Study On Mechanism Of Alleviating Cow’s Milk Allergy By Lactobacillus Acidophilus Based On TLR4/NF-κB Pathway

In recent years,the incidence of food allergies has gradually increased,and it has become a serious public health problem,but its pathogenesis is not fully understood.TLR4 is an important pattern recognition receptor that recognizes pathogen-associated molecular patterns,then it triggers cascades of cellular signaling and subsequently activates nuclear factor NF-κB,leading to release of a variety of cytokines,thereby regulating the immune response.The TLR4/NF-κB signaling pathway has been shown to play an important role in a variety of inflammatory diseases.However,the role of the TLR4/NF-κB pathway in beta-lactoglobulin(β-Lg)allergy inflammation has not yet been investigated.Our previous study found a strain of Lactobacillus acidophilus KLDS 1.0738 with anti-allergic effects.However,whether the TLR4/NF-κB signaling pathway is also involved in alleviating β-Lg allergy inflammation by Lactobacillus acidophilus is unknown.Based on this,in vitro cell experiments,this study established β-Lg-induced macrophage RAW264.7 allergic model,and studied the role of TLR4/NF-κB signaling pathway in β-Lg-induced allergic inflammation,and compared and analyzed the role of viable Lactobacillus acidophilus and its heated-killed bacteria on the regulation of β-Lg allergy.Furthermore,this study further explained the mechanism of alleviating β-Lg allergy by Lactobacillus acidophilus based on the key role of TLR4/NF-κB in allergic reactions.Finally,this study provides new targets for the treatment and prevention of β-Lg allergy.(1)The role of TLR4/NF-κB signaling pathway in β-Lg-induced macrophage allergyIn order to analyze the role of TLR4/NF-κB pathway in β-Lg-induced macrophage hypersensitivity,RAW264.7 cells were treated with 1 mg/m L β-Lg(β-Lg allergic group)and 10 μg/m L TLR4 inhibitor C34(TLR4 inhibitor C34 group),The cells in TLR4 inhibitor C34 + β-Lg group were cultured with 10 μg/m L TLR4 inhibitor C34 for 30 min,then added 1 mg/m L β-Lg.The cell cultured with medium alone was used as normal group.Then,the content of specific Ig E in the supernatant was detected by ELISA.The m RNA expression s level of TLR4,NF-κB,My D88,IRAK1,TRAF6 and cytokines IL-1β,IL-6,TNF-α were detected by q RT-PCR.The expressions of TLR4 and NF-κB p65 protein were detected by Western blot.The results showed that β-Lg increased the content of β-Lg-specific Ig E in macrophages.Compared with the allergic group,the results showed that the content of specific Ig E in the cell supernatant was significantly decreased after inhibition of the TLR4/NF-κB pathway.At the same time,the m RNA expression levels of TLR4,NF-κB,My D88,IRAK1 and TRAF6 in TLR4/NF-κB pathway related molecules and cytokines were down-regulated.Western blot analysis showed that the expression of TLR4 and NF-κB p65 protein was also significantly decreased after inhibition of TLR4 pathway(P<0.05).The study found that the TLR4/NF-κB pathway played an important positive regulatory role in β-Lg allergy.(2)Regulatory effect of viable and heated-killed Lactobacillus acidophilus on allergic inflammation in β-Lg-induced macrophageIn order to compare the regulatory effects of different viable Lactobacillus acidophilus and its heated-killed bacteria on allergic inflammation in β-Lg-induced macrophage,the cells cultured with medium alone was used as normal group.The cells were stimulated with β-Lg in the absence(β-Lg allergic group)or presence of different viable probiotics and its heated-killed bacteria(viable/heated-killed CP group,viable/heated-killed La 1.0738 group,viable/heated-killed La 1.0210 group,viable/heated-killed La 08001 group).Then,β-Lg-induced macrophage proliferation by Lactobacillus acidophilus and the contents of specific Ig E,IL-1β,IL-6 and TNF-α in cell supernatant were determined by MTT and ELISA,respectively.Compared with the allergic group,The results showed that viable Lactobacillus acidophilus and their heat-killed bacteria reduced the content of specific Ig E,and increased the survival rate of macrophages and reduced the content of cytokines IL-1β,IL-6 and TNF-α in the cell supernatant(P <0.05).The results also showed that viable probiotic groups were better than the heat-killed bacteria groups,especially the viable Lactobacillus acidophilus 1.0738 group and the viable commercial probiotic group.(3)Effects of viable Lactobacillus acidophilus on TLR4/NF-κB signaling pathway in β-Lg-induced macrophagesIn order to analyze the effect of viable Lactobacillus acidophilus on TLR4/NF-κB pathway in β-Lg-induced macrophages,the cell cultured with medium alone was used as normal group.The cells were stimulated with β-Lg in the absence(β-Lg allergic group)or presence of different viable probiotics(viable CP group,viable La 1.0738 group,viable La 1.0210 group,viable La 08001 group),and the cells in TLR4 inhibitor C34 + β-Lg group were cultured with 10 μg/m L TLR4 inhibitor C34 for 30 min,then added 1 mg/m L β-Lg.Then,the m RNA expressions of related molecules TLR4/NF-κB signaling pathway and cytokines were detected by q RT-PCR,and the expressions of TLR4,NF-κB p65 protein were determined by Western blot.Compared with the allergic group,The results showed that the four probiotics significantly reduced the m RNA expression of TLR4,NF-κB,My D88,IRAK1 and TRAF6(P<0.05),Moreover,Western blot results also showed that probiotics could reduce the expression of TLR4 and NF-κB p65 protein,thereby inhibiting the transcriptional activity of NF-κB and further inhibiting the m RNA expression of IL-1β,IL-6 and TNF-α(P<0.05).These results indicated that the TLR4/NF-κB signaling pathway played a positive role in β-Lg allergy.Moreover,the effect of Lactobacillus acidophilus on β-Lg allergy may be to inhibit the production of inflammatory cytokines by regulating TLR4/NF-κB signaling pathway.The mechanism may be that Lactobacillus acidophilus down-regulated the expression of TLR4,inhibited the activation of IRAK1 and TRAF6 through My D88-dependent pathway,thereby inhibiting the activation of nuclear factor NF-κB and transfer of NF-κB into the nucleus to regulate cytokines.These results are beneficial for a better understanding of the mechanism by which Lactobacillus acidophilus participate in attenuating food allergies,these new findings provide new strategies for the prevention and treatment of food allergic diseases and provide a strong basis for clinical applications of Lactobacillus acidophilus.