Immune Regulation Effect And Mechanism Of Four Milk-borne Probiotics On Balb/c Mice

Objective: In the previous study,we confirmed that the immune active peptides which isolated from Xinjiang traditional fermented camel milk(shubat)could promote the proliferation of mouse spleen lymphocytes,and the mRNA relative expression of NF-kappa B,IL-6 were decreased,the relative experession of IL-12,IL-23 were increased.It could promote the immunity of mice spleen.This effect may be related to regulating the balance of Th1/Th2.In addition,fermented dairy may have many probiotics,so we wanted to investigate the immunoregulatory of four probiotics from Xinjiang traditional fermented camel milk and cheese whey,reveal its immune regulation mechanisms.Methods: 1)We cultured the four probiotics with its freeze dried powders and expended,prepared the high dose and low dose suspension;2)Established the immunocompromised model with intraperitoneal injection of 80mg/kg cyclophosphamide for three days;3)Animal test: the Balb/c mice were randomly divided into eleven groups,were given high and low dose suspensions of Lactobacillus kefiranofaciens,Oriental Issa yeast,Lactobacillus Kefir and Lactobacillus pentosus bacterials and positive drug group were given high dose of Bifidobacterium suspension,normal group and model group were given the same volume of saline;4)Determination index: after 4 weeks,we took spleen,thymus,colon of mice and calculated organ indexes,used CCK-8 to detected spleen lymphocyte activity,serum biochemical indicators were detected by automatic biochemical analyzer,used flow cytometry CBA kit to detect serum cytokines level;5)Determination of genes relative expression level: extracted mouse spleen total RNA,reverse transcription and detected the concentration of cDNA,used real-time quantitative PCR(RT-PCR)to detect the mRNA relative expression of IFN-?,IL-4,IL-10,MyD88,TLR-7,to investigate the immunoregulatory function andsignaling pathway of 4 kinds of probiotics on immunosuppressed mice.Results: 1)the weight of mice in the model group was significantly decreased(P<0.05)after one week,and four probiotic groups were both increased;2)The viscera index of spleen and thymus were significantly increased(P < 0.01),and there was no significant difference in the colon.The spleen index was significantly increased(P<0.01);3)The splenic lymphocyte viability of the model group decreased significantly(P < 0.01),and the splenic lymphocyte activities of the four probiotics groups increased significantly(P<0.01),4)Serum biochemical test showed that probiotics can reduce the levels of AST,ALT and UREA,and there was no significant difference in the level of CREA;5)Detection of serum cytokines showed that,compared with the model group,the positive drug group made the significantly increased of IL-2,IFN-?,and significantly decreased of IL-10.Probiotics groups made significantly increased of IL-2,IFN-?,significantly reduced the serum level of IL-6,IL-10,IL-17A(P < 0.05);6)The results of signal pathway: the RT-PCR results showed that compared with the normal group,the IFN-?,IL-4,IL-10,MyD88 and TLR-7 of the model group were significantly higher(P<0.05),compared with the model group,the five groups of these four probiotics groups were significantly decreased(P < 0.01).Conclusion: Gave the probiotic bacteria suspensions in immunocompromised mice,the body weight,liver and kidney function were all maintained at the normal level,they improved the spleen index,spleen lymphocyte activity of the immunocompromised mice,we can see the immune response was partial to Th2 after modeling,and then moved to Th1.It is concluded that these four probiotic bacteria can be regulated by MyD88 dependent TLR-7 signaling pathway,and they have some protective effects on the immune disorder.