{'fraction_non_alphanumeric': 0.031955452994883116, 'fraction_numerical': 0.0263369118089696, 'mean_word_length': 5.278740157480315, 'pattern_counts': {'":': 0, '<': 11, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Improvement of Gut Microbiota by Inhibition of P38 Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Rats with Severe Acute Pancreatitis

Background Gut microbiota dysbiosis plays a key role in pathogenesis of severe acute pancreatitis (SAP). In this study, we explored the protective effects of the p38 MAPK inhibitor, SB203580, against gut inflammation and microbiota dysbiosis induced by pancreatic duct injection with 3.5% sodium taurocholate in an SAP rat model. Material/Methods Ninety male Sprague-Dawley rats were randomly assigned to sham-operated, SAP model, and SAP plus SB203580 groups (n=30/group). Histological examination was conducted to assess gut and pancreatitis injury. The levels of amylase, D-lactate, diamine oxidase, tumor necrosis factor α, IL-6, IL-1β, and phospho-p38MAPK in the plasma and intestine were evaluated at 3, 6, or 12 h after SAP induction. The gut microbiome was investigated based on16S rDNA gene sequencing at 12 h after SAP induction. Results Histological examination revealed edema and inflammatory infiltrations in the pancreas and distal ileum. The expression of tumor necrosis factor α, IL-1β, and IL-6 in plasma and distal ileum was increased in the SAP group, which were restored after treatment with SB203580. Significantly lower bacterial diversity and richness was found in the SAP group. In the SAP group, the abundance of Bacteroidetes and Firmicutes was decreased, and there was a higher proportion of Proteobacteria at the phylum level. The SAP plus SB203580 group exhibited significantly less damage to the gut microbiota, with higher bacterial diversity and a more normal proportion of intestinal microbiota. Conclusions SB203580 mediated suppression of the p38 MAPK signaling pathway via reduced gut inflammatory response and microbiota dysbiosis.


Background
Acute pancreatitis is one of the most common gastrointestinal disorders and is associated with a high risk of mortality [1]. Several interventions have been employed to improve the prognosis of severe acute pancreatitis (SAP); however, clinical trials involving various therapies, including intravenous antibiotic prophylaxis, selective gut decontamination [2], and probiotic prophylaxis [3], have failed to demonstrate consistent benefits. Thus, development of a more effective approach to SAP care is urgently needed.
Changes in the diversity and structure of the gut microbiota have been implicated in the pathogenesis of several metabolic and inflammatory disorders, including diabetes, obesity, and atherosclerosis [4]. Microbiota community structure has been associated with the expressions of TNF-a and IL-6 in the intestines [5]. Our previous study [6] found that bacterial diversity was decreased and the abundance of 1 or 2 rare bacterial species was increased in septic shock patients. A recent study showed that intestinal microbiota structure was changed in SAP rats with decreased abundance of Saccharibacteria and Tenericutes at the phyla level [7]. Hamada et al. [8] conducted comprehensive analysis of gut microbiota between patients with type 1 autoimmune pancreatitis and those with chronic pancreatitis, and found that the proportions of Bacteroides, Streptococcus, and Clostridium species were higher in patients with chronic pancreatitis. Using pancreatic enzyme replacement therapy, Nishiyama et al. [9] demonstrated that the abundance of key beneficial bacterium in the intestinal tract, including Akkermansia muciniphila and Lactobacillus reuteri, was increased in chronic pancreatitis patients. However, the evidence was still insufficient because of the limited studies related to the change in gut microbiota in SAP patients.
Excessive systemic and intestinal release of inflammatory cytokines results in generation of SAP. Mitogen-activated protein kinases (MAPKs) are a class of serine/threonine protein kinases and signal transduction mediators. Activation of the p38MAPK signaling pathway is involved in regulation of inflammation. Previous findings supported the inhibition of p38 MAPK signaling as a selective intervention to reduce neutrophilic inflammation [10], and SB203580 has been indicated to reduce the injury of the intestinal mucosal barrier in SAP patients [11]. However, the effects of a p38 MAPK inhibitor, SB203580, in gut inflammation and microbiota dysbiosis remain unknown.
The goal of this study was to elucidate the role of the p38MAPK signaling pathway in SAP-induced intestinal microbiota dysbiosis and to investigate the effect of SB203580 treatment on gut inflammation.

