Protective Effects of Amoxicillin and Probiotics on Colon Disorders in an Experimental Model of Acute Diverticulitis Disease

Protective Effects of Amoxicillin and Probiotics on Colon Disorders in an Experimental Model of Acute Diverticulitis Disease | 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 Research Article Protective Effects of Amoxicillin and Probiotics on Colon Disorders in an Experimental Model of Acute Diverticulitis Disease Eman Shawky, Maha Ghazy Solaiman, Hanaa Abd-Elfattah Mansour, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1694157/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Acute diverticulitis disease is associated with inflammation and infection in the colon diverticula and may lead to severe morbidity. This study aimed to evaluate and compare the protective effects of the antibiotic (amoxicillin) and the probiotics ( Lactobacillus acidophilus and Bifidobacterium lactis ) given concurrently in addition to amoxicillin; in a rat model of acute diverticulitis disease. Acute diverticulitis was induced, in albino rats, by adding 3% weight/volume of dextran sulfate sodium (DSS), to the rats ’ drinking water; daily for seven days. Furthermore, injection of lipopolysaccharide (LPS) enema (4 mg/kg). The impact of treatment was assessed by measuring physiological and immunological parameters and evaluating colon macroscopic and microscopic lesions. The results showed that both treatments (especially probiotics with amoxicillin) alleviated the adverse effects of DSS and LPS, where the rats’ body weights and the colon weight-to-length ratio were modulated. Also, the colon macroscopic damage score was significantly ( p < 0.001) decreased. The pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF)-α, interleukin-1 beta (IL)-1β, interferon-gamma (IFN-γ), and interleukin-18 (IL)-18]; in the colon tissue, were significantly ( p < 0.001) decreased. Also, the elevation of myeloperoxidase (MPO) activity and C-reactive protein (CRP) level was ameliorated. Moreover, the histopathological alterations of the colon tissue were improved. In conclusion, amoxicillin and probiotics-amoxicillin were effective in preventing the development of the experimentally induced acute diverticulitis through the anti-inflammatory and the immunomodulatory effects. Additionally, this study showed the role of probiotics in preventing DSS/LPS-induced acute diverticulitis, so it can be employed as a promising treatment option for acute diverticulitis. Amoxicillin Probiotics DSS LPS Colon disorders Acute diverticulitis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Acute diverticulitis is a gastrointestinal disease, which is characterized by inflammation of the colon diverticulum and its surrounding mucosa (Strate et al. 2015 ). Diverticulitis refers to the diverticular disease as well as the spectrum of complications associated with colon diverticulosis, such as bleeding (Cohen et al. 2013 ). The colon diverticulum is formed as a consequence of herniation of colon mucosa and submucosa; through the perivascular connective tissue sheath, which surrounds the intramural vasa recta (Wedel et al. 2015 ). Part of the pathogenesis is returned to deterioration in the colon wall strength in aged individuals, which could be returned to the increased collagen cross-linkage of the colon wall (Wess et al. 1996 ). Yamamichi et al. ( 2015 ) demonstrated age, gender, tobacco usage, obesity, pre-diabetic conditions, alcohol intake, as well as increased serum triglyceride concentrations, as risk factors for diverticular disease. Acute diverticulitis is believed to develop when a colon diverticulum becomes obstructed by feces, resulting in fecal stasis, mucosal damage, and ischemia. The genetic factors, as well as alterations in the colon neuromusculature, take part in the development of diverticulitis (Strate and Morris 2019 ). Furthermore, it was hypothesized that; diverticular disease results from the deficiency of dietary fibers. According to this theory, the low-fiber diet causes small-caliber stools, increases the pressure inside the colon, and ultimately, herniation of the colon mucosa and submucosa through the muscle layers of the colon wall, which are adjacent to the vasa recta (Painter and Burkitt 1971 ). Diverticular disease has been associated with alterations of bacterial flora; in the peri-diverticula, which cause diverticular inflammation as well as abdominal symptoms (Lahner et al. 2016 ). The alterations of bacterial flora, which occur as a result of slow colonic transit and fecal stagnation, impair the mucosal barrier function and enhance the release of inflammatory cytokines, which ultimately lead to inflammation of the colon (Tursi 2007 ). In response to bacterial invasion, the colon’s epithelial cells release inflammatory cytokines. The inflammatory cytokines, such as tumor necrosis factor-alpha (TNF)-α and interferon-gamma (IFN-γ), have been shown to increase the intestinal permeability (Corridoni et al. 2012 ). Due to the concept that diverticulitis is inflammatory as well as an infection-associated disease (Strate and Morris 2019 ); and since bacteria are responsible for the inflammation, antibiotics have been the cornerstone for the treatment of acute diverticulitis. Moreover, because the colon harbors so many bacterial species, broad-spectrum antibiotics, are prescribed treatment that targets a broad range of bacteria, including Bacteroides as well as other anaerobic bacteria. Amoxicillin-clavulanic acid is effective against both types of bacteria (Feingold et al. 2014 ). Inpatient treatment of acute diverticulitis encompasses intravenous fluid resuscitation, intravenous antibiotics, and bowel rest (Daniels et al. 2014 ). The outpatient treatment of mild or uncomplicated acute diverticulitis follows new guidelines, for the management of acute diverticulitis, which suggests that; antibiotics can be used selectively rather than routinely (Balasubramanian et al. 2017 ). On the other hand, the probiotics have been reviewed, by different authors, in the management of diverticular disease (Ojetti et al. 2018 ). The authors have demonstrated that; probiotics exert beneficial effects in the prevention and treatment of several gastrointestinal diseases through modulation of the gut microbiota composition (Rondanelli et al. 2017 ). Lactobacillus and Bifidobacterium are two types of probiotics that extensively observed in the human intestine. The previous studies demonstrated that; these probiotics strains have antiinflammatory and immunomodulatory activities (Yao et al 2017 ). Furthermore, It was observed that; Bifidobacterium adolescentis , Lactobacillus , and Phascolarctobacterium were reduced in patients with intestinal inflammation. Interestingly, when they were present, they reduced inflammation by acting on C-reactive protein (CRP), interleukin (IL)-6, and tumor necrosis factor (TNF)-α (Al Bander et al. 2020 ). In this study, data from diverticulitis rat model highlight the critical involvement of dysregulated immune responses and impaired colonic epithelial defense system in the pathogenesis of acute diverticulitis disease. In addition, this study aimed to investigate the preventive and potential therapeutic value of antibiotics and probiotics in a rat model of acute diverticulitis disease. 2. Materials And Methods 2.1. Chemicals and Drugs Dextran sulfate sodium (DSS) salt, (molecular weight: 40,000), was purchased from Alfa Aesar (ThermoFisher, Kandel, GmbH, Germany); Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA); Probiotics ( Lactobacillus acidophilus and Bifidobacterium lactis ) were purchased from PharmaCare Europe Ltd (West Sussex, RH10 9NQ, UK). Amoxicillin antibiotic was purchased from Egyptian International Pharmaceutical Industries Company (Industrial Area, Egypt). DSS and LPS were dissolved in distilled water. Amoxicillin was suspended in distilled water, and shaken well before treatment. The probiotics pellets were dilacerated using a ceramic mortar and pestle; then, suspended in distilled water, and shaken well before treatment. 2.2. Experimental Animals Male albino rats (Sprague Dawley), weighing 150–160 g, and obtained from the animal house of the National Organization for Drug Control and Research (NODCAR, Giza, Egypt); were used in the present study. The rats were housed in the laboratory room for one week before the commencement of the experiment, under controlled environmental conditions; constant temperature (25 ± 2 ° C), humidity (60 ± 10%), and alternating 12 hours of light/dark cycles. Standard pellet diet and water were allowed ad libitum. All animal procedures were performed following the Institutional Ethics Committee and under the recommendations for the proper care and use of laboratory animals. The study was approved by the Ethics Committee for Animal Experimentation of Cairo University with approval number (CUIF6219). Unnecessary disturbance of animals was avoided. Animals were treated gently; squeezing, pressure, and tough maneuvers were avoided. 2.3. Experimental Design The rats were randomly divided into seven groups, with 6 rats in each group, for a study period of seven days, as follows: group I , control group: rats only received water and food (no treatment); group II , DSS group: rats were received 3% DSS solution, added to their drinking water, daily for seven days; group III , LPS group: rats were injected with LPS enema, by a catheter, at the dose of (4 mg/kg), 48 hours before sacrificing them at the end of the experiment; group IV , amoxicillin-treated group: rats were treated with amoxicillin (0.162 g suspended in 2 ml distilled water/rat, by oral gavage, once daily for seven days; group V , DSS/LPS group: in which acute diverticulitis was induced, rats were received DSS and LPS enema, similar to the ways mentioned above, in the DSS and LPS groups; group VI , DSS/LPS-amoxicillin-treated group: rats were received DSS, LPS, and amoxicillin; at the doses and ways mentioned in DSS, LPS, and amoxicillin groups; group VII , DSS/LPS-probiotics-amoxicillin-treated group: rats were received DSS, LPS, amoxicillin (at the doses and ways mentioned in DSS, LPS, and amoxicillin groups), as well as probiotics ( Lactobacillus acidophilus and Bifidobacterium lactis ) each of which (4 x 10 8 CFU suspended in 2 ml distilled water) orally, once daily for seven days. 2.4. Induction of Diverticulitis Acute diverticulitis was experimentally induced by adding 3% weight/volume of DSS, dissolved in distilled water, to the rats’ drinking water; daily for seven days (Masubuchi and Horie 2004 ). Furthermore, the rats were injected with LPS enema, at the dose of (4 mg/kg), by a Nelaton catheter 8 FG, 48 hours before sacrificing the rats; at the end of the experiment, where local immune reaction by LPS seems to play an important role in the perpetuation of experimental diverticulitis (Hotta et al. 1986 ) and aggravating colon inflammation (Zhang et al. 2011 ). 