Hyperammonemia leads to intestinal barrier function impairment and gut microbiota dysbiosis associated with PERK signaling pathway-induced endoplasmic reticulum stress

Hyperammonemia leads to intestinal barrier function impairment and gut microbiota dysbiosis associated with PERK signaling pathway-induced endoplasmic reticulum stress | 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 Short Report Hyperammonemia leads to intestinal barrier function impairment and gut microbiota dysbiosis associated with PERK signaling pathway-induced endoplasmic reticulum stress Hui Su, Ming Zhuang, He Wei, Shu-qin Ren, Si-jing Han, Li-ping Zhou, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6114564/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background/Objective: L-Ornithine L-aspartate (LOLA) can effectively alleviate hepatic encephalopathy by reducing blood ammonia levels. Recent studies have shown that amino acids can affect via the gut‒liver axis has a significant effect on the progression of various liver diseases, but the effects of LOLA on the intestine have not been reported. Methods Rats were given intraperitoneal injections of 0.5% N‒E dimethylnitrosamine (DMN) at 10 mg/kg while being simultaneously administered LOLA (1000 mg/kg) via gavage three times per week for 4 weeks, after which the animals were euthanized. Serum was collected to assess liver function. Rat feces were collected for 16S rRNA sequencing of the microbiota, and biochemical and histological evaluations of ileal damage were performed.The expression of tight junction proteins (ZO-1), lysozyme (Lyz), and endoplasmic reticulum stress (ER) pathway-related markers were detected in the rat ileal epithelium. Results LOLA gavage did not significantly restore gut microbiota diversity or the abundance of dominant intestinal bacteria such as Prevotella, but it significantly reduced the abundance of harmful bacteria such as Clostridium. Additionally, LOLA gavage increased ZO-1 expression in the rat small intestinal mucosa, reduced plasma DAO and LPS levels, and restored Lyz secretion. Furthermore, the expression of ER stress pathway-related markers (BIP, p-PERK, p-eIF2a, ATF4, and CHOP) decreased after LOLA gavage. Conclusion LOLA may ameliorate gut microbiota dysbiosis in cirrhotic rats by affecting the ER stress response and restoring intestinal barrier function, revealing a new mechanism by which LOLA controls liver disease. LOLA cirrhosis intestinal barrier gut microbiota endoplasmic reticulum stress hyperammonemia Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cirrhosis is a chronic and irreversible disease caused by various types of liver injury characterized by persistent inflammation, necrosis, and fibrous tissue proliferation, leading to end-stage liver failure. This pathological process is characterized by the replacement of normal liver cell structure with extensive nodular hyperplasia and dense fibrous septa, resulting in liver structure atrophy and vascular compression. These changes increase portal vein blood flow resistance, leading to portal hypertension and liver dysfunction. Cirrhosis is one of the leading causes of death worldwide, with high mortality rates in the United States, Europe, and China. Moreover, cirrhosis can damage the intestinal mucosa, weakening its barrier function. Owing to their anatomical proximity, the intestine and liver are closely connected, with intestinal blood flowing directly into the liver through the portal vein, exposing the liver to microorganisms and their metabolites from the intestine. Clinical observations have shown that as portal vein pressure increases in cirrhosis patients, the amount of bacterial products in the systemic circulation also increases. Portal hypertension leads to gut‒liver axis dysfunction, and as liver disease progresses, gut microbiota imbalance further worsens, increasing the entry of harmful bacteria and their metabolites into the bloodstream, which further activates the liver immune response, exacerbating fibrosis and portal hypertension. L-Ornithine L-aspartate (LOLA) consists of aspartic acid and ornithine. These groups directly participate in hepatocyte metabolism and activate ornithine carbamoyltransferase and carbamoyl phosphate synthetase to facilitate liver detoxification function. These enzymes can remove free radicals and reduce the blood ammonia concentration. Although LOLA has been used to treat cirrhosis and acute liver injury, it is unclear whether LOLA enhances the intestinal mucosal barrier function in patients with cirrhosis. Our research revealed that in a cirrhotic rat model, LOLA ameliorated intestinal ER stress, supported the repair of the intestinal mucosal barrier, and reduced the number of harmful intestinal bacteria. These results provide a new mechanism for the alleviation of cirrhosis by LOLA. Materials and Methods Animals Twenty-four 6-week-old male Sprague–Dawley (SD) rats weighing 200 ± 15 g were housed in a pathogen-free animal room at the Animal Care Center of the First Affiliated Hospital of Chengdu Medical College. The temperature was controlled at 24 ± 2°C, the average humidity was 55 ± 5.0%, and the light:dark cycle was 12:12 h. All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Chengdu Medical College. After 5 days of adaptive feeding, the rats were randomly divided into 3 groups: the cirrhosis group, LOAL group, and control group. For the rats in the cirrhosis group (n = 8), 0.5% DMN (McLean Biochemical Technology Co., Ltd., Shanghai, China) was injected intraperitoneally at a dose of 10 mg/kg for four consecutive weeks (Monday, Tuesday, Wednesday), with saline gavage of the same volume as the LOLA group. For the rats in the LOLA group (n = 8), DMN was injected intraperitoneally at 10 mg/kg for four consecutive weeks (Monday, Tuesday, Wednesday), while 1000 mg/kg LOLA (Qirui Pharmaceutical Co., Ltd., Wuhan, China) diluted 10% with saline was simultaneously administered via gavage. For the rats in the control group (n = 8), saline gavage and intraperitoneal injection were administered according to each rat's body weight. The rats were fed normally at other times. On day 21 of the experiment, liver samples were taken after the rats in all groups were euthanized to observe liver damage. Blood Tests On day 21 of the experiment, blood samples were taken from the hearts of rats in the cirrhosis, LOLA, and control groups after euthanasia. ELISA kits were used to determine the serum levels of DAO and lipopolysaccharide (LPS) in each sample (Camilo Biological Engineering Company). Western Blotting Western Blotting On day 21 of the experiment, ileum tissue was collected from rats in the model, treatment, and control groups after euthanasia. Ileum samples were homogenized, and the proteins were separated via 12% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis. After blotting, polyclonal antibodies against ZO-1, p-PERK, and p-eIF2α (Affinity Biosciences); BIP (Bioaosen Biotechnology Company); and ATF4 and CHOP (Santa Cruz Biotechnology) were used. Relative protein expression levels were normalized to those of β-actin on the basis of the optical density of the protein bands and quantified via ImageJ (NIH) software. H&E Staining and Immunohistochemistry (IHC) The excised ileum was immediately fixed in 10% neutral buffered formalin, paraffin embedded, and cut into 5 µm sections. After deparaffinization with xylene and ethanol, the sections were stained with hematoxylin‒eosin (H&E) and oil red O for morphological evaluation under a microscope. Ileum tissues were subjected to paraffin embedding, sectioning, antigen retrieval, and immunostaining. Specific antibodies against Lyz, ZO-1, BIP, p-PERK, p-eIF2α, ATF4, and CHOP were used. The signals for DAB (antigen-positive expression) and hematoxylin (nuclear staining) were captured under a microscope (NIKON DS-U3) and processed via Image Pro Plus 6.0 software (CAD/CAM Services Inc.). Immunofluorescence The ileum was fixed with paraformaldehyde, cryosectioned, washed 3 times with 1x PBS, and blocked with 0.3% Triton X-100 and 5% goat serum (#SL038, Solarbio) for 2 hours. The blocked sections were washed 3 times with 1x PBS, and primary antibodies targeting BIP (1:100), p-PERK (1:100), p-eIF2α (1:100), ATF4 (1:100), and CHOP (1:100) were incubated overnight at 4°C, followed by incubation with the secondary antibodies AlexaFluor488-labeled goat anti-rabbit IgG (H + L) (#ZF-0511, ZSGB-BIO) and AlexaFluor594-labeled goat anti-mouse IgG (H + L) (#ZF-0513, ZSGB-BIO). The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Images were captured via a Leica TCS SP8 confocal microscope (Leica Microsystems, Germany). 16S rRNA Data Analysis The raw sequencing data for all the samples were deposited in the NCBI Sequence Read Archive database (accession number: PRJNA540574). QIIME2 (v2018.11.4) was used for quality filtering of the raw reads. Noisy sequencing data, including erroneous barcodes, chimeras, and low-quality sequences, were excluded. Clean data were clustered into operational taxonomic units (OTUs) at a 97% threshold. OTU screening was performed against the Greengenes database (release 13.8), filtering out rare OTUs (≤ 0.001%). The α and β diversities were calculated on the basis of the relative abundance of OTUs via the QIIME and MicrobiomeAnalyst platforms, including principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS). Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify differential taxonomies. Additionally, redundancy analysis (RDA) was performed via the R vegan package on the basis of different key clinical factors. Statistical Analysis SPSS 21.0 software was used. Data conforming to a normal distribution are expressed as the mean ± standard deviation and were analyzed by one-way ANOVA. Data conforming to a nonnormal distribution are expressed as the median (p25, p75) and were analyzed via the H test. P < 0.05 was considered to indicate statistical significance. ImageJ software was used for semiquantitative statistical analysis of the immunohistochemistry data and images of other sections, which are expressed as the average optical density (AOD). For all the fecal gut microbiota sequencing data, rarefaction curves and hierarchical clustering curves were plotted via R language on the basis of the OTU information from each group of samples. The Chao1 index and ACE index were calculated via amplicon analysis software (QIIME) to compare intragroup microbial diversity. Principal component analysis (PCA) and PCoA were performed via R language to compare intergroup microbial differences. The multiresponse permutation procedure (MRPP) test was used to compare microbial differences between groups. Results 1.LOLA reduce ammonia and liver injury, does not improve liver index The liver index values and levels of ammonia, aspartate transaminase (AST), alanine aminotransferase (ALT), and other related indicators of the rats in each group were not significantly different in terms of the overall distributions of the three groups (p > 0.05). The cirrhosis group rats had significantly elevated levels of blood ammonia, AST, and ALT. In the LOLA group, the levels of blood ammonia, AST, and ALT significantly decreased but were still notably greater than those in the control group(shown in Table.1). The liver cells of the cirrhosis group rats exhibited point-fragmented necrosis similar to that in the LOLA group, with a large accumulation of inflammatory cells in the necrotic areas (shown in Fig. 1 ). There was no difference in liver fibrosis between the LOLA and the cirrhosis groups (shown in Fig. 2 , 3 ). 2. LOLA alters the composition of the gut microbiota in cirrhotic rats 16S rDNA sequencing of 18 samples revealed a total of 1,424,211 reads, with 864,955 reads remaining after filtering, which were clustered into 1,276 OTUs. The rarefaction curves drawn from the sequencing of the three groups of rat samples tended to plateau, indicating that the sequencing depth met the requirements (shown in Fig. 1 .A). The species abundance curve revealed that the species in each group had good richness and evenness (shown in Fig. 1 .B). The species accumulation box plot shows that the estimated cumulative genus richness approached the asymptotic value, indicating sufficient sampling (shown in Fig. 1 .C). The total number of OTUs in each group and the analysis of unique and shared OTUs between groups are presented in a Venn diagram (shown in Fig. 1 .D). The control group had 1,062 OTUs, the LOLA group had 1,046 OTUs, and the cirrhosis group had 1,021 OTUs. Among these, 792 OTUs were shared by all three groups, while the control group had 72 unique OTUs, the LOLA group had 62 unique OTUs, and the cirrhosis group had 74 unique OTUs (shown in Fig. 1 .D). ACE, Chao1, and observed species indices were used to reflect the diversity of the fecal microbiota in the three groups of rats (shown in Fig. 1 .E-G), suggesting that in the cirrhosis model, the diversity of the rat gut microbiota was impaired (P 0.05). The Shannon index and Simpson index were used to evaluate the evenness and richness of the gut microbiota. The results showed that LOLA ammonia-lowering treatment increased the evenness and richness of the gut microbiota in cirrhotic rats (shown in Fig. 1 .H-I). The MRPP test combined with PCA and PCoA was used for comprehensive analysis of the intergroup differences in the fecal microbiota among the three groups of rats. The results revealed significant differences in the structure of the fecal microbiota among the three groups of rats (shown in Fig. 1 .J‒L and Table 2 ), indicating that there were indeed significant changes in the microbiota in the cirrhotic rat model and that LOLA ammonia-lowering treatment altered the composition of the gut microbiota in cirrhotic rats. Table 1 Biochemical indicators of the rats in each group Group Liver index Ammonia AST ALT Control (n = 7) 2.88(3.01, 3.08) 70.5(45.8, 114.7) 132.0(118.0, 171.0) 43.0(39.0, 54.0) Cirrhosis (n = 7) 2.86(2.68, 3.38) 1151.6(415.9, 1145.1) 318.0(310.0, 507.0) 189.0(181.0, 212.0) LOLA (n = 7) 2.81(2.18, 3.19) 397.4(334.6, 417.6) 189.0(146.0, 240.0) 95.0(83.0, 102.0) H value 0.745 17.818 13.291 17.121 P value P = 0.689 P<0.001 P<0.001 P<0.001 Table 2 MRPP tests Groups θ Observed variables Expected variables P value A-C 0.147 0.5215 0.6114 0.001 A-B 0.1461 0.5372 0.6291 0.004 B-C 0.03333 0.5778 0.5978 0.025 Table 3 Stereological estimates of IDAO and LPS levels in each group DAO(U/ml) LPS(U/ml) Control(n = 7) 60.06 ± 14.79 131.78(115.44, 191.27) Cirrhosis(n = 7) 158.43 ± 30.45 324.34(265.67, 404.46) LOLA(n = 7) 98.34 ± 16.28 162.65(115.44, 255.66) F/H value 36.588 11.208 P value P <0.001 P = 0.004 3. LOLA reduces the relative abundance of harmful bacteria in the gut microbiota of cirrhotic rats Furthermore, we examined the differences in the gut microbiota composition at the phylum and genus levels among the groups. The dominant phyla in all the groups were Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. Blood ammonia levels were significantly elevated in the cirrhosis group and markedly decreased in the LOLA group compared with those in the cirrhosis group but were still significantly greater than those in the control group. In the LOLA group, the abundance of Firmicutes increased significantly, whereas the abundance of Bacteroidetes decreased notably. The abundances of Firmicutes and Proteobacteria in the intestine did not significantly increase. The abundance of Actinobacteria decreased in the gut of cirrhotic model rats but increased somewhat in the LOLA-gavaged rats (shown in Fig. 2 .A‒C). After the composition of the fecal microbiota in each group of rats was determined, the top 10 most abundant genera were ranked as follows: Prevotellaceae_UCG-003; Lactobacillus; Staphylococcus; Alloprevotella; Allobaculum; Bacteroides; Dubosiella; unidentified_[Eubacterium]_coprostanoligenes_group; Clostridium_sensu_stricto_1; and Coriobacteriaceae_UCG-002. The abundance of Prevotella decreased significantly in the cirrhosis group but did not increase notably after LOLA gavage. The abundance of Lactobacillus increased in both the cirrhosis and LOLA groups, with a more pronounced increase in the LOLA group. The abundance of Clostridium increased significantly in the cirrhosis group but decreased markedly in the LOLA group (shown in Fig. 2 .D-E). The LEfSe algorithm was used to identify characteristic bacteria specific to each group and to identify the taxonomic biomarkers with the greatest degree of change in levels between groups (shown in Fig. 2 .F-H). The results revealed that Micrococcus, Prevotella, Helicobacter, and Campylobacter were significantly enriched in the control group; Lactobacillus, Staphylococcus, and Bacillus were significantly enriched in the LOLA group; and Lactobacillus and anaerobic Clostridium were significantly enriched in the cirrhosis group. Notably, the abundance of anaerobic Clostridium increased significantly in the cirrhosis group and decreased markedly after LOLA gavage (shown in Fig. 2 .G). 4. LOLA enhances intestinal barrier function in cirrhotic rats Examination of rat intestinal tissue revealed that the ileal epithelial structure in the control group was continuous and intact, with no significant inflammatory infiltration. The ileal epithelium of the cirrhosis group presented many swollen and detached villi, with extensive infiltration of eosinophils and lymphocytes. Compared with those in the cirrhosis group, the degree of villous structure destruction and eosinophil and lymphocyte infiltration in the ileal epithelium in the LOLA group were significantly lower (shown in Fig. 3 .A). These findings indicate that the ileal epithelial cells of cirrhotic model rats established by intraperitoneal injection of the DMN underwent morphological changes, such as necrosis and inflammatory infiltration, which were significantly improved after LOLA gavage-induced ammonia-lowering treatment. ELISA kits were used to determine the serum DAO and LPS levels in the three groups of rats (shown in Table.3). The serum DAO and LPS levels in the cirrhosis group were significantly elevated, whereas they were decreased markedly in the LOLA group. These results suggest that LOLA enhances the integrity of the intestinal mucosal barrier. To further understand the effect of LOLA on intestinal Paneth cells (the main secretory cells), we examined the ability of ileal Paneth cells to secrete Lyz via IHC. All ileal tissues were observed under 200x magnification, with Paneth cells located at the base of the ileal glands and having a conical shape. Compared with the control group, the cirrhosis group presented significantly reduced Lyz secretion by Paneth cells, whereas the LOLA group presented a notable increase in Lyz