Comparative Study On The Anti-tumor, Anti-inflammatory, Immunomodulatory Effects Of Lfcin Derived From Different Sources

Bovine lactoferrcin(Lfcin B) is an active polypeptide which is produced in the hydrolyzation process of lactoferrin of milk under the environment of pepsin, while human lactoferricin(Lfcin H) is similar to Lfcin B but a product of human milk.Many study focused on the biological activity of Lfcin B,butless research on Lfcin H.In this study,we treated Jurkat cell,mouse macrophages and mouse lymphocyte derived from spleen with different concentrations of Lfcin B and Lfcin H,then identified the anti-tumor,anti-inflammatory and immnomodulatory effect of both Lfcin B and Lfcin H and analyzed the difference and correlation between Lfcin B and Lfcin H. This could provide the theoretical and experimental basis of developing Lfcin as biological anti-tumor drug,infant formula and dairy product.The effects of Lfcin B and Lfcin H on the proliferation of Jurkat cells were assayed by MTT.The nucleus changes of the cells were observed by Hoechst33258 staining fluorescence microscopy.Double-labeled Annexin V-FITC/PI was used to distinguish the apoptosis of Jurkat cells which was promoted by Lfcin H and Lfcin B.The changes of mitochondrial membrane potential of Jurkat cells were observed by JC-1 staining.The amount of NO released by mouse macrophages after stimulating with LPS was measured using Griess. Both the effects on proliferation of spleen lymphocyte treated with different concentrations of Lfcin and the effects on proliferation of T and B lymphocytes with the treatment of different mass concen trations of Lfcin and motigen together were determined by CCK-8.The concentrations of TNF-α secreted by mouse macrophages and IL-2、IL-4、TNF-α spleen lymphocytes were detected by ELISA.1.The results showed that Lfcin B and Lfcin H had a significant inhibitory effect on Jurkat cells proliferation(P<0.05), in a dose-dependent manner. It presented the characteristic of apoptosis such as nuclear shrinkage and cell debris after being treated by Lfcin B and Lfcin H using fluorescence microscopy.The decreased mitochondrial membrane potential of Jurkat cells was observed by laser scanning confocal microscope.After 48 h treatment of Lfcin H and Lfcin B on Jurkat cells,it mainly promoted the early phase of apoptosis.Above all,both Lfcin B and Lfcin H with the concentration of 100-300μg/m L could significantly inhibit cell proliferation and induce apoptosis, Optimal concentration of Lfcin H and Lfcin B was respectively 150μg/mL and 250μg/mL to inhibit the cell proliferation of Jurkat.So we can inferred that the mechanism of apoptosis might inhibit cell proliferation by affecting mitochondrial membrane potential, furthermore,lead to apoptosis.2.According to Griess and cck-8,Lfcin B and Lfcin H could dose-dependently inhibit NO and TNF-α released by Balb/c mouse macrophage stimulated with LPS.The level of NO and TNF-α by experimental group releasing was significantly different from blank group but both experimental group and blank group were lower than control group only with treatment of 1 μg/mL LPS(LPS control group).The optimal concentration for inhibiting macrophages to release NO and TNF-α was 100μg/mL and 150μg/mL of Lfcin H and Lfcin B.Activated LPS could induce macrophages to produce NO and to secret proinflammatory cytokine TNF-α.It can be inferred that Lfcin B and Lfcin H could avoid body damage and present their anti-inflammatory effect by inhibiting NO releasing and TNF-α secretion.3.Lfcin B and Lfcin H could effectively promote cell proliferation of lymphocytes derived from spleen according to CCK-8.Lfcin B and Lfcin H revealed synergistic effect with Con A and LPS to cell proliferation in T and B lymphocytes and then significantly promoted lymphocytes derived from spleen to secret IL-2、IL-4、IL-10 and TNF-α in vitro compared with blank group(P<0.05).The optimal concentration of Lfcin B and Lfcin H was respectively 150 μg/m L and 100 μg/mL for promoting cell proliferation in lymphocytes derived from spleen,meanwhile,the secretion of cytokines,including IL-2,IL-4,IL-10 and TNF-α reached the highest.Therefor e, Lfcin B and Lfcin H could enhance the immunity via promoting cell proliferation in lymphocytes derived from spleen and promoting the secretion of cytokines.Given above,both Lfcin B and LfcinH could inhibit cell proliferation of Jurkat and induce Jurkat cell apoptosis,also could inhibit mouse macrophages treated with LPS to release NO and to secret TNF-α.Both Lfcin B and Lfcin H promoted cell proliferation in mouse lymphocytes derived from spleen and regulated the secretion of IL-2、IL-4、IL-10 and TNF-α.Therefore,both Lfcin B and Lfcin H presented their anti-tumor,anti-inflammatory and immunomodulatory effects,then these three effects of Lfcin B and Lfcin H were basically identical but the optimal concentrations of Lfcin B and Lfcin H were not the same.This study provided a good basis for subsequent experiment in vivo and also provided a support that ingredients of milk from wide origins could substitute the mother’s milk.Furthermore,this study could enrich the research on naturally anti-tumor drug and open up a new way to the development of infant fomula and immunomodulatory food.