Effects Of T.Spiralis-exosomes And MiR-153 On IPEC-J2 Cell Barrier Function And Protection Of Recombinant Lactococcus Lactis Secreting Bcl2

As a zoonotic disease,trichinellosis entails a threat to the health and well-being of both humans and animals,and challenges public health systems.Upon invasion into the host,the infectious muscle larvae parasitize in the intestinal epithelial cells to complete a series of life activities including colonization and reproduction.Recent research has highlighted the importance of parasite-derived exosomes in intercellular communication and interactions with host cells,as well as in evading immune surveillance.The communication between secreting and receiving cells mediated by exosomes depends on the bioactive molecules carried by exosomes,such as miRNA.The miRNA shuttles between cells and functions in new locations,modifying the gene expression of recipient cells and regulating protein production,and providing necessary signals for regulating recipient cells.In this study,we aimed to investigate the effects of exosomes derived from Trichinella muscle larvae（TsExos）and miRNAs within TsExos on intestinal epithelial barrier function.Lactococcus lactis,with excellent characteristics such as non-toxicity,safety,and probiotic effects,have been widely used as expression hosts for heterologous genes.Using Lactococcus lactis as oral vaccines or drug delivery vehicles to control trichinellosis is a promising approach.Based on our mechanistic studies,we planned to construct a secretion-type recombinant Lactococcus lactis and evaluated its ability to interfere with trichinella invasion.The main research are as follows:TsExos were successfully obtained using ultracentrifugation,which were circular or elliptical in shape,had a membrane structure,particle diameters between 30-150 nm,and expressed the specific surface protein CD63.It was also confirmed that IPEC-J2 intestinal epithelial cells could uptake TsExos.It was then studied the effects of TsExos on the barrier function of IPEC-J2 cells.By measuring cell viability,150μg/m L TsExos were selected as the working concentration of subsequent experiments and co-cultured with cells for 12 or 24 h,reducing the cell proliferation by30%.Based on the above experiments,the effects of TsExos on cellular biological processes,innate immunity,and tight junction state were sequentially explored.After co-incubation of TsExos with IPEC-J2 cells for 12 or 24 h,the content of FITC-Dextran in the culture medium,as well as the levels of LDH and ROS in the cells,significantly increased.The apoptosis rate of cells increased by 4.77%（12 h）and 12.57%（24 h）,and an increase in the number of apoptotic cells with nuclear condensation and fragmentation was observed.In addition,the expression of the anti-apoptotic factor Bcl2 was decreased,while the expression of the pro-apoptotic factor Bax was increased.Following TsExos induction,protein expression changes involved in innate immunity of the IPEC-J2 cells were discussed.The results showed that the expression of the inflammatory cytokine IL-1 was upregulated,while the expression of the inflammatory cytokine IL-10,TGF-β,Toll-like receptor TLR-5,and the mucins MUC-1 and MUC-2 were downregulated.Similarly,the levels of tight junction proteins ZO-1,Claudin-3,and Occludin in the cells were decreased following TsExos induction.We investigated the mechanism of interaction between miRNAs within TsExos and intestinal epithelial cells.First,high-throughput sequencing was used to identify miRNAs in TsExos.Based on the results of miRNA high-throughput sequencing,target gene enrichment analysis,the assay of miRNA contained in TsExos,and the assay of miRNA delivery,the miR-153 and its predicted target genes Agap2,Bcl2,and Pten were selected for further study.Dual-luciferase reporter gene assay were used to verify the targeting relationship between miR-153 and the three candidate target genes.The results showed that co-transfection of miR-153 with the recombinant plasmids pmir GLO-Bcl2-WT and pmir GLO-Pten-WT led to a significant decrease in relative luciferase activity in 293T cells,indicating that miR-153 could downregulate the transcription levels of Bcl2and Pten.Next,the transfection efficiency of miR-153 in IPEC-J2 cells was determined,and the effect of TsExos-delivered miR-153 on the expression of the target genes Bcl2 and Pten was discussed.The results showed that 100 n M miR-153 mimics could reduce the m RNA levels of Bcl2 and Pten in IPEC-J2 cells,and therefore,100 n M was chosen as the working concentration of miR-153 mimics.Furthermore,RT-q PCR and Western blot confirmed that miR-153 in TsExos only degraded Bcl2 levels in IPEC-J2 cells.Subsequent experiments focused on miR-153 and its target gene Bcl2.MiR-153 is an important tumor