secretion (shown in Fig. 3 .B). These findings suggest that Lyz, an important antibacterial substance in the intestine, plays a crucial role in maintaining gut microbiota stability. Moreover, these results indicate that LOLA may regulate the gut microbiota by affecting Lyz secretion. IHC analysis of ileal tissue revealed that the control group presented increased ZO-1 expression, with darker mucosal epithelial cell staining and a brown color. The ileal tissue of the cirrhosis group presented relatively low levels of ZO-1, with relatively weak staining. Compared with that of the cirrhosis group, the ileal tissue of the LOLA group presented increased ZO-1expression, with varying degrees of darker staining (shown in Fig. 3 .C). Compared with that in the blank group, ZO-1 protein expression in the ileal tissue of the cirrhosis group was lower (P < 0.01); compared with that in the cirrhosis group, ZO-1 expression in the ileal tissue of the LOLA group was greater (P < 0.01) (shown in Table 4 ). Table 4 Average optical density (AOD) values determined via immunohistochemistry Lyz(AOD) ZO-1(AOD) Control(n = 7) 162.60 ± 13.34 148.50 ± 13.36 Cirrhosis(n = 7) 99.75 ± 19.72 104.54 ± 11.81 LOLA(n = 7) 134.57 ± 20.22 123.45 ± 11.76 F value 21.336 22.385 P value P <0.001 P <0.001 5. LOLA affects the endoplasmic reticulum stress signaling pathway To investigate the mechanism by which LOLA influences intestinal homeostasis, we examined the expression of the ER stress marker BIP in the ileal mucosa. Compared with that in the cirrhosis group, BIP expression was significantly upregulated in the intestinal mucosa of the cirrhosis group, whereas it was downregulated after LOLA gavage. These results suggest that ER stress in the intestinal mucosa is increased in the cirrhotic rat model and that LOLA can alleviate this stress. The PERK/eIF2α/CHOP pathway is an important cellular stress signaling pathway that is involved mainly in regulating the intestinal mucosal response to internal and external environmental pressures and regulating cell apoptosis. Using Western blotting to measure the PERK protein content, we found that the PERK protein level was significantly increased in the small intestinal epithelial cells of the cirrhosis group, whereas it was decreased in the LOLA group (shown in Fig. 4 .A). IHC and IF methods were used to verify the changes in PERK protein expression in the cirrhosis and LOLA groups. To understand the changes in downstream molecules of the PERK pathway, we measured the changes in p-eIF2α, ATF4, and CHOP protein levels. The results revealed that p-eIF2α, ATF4, and CHOP were significantly upregulated in the cirrhosis group compared with the control group, whereas they were downregulated in the LOLA group (shown in Fig. 4 .B). These results were consistent with the trends in p-eIF2α, ATF4, and CHOP protein expression determined by IHC and IF (shown in Fig. 4 .C). These findings suggest that the effects of LOLA on the gut microbiota and protection of the intestinal mucosal barrier may be related to its regulation of ER stress in intestinal cells. Discussion In this study, using a rat cirrhosis model induced by intraperitoneal DMN injection, we found that LOLA gavage treatment along with DMN injection did not significantly alleviate liver fibrosis or hepatocyte necrosis and inflammatory infiltration but did significantly reduce blood ammonia levels (P < 0.001). LOLA promotes ammonia metabolism in the body by providing a source of aspartic acid and ornithine, thereby lowering blood ammonia levels, and is mainly used to treat hepatic encephalopathy. This study is the first to reveal that LOLA can ameliorate cirrhosis-related intestinal barrier dysfunction. The impact of signaling via the gut‒liver axis on liver diseases has become a research hotspot in recent years. The mechanism of interaction and communication between the intestine and liver involves various biological processes, including the exchange of metabolites, the regulation of immune responses, and the occurrence of inflammatory responses. Our experimental results also revealed that LOLA can increase Lys secretion in the intestinal mucosa of cirrhotic rats, affect the gut microbiota, and enhance intestinal mucosal tight junctions, leading to improved intestinal mucosal barrier function. Intestinal mucosal cells secrete active substances such as antimicrobial peptides (AMPs) and Lyz into the intestinal lumen through degranulation. These active substances are important components of the intestinal immune barrier and can effectively inhibit excessive growth of intestinal bacteria, maintaining the balance of the gut microbiota [ 16 ]. In the cirrhotic rat model, the ability of the small intestine to secrete Lyz was partially restored after LOLA treatment. In this study, we used 16S rDNA sequencing to identify the composition of the gut microbiota in each group of rats. We detected differences in the gut microbiota composition among the three groups of rats, with a significant decrease in the alpha diversity of the intestinal microbiota of the cirrhosis group. Even after LOLA ammonia-lowering treatment, the alpha diversity did not increase significantly (shown in Fig. 1 .E-G). Furthermore, compared with that in the control group, the abundance of anaerobic Clostridium was significantly greater in the cirrhosis group but markedly lower in the LOLA group than in the cirrhosis group. Anaerobic Clostridium is a major pathogen that can produce exotoxins and invasion-promoting enzymes that damage the body. These findings indicate that the gut microbiota of cirrhotic rats changes and that LOLA can improve the gut microbiota community structure. Through morphological examination of the small intestinal epithelium of cirrhotic rats, we detected significant shedding of villi and extensive inflammatory cell infiltration in the cirrhosis group. Compared with the cirrhosis group, the LOLA group presented more intact small intestinal epithelial villi and significantly reduced inflammatory cell infiltration (shown in Fig. 3 .A). LPS and DAO are rarely secreted extracellularly under normal conditions, making them reliable indicators of intestinal mucosal integrity [ 35 – 36 ]. ZO-1 is a peripheral membrane protein and one of the most important proteins for maintaining tight junctions between cells. Additionally, it can act as a signal transduction molecule, participating in cellular signal transduction [ 37 ]. ZO-1 has been proven to reliably reflect the integrity of the intestinal mucosal barrier in sepsis models and inflammatory bowel disease models [ 38 – 39 ]. Therefore, we further selected related factors, such as LPS, DAO, and ZO-1 protein, to assess the impact of blood ammonia on the integrity of the small intestinal mucosal barrier. The results suggest that in the cirrhotic rat model, after LOLA treatment, ZO-1 expression was significantly upregulated, and serum LPS and DAO levels were significantly decreased, indicating that LOLA may affect the integrity of the small intestinal mucosal barrier. When the intestine is subjected to external stimuli, it can induce cell apoptosis by stimulating signaling via the ER stress pathway [ 41 – 42 ]. Therefore, we hypothesized that ammonia causes small intestinal epithelial cell dysfunction and destruction of intestinal mucosal integrity by mediating ER stress. We first examined BIP expression (shown in Fig. 4 ) and found that BIP expression in the cirrhosis group was significantly greater than that in the control group; after LOLA ammonia-lowering treatment, BIP expression in the LOLA group was lower than that in the cirrhosis group, indicating that ER stress indeed occurred in the small intestinal epithelial cells of cirrhotic rats. Therefore, we detected proteins related to the PERK signaling pathway, the most important pathway in ER stress, through WB, IHC, and IF. We found that ER stress-related protein expression was significantly upregulated in the cirrhosis group but downregulated in the LOLA group compared with that in the cirrhosis group. These findings suggest that ammonia may mediate ER stress through signaling via the PERK pathway, leading to small intestinal epithelial cell secretion and mucosal barrier dysfunction, thereby causing gut microbiota dysbiosis and increasing the abundance of harmful bacteria. Conclusion Our experimental results reveal a new mechanism by which LOLA ameliorates cirrhosis progression, providing experimental evidence for further clinical use of LOLA and offering new insights into controlling liver diseases by targeting the gut‒liver axis. LOLA may enhance the gut microbiota community structure and enhance intestinal barrier integrity. Declarations Acknowledgements We thanked all subjects who participated in this study. Ethics approval and consent to participate This study was approved by the Ethics Review Committee of the First Affiliated Hospital of Chengdu Medical College (permit number: CYYFYEC2019002). Conflict of interest The authors declare that they have no competing interests. Funding The research was supported by Chengdu Science and Technology Department (grant No. 2021-YF0500666-SN). Authors' contributions Luo Zuo designed the study. Hui Su, Ming Zhuang, He Wei, Su-qing Ren, Si-jing Han, Li-ping Zhou, Wen-Yang, Hui-yue Zhang performed the experiments. Luo Zuo, Hui Su, Ming Zhuang analyzed the results. Luo Zuo and Hui Su explained the findings and wrote the paper. All authors read and approved the final manuscript. Data Availability Statement If you need raw data generated in the current study, you can ask the corresponding author by E-mail. Consent for publication Not Applicable. References Trebicka Jonel,Macnaughtan Jane,Schnabl Bernd et al. The microbiota in cirrhosis and its role in hepatic decompensation.