2.5. Assessment of Body Weight Loss, Colon Weight/Length Ratio, and Stool Score The alteration in rats’ body weights was monitored from the beginning to the end of the experiment. The colons were excised, opened longitudinally, and washed with phosphate-buffered saline. The length of the colon was measured from the ileocecal junction to the anal verge. The colonic weight-to-length ratio was calculated for each rat. The stool scoring criteria were performed according to Yamada et al. ( 2014 ) as following: stool consistency (0 = normal; 2 = loose stools; 4 = watery diarrhea) and the occurrence of gross blood in the stool (0 = negative; 4 = positive). 2.6. Colon Macroscopic Lesions Evaluation Colonic damage evaluation was performed according to Cuzzocrea et al. ( 2003 ) as following: (0 = no injury; 1 = localized hyperemia; 2 = ulcers; 3 = ulcers with inflammation; 4 = extending ulcers with inflammation (more than 1 centimeter along the length of the colon); and 5 = ulceration covering 2 centimeters, the score is increased 1 point for each additional centimeter of ulceration covering the length of the colon. 2.7. Blood Sampling and Colon Tissue Preparation At the completion of the days of the experiment, the rats were anesthetized with 4% isofluorane and sacrificed to enable blood and tissue collection. The serum was separated from the rats’ blood by centrifugation of blood at 4000 R.P.M. for 10 minutes, divided into aliquots, and stored at − 70 ° C until used for analysis. The colons were excised from the ileocecal junction to the anus and the colon damage was evaluated macroscopically for each rat. Part of the colon from each rat (unique for all rats, 8 cm away from the anus) was separated and fixed in 10% formaldehyde for use in the histopathological examination. The rest of the colon tissues were wrapped in aluminum foil and kept frozen at − 70 ° C until used for analysis. 2.8. Determination of Myeloperoxidase (MPO) Activity in the Colon Tissue MPO activity was evaluated using a kinetic colorimetric technique described by Bradley et al. ( 1982 ). The colon tissues’ homogenates were subjected to three cycles of freezing and thawing (–70 ° C/37 ° C). Then, the homogenates were centrifuged at 10000 rpm at 4 ⸰ C for 15 minutes. 50 microliter of the supernatant were separated for use in the MPO assay. MPO activity was evaluated by adding and incubating 50 microliters of the supernatant for 5 minutes at 37 ° C to 2.4 milliliters of 50 mM potassium phosphate buffer (K 2 HPO 4 ), PH 6.0, containing 0.167 mg/ml of ortho-dianisidine dihydrochloride, and 4.0 microliter of 30% hydrogen peroxide (H 2 O 2 ). Ortho-dianisidine is oxidized by MPO in the presence of H 2 O 2 and produces a yellowish-orange product that can be absorbed at 460 nm. One unit of MPO activity is defined as that required to degrade 1 µmol of H 2 O 2 per minute at 25 ° C. The results were expressed as (U/g of tissue). 2.9. Determination of Serum C-Reactive Protein (CRP) Level Serum CRP was measured using a Spinreact® kit (Girona, Spain). The test is based on the principle of latex agglutination. 2.10. Measurement of Pro-Inflammatory Cytokines in the Colon Tissue The levels of the colonic TNF-α, IL-1𝛽, IFN-γ, and IL-18 were measured using a colorimetric sandwich enzyme-linked immunosorbent assay (ELISA) kit (MyBiosource, Inc., San Deigo, USA), according to the manufacturer instructions. 2.11. Colon Microscopic Lesions Evaluation Colon tissues fixed in 10% formalin were washed and dehydrated in ascending grades of alcohol. Next, the specimens were cleared in xylene, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) for examination. Histological scores were assigned according to Vowinkel et al. ( 2004 ) as following: severity of inflammation (0 = no inflammation; 1 = slight inflammation; 2 = moderate inflammation; 3 = severe inflammation), depth of injury (0 = no injury; 1 = mucosal damage; 2 = mucosal and submucosal damage; 3 = transmural injury), and crypt damage (0 = no crypt damage; 1 = basal one-third damaged; 2 = basal two-thirds damaged; 3 = only surface epithelium intact; 4 = entire crypt and epithelium lost, congested dilated vessels. 2.12. Statistical Analysis Statistical differences between groups were computed by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test for multiple comparisons. The results were analyzed using the GraphPad Prism program (GraphPad software 5, San Diego, CA, USA). P < 0.05 was considered statistically significant. Data were expressed as mean ± standard error of the mean (SEM). 3. Results 3.1. Effect of Amoxicillin and Probiotics-Amoxicillin on Rats’ Body Weight, Colon Weight/Length Ratio, and Stool Score As shown in ( Figs. 1 A, B, and C) , the significance of rats’ body weight loss ( p < 0.001), high colon weight-to-length ratio ( p < 0.01), and a high stool score ( p < 0.01); was demonstrated in the DSS/LPS group, compared to the control group. Otherwise, treatment with amoxicillin; in DSS/LPS manipulated rats; showed a significant decrease in colon weight-to-length ratio ( p 0.05) in the rats’ body weight loss and stool score; when compared to DSS/LPS group. Furthermore, treatment with probiotics-amoxicillin markedly increased the rats’ body weight ( p < 0.001) and decreased the colon weight-to-length ratio ( p 0.05) in the stool score; compared to DSS/LPS group. 3.2. Effect of Amoxicillin; Probiotics-Amoxicillin on Macroscopic Lesions of Colon Tissue The rats, in the control group, showed normal colon tissues, while the rats treated with amoxicillin showed localized hyperemia. Administration of DSS or LPS enema; caused localized hyperemia and ulcers. In addition, the combination of DSS administration with LPS enema caused localized hyperemia as well as extending ulcers with inflammation. Otherwise, the colons of amoxicillin-treated rats, which were subjected to DSS/LPS, didn’t show any inflammation; however, the localized hyperemia and ulcers were still present. Moreover, probiotics-amoxicillin treatment, in the DSS/LPS-probiotics-amoxicillin group, markedly reduced the colon macroscopic damage, since the colons didn’t show any ulcers or inflammation, and there was minimal hyperemia ( Fig. 1 D ) . 3.3. Effect of Amoxicillin and Probiotics-Amoxicillin on MPO Activity and CRP Level MPO activity; in the colon tissue; was significantly elevated ( p < 0.001) in DSS and DSS/LPS groups; and ( p < 0.01) in the LPS group; compared to the control group. On the other hand, MPO activity significantly decreased ( p < 0.05) with amoxicillin; and ( p 0.05) in the serum CRP level; compared to the control group. Contrariwise, the serum CRP level was significantly increased ( p < 0.001) in the DSS/LPS group; compared to the control group. Amoxicillin, with or without probiotics, could reverse the increase in the serum CRP level; in comparison to DSS/LPS group ( Fig. 2 B ) . 3.4. Effect of Amoxicillin and Probiotics-Amoxicillin on Pro-Inflammatory Cytokines in the Colon Tissue Either administration of DSS or DSS/LPS; caused a significant increase ( p < 0.001) in the inflammatory markers (TNF-α, IL-1β, IFN-γ, and IL-18 levels); compared to the control group. The LPS enema only resulted in raising TNF-α level ( p < 0.01); compared to the control group. Treatment of rats with amoxicillin or probiotics-amoxicillin significantly ameliorated this increase ( p < 0.001); compared to the DSS/LPS group ( Figs. 3 A, B, C, and D) . 3.5. Effect of Amoxicillin and Probiotics-Amoxicillin on Colon Histopathology As shown in ( Figs. 4 A, and B) , colon tissues of the control and amoxicillin groups didn’t show any observable histopathological changes. Contrariwise, colon tissues of the DSS group showed moderate inflammation, mucosal and submucosal injury, and only surface intact ( Fig. 5 A ) . The colon tissues of the LPS group displayed slight inflammation, mucosal damage, and basal crypt damage ( Fig. 5 B ) . Furthermore, the colon tissues of the DSS/LPS group showed severe inflammation and lymphocytes infiltration, entire crypt and epithelium lost, and also there were congested dilated vessels ( Figs. 5 C, and D) . Conversely, treatment with amoxicillin revealed mucosal and submucosal inflammatory cells infiltration, dilated blood vessels, and degenerative mucosal changes with damaged crypts ( Fig. 6 A ) . The colon tissues of DSS/LPS-probiotics-amoxicillin-treated rats showed amelioration of the colon injury, slight inflammation, and few mucosal lymphocytes infiltration ( Fig. 6 B ) . The total histological scores were shown in ( Fig. 7 ) based on the histological grading criteria. 