Lacidophilin Modulated Gut Microbiota and Ameliorated Dextran Sulfate Sodium-Induced Mouse Colitis

Lacidophilin Modulated Gut Microbiota and Ameliorated Dextran Sulfate Sodium-Induced Mouse Colitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lacidophilin Modulated Gut Microbiota and Ameliorated Dextran Sulfate Sodium-Induced Mouse Colitis Yu Jingting, Cheng Xiaoying, Zhan Yang, Zhang Jingwen, Li Yingmeng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4684193/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract The prevalence of inflammatory bowel disease (IBD) has been rising significantly in recent years. It is widely accepted that gut microbes play an essential role in the development of IBD. Lacidophilin is a product of milk fermentation by lactobacillus acidophilus. The aim of this study was to investigate the effect of Lacidophilin on colitis induced by dextran sulfate sodium (DSS). 16s RNA sequencing was performed to determine the changes of species composition and community structure of the intestinal microflora, and transcriptome sequencing was conducted to find out the gene or protein which may be affected by Lactobacillus on colitis development potentially. It was observed that the 7 days administration of Lacidophilin protected the intestinal mucosal barrier from damage, and thereby enabled the remission of colitis severity. Compared to the model group, Lacidophilin could restore the shortened colon length and marked decrease levels of TNF-α and IL-6 in serum. More importantly, Lacidophilin significantly increased the abundance of beneficial bacteria such as Lactobacillus , Bifidobacterium and Lachnospiraceae_NK4A136_group , decreased the abundance of harmful bacteria such as Escherichia-Shigella and Parvibacter. Transcriptomic analysis shows that IL-17 signaling pathway, BCR signaling pathway, Toll-like receptor signaling pathway, and TNF signaling pathway was enriched, and we found that Lcn2, Ccl3, Mmp8, Slc11a1, Spp1, and Serpine1 might be potential targets of Lacidophilin treatment. These studies indicate that Lacidophilin can ameliorate colitis in mice through maintaining the integrity of intestinal structure and improving intestinal microbiota, and its mechanism may be involved in immune-related proteins and pathways. IBD Lacidophilin DSS microbiota transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Inflammatory bowel disease (IBD) is a chronic, recurrent, long-term inflammatory disease that eventually leads to colon and rectal ulcers, including Crohn's disease (CD) and Ulcerative colitis (UC) [ 1 ] . The difference between UC and CD lies in the location of inflammation. Ulcerative colitis is a persistent inflammation of the mucosa that begins in the rectum and extends proximally [ 2 ] , but in Crohn's disease, inflammation is widespread throughout the gastrointestinal tract [ 3 ] . The pathogenesis of UC involves genetic susceptibility components and environmental factors, but its specific pathogenesis remains unclear. Recent reports have documented the number of individuals who suffer from IBD has increased from 3.7 million to more than 6.8 million during the past three decades, suggesting the prevalence of IBD has risen sharply worldwide [ 4 ] . Up to now, first-line medications for IBD include anti-inflammatory drugs, corticosteroids and immunosuppressants, whereas these medications are effective in the early stages of the disease with coming at the cost of severe side effects [ 5 ] . Searching for IBD effective treatments with minimal side effects, therefore, has generated considerable recent research interest. The intestinal microbiome is a complex community of microorganisms that influence human nutrient metabolism, physical development, immune defense and oncogenesis [ 6 ] . Numerous experiments have so far revealed the associations between microflora with intestinal health and disorders, especially IBD. Research have shown a significant decrease of microbial diversity as well as an imbalance of microbial composition observed in IBD patients, often referred to as dysbiosis, mainly characterized by a reduction of beneficial bacteria and an increase of harmful bacteria [ 7 , 8 ] . Therefore, much research in recent years has focused on maintaining gut homeostasis to improve IBD. For example, it was shown that the role in improving UC of Panax quinquefolius polysaccharides was conducted by regulating the structure of gut microbiota [ 9 ] . In contrast, when intestinal mucosal was disrupted, the colonic homeostasis would be perturbated by E. coli capable of producing colistin [ 10 ] . Apart from intestinal microflora itself, its metabolites, short-chain fatty-acid (SCFA), also play a key role in maintaining the balance of gut homeostasis, including downregulate the levels of proinflammatory cytokines [ 11 ] , regulate intestinal barrier [ 12 ] as well as control gut homeostasis [ 13 ] . Also, the intestinal microbiota is involved in regulating the integrity and function of the intestinal barrier for maintaining homeostatic balance. Probiotic activity produced by lactic acid bacteria (LAB) has been expected to improve the intestinal environment, which can prevent some diseases such as cancers [ 14 ] and intestinal inflammation [ 15 ] . Lactobacillus acidophilus is an important member of the lactic acid bacteria family, and there is extensive literature on its amelioration of ulcerative colitis [ 16 – 19 ] . However, it takes a significant risk of bacteremia or other probiotic infections in direct ingesting live bacteria [ 20 ] , especially for ulcerative colitis patients whose intestinal barrier was impaired [ 21 , 22 ] . Lacidophilin (LP) is derived from the fermentation of skim milk by lactobacillus acidophilus. Its composition is relatively complex, containing lactic acid, antimicrobial substances, amino acids several trace elements. Different from probiotics which are live microorganisms, LP is a collection of metabolites secreted by live bacteria which is non-viable. Therefore, the safety of LP is more predictable than live Lactobacillus acidophilus. As early as 1930, Okayasu began introducing the model of DSS (dextran sulfate sodium) to induce colitis in mice, since its morphological damage and symptoms of intestinal epithelium were similar to those of UC in humans [ 23 ] . In this paper, we applied the DSS-induced acute ulcer model in mice to investigate the effect of LP on UC, and performed 16S rRNA and transcriptome sequencing to find out its possible action mechanism. Materials and Methods 2.1 Materials Lacidophilin was purchased from Jiangzhong Pharmaceutical Co., Ltd.(Jiangxi, China); Dextran sulfate sodium (DSS) was purchased from MP Biomedicals (California, USA). 2.2 Mice Male C57 mice (6–8 weeks) were provided by Gempharmatech Co., Ltd (Jiangsu, China). All mice (four per cage) housed under the conditions of a controlled temperature of 24 ± 2°C and relative humidity of 45–65% with 12 hours of light/dark cycle. Mice had free access to food and water throughout the experiment. The experiment protocol was approved by the Jiangzhong Pharmaceutical Co., Ltd. Ethics Committee. 2.3 DSS-induced experimental colitis After one week of adaptive feeding, the mice were randomly divided into four groups (n = 8): (1) Control group; (2) Model group; (3) Low dose lactobacillus group (LPL). (4) High dose lactobacillus group (LPH). The mice in the Model, LPL, and LPH group were given water containing 3% DSS (w/v) for 5 days, followed by 2 days of sterile water. Meanwhile, the mice in the Control groups only received sterile water for 7 days. The LPL group was orally administrated with 0.6g/kg Lacidophilin for 7 consecutive days, while the LPH group was given 1.2g/kg Lacidophilin, and the mice in the Control and Model groups were given the equivalent volume of saline. At day 8, all mice were euthanized using the method of cervical dislocation (Fig. 1 A). At the endpoint of experiment, blood samples were collected and then serum was stored at -80°C after centrifugation at 3000 rpm for 10 min. Mice were sacrificed by cervical dislocation under anaesthesia, then the colon tissue was removed as soon as possible. The colon tissue was unfolded without forceful pulling, a straightedge was placed underneath the colon and photographed under a stereomicroscope at the same height, light and magnification. Meanwhile, the length of the colon was recorded, and then the proximal colon was stored in 4% paraformaldehyde for histopathological examination. The cecum contents and residual colon tissue were frozen in liquid nitrogen for 16sRNA sequencing and transcriptome sequencing separately. 2.4 Histopathological, immunohistochemistry and immunofluorescence staining The colons were fixed in 4% paraformaldehyde for paraffin preparation, or embedded in tissue OCT freezing medium (Leica, Germany) for frozen block preparation. Paraffin-embedded colons were cut by ultra-thin semiautomatic microtome (Leica, Germany) to prepared 5µm tissue sections which were used for H&E or immunohistochemical staining of occludin and claudin-4. Hematoxylin counterstain was applied to visualize nuclei. The sections were observed under a light microscope (Leica, Germany). Frozen tissue sections with a thickness of 5µm were prepared with a frozen slicer (Leica, Germany) and subsequently stained with MUC-2 antibodies. DAPI counterstain was applied to visualize nuclei. The sections were observed under a fluorescence microscope (Leica, Germany). The expression of occludin, claudin-4, and MUC-2 was calculated using the 4 random view fields in each colon sample (n = 3). The fields were analyzed using ImageJ (The University of Nottingham, UK). The area was used to represent the protein expression, and the results of occludin and claudin-4 expression in each group were compared with the control group, while the results of MUC-2 were represented as the ratio of MUC-2 to DAPI positive area. 2.5 Enzyme-Linked Immunosorbent Assay (ELISA) The concentrations of inflammatory factors were detected by mouse immunoassay kits according to the manufacturer’s instructions. ELISA was used to assay the levels of TNF-α and IL-6 in the serum by commercial ELISA kits (NanJing JianCheng Bioengineering Institute, China). 2.6 16sRNA sequencing The contents of the cecum were taken for total microbiota DNA extraction and PCR amplification of the V3-V4 region of the 16S rRNA gene. The PCR products were detected and quantified by QuantiFluorTM-ST blue fluorescence quantitative system (Promega, USA), and then mixed in proportion according to the sequencing requirements of each sample. Then, Illumina library was constructed using TruSeqTM DNA Sample Prep Kit (Illumina, USA). Sequencing was performed using Illumina's MiseqPE300 platform(Illumina, USA). 2.7 Transcriptome sequencing Total RNA was extracted from the colons using MJZol total RNA extraction kit ( Majorbio, China) following the manufacturer’s instructions. All mRNA extracted from colon tissue were sequenced, and the library was constructed according to Illumina® Stranded mRNA Prep, Ligation (Illumina, USA) in the sequencing experiment. According to Illumina’s library construction protocol, the adaptor was connected to the fragments, then the products were purified, sorted and amplified to purify and enrich the library. The amplicons were quantified and then mixed in proportions, and paired-end sequencing was conducted on NovaSeq 6000 sequencer (Illumina, USA). 2.8 Statistics Results were shown as mean ± standard error of the mean (SEM). Differences in expression of colonic length, inflammatory factor and Chao, ACE, Shannon, Simpson indexes were analyzed by one-way analysis of variance (ANOVA), as differences of weight, food intake and water intake were analyzed by two-way ANOVA using GraphPad Prism 8.0 software. P < 0.05 is considered as statistically significant for the results, while P ≥ 0.05 represents no significant difference (NS). Sequencing data analysis and plotting were analyzed on the online platform of Majorbio Cloud Platform.( https://cloud.majorbio.com/ ). Results 3.1 Lacidophilin relieved DSS-induced ulcerative colitis To investigate the efficacy and action mechanism of Lacidophilin on UC, DSS-induced colitis mice were administered different dose Lacidophilin or normal saline. Figure 1 A shows the experimental timeline. In the present study, the body weight (Fig. 1 B) of DSS-induced colitis mice considerably reduced from day 6 to day 8 of the experiment, while this loss was alleviated by LPH group to a greater extent than model group. The food intake (Fig. 1 C) and water intake (Fig. 1 D) of mice had been recorded within seven days after administrated with DSS. Intake of DSS decreased food intake (P < 0.01)and water intake (NS) of mice compared with control group, however, there was no significant difference between those group which was interfered by DSS. After treatment with different dose Lacidophilin, effective reversal of the significant changes in colon length、the level of TNF-α、IL-6 in serum of mice with UC was achieved. Compared with control group, the model group had significantly shorter colonic length, while it was significantly higher in the LPH group than in the model group(Fig. 2 A and 2 B). The concentrations of TNF-α and IL-6 in the serum were measured by ELISA to assess the effects of Lacidophilin on inflammatory cytokines. We found that DSS-induced upregulations of TNF-α (Fig. 2 C) and IL-6 (Fig. 2 D) were significantly downregulated by Lacidophilin. H&E staining was performed to evaluate the histopathology of colon injury. Pathological analysis suggested that there were no ulcer on the surface of the mucosa and the structure of the crypt was regular and clear without obvious inflammatory infiltration in the control group, while the mucosal structure of the model group was disorganized, with abnormal crypt and a large number of lymphocyte infiltration. Compared with the model group, the inflammatory reaction of the LPL and LPH groups were reduced, and the mucosal surface was more intact, which tended to be normal (Fig. 2 E). 