[J] .J Hepatol, 2021, null: S67-S81. Pinzone MR, Celesia BM, Di Rosa M, et al. Microbial translocation in chronic liver diseases. Int J Microbiol 2012;2012:694629 Xue Tao,Qiu Jian-hua,Qiao Li,Rifaximin treatment in hepatic encephalopathy.[J] .N. Engl. J. Med., 2010, 362: 2424; author reply 2424-5. Dalal Rohan,McGee Richard G,Riordan Stephen M et al. 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Butyrivibrio hungatei sp. nov. and Pseudobutyrivibrio xylanivorans sp. nov., butyrate-producing bacteria from the rumen.[J] .Int J Syst Evol Microbiol, 2003, 53: 201-209. Cignarella Francesca,Cantoni Claudia,Ghezzi Laura et al. Intermittent Fasting Confers Protection in CNS Autoimmunity by Altering the Gut Microbiota.[J] .Cell Metab, 2018, 27: 1222-1235.e6. Ooijevaar R E,van Beurden Y H,Terveer E M et al. Update of treatment algorithms for Clostridium difficile infection.[J] .Clin Microbiol Infect, 2018, 24: 452-462. Antonelli Massimo,Martin-Loeches Ignacio,Dimopoulos George et al. Clostridioides difficile (formerly Clostridium difficile) infection in the critically ill: an expert statement.[J] .Intensive Care Med, 2020, 46: 215-224. Chen Yunbo,Gu Hongqin,Lv Tao et al. Longitudinal investigation of carriage rates and genotypes of toxigenic Clostridium difficile in hepatic cirrhosis patients.[J] .Epidemiol Infect, 2019, 147: e166. Zhao Aiping,Lu Wuyuan,de Leeuw Erik,Functional synergism of Human Defensin 5 and Human Defensin 6.[J] .Biochem Biophys Res Commun, 2015, 467: 967-72. Chu Hiutung,Pazgier Marzena,Jung Grace et al. Human α-defensin 6 promotes mucosal innate immunity through self-assembled peptide nanonets.[J] .Science, 2012, 337: 477-81. Chairatana Phoom,Nolan Elizabeth M,Molecular basis for self-assembly of a human host-defense peptide that entraps bacterial pathogens.[J] .J Am Chem Soc, 2014, 136: 13267-76. Chairatana Phoom,Chiang I-Ling,Nolan Elizabeth M,Human α-Defensin 6 Self-Assembly Prevents Adhesion and Suppresses Virulence Traits of Candida albicans.[J] .Biochemistry, 2017, 56: 1033-1041. Lindhauer Nora S,Bertrams Wilhelm,Pöppel Anne et al. Tribolium castaneumAntibacterial activity of a defensin in an infection model of .[J] .Virulence, 2019, 10: 902-909. Shukla Pradeep K,Meena Avtar S,Rao Vaishnavi et al. Human Defensin-5 Blocks Ethanol and Colitis-Induced Dysbiosis, Tight Junction Disruption and Inflammation in Mouse Intestine.[J] .Sci Rep, 2018, 8: 16241. Gyongyosi B, Cho Y, Lowe P, Calenda CD, Iracheta-Vellve A, Satishchandran A, et al. Alcohol-induced IL-17A production in paneth cells amplifies endoplasmic reticulum stress, apoptosis, and inflammasome-IL-18 activation in the proximal small intestine in mice. Mucosal Immunol. 2019;12:930–44. Yan J, Zheng Y, Min Z, Ning Q, Lu S. Selenium effect on selenoprotein transcriptome in chondrocytes. Biometals. 2013; 26:285–96 Additional Declarations No competing interests reported. Supplementary Files file.pptx Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-6114564","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":422023097,"identity":"9924b9c3-abbf-4d3c-8a2f-34a04c9577a8","order_by":0,"name":"Hui Su","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chengdu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Su","suffix":""},{"id":422023098,"identity":"5e9d44ad-9376-4bc6-ae17-9f2d2e664678","order_by":1,"name":"Ming Zhuang","email":"","orcid":"","institution":"Sichuan Mianyang 404 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Zhuang","suffix":""},{"id":422023099,"identity":"11c53dc2-c5f7-4177-a60e-2ee3f840c76a","order_by":2,"name":"He Wei","email":"","orcid":"","institution":"Chengdu Medical College","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Wei","suffix":""},{"id":422023101,"identity":"a8ddd2a8-8a05-45ee-a173-b1ce9eb16eff","order_by":3,"name":"Shu-qin Ren","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chengdu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Shu-qin","middleName":"","lastName":"Ren","suffix":""},{"id":422023103,"identity":"ae36e820-5332-4762-b047-9abf02070f02","order_by":4,"name":"Si-jing Han","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chengdu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Si-jing","middleName":"","lastName":"Han","suffix":""},{"id":422023105,"identity":"69256595-4be3-4476-b7a8-ae3be76f53a7","order_by":5,"name":"Li-ping Zhou","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chengdu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Li-ping","middleName":"","lastName":"Zhou","suffix":""},{"id":422023107,"identity":"7f64e93c-2bf4-4499-8ca6-7205ee818357","order_by":6,"name":"Wen Yang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chengdu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Yang","suffix":""},{"id":422023109,"identity":"f2da1271-c0d7-4d28-960f-bb1a6cf2daf6","order_by":7,"name":"Hui-yue Zhang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chengdu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Hui-yue","middleName":"","lastName":"Zhang","suffix":""},{"id":422023111,"identity":"796c1306-b67e-4bd0-a2e5-e05847bc163b","order_by":8,"name":"Luo Zuo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBACNvaGxMc/KiTk+InWwsdz4LExwxkLY8kGYrXISSQ+k2Zsq0g0OEC0wySS06QL2CQSjI8nb2D4UbGNCC08z5KtZ/BI5JmdeVbA2HPmNhFa2HMSb/BISBSb3cgxYGZsI0YLQ/4HCR4DicTNM4jWwpGQJM2TIJG4QYJoLTwHkg1nHJAwlgD65SBRfpFvb0h88PFfnRx/e/LGBz8qiNCCBBKIjxqEFlJ1jIJRMApGwQgBAEvtPAMPT6UTAAAAAElFTkSuQmCC","orcid":"","institution":"The Second Affiliated Hospital of Chengdu Medical College","correspondingAuthor":true,"prefix":"","firstName":"Luo","middleName":"","lastName":"Zuo","suffix":""}],"badges":[],"createdAt":"2025-02-26 15:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6114564/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6114564/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77576659,"identity":"4969ca55-88b2-40e2-a529-c6202137dd6d","added_by":"auto","created_at":"2025-03-03 09:09:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4295328,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LOLA on the composition of the intestinal microbiota in cirrhotic rats. (A) The dilution curve trends after sequencing the samples from the three groups of rats. (B) Species abundance curve of the samples from the three groups of rats. (C) The species accumulation box plot shows that the estimated value of cumulative genus richness is close to the asymptote. (D) Circles A, B, and C represent the total OTUs of the control group, LOLA group, and cirrhosis group, respectively. (E) ACE index in each group. (F) Chao1 index in each group. (G) Observed species index in the three groups of rats. (H) Shannon index in each group. (I) Simpson index in the three groups of rats. (J) PCOA determined via an unweighted UniFrac algorithm. (K) PCoA determined via a weighted UniFrac algorithm. (L) PCA determined via the Euclidean distance algorithm.\u003c/p\u003e","description":"","filename":"Fig1R.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6114564/v1/1e657d7cdcaa4ae904387be7.jpg"},{"id":77577147,"identity":"2f9ee1a3-d859-497a-b529-7fdf8752cfa0","added_by":"auto","created_at":"2025-03-03 09:17:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6268061,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LOLA on harmful bacteria in the intestinal microbiota of cirrhotic rats. (A) Differences in the composition of the intestinal microbiota at the phylum level among the fecal contents of each group. (B) Differences in the composition of the intestinal microbiota at the phylum level among the fecal contents of each group. (C) Bar chart showing the proportions of Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes among the top 10 bacterial taxa at the phylum level. (D) After the fecal flora of each rat in each group was quantified, the subordinate level was determined. (E) Analysis of the composition of the intestinal flora of the three groups of rats at the subordinate level. (F) The circle radiating from inside to outside represents the classification level from phylum to genus (or species). Each small circle at a different classification level represents a classification at that level, and the diameter of the small circle is proportional to the relative abundance. (H) The histogram of the LDA value distribution revealed that the species with LDA scores greater than the set value (the default setting was 4) were biomarkers with significant differences between groups. (G) Abundance of Clostridium-sensu-stricto-1 in the intestines of rats in the different groups. The solid line in the figure represents the average relative abundance for all samples in each group at the genus level; the dotted line in the figure represents the median relative abundance for all samples in each group at the species level.\u003c/p\u003e","description":"","filename":"Fig2R.