelial barrier permeability in the co-culture system of THP-1and Caco-2 cells(P<0.01).Ginsenosides Rg_(1) and Rb_(1) antagonized LPS-induced increased expression of IL-6,IL-1β,and TNF-αin THP-1cells(P<0.05).When the supernatant of THP-1cells treated with ginsenosides Rg_(1) and Rb_(1) was co-cultured with Caco-2 cells,the expression of IL-6,IL-8,and TNF-αin Caco-2 cells was significantly reduced(P<0.01),and the expression of tight junction protein Occludin was up-regulated.Conclusion:In the co-culture system of THP-1and Caco-2 cells simulating the intestinal epithelial barrier function in vitro,ginsenosides Rg_(1)and Rb_(1) play a protective role against LPS-induced intestinal epithelial barrier injury by regulating the release of inflammatory cytokines by THP-1cells,thereby regulating the inflammatory response and cell barrier integrity of Caco-2 cells.