3.2 Lacidophilin improved gut barrier integrity in ulcerative colitis mouse Occludin and Claudin-4 are important components of tight junctions. Once the function of tight junction proteins is dysfunctional, it weakens intercellular adhesion, promotes intestinal permeability and inflammation. Immunolocalization of Occludin and Claudin-4 were investigated using immunohistochemical staining. Occludin was localized at mucosa layer and the whole crypts, while Claudin-4 was predominantly distributed at the surface and tip of crypts. Intense Occludin and claudin-4 immunostaining was observed in controls, whereas reduced in models. Compared with the model group, LPH group has more intense immunostaining, whereas there appeared to be little change in the LPL group(Fig. 3 A, B). Using ImageJ to scan the slides, the staining intensity of Occludin and Claudin-4, expressed as the ratio of the positively stained area of the entire scanned specimen, were higher in the treatment group than model group, especially in LPH group (p < 0.001). (Fig. 3 C, D). Loss of goblet cells characterizes mice with UC. As the most abundant mucins, MUC-2 is which is used as a marker of goblet cell homeostasis. Therefore, we further performed Immunofluorescence staining to examine muc-2 expression in the colon of mice. We observed a significant decrease in muc-2 positive cells in the colon of model group compared to control, while treatment with high dose Lacidophilin moderately enhanced MUC-2 immunostaining (Model: 32.1% positive, LPH: 47.5. p < 0.05) (Fig. 4 ). 3.3 Lacidophilin improved gut microbial dysbiosis induced by ulcerative colitis The intestinal microbiota plays an essential role in maintaining gut homeostasis to shield us from diseases associated with dysbiosis. To determine changes in the intestinal microbial community, 16S rRNA gene sequencing was used to evaluate the microbiota in cecal contents in the control, model and LPH group. The species abundance was evaluated on the number of OTUs. The OTUs from the samples of control, model and LPH group are shown with a Venn diagram (Fig. 5 A). The model group has fewer OTUs than the controls, while the LPH group has more OTUs than the models, which indicated Lacidophilin could be able to increase the abundance of intestinal flora in mice with ulcerative colitis. Moreover, the curve of the pan/core analysis on OTU level implies sufficient samples and reliable sequencing results in the study (Fig. 5 B). Diversity of the gut flora was estimated using the Chao、ACE、Shannon and Shannon indices(Fig. 5 C). Microbial community diversity was significantly altered by Lacidophilin treatment on Chao、ACE and Shannon indices. Beta diversity of each group was calculated through PCoA. PCoA based on the relative abundance of OTUs revealed a separation of the control、model、LPH group(Fig. 5 D), suggesting that ulcerative colitis and Lacidophilin may be the important factors accounting for the change in structure of microbial community. Then, Partial Least Squares Discriminant Analysis (PLS-DA) was performed for the further evidence of significant differences between the bacterial communities in the groups(Fig. 5 E). The bacterial communities in the model samples and the matched controls as well as the LPH group clustered separately, indicating the overall structures of the microbial communities in the groups were remarkable different. The gut flora were analyzed at the phylum (Fig. 6 A) and genus (Fig. 6 C) levels in the groups, as well as differences in the relative abundance of at the phylum (Fig. 6 B) and genus (Fig. 6 D) levels among groups. The top six most abundant phyla are Firmicutes、Bacteroidetes、Verrucomicrobiota、Actinobacteriota、Proteobacteria and Desulfobacterota, and Firmicutes and Bacteroidetes, the dominant phylum in the gut, comprised more than 67% of all sequences. In addition, Proteobacteria, referred to as detrimental bacteria, were observed an increase relative abundance in the model compared to the control group, but a decrease in the LPH group. The relative abundance of Actinobacteriota was significantly lower in the model group than in the control group, while it was higher in the LPH group. At the genus level, Lactobacillus 、Dubosiella、Lachnospiraceae_NK4A136_group、Bifidobacterium、norank-f-norank-o-RF39 、 norank-f-Lachnospiraceae and norank_f_norank_o_Gastranaerophilales were of decreased abundance in model group, whereas Lacidophilin treatment showed the opposite change. Notably, Lacidophilin-treated mice showed a significant contraction of Escherichia-Shigella and Parvibacter in the gut microbiota. 3.4 Transcriptional changes induced by Lacidophilin treatment in ulcerative colitis mouse To deeply investigate the underlying mechanisms responsible for Lacidophilin on ulcerative colitis, the transcriptome was conducted. As shown in Fig. 7 A, we found that the distribution of samples from the model group and the controls were clearly separated by applying principal component analysis (PCA), so did the control group and LPH group. Then, we defined 1393 significantly differentially expressed genes by setting absolute fold change ≥ 2 and P < 0.05 as thresholds, in which 687 upregulated genes and 706 downregulated genes were identified in the model group relative to control. As for the LPH group versus the models, 67 significantly differentially expressed were determined, among which 63 genes were downregulated and 4 genes were upregulated (Fig. 7 B). The Venn diagram shown in Fig. 7 C indicated that there occurred significant discrepancy of 50 genes between control vs model group and model vs LPH group. Based on these differentially expressed genes (DEGs), a clustering analysis was performed. As shown in Fig. 7 D, the control and LPH groups showed similar trends in DEGs, while those in the model group showed the reverse trend. Then, to gain a better understanding of how Lacidophilin effect on UC, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis showed that these 50 genes were associated with immune system (Fig. 7 E). Further KEGG pathway enrichment analysis of identified DEGs was performed, and the 20 most significantly enriched pathways were presented in Fig. 7 F. Remarkably, cytokine-cytokine receptor interaction、IL-17 signaling pathway、complement and coagulation cascades、B cell receptor signaling pathway、Toll-like receptor signaling pathway and TNF signaling pathway were clearly enriched. Moreover, these 50 DEGs were used for the proteinprotein interactions network analysis (Fig. 7 G). We noticed greater degree and more edge connections in the nodes of Ccl3, Lcn2, SPP1 and Serpine1, which involved in immune functions, indicating greater importance in these protein in the network. Discussion In this study, mice were induced acute ulcerative colitis by DSS, while Lacidophilin was administered for 7 days. We observed that Lacidophilin can prevent the changes of the body weight, colonic length, serum inflammatory factor levels, and histopathological damage in colitis mice effectively. In addition, tight junctions and mucin showed enhanced expression following Lacidophilin treatment compared to controls. Then we further investigated the action mechanism of Lacidophilin in alleviating colitis. Patients with IBD suffer from dysbacteriosis which are defined as decreased diversity and abundance of gut microbial, with an imbalance between commensal and potentially pathogenic microorganisms [ 24 ] . We performed 16S ribosomal RNA tagged sequencing to identify alterations of intestinal microflora. At the phylum level, compared with the control group, the abundance of Bacteroidetes and Proteobacteria was decreased in the model group, whereas that Actinobacteriota of increased. However, Lacidophilin administration reversed this trend, aligning with the research of Zhu [ 25 ] and Zhou [ 26 ] . In addition, we revealed that Lactobacillus、Dubosiella、Lachnospiraceae_NK4A136_group、Bifidobacterium、norank-f-norank-o-RF39、norank-f-Lachnospiraceae and norank_f_norank_o_Gastranaerophilales was significantly increased in UC mice with treatment of Lacidophilin. Lactobacillus 、 Bifidobacterium and Lachnospiraceae_NK4A136_group have potential benefits in improving IBD due to their ability to produce SCFA [ 26 , 27 ] . Moreover, it had been reported that Lachnospiraceae_NK4A136_group were capable of maintaining epithelial health and immune balance of the intestine [ 27 ] . Toll-like receptors (TLRs) activate inflammatory cells and release pro-inflammatory cytokines, which are the key mediators of inflammatory routes in the intestine [ 28 , 29 ] . It has been shown that norank-f-norank-o-RF39 were negatively correlated with TLR2 receptor expression and proinflammatory factors IL-4 and IL-17 levels, but positively correlated with anti-inflammatory factors IL-10 levels [ 30 ] , which means these intestinal flora might be beneficial to attenuate intestinal inflammation of DSS colitis. Dubosiella thought to be improved colitis symptoms have been explored in several studies [ 31 – 33 ] . Nevertheless, some contrary research was also observed with more prominent relative abundance in colitis mice than the normal ones [ 34 , 35 ] , which is inconsistent with our results. Hence a further study of Dubosiella function on colitis in vivo will be required in vivo. Conversely, the abundance of Escherchia-Shigella and Parvibacter was significantly decreased in the LPH group than in UC mice. An increase in the abundance of Escherchia-Shigella was observed in the mice accompanied with aggravating DSS-induced colitis [ 36 , 37 ] . Moreover, it is demonstrated that Parvibacter might have potential benefit effects on colitis cause its positive correlation with IL-10 [ 38 ] , whereas, some research had demonstrated that DSS induced a significant reduction of Parvibacter in mice with colitis [ 39 ] . Therefore, whether it is beneficial for colitis remains further evidence. Taken together, Lacidophilin might restrain the growth of potentially harmful bacteria and enrich potentially beneficial bacteria to improve DSS-induced ulcerative colitis. To deeply investigate the molecular mechanism of Lacidophilin to UC, transcriptome of mice colon was performed after high-dose Lacidophilin treatment. In the present study, 50 significantly changed genes were observed in Control versus Model and Model versus LPH groups. It is worth mentioning that immune system dominated KEGG pathway, suggesting its mechanism was mainly correlated with immunity. The IL-17 signaling pathway which could mediate the promotion of immune response and activation of various inflammatory pathways [ 40 ] , was originally described in the autoimmune disease, and it has been a drug target for many autoimmune and chronic inflammatory diseases. There is evidence for IL-17 signaling pathway in promoting inflammation that damages the gut mucosa, while it also plays protective roles in regulating intestinal flora [ 41 ] . The B cell receptor (BCR) signaling pathway is a key signaling pathway for the development and maturation of B cells [ 42 ] . Most studies in the field of B cell receptor signaling pathway have only focused on lymphoma [ 43 , 44 ] , but there was little research on B-cell receptor pathways and UC. Thus, our experiment might provide more evidence for the relationship between the B-cell receptor pathway and UC. Moreover, following our research, Toll-like receptor signaling pathway and TNF signaling pathway which were involved in driving inflammation might be valuable for the molecular mechanisms of Lacidophilin improving UC. PPI network have demonstrated that the protein, Lcn2, Ccl3, Mmp8, Slc11a1, Spp1 and Serpine1, are involved in mediating multiple pathways to improve DSS-induced intestinal injury. Lipocalin 2 (Lcn2), a multifunctional immune protein, was closely related to the intestinal inflammation. Reports are rather controversial, and there is no general agreement about whether Lcn2 mediate anti-inflammatory or pro-inflammatory functions. Researchers reported that Lcn2 released in a partial MyD88-dependent manner, and UC would be aggravated significantly in