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6114564/v1/b263c906d2785d3c7c271b3d.jpg"},{"id":77576671,"identity":"467720be-f40c-441f-b948-215e70f8d0eb","added_by":"auto","created_at":"2025-03-03 09:09:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9830140,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LOLA on the intestinal mucosal barrier and secretory function. (A) HE staining of rat ileal epithelial cells (observed under a 400x light microscope). (B) IHC expression of Lyz in the rat ileal epithelium (observed under a 200x light microscope). (C) IHC expression of ZO-1 in the rat ileal epithelium (observed under a 200x light microscope). (D) Expression levels of ZO-1 protein in the ileal epithelium of each group of rats. #: P \u0026lt; 0.05, ##: P \u0026lt; 0.01, ###: P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig3R.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6114564/v1/ad6fe362533dd40a11886883.jpg"},{"id":77576670,"identity":"d8218c3c-cd01-41f8-a4f6-821eae294d44","added_by":"auto","created_at":"2025-03-03 09:09:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8129131,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ammonia on endoplasmic reticulum stress. (A) Western blotting was used to determine the expression levels of BIP, p-PERK, p-eIF2a, ATF4, and CHOP in different groups of rats. #: P \u0026lt; 0.05, ##: P \u0026lt; 0.01, ###: P \u0026lt; 0.001. (B) IHC was used to determine the expression levels of PERK signaling pathway-related proteins in the ileal epithelial cells of each group of rats, which were observed under a 400x light microscope. (C) The expression levels of PERK signaling pathway-related proteins in each group of rats were determined via IF and observed under a 400x inverted fluorescence microscope.\u003c/p\u003e","description":"","filename":"Fig4R.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6114564/v1/7f7d197fcbfc724ed894367f.jpg"},{"id":81054985,"identity":"daced592-25da-48e2-aafa-9c1aca991267","added_by":"auto","created_at":"2025-04-21 17:16:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29327053,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6114564/v1/86ed4471-0384-4d35-9eec-d1272894b663.pdf"},{"id":77576681,"identity":"42f2c8a8-e1bc-4ee3-a795-5a1e3d5e4414","added_by":"auto","created_at":"2025-03-03 09:09:46","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":111889192,"visible":true,"origin":"","legend":"","description":"","filename":"file.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6114564/v1/a7079c47ee9872fb8209c301.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hyperammonemia leads to intestinal barrier function impairment and gut microbiota dysbiosis associated with PERK signaling pathway-induced endoplasmic reticulum stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCirrhosis is a chronic and irreversible disease caused by various types of liver injury characterized by persistent inflammation, necrosis, and fibrous tissue proliferation, leading to end-stage liver failure. This pathological process is characterized by the replacement of normal liver cell structure with extensive nodular hyperplasia and dense fibrous septa, resulting in liver structure atrophy and vascular compression. These changes increase portal vein blood flow resistance, leading to portal hypertension and liver dysfunction. Cirrhosis is one of the leading causes of death worldwide, with high mortality rates in the United States, Europe, and China.\u003c/p\u003e \u003cp\u003eMoreover, cirrhosis can damage the intestinal mucosa, weakening its barrier function. Owing to their anatomical proximity, the intestine and liver are closely connected, with intestinal blood flowing directly into the liver through the portal vein, exposing the liver to microorganisms and their metabolites from the intestine. Clinical observations have shown that as portal vein pressure increases in cirrhosis patients, the amount of bacterial products in the systemic circulation also increases. Portal hypertension leads to gut‒liver axis dysfunction, and as liver disease progresses, gut microbiota imbalance further worsens, increasing the entry of harmful bacteria and their metabolites into the bloodstream, which further activates the liver immune response, exacerbating fibrosis and portal hypertension.\u003c/p\u003e \u003cp\u003eL-Ornithine L-aspartate (LOLA) consists of aspartic acid and ornithine. These groups directly participate in hepatocyte metabolism and activate ornithine carbamoyltransferase and carbamoyl phosphate synthetase to facilitate liver detoxification function. These enzymes can remove free radicals and reduce the blood ammonia concentration. Although LOLA has been used to treat cirrhosis and acute liver injury, it is unclear whether LOLA enhances the intestinal mucosal barrier function in patients with cirrhosis. Our research revealed that in a cirrhotic rat model, LOLA ameliorated intestinal ER stress, supported the repair of the intestinal mucosal barrier, and reduced the number of harmful intestinal bacteria. These results provide a new mechanism for the alleviation of cirrhosis by LOLA.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e Twenty-four 6-week-old male Sprague\u0026ndash;Dawley (SD) rats weighing 200\u0026thinsp;\u0026plusmn;\u0026thinsp;15 g were housed in a pathogen-free animal room at the Animal Care Center of the First Affiliated Hospital of Chengdu Medical College. The temperature was controlled at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, the average humidity was 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0%, and the light:dark cycle was 12:12 h. All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Chengdu Medical College. After 5 days of adaptive feeding, the rats were randomly divided into 3 groups: the cirrhosis group, LOAL group, and control group. For the rats in the cirrhosis group (n\u0026thinsp;=\u0026thinsp;8), 0.5% DMN (McLean Biochemical Technology Co., Ltd., Shanghai, China) was injected intraperitoneally at a dose of 10 mg/kg for four consecutive weeks (Monday, Tuesday, Wednesday), with saline gavage of the same volume as the LOLA group. For the rats in the LOLA group (n\u0026thinsp;=\u0026thinsp;8), DMN was injected intraperitoneally at 10 mg/kg for four consecutive weeks (Monday, Tuesday, Wednesday), while 1000 mg/kg LOLA (Qirui Pharmaceutical Co., Ltd., Wuhan, China) diluted 10% with saline was simultaneously administered via gavage. For the rats in the control group (n\u0026thinsp;=\u0026thinsp;8), saline gavage and intraperitoneal injection were administered according to each rat's body weight. The rats were fed normally at other times. On day 21 of the experiment, liver samples were taken after the rats in all groups were euthanized to observe liver damage.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBlood Tests\u003c/h3\u003e\n\u003cp\u003eOn day 21 of the experiment, blood samples were taken from the hearts of rats in the cirrhosis, LOLA, and control groups after euthanasia. ELISA kits were used to determine the serum levels of DAO and lipopolysaccharide (LPS) in each sample (Camilo Biological Engineering Company).\u003c/p\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern Blotting\u003c/div\u003e \u003cp\u003eOn day 21 of the experiment, ileum tissue was collected from rats in the model, treatment, and control groups after euthanasia. Ileum samples were homogenized, and the proteins were separated via 12% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis. After blotting, polyclonal antibodies against ZO-1, p-PERK, and p-eIF2α (Affinity Biosciences); BIP (Bioaosen Biotechnology Company); and ATF4 and CHOP (Santa Cruz Biotechnology) were used. Relative protein expression levels were normalized to those of β-actin on the basis of the optical density of the protein bands and quantified via ImageJ (NIH) software.\u003c/p\u003e\n\u003ch3\u003eH\u0026E Staining and Immunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eThe excised ileum was immediately fixed in 10% neutral buffered formalin, paraffin embedded, and cut into 5 \u0026micro;m sections. After deparaffinization with xylene and ethanol, the sections were stained with hematoxylin‒eosin (H\u0026amp;E) and oil red O for morphological evaluation under a microscope. Ileum tissues were subjected to paraffin embedding, sectioning, antigen retrieval, and immunostaining. Specific antibodies against Lyz, ZO-1, BIP, p-PERK, p-eIF2α, ATF4, and CHOP were used. The signals for DAB (antigen-positive expression) and hematoxylin (nuclear staining) were captured under a microscope (NIKON DS-U3) and processed via Image Pro Plus 6.0 software (CAD/CAM Services Inc.).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eThe ileum was fixed with paraformaldehyde, cryosectioned, washed 3 times with 1x PBS, and blocked with 0.3% Triton X-100 and 5% goat serum (#SL038, Solarbio) for 2 hours. The blocked sections were washed 3 times with 1x PBS, and primary antibodies targeting BIP (1:100), p-PERK (1:100), p-eIF2α (1:100), ATF4 (1:100), and CHOP (1:100) were incubated overnight at 4\u0026deg;C, followed by incubation with the secondary antibodies AlexaFluor488-labeled goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (#ZF-0511, ZSGB-BIO) and AlexaFluor594-labeled goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (#ZF-0513, ZSGB-BIO). The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Images were captured via a Leica TCS SP8 confocal microscope (Leica Microsystems, Germany).