Stuy On Anti Inflammatory Effect Of Extract From Piper Sarmentosum Roxb On Porcine Intestinal Epithelial Cells

Objective:in vitro and cell tests,qPCR,Western-blot and other techniques were used to study the anti-oxidation effect of extracts from the Piper sarmentosum roxb and its anti-inflammatory effects on porcine intestinal epithelial cells.In this experiment,Piper sarmentosum roxb was selected as the research object,in order to provide scientific basis for its development as a feed additive of Chinese veterinary medicine for preventing and curing diarrhea in livestock and poultry.Methods:(1)The antioxidant capacity of three extracts of Piper sarmentosum(petroleum ether,n-butanol and ethyl acetate)was studied by antioxidant test in vitro.The antioxidant capacity of the three extracts was tested by ABTS and the active parts were screened.(2)The inflammation model of IPEC-J2 cell was established with LPS in vitro,after pretreatment with Piper sarmentosum extract for 24 hours,collected cell.The content of inflammatory cytokines IL-6,IL-1 and TNF-a was detected by ELISA,and the effective anti-inflammatory fractions were screened.(3)The inflammation model of IPEC-J2 cells in vitro was established by LPS method.The levels of IL-6,IL-1 and TNF-alpha gene and TLR2,TLR4 and TLR9 gene were detected by qRT-PCR.The optimal concentration of Arctium pseudoacacia extract was selected for subsequent Western blotting test.(4)The inflammation model of IPEC-J2 cells in vitro was established by LPS method and after pretreatment with Piper sarmentosum extract for 24 hours,collected cell.Western blotting was used to detect the phosphorylation of NF-kB signaling pathways p65 and i-kb alpha,p38 and its phosphorylation level in p38-MAPK signaling pathway,ERK and its phosphorylation level in erkl/2 signaling pathway.The effect extract on IPEC-J2 cell signaling pathway was studied and verify signaling pathways.Result:(1)The results showed that the total antioxidant activity of Piper sarmentosum extract in descending order were n-butanol extract,ethyl acetate extract and petroleum ether extract.The contents of total alkaloids and total phenols of Piper sarmentosum n-butanol extract were the highest,which were 2.818 mEq/100g and 161.82 mg GAE/g,respectively.(2)In anti-inflammatory experiments,compared with the blank group,the levels of TNF-α and IL in IPEC-J2 cells increased significantly in the LPS group(P<0.05).Meanwhile,compared with the Piper sarmentosum pretreatment group,the levels of TNF-α and IL decreased significantly(P<0.05),and the concentrations of cytokines from high to low were ethyl acetate extract,petroleum ether extract and n-butanol extract,respectively.The results showed that JZ group had the best anti-inflammatory effect among the three extracts,which could affect the secretion of inflammatory factors in IPEC-J2 cells and effectively inhibit the inflammatory response.The results showed that JZ group had the best anti-inflammatory effect among the three extracts,which could affect the secretion of inflammatory factors in IPEC-J2 cells and effectively inhibit the inflammatory response.(3)The results of qPCR showed that the expression of IL-1,IL-6 and TNF-a increased significantly 24 hours after LPS treatment at 1μg/ml compared with the blank control group.After JZ treatment at 10μg/ml,the expression of IL-1,IL-6 and TNF-α decreased.These results sμggest that JZ at 10μg/ml can significantly reduce LPS-induced inflammation and down-regulate the expression of inflammatory factors.After LPS induction,the expression of TLR2 and TLR9 increased significantly,while JZ treatment decreased the expression of TLR9.These results s u ggest that JZ can inhibit LPS-mediated TLR-induced inflammation.(4)Westem blotting results showed that LPS could significantly increase the level of p65 in the NF-kappa B pathway,while the levels of p38 phosphorylation,p65 and its phosphorylation,ERK and their phosphorylation were significantly decreased after JZ pretreatment at different concentrations.The results of inhibitor validation showed that the dR μ gs could play an anti-inflammatory role by inhibiting the signal pathways of NF-kappa B and p38 MAPK in intestinal epithelial cells.Conclusion:The extract of Piper sarmentosum has antioxidant activity,and it can affect the secretion of cytokines in IPEC-J2 cells and inhibit the inflammation;Piper sarmentosum extract can down-regulate the expression of inflammatory factors,and inhibit inflammation by inhibiting the signal pathway of NF-kB and p38MAPK in intestinal epithelial cells.