Lcn2 knockout mice [ 45 ] . Lcn2 deletion could augment pro-inflammatory response in the model challenged with LPS [ 46 ] . However, other authors question the function of Lcn2, who suggested that Lcn2 with the activation of NF-κB pathway while enhancing inflammasome assembly and IL-1β secretion, could lead to more severe inflammation [ 47 ] . Our results show that an elevated level of Lcn2 in colon tissue of mice with UC, while it declined in mice administrated with Lacidophilin, consistent with increase of intestinal mucosal Lcn2 in patients with IBD [ 48 ] . Notably, Ccl3, belongs to chemokines family, which could induce macrophages and granulocytes migration to sites of acute inflammation [ 49 ] , has up-regulated during intestinal inflammation, while oral administration of Lacidophilin could significantly reverse its increase. Consistent with our findings, several studies have shown a strongly up-regulation of Ccl3 expression was observed in IL-10 -/- mice [ 50 ] , TNBS model [ 51 ] , as well as DSS model [ 52 ] , all of which are known as common animal models for IBD. It would seem to suggest that Ccl3 was a possible biomarkers and potential therapeutic target for IBD, and our results might provide a rationale for further studies. It has been accepted that regulated expression of matrix metalloproteinases (MMPs) plays multifaced roles in the pathogenesis of IBD. Previous findings showing that MMP8, which is predominantly expressed by macrophages, led to increased inflammatory cells infiltration after mice exposure to DSS [ 53 ] , are compatible with our results. The latest research pointed to a significant role of the Slc11a1 gene (formerly NRAMP1) on the DSS-induced colitis phenotype [ 54 ] . In addition, a strong relationship between the secreted phosphoprotein 1 (Spp1) and CD susceptibility was observed [ 55 ] . The Serpine1 encoding a protein called plasminogen activator inhibitor-1 (PAI-1) which can promote peripheral angiogenesis [ 56 ] , was elevated during the disease activity cycle in the inflamed colon, contributing to an aggravation of mucosal damage in colitis [ 57 ] . Taken together, our findings suggest the role of Lacidophilin in alleviating DSS-induced colitis, identifying the enhancement of colon barrier integrity and improvement of intestinal microflora as a possible action mechanism. Lacidophilin could drive the immune system, reduce the expression of inflammatory cytokines, and decrease the protein expression of Lcn2, Ccl3, Mmp8, Slc11a1, Spp1 and Serpine1, which were associated with immune response or neo-angiogenesis. However, this study was limited by the absence the experimental methods of polymerase chain reaction (PCR) and western blot (WB) to verify its changes at molecular and protein levels, and a more precise mechanism how Lacidophilin mediate immune system in colitis remains to be elucidated. In spite of its limitations, this work offers guidance on future research and clinical application of postbiotics to improve acute ulcerative colitis. Declarations Ethics Statement The animal study was reviewed and approved by the Experimental Animal Ethics Committee of Jiangzhong Pharmaceutical Co., Ltd. The Ethical Commitee protocol number was 20220407. The reporting in this study follows the recommendations in the ARRIVE guidelines. We have strictly abided by the international animal welfare and ethical standards, carry out the relevant laws, regulations and policies on the management of laboratory animals, and care laboratory animals. Data availability Sequence data that support the findings of this study have been deposited in the National Center for Biotechnology Information Archive with the link of http://www.ncbi.nlm.nih.gov/bioproject/1130646 and the China National Gene Bank Nucleotide Sequence Archive with the link of http://db.cngb.org/cnsa/project/CNP0005936_92303499/reviewlink/. Funding This study was supported by Nanchang City High-level Scientific and Technological Innovation Talents “Double Hundred Plan” (Grant No. [2022]321). Author Contributions WJ-L conceived and designed the experiments. JT-Y, XY-C and ZY performed the experiments. WJ-L, YM-L, LJ-Z contributed reagents/materials/analytical tools. JT-Y, JW-Z and DL-S analyzed the data. JT-Y wrote the manuscript. References Panaccione R, Lee WJ, Clark R, et al. Dose escalation patterns of advanced therapies in crohn's disease and ulcerative colitis: A systematic literature review [J]. Adv Ther, 2023,40(5):2051-2081. DOI:10.1007/s12325-023-02457-6. Feuerstein JD, Cheifetz AS. Ulcerative colitis: Epidemiology, diagnosis, and management [J]. Mayo Clin Proc, 2014,89(11):1553-1563. DOI:10.1016/j.mayocp.2014.07.002. Feuerstein JD, Cheifetz AS. Crohn disease: Epidemiology, diagnosis, and management [J]. Mayo Clin Proc, 2017,92(7):1088-1103. DOI:10.1016/j.mayocp.2017.04.010. Alatab S, Sepanlou S, Ikuta KS, et al. The global, regional, and national burden of inflammatory bowel disease in 195 countries and territories, 1990–2017: A systematic analysis for the global burden of disease study 2017 [J]. The Lancet Gastroenterology & Hepatology, 2020,5(1):17-30. Saniabadi AR, Tanaka T, Ohmori T, et al. Treating inflammatory bowel disease by adsorptive leucocytapheresis: A desire to treat without drugs [J]. World J Gastroenterol, 2014,20(29):9699-9715. DOI:10.3748/wjg.v20.i29.9699. Chen Z, Jin W, Hoover A, et al. Decoding the microbiome: Advances in genetic manipulation for gut bacteria [J]. Trends Microbiol, 2023. DOI:10.1016/j.tim.2023.05.007. Knights D, Lassen KG, Xavier RJ. Advances in inflammatory bowel disease pathogenesis: Linking host genetics and the microbiome [J]. Gut, 2013,62(10):1505-1510. DOI:10.1136/gutjnl-2012-303954. Imhann F, Vich Vila A, Bonder MJ, et al. Interplay of host genetics and gut microbiota underlying the onset and clinical presentation of inflammatory bowel disease [J]. Gut, 2018,67(1):108-119. DOI:10.1136/gutjnl-2016-312135. Ren DD, Chen KC, Li SS, et al. Panax quinquefolius polysaccharides ameliorate ulcerative colitis in mice induced by dextran sulfate sodium [J]. Front Immunol, 2023,14(1161625. DOI:10.3389/fimmu.2023.1161625. Harnack C, Berger H, Liu L, et al. Short-term mucosal disruption enables colibactin-producing e. Coli to cause long-term perturbation of colonic homeostasis [J]. Gut Microbes, 2023,15(1):2233689. DOI:10.1080/19490976.2023.2233689. Chang PV, Hao L, Offermanns S, et al. The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition [J]. Proc Natl Acad Sci U S A, 2014,111(6):2247-2252. DOI:10.1073/pnas.1322269111. Yang W, Yu T, Huang X, et al. Intestinal microbiota-derived short-chain fatty acids regulation of immune cell il-22 production and gut immunity [J]. Nat Commun, 2020,11(1):4457. DOI:10.1038/s41467-020-18262-6. Macia L, Tan J, Vieira AT, et al. Metabolite-sensing receptors gpr43 and gpr109a facilitate dietary fibre-induced gut homeostasis through regulation of the inflammasome [J]. Nat Commun, 2015,6(6734. DOI:10.1038/ncomms7734. Kwak SH, Cho YM, Noh GM, et al. Cancer preventive potential of kimchi lactic acid bacteria (weissella cibaria, lactobacillus plantarum) [J]. J Cancer Prev, 2014,19(4):253-258. DOI:10.15430/JCP.2014.19.4.253. Park JS, Joe I, Rhee PD, et al. A lactic acid bacterium isolated from kimchi ameliorates intestinal inflammation in dss-induced colitis [J]. J Microbiol, 2017,55(4):304-310. DOI:10.1007/s12275-017-6447-y. Wang MX, Lin L, Chen YD, et al. Evodiamine has therapeutic efficacy in ulcerative colitis by increasing lactobacillus acidophilus levels and acetate production [J]. Pharmacol Res, 2020,159(104978. DOI:10.1016/j.phrs.2020.104978. Kye YJ, Lee SY, Kim HR, et al. Lactobacillus acidophilus pin7 paraprobiotic supplementation ameliorates dss-induced colitis through anti-inflammatory and immune regulatory effects [J]. J Appl Microbiol, 2022,132(4):3189-3200. DOI:10.1111/jam.15406. Huang Z, Gong L, Jin Y, et al. Different effects of different lactobacillus acidophilus strains on dss-induced colitis [J]. Int J Mol Sci, 2022,23(23). DOI:10.3390/ijms232314841. Kim WK, Han DH, Jang YJ, et al. Alleviation of dss-induced colitis via lactobacillus acidophilus treatment in mice [J]. Food Funct, 2021,12(1):340-350. DOI:10.1039/d0fo01724h. Kullar R, Goldstein EJC, Johnson S, et al. Lactobacillus bacteremia and probiotics: A review [J]. Microorganisms, 2023,11(4). DOI:10.3390/microorganisms11040896. Vahabnezhad E, Mochon AB, Wozniak LJ, et al. Lactobacillus bacteremia associated with probiotic use in a pediatric patient with ulcerative colitis [J]. J Clin Gastroenterol, 2013,47(5):437-439. DOI:10.1097/MCG.0b013e318279abf0. Meini S, Laureano R, Fani L, et al. Breakthrough lactobacillus rhamnosus gg bacteremia associated with probiotic use in an adult patient with severe active ulcerative colitis: Case report and review of the literature [J]. Infection, 2015,43(6):777-781. DOI:10.1007/s15010-015-0798-2. Okayasu I, Hatakeyama S, Yamada M, et al. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice [J]. Gastroenterology, 1990,98(3):694-702. DOI:10.1016/0016-5085(90)90290-h. Verbeke KA, Boesmans L, Boets E. Modulating the microbiota in inflammatory bowel diseases: Prebiotics, probiotics or faecal transplantation? [J]. Proc Nutr Soc, 2014,73(4):490-497. DOI:10.1017/S0029665114000639. Zhu Y, Zhao Q, Huang Q, et al. Nuciferine regulates immune function and gut microbiota in dss-induced ulcerative colitis [J]. Front Vet Sci, 2022,9(939377. DOI:10.3389/fvets.2022.939377. Zhou Y, Xu ZZ, He Y, et al. Gut microbiota offers universal biomarkers across ethnicity in inflammatory bowel disease diagnosis and infliximab response prediction [J]. mSystems, 2018,3(1). DOI:10.1128/mSystems.00188-17. Wang K, Qin L, Cao J, et al. Kappa-selenocarrageenan oligosaccharides prepared by deep-sea enzyme alleviate inflammatory responses and modulate gut microbiota in ulcerative colitis mice [J]. Int J Mol Sci, 2023,24(5). DOI:10.3390/ijms24054672. Asadzadeh Aghdaei H, Rezasoltani S, Olfatifar M, et al. Expression of toll-like receptors 2, 4 and 5 in relation to gut microbiota in colon neoplasm patients with and without inflammatory bowel disease [J]. Avicenna J Med Biotechnol, 2022,14(3):188-195. DOI:10.18502/ajmb.v14i3.9825. Honjo H, Watanabe T, Kamata K, et al. Ripk2 as a new therapeutic target in inflammatory bowel diseases [J]. Front Pharmacol, 2021,12(650403. DOI:10.3389/fphar.2021.650403. Jia W, Xu C, Zhao T, et al. Integrated network pharmacology and gut microbiota analysis to explore the mechanism of sijunzi decoction involved in alleviating airway inflammation in a mouse model of asthma [J]. Evid Based Complement Alternat Med, 2023,2023(1130893. DOI:10.1155/2023/1130893. Zhai Z, Zhang F, Cao R, et al. Cecropin a alleviates inflammation through modulating the gut microbiota of c57bl/6 mice with dss-induced ibd [J]. Front Microbiol, 2019,10(1595. DOI:10.3389/fmicb.2019.01595. Wan F, Wang M, Zhong R, et al. Supplementation with chinese medicinal plant extracts from lonicera hypoglauca and scutellaria baicalensis mitigates colonic inflammation by regulating oxidative stress and gut microbiota in a colitis mouse model [J]. Front Cell Infect Microbiol, 2021,11(798052. DOI:10.3389/fcimb.2021.798052. Wan F, Zhong R, Wang M, et al. Caffeic acid supplement alleviates colonic inflammation and oxidative stress potentially through improved gut microbiota community in mice [J]. Front Microbiol, 2021,12(784211. DOI:10.3389/fmicb.2021.784211. Fu YP, Li CY, Peng X, et al. Pectic polysaccharides from aconitum carmichaelii leaves protects against dss-induced ulcerative colitis in mice through modulations of metabolism and microbiota composition [J]. Biomed Pharmacother, 2022,155(113767. DOI:10.1016/j.biopha.2022.113767. Li Q, Chen G, Zhu D, et al. Effects of dietary phosphatidylcholine and sphingomyelin on dss-induced colitis by regulating metabolism and gut microbiota in mice [J]. J Nutr Biochem, 2022,105(109004. DOI:10.1016/j.jnutbio.2022.109004. Huang H, Li M, Wang Y, et al. Excessive intake of longan arillus alters gut homeostasis and aggravates colitis in mice [J]. Front Pharmacol, 2021,12(640417. DOI:10.3389/fphar.2021.640417. Wang SL, Shao BZ, Zhao SB, et al. Intestinal autophagy links psychosocial stress with gut microbiota to promote inflammatory bowel disease [J]. Cell Death Dis, 2019,10(6):391. DOI:10.1038/s41419-019-1634-x. Ge W, Zhou BG, Zhong YB, et al. Sishen pill ameliorates dextran sulfate sodium (dss)-induced colitis with spleen-kidney yang deficiency syndromes: Role of gut microbiota, fecal metabolites, inflammatory dendritic cells, and tlr4/nf-kappab pathway [J]. Evid Based Complement Alternat Med, 2022,2022(6132289. DOI:10.1155/2022/6132289. Cheng H, Zhang D, Wu J, et al. Atractylodes macrocephala koidz. Volatile oil relieves acute ulcerative colitis via regulating gut microbiota and gut microbiota metabolism [J]. Front Immunol, 2023,14(1127785. DOI:10.3389/fimmu.2023.1127785. Bianchi E, Vecellio M, Rogge L. Editorial: Role of the il-23/il-17 pathway in chronic immune-mediated inflammatory diseases: Mechanisms and targeted therapies [J]. Front Immunol, 