Study On The Mechanism Of Cadmium Contamination In Feed Promoting Intestinal Pathogens To Damage Intestinal Mucosal Barrier

In recent years,the increasing incidence of foodborne diseases,such as Salmonella infection,has highlighted the critical role of environmental pollutants as causative factors in their pathophysiology.Cadmium is a toxic,widely distributed heavy metal pollutant which can lead to worldwide environmental health problems.Due to the development of modern industry,metal mining,smelting,processing,chemical production,automobile exhaust emissions and other human activities of cadmium emissions,the living environment has been polluted by a wide range of cadmium.Cadmium can be ingested through inhaled dust,polluted water supplies,and the food chain.Because of its long half-life,cadmium is extremely difficult to expel from the body and can cause toxic accumulation and damage to multiple organs.Cadmium pollution and cadmium poisoning have become important public health safety issues.However,whether cadmium ingestion affects the susceptibility to infection and the detailed mechanism has not been investigated.In this study,we aimed to evaluate the effects of cadmium on the intestinal mucosal barrier and Salmonella infection.We demonstrated that oral administration of 12.5mg/kg body weight cadmium caused damage to the intestinal mucosal barrier,with bodyweight loss,an increase in inflammation,significantly reduced Muc2 expression and goblet cell loss in the intestine.The effect of cadmium on secretory cell differentiation was further demonstrated to be regulated by the over-activation of the Notch signaling pathway by increased production of ROS.The damage of cadmium to the intestinal barrier and goblet cell loss dramatically increased susceptibility to S.Typhimurium infection at a low dose（10² CFU）,with a high death ratio,body weight loss and severe intestinal inflammation.However,Salmonella susceptibility and intestinal barrier damage enhanced by cadmium could be alleviated by inhibiting ROS production and Notch pathway activation,with the reversion of goblet cell loss.This study indicated that cadmium ingestion not only affected the integrity of the intestinal barrier and intestinal epithelial differentiation but also increased the risk of enteropathogenic infection from food contamination or environmental pollution,which signals an alarm for public health.This study is divided into the following six parts:1 Cadmium ingestion caused damage to the intestinal mucosal barrier in mice C57BL/6 mice were fed for 2 weeks with PBS or cadmium at a concentration of 12.5mg of CdCl₂ kg^-1 per day.Body weight loss significantly occurred on the 7th day after cadmium treatment.The integrity of the intestinal mucosal barrier was assessed by macroscopic,histological,and molecular parameters.The intestine tract was significantly shorter and thinner in cadmium-treated mice than in PBS-treated mice.Consistently,at the microscopic level,the histological score of cadmium-treated mice was more severe than that of PBS-treated mice,the villi tended to be shorter and thicker,and the fusion of some villi was visible.In contrast to those in PBS-treated mice,the levels of LPS and TNF-α,reflecting inflammation,were also significantly higher following cadmium consumption.Mice treated with cadmium had a high level of cadmium accumulation,especially in the liver,while cadmium in the control group was undetected.Mucin 2（Muc2）and lysozyme are two important antibacterial peptides in protecting the intestinal mucosal barrier against pathogens invasion.Interestingly,mice ingesting cadmium had fewer goblet cells and decreased gene expression of Muc2 compared with those of PBS-treated mice.Cadmium exposure also showed similar inhibition effect on Paneth cells induction and Lysozyme expression.These results demonstrated that cadmium exposure had a damaging effect on the intestinal mucosal barrier.2 Cadmium exposure increased ROS production and activated the Notch signaling pathway.NAC was given at the same time for 2 weeks to explore the effect of reactive oxygen species（ROS）on the Notch pathway and goblet cell formation.Mice treated with cadmium continued to lose weight,while mice treated with NAC and cadmium simultaneously showed less severe body weight loss.The concentration of GSH/GSSG was detected to evaluate the effect of cadmium on the redox status.Cadmium exposure significantly reduced the level of GSH and the GSH/GSSG ratio,which was alleviated by NAC treatment.Intestinal secretory cell differentiation is regulated by the Notch signaling pathway.D114 and Jagged l are two Notch ligands,and regulate the Notch target genes Hes1 expression.In this study,Cadmium could upregulate Jagl,D114,Notchl,and Hesl expression and downregulate Mathl gene expression,which indicated that cadmium activated the Notch pathway.However,the expression of related genes stimulated by cadmium was reversed after treatment with NAC.Furthermore,the increased expression level of NICD detected by immunohistochemistry（IHC）further demonstrated the activation of the Notch pathway.Changes in the number of goblet cells and the amount of mucus were assessed by PAS and UEA-1 staining.The results showed that the loss of goblet cells and intestinal mucus caused by cadmium exposure could be improved after treatment with NAC.Consistently,the trend of Muc2 and Lysozyme gene expression was the same as that of goblet cell changes.The depletion of Paneth cells caused by cadmium exposure could also be improved after treatment with NAC.These data indicated that cadmium activated the Notch pathway by ROS,leading to the loss of goblet cells.To further explore the effect of cadmium on intestinal stem cells differentiation,intestinal organoids were cultured and treated with cadmium or NAC.Obvious damage to intestinal organoids was observed after cadmium treatment,and more than 80% of organoids were disrupted within 8 h.However,NAC treatment significantly reduced the percentage of disrupted organoids.This damaged intestinal