Dietary Emulsifier Carboxymethylcellulose-induced Gut Dysbiosis Aggravates Acute Pancreatitis

Acute pancreatitis(AP)is an inflammatory response characterized by the autodigestion,edema,hemorrhage,and even necrosis of pancreatic tissue due to the abnormal activation of pancreatic enzymes within the pancreas,triggered by various etiologies.It represents the most common cause for gastrointestinal disease-related hospital admissions,imposing a significant burden on the current healthcare system.The severity of AP varies,with most cases being mild,predominantly featuring pancreatic edema,self-limiting in nature,and generally prognosticating well.However,it is noteworthy that approximately one-fifth of AP patients develop severe acute pancreatitis,characterized by severe systemic inflammation leading to persistent organ failure,with a mortality rate of about 20%.In recent years,there has been an annual increase in the incidence of AP,possibly related to dietary shifts.Ultra-processed foods,characterized by industrial production and the addition of numerous ingredients and additives,have seen a dramatic increase in consumption since the mid-20th century.This rise in consumption correlates with an increased incidence of various chronic inflammatory diseases,thereby drawing increasing concern over the adverse effects of ultra-processed foods on health.Notably,the rise in AP incidence runs parallel to the increased consumption of ultra-processed foods,yet there is a current lack of research on the relationship between ultra-processed foods and AP.A common feature of ultra-processed foods is the inclusion of one or more emulsifiers,which are used to disperse immiscible components,ensuring the stability of functional ingredients and flavors in food and beverages while extending their shelf life.Carboxymethylcellulose(CMC)is among the most commonly used dietary emulsifiers in the food industry.Numerous studies have confirmed that CMC can alter the composition of the human and murine gut microbiome,potentially promoting the development of colitis or metabolic syndrome.Despite the lack of direct evidence,a variety of indications strongly suggest that CMC may contribute to the onset of AP.Firstly,evidence exists that alterations in the gut microbiota play a role in the development of AP,with CMC exerting its deleterious effects primarily through inducing dysbiosis.Secondly,exposure to CMC can alter the fecal metabolome,such as changes in short-chain fatty acids(SCFA)and bile acids,whose composition and concentration variations are related to changes in the condition of AP.This study aims to elucidate the impact and mechanisms of CMC on the severity of AP.To investigate the effect of CMC on AP,this study induced AP in mice through intraperitoneal injection of cerulein.Male C57BL/6 mice were given 1%(w/v)CMC solution as drinking water,while the control group consumed pure water.After a 4-week water intervention,AP was induced by cerulein injection,and mice were euthanized one day post-modeling for subsequent tissue collection and analysis.Pancreatic tissue weight was measured,and histological examination was performed after paraffin embedding and H&E staining,revealing that mice exposed to CMC exhibited a higher pancreas/body weight ratio and AP pathological scores,indicating that CMC exposure exacerbated the severity of AP in mice.Given that CMC is not absorbed by the digestive tract,previous research has confirmed that CMC primarily exerts its negative effects on the human body through its impact on the gut microbiota.Therefore,this study utilized 16 s rRNA sequencing technology to assess the gut microbiota in mice,finding significant alterations in the composition of the gut microbiota in AP mice exposed to CMC.At the phylum level,compared to the control group,mice exposed to CMC showed increased abundance of Proteobacteria,Campilobactre,and Bacteroidota,while the abundance of Verrucomicrobiota and Deferribacterota decreased.At the genus level,the abundance of Escherichia-Shigella,Bacteroides,and Clostridium＿sensu＿stricto＿1 increased in mice exposed to CMC,whereas the abundance of Akkermansia,Muribaculaceae,and Lactobacillus decreased.Moreover,the abundance of potential pathogenic bacteria in the intestines of mice exposed to CMC increased.To explore whether the changes in the gut microbiota mediated the exacerbating effect of CMC on AP in mice,this study conducted fecal microbiota transplantation(FMT)experiments using specific pathogen-free(SPF)and germ-free(GF)mice as models.Donor mice were given either pure water or CMC solution,and all recipient mice drank pure water.After a 2-week intervention,fecal material from the donor mice was transplanted into the recipient mice(SPF and GF mice).Following FMT,AP was induced in the recipient mice using cerulein,and they were euthanized one day later for further tissue analysis.The effect of donor microbiota on the AP phenotype in recipient mice was observed.To verify the effectiveness of the FMT,16 s rRNA sequencing of the feces of recipient mice was conducted,showing that the gut microbiota of recipient mice resembled that of the donor mice,confirming the efficacy of the FMT procedure in this study.Compared to recipient mice that received gut microbiota from mice drinking pure water,those that received gut microbiota from mice exposed to CMC showed a significant exacerbation of AP.This indicates that the altered gut microbiota mediated the adverse effects of CMC on AP.Given that the gut microbiota can affect systemic inflammation levels through its impact on the immune system,we hypothesized that certain types of immune cells in the circulation of mice mediated the exacerbating effect of CMC on AP.This study performed single-cell RNA sequencing(sc RNA-seq)on peripheral blood mononuclear