4. Discussion Diverticular disease refers to a spectrum of alterations in the intestinal tract; that begins with diverticulosis, or herniation of mucosa and submucosa through the muscle layer of the colon wall, commonly in regions where the mural blood vessels penetrate through the muscle layer of the colon wall (Kleessen et al. 1999 ). In terms of diverticulitis pathogenesis, it has been proposed that; diverticulitis develops from obstruction of a diverticulum's neck, resulting in bacterial proliferation, local ischemia, and micro-perforation (Jacobs 2007 ). Thus, dysbiosis is considered to be an important determinant in the pathogenesis of diverticulitis; since the imbalance in the microbial milieu leads to disruption of the immune homeostasis and causes intestinal diseases (Chassaing and Darfeuille-Michaud 2011 ). Otherwise, the interactions between the intestinal flora and the host immune system have a critical role in the prevention of intestinal diseases, where the commensal microbiome enhances the maturation of the mucosal immune system, whereas the pathogenic microbiome causes immunity dysfunction; which leads to disease development (Shi et al. 2017 ). As a consequence, using antibiotics or probiotics to manipulate the gut microbiota composition has recently been recommended as a therapeutic strategy for acute diverticulitis disease. Antibiotics have long been the cornerstone for the management of acute diverticulitis, intending to prevent inflammation and alleviate associated symptoms (Hanna and Kaiser 2021 ). In this study, we showed that amoxicillin (especially together with probiotics) had protective effects on DSS/LPS-induced diverticulitis. DSS induces intestinal inflammation, which damages the epithelial monolayer lining the large intestine, allowing the dissemination of pro-inflammatory intestinal contents (e.g. bacteria and their products) into the underlying tissue (Chassaing et al. 2014 ). Furthermore, LPS aggravates colon inflammation (Zhang et al. 2011 ). In this study, DSS together with LPS enema gave rise to a significant decrease in the rats’ body weights. This loss in body weight is due to a deficiency of nutrients resulting from food aversion, reduced appetite, malabsorption, as well as loss of body fluids through colorectal bleeding and diarrhea (Hunschede et al. 2017 ). Also, there was an increase in the colon weight-to-length ratio due to tissue edema, necrosis, and inflammatory cell infiltration confirmed in our study through the high macroscopic and microscopic lesion scores. These results agreed with Peran et al. ( 2007 ) and Khodir et al. ( 2019 ). Also, Mahoro et al. ( 2021 ) found that; DSS induced bloody diarrhea, weight loss, shortening of the colon, and mucosal deterioration in a rat model of DSS-induced colitis. Otherwise, only treatment with amoxicillin together with probiotics could significantly ameliorate the body weight loss caused by DSS/LPS, while both treatments (amoxicillin and probiotics-amoxicillin) showed inhibitory effects on the high colon weight-to-length ratio ( p < 0.01), as well as colon macroscopic lesion score ( p < 0.001). Applegate et al. ( 2010 ) clarified that; treatment with probiotics results in bacterial antagonism, colonization competition, and emulation for nutrients. These actions lead to amelioration of toxic compounds, modulation of the immune system, increasing nutrient absorption and digestibility, and ultimately decline in the body weight loss. Lactobacillus and Bifidobacterium strains have anti-inflammatory and immunomodulatory activities. Moreover, certain Lactobacillus strains can upregulate the expression of mucin-3 and enhance the intestinal mucus layer (Yao et al 2017 ), so they could inhibit DSS/LPS-induced damage in the colon tissue. MPO is a member of the peroxidases subfamily and has more expression in immune cells such as neutrophils, lymphocytes, monocytes, and macrophages. The enhanced level of MPO activity is a well diagnostic marker of inflammation and oxidative stress (Khan et al. 2018 ). In this study, combined treatment of probiotics and amoxicillin was more effective than amoxicillin alone in ameliorating the elevated MPO activity caused by DSS/LPS. This amelioration is due to the anti-inflammatory properties. The decrease in this enzyme activity reveals a lower infiltration of neutrophils in the inflamed colon tissue; since treatment with probiotics caused a reduction in the colonic production of the chemotactic eicosanoid LTB4. It was observed that; Lactobacillus acidophilus administration reduced colonic MPO activity in the trinitrobenzene sulfonic acid (TNBS) model of rat colitis (Peran et al. 2007 ). CRP is one of the most prominent proteins in acute inflammation (Norouzinia et al. 2017 ). It has been found that; higher levels of CRP, as well as the inflammatory markers, have been detected in patients with severe acute diverticulitis disease (Lahat et al. 2019 ). In the present study, DSS/LPS administration caused a significant elevation ( p < 0.001) in the serum level of CRP. This elevation was declined on either treatment with amoxicillin or probiotics-amoxicillin due to the inhibition of inflammation and disease progression. According to Sartelli et al. ( 2020 ), CRP is a valuable biomarker of inflammation and can be used in the prediction of the severity of acute diverticulitis. Furthermore, the current study revealed that DSS/LPS resulted in an elevation of the levels of TNF-α, IL-1β, IFN-γ, and IL-18. Pro-inflammatory cytokines such as IL-1β and TNF-α have been involved in the inflammatory process in DSS-induced colitis (Triantafillidis et al. 2011 ). Lahat et al. ( 2019 ) demonstrated that; patients with severe acute diverticulitis have higher tissue inflammatory cytokine levels including TNF-α, IL-6, IL-1β, and more inflammatory infiltrates in diverticular colon tissue. On the other hand, the elevation in TNF-α, IL-1β, IFN-γ, and IL-18 levels was significantly declined on treatment with amoxicillin or probiotics-amoxicillin. This inhibitory effect is attributed to the existence of a cross-talk between probiotics and mucosal cells. Bifidobacterium lactis , either alone or in combination with other probiotics, was able to downregulate the degree of activation of intestinal immune cells in the TNBS model of rat colitis (Peran et al. 2007 ). Probiotics may restore the balance of gut flora by decreasing pathogenic gram-negative bacteria; that may have been altered in diverticular disease due to stasis and reduced colonic transit time, and have been proposed to be used in diverticular disease to prevent inflammation (Boynton and Floch 2013 ). The biochemical analysis and the colonic macroscopic lesions evaluation were confirmed by the histological study, where DSS/LPS caused mucosal and submucosal inflammatory cells infiltration, dilated blood vessels, and degenerative mucosal changes with damaged crypts. Both Schieffer et al. ( 2018 ) and Tursi et al. ( 2020 ) demonstrated similar changes that occurred in the architecture of the colon wall, including loss of elasticity function and deposition of immature collagen fibers in the extracellular matrix, which are implicated in the formation of diverticula as part of the pathophysiology of diverticular disease. Treatment with probiotics-amoxicillin was more effective than treatment with amoxicillin alone in improving the architecture of the colon tissue since the colon tissues showed slight inflammation and few mucosal lymphocytes infiltration on probiotics-amoxicillin treatment while showed few mucosal and submucosal inflammatory cells infiltration and degenerative mucosal changes with few damaged crypts on amoxicillin treatment. Conclusively, antibiotics have been used to treat acute diverticulitis in all patients. Recent findings have indicated that; the manipulation of antibiotics is not necessary for mild or moderate uncomplicated acute diverticulitis management, as was initially thought (Feuerstein and Falchuk 2016 ). Treatment of acute diverticulitis generally comprises dietary fiber supplementation, anti-inflammatory drugs, pharmacological therapies such as antibiotics, as well as probiotics, either alone or in combination (Tursi et al. 2015 ). The present study revealed that each of the treatments attenuated the severity of DSS/LPS-induced acute diverticulitis in rats and displayed differing effectiveness in disease parameters. Combined probiotics-amoxicillin therapy was more effective in restoring the rats’ body weight, inhibiting the inflammatory markers tested in this study, and improving the epithelium damage score. Further studies are required in order to understand how probiotics can be employed in treating acute diverticulitis. The impact of the intestinal milieu, especially enteric microbiota, appears to be of great significance. Our study demonstrates that; probiotics enhanced the prevention of DSS/LPS-induced acute diverticulitis, so we suggest employing probiotics in treating acute diverticulitis disease. Declarations Authors Contributions Maha G Solaiman— Administrative support, interpretation, and review of the article. Hanna A Mansour— Conception and design, interpretation, review of the article, and editing. Wedad A Hassan— Provision of study materials, design, and data analysis. Eman Shawky— Collection and assembly of data, data analysis, and writing of the article. Funding This study didn ’ t receive any specific grant from funding agencies. Conflict of Interests The authors declare that there are no conflicts of interest. Data Availability The data used to support the findings of this study are available from the corresponding author when requested. References Al Bander, Z, Nitert MD, Mousa A, Naderpoor N (2020) The Gut Microbiota and Inflammation: An Overview. Int J Environ Res Public Health 17:7618. Applegate T, Klose V, Steiner T, Ganner A, Schatzmayr G (2010) Probiotics and phytogenics for poultry: Myth or reality? J Appl Poult Res 19:194-210. Balasubramanian I, Fleming C, Mohan HM, Schmidt K, Haglind E, Winter DC (2017) Out-Patient Management of Mild or Uncomplicated Diverticulitis: A Systematic Review. Dig Surg 34:151-160. 