Study On The Immunological Activity Of Apple Pomace Pectin In Vivo And In Vitro

With the development of the juice industry,a large number of peels and pomace are corrupted and acidified,resulting in environmental pollution and waste of resources.The development and utilization of apple pomace resources has become a major development strategy for the fruit industry today.Apple pomace is rich in nutrients and dietary fiber.It is a good feed resource,and can also be used to extract pectin,produce jam,and wine.In recent years,a large number of studies have found that pectin has biological activities such as improving human immunity,anti-aging,and anti-tumor.At the same time,our laboratory found that the extraction of pectin by subcritical water method can give new functional activity to pectin fructose.Therefore,this paper hopes to study the pro-immunological activity of apple pomace pectin prepared by subcritical water method and analyze its Mechanism of action,and its difference in bioactivity from commercial pectin.This study explored the immunological activity of pectin in in vivo and in vitro assays.The commercial pectin(CAP),crude pectin(NT-APP)and the four purified pectin polysaccharides isolated and purified were used as samples.The immune function of macrophage RAW264.7 and colon cancer cell line HT were tested in vitro.-29 The effects of proliferative viability were analyzed.The effects of pectin on the immunity of colon cancer mice were analyzed in vivo.At the same time,the main signal pathways for the immunological activity of pectin were determined and the related research results were as follows:(1)CCK-8 and EDU assays showed that NT-APP significantly promoted the proliferation of RAW264.7.Flow cytometry and Western Blotting assay showed that NT-APP promoted RAW264.7 proliferation by regulating cell cycle.The study found that NT-APP positively regulates the expression of PCNA protein in RAW264.7 cells,and the number of RAW264.7 cells in S phase is as high as 41% after treatment with high concentration of NTAPP,which is significantly higher than the control group.At the same time,NT-APP can Significantly promoted the phagocytic ability of RAW264.7,TNF-a and NO release,however,it has no effect on the release of IL-10;commercialized pectin did not significantly promote macrophage proliferation and phagocytosis in the analysis.Therefore,Compared with commercial pectin,NT-APP prepared by subcritical water method has better immunological activity and exhibits a significant concentration dependence.(2)Study on the immunological activity of four purified polysaccharides isolated from NT-APP,the operation was consistent with NT-APP,and the four purified polysaccharides with concentration of 50 ug/ml,100 ug/ml,200 ug/ml were selected to treat RAW264.7.Detecting its proliferative capacity and phagocytic change.Then,treating RAW264.7 with four purified polysaccharides at a concentration of 200 ug/ml,and detecting the release ability of cytokines and inflammatory mediators.The results showed that NT-APP-1 is not immunologically active,NT-APP-2 and NT-APP-4 have significant immunological activities.NT-APP-5 can only increase the phagocytic function of macrophages and cannot promote the elevation of other immune indicators.(3)To test the immunological activity of normal mice and colon cancer cells,the mice were divided into five groups,weighed before sacrifice,and the spleens and thymus were weighed,and the spleen and thymus index were calculated.It was found that CRC induction can lead to small Inflammatory immunity in mice,dietary intervention in CRC mice can improve inflammatory immunity in mice with colorectal cancer,and the greater the concentration,the more obvious the inflammatory immunity.Oral mice fed high doses of pectin can increase inflammatory immunity in mice.The release of cytokines in serum was then detected by ELISA.The results of this study demonstrate that high-dose pectin dietary intervention can improve inflammatory immunity in normal mice,and its effect is more severe than inflammatory immune response caused by CRC intervention,while low dose Pectin intervention can inhibit inflammatory immunity in CRC mice,and the inflammatory immune response of CRC mice reaches normal levels.The effect of pectin on HT-29 was detected by CCK-8 method.It was found that pectin could not directly act on HT-29,but it could promote the release of TNF-a by RAW264.7 and inhibit the proliferation of HT-29.(4)Detection of proliferation-related P-ERK signaling pathways,receptor-receptor protein TLRs on macrophages,and P-NF-KB signaling proteins involved in the regulation of inflammation,analysis showed that NT-APP is recognized by TLR4 on the surface of RAW264.7 and stimulates TLR4 expression,and activate the phosphorylation of the downstream signaling protein ERK,thereby promoting cell proliferation,then activating the phosphorylation of the inflammatory signaling protein NF-KB,and promoting inflammation-related Release of cytokines.