The Impact Of Supernatant Fluid Of Rhubarb Combined With Human Breast Milk On The Expression Of Caco-2Inflammatory Cell’s Toll-like Receptor And Nuclear Transcription Factor

Objective:Neonatal necrotizing enterocolitis is a disease which is caused by the ischemia reperfusion injury of intestine and systemic inflammatory response induced by breeding of pathogens. Relevant clinical and experimental research has confirmed the nice effectiveness of rhubarb and breast feeding on preventing NEC. To Investigate the correlative mechanism of rhubarb and breast milk on preventing NEC, we induced Intestinal inflammation cell model of NEC with inflammatory factor TNF-a and studied the impact of rhubarb and breast milk on the expression of toll-like receptor mRNA and NF-κB P65of inflammatory Caco-2cells, providing the evidence for clinical application.Methods:The experiment is divided into blank control group and model group induced by TNF-a.The blank control group is normal Caco-2cell added conventional culture. We induced intestinal inflammation cell model of NEC with inflammatory factor TNF-α.We detected the content of NF-κB p65with Trans AM NF-κB p65kit and determined the optimal stimulating time-point with dual luciferase report system. The model group induced by TNF-α was divided into four groups:we added conventional culture,1%rhubarb supernatant fluid,1%breast milk supernatant fluid and the combination of the two into the four groups respectively. We detected the expression of toll-like receptor mRNA with RT-PCR method,the expression of NF-αB P65with immunocytochemical method and the relative expression of IL-1βmRNA which is downstream product of NF-κB P65with RT-PCR method, and compared the differences among the detection results above. We revealed the impact of rhubarb supernatant fluid、breast milk supernatant fluid and the combination of the two on the expression of toll-like receptor mRNA and NF-κB P65of inflammatory Caco-2cells.Results:In this experiment:we discovered1%rhubarb supernatant fluid,1%breast milk supernatant fluid and the combination of the two could obviously inhibit the expression of toll-like receptor mRNA, compared with inflammatory Caco-2cells added conventional culture, obvious difference was significant (P<0.01);compared with1%rhubarb supernatant fluid group,the combination group had better effect, obvious difference was significant (P<0.01); compared with1%breast milk supernatant fluid group, the combination group had better effect, the difference was statistically significant (P<0.05); compared with1%rhubarb supernatant fluid group,1%breast milk supernatant fluid group had better effect, but the difference had no statistical significance (P>0.05). We discovered1%rhubarb supernatant fluid,1%breast milk supernatant fluid and the mixture of the two could obviously inhibit the expression of NF-κB P65, compared with inflammatory Caco-2cells added conventional culture, the inhibition effect of the combined group was best, the breast milk supernatant fluid group was second and the rhubarb supernatant fluid was last. We discovered1%rhubarb supernatant fluid,1%breast milk supernatant fluid and the combination of the two could obviously inhibit the expression of IL-1βmRNA which is downstream product of NF-κB P65, compared with inflammatory Caco-2cells added conventional culture, obvious difference was significant (P<0.01); compared with1%rhubarb supernatant fluid group, the combination group had better effect, obvious difference was significant (P<0.01); compared with1%breast milk supernatant fluid group, the combination group had better effect, the difference was statistically significant (P<0.05); compared with1%rhubarb supernatant fluid group,1%breast milk supernatant fluid group had better effect, but the difference had no statistical significance (P>0.05). Conclusions:Rhubarb supernatant fluid, breast milk supernatant fluid and the combination of the two can inhibit the expression of toll-like receptor, NF-κB P65and its downstream product IL-1βmRNA of inflammatory Caco-2cells, that is to reduce inflammatory reaction; It proves that toll-like receptor and nuclear transcription factor participate in the pathogenesis of NEC, this may be one of the mechanisms of rhubarb and breast milk preventing NEC. This study clarifies the effectiveness and the mechanisms of rhubarb and breast milk preventing NEC through the level of cellular and molecular biology and provides the evidence for clinical application of using rhubarb and breast milk.