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e16S rRNA Data Analysis\u003c/h2\u003e \u003cp\u003eThe raw sequencing data for all the samples were deposited in the NCBI Sequence Read Archive database (accession number: PRJNA540574). QIIME2 (v2018.11.4) was used for quality filtering of the raw reads. Noisy sequencing data, including erroneous barcodes, chimeras, and low-quality sequences, were excluded. Clean data were clustered into operational taxonomic units (OTUs) at a 97% threshold. OTU screening was performed against the Greengenes database (release 13.8), filtering out rare OTUs (\u0026le;\u0026thinsp;0.001%). The α and β diversities were calculated on the basis of the relative abundance of OTUs via the QIIME and MicrobiomeAnalyst platforms, including principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS). Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify differential taxonomies. Additionally, redundancy analysis (RDA) was performed via the R vegan package on the basis of different key clinical factors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eSPSS 21.0 software was used. Data conforming to a normal distribution are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and were analyzed by one-way ANOVA. Data conforming to a nonnormal distribution are expressed as the median (p25, p75) and were analyzed via the H test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance. ImageJ software was used for semiquantitative statistical analysis of the immunohistochemistry data and images of other sections, which are expressed as the average optical density (AOD). For all the fecal gut microbiota sequencing data, rarefaction curves and hierarchical clustering curves were plotted via R language on the basis of the OTU information from each group of samples. The Chao1 index and ACE index were calculated via amplicon analysis software (QIIME) to compare intragroup microbial diversity. Principal component analysis (PCA) and PCoA were performed via R language to compare intergroup microbial differences. The multiresponse permutation procedure (MRPP) test was used to compare microbial differences between groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e1.LOLA reduce ammonia and liver injury, does not improve liver index\u003c/h2\u003e \u003cp\u003eThe liver index values and levels of ammonia, aspartate transaminase (AST), alanine aminotransferase (ALT), and other related indicators of the rats in each group were not significantly different in terms of the overall distributions of the three groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The cirrhosis group rats had significantly elevated levels of blood ammonia, AST, and ALT. In the LOLA group, the levels of blood ammonia, AST, and ALT significantly decreased but were still notably greater than those in the control group(shown in Table.1). The liver cells of the cirrhosis group rats exhibited point-fragmented necrosis similar to that in the LOLA group, with a large accumulation of inflammatory cells in the necrotic areas (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There was no difference in liver fibrosis between the LOLA and the cirrhosis groups (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2. LOLA alters the composition of the gut microbiota in cirrhotic rats\u003c/h2\u003e \u003cp\u003e16S rDNA sequencing of 18 samples revealed a total of 1,424,211 reads, with 864,955 reads remaining after filtering, which were clustered into 1,276 OTUs. The rarefaction curves drawn from the sequencing of the three groups of rat samples tended to plateau, indicating that the sequencing depth met the requirements (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.A). The species abundance curve revealed that the species in each group had good richness and evenness (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.B). The species accumulation box plot shows that the estimated cumulative genus richness approached the asymptotic value, indicating sufficient sampling (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.C). The total number of OTUs in each group and the analysis of unique and shared OTUs between groups are presented in a Venn diagram (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.D). The control group had 1,062 OTUs, the LOLA group had 1,046 OTUs, and the cirrhosis group had 1,021 OTUs. Among these, 792 OTUs were shared by all three groups, while the control group had 72 unique OTUs, the LOLA group had 62 unique OTUs, and the cirrhosis group had 74 unique OTUs (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.D).\u003c/p\u003e \u003cp\u003eACE, Chao1, and observed species indices were used to reflect the diversity of the fecal microbiota in the three groups of rats (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.E-G), suggesting that in the cirrhosis model, the diversity of the rat gut microbiota was impaired (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). After LOLA ammonia-lowering treatment, there was no significant improvement in the diversity of the gut microbiota in cirrhotic rats (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The Shannon index and Simpson index were used to evaluate the evenness and richness of the gut microbiota. The results showed that LOLA ammonia-lowering treatment increased the evenness and richness of the gut microbiota in cirrhotic rats (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.H-I).\u003c/p\u003e \u003cp\u003eThe MRPP test combined with PCA and PCoA was used for comprehensive analysis of the intergroup differences in the fecal microbiota among the three groups of rats. The results revealed significant differences in the structure of the fecal microbiota among the three groups of rats (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.J‒L and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating that there were indeed significant changes in the microbiota in the cirrhotic rat model and that LOLA ammonia-lowering treatment altered the composition of the gut microbiota in cirrhotic rats.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBiochemical indicators of the rats in each group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLiver index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmmonia\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAST\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eALT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.88(3.01, 3.08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.5(45.8, 114.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e132.0(118.0, 171.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.0(39.0, 54.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCirrhosis (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.86(2.68, 3.38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1151.6(415.9, 1145.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e318.0(310.0, 507.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e189.0(181.0, 212.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOLA (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.81(2.18, 3.19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e397.4(334.6, 417.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e189.0(146.0, 240.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95.0(83.0, 102.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.745\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.818\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;0.689\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMRPP tests\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eθ\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eObserved variables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExpected variables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA-C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA-B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5372\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB-C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.03333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5778\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5978\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStereological estimates of IDAO and LPS levels in each group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDAO(U/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLPS(U/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.06\u0026thinsp;\u0026plusmn;\u0026thinsp;14.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e131.78(115.44, 191.27)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCirrhosis(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e158.43\u0026thinsp;\u0026plusmn;\u0026thinsp;30.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e324.34(265.67, 404.46)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOLA(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.34\u0026thinsp;\u0026plusmn;\u0026thinsp;16.