2021,12(770275. DOI:10.3389/fimmu.2021.770275. Zhang HJ, Xu B, Wang H, et al. Il-17 is a protection effector against the adherent-invasive escherichia coli in murine colitis [J]. Mol Immunol, 2018,93(166-172. DOI:10.1016/j.molimm.2017.11.020. Chang S, Li B, Xie Y, et al. Dcz0014, a novel compound in the therapy of diffuse large b-cell lymphoma via the b cell receptor signaling pathway [J]. Neoplasia, 2022,24(1):50-61. DOI:10.1016/j.neo.2021.12.001. Krysiak K, Gomez F, White BS, et al. Recurrent somatic mutations affecting b-cell receptor signaling pathway genes in follicular lymphoma [J]. Blood, 2017,129(4):473-483. DOI:10.1182/blood-2016-07-729954. Buchner M, Muschen M. Targeting the b-cell receptor signaling pathway in b lymphoid malignancies [J]. Curr Opin Hematol, 2014,21(4):341-349. DOI:10.1097/MOH.0000000000000048. Singh V, Yeoh BS, Chassaing B, et al. Microbiota-inducible innate immune, siderophore binding protein lipocalin 2 is critical for intestinal homeostasis [J]. Cell Mol Gastroenterol Hepatol, 2016,2(4):482-498 e486. DOI:10.1016/j.jcmgh.2016.03.007. Du H, Liang L, Li J, et al. Lipocalin-2 alleviates lps-induced inflammation through alteration of macrophage properties [J]. J Inflamm Res, 2021,14(4189-4203. DOI:10.2147/JIR.S328916. Kim SL, Shin MW, Kim SW. Lipocalin 2 activates the nlrp3 inflammasome via lps‑induced nf‑κb signaling and plays a role as a pro‑inflammatory regulator in murine macrophages [J]. Mol Med Rep, 2022,26(6). DOI:10.3892/mmr.2022.12875. Carlson M, Raab Y, Seveus L, et al. Human neutrophil lipocalin is a unique marker of neutrophil inflammation in ulcerative colitis and proctitis [J]. Gut, 2002,50(4):501-506. DOI:10.1136/gut.50.4.501. Trivedi PJ, Adams DH. Chemokines and chemokine receptors as therapeutic targets in inflammatory bowel disease; pitfalls and promise [J]. J Crohns Colitis, 2018,12(suppl_2):S641-S652. DOI:10.1093/ecco-jcc/jjx145. Hagenlocher Y, Hosel A, Bischoff SC, et al. Cinnamon extract reduces symptoms, inflammatory mediators and mast cell markers in murine il-10(-/-) colitis [J]. J Nutr Biochem, 2016,30(85-92. DOI:10.1016/j.jnutbio.2015.11.015. Prados ME, Garcia-Martin A, Unciti-Broceta JD, et al. Betulinic acid hydroxamate prevents colonic inflammation and fibrosis in murine models of inflammatory bowel disease [J]. Acta Pharmacol Sin, 2021,42(7):1124-1138. DOI:10.1038/s41401-020-0497-0. Ding P, Li L, Huang T, et al. Complement component 6 deficiency increases susceptibility to dextran sulfate sodium-induced murine colitis [J]. Immunobiology, 2016,221(11):1293-1303. DOI:10.1016/j.imbio.2016.05.014. Koelink PJ, Overbeek SA, Braber S, et al. Collagen degradation and neutrophilic infiltration: A vicious circle in inflammatory bowel disease [J]. Gut, 2014,63(4):578-587. DOI:10.1136/gutjnl-2012-303252. de Andrade STQ, Guidugli TI, Borrego A, et al. Slc11a1 gene polymorphism influences dextran sulfate sodium (dss)-induced colitis in a murine model of acute inflammation [J]. Genes Immun, 2023,24(2):71-80. DOI:10.1038/s41435-023-00199-7. Glas J, Seiderer J, Bayrle C, et al. The role of osteopontin (opn/spp1) haplotypes in the susceptibility to crohn's disease [J]. PLoS One, 2011,6(12):e29309. DOI:10.1371/journal.pone.0029309. Duan ZL, Wang YJ, Lu ZH, et al. Wumei wan attenuates angiogenesis and inflammation by modulating rage signaling pathway in ibd: Network pharmacology analysis and experimental evidence [J]. Phytomedicine, 2023,111(154658. DOI:10.1016/j.phymed.2023.154658. Kaiko GE, Chen F, Lai CW, et al. Pai-1 augments mucosal damage in colitis [J]. Sci Transl Med, 2019,11(482). DOI:10.1126/scitranslmed.aat0852. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.tif Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 13 Aug, 2024 Reviews received at journal 12 Aug, 2024 Reviews received at journal 11 Aug, 2024 Reviewers agreed at journal 03 Aug, 2024 Reviewers agreed at journal 02 Aug, 2024 Reviewers invited by journal 01 Aug, 2024 Editor assigned by journal 27 Jul, 2024 Editor invited by journal 22 Jul, 2024 Submission checks completed at journal 17 Jul, 2024 First submitted to journal 04 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4684193","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":335515370,"identity":"684f088c-7e94-41b4-b414-e3e856320f04","order_by":0,"name":"Yu Jingting","email":"","orcid":"","institution":"State Key Laboratory for the Modernization of Classical and Famous Prescriptions of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Jingting","suffix":""},{"id":335515371,"identity":"438741cf-7854-4d31-a58e-e53371d8f925","order_by":1,"name":"Cheng Xiaoying","email":"","orcid":"","institution":"State Key Laboratory for the Modernization of Classical and Famous Prescriptions of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Xiaoying","suffix":""},{"id":335515372,"identity":"b074530a-29d6-4513-974d-755935ec27aa","order_by":2,"name":"Zhan Yang","email":"","orcid":"","institution":"State Key Laboratory for the Modernization of Classical and Famous Prescriptions of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhan","middleName":"","lastName":"Yang","suffix":""},{"id":335515373,"identity":"79641a21-7194-4335-a14b-67340220d101","order_by":3,"name":"Zhang Jingwen","email":"","orcid":"","institution":"State Key Laboratory for the Modernization of Classical and Famous Prescriptions of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Jingwen","suffix":""},{"id":335515374,"identity":"919cd727-36ad-45b4-91e5-90f286a4bbf6","order_by":4,"name":"Li Yingmeng","email":"","orcid":"","institution":"State Key Laboratory for the Modernization of Classical and Famous Prescriptions of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Yingmeng","suffix":""},{"id":335515375,"identity":"c703ccff-7dc8-47b2-b739-0d11d3c9a8ac","order_by":5,"name":"Sun Denglong","email":"","orcid":"","institution":"State Key Laboratory for the Modernization of Classical and Famous Prescriptions of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sun","middleName":"","lastName":"Denglong","suffix":""},{"id":335515376,"identity":"3cd172d1-a20e-43f9-a54e-1357b024569b","order_by":6,"name":"Zheng Longjin","email":"","orcid":"","institution":"State Key Laboratory for the Modernization of Classical and Famous Prescriptions of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Longjin","suffix":""},{"id":335515377,"identity":"f597c272-7683-46f9-bd19-414466ed8477","order_by":7,"name":"Liu Wenjun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYHCCBIYHBlAGQ4WEnDxRWhLgWs5YGBs2EGcPFDC2VSQyHCCgmn/2gccfEgoOJ/ZLNzyTLpwnkcDYwPzw0Q08WiTOJSQYJBgcTpw550Ca9MxtEnnsDGzGxjl4tBjwMCQANR3O3XAjIU2ad5tEMWMDD5s0IS0HQFr2g7XMkUhsOEBYS2ID2BYJkJYGIrRInGFIBgZyev2MGwnJ1jzHJIwNmwn4hb+HJ/nDhz/WxvwzchJv89TUycmzNz98jE8LAwNPAhqDGa9yEGA/gM4YBaNgFIyCUYAKAAlpSWpOXalgAAAAAElFTkSuQmCC","orcid":"","institution":"State Key Laboratory for the Modernization of Classical and Famous Prescriptions of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Liu","middleName":"","lastName":"Wenjun","suffix":""}],"badges":[],"createdAt":"2024-07-04 06:27:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4684193/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4684193/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62102853,"identity":"b11a5fe2-c8a2-4c09-b811-916930f2533d","added_by":"auto","created_at":"2024-08-09 10:07:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":179311,"visible":true,"origin":"","legend":"\u003cp\u003eThe appearance index of Lacidophilin on DSS-induced colitis in mice Lacidophilin influenced appearance index in mice with colitis. \u003cstrong\u003e(A) \u003c/strong\u003eExperimental timeline for DSS-induced ulcerative colitis mouse models of with interventional administration of the Lacidophilin. \u003cstrong\u003e(B) \u003c/strong\u003eBody weight evolution (n=9). \u003cstrong\u003e(C)\u003c/strong\u003e Food intake evolution (n=3). \u003cstrong\u003e(D)\u003c/strong\u003e Water intake evolution (n=3). Data are expressed as mean ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, compared to the Control group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4684193/v1/831a0d770be0b6e3b14618d4.png"},{"id":62102855,"identity":"fd0bca4f-e4cf-42ea-9777-908bd25ade0c","added_by":"auto","created_at":"2024-08-09 10:07:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2008443,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic evaluation of Lacidophilin on DSS-induced colitis in mice. \u003cstrong\u003e(A)\u003c/strong\u003e Changes in colonic length by naked eye. \u003cstrong\u003e(B)\u003c/strong\u003e Statistical graph for colonic length (n=9). \u003cstrong\u003e(C) \u003c/strong\u003eThe level of TNF-α in the serum (n=8). \u003cstrong\u003e(D) \u003c/strong\u003eThe level of IL-6 in the serum (n=8). \u003cstrong\u003e(E)\u003c/strong\u003e Hematoxylin and eosin staining of the colons. Data are representative images or expressed as mean ± SEM. *P \u0026lt; 0.05, ***P \u0026lt; 0.001, compared to the Control group. \u003csup\u003e#\u003c/sup\u003e P \u0026lt; 0.05, \u003csup\u003e## \u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e P \u0026lt; 0.001 compared to the Model group.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4684193/v1/14b23983942b6da82a4aa372.png"},{"id":62102858,"identity":"87fe0222-1e6a-4ce1-be65-7caa2fcdd63a","added_by":"auto","created_at":"2024-08-09 10:07:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7499153,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry staining for tight junctions in the colon of healthy (Control) and ulcerative colitis (Model) and Treated（LPL、LPH）mice. (A) Occludinexpression (200X). (B) Claudin-4 expression (200X). (C) Quantification of Occludin expression. (D) Quantification of Claudin-4 expression.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4684193/v1/09426cc3bec327bf45aec6c5.png"},{"id":62103718,"identity":"a6ec70c2-8448-434d-b083-f874385e3cf1","added_by":"auto","created_at":"2024-08-09 10:15:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4965466,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence staining for intestinal mucin in the colon of healthy (Control) and ulcerative colitis (Model) and Treated（LPL、LPH）mice (200X). (A) MUC-2 expression. (B) Quantification of MUC-2 expression.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4684193/v1/c77821ace412c897cee4ad9a.png"},{"id":62102856,"identity":"86f674bc-ce6d-4501-ac15-a4766e212ca5","added_by":"auto","created_at":"2024-08-09 10:07:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":295349,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of intestinal microbiota structures in the colon of healthy (Control) and acute colitis (Model) and Treated（LPH）mice.\u003cstrong\u003e (A) \u003c/strong\u003eVenn diagram on OTU level. \u003cstrong\u003e(B)\u003c/strong\u003e Pan/Core curve on OTU level. \u003cstrong\u003e(C) \u003c/strong\u003eAlpha diversities comparisons of microbial communities, Chao, ACE, Shannon, Simpson index (n=8). \u003cstrong\u003e(D)\u003c/strong\u003e Principal coordinate analysis of bacterial community composition (beta diversity).\u003cstrong\u003e (E)\u003c/strong\u003e Partial least square discriminant score plot of cecal microbiota. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, compared to the Control group. \u003csup\u003e#\u003c/sup\u003e P \u0026lt; 0.05, \u003csup\u003e## \u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e P \u0026lt; 0.001 compared to the Model group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4684193/v1/676f6cc5cdc8dd0cc10607e5.png"},{"id":62103717,"identity":"2228665d-6556-46a2-9f1d-2323b03c82ab","added_by":"auto","created_at":"2024-08-09 10:15:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":177076,"visible":true,"origin":"","legend":"\u003cp\u003eLacidophilin alters the community composition of intestinal microbiota in models of ulcerative colitis.\u003cstrong\u003e (A)\u003c/strong\u003e Bacterial composition of the different communities at the phylum level. \u003cstrong\u003e(B) \u003c/strong\u003eRelative abundance of intestinal microbiota communities at the phylum level. \u003cstrong\u003e(C) \u003c/strong\u003eBacterial composition of the different communities at the genus level. \u003cstrong\u003e(D)\u003c/strong\u003e Relative abundance of intestinal microbiota communities at the genus level.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4684193/v1/6788c36bf016d75b0850eb2b.png"},{"id":62102859,"identity":"8d8dd661-c1c2-4e5c-803c-0db73c4c5594","added_by":"auto","created_at":"2024-08-09 10:07:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2580394,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq analysis in the colon of healthy (Control) and acute colitis (Model) and Treated（LPH）mice. \u003cstrong\u003e(A) \u003c/strong\u003eThe PCA plot shows a distinct separation based on the gene expression profiles among the Control vs Model group and Model vs LPH groups. \u003cstrong\u003e(B)\u003c/strong\u003e Volcano plot showing the differentially expressed genes (DEGs) in the Control vs Model group and Model vs LPH groups. (P＜0.05, Fold change ≥ 2). \u003cstrong\u003e(C) \u003c/strong\u003eVenn diagram analyze of gene or transcript expression among the Control vs Model group and Model vs LPH groups. \u003cstrong\u003e(D)\u003c/strong\u003e DEGs heatmap analysis. Blue indicates downregulated genes. Red indicates upregulated genes.