| The liver is one of the immune organs on the front line of the animal’s immune system.Most of the blood in the liver is supplied by the portal vein,and the blood from the gastrointestinal tract enters the liver through the portal vein before entering the systemic circulation.Moreover,the liver and the intestines originate from a common embryo,thus,there is an inseparable relationship betwee n the liver and intestines on growth,development and immune responses.Due to the possible existence of foreign harmless microorganisms in portal vein blood,the liver is generally believed to have anti-inflammatory or immune tolerance functions.The symbiotic beneficial bacteria in the intestines can enter the circulatory system,and then their metabolic products might affect the homeostasis of the animal body through the blood to other tissues and organs.Recent studies have found that various microorganisms can be detected in liver cancer tissue,in which the content of probiotics lactobacilli is the highest.It was found that a large number of probiotics colonized in damaged liver tissues of the immune system,but whether probiotics can colonize in healthy liver tissues is still unclear.This study aims to explore whether Lactobacillus casei(L.casei)can colonize and exert biological and immunological effects in healthy liver tissues through intraperitoneal injection,and then investigate the potential molecular mechanisms of how L.casei escapes from immune phagocytosis by macrophages and eventually colonizes in liver tissues.Firstly,mice were injected intraperitoneally with different concentrations of L.casei,and then the liver tissue suspension was collected for in vitro bacterial culture and dynamic analysis of bacterial colonization in the liver.Results showed that intraperitoneal injection of L.casei can colonize in the liver with a significant dosedependent effect,and the number of colonizing bacteria did not decrease significantly over time.Meanwhile,pathological observations of mouse liver and colon tissues through H&E staining and transmission electron microscopy showed that low concentration of L.casei had no significant pathological changes to the liver and colon tissues after colonization.However,high concentration of L.casei led to mild liver damage,and enlarged cell gap,swelling,tissue fibrosis and lipid droplets,accompanied with mitochondrial structural damage;the patholo gical changes in the colon tissues included crypts degeneration and mitochondrial injury.The changes in the expression of inflammatory-related factors in the liver tissue after colonization of L.casei were analyzed using real-time fluorescent PCR(q RT-PCR)method.The results showed that after colonization,the m RNA expression levels of pro-inflammatory factors such as IL-6,IL-1β,TNF-α,IFN-γ,IL-17 f and JAK in the liver tissue decreased,while the m RNA expression levels of anti-inflammatory factors such as IL-10,IL-22 and growth factor Wnt increased,indicating that colonization of L.casei in the liver can inhibit the expression of pro-inflammatory cytokines and promote the growth and development of liver tissue.Flow cytometry was used to analyze the effect of L.casei colonization hepatocyte proliferation and differentiation.The results showed that L.casei colonization in the liver can promote the proliferation and differentiation of hepatocytes.Subsequently,this study investigated how L.casei overcome immune evasion and colonize in liver tissueat the cellular and molecular level.L.casei and mouse peritoneal macrophages were co-cultured in vitro,and the interaction between L.casei and mouse peritoneal macrophages was determined by immu nofluorescence and laser confocal microscopy techniques.It was observed that L.casei was tightly attached to the surface of mouse peritoneal macrophages and was engulfed or degraded by macrophages under immunofluorescence microscopy and laser confocal mic roscopy.The results confirmed that L.casei has immune evasion properties against peritoneal macrophages.Flow cytometry analysis showed that L.casei can evade the immune phagocytosis of mouse peritoneal macrophages.Moreover,flow cytometry and q RTPCR were used to analyze the differentiation subtypes mouse peritoneal macrophages induced L.casei.Flow cytometry results showed that L.casei can break the balance of M1/M2 macrophages and induce macrophages to proliferate and differentiate into M2-type macrophages.q RT-PCR results showed that L.casei can induce the expression of M2-type macrophage-related cytokines and chemokines such as IL-10,Arg-1,TGF-β,CD206 and IL-4,and inhibit the expression of M1-type macrophagerelated cell molecules such as i NOS,IL-1β and IFN-γ.The results above indicate that L.casei has an immune evasion mechanism against mouse peritoneal macrophages,and L.casei induces mouse peritoneal macrophages to proliferate and differentiate into M2-type macrophages by promoting the expression of M2-type macrophagerelated cytokines and chemokines.To further explore the application of L.casei in livestock production,we investigated the cellular and molecular mechanisms of how L.casei overcomes porcine macrophages’ immune evasion using in vitro co-culture techniques.Through flow cytometry and laser confocal microscopy,we explored the effect of L.casei on the activity and phagocytic ability of porcine macrophages.The results showed that L.casei can evade the phagocytosis of porcine macrophages and promote porcine macrophage proliferation and differentiation.Immunofluorescence microscopy,q RTPCR,and Western Blot were used to analyze the differentiation subtypes of L.caseiinduced porcine macrophages,the results confirmed that L.casei breaks the M1/M2 immune balance of macrophages and induces M0 macrophages to differentiate into M2 macrophages,and then plays an immune evasion effect.Under immunofluorescence microscopy,it can be observed that L.casei promotes the expression of surface markers CD206 in M2 porcine macrophages.q RT-PCR results showed that L.casei stimulated the expression of M2 porcine macrophage-related cell molecules Arg1,TNF-α,CD206,IL-10 and TGF-β.Western Blot results showed that L.casei can activate IL-4 and Arg-1 protein expression to inhibit i NOS protein expression.The results above show that L.casei induces porcine macrophages to differentiate into M2 type by activating the IL-4 signaling pathway,inducing macrophages to play an anti-inflammatory role and L.casei evading macrophage phagocytosis thereby.In summary,this research shows that L.casei can colonize in liver tissue through intraperitoneal injection,exhibiting certain dose-dependent effects in regulating liver immune function and promoting liver tissue growth and development.Furthermore,we have discovered that L.casei can break the M1/M2 immune balance of macrophages in mice and pigs,adhering to the surface of macrophages and inducing them to differentiate to anti-inflammatory M2 macrophages,then exhibiting the immune evasion characteristics of macrophages.This study provides a detailed understanding of the immune evasion mechanism of L.casei in liver colonization by analyzing the interaction and signaling molecule mechanisms betw een the probiotic bacteria and host cells.This is the first study to provide innovative insights into the immune,cellular,and molecular mechanisms underlying many of the probiotic effects of L.casei,including liver colonization,immune evasion and anti-inflammatory effects.Our results provide new scientific directions for studying emerging scientific issues such as the gut-liver axis and offer new scientific references for further exploring the effects of L.casei on the innate immunity of animal bodies. |
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