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e162.65(115.44, 255.66)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF/H value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.208\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3. LOLA reduces the relative abundance of harmful bacteria in the gut microbiota of cirrhotic rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we examined the differences in the gut microbiota composition at the phylum and genus levels among the groups. The dominant phyla in all the groups were Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. Blood ammonia levels were significantly elevated in the cirrhosis group and markedly decreased in the LOLA group compared with those in the cirrhosis group but were still significantly greater than those in the control group. In the LOLA group, the abundance of Firmicutes increased significantly, whereas the abundance of Bacteroidetes decreased notably. The abundances of Firmicutes and Proteobacteria in the intestine did not significantly increase. The abundance of Actinobacteria decreased in the gut of cirrhotic model rats but increased somewhat in the LOLA-gavaged rats (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A‒C).\u003c/p\u003e \u003cp\u003eAfter the composition of the fecal microbiota in each group of rats was determined, the top 10 most abundant genera were ranked as follows: Prevotellaceae_UCG-003; Lactobacillus; Staphylococcus; Alloprevotella; Allobaculum; Bacteroides; Dubosiella; unidentified_[Eubacterium]_coprostanoligenes_group; Clostridium_sensu_stricto_1; and Coriobacteriaceae_UCG-002. The abundance of Prevotella decreased significantly in the cirrhosis group but did not increase notably after LOLA gavage. The abundance of Lactobacillus increased in both the cirrhosis and LOLA groups, with a more pronounced increase in the LOLA group. The abundance of Clostridium increased significantly in the cirrhosis group but decreased markedly in the LOLA group (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.D-E).\u003c/p\u003e \u003cp\u003eThe LEfSe algorithm was used to identify characteristic bacteria specific to each group and to identify the taxonomic biomarkers with the greatest degree of change in levels between groups (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.F-H). The results revealed that Micrococcus, Prevotella, Helicobacter, and Campylobacter were significantly enriched in the control group; Lactobacillus, Staphylococcus, and Bacillus were significantly enriched in the LOLA group; and Lactobacillus and anaerobic Clostridium were significantly enriched in the cirrhosis group. Notably, the abundance of anaerobic Clostridium increased significantly in the cirrhosis group and decreased markedly after LOLA gavage (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.G).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4. LOLA enhances intestinal barrier function in cirrhotic rats\u003c/h2\u003e \u003cp\u003eExamination of rat intestinal tissue revealed that the ileal epithelial structure in the control group was continuous and intact, with no significant inflammatory infiltration. The ileal epithelium of the cirrhosis group presented many swollen and detached villi, with extensive infiltration of eosinophils and lymphocytes. Compared with those in the cirrhosis group, the degree of villous structure destruction and eosinophil and lymphocyte infiltration in the ileal epithelium in the LOLA group were significantly lower (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A). These findings indicate that the ileal epithelial cells of cirrhotic model rats established by intraperitoneal injection of the DMN underwent morphological changes, such as necrosis and inflammatory infiltration, which were significantly improved after LOLA gavage-induced ammonia-lowering treatment. ELISA kits were used to determine the serum DAO and LPS levels in the three groups of rats (shown in Table.3). The serum DAO and LPS levels in the cirrhosis group were significantly elevated, whereas they were decreased markedly in the LOLA group. These results suggest that LOLA enhances the integrity of the intestinal mucosal barrier.\u003c/p\u003e \u003cp\u003eTo further understand the effect of LOLA on intestinal Paneth cells (the main secretory cells), we examined the ability of ileal Paneth cells to secrete Lyz via IHC. All ileal tissues were observed under 200x magnification, with Paneth cells located at the base of the ileal glands and having a conical shape. Compared with the control group, the cirrhosis group presented significantly reduced Lyz secretion by Paneth cells, whereas the LOLA group presented a notable increase in Lyz secretion (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.B). These findings suggest that Lyz, an important antibacterial substance in the intestine, plays a crucial role in maintaining gut microbiota stability. Moreover, these results indicate that LOLA may regulate the gut microbiota by affecting Lyz secretion.\u003c/p\u003e \u003cp\u003eIHC analysis of ileal tissue revealed that the control group presented increased ZO-1 expression, with darker mucosal epithelial cell staining and a brown color. The ileal tissue of the cirrhosis group presented relatively low levels of ZO-1, with relatively weak staining. Compared with that of the cirrhosis group, the ileal tissue of the LOLA group presented increased ZO-1expression, with varying degrees of darker staining (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.C). Compared with that in the blank group, ZO-1 protein expression in the ileal tissue of the cirrhosis group was lower (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01); compared with that in the cirrhosis group, ZO-1 expression in the ileal tissue of the LOLA group was greater (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAverage optical density (AOD) values determined via immunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLyz(AOD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZO-1(AOD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e162.60\u0026thinsp;\u0026plusmn;\u0026thinsp;13.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e148.50\u0026thinsp;\u0026plusmn;\u0026thinsp;13.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCirrhosis(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.75\u0026thinsp;\u0026plusmn;\u0026thinsp;19.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e104.54\u0026thinsp;\u0026plusmn;\u0026thinsp;11.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOLA(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e134.57\u0026thinsp;\u0026plusmn;\u0026thinsp;20.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e123.45\u0026thinsp;\u0026plusmn;\u0026thinsp;11.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.336\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.385\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e5. LOLA affects the endoplasmic reticulum stress signaling pathway\u003c/h2\u003e \u003cp\u003eTo investigate the mechanism by which LOLA influences intestinal homeostasis, we examined the expression of the ER stress marker BIP in the ileal mucosa. Compared with that in the cirrhosis group, BIP expression was significantly upregulated in the intestinal mucosa of the cirrhosis group, whereas it was downregulated after LOLA gavage. These results suggest that ER stress in the intestinal mucosa is increased in the cirrhotic rat model and that LOLA can alleviate this stress.\u003c/p\u003e \u003cp\u003eThe PERK/eIF2α/CHOP pathway is an important cellular stress signaling pathway that is involved mainly in regulating the intestinal mucosal response to internal and external environmental pressures and regulating cell apoptosis. Using Western blotting to measure the PERK protein content, we found that the PERK protein level was significantly increased in the small intestinal epithelial cells of the cirrhosis group, whereas it was decreased in the LOLA group (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.A). IHC and IF methods were used to verify the changes in PERK protein expression in the cirrhosis and LOLA groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo understand the changes in downstream molecules of the PERK pathway, we measured the changes in p-eIF2α, ATF4, and CHOP protein levels. The results revealed that p-eIF2α, ATF4, and CHOP were significantly upregulated in the cirrhosis group compared with the control group, whereas they were downregulated in the LOLA group (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.B). These results were consistent with the trends in p-eIF2α, ATF4, and CHOP protein expression determined by IHC and IF (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.C). These findings suggest that the effects of LOLA on the gut microbiota and protection of the intestinal mucosal barrier may be related to its regulation of ER stress in intestinal cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, using a rat cirrhosis model induced by intraperitoneal DMN injection, we found that LOLA gavage treatment along with DMN injection did not significantly alleviate liver fibrosis or hepatocyte necrosis and inflammatory infiltration but did significantly reduce blood ammonia levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). LOLA promotes ammonia metabolism in the body by providing a source of aspartic acid and ornithine, thereby lowering blood ammonia levels, and is mainly used to treat hepatic encephalopathy. This study is the first to reveal that LOLA can ameliorate cirrhosis-related intestinal barrier dysfunction.