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Norouzinia M, Chaleshi V, Mohammad Alizadeh AH, Zali MR (2017) Biomarkers in inflammatory bowel diseases: insight into diagnosis, prognosis and treatment. Gastroenterol Hepatol Bed Bench 10:155-167. Ojetti V, Petruzziello C, Cardone S, et al (2018) The use of probiotics in different phases of diverticular disease. Rev Recent Clin Trial 13:89-96. Paget GE, Barnes JM (1964) Interspecies dosage conversion scheme in evaluation of results and quantitative application in different species. In: Evaluation of Drug Activities: Pharmacometrics” vol. 1, Laurence Dr, Bacharach AL. Eds.; Acamemic Press, London and New York, 1964:160-162. Painter NS, Burkitt DP (1971) Diverticular disease of the colon: a deficiency disease of Western civilization. Br Med J 2:450-454. Peran L, Sierra S, Comalada M, et al (2007) A comparative study of the preventative effects exerted by two probiotics, Lactobacillus reuteri and Lactobacillus fermentum, in the trinitrobenzenesulfonic acid model of rat colitis. Br J Nutr 97:96-103. Rondanelli M, Faliva MA, Perna S, et al (2017) Using probiotics in clinical practice: Where are we now? A review of existing metaanalyses Gut Microbes 8:521-43. Sartelli M, Weber DG, Kluger Y, Ansaloni L, et al (2020) 2020 update of the WSES guidelines for the management of acute colonic diverticulitis in the emergency setting. World J Emerg Surg 15:32. Schieffer KM, Kline BP, Yochum GS, Koltun WA (2018) Pathophysiology of diverticular disease. Expert Rev Gastroenterol Hepatol 12:683-692. Shi N, Li N, Duan X, Niu H (2017) Interaction between the gut microbiome and mucosal immune system. Mil Med Res 4:14. Strate LL, Morris AM (2019) Epidemiology, Pathophysiology, and Treatment of Diverticulitis. Gastroenterology 156:1282-1298. Strate LL, Peery AF, Neumann I (2015) American Gastroenterological Association Institute technical review on the management of acute diverticulitis. Gastroenterology 149:1950-1976. Triantafillidis JK, Merikas E, Georgopoulos F (2011) Current and emerging drugs for the treatment of inflammatory bowel disease. Drug Des Devel Ther 5:185-210. Tursi A (2007) New physiopathological and therapeutic approaches to diverticular disease of the colon. Expert Opin Pharmacother 8:299-307. Tursi A, Papa A, Danese S (2015) Review article: the pathophysiology and medical management of diverticulosis and diverticular disease of the colon. Aliment Pharmacol Ther 42:664-684. Tursi A, Scarpignato C, Strate LL, et al (2020) Colonic diverticular disease. Nat Rev Dis Primers 6:20. Vowinkel T, Kalogeris TJ, Mori M. et al (2004) Impact of Dextran Sulfate Sodium Load on the Severity of Inflammation in Experimental Colitis. Dig Dis Sci 49:556-564. Wedel T, Barrenschee M, Lange C, Cossais F, Böttner M (2015) Morphologic basis for developing diverticular disease, diverticulitis, and diverticular bleeding. Viszeralmedizin 31:76-82. Wess L, Eastwood M, Busuttil A, Edwards C, Miller A (1996) An association between maternal diet and colonic diverticulosis in an animal model. Gut 39:423-427. Yamada S, Koyama T, Noguchi H, et al (2014) Marine hydroquinone zonarol prevents inflammation and apoptosis in dextran sulfate sodium-induced mice ulcerative colitis. PLoS One 9:e113509. Yamamichi N, Shimamoto T, Takahashi Y, et al (2015) Trend and risk factors of diverticulosis in Japan: age, gender, and lifestyle/metabolic-related factors may cooperatively affect on the colorectal diverticula formation. PLoS One 10:e0123688. Yao P, Tan F, Gao H, Wang L, Yang T, et al (2017) Effects of probiotics on Toll like receptor expression in ulcerative colitis rats induced by 2,4,6 trinitro benzene sulfonic acid. Molecular Medicine Reports 15:1973-1980. Zhang M , Long Y , Sun Y , et al (2011) Evidence for the complementary and synergistic effects of the three-alkaloid combination regimen containing berberine, hypaconitine and skimmianine on the ulcerative colitis rats induced by trinitrobenzene-sulfonic acid. Eur J Pharmacol 651:187-196. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 27 May, 2022 Reviewers invited by journal 26 May, 2022 Editor invited by journal 26 May, 2022 Editor assigned by journal 26 May, 2022 First submitted to journal 25 May, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1694157","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":109049946,"identity":"a4740391-80a2-451c-b582-3a8fecd722d9","order_by":0,"name":"Eman Shawky","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBAC+wYeIHkAxGRsfABl4QcGDAgtzQakamFgkyBOC//Zg595zhxmkG8/3FbNU3NHjp+B+eGjG3i02EvkJUvz3DjMYHAmse02z7FnxpINbMbGOfhskeAxkM75ANQiwQjUwnY4ccMBHjZpvFr4zxj/BmmRn8HYVszzjxgtDDlm0jlAhzHcYGxj5m0jRotEjpn1nzPpIL80S87tO2ws2UzAL/b9Z4xvzjhmDQyx4w8/vPl2WI6fvfnhY3xaoKC5vgFIMoHiiIGZsHIQqAOTjD+IUz0KRsEoGAUjDAAAemlO4nCCLC0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3321-0384","institution":"Al-Azhar University Faculty of Science","correspondingAuthor":true,"prefix":"","firstName":"Eman","middleName":"","lastName":"Shawky","suffix":""},{"id":109049947,"identity":"5db9ca09-f69c-48fa-acd2-df79ea5ede9e","order_by":1,"name":"Maha Ghazy Solaiman","email":"","orcid":"","institution":"Al-Azhar University Faculty of Science","correspondingAuthor":false,"prefix":"","firstName":"Maha","middleName":"Ghazy","lastName":"Solaiman","suffix":""},{"id":109049948,"identity":"26ca40ed-0206-4677-a882-e2f85acc9b8c","order_by":2,"name":"Hanaa Abd-Elfattah Mansour","email":"","orcid":"","institution":"National Organization for Drug Control and Research Pyramids Branch: Egyptian Drug Authority Pyramids Branch","correspondingAuthor":false,"prefix":"","firstName":"Hanaa","middleName":"Abd-Elfattah","lastName":"Mansour","suffix":""},{"id":109049949,"identity":"a559c453-8ae6-4185-af7d-31427b6db21b","order_by":3,"name":"Wedad Abdallah Hassan","email":"","orcid":"","institution":"National Organization for Drug Control and Research Pyramids Branch: Egyptian Drug Authority Pyramids Branch","correspondingAuthor":false,"prefix":"","firstName":"Wedad","middleName":"Abdallah","lastName":"Hassan","suffix":""}],"badges":[],"createdAt":"2022-05-25 21:50:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1694157/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1694157/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":22099166,"identity":"3d7f3a53-8e7c-4aec-bc2e-8f7976401e67","added_by":"auto","created_at":"2022-05-31 21:25:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":337811,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of amoxicillin and probiotics-amoxicillin on DSS/LPS-induced acute diverticulitis. Levels of the pro-inflammatory cytokines \u003cstrong\u003e(A)\u003c/strong\u003e Rats’ body weight, \u003cstrong\u003e(B)\u003c/strong\u003e colon weight-to-length ratio, \u003cstrong\u003e(C)\u003c/strong\u003e stool score, and \u003cstrong\u003e(D)\u003c/strong\u003e colon macroscopic lesions score. Data are means, with their standard errors; represented by vertical bars. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer test. *,** and *** represent significant differences from the control group (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0·05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0·01,\u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·001). \u003csup\u003e\u003cstrong\u003e##\u003c/strong\u003e\u003c/sup\u003e and \u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e represent significant differences from DSS/LPS group (\u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·05,\u003csup\u003e \u003cstrong\u003e##\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·01, and \u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·001).\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1694157/v1/7015bb44433d5bbd986ba21b.png"},{"id":22100097,"identity":"da704d85-ba6c-4a1b-bc1b-b15a5366b116","added_by":"auto","created_at":"2022-05-31 21:35:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":92305,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of amoxicillin and probiotics-amoxicillin on DSS/LPS-induced acute diverticulitis. \u003cstrong\u003e(A)\u003c/strong\u003e MPO (myeloperoxidase) activity, in the colon tissue, \u003cstrong\u003e(B)\u003c/strong\u003e serum CRP (C-reactive protein) concentration. Data are means, with their standard errors; represented by vertical bars. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer test. ** and *** represent significant differences from the control group \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;(\u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0·01,\u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·001). \u003csup\u003e\u003cstrong\u003e#, ##,\u003c/strong\u003e\u003c/sup\u003e and \u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e represent significant differences from DSS/LPS group (\u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·05,\u003csup\u003e \u003cstrong\u003e##\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·01, and \u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·001).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1694157/v1/169d1abfd4b9aceb362cffce.png"},{"id":22099721,"identity":"c1deb9cc-9a15-4f95-b117-fd190254647b","added_by":"auto","created_at":"2022-05-31 21:30:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":233363,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of amoxicillin and probiotics-amoxicillin on DSS/LPS-induced acute diverticulitis. Levels of the pro-inflammatory cytokines \u003cstrong\u003e(A)\u003c/strong\u003e TNF-α (tumor necrosis factor alpha), \u003cstrong\u003e(B)\u003c/strong\u003e IL-1β (interleukin-1 beta), \u003cstrong\u003e(C)\u003c/strong\u003e IFN-γ (interferon gamma), and \u003cstrong\u003e(D)\u003c/strong\u003e IL-18 (interleukin-18), in the colon tissue, were determined by ELISA. Data are means, with their standard errors; represented by vertical bars. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer test. *,** and *** represent significant differences from the control group (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0·05,\u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0·01,\u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·001). \u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e represents significant differences from DSS/LPS group (\u003csup\u003e\u003cstrong\u003e###\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·001).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1694157/v1/180d68f2032f6a1f62c0e3bc.png"},{"id":22099164,"identity":"0b72bfd7-a7e9-4ccb-a81a-06f3c54ab991","added_by":"auto","created_at":"2022-05-31 21:25:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":445884,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of rat colonic tissue from \u003cstrong\u003e(A)\u003c/strong\u003e control group showing no histopathological alterations, \u003cstrong\u003e(B)\u003c/strong\u003e amoxicillin-treated group showing almost normal histopathological appearance with few cellular infiltrations (black arrow) (H \u0026amp; E 100 x).