Animal model
This study was performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Qingdao University. The protocol was approved by the Animal Experiments Ethics Committee of Qingdao University. All the surgeries were performed under sodium pentobarbital anesthesia, and all possible efforts were made to minimize suffering. Healthy male Sprague-Dawley (SD) rats weighing between 250 and 300 g were supplied by the Laboratory Animal Center of Qingdao University (Qingdao, China). These animals were housed on a 12-h light/dark cycle under controlled temperature (23±1°C) and relative humidity (65-70%). All animals were allowed free access to standard rat chow and water, and were then acclimatized to these conditions 2 weeks before experimentation. As described previously [12], model and experimental rats received a retrograde infusion of 5% sodium taurocholate (Sigma, St Louis, MO) (1 mg/kg) through the biliopancreatic duct to induce acute pancreatitis. After the surgery, the rats were injected subcutaneously with 5mL of 0.9% normal saline to supplement blood volume. Rats were sacrificed at 3, 6, and 12 h after SAP induction. Ninety SD rats were randomly divided into 3 groups as follows: a sham-operated group (the SO group), an SAP group treated with the p38MAPK inhibitor SB203580 (MedChemExpress, NJ) (the SB203580 group), and an untreated SAP group, with 30 rats in each group. Each group was further divided into 3-h, 6-h, and 12-h subgroups. One hour before establishing the animal model, the p38MAPK inhibitor SB203580 was intraperitoneally injected (5 mg/kg). Blood was collected by cardiac puncture and centrifuged at 15 000 rpm for 15 min. The extracted serum was frozen at -20°C until further testing. Distal ileum and pancreatic tissues were harvested individually and fixed in 40 g/L formaldehyde. Samples were then embedded in paraffin and continually sectioned. We collected fresh stool samples before the rats were sacrificed and stored them at -80°C.

Histological examination
Pancreas and distal ileum were stained with hematoxylin and eosin (HE), and examined and scored using a light microscope (Olympus, Japan) by 2 pathologists who were blinded to the treatment protocol. We observed the morphological changes in the pancreas, including pancreatic tissue edema, inflammatory cell infiltration, pancreatic adipose, and substance necrosis. Histological ileum damage was classified according to the grading of intestinal tissue injury using a system described by Chiu et al. [13].

Measurement of plasma and ascites amylase levels
Amylase activity in serum and ascites was measured using enzyme assay kits (Shanghai Hengfei Bioscience, China).

Measurement of D-lactate levels
Plasma D-lactate level was measured using an ELISA kit (Shanghai Hengfei Bioscience, China). Diamine oxidase activity was assessed using a commercial kit (EterLife, Birmingham, UK).

Measurement of levels of inflammatory cytokines
The levels of IL-1b, TNF-a, and IL-6 in plasma and intestinal tissue were measured using an ELISA kit following the manufacturer's instructions (LMAIBio Biotech, China).

Western blot analysis of p-p38MAPK and p38MAPK
Intestinal tissue was isolated and homogenized in lysis buffer (Beyotime Biotechnology, China) on ice. The homogenate was centrifuged (14 000 rpm, 15 min, 4°C) and the level of total supernatant protein was measured by BCA protein assay kit (Beyotime Biotechnology, China). The protein samples (20 μg) were boiled in Laemmle's sampling buffer and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels. Next, the proteins were transferred to a polyvinylidene difluoride membrane and blocked with Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20. They were then incubated with anti-p38 MAPK or the anti-phospho-p38 MAPK antibody (1: 1000; Cell Signaling Technology, Danvers, MA) for 16 h at 4°C, followed by incubation with Alexa Fluor 680-conjugated anti-rabbit IgG (Molecular Probes). After washing 5 times for 5 min each time, the membranes were visualized by NBT/BCIP (Beyotime Institute of Biotechnology, Nantong, China). The protein bands were scanned and quantified using ImageJ software version 1.34 s (Wayne Rasband National Institute of Health) using b-actin for normalization.

Intestinal microbiota analysis
Samples were analyzed using 16S rDNA gene sequencing according to the method described by Zhang et al. [14]. We used a bead-beating protocol to extract DNA according to the manufacturer's instructions [15]. Libraries were prepared according to a previously described protocol [16]. Briefly, the libraries were constructed from the PCR amplicons of the V3-V4 region of the 16S rDNA gene generated using unique dual-index primers for each sample and TransGen AP221-02 (TransGen Biotech, Beijing, China), followed by sequencing on the Illumina MiSeq sequencing platform (Illumina, San Diego, CA). Data were analyzed using QIIME and Mothur software.

Statistical analysis
Data were subjected to statistical analysis using SPSS 15 software (SPSS, Chicago, IL), and the results were expressed as mean ±SD. Data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test, as the data were predominantly non-parametric and could not be normalized using standard techniques. P<0.05 was considered statistically significant.