\u003cstrong\u003e (E) \u003c/strong\u003eKEGG functional annotation \u0026nbsp;analysis among DEGs. \u003cstrong\u003e(F)\u003c/strong\u003e KEGG enrichment functional analysis reveals the biological functions that are enriched in the DEGs. \u003cstrong\u003e(G) \u003c/strong\u003eThe protein–protein interaction (PPI) network based on DEGs.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4684193/v1/f92584edf8dd660dab53a592.png"},{"id":62104604,"identity":"c725b40c-1ad9-4c4b-86a3-691888421298","added_by":"auto","created_at":"2024-08-09 10:23:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21266366,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4684193/v1/9a5c8492-e754-450c-b2bd-cff59a3131ea.pdf"},{"id":62102861,"identity":"d71f6f54-ccc5-41c1-88e2-e958e02a8b02","added_by":"auto","created_at":"2024-08-09 10:07:22","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29465180,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-4684193/v1/f74b87b8f076675555ecaefa.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lacidophilin Modulated Gut Microbiota and Ameliorated Dextran Sulfate Sodium-Induced Mouse Colitis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInflammatory bowel disease (IBD) is a chronic, recurrent, long-term inflammatory disease that eventually leads to colon and rectal ulcers, including Crohn's disease (CD) and Ulcerative colitis (UC)\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The difference between UC and CD lies in the location of inflammation. Ulcerative colitis is a persistent inflammation of the mucosa that begins in the rectum and extends proximally\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, but in Crohn's disease, inflammation is widespread throughout the gastrointestinal tract\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The pathogenesis of UC involves genetic susceptibility components and environmental factors, but its specific pathogenesis remains unclear.\u003c/p\u003e \u003cp\u003eRecent reports have documented the number of individuals who suffer from IBD has increased from 3.7\u0026nbsp;million to more than 6.8\u0026nbsp;million during the past three decades, suggesting the prevalence of IBD has risen sharply worldwide\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Up to now, first-line medications for IBD include anti-inflammatory drugs, corticosteroids and immunosuppressants, whereas these medications are effective in the early stages of the disease with coming at the cost of severe side effects\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Searching for IBD effective treatments with minimal side effects, therefore, has generated considerable recent research interest.\u003c/p\u003e \u003cp\u003eThe intestinal microbiome is a complex community of microorganisms that influence human nutrient metabolism, physical development, immune defense and oncogenesis\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Numerous experiments have so far revealed the associations between microflora with intestinal health and disorders, especially IBD. Research have shown a significant decrease of microbial diversity as well as an imbalance of microbial composition observed in IBD patients, often referred to as dysbiosis, mainly characterized by a reduction of beneficial bacteria and an increase of harmful bacteria\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Therefore, much research in recent years has focused on maintaining gut homeostasis to improve IBD. For example, it was shown that the role in improving UC of \u003cem\u003ePanax quinquefolius\u003c/em\u003e polysaccharides was conducted by regulating the structure of gut microbiota\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. In contrast, when intestinal mucosal was disrupted, the colonic homeostasis would be perturbated by \u003cem\u003eE. coli\u003c/em\u003e capable of producing colistin\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Apart from intestinal microflora itself, its metabolites, short-chain fatty-acid (SCFA), also play a key role in maintaining the balance of gut homeostasis, including downregulate the levels of proinflammatory cytokines\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, regulate intestinal barrier\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e as well as control gut homeostasis\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Also, the intestinal microbiota is involved in regulating the integrity and function of the intestinal barrier for maintaining homeostatic balance.\u003c/p\u003e \u003cp\u003eProbiotic activity produced by lactic acid bacteria (LAB) has been expected to improve the intestinal environment, which can prevent some diseases such as cancers\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e and intestinal inflammation\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Lactobacillus acidophilus is an important member of the lactic acid bacteria family, and there is extensive literature on its amelioration of ulcerative colitis \u003csup\u003e[\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. However, it takes a significant risk of bacteremia or other probiotic infections in direct ingesting live bacteria\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, especially for ulcerative colitis patients whose intestinal barrier was impaired\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Lacidophilin (LP) is derived from the fermentation of skim milk by lactobacillus acidophilus. Its composition is relatively complex, containing lactic acid, antimicrobial substances, amino acids several trace elements. Different from probiotics which are live microorganisms, LP is a collection of metabolites secreted by live bacteria which is non-viable. Therefore, the safety of LP is more predictable than live Lactobacillus acidophilus.\u003c/p\u003e \u003cp\u003eAs early as 1930, Okayasu began introducing the model of DSS (dextran sulfate sodium) to induce colitis in mice, since its morphological damage and symptoms of intestinal epithelium were similar to those of UC in humans\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In this paper, we applied the DSS-induced acute ulcer model in mice to investigate the effect of LP on UC, and performed 16S rRNA and transcriptome sequencing to find out its possible action mechanism.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eLacidophilin was purchased from Jiangzhong Pharmaceutical Co., Ltd.(Jiangxi, China); Dextran sulfate sodium (DSS) was purchased from MP Biomedicals (California, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Mice\u003c/h2\u003e \u003cp\u003eMale C57 mice (6\u0026ndash;8 weeks) were provided by Gempharmatech Co., Ltd (Jiangsu, China). All mice (four per cage) housed under the conditions of a controlled temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and relative humidity of 45\u0026ndash;65% with 12 hours of light/dark cycle. Mice had free access to food and water throughout the experiment. The experiment protocol was approved by the Jiangzhong Pharmaceutical Co., Ltd. Ethics Committee.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 DSS-induced experimental colitis\u003c/h2\u003e \u003cp\u003eAfter one week of adaptive feeding, the mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;8): (1) Control group; (2) Model group; (3) Low dose lactobacillus group (LPL). (4) High dose lactobacillus group (LPH). The mice in the Model, LPL, and LPH group were given water containing 3% DSS (w/v) for 5 days, followed by 2 days of sterile water. Meanwhile, the mice in the Control groups only received sterile water for 7 days. The LPL group was orally administrated with 0.6g/kg Lacidophilin for 7 consecutive days, while the LPH group was given 1.2g/kg Lacidophilin, and the mice in the Control and Model groups were given the equivalent volume of saline. At day 8, all mice were euthanized using the method of cervical dislocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eAt the endpoint of experiment, blood samples were collected and then serum was stored at -80\u0026deg;C after centrifugation at 3000 rpm for 10 min. Mice were sacrificed by cervical dislocation under anaesthesia, then the colon tissue was removed as soon as possible. The colon tissue was unfolded without forceful pulling, a straightedge was placed underneath the colon and photographed under a stereomicroscope at the same height, light and magnification. Meanwhile, the length of the colon was recorded, and then the proximal colon was stored in 4% paraformaldehyde for histopathological examination. The cecum contents and residual colon tissue were frozen in liquid nitrogen for 16sRNA sequencing and transcriptome sequencing separately.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Histopathological, immunohistochemistry and immunofluorescence staining\u003c/h2\u003e \u003cp\u003eThe colons were fixed in 4% paraformaldehyde for paraffin preparation, or embedded in tissue OCT freezing medium (Leica, Germany) for frozen block preparation. Paraffin-embedded colons were cut by ultra-thin semiautomatic microtome (Leica, Germany) to prepared 5\u0026micro;m tissue sections which were used for H\u0026amp;E or immunohistochemical staining of occludin and claudin-4. Hematoxylin counterstain was applied to visualize nuclei. The sections were observed under a light microscope (Leica, Germany). Frozen tissue sections with a thickness of 5\u0026micro;m were prepared with a frozen slicer (Leica, Germany) and subsequently stained with MUC-2 antibodies. DAPI counterstain was applied to visualize nuclei. The sections were observed under a fluorescence microscope (Leica, Germany).\u003c/p\u003e \u003cp\u003eThe expression of occludin, claudin-4, and MUC-2 was calculated using the 4 random view fields in each colon sample (n\u0026thinsp;=\u0026thinsp;3). The fields were analyzed using ImageJ (The University of Nottingham, UK). The area was used to represent the protein expression, and the results of occludin and claudin-4 expression in each group were compared with the control group, while the results of MUC-2 were represented as the ratio of MUC-2 to DAPI positive area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Enzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe concentrations of inflammatory factors were detected by mouse immunoassay kits according to the manufacturer\u0026rsquo;s instructions. ELISA was used to assay the levels of TNF-α and IL-6 in the serum by commercial ELISA kits (NanJing JianCheng Bioengineering Institute, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 16sRNA sequencing\u003c/h2\u003e \u003cp\u003eThe contents of the cecum were taken for total microbiota DNA extraction and PCR amplification of the V3-V4 region of the 16S rRNA gene. The PCR products were detected and quantified by QuantiFluorTM-ST blue fluorescence quantitative system (Promega, USA), and then mixed in proportion according to the sequencing requirements of each sample. Then, Illumina library was constructed using TruSeqTM DNA Sample Prep Kit (Illumina, USA). Sequencing was performed using Illumina's MiseqPE300 platform(Illumina, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Transcriptome sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the colons using MJZol total RNA extraction kit ( Majorbio, China) following the manufacturer\u0026rsquo;s instructions. All mRNA extracted from colon tissue were sequenced, and the library was constructed according to Illumina\u0026reg; Stranded mRNA Prep, Ligation (Illumina, USA) in the sequencing experiment. According to Illumina\u0026rsquo;s library construction protocol, the adaptor was connected to the fragments, then the products were purified, sorted and amplified to purify and enrich the library. The amplicons were quantified and then mixed in proportions, and paired-end sequencing was conducted on NovaSeq 6000 sequencer (Illumina, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistics\u003c/h2\u003e \u003cp\u003eResults were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Differences in expression of colonic length, inflammatory factor and Chao, ACE, Shannon, Simpson indexes were analyzed by one-way analysis of variance (ANOVA), as differences of weight, food intake and water intake were analyzed by two-way ANOVA using GraphPad Prism 8.0 software. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered as statistically significant for the results, while P\u0026thinsp;\u0026ge;\u0026thinsp;0.05 represents no significant difference (NS).\u003c/p\u003e \u003cp\u003eSequencing data analysis and plotting were analyzed on the online platform of Majorbio Cloud Platform.( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com/\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Lacidophilin relieved DSS-induced ulcerative colitis\u003c/h2\u003e \u003cp\u003eTo investigate the efficacy and action mechanism of Lacidophilin on UC, DSS-induced colitis mice were administered different dose Lacidophilin or normal saline. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows the experimental timeline. In the present study, the body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) of DSS-induced colitis mice considerably reduced from day 6 to day 8 of the experiment, while this loss was alleviated by LPH group to a greater extent than model group. The food intake (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and water intake (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) of mice had been recorded within seven days after administrated with DSS. Intake of DSS decreased food intake (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01)and water intake (NS) of mice compared with control group, however, there was no significant difference between those group which was interfered by DSS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter treatment with different dose Lacidophilin, effective reversal of the significant changes in colon length、the level of TNF-α、IL-6 in serum of mice with UC was achieved. Compared with control group, the model group had significantly shorter colonic length, while it was significantly higher in the LPH group than in the model group(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The concentrations of TNF-α and IL-6 in the serum were measured by ELISA to assess the effects of Lacidophilin on inflammatory cytokines. We found that DSS-induced upregulations of TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) were significantly downregulated by Lacidophilin. H\u0026amp;E staining was performed to evaluate the histopathology of colon injury. Pathological analysis suggested that there were no ulcer on the surface of the mucosa and the structure of the crypt was regular and clear without obvious inflammatory infiltration in the control group, while the mucosal structure of the model group was disorganized, with abnormal crypt and a large number of lymphocyte infiltration. Compared with the model group, the inflammatory reaction of the LPL and LPH groups were reduced, and the mucosal surface was more intact, which tended to be normal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Lacidophilin improved gut barrier integrity in ulcerative colitis mouse\u003c/h2\u003e \u003cp\u003eOccludin and Claudin-4 are important components of tight junctions. Once the function of tight junction proteins is dysfunctional, it weakens intercellular adhesion, promotes intestinal permeability and inflammation. Immunolocalization of Occludin and Claudin-4 were investigated using immunohistochemical staining. Occludin was localized at mucosa layer and the whole crypts, while Claudin-4 was predominantly distributed at the surface and tip of crypts. Intense Occludin and claudin-4 immunostaining was observed in controls, whereas reduced in models. Compared with the model group, LPH group has more intense immunostaining, whereas there appeared to be little change in the LPL group(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Using ImageJ to scan the slides, the staining intensity of Occludin and Claudin-4, expressed as the ratio of the positively stained area of the entire scanned specimen, were higher in the treatment group than model group, especially in LPH group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLoss of goblet cells characterizes mice with UC. As the most abundant mucins, MUC-2 is which is used as a marker of goblet cell homeostasis. Therefore, we further performed Immunofluorescence staining to examine muc-2 expression in the colon of mice. We observed a significant decrease in muc-2 positive cells in the colon of model group compared to control, while treatment with high dose Lacidophilin moderately enhanced MUC-2 immunostaining (Model: 32.1% positive, LPH: 47.5. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Lacidophilin improved gut microbial dysbiosis induced by ulcerative colitis\u003c/h2\u003e \u003cp\u003eThe intestinal microbiota plays an essential role in maintaining gut homeostasis to shield us from diseases associated with dysbiosis. To determine changes in the intestinal microbial community, 16S rRNA gene sequencing was used to evaluate the microbiota in cecal contents in the control, model and LPH group.\u003c/p\u003e \u003cp\u003eThe species abundance was evaluated on the number of OTUs. The OTUs from the samples of control, model and LPH group are shown with a Venn diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The model group has fewer OTUs than the controls, while the LPH group has more OTUs than the models, which indicated Lacidophilin could be able to increase the abundance of intestinal flora in mice with ulcerative colitis. Moreover, the curve of the pan/core analysis on OTU level implies sufficient samples and reliable sequencing results in the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Diversity of the gut flora was estimated using the Chao、ACE、Shannon and Shannon indices(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Microbial community diversity was significantly altered by Lacidophilin treatment on Chao、ACE and Shannon indices. Beta diversity of each group was calculated through PCoA. PCoA based on the relative abundance of OTUs revealed a separation of the control、model、LPH group(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), suggesting that ulcerative colitis and Lacidophilin may be the important factors accounting for the change in structure of microbial community. Then, Partial Least Squares Discriminant Analysis (PLS-DA) was performed for the further evidence of significant differences between the bacterial communities in the groups(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The bacterial communities in the model samples and the matched controls as well as the LPH group clustered separately, indicating the overall structures of the microbial communities in the groups were remarkable different.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe gut flora were analyzed at the phylum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) and genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) levels in the groups, as well as differences in the relative abundance of at the phylum (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) levels among groups. The top six most abundant phyla are Firmicutes、Bacteroidetes、Verrucomicrobiota、Actinobacteriota、Proteobacteria and Desulfobacterota, and Firmicutes and Bacteroidetes, the dominant phylum in the gut, comprised more than 67% of all sequences. In addition, Proteobacteria, referred to as detrimental bacteria, were observed an increase relative abundance in the model compared to the control group, but a decrease in the LPH group. The relative abundance of Actinobacteriota was significantly lower in the model group than in the control group, while it was higher in the LPH group. At the genus level, \u003cem\u003eLactobacillus 、Dubosiella、Lachnospiraceae_NK4A136_group、Bifidobacterium、norank-f-norank-o-RF39\u003c/em\u003e、\u003cem\u003enorank-f-Lachnospiraceae\u003c/em\u003e and \u003cem\u003enorank_f_norank_o_Gastranaerophilales\u003c/em\u003e were of decreased abundance in model group, whereas Lacidophilin treatment showed the opposite change. Notably, Lacidophilin-treated mice showed a significant contraction of \u003cem\u003eEscherichia-Shigella\u003c/em\u003e and \u003cem\u003eParvibacter\u003c/em\u003e in the gut microbiota.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Transcriptional changes induced by Lacidophilin treatment in ulcerative colitis mouse\u003c/h2\u003e \u003cp\u003eTo deeply investigate the underlying mechanisms responsible for Lacidophilin on ulcerative colitis, the transcriptome was conducted. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, we found that the distribution of samples from the model group and the controls were clearly separated by applying principal component analysis (PCA), so did the control group and LPH group. Then, we defined 1393 significantly differentially expressed genes by setting absolute fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as thresholds, in which 687 upregulated genes and 706 downregulated genes were identified in the model group relative to control. As for the LPH group versus the models, 67 significantly differentially expressed were determined, among which 63 genes were downregulated and 4 genes were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The Venn diagram shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC indicated that there occurred significant discrepancy of 50 genes between control vs model group and model vs LPH group. Based on these differentially expressed genes (DEGs), a clustering analysis was performed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, the control and LPH groups showed similar trends in DEGs, while those in the model group showed the reverse trend.\u003c/p\u003e \u003cp\u003eThen, to gain a better understanding of how Lacidophilin effect on UC, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis showed that these 50 genes were associated with immune system (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Further KEGG pathway enrichment analysis of identified DEGs was performed, and the 20 most significantly enriched pathways were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF. Remarkably, cytokine-cytokine receptor interaction、IL-17 signaling pathway、complement and coagulation cascades、B cell receptor signaling pathway、Toll-like receptor signaling pathway and TNF signaling pathway were clearly enriched. Moreover, these 50 DEGs were used for the proteinprotein interactions network analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). We noticed greater degree and more edge connections in the nodes of Ccl3, Lcn2, SPP1 and Serpine1, which involved in immune functions, indicating greater importance in these protein in the network.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, mice were induced acute ulcerative colitis by DSS, while Lacidophilin was administered for 7 days. We observed that Lacidophilin can prevent the changes of the body weight, colonic length, serum inflammatory factor levels, and histopathological damage in colitis mice effectively. In addition, tight junctions and mucin showed enhanced expression following Lacidophilin treatment compared to controls.\u003c/p\u003e \u003cp\u003eThen we further investigated the action mechanism of Lacidophilin in alleviating colitis. Patients with IBD suffer from dysbacteriosis which are defined as decreased diversity and abundance of gut microbial, with an imbalance between commensal and potentially pathogenic microorganisms \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. We performed 16S ribosomal RNA tagged sequencing to identify alterations of intestinal microflora. At the phylum level, compared with the control group, the abundance of Bacteroidetes and Proteobacteria was decreased in the model group, whereas that Actinobacteriota of increased. However, Lacidophilin administration reversed this trend, aligning with the research of Zhu\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e and Zhou\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In addition, we revealed that \u003cem\u003eLactobacillus、Dubosiella、Lachnospiraceae_NK4A136_group、Bifidobacterium、norank-f-norank-o-RF39、norank-f-Lachnospiraceae\u003c/em\u003e and \u003cem\u003enorank_f_norank_o_Gastranaerophilales\u003c/em\u003e was significantly increased in UC mice with treatment of Lacidophilin. \u003cem\u003eLactobacillus\u003c/em\u003e、\u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e have potential benefits in improving IBD due to their ability to produce SCFA\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Moreover, it had been reported that \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e were capable of maintaining epithelial health and immune balance of the intestine\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eToll-like receptors (TLRs) activate inflammatory cells and release pro-inflammatory cytokines, which are the key mediators of inflammatory routes in the intestine \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. It has been shown that norank-f-norank-o-RF39 were negatively correlated with TLR2 receptor expression and proinflammatory factors IL-4 and IL-17 levels, but positively correlated with anti-inflammatory factors IL-10 levels\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, which means these intestinal flora might be beneficial to attenuate intestinal inflammation of DSS colitis. Dubosiella thought to be improved colitis symptoms have been explored in several studies\u003csup\u003e[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, some contrary research was also observed with more prominent relative abundance in colitis mice than the normal ones \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, which is inconsistent with our results. Hence a further study of Dubosiella function on colitis in vivo will be required in vivo. Conversely, the abundance of \u003cem\u003eEscherchia-Shigella\u003c/em\u003e and \u003cem\u003eParvibacter\u003c/em\u003e was significantly decreased in the LPH group than in UC mice. An increase in the abundance of \u003cem\u003eEscherchia-Shigella\u003c/em\u003e was observed in the mice accompanied with aggravating DSS-induced colitis\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Moreover, it is demonstrated that \u003cem\u003eParvibacter\u003c/em\u003e might have potential benefit effects on colitis cause its positive correlation with IL-10\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, whereas, some research had demonstrated that DSS induced a significant reduction of \u003cem\u003eParvibacter\u003c/em\u003e in mice with colitis \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Therefore, whether it is beneficial for colitis remains further evidence. Taken together, Lacidophilin might restrain the growth of potentially harmful bacteria and enrich potentially beneficial bacteria to improve DSS-induced ulcerative colitis.