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1694157/v1/0b72a9934e2dc016df47bb43.png"},{"id":22099723,"identity":"b40b46f2-eda8-49ee-acc6-17b0e1a181ed","added_by":"auto","created_at":"2022-05-31 21:30:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":960734,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of rat colonic tissue from \u003cstrong\u003e(A)\u003c/strong\u003e DSS group showing degenerative mucosal changes (blue arrow), hypertrophy of muscularis mucosa (black arrow), and congestion of submucosal blood vessel (red arrow), \u003cstrong\u003e(B)\u003c/strong\u003e LPS group showing few mucosal inflammatory cells infiltration (black arrow) and slight submucosal edema (blue arrow), \u003cstrong\u003e(C)\u003c/strong\u003e DSS/LPS group showing mucosal degenerative changes (black arrow) with; damaged crypt, shortening of the mucosa, depletion of mucous glands, and inflammatory cells infiltration (white arrow); degeneration of the muscularis externa (red arrow), and formation of crypt abscesses (blue arrows), \u003cstrong\u003e(D)\u003c/strong\u003e DSS/LPS group showing degenerative mucosal changes with massive lymphocytes infiltration (black arrows) which extended to the submucosa (red arrow), (H \u0026amp; E 100x).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-1694157/v1/5768b281f03ca6578c6f18a8.png"},{"id":22099165,"identity":"17455604-f06b-4b46-8cfb-e96539d59a95","added_by":"auto","created_at":"2022-05-31 21:25:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":468604,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of rat colonic tissue from \u003cstrong\u003e(A) \u003c/strong\u003eDSS/LPS-amoxicillin-treated group showing few mucosal (black arrow) and submucosal (blue arrow) inflammatory cells infiltration, and degenerative mucosal changes with damaged crypt (red arrow), \u003cstrong\u003e(B)\u003c/strong\u003e DSS/LPS-probiotics-amoxicillin-treated group showing few mucosal lymphocytes infiltration (black arrow) (H\u0026amp;E 100x).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-1694157/v1/d7100f1941600c35da7cf56a.png"},{"id":22099170,"identity":"4c7aa290-2880-40a7-b698-ca1cd42fa575","added_by":"auto","created_at":"2022-05-31 21:25:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":65062,"visible":true,"origin":"","legend":"\u003cp\u003eTotal histological scores based on the histological grading criteria. Data are means, with their standard errors. Statistical analysis was performed using one-way ANOVA followed by the Tukey–Kramer test. *, and *** represent significant differences from the control group (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0·05, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·001). \u003csup\u003e##, ###\u003c/sup\u003e represent significant differences from DSS/LPS group \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;(\u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0·001).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-1694157/v1/46f9e885fe6a58ff077b8fab.png"},{"id":22100098,"identity":"418cf3cb-5454-4070-ae8e-405144768090","added_by":"auto","created_at":"2022-05-31 21:35:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1952774,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1694157/v1/df6b5000-4bcc-4277-848b-82f7b5fe85d2.pdf"}],"financialInterests":"","formattedTitle":"Protective Effects of Amoxicillin and Probiotics on Colon Disorders in an Experimental Model of Acute Diverticulitis Disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAcute diverticulitis is a gastrointestinal disease, which is characterized by inflammation of the colon diverticulum and its surrounding mucosa (Strate et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Diverticulitis refers to the diverticular disease as well as the spectrum of complications associated with colon diverticulosis, such as bleeding (Cohen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The colon diverticulum is formed as a consequence of herniation of colon mucosa and submucosa; through the perivascular connective tissue sheath, which surrounds the intramural vasa recta (Wedel et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Part of the pathogenesis is returned to deterioration in the colon wall strength in aged individuals, which could be returned to the increased collagen cross-linkage of the colon wall (Wess et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Yamamichi et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) demonstrated age, gender, tobacco usage, obesity, pre-diabetic conditions, alcohol intake, as well as increased serum triglyceride concentrations, as risk factors for diverticular disease.\u003c/p\u003e \u003cp\u003eAcute diverticulitis is believed to develop when a colon diverticulum becomes obstructed by feces, resulting in fecal stasis, mucosal damage, and ischemia. The genetic factors, as well as alterations in the colon neuromusculature, take part in the development of diverticulitis (Strate and Morris \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, it was hypothesized that; diverticular disease results from the deficiency of dietary fibers. According to this theory, the low-fiber diet causes small-caliber stools, increases the pressure inside the colon, and ultimately, herniation of the colon mucosa and submucosa through the muscle layers of the colon wall, which are adjacent to the vasa recta (Painter and Burkitt \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). Diverticular disease has been associated with alterations of bacterial flora; in the peri-diverticula, which cause diverticular inflammation as well as abdominal symptoms (Lahner et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The alterations of bacterial flora, which occur as a result of slow colonic transit and fecal stagnation, impair the mucosal barrier function and enhance the release of inflammatory cytokines, which ultimately lead to inflammation of the colon (Tursi \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In response to bacterial invasion, the colon\u0026rsquo;s epithelial cells release inflammatory cytokines. The inflammatory cytokines, such as tumor necrosis factor-alpha (TNF)-α and interferon-gamma (IFN-γ), have been shown to increase the intestinal permeability (Corridoni et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to the concept that diverticulitis is inflammatory as well as an infection-associated disease (Strate and Morris \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); and since bacteria are responsible for the inflammation, antibiotics have been the cornerstone for the treatment of acute diverticulitis. Moreover, because the colon harbors so many bacterial species, broad-spectrum antibiotics, are prescribed treatment that targets a broad range of bacteria, including \u003cem\u003eBacteroides\u003c/em\u003e as well as other anaerobic bacteria. Amoxicillin-clavulanic acid is effective against both types of bacteria (Feingold et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Inpatient treatment of acute diverticulitis encompasses intravenous fluid resuscitation, intravenous antibiotics, and bowel rest (Daniels et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The outpatient treatment of mild or uncomplicated acute diverticulitis follows new guidelines, for the management of acute diverticulitis, which suggests that; antibiotics can be used selectively rather than routinely (Balasubramanian et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). On the other hand, the probiotics have been reviewed, by different authors, in the management of diverticular disease (Ojetti et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The authors have demonstrated that; probiotics exert beneficial effects in the prevention and treatment of several gastrointestinal diseases through modulation of the gut microbiota composition (Rondanelli et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e are two types of probiotics that extensively observed in the human intestine. The previous studies demonstrated that; these probiotics strains have antiinflammatory and immunomodulatory activities (Yao et al \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, It was observed that; \u003cem\u003eBifidobacterium adolescentis\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, and \u003cem\u003ePhascolarctobacterium\u003c/em\u003e were reduced in patients with intestinal inflammation. Interestingly, when they were present, they reduced inflammation by acting on C-reactive protein (CRP), interleukin (IL)-6, and tumor necrosis factor (TNF)-α (Al Bander et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, data from diverticulitis rat model highlight the critical involvement of dysregulated immune responses and impaired colonic epithelial defense system in the pathogenesis of acute diverticulitis disease. In addition, this study aimed to investigate the preventive and potential therapeutic value of antibiotics and probiotics in a rat model of acute diverticulitis disease.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Chemicals and Drugs\u003c/h2\u003e\n \u003cp\u003eDextran sulfate sodium (DSS) salt, (molecular weight: 40,000), was purchased from Alfa Aesar (ThermoFisher, Kandel, GmbH, Germany); Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA); Probiotics (\u003cem\u003eLactobacillus acidophilus\u003c/em\u003e and \u003cem\u003eBifidobacterium lactis\u003c/em\u003e) were purchased from PharmaCare Europe Ltd (West Sussex, RH10 9NQ, UK). Amoxicillin antibiotic was purchased from Egyptian International Pharmaceutical Industries Company (Industrial Area, Egypt). DSS and LPS were dissolved in distilled water. Amoxicillin was suspended in distilled water, and shaken well before treatment. The probiotics pellets were dilacerated using a ceramic mortar and pestle; then, suspended in distilled water, and shaken well before treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Experimental Animals\u003c/h2\u003e\n \u003cp\u003eMale albino rats (Sprague Dawley), weighing 150\u0026ndash;160 g, and obtained from the animal house of the National Organization for Drug Control and Research (NODCAR, Giza, Egypt); were used in the present study. The rats were housed in the laboratory room for one week before the commencement of the experiment, under controlled environmental conditions; constant temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003e\u0026deg;\u003c/sup\u003eC), humidity (60\u0026thinsp;\u0026plusmn;\u0026thinsp;10%), and alternating 12 hours of light/dark cycles. Standard pellet diet and water were allowed ad libitum. All animal procedures were performed following the Institutional Ethics Committee and under the recommendations for the proper care and use of laboratory animals. The study was approved by the Ethics Committee for Animal Experimentation of Cairo University with approval number (CUIF6219). Unnecessary disturbance of animals was avoided. Animals were treated gently; squeezing, pressure, and tough maneuvers were avoided.