Results
Establishment and assessment of SAP models SAP was induced by pancreatic duct injection of 3.5% sodium taurocholate, and was assessed using amylase quantification and histopathological changes in pancreatic and ileum tissue. As shown in Figure 1A, no significant abnormalities were found in the pancreatic tissues in the SO group. Pancreatic inflammatory cell infiltration, hemorrhage, damage to the pancreatic lobular structure, and high degree of acinar cell necrosis were observed in the SAP group ( Figure 1A). In addition, the histological structures of the ileum revealed edema, shortened villi, and infiltration of inflammatory cells ( Figure 1A). Compared with the SO group, the levels of amylase in serum and ascites were significantly increased in the SAP group at all time points (P<0.05) ( Figure 1B). The above results indicated that the SAP model was established successfully, and the tissue damage was increasingly aggravated over time.

Morphological changes in the pancreas and intestine
Histopathological changes in pancreas were characterized by interstitial edema, leukocyte infiltration, and acinar cell necrosis in SAP rats ( Figure 1A). Similarly, histopathological changes in ileum were characterized by edema, shortened villi, and infiltration of inflammatory cells. Compared with the SAP group, the SB203580 treatment group showed less severe morphological changes, which indicated that SB203580 ameliorated pancreatic and ileal pathological damages induced during SAP.

SB203580 attenuated gut inflammatory cytokines
Plasma D-lactate and diamine oxidase levels were measured as indicators of intestinal barrier function. As shown in Figure 2A, levels of plasma diamine oxidase and D-lactate were both significantly increased in the SAP group at 3 h, 6 h, and 12 h compared with the SO group (all p<0.05). Treatment with SB203580 significantly decreased the levels of diamine oxidase and D-lactate at 6 h and 12 h. These results revealed that SAP leads to damage to the intestinal barrier, which can be alleviated treatment with the p38MAPK inhibitor SB203580.

4611
As shown in Figure 2B, there was a significant increase in plasma levels of TNF-a, IL-1b, and IL-6 in the SAP group at 3 h, 6 h, and 12 h (all p<0.05), consistent with that for distal ileum ( Figure 2C). Treatment with SB203580 significantly decreased the plasma levels of TNF-a, IL-1b, and IL-6 at 6 h and 12 h, although their levels remained higher than those in the SO group ( Figure 2B, 2C).

Activities of p38MAPK in intestinal tissue
To investigate whether p38MAPK proteins are involved in the induction of mucosal injury in the intestinal barrier of SAP patients, the activities of p38MAPK in the intestinal tissues were investigated using Western blotting. Compared with the SO group, the levels of p-p38MAPK were significantly increased in the SAP group at all time points (P<0.05). Compared with the SAP group, the SB203580 treatment group exhibited a significant decrease in p-p38MAPK levels (P<0.05) (Figure 3).

Effect of SB203580 treatment on gut microbiota
To determine the effect of SB203580 treatment on gut microbiota composition, we conducted 16S rDNA amplicon sequencing at 12 h, producing 1 381 347 valid sequences. Compared with the SO group, the SAP group showed decreased richness and diversity. Compared with the SAP group, the Chao1 richness estimator revealed that SB203580 treatment significantly increased microbial richness in the intestines (P<0.05) ( Figure 4A). Gut microbiota diversity was also markedly increased by SB203580 treatment, as evaluated by Shannon index (P<0.05) ( Figure 4A), although these levels remained lower than those in the SO group. In SO group, Bacteroidetes and Firmicutes ( Figure 4B) were the most abundant microbes at the phylum level in the fecal samples. Compared to the SO group, the stool microbiota composition of SAP showed a remarkable variation. Although Bacteroidetes and Firmicutes were the primary bacteria, their mean proportion decreased significantly (P<0.05) ( Figure 4B). A significantly higher proportion of Proteobacteria was found in the SAP group as compared  to the SO group (P<0.05) ( Figure 4B). In the SB203580 group, the abundances of Bacteroidetes, Firmicutes, and Proteobacteria increased (P<0.05). At the genus level, the individual variation in the stool composition was enhanced ( Figure 4C). Bacteroides was the main bacteria found in the SO, SAP, and SB203580 groups, and its proportion was comparable among all 3 groups. The abundance of Escherichia-Shigella increased in the SAP and SB203580 groups, but it was low in the SO group. The abundance of Parabacteroides was significantly increased in the SAP group.