suppressor factor that plays a crucial role in various biological processes within cells.As an important anti-apoptotic protein,Bcl2 plays a crucial role in cell apoptosis and is a common crossroads of various signaling pathways.Therefore,we speculated that miR-153 from TsExos induces cell apoptosis by targeting Bcl2.The results showed that after transfection with miR-153,the number of apoptotic cells was increased,mitochondrial membrane potential was decreased,cell proliferation was affected,and the levels of LDH and ROS in the cells was increased.Additionally,the accumulation of Bax and Bad in the cells was increased,and the expression of the apoptotic execution proteins Caspase9 and Caspase3 was higher.We further validated the regulatory effects of TsExos-cargoed miR-153 on the apoptotic pathways MAPK,p53,and PI3K/AKT.The results showed that TsExos-delivered miR-153 could decrease the expression levels of ERK2 and MEK1 and increase the expression levels of p38 and p53.On the basis of mechanism research,we identified that miR-153 carried by the exosomes of T.spiralis could inhibit the transcription and translation of target gene Bcl2,and induced apoptosis in intestinal epithelial cells.We hypothesized that Bcl2 was a key protein in the interaction between T.spiralis and the host,and increasing the amount of Bcl2 protein could partially block T.spiralis invasion,thereby achieving the goal of preventing and treating trichinellosis.In this study,we constructed a novel secretion-type Lactococcus lactis expressing strain as a Bcl2 delivery carrier to explore the effects of Bcl2 protein in antagonizing T.spiralis invasion.Through analyzing the biological information of Bcl2 and optimizing the codons according to the codon preference of Lactococcus lacits,we constructed a recombinant plasmid p NZ8148 carrying SP_USP45 and Bcl2.After electroporation of the plasmid p NZ-Bcl2 into Lactococcus lactis NZ9000,the qualified strains were obtained and the expression of the target protein in the bacterial body and culture supernatant was confirmed by Western blot analysis after induction with Nisin,indicating successful construction of recombinant Lactococcus lactis NZ-Bcl2 that could secrete Bcl2.Stability tests of the plasmid showed that after serial passages,Lactococcus lactis still contained the recombinant plasmid p NZ-Bcl2 at the 24th generation.Retention tests showed that the recombinant Lactococcus lactis could survive in the host on the 7th day.Based on the above work and combined with the life cycle of T.spiralis,after orally administering NZ-Bcl2 every 2 days for2 weeks,mice were infected orally with 150 muscle larvae of T.spiralis as lower challenge and600 larvae as higher challenge.Then,the antagonism of NZ-Bcl2 on the invasion of T.spiralis was analyzed at day 3 and 7 post challenge.Animal experiments were conducted to comprehensively evaluate the growth performance of mice,immune organ index,small intestinal pathological sections,adult worm reduction rate,and changes in intestinal cell cytokines,tight junction proteins,and mucins.For mice infected with 600 larvae,whether on the 3rd day or the 7th day of infection,recombinant Lactococcus lactis NZ-Bcl2 did not improve the weight loss,immune organ enlargement,severe small intestine damage,decreased expression of tight junction proteins ZO-1,Occludin,and Claudin-3,and decreased expression of mucin MUC-1 caused by T.spiralis.For mice infected with 150 larvae of T.spiralis on the 3rd day of infection,recombinant Lactococcus lactis NZ-Bcl2 could reduce the mesenteric lymph node index of infected mice,increase the protein expression of Claudin-3 and MUC-1,and decrease the number of muscle larvae.In summary,TsExos participated in regulating various host cell activities after entering intestinal epithelial cells IPEC-J2.It was mainly manifested by decreasing cell viability;increasing cell permeability;causing cell damage,oxidative stress,and abnormal apoptosis;promoting cell inflammation and affecting Toll receptor-mediated signal transduction;disrupting cell mucin defense system and tight junction status.The miR-153 within TsExos could result in Bcl2degradation,induce cell apoptosis,decrease cell viability,exacerbate cell damage,and induce excessive oxidative stress.In addition,miR-153 carried by TsExos could mediate MAPK and p53signal transduction,promoting cell apoptosis.Secreted recombinant Lactococcus lactis NZ-Bcl2could enhance the intestinal barrier function and mucosal immunity of hosts with low-intensity T.spiralis infection,reduce the number of muscle larvae,and protect hosts against T.spiralis invasion.This study provides a theoretical basis for further exploring the pathogenic mechanism of T.spiralis and provides a new direction for the prevention and treatment of Trichomoniasis.