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3. Experimental Design\u003c/h2\u003e\n \u003cp\u003eThe rats were randomly divided into seven groups, with 6 rats in each group, for a study period of seven days, as follows: \u003cstrong\u003egroup I\u003c/strong\u003e, control group: rats only received water and food (no treatment); \u003cstrong\u003egroup II\u003c/strong\u003e, DSS group: rats were received 3% DSS solution, added to their drinking water, daily for seven days; \u003cstrong\u003egroup III\u003c/strong\u003e, LPS group: rats were injected with LPS enema, by a catheter, at the dose of (4 mg/kg), 48 hours before sacrificing them at the end of the experiment; \u003cstrong\u003egroup IV\u003c/strong\u003e, amoxicillin-treated group: rats were treated with amoxicillin (0.162 g suspended in 2 ml distilled water/rat, by oral gavage, once daily for seven days; \u003cstrong\u003egroup V\u003c/strong\u003e, DSS/LPS group: in which acute diverticulitis was induced, rats were received DSS and LPS enema, similar to the ways mentioned above, in the DSS and LPS groups; \u003cstrong\u003egroup VI\u003c/strong\u003e, DSS/LPS-amoxicillin-treated group: rats were received DSS, LPS, and amoxicillin; at the doses and ways mentioned in DSS, LPS, and amoxicillin groups; \u003cstrong\u003egroup VII\u003c/strong\u003e, DSS/LPS-probiotics-amoxicillin-treated group: rats were received DSS, LPS, amoxicillin (at the doses and ways mentioned in DSS, LPS, and amoxicillin groups), as well as probiotics (\u003cem\u003eLactobacillus acidophilus\u003c/em\u003e and \u003cem\u003eBifidobacterium lactis\u003c/em\u003e) each of which (4 x 10\u003csup\u003e8\u003c/sup\u003e CFU suspended in 2 ml distilled water) orally, once daily for seven days.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4. Induction of Diverticulitis\u003c/h2\u003e\n \u003cp\u003eAcute diverticulitis was experimentally induced by adding 3% weight/volume of DSS, dissolved in distilled water, to the rats\u0026rsquo; drinking water; daily for seven days (Masubuchi and Horie \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e). Furthermore, the rats were injected with LPS enema, at the dose of (4 mg/kg), by a Nelaton catheter 8 FG, 48 hours before sacrificing the rats; at the end of the experiment, where local immune reaction by LPS seems to play an important role in the perpetuation of experimental diverticulitis (Hotta et al. \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e) and aggravating colon inflammation (Zhang et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5. Assessment of Body Weight Loss, Colon Weight/Length Ratio, and Stool Score\u003c/h2\u003e\n \u003cp\u003eThe alteration in rats\u0026rsquo; body weights was monitored from the beginning to the end of the experiment. The colons were excised, opened longitudinally, and washed with phosphate-buffered saline. The length of the colon was measured from the ileocecal junction to the anal verge. The colonic weight-to-length ratio was calculated for each rat. The stool scoring criteria were performed according to Yamada et al. (\u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) as following: stool consistency (0\u0026thinsp;=\u0026thinsp;normal; 2\u0026thinsp;=\u0026thinsp;loose stools; 4\u0026thinsp;=\u0026thinsp;watery diarrhea) and the occurrence of gross blood in the stool (0\u0026thinsp;=\u0026thinsp;negative; 4\u0026thinsp;=\u0026thinsp;positive).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6. Colon Macroscopic Lesions Evaluation\u003c/h2\u003e\n \u003cp\u003eColonic damage evaluation was performed according to Cuzzocrea et al. (\u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e) as following: (0\u0026thinsp;=\u0026thinsp;no injury; 1\u0026thinsp;=\u0026thinsp;localized hyperemia; 2\u0026thinsp;=\u0026thinsp;ulcers; 3\u0026thinsp;=\u0026thinsp;ulcers with inflammation; 4\u0026thinsp;=\u0026thinsp;extending ulcers with inflammation (more than 1 centimeter along the length of the colon); and 5\u0026thinsp;=\u0026thinsp;ulceration covering 2 centimeters, the score is increased 1 point for each additional centimeter of ulceration covering the length of the colon.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.7. Blood Sampling and Colon Tissue Preparation\u003c/h2\u003e\n \u003cp\u003eAt the completion of the days of the experiment, the rats were anesthetized with 4% isofluorane and sacrificed to enable blood and tissue collection. The serum was separated from the rats\u0026rsquo; blood by centrifugation of blood at 4000 R.P.M. for 10 minutes, divided into aliquots, and stored at \u0026minus;\u0026thinsp;70\u003csup\u003e\u0026deg;\u003c/sup\u003eC until used for analysis. The colons were excised from the ileocecal junction to the anus and the colon damage was evaluated macroscopically for each rat. Part of the colon from each rat (unique for all rats, 8 cm away from the anus) was separated and fixed in 10% formaldehyde for use in the histopathological examination. The rest of the colon tissues were wrapped in aluminum foil and kept frozen at \u0026minus;\u0026thinsp;70\u003csup\u003e\u0026deg;\u003c/sup\u003eC until used for analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e2.8. Determination of Myeloperoxidase (MPO) Activity in the Colon Tissue\u003c/h2\u003e\n \u003cp\u003eMPO activity was evaluated using a kinetic colorimetric technique described by Bradley et al. (\u003cspan class=\"CitationRef\"\u003e1982\u003c/span\u003e). The colon tissues\u0026rsquo; homogenates were subjected to three cycles of freezing and thawing (\u0026ndash;70\u003csup\u003e\u0026deg;\u003c/sup\u003eC/37\u003csup\u003e\u0026deg;\u003c/sup\u003eC). Then, the homogenates were centrifuged at 10000 rpm at 4\u003csup\u003e⸰\u003c/sup\u003eC for 15 minutes. 50 microliter of the supernatant were separated for use in the MPO assay. MPO activity was evaluated by adding and incubating 50 microliters of the supernatant for 5 minutes at 37\u003csup\u003e\u0026deg;\u003c/sup\u003eC to 2.4 milliliters of 50 mM potassium phosphate buffer (K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), PH 6.0, containing 0.167 mg/ml of ortho-dianisidine dihydrochloride, and 4.0 microliter of 30% hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). Ortho-dianisidine is oxidized by MPO in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and produces a yellowish-orange product that can be absorbed at 460 nm. One unit of MPO activity is defined as that required to degrade 1 \u0026micro;mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e per minute at 25\u003csup\u003e\u0026deg;\u003c/sup\u003eC. The results were expressed as (U/g of tissue).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003e2.9. Determination of Serum C-Reactive Protein (CRP) Level\u003c/h2\u003e\n \u003cp\u003eSerum CRP was measured using a Spinreact\u0026reg; kit (Girona, Spain). The test is based on the principle of latex agglutination.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003e2.10. Measurement of Pro-Inflammatory Cytokines in the Colon Tissue\u003c/h2\u003e\n \u003cp\u003eThe levels of the colonic TNF-\u0026alpha;, IL-1𝛽, IFN-\u0026gamma;, and IL-18 were measured using a colorimetric sandwich enzyme-linked immunosorbent assay (ELISA) kit (MyBiosource, Inc., San Deigo, USA), according to the manufacturer instructions.\u003c/p\u003e\n \u003ch2\u003e\u003cstrong\u003e2.11. Colon Microscopic Lesions Evaluation\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eColon tissues fixed in 10% formalin were washed and dehydrated in ascending grades of alcohol. Next, the specimens were cleared in xylene, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H\u0026amp;E) for examination. Histological scores were assigned according to Vowinkel et al. (\u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e) as following: severity of inflammation (0\u0026thinsp;=\u0026thinsp;no inflammation; 1\u0026thinsp;=\u0026thinsp;slight inflammation; 2\u0026thinsp;=\u0026thinsp;moderate inflammation; 3\u0026thinsp;=\u0026thinsp;severe inflammation), depth of injury (0\u0026thinsp;=\u0026thinsp;no injury; 1\u0026thinsp;=\u0026thinsp;mucosal damage; 2\u0026thinsp;=\u0026thinsp;mucosal and submucosal damage; 3\u0026thinsp;=\u0026thinsp;transmural injury), and crypt damage (0\u0026thinsp;=\u0026thinsp;no crypt damage; 1\u0026thinsp;=\u0026thinsp;basal one-third damaged; 2\u0026thinsp;=\u0026thinsp;basal two-thirds damaged; 3\u0026thinsp;=\u0026thinsp;only surface epithelium intact; 4\u0026thinsp;=\u0026thinsp;entire crypt and epithelium lost, congested dilated vessels.\u003c/p\u003e\n \u003ch2\u003e\u003cstrong\u003e2.12. Statistical Analysis\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eStatistical differences between groups were computed by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test for multiple comparisons. The results were analyzed using the GraphPad Prism program (GraphPad software 5, San Diego, CA, USA). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Effect of Amoxicillin and Probiotics-Amoxicillin on Rats\u0026rsquo; Body Weight, Colon Weight/Length Ratio, and Stool Score\u003c/h2\u003e \u003cp\u003eAs shown in \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, \u003cb\u003eand C)\u003c/b\u003e, the significance of rats\u0026rsquo; body weight loss (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), high colon weight-to-length ratio (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and a high stool score (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); was demonstrated in the DSS/LPS group, compared to the control group. Otherwise, treatment with amoxicillin; in DSS/LPS manipulated rats; showed a significant decrease in colon weight-to-length ratio (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); while didn\u0026rsquo;t show any significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the rats\u0026rsquo; body weight loss and stool score; when compared to DSS/LPS group. Furthermore, treatment with probiotics-amoxicillin markedly increased the rats\u0026rsquo; body weight (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and decreased the colon weight-to-length ratio (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); while didn\u0026rsquo;t show a significant decrease (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the stool score; compared to DSS/LPS group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Effect of Amoxicillin; Probiotics-Amoxicillin on Macroscopic Lesions of Colon Tissue\u003c/h2\u003e \u003cp\u003eThe rats, in the control group, showed normal colon tissues, while the rats treated with amoxicillin showed localized hyperemia. Administration of DSS or LPS enema; caused localized hyperemia and ulcers. In addition, the combination of DSS administration with LPS enema caused localized hyperemia as well as extending ulcers with inflammation. Otherwise, the colons of amoxicillin-treated rats, which were subjected to DSS/LPS, didn\u0026rsquo;t show any inflammation; however, the localized hyperemia and ulcers were still present. Moreover, probiotics-amoxicillin treatment, in the DSS/LPS-probiotics-amoxicillin group, markedly reduced the colon macroscopic damage, since the colons didn\u0026rsquo;t show any ulcers or inflammation, and there was minimal hyperemia \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Effect of Amoxicillin and Probiotics-Amoxicillin on MPO Activity and CRP Level\u003c/h2\u003e \u003cp\u003eMPO activity; in the colon tissue; was significantly elevated (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in DSS and DSS/LPS groups; and (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the LPS group; compared to the control group. On the other hand, MPO activity significantly decreased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with amoxicillin; and (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) with probiotics-amoxicillin compared to the DSS/LPS group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Administration of DSS or LPS enema showed no significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the serum CRP level; compared to the control group. Contrariwise, the serum CRP level was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the DSS/LPS group; compared to the control group. Amoxicillin, with or without probiotics, could reverse the increase in the serum CRP level; in comparison to DSS/LPS group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Effect of Amoxicillin and Probiotics-Amoxicillin on Pro-Inflammatory Cytokines in the Colon Tissue\u003c/h2\u003e \u003cp\u003eEither administration of DSS or DSS/LPS; caused a significant increase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the inflammatory markers (TNF-α, IL-1β, IFN-γ, and IL-18 levels); compared to the control group. The LPS enema only resulted in raising TNF-α level (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); compared to the control group. Treatment of rats with amoxicillin or probiotics-amoxicillin significantly ameliorated this increase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001); compared to the DSS/LPS group \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, C, \u003cb\u003eand D)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Effect of Amoxicillin and Probiotics-Amoxicillin on Colon Histopathology\u003c/h2\u003e \u003cp\u003eAs shown in \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cb\u003eand B)\u003c/b\u003e, colon tissues of the control and amoxicillin groups didn\u0026rsquo;t show any observable histopathological changes. Contrariwise, colon tissues of the DSS group showed moderate inflammation, mucosal and submucosal injury, and only surface intact \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The colon tissues of the LPS group displayed slight inflammation, mucosal damage, and basal crypt damage \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Furthermore, the colon tissues of the DSS/LPS group showed severe inflammation and lymphocytes infiltration, entire crypt and epithelium lost, and also there were congested dilated vessels \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cb\u003eand D)\u003c/b\u003e. Conversely, treatment with amoxicillin revealed mucosal and submucosal inflammatory cells infiltration, dilated blood vessels, and degenerative mucosal changes with damaged crypts \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The colon tissues of DSS/LPS-probiotics-amoxicillin-treated rats showed amelioration of the colon injury, slight inflammation, and few mucosal lymphocytes infiltration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The total histological scores were shown in \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e based on the histological grading criteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDiverticular disease refers to a spectrum of alterations in the intestinal tract; that begins with diverticulosis, or herniation of mucosa and submucosa through the muscle layer of the colon wall, commonly in regions where the mural blood vessels penetrate through the muscle layer of the colon wall (Kleessen et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). In terms of diverticulitis pathogenesis, it has been proposed that; diverticulitis develops from obstruction of a diverticulum's neck, resulting in bacterial proliferation, local ischemia, and micro-perforation (Jacobs \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Thus, dysbiosis is considered to be an important determinant in the pathogenesis of diverticulitis; since the imbalance in the microbial milieu leads to disruption of the immune homeostasis and causes intestinal diseases (Chassaing and Darfeuille-Michaud \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Otherwise, the interactions between the intestinal flora and the host immune system have a critical role in the prevention of intestinal diseases, where the commensal microbiome enhances the maturation of the mucosal immune system, whereas the pathogenic microbiome causes immunity dysfunction; which leads to disease development (Shi et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). As a consequence, using antibiotics or probiotics to manipulate the gut microbiota composition has recently been recommended as a therapeutic strategy for acute diverticulitis disease. Antibiotics have long been the cornerstone for the management of acute diverticulitis, intending to prevent inflammation and alleviate associated symptoms (Hanna and Kaiser \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we showed that amoxicillin (especially together with probiotics) had protective effects on DSS/LPS-induced diverticulitis. DSS induces intestinal inflammation, which damages the epithelial monolayer lining the large intestine, allowing the dissemination of pro-inflammatory intestinal contents (e.g. bacteria and their products) into the underlying tissue (Chassaing et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Furthermore, LPS aggravates colon inflammation (Zhang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In this study, DSS together with LPS enema gave rise to a significant decrease in the rats\u0026rsquo; body weights. This loss in body weight is due to a deficiency of nutrients resulting from food aversion, reduced appetite, malabsorption, as well as loss of body fluids through colorectal bleeding and diarrhea (Hunschede et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Also, there was an increase in the colon weight-to-length ratio due to tissue edema, necrosis, and inflammatory cell infiltration confirmed in our study through the high macroscopic and microscopic lesion scores. These results agreed with Peran et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and Khodir et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Also, Mahoro et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that; DSS induced bloody diarrhea, weight loss, shortening of the colon, and mucosal deterioration in a rat model of DSS-induced colitis. Otherwise, only treatment with amoxicillin together with probiotics could significantly ameliorate the body weight loss caused by DSS/LPS, while both treatments (amoxicillin and probiotics-amoxicillin) showed inhibitory effects on the high colon weight-to-length ratio (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), as well as colon macroscopic lesion score (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Applegate et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) clarified that; treatment with probiotics results in bacterial antagonism, colonization competition, and emulation for nutrients. These actions lead to amelioration of toxic compounds, modulation of the immune system, increasing nutrient absorption and digestibility, and ultimately decline in the body weight loss. \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e strains have anti-inflammatory and immunomodulatory activities. Moreover, certain \u003cem\u003eLactobacillus\u003c/em\u003e strains can upregulate the expression of mucin-3 and enhance the intestinal mucus layer (Yao et al \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), so they could inhibit DSS/LPS-induced damage in the colon tissue.