epithelium may be attributed to the reduced level of GSH and GSH/GSSG ratio,which was alleviated by NAC treatment.Cadmium significantly increased Jagl,D114,Notch 1,and Hes1 expression and downregulated Mathl gene expression,which were also reversed after treatment with NAC.NAC also saved the loss of UEA-1+cells caused by cadmium,as well as reversed the expression of Muc2 and Lyz-1.3 Cadmium intake enhanced susceptibility to Salmonella infection,with goblet cell lossTo explore the impact of cadmium exposure on susceptibility to S.Typhimurium and severity of infection,mice were concomitantly treated with cadmium gavage for 2 weeks and challenged with 10² or 10⁸ CFU of S.Typhimurium on the 10th day of the experiment.Interestingly,a significantly increased mortality was observed in cadmium treated mice with S.Typhimurium infection compared with that in mice treated with an equal amount of S.Typhimurium only.Moreover,mice treated with S.Typhimurium and cadmium simultaneously had much more body weight loss than S.Typhimurium and PBS-treated mice.Cadmium ingestion also significantly increased the S.Typhimurium burden in liver tissues.Consistently,at the macroscopic and microscopic levels,S.Typhimurium infection-induced general pathological changes,while cadmium exposure significantly enhanced the intensity of intestinal inflammation,with obvious bleeding of intestinal tissue.The pathological score of intestinal tissue for mice treated with cadmium and S.simultaneously was significantly increased compared with that for S.Typhimurium only-challenged mice.Furthermore,the levels of LPS and TNF-α were also increased in cadmium and S.Typhimurium-treated mice compared with the S.Typhimurium only infection group.It is noteworthy that a low dose of S.Typhimurium（10² CFU）can also cause serious disease under cadmium exposure.4 Inhibition of ROS production alleviated S.Typhimurium-induced intestinal inflammation and the death ratioTo explore the effect of ROS induced by cadmium intake on S.Typhimurium infection,mice received continuous cadmium and NAC by oral gavage for 2 weeks,followed by challenge with 10² CFU of S.Typhimurium on the 10th day.NAC treatment showed a protective role against Salmonella infection in cadmium intake mice,with decreased mortality and body weight loss.Additionally,mice treated with NAC had less erythrocyte infiltration and alleviated epithelial damage in line with decreased pathological scores compared to those that did not receive NAC.The levels of LPS and TNF-α,combined with the bacterial loads in the liver,were all also decreased in NAC-treated mice compared with those in mice receiving cadmium and S.Typhimurium simultaneously.In addition,the protective function of NAC was further proven by an increase in the GSH/GSSG ratio,which indicated that NAC can restore the balance of redox status.The results indicated that ROS induced by cadmium aggravated S.Typhimurium infection.5 Notch pathway inhibition or goblet cells recovery rescued the death of mice challenged with S.TyphimuriumTo confirm whether cadmium-induced goblet cell loss aggravated S.Typhimurium infection,mice were orally administered cadmium for 2 weeks,intraperitoneally injected with DBZ or PGE2 for 5 consecutive days on the 9th day and simultaneously challenged with 10² CFU of S.Typhimurium on the 10th day.Starting from the 2nd day of S.Typhimurium infection,mice treated with cadmium were subject to death,while mice treated with DBZ and PGE2 had low mortality or no death.Consistently,mice receiving cadmium with S.Typhimurium infection presented persiste-nt body weight loss.In contrast,body weight changes were almost paralleled after day 4 in mice receiving DBZ and PGE2,Mice with cadmium ingestion after S.Typhimurium challenge had significantly increased S.Typhimurium burden in the liver and LPS and TNF-α concentrations in serum,while this intestinal mucosal barrier damage could be markedly improved in mice receiving DBZ or PGE2 treatment.DBZ or PGE2 treatment could both alleviate the activation of the Notch signaling pathway by downregulation of related gene mRNA expression levels（Jagl,D114,Notchl,and Hesl）to avoid over-activation of the Notch pathway induced by cadmium ingestion.Furthermore,the reduced NICD expression detected by immunohistology verified the inhibitory effect of the Notch pathway.The number of goblet cells and the amount of mucus in the mice treated with DBZ and PGE2 were increased compared with those in mice receiving cadmium.Meanwhile,the trend of the Lysozyme gene expression and the number of Paneth cell were the same’ as that of goblet cell changes.These data suggested that the Notch pathway and goblet cells were involved in aggravating the infection of S.Typhimurium.6 Cadmium exposure increased susceptibility to L.monocytogenes and Enterohemorrhagic E coli（EHEC）infectionTo further explore the impact of cadmium exposure on susceptibility to L.monocytogenes and EHEC,mice were treated with cadmium and NAC by oral gavage for 2 weeks and then challenged with 10² or 10⁸ CFU of L.monocytogenes or EHEC on the 10th day of the experiment respectively.Significantly increased mortality and body weight loss were observed in cadmium-treated mice with L.monocytogenes and EHEC infection compared with that in mice treated with an equal amount of L.monocytogenes or EHEC only,NAC treatment showed a protective role against L.monocytogenes or EHEC infection in cadmium intake mice,with decreased mortality and body weight loss.Cadmium ingestion also significantly increased the L.monocytogenes or EHEC burden in liver tissues,enhanced the intensity of intestinal inflammation and increased pathological score compared with that for L.monocytogenes or EHEC only-challenged mice.However,mice treated with NAC had fewer bacteria burden and alleviated epithelial damage in line with decreased pathological scores.The protective effect of NAC treatment after cadmium exposure is consistent with the reduced level of LPS and TNF-α compared with those in mice receiving cadmium and pathogens simultaneously.