Study On The Intestinal Immunomodulatory Function And Mechanism Of Lactobacillus Surface-layer Proteins

Food-based interventions that help promote the gastrointestinal tract health,suppress inflammation and regulate immune functions have received a great deal of attention in recent years.Lactobacillus,a type of GRAS-grade intestinal probiotic,can offer numerous health benefits and regulate the human immune system by competing with adhesion sites in intestinal epithelial cells?IECs?,interfering with the pathogens colonization,producing bacteriocin,and lowering pH.Several studies have shown that surface-layer protein?Slp?extracted from Lactobacillus strains is closely related to the probiotic properties of Lactobacillus.Slp has been confirmed to possess multiple biological properties,but its intestinal health protection effects via modulation of the immune system are unclear.Therefore,the objective of this research was to evaluate the related molecular mechanism of immnoregulation effect of Slp,explore its contribution to the Lactobacillus,which widening and enriching people's observation on Slp.The main results were as follows:Firstly,L.acidophilus NCFM carried Slp with a relative molecular mass of 46,000 was selected as control,six Lactobacillus strains that carried Slp were selected from fifteen Lactobacillus strains.The purified Slps?relative molecular weight of 46,000?were obtained through cation chromatography purification.After analyzing thermodynamic properties of these Slps using differential scanning calorimeter,it was concluded that all Slps had only one endothermic peak and their thermal denaturation temperature was range from 58°C to 77°C.L.crispatus JCM 2009 presented the highest thermal denaturation temperature,L.acidophilus NCFM was the second,while L.fermentum CCFM Y40 showed the lowest thermal denaturation temperature.That of Slps from other three Lactobacillus strains was around60°C.The molecular mechanism of the anti-inflammatory effects of Slps in lipopolysaccharide?LPS?-induced RAW264.7 cells was investigated.The results presented that Slps at the concentration from 1 to 10?g/mL had no cytotoxicity effects on RAW264.7 cells and Slps?5-10?g/mL?remarkably inhibited the reactive oxygen species?ROS?production in LPS-induced RAW 264.7 cells?P<0.05?.Except for Slps from L.fermentum,other Slps significantly attenuated the excretion of pro-inflammatory cytokines such as tumor necrosis factor-??TNF-??,interleukin-1??IL-1??,NO and prostaglandin E₂?PGE₂??P<0.05?.Combining Western blot and immunofluorescence to investigate their immune regulation mechanism,we found that Slps?including Slp-1,Slp-2 and Slp-4?exerted their anti-inflammatory actions by inhibiting mitogen-activated protein kinase?MAPK?and nuclear factor-?B?NF-?B?signaling pathways in LPS-stimulated RAW264.7 cells.These results indicated that Slp exerted a positive immunoregulation effect in LPS-induced RAW264.7 cells.Furthermore,we found that Slp-4 derived from L.acidophilus NCFM could exert the strongest anti-inflammatory effect among these Lactobacillus Slps.Then,we investigated the effect of Slp-4 from L.acidophilus NCFM on TNF-?-elicited intestinal barrier dysfunction and explored the underlying molecular mechanism.We first examined the transepithelial electrical resistance and cells paracellular permeability,and found that Slp had no capacity to increase Caco-2 cell permeability.Pre-incubation of Caco-2cells with Slp-4?50-100?g/mL?for 6 h improved intestinal epithelial cell integrity and permeability,restored epithelial tight junction protein?such as ZO-1,Occludin and Claudin-1?expressions and reduced the secretion of interleukin 8?IL-8?in TNF-?-induced intestinal epithelial barrier dysfunction.Furthermore,the flow cytometry and immunofluorescence's results showed that the addition of Slp-4 to Caco-2 cell monolayers attenuated cell apoptosis and inhibited NF-?B p65 nucleus translocation by suppressing the activation of NF-?B.Collectively,the ability of Slp-4 to block TNF-?from penetrating the intestinal mucosal barrier and attenuate dysfunction of the intestinal epithelial barrier stimulated by TNF-?exerts its anti-inflammatory effect.Based on the immunoregulation effect and suppression of tumor cell activity of food-derived bioactive proteins,we first evaluated the effect of Slp-4 on the intestinal tumor cell activity.In this research,normal colonic epithelial CCD841 CoN cells were selected as control,and HCT116 cells were used as the intestinal tumor cell.It was confirmed that Slp-4suppressed cell proliferation in a dose-dependent manner.Acridine orange staining revealed that Slp-4 promoted the autophagic vesicles formation in HCT116 cells.Combining Western blot and immunofluorescence techniques to research the pathway of Slp-4 induced HCT116cells death,we found Slp-4 did not induce apoptosis,but induced autophagy in HCT116 cells.Accumulation of Beclin-1 and microtubule-associated protein 1 light chain 3 from II?LC3-II?,and the degradation of p62 were observed when cells were treated with Slp-4 for 24 h.We also found that the mammalian targets of PI3K/AKT/mTOR and c-Jun N-terminal kinase?JNK?signaling pathways were crucial mediators regulating Slp-4-induced autophagic cell death.Additionally,Slp was likely to induce HCT116 colon cancer cell cycle arrest at the G0/G1 phase through up-regulation of p53 and p21 protein expression levels.Treatment with Slp-4 resulted in the obvious formation of ROS?P<0.05?,which attenuated Slp-4 induced autophagic cell death in HCT116 cells.Meanwhile a novel mechanism of action of Slp-4induced autophagy,acting simultaneously through the ROS-mediated PI3K/AKT/mTOR and JNK signaling pathways in HCT116 colon cancer cells.Taken together,our results suggest that Slp-4 inhibits the HCT116 cells proliferation,and then induces autophagy,which maintains intestinal health,and prevents the transformation of intestinal inflammation to intestinal tumors.Slp has anti-inflammatory effect in LPS-induced macrophages cells.In this study,we also found that Slp-1 extrated from L.crispatus JCM 2009 alleviated the inflammatory response by regulating PI3K/AKT/mTOR signaling pathway and activating autophagy in LPS-induced RAW 264.7 cells.These results from ELISA and Western blot presented NF-?B signaling pathway positively regulated the autophagy activity to inhibit the production of pro-inflammatory cytokines?PGE₂ and NO?in this inflammatory response,and the p65 was an important molecule in NF-?B signaling pathway for relieving inflammatory response activated by Slp-1 in LPS-induced RAW264.7 cells.In summary,this study selected six Lactobacillus strains carried Slp.We also demonstrated Slp exerted its positive immunoregulation action in LPS-induced macrophages cells,and blocked TNF-?from penetrating the intestinal mucosal barrier to relieve intestinal epithelial barrier dysfunction,and induced the autophagic death in HCT116 cancer cells to maintain intestinal health.The related molecular mechanism of intestinal immune regulation of Lactobacillus Slps was systematic study in this research,making it a promising ingredient in the development of functional foods.