cells(PBMC)from SPF-level recipient mice that underwent FMT.Sc RNA-seq analysis indicated that among the six main cell types in PBMC(monocytes,dendritic cells,T cells,basophils,NK cells,and B cells),only monocytes showed significant differences between the control group(FMT-Ctrl)and the CMC-exposed fecal microbiota transplantation group(FMT-CMC).Compared to control recipient mice,recipient mice that received gut microbiota from mice exposed to CMC had a significantly increased proportion of monocytes in their PBMC.Further analysis revealed a significant increase in classical monocytes,a subtype that can promote inflammation and has the capacity to differentiate into macrophages in tissues.These findings suggest that classical monocytes play a role in the exacerbating effect of CMC on AP.To further explore the molecular mechanisms of classical monocyte activation,considering the critical role of transcription factors in cell differentiation,this study conducted transcription factor analysis on mouse classical monocytes.The results suggested that C/EBPδ is the transcription factor most specifically associated with classical monocytes.To verify the function of C/EBPδ,this study conducted overexpression and knockdown experiments on C/EBPδ in RAW264.7 cells(mouse monocyte-macrophage leukemia cells).Overexpression of C/EBPδ significantly upregulated the marker molecules of classical monocytes,while knockdown of C/EBPδ produced the opposite effect,proving the key role of C/EBPδ in the activation of classical monocytes.This study used gene set enrichment analysis(GSEA)to identify upstream factors leading to the upregulation of C/EBPδ.Compared to the FMT-Ctrl group,the FMT-CMC group showed significant enrichment of signaling pathways related to lipopolysaccharide(LPS).Using enzyme-linked immunosorbent assay(ELISA),this study measured the serum LPS concentration in the two groups of mice,finding that the FMT-CMC group had a significantly higher LPS concentration than the FMT-Ctrl group.To verify whether LPS mediated the exacerbating effect of CMC on AP,this study conducted a recovery experiment using eritoran(an LPS antagonist)to successfully alleviate the exacerbation of AP induced by CMC,significantly reducing the frequency of classical monocytes.This highlights the key role of LPS in mediating the harmful effects of CMC on AP.An increase in serum LPS is often a result of impaired intestinal barrier function.Therefore,this study assessed the intestinal barrier function in mice,using immunofluorescence,Western blot,and q PCR methods to detect the expression of intestinal barrier proteins ZO-1 and Claudin-1.The study found that both direct exposure to CMC and transplantation of gut microbiota from mice exposed to CMC could impair the intestinal barrier function in mice.Mechanistically,CMC exposure increases intestinal permeability,facilitating the entry of LPS into the bloodstream,thereby upregulating C/EBPδ,activating classical monocytes,and ultimately exacerbating AP.Short-chain fatty acids(SCFA)are a class of saturated fatty acids produced in the colon through the microbial fermentation of indigestible food components(mainly dietary fiber),with carbon chain lengths typically not exceeding six carbon atoms.The most common SCFA include butyrate,propionate,and acetate.SCFA are an important energy source for colon cells(especially colonic mucosal cells)and play a significant role in maintaining intestinal health,regulating the immune system,influencing metabolism,and potentially reducing the risk of chronic diseases.Considering the importance of SCFA in maintaining intestinal barrier function,this study measured the SCFA content in mouse feces.Both direct exposure to CMC and transplantation of gut microbiota from mice exposed to CMC reduced the SCFA content in mouse feces.Correlation analysis between SCFA subclass content levels and AP phenotype parameters found that butyrate content was most closely related to the severity of AP.A recovery experiment targeting butyrate demonstrated that butyrate could alleviate AP exacerbated by CMC,restoring intestinal barrier function and reducing the proportion of peripheral blood classical monocytes.To further identify the specific bacterial species mediating the exacerbation of AP by CMC,this study selected the top five bacterial species that differed between the CMCexposed group and the control group,including Akkermansia muciniphila,Escherichia coli,Clostridium perfringens,Bacteroides vulgatus,and an unclassified Alistipes species.Akkermansia muciniphila was strongly correlated with various AP severity parameters and intestinal barrier function parameters,indicating its potential involvement in the exacerbation of AP related to CMC.Supplementing Akkermansia muciniphila alleviated the exacerbation of AP induced by CMC.Supplementing Akkermansia muciniphila restored the colonic SCFA levels in mice,consistent with its role as an SCFA producer,revealing that the reduced butyrate levels following CMC exposure were due to a decrease in Akkermansia muciniphila.In summary,this study is the first to demonstrate that CMC exposure exacerbates AP in a mouse model,mediated by the gut microbiota.The reduction in the abundance of bacterial species such as Akkermansia muciniphila leads to decreased SCFA,impaired intestinal barrier function,and LPS leakage,which in turn activates classical monocytes,ultimately exacerbating AP.Given the widespread use of CMC in processed foods,our study reveals an important risk factor contributing to the recent increase in AP incidence.Furthermore,this research has direct translational significance for AP patients exposed to CMC in their daily diet,suggesting that supplementing Akkermansia muciniphila or butyrate may prevent AP attacks or mitigate their severity.