Discussion
We examined the protective effects of the p38 MAPK inhibitor SB203580 against gut inflammation and microbiota dysbiosis induced in an SAP rat model. We found that SAP rats exhibited severe histopathological damage to the pancreas and ileum, and increased expression of tumor necrosis factor a, IL-1b, and IL-6 in plasma and ileum. Significantly lower bacterial diversity and richness was found in the SAP group. The abundances of Bacteroidetes and Firmicutes decreased and a higher proportion of Proteobacteria was found in the SAP group. Treatment with SB203580 significantly reduced inflammatory reaction and damage to the gut microbiota and induced higher bacterial diversity and more normal intestinal microbiota composition. SAP is associated with high morbidity and mortality due to the development of pancreatic and extra-pancreatic necrosis, subsequent infection, and multisystem organ failure. Despite reduction in SAP-related mortality in the last decade, it is still a devastating disease that is associated with mortality rates ranging from less than 10% to as high as 85%, according to various studies [17]. The management of SAP is complicated due to limited understanding of the pathogenesis and multi-causality of the disease, uncertainties in outcome prediction, and low number of effective treatment modalities. Inflammation plays a key role in development of SAP [18]. In our study, we found a continuous increase in levels of inflammatory cytokines, including TNF-a, IL-1b, and IL-6, in plasma and ileum. The results from histopathological examination were consistent with these results. Histopathological changes in pancreatic and distal ileum were characterized by leukocyte infiltration. Mitogen-activated protein kinases (MAPKs) are a class of serine/threonine protein kinases and signal transduction mediators. In mammalian cells, 3 major families of MAPKs have been found, including p38 MAPK, extracellular signalregulated kinase (Erk 1/2), and c-Jun N-terminal protein kinase (JNK). Among these, p38 MAPK affects the severity of pancreatitis. During the progression of SAP, the p38 MAPK signaling pathway is involved in the regulation of NF-kB activation, which plays a crucial role in the inflammatory cascade [19]. Activation of p38 MAPK results in phosphorylation of MK2, a downstream protein kinase of p38 MAPK, which triggers NF-kB expression and increases inflammation. Several studies have revealed that the p38 inhibitor SB203580 reduces p38 MAPK activity in cerulein-induced SAP, and attenuates the inflammatory response and cytokine release in the pancreas [10,20]. In the present study, we found that treatment with SB203580 significantly decreased levels of TNF-a, IL-1b, and IL-6 at 3 h, 6 h, and 12 h in SAP rats, consistent with the results of previous studies.
The human gut microbiota is a diverse microbial community, estimated to contain over 1000 different bacterial species, as well as commensal fungi and viruses [21]. The total number of microbial organisms in the human gut is estimated to about 100 trillion, up to 10 times the number of cells constituting the human body, while the collective microbial genome, the microbiome, contains approximately 100 times more genes than the entire human genome [22]. Microbiota substantially enhances host metabolic capacity and actively contributes to maintaining gut homeostasis [23]. There is still limited knowledge about change in gut microbiota and its effect. Chen et al. [7], using 16SrDNA gene sequencing on fecal samples to study the changes of intestinal microecology in SAP rat, found decreased microbiota diversity, especially decreased abundances of Saccharibacteria and Tenericutes at phyla level. However, in our study, although Bacteroidetes and Firmicutes were the primary bacteria in the intestinal gut, their mean abundances were still decreased, whereas the abundances of Escherichia-Shigella and Parabacteroides increased significantly. Tan et al. [24] enrolled 108 participants and found that the rates of multi-organ failures and infectious complications in patients with SAP with altered intestinal microbiota were significantly higher than in those with unaltered intestinal microbiota. Enterococcus abundance increased and Bifidobacterium abundance decreased in the patients with SAP. Hamada et al. [8] conducted a comprehensive analysis and comparison of gut microbiota between patients with type 1 autoimmune pancreatitis and chronic pancreatitis, and found that Bacteroides ovatus, Streptococcus australis, Streptococcus gordonii, Clostridium lactatifermentans, and Clostridium lavalense were more abundant in chronic pancreatitis patients. However, the effect of anti-inflammation treatment on gut microbiota in SAP patients remains unknown. We found that the proportion of Bacteroidetes, Firmicutes, and

Conclusions
SAP rats exhibited aggravated systemic inflammation, intestinal barrier injury, and intestinal microbiota disorder. Inhibition of the p38 MAPK signaling pathway by SB203580 reduced gut inflammatory response and microbiota dysbiosis. SB203580 thus has potential as a treatment for pancreatic and intestinal injury during SAP. These results provide fresh insight into exploring novel therapeutic strategies for SAP.