\u003c/p\u003e \u003cp\u003eTo deeply investigate the molecular mechanism of Lacidophilin to UC, transcriptome of mice colon was performed after high-dose Lacidophilin treatment. In the present study, 50 significantly changed genes were observed in Control versus Model and Model versus LPH groups. It is worth mentioning that immune system dominated KEGG pathway, suggesting its mechanism was mainly correlated with immunity. The IL-17 signaling pathway which could mediate the promotion of immune response and activation of various inflammatory pathways\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, was originally described in the autoimmune disease, and it has been a drug target for many autoimmune and chronic inflammatory diseases. There is evidence for IL-17 signaling pathway in promoting inflammation that damages the gut mucosa, while it also plays protective roles in regulating intestinal flora\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. The B cell receptor (BCR) signaling pathway is a key signaling pathway for the development and maturation of B cells\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Most studies in the field of B cell receptor signaling pathway have only focused on lymphoma\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, but there was little research on B-cell receptor pathways and UC. Thus, our experiment might provide more evidence for the relationship between the B-cell receptor pathway and UC. Moreover, following our research, Toll-like receptor signaling pathway and TNF signaling pathway which were involved in driving inflammation might be valuable for the molecular mechanisms of Lacidophilin improving UC.\u003c/p\u003e \u003cp\u003ePPI network have demonstrated that the protein, Lcn2, Ccl3, Mmp8, Slc11a1, Spp1 and Serpine1, are involved in mediating multiple pathways to improve DSS-induced intestinal injury. Lipocalin 2 (Lcn2), a multifunctional immune protein, was closely related to the intestinal inflammation. Reports are rather controversial, and there is no general agreement about whether Lcn2 mediate anti-inflammatory or pro-inflammatory functions. Researchers reported that Lcn2 released in a partial MyD88-dependent manner, and UC would be aggravated significantly in Lcn2 knockout mice\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Lcn2 deletion could augment pro-inflammatory response in the model challenged with LPS\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. However, other authors question the function of Lcn2, who suggested that Lcn2 with the activation of NF-κB pathway while enhancing inflammasome assembly and IL-1β secretion, could lead to more severe inflammation\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Our results show that an elevated level of Lcn2 in colon tissue of mice with UC, while it declined in mice administrated with Lacidophilin, consistent with increase of intestinal mucosal Lcn2 in patients with IBD\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNotably, Ccl3, belongs to chemokines family, which could induce macrophages and granulocytes migration to sites of acute inflammation\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e, has up-regulated during intestinal inflammation, while oral administration of Lacidophilin could significantly reverse its increase. Consistent with our findings, several studies have shown a strongly up-regulation of Ccl3 expression was observed in IL-10\u003csup\u003e-/-\u003c/sup\u003e mice \u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e, TNBS model\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e, as well as DSS model\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e, all of which are known as common animal models for IBD. It would seem to suggest that Ccl3 was a possible biomarkers and potential therapeutic target for IBD, and our results might provide a rationale for further studies.\u003c/p\u003e \u003cp\u003eIt has been accepted that regulated expression of matrix metalloproteinases (MMPs) plays multifaced roles in the pathogenesis of IBD. Previous findings showing that MMP8, which is predominantly expressed by macrophages, led to increased inflammatory cells infiltration after mice exposure to DSS\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e, are compatible with our results. The latest research pointed to a significant role of the Slc11a1 gene (formerly NRAMP1) on the DSS-induced colitis phenotype\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. In addition, a strong relationship between the secreted phosphoprotein 1 (Spp1) and CD susceptibility was observed\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. The Serpine1 encoding a protein called plasminogen activator inhibitor-1 (PAI-1) which can promote peripheral angiogenesis\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e, was elevated during the disease activity cycle in the inflamed colon, contributing to an aggravation of mucosal damage in colitis \u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTaken together, our findings suggest the role of Lacidophilin in alleviating DSS-induced colitis, identifying the enhancement of colon barrier integrity and improvement of intestinal microflora as a possible action mechanism. Lacidophilin could drive the immune system, reduce the expression of inflammatory cytokines, and decrease the protein expression of Lcn2, Ccl3, Mmp8, Slc11a1, Spp1 and Serpine1, which were associated with immune response or neo-angiogenesis. However, this study was limited by the absence the experimental methods of polymerase chain reaction (PCR) and western blot (WB) to verify its changes at molecular and protein levels, and a more precise mechanism how Lacidophilin mediate immune system in colitis remains to be elucidated. In spite of its limitations, this work offers guidance on future research and clinical application of postbiotics to improve acute ulcerative colitis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics Statement\u003c/p\u003e\n\u003cp\u003eThe animal study was reviewed and approved by the Experimental Animal Ethics Committee of Jiangzhong Pharmaceutical Co., Ltd. The Ethical Commitee protocol number was 20220407. The reporting in this study follows the recommendations in the ARRIVE guidelines. We have strictly abided by the international animal welfare and ethical standards, carry out the relevant laws, regulations and policies on the management of laboratory animals, and care laboratory animals. \u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eSequence data that support the findings of this study have been deposited in the National Center for Biotechnology Information Archive with the link of http://www.ncbi.nlm.nih.gov/bioproject/1130646 and the China National Gene Bank Nucleotide Sequence Archive with the link of http://db.cngb.org/cnsa/project/CNP0005936_92303499/reviewlink/.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by Nanchang City High-level Scientific and Technological Innovation Talents \u0026ldquo;Double Hundred Plan\u0026rdquo; (Grant No. [2022]321).\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eWJ-L conceived and designed the experiments. JT-Y, XY-C and ZY performed the experiments. WJ-L, YM-L, LJ-Z contributed reagents/materials/analytical tools. JT-Y, JW-Z and DL-S analyzed the data. JT-Y wrote the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePanaccione R, Lee WJ, Clark R, et al. Dose escalation patterns of advanced therapies in crohn\u0026apos;s disease and ulcerative colitis: A systematic literature review [J]. Adv Ther, 2023,40(5):2051-2081. DOI:10.1007/s12325-023-02457-6.\u003c/li\u003e\n\u003cli\u003eFeuerstein JD, Cheifetz AS. Ulcerative colitis: Epidemiology, diagnosis, and management [J]. Mayo Clin Proc, 2014,89(11):1553-1563. DOI:10.1016/j.mayocp.2014.07.002.\u003c/li\u003e\n\u003cli\u003eFeuerstein JD, Cheifetz AS. Crohn disease: Epidemiology, diagnosis, and management [J]. Mayo Clin Proc, 2017,92(7):1088-1103. DOI:10.1016/j.mayocp.2017.04.010.\u003c/li\u003e\n\u003cli\u003eAlatab S, Sepanlou S, Ikuta KS, et al. The global, regional, and national burden of inflammatory bowel disease in 195 countries and territories, 1990\u0026ndash;2017: A systematic analysis for the global burden of disease study 2017 [J]. The Lancet Gastroenterology \u0026amp; Hepatology, 2020,5(1):17-30.\u003c/li\u003e\n\u003cli\u003eSaniabadi AR, Tanaka T, Ohmori T, et al. Treating inflammatory bowel disease by adsorptive leucocytapheresis: A desire to treat without drugs [J]. World J Gastroenterol, 2014,20(29):9699-9715. DOI:10.3748/wjg.v20.i29.9699.\u003c/li\u003e\n\u003cli\u003eChen Z, Jin W, Hoover A, et al. Decoding the microbiome: Advances in genetic manipulation for gut bacteria [J]. Trends Microbiol, 2023. DOI:10.1016/j.tim.2023.05.007.\u003c/li\u003e\n\u003cli\u003eKnights D, Lassen KG, Xavier RJ. Advances in inflammatory bowel disease pathogenesis: Linking host genetics and the microbiome [J]. Gut, 2013,62(10):1505-1510. DOI:10.1136/gutjnl-2012-303954.\u003c/li\u003e\n\u003cli\u003eImhann F, Vich Vila A, Bonder MJ, et al. Interplay of host genetics and gut microbiota underlying the onset and clinical presentation of inflammatory bowel disease [J]. Gut, 2018,67(1):108-119. DOI:10.1136/gutjnl-2016-312135.\u003c/li\u003e\n\u003cli\u003eRen DD, Chen KC, Li SS, et al. Panax quinquefolius polysaccharides ameliorate ulcerative colitis in mice induced by dextran sulfate sodium [J]. Front Immunol, 2023,14(1161625. DOI:10.3389/fimmu.2023.1161625.\u003c/li\u003e\n\u003cli\u003eHarnack C, Berger H, Liu L, et al. Short-term mucosal disruption enables colibactin-producing e. Coli to cause long-term perturbation of colonic homeostasis [J]. Gut Microbes, 2023,15(1):2233689. DOI:10.1080/19490976.2023.2233689.\u003c/li\u003e\n\u003cli\u003eChang PV, Hao L, Offermanns S, et al. The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition [J]. Proc Natl Acad Sci U S A, 2014,111(6):2247-2252. DOI:10.1073/pnas.1322269111.\u003c/li\u003e\n\u003cli\u003eYang W, Yu T, Huang X, et al. Intestinal microbiota-derived short-chain fatty acids regulation of immune cell il-22 production and gut immunity [J]. Nat Commun, 2020,11(1):4457. DOI:10.1038/s41467-020-18262-6.\u003c/li\u003e\n\u003cli\u003eMacia L, Tan J, Vieira AT, et al. Metabolite-sensing receptors gpr43 and gpr109a facilitate dietary fibre-induced gut homeostasis through regulation of the inflammasome [J]. Nat Commun, 2015,6(6734. DOI:10.1038/ncomms7734.\u003c/li\u003e\n\u003cli\u003eKwak SH, Cho YM, Noh GM, et al. Cancer preventive potential of kimchi lactic acid bacteria (weissella cibaria, lactobacillus plantarum) [J]. J Cancer Prev, 2014,19(4):253-258. DOI:10.15430/JCP.2014.19.4.253.\u003c/li\u003e\n\u003cli\u003ePark JS, Joe I, Rhee PD, et al. A lactic acid bacterium isolated from kimchi ameliorates intestinal inflammation in dss-induced colitis [J]. J Microbiol, 2017,55(4):304-310. DOI:10.1007/s12275-017-6447-y.\u003c/li\u003e\n\u003cli\u003eWang MX, Lin L, Chen YD, et al. Evodiamine has therapeutic efficacy in ulcerative colitis by increasing lactobacillus acidophilus levels and acetate production [J]. Pharmacol Res, 2020,159(104978. DOI:10.1016/j.phrs.2020.104978.\u003c/li\u003e\n\u003cli\u003eKye YJ, Lee SY, Kim HR, et al. Lactobacillus acidophilus pin7 paraprobiotic supplementation ameliorates dss-induced colitis through anti-inflammatory and immune regulatory effects [J]. J Appl Microbiol, 2022,132(4):3189-3200. DOI:10.1111/jam.15406.\u003c/li\u003e\n\u003cli\u003eHuang Z, Gong L, Jin Y, et al. Different effects of different lactobacillus acidophilus strains on dss-induced colitis [J]. Int J Mol Sci, 2022,23(23). DOI:10.3390/ijms232314841.\u003c/li\u003e\n\u003cli\u003eKim WK, Han DH, Jang YJ, et al. Alleviation of dss-induced colitis via lactobacillus acidophilus treatment in mice [J]. Food Funct, 2021,12(1):340-350. DOI:10.1039/d0fo01724h.