\u003c/p\u003e \u003cp\u003eThe impact of signaling via the gut‒liver axis on liver diseases has become a research hotspot in recent years. The mechanism of interaction and communication between the intestine and liver involves various biological processes, including the exchange of metabolites, the regulation of immune responses, and the occurrence of inflammatory responses. Our experimental results also revealed that LOLA can increase Lys secretion in the intestinal mucosa of cirrhotic rats, affect the gut microbiota, and enhance intestinal mucosal tight junctions, leading to improved intestinal mucosal barrier function.\u003c/p\u003e \u003cp\u003eIntestinal mucosal cells secrete active substances such as antimicrobial peptides (AMPs) and Lyz into the intestinal lumen through degranulation. These active substances are important components of the intestinal immune barrier and can effectively inhibit excessive growth of intestinal bacteria, maintaining the balance of the gut microbiota [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the cirrhotic rat model, the ability of the small intestine to secrete Lyz was partially restored after LOLA treatment.\u003c/p\u003e \u003cp\u003eIn this study, we used 16S rDNA sequencing to identify the composition of the gut microbiota in each group of rats. We detected differences in the gut microbiota composition among the three groups of rats, with a significant decrease in the alpha diversity of the intestinal microbiota of the cirrhosis group. Even after LOLA ammonia-lowering treatment, the alpha diversity did not increase significantly (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.E-G). Furthermore, compared with that in the control group, the abundance of anaerobic Clostridium was significantly greater in the cirrhosis group but markedly lower in the LOLA group than in the cirrhosis group. Anaerobic Clostridium is a major pathogen that can produce exotoxins and invasion-promoting enzymes that damage the body. These findings indicate that the gut microbiota of cirrhotic rats changes and that LOLA can improve the gut microbiota community structure.\u003c/p\u003e \u003cp\u003eThrough morphological examination of the small intestinal epithelium of cirrhotic rats, we detected significant shedding of villi and extensive inflammatory cell infiltration in the cirrhosis group. Compared with the cirrhosis group, the LOLA group presented more intact small intestinal epithelial villi and significantly reduced inflammatory cell infiltration (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.A). LPS and DAO are rarely secreted extracellularly under normal conditions, making them reliable indicators of intestinal mucosal integrity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. ZO-1 is a peripheral membrane protein and one of the most important proteins for maintaining tight junctions between cells. Additionally, it can act as a signal transduction molecule, participating in cellular signal transduction [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. ZO-1 has been proven to reliably reflect the integrity of the intestinal mucosal barrier in sepsis models and inflammatory bowel disease models [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, we further selected related factors, such as LPS, DAO, and ZO-1 protein, to assess the impact of blood ammonia on the integrity of the small intestinal mucosal barrier. The results suggest that in the cirrhotic rat model, after LOLA treatment, ZO-1 expression was significantly upregulated, and serum LPS and DAO levels were significantly decreased, indicating that LOLA may affect the integrity of the small intestinal mucosal barrier.\u003c/p\u003e \u003cp\u003eWhen the intestine is subjected to external stimuli, it can induce cell apoptosis by stimulating signaling via the ER stress pathway [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Therefore, we hypothesized that ammonia causes small intestinal epithelial cell dysfunction and destruction of intestinal mucosal integrity by mediating ER stress. We first examined BIP expression (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and found that BIP expression in the cirrhosis group was significantly greater than that in the control group; after LOLA ammonia-lowering treatment, BIP expression in the LOLA group was lower than that in the cirrhosis group, indicating that ER stress indeed occurred in the small intestinal epithelial cells of cirrhotic rats. Therefore, we detected proteins related to the PERK signaling pathway, the most important pathway in ER stress, through WB, IHC, and IF. We found that ER stress-related protein expression was significantly upregulated in the cirrhosis group but downregulated in the LOLA group compared with that in the cirrhosis group. These findings suggest that ammonia may mediate ER stress through signaling via the PERK pathway, leading to small intestinal epithelial cell secretion and mucosal barrier dysfunction, thereby causing gut microbiota dysbiosis and increasing the abundance of harmful bacteria.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur experimental results reveal a new mechanism by which LOLA ameliorates cirrhosis progression, providing experimental evidence for further clinical use of LOLA and offering new insights into controlling liver diseases by targeting the gut‒liver axis. LOLA may enhance the gut microbiota community structure and enhance intestinal barrier integrity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thanked all subjects who participated in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Review Committee of the First Affiliated Hospital of Chengdu Medical College (permit number: CYYFYEC2019002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by Chengdu Science and Technology Department (grant No. 2021-YF0500666-SN).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuo Zuo designed the study. Hui Su, Ming Zhuang, He Wei, Su-qing Ren, Si-jing Han, Li-ping Zhou, Wen-Yang, Hui-yue Zhang performed the experiments. Luo Zuo, Hui Su, Ming Zhuang analyzed the results. Luo Zuo and Hui Su explained the findings and wrote the paper. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIf you need raw data generated in the current study, you can ask the corresponding author by E-mail.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTrebicka Jonel,Macnaughtan Jane,Schnabl Bernd et al. The microbiota in cirrhosis and its role in hepatic decompensation.[J] .J Hepatol, 2021, null: S67-S81.\u003c/li\u003e\n\u003cli\u003ePinzone MR, Celesia BM, Di Rosa M, et al. 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Biometals. 2013; 26:285\u0026ndash;96\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"LOLA, cirrhosis, intestinal barrier, gut microbiota, endoplasmic reticulum stress, hyperammonemia","lastPublishedDoi":"10.21203/rs.3.rs-6114564/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6114564/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground/Objective:\u003c/h2\u003e \u003cp\u003eL-Ornithine L-aspartate (LOLA) can effectively alleviate hepatic encephalopathy by reducing blood ammonia levels. Recent studies have shown that amino acids can affect via the gut‒liver axis has a significant effect on the progression of various liver diseases, but the effects of LOLA on the intestine have not been reported.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eRats were given intraperitoneal injections of 0.5% N‒E dimethylnitrosamine (DMN) at 10 mg/kg while being simultaneously administered LOLA (1000 mg/kg) via gavage three times per week for 4 weeks, after which the animals were euthanized. Serum was collected to assess liver function. Rat feces were collected for 16S rRNA sequencing of the microbiota, and biochemical and histological evaluations of ileal damage were performed.The expression of tight junction proteins (ZO-1), lysozyme (Lyz), and endoplasmic reticulum stress (ER) pathway-related markers were detected in the rat ileal epithelium.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eLOLA gavage did not significantly restore gut microbiota diversity or the abundance of dominant intestinal bacteria such as Prevotella, but it significantly reduced the abundance of harmful bacteria such as Clostridium. Additionally, LOLA gavage increased ZO-1 expression in the rat small intestinal mucosa, reduced plasma DAO and LPS levels, and restored Lyz secretion. Furthermore, the expression of ER stress pathway-related markers (BIP, p-PERK, p-eIF2a, ATF4, and CHOP) decreased after LOLA gavage.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eLOLA may ameliorate gut microbiota dysbiosis in cirrhotic rats by affecting the ER stress response and restoring intestinal barrier function, revealing a new mechanism by which LOLA controls liver disease.\u003c/p\u003e","manuscriptTitle":"Hyperammonemia leads to intestinal barrier function impairment and gut microbiota dysbiosis associated with PERK signaling pathway-induced endoplasmic reticulum stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 09:09:34","doi":"10.21203/rs.3.rs-6114564/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9a87869c-94ae-431a-a6c7-76aa3112db1e","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-21T17:08:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-03 09:09:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6114564","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6114564","identity":"rs-6114564","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}