\u003c/li\u003e\n\u003cli\u003eKullar R, Goldstein EJC, Johnson S, et al. Lactobacillus bacteremia and probiotics: A review [J]. Microorganisms, 2023,11(4). DOI:10.3390/microorganisms11040896.\u003c/li\u003e\n\u003cli\u003eVahabnezhad E, Mochon AB, Wozniak LJ, et al. Lactobacillus bacteremia associated with probiotic use in a pediatric patient with ulcerative colitis [J]. J Clin Gastroenterol, 2013,47(5):437-439. DOI:10.1097/MCG.0b013e318279abf0.\u003c/li\u003e\n\u003cli\u003eMeini S, Laureano R, Fani L, et al. Breakthrough lactobacillus rhamnosus gg bacteremia associated with probiotic use in an adult patient with severe active ulcerative colitis: Case report and review of the literature [J]. Infection, 2015,43(6):777-781. DOI:10.1007/s15010-015-0798-2.\u003c/li\u003e\n\u003cli\u003eOkayasu I, Hatakeyama S, Yamada M, et al. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice [J]. Gastroenterology, 1990,98(3):694-702. DOI:10.1016/0016-5085(90)90290-h.\u003c/li\u003e\n\u003cli\u003eVerbeke KA, Boesmans L, Boets E. Modulating the microbiota in inflammatory bowel diseases: Prebiotics, probiotics or faecal transplantation? [J]. Proc Nutr Soc, 2014,73(4):490-497. DOI:10.1017/S0029665114000639.\u003c/li\u003e\n\u003cli\u003eZhu Y, Zhao Q, Huang Q, et al. Nuciferine regulates immune function and gut microbiota in dss-induced ulcerative colitis [J]. Front Vet Sci, 2022,9(939377. DOI:10.3389/fvets.2022.939377.\u003c/li\u003e\n\u003cli\u003eZhou Y, Xu ZZ, He Y, et al. Gut microbiota offers universal biomarkers across ethnicity in inflammatory bowel disease diagnosis and infliximab response prediction [J]. mSystems, 2018,3(1). DOI:10.1128/mSystems.00188-17.\u003c/li\u003e\n\u003cli\u003eWang K, Qin L, Cao J, et al. Kappa-selenocarrageenan oligosaccharides prepared by deep-sea enzyme alleviate inflammatory responses and modulate gut microbiota in ulcerative colitis mice [J]. Int J Mol Sci, 2023,24(5). DOI:10.3390/ijms24054672.\u003c/li\u003e\n\u003cli\u003eAsadzadeh Aghdaei H, Rezasoltani S, Olfatifar M, et al. Expression of toll-like receptors 2, 4 and 5 in relation to gut microbiota in colon neoplasm patients with and without inflammatory bowel disease [J]. Avicenna J Med Biotechnol, 2022,14(3):188-195. DOI:10.18502/ajmb.v14i3.9825.\u003c/li\u003e\n\u003cli\u003eHonjo H, Watanabe T, Kamata K, et al. Ripk2 as a new therapeutic target in inflammatory bowel diseases [J]. Front Pharmacol, 2021,12(650403. DOI:10.3389/fphar.2021.650403.\u003c/li\u003e\n\u003cli\u003eJia W, Xu C, Zhao T, et al. Integrated network pharmacology and gut microbiota analysis to explore the mechanism of sijunzi decoction involved in alleviating airway inflammation in a mouse model of asthma [J]. Evid Based Complement Alternat Med, 2023,2023(1130893. DOI:10.1155/2023/1130893.\u003c/li\u003e\n\u003cli\u003eZhai Z, Zhang F, Cao R, et al. Cecropin a alleviates inflammation through modulating the gut microbiota of c57bl/6 mice with dss-induced ibd [J]. Front Microbiol, 2019,10(1595. DOI:10.3389/fmicb.2019.01595.\u003c/li\u003e\n\u003cli\u003eWan F, Wang M, Zhong R, et al. Supplementation with chinese medicinal plant extracts from lonicera hypoglauca and scutellaria baicalensis mitigates colonic inflammation by regulating oxidative stress and gut microbiota in a colitis mouse model [J]. Front Cell Infect Microbiol, 2021,11(798052. DOI:10.3389/fcimb.2021.798052.\u003c/li\u003e\n\u003cli\u003eWan F, Zhong R, Wang M, et al. Caffeic acid supplement alleviates colonic inflammation and oxidative stress potentially through improved gut microbiota community in mice [J]. Front Microbiol, 2021,12(784211. DOI:10.3389/fmicb.2021.784211.\u003c/li\u003e\n\u003cli\u003eFu YP, Li CY, Peng X, et al. Pectic polysaccharides from aconitum carmichaelii leaves protects against dss-induced ulcerative colitis in mice through modulations of metabolism and microbiota composition [J]. Biomed Pharmacother, 2022,155(113767. DOI:10.1016/j.biopha.2022.113767.\u003c/li\u003e\n\u003cli\u003eLi Q, Chen G, Zhu D, et al. Effects of dietary phosphatidylcholine and sphingomyelin on dss-induced colitis by regulating metabolism and gut microbiota in mice [J]. J Nutr Biochem, 2022,105(109004. DOI:10.1016/j.jnutbio.2022.109004.\u003c/li\u003e\n\u003cli\u003eHuang H, Li M, Wang Y, et al. Excessive intake of longan arillus alters gut homeostasis and aggravates colitis in mice [J]. Front Pharmacol, 2021,12(640417. DOI:10.3389/fphar.2021.640417.\u003c/li\u003e\n\u003cli\u003eWang SL, Shao BZ, Zhao SB, et al. Intestinal autophagy links psychosocial stress with gut microbiota to promote inflammatory bowel disease [J]. Cell Death Dis, 2019,10(6):391. DOI:10.1038/s41419-019-1634-x.\u003c/li\u003e\n\u003cli\u003eGe W, Zhou BG, Zhong YB, et al. Sishen pill ameliorates dextran sulfate sodium (dss)-induced colitis with spleen-kidney yang deficiency syndromes: Role of gut microbiota, fecal metabolites, inflammatory dendritic cells, and tlr4/nf-kappab pathway [J]. Evid Based Complement Alternat Med, 2022,2022(6132289. DOI:10.1155/2022/6132289.\u003c/li\u003e\n\u003cli\u003eCheng H, Zhang D, Wu J, et al. Atractylodes macrocephala koidz. Volatile oil relieves acute ulcerative colitis via regulating gut microbiota and gut microbiota metabolism [J]. Front Immunol, 2023,14(1127785. DOI:10.3389/fimmu.2023.1127785.\u003c/li\u003e\n\u003cli\u003eBianchi E, Vecellio M, Rogge L. Editorial: Role of the il-23/il-17 pathway in chronic immune-mediated inflammatory diseases: Mechanisms and targeted therapies [J]. Front Immunol, 2021,12(770275. DOI:10.3389/fimmu.2021.770275.\u003c/li\u003e\n\u003cli\u003eZhang HJ, Xu B, Wang H, et al. Il-17 is a protection effector against the adherent-invasive escherichia coli in murine colitis [J]. Mol Immunol, 2018,93(166-172. DOI:10.1016/j.molimm.2017.11.020.\u003c/li\u003e\n\u003cli\u003eChang S, Li B, Xie Y, et al. Dcz0014, a novel compound in the therapy of diffuse large b-cell lymphoma via the b cell receptor signaling pathway [J]. Neoplasia, 2022,24(1):50-61. DOI:10.1016/j.neo.2021.12.001.\u003c/li\u003e\n\u003cli\u003eKrysiak K, Gomez F, White BS, et al. Recurrent somatic mutations affecting b-cell receptor signaling pathway genes in follicular lymphoma [J]. Blood, 2017,129(4):473-483. DOI:10.1182/blood-2016-07-729954.\u003c/li\u003e\n\u003cli\u003eBuchner M, Muschen M. Targeting the b-cell receptor signaling pathway in b lymphoid malignancies [J]. Curr Opin Hematol, 2014,21(4):341-349. DOI:10.1097/MOH.0000000000000048.\u003c/li\u003e\n\u003cli\u003eSingh V, Yeoh BS, Chassaing B, et al. Microbiota-inducible innate immune, siderophore binding protein lipocalin 2 is critical for intestinal homeostasis [J]. Cell Mol Gastroenterol Hepatol, 2016,2(4):482-498 e486. DOI:10.1016/j.jcmgh.2016.03.007.\u003c/li\u003e\n\u003cli\u003eDu H, Liang L, Li J, et al. Lipocalin-2 alleviates lps-induced inflammation through alteration of macrophage properties [J]. J Inflamm Res, 2021,14(4189-4203. DOI:10.2147/JIR.S328916.\u003c/li\u003e\n\u003cli\u003eKim SL, Shin MW, Kim SW. Lipocalin 2 activates the nlrp3 inflammasome via lps‑induced nf‑\u0026kappa;b signaling and plays a role as a pro‑inflammatory regulator in murine macrophages [J]. Mol Med Rep, 2022,26(6). DOI:10.3892/mmr.2022.12875.\u003c/li\u003e\n\u003cli\u003eCarlson M, Raab Y, Seveus L, et al. Human neutrophil lipocalin is a unique marker of neutrophil inflammation in ulcerative colitis and proctitis [J]. Gut, 2002,50(4):501-506. DOI:10.1136/gut.50.4.501.\u003c/li\u003e\n\u003cli\u003eTrivedi PJ, Adams DH. Chemokines and chemokine receptors as therapeutic targets in inflammatory bowel disease; pitfalls and promise [J]. J Crohns Colitis, 2018,12(suppl_2):S641-S652. DOI:10.1093/ecco-jcc/jjx145.\u003c/li\u003e\n\u003cli\u003eHagenlocher Y, Hosel A, Bischoff SC, et al. Cinnamon extract reduces symptoms, inflammatory mediators and mast cell markers in murine il-10(-/-) colitis [J]. J Nutr Biochem, 2016,30(85-92. DOI:10.1016/j.jnutbio.2015.11.015.\u003c/li\u003e\n\u003cli\u003ePrados ME, Garcia-Martin A, Unciti-Broceta JD, et al. Betulinic acid hydroxamate prevents colonic inflammation and fibrosis in murine models of inflammatory bowel disease [J]. Acta Pharmacol Sin, 2021,42(7):1124-1138. DOI:10.1038/s41401-020-0497-0.\u003c/li\u003e\n\u003cli\u003eDing P, Li L, Huang T, et al. Complement component 6 deficiency increases susceptibility to dextran sulfate sodium-induced murine colitis [J]. Immunobiology, 2016,221(11):1293-1303. DOI:10.1016/j.imbio.2016.05.014.\u003c/li\u003e\n\u003cli\u003eKoelink PJ, Overbeek SA, Braber S, et al. Collagen degradation and neutrophilic infiltration: A vicious circle in inflammatory bowel disease [J]. Gut, 2014,63(4):578-587. DOI:10.1136/gutjnl-2012-303252.\u003c/li\u003e\n\u003cli\u003ede Andrade STQ, Guidugli TI, Borrego A, et al. Slc11a1 gene polymorphism influences dextran sulfate sodium (dss)-induced colitis in a murine model of acute inflammation [J]. Genes Immun, 2023,24(2):71-80. DOI:10.1038/s41435-023-00199-7.\u003c/li\u003e\n\u003cli\u003eGlas J, Seiderer J, Bayrle C, et al. The role of osteopontin (opn/spp1) haplotypes in the susceptibility to crohn\u0026apos;s disease [J]. PLoS One, 2011,6(12):e29309. DOI:10.1371/journal.pone.0029309.\u003c/li\u003e\n\u003cli\u003eDuan ZL, Wang YJ, Lu ZH, et al. Wumei wan attenuates angiogenesis and inflammation by modulating rage signaling pathway in ibd: Network pharmacology analysis and experimental evidence [J]. Phytomedicine, 2023,111(154658. DOI:10.1016/j.phymed.2023.154658.\u003c/li\u003e\n\u003cli\u003eKaiko GE, Chen F, Lai CW, et al. Pai-1 augments mucosal damage in colitis [J]. Sci Transl Med, 2019,11(482). DOI:10.1126/scitranslmed.aat0852.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"IBD, Lacidophilin, DSS, microbiota, transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-4684193/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4684193/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe prevalence of inflammatory bowel disease (IBD) has been rising significantly in recent years. It is widely accepted that gut microbes play an essential role in the development of IBD. Lacidophilin is a product of milk fermentation by lactobacillus acidophilus. The aim of this study was to investigate the effect of Lacidophilin on colitis induced by dextran sulfate sodium (DSS). 16s RNA sequencing was performed to determine the changes of species composition and community structure of the intestinal microflora, and transcriptome sequencing was conducted to find out the gene or protein which may be affected by Lactobacillus on colitis development potentially. It was observed that the 7 days administration of Lacidophilin protected the intestinal mucosal barrier from damage, and thereby enabled the remission of colitis severity. Compared to the model group, Lacidophilin could restore the shortened colon length and marked decrease levels of TNF-α and IL-6 in serum. More importantly, Lacidophilin significantly increased the abundance of beneficial bacteria such as \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e, decreased the abundance of harmful bacteria such as \u003cem\u003eEscherichia-Shigella\u003c/em\u003e and Parvibacter. Transcriptomic analysis shows that IL-17 signaling pathway, BCR signaling pathway, Toll-like receptor signaling pathway, and TNF signaling pathway was enriched, and we found that Lcn2, Ccl3, Mmp8, Slc11a1, Spp1, and Serpine1 might be potential targets of Lacidophilin treatment. These studies indicate that Lacidophilin can ameliorate colitis in mice through maintaining the integrity of intestinal structure and improving intestinal microbiota, and its mechanism may be involved in immune-related proteins and pathways.\u003c/p\u003e","manuscriptTitle":"Lacidophilin Modulated Gut Microbiota and Ameliorated Dextran Sulfate Sodium-Induced Mouse Colitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 10:07:16","doi":"10.21203/rs.3.rs-4684193/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-13T08:11:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-12T20:43:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-11T08:47:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228697074651434036625017166029908180575","date":"2024-08-03T09:31:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213261408936816616290478196300448983788","date":"2024-08-02T06:58:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-01T12:39:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-27T12:16:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-22T16:40:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-17T09:08:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-04T06:26:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b22e1fad-230d-4ea4-ae9f-43fc717a7456","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-09-24T08:23:55+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-09 10:07:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4684193","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4684193","identity":"rs-4684193","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}