\u003c/p\u003e \u003cp\u003eMPO is a member of the peroxidases subfamily and has more expression in immune cells such as neutrophils, lymphocytes, monocytes, and macrophages. The enhanced level of MPO activity is a well diagnostic marker of inflammation and oxidative stress (Khan et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, combined treatment of probiotics and amoxicillin was more effective than amoxicillin alone in ameliorating the elevated MPO activity caused by DSS/LPS. This amelioration is due to the anti-inflammatory properties. The decrease in this enzyme activity reveals a lower infiltration of neutrophils in the inflamed colon tissue; since treatment with probiotics caused a reduction in the colonic production of the chemotactic eicosanoid LTB4. It was observed that; \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e administration reduced colonic MPO activity in the trinitrobenzene sulfonic acid (TNBS) model of rat colitis (Peran et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCRP is one of the most prominent proteins in acute inflammation (Norouzinia et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It has been found that; higher levels of CRP, as well as the inflammatory markers, have been detected in patients with severe acute diverticulitis disease (Lahat et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the present study, DSS/LPS administration caused a significant elevation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the serum level of CRP. This elevation was declined on either treatment with amoxicillin or probiotics-amoxicillin due to the inhibition of inflammation and disease progression. According to Sartelli et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), CRP is a valuable biomarker of inflammation and can be used in the prediction of the severity of acute diverticulitis. Furthermore, the current study revealed that DSS/LPS resulted in an elevation of the levels of TNF-α, IL-1β, IFN-γ, and IL-18. Pro-inflammatory cytokines such as IL-1β and TNF-α have been involved in the inflammatory process in DSS-induced colitis (Triantafillidis et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Lahat et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) demonstrated that; patients with severe acute diverticulitis have higher tissue inflammatory cytokine levels including TNF-α, IL-6, IL-1β, and more inflammatory infiltrates in diverticular colon tissue. On the other hand, the elevation in TNF-α, IL-1β, IFN-γ, and IL-18 levels was significantly declined on treatment with amoxicillin or probiotics-amoxicillin. This inhibitory effect is attributed to the existence of a cross-talk between probiotics and mucosal cells. \u003cem\u003eBifidobacterium lactis\u003c/em\u003e, either alone or in combination with other probiotics, was able to downregulate the degree of activation of intestinal immune cells in the TNBS model of rat colitis (Peran et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Probiotics may restore the balance of gut flora by decreasing pathogenic gram-negative bacteria; that may have been altered in diverticular disease due to stasis and reduced colonic transit time, and have been proposed to be used in diverticular disease to prevent inflammation (Boynton and Floch \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe biochemical analysis and the colonic macroscopic lesions evaluation were confirmed by the histological study, where DSS/LPS caused mucosal and submucosal inflammatory cells infiltration, dilated blood vessels, and degenerative mucosal changes with damaged crypts. Both Schieffer et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and Tursi et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) demonstrated similar changes that occurred in the architecture of the colon wall, including loss of elasticity function and deposition of immature collagen fibers in the extracellular matrix, which are implicated in the formation of diverticula as part of the pathophysiology of diverticular disease. Treatment with probiotics-amoxicillin was more effective than treatment with amoxicillin alone in improving the architecture of the colon tissue since the colon tissues showed slight inflammation and few mucosal lymphocytes infiltration on probiotics-amoxicillin treatment while showed few mucosal and submucosal inflammatory cells infiltration and degenerative mucosal changes with few damaged crypts on amoxicillin treatment. Conclusively, antibiotics have been used to treat acute diverticulitis in all patients. Recent findings have indicated that; the manipulation of antibiotics is not necessary for mild or moderate uncomplicated acute diverticulitis management, as was initially thought (Feuerstein and Falchuk \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Treatment of acute diverticulitis generally comprises dietary fiber supplementation, anti-inflammatory drugs, pharmacological therapies such as antibiotics, as well as probiotics, either alone or in combination (Tursi et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study revealed that each of the treatments attenuated the severity of DSS/LPS-induced acute diverticulitis in rats and displayed differing effectiveness in disease parameters. Combined probiotics-amoxicillin therapy was more effective in restoring the rats\u0026rsquo; body weight, inhibiting the inflammatory markers tested in this study, and improving the epithelium damage score. Further studies are required in order to understand how probiotics can be employed in treating acute diverticulitis. The impact of the intestinal milieu, especially enteric microbiota, appears to be of great significance. Our study demonstrates that; probiotics enhanced the prevention of DSS/LPS-induced acute diverticulitis, so we suggest employing probiotics in treating acute diverticulitis disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaha G Solaiman\u0026mdash;\u0026nbsp;Administrative support, interpretation, and review of the article.\u003c/p\u003e\n\u003cp\u003eHanna A Mansour\u0026mdash;\u0026nbsp;Conception and design, interpretation, review of the article, and editing.\u003c/p\u003e\n\u003cp\u003eWedad A Hassan\u0026mdash;\u0026nbsp;Provision of study materials, design, and data analysis.\u003c/p\u003e\n\u003cp\u003eEman Shawky\u0026mdash;\u0026nbsp;Collection and assembly of data, data analysis, and writing of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study\u0026nbsp;didn\u003cspan dir=\"RTL\"\u003e\u0026rsquo;\u003c/span\u003et\u0026nbsp;receive any specific grant from funding agencies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the findings of this study are available from the corresponding author when requested.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAl Bander, Z, Nitert MD, Mousa A, Naderpoor N (2020) The Gut Microbiota and Inflammation: An Overview. 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Dig Dis Sci 49:556-564.\u003c/li\u003e\n\u003cli\u003eWedel T, Barrenschee M, Lange C, Cossais F, B\u0026ouml;ttner M (2015) Morphologic basis for developing diverticular disease, diverticulitis, and diverticular bleeding. Viszeralmedizin 31:76-82.\u003c/li\u003e\n\u003cli\u003eWess L, Eastwood M, Busuttil A, Edwards C, Miller A (1996) An association between maternal diet and colonic diverticulosis in an animal model. Gut 39:423-427. \u003c/li\u003e\n\u003cli\u003eYamada S, Koyama T, Noguchi H, et al (2014) Marine hydroquinone zonarol prevents inflammation and apoptosis in dextran sulfate sodium-induced mice ulcerative colitis. PLoS One 9:e113509. \u003c/li\u003e\n\u003cli\u003eYamamichi N, Shimamoto T, Takahashi Y, et al (2015) Trend and risk factors of diverticulosis in Japan: age, gender, and lifestyle/metabolic-related factors may cooperatively affect on the colorectal diverticula formation. PLoS One 10:e0123688. \u003c/li\u003e\n\u003cli\u003eYao P, Tan F, Gao H, Wang L, Yang T, et al (2017) Effects of probiotics on Toll like receptor expression in ulcerative colitis rats induced by 2,4,6 trinitro benzene sulfonic acid. Molecular Medicine Reports 15:1973-1980. \u003c/li\u003e\n\u003cli\u003eZhang M , Long Y , Sun Y , et al (2011) Evidence for the complementary and synergistic effects of the three-alkaloid combination regimen containing berberine, hypaconitine and skimmianine on the ulcerative colitis rats induced by trinitrobenzene-sulfonic acid. Eur J Pharmacol 651:187-196.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Amoxicillin, Probiotics, DSS, LPS, Colon disorders, Acute diverticulitis","lastPublishedDoi":"10.21203/rs.3.rs-1694157/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1694157/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcute diverticulitis disease is associated with inflammation and infection in the colon diverticula and may lead to severe morbidity. This study aimed to evaluate and compare the protective effects of the antibiotic (amoxicillin) and the probiotics (\u003cem\u003eLactobacillus acidophilus\u003c/em\u003e and \u003cem\u003eBifidobacterium lactis\u003c/em\u003e) given concurrently in addition to amoxicillin; in a rat model of acute diverticulitis disease. Acute diverticulitis was induced, in albino rats, by adding 3% weight/volume of dextran sulfate sodium (DSS), to the rats\u003cem\u003e\u0026rsquo;\u003c/em\u003e drinking water; daily for seven days. Furthermore, injection of lipopolysaccharide (LPS) enema (4 mg/kg). The impact of treatment was assessed by measuring physiological and immunological parameters and evaluating colon macroscopic and microscopic lesions. The results showed that both treatments (especially probiotics with amoxicillin) alleviated the adverse effects of DSS and LPS, where the rats\u0026rsquo; body weights and the colon weight-to-length ratio were modulated. Also, the colon macroscopic damage score was significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decreased. The pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF)-α, interleukin-1 beta (IL)-1β, interferon-gamma (IFN-γ), and interleukin-18 (IL)-18]; in the colon tissue, were significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decreased. Also, the elevation of myeloperoxidase (MPO) activity and C-reactive protein (CRP) level was ameliorated. Moreover, the histopathological alterations of the colon tissue were improved. In conclusion, amoxicillin and probiotics-amoxicillin were effective in preventing the development of the experimentally induced acute diverticulitis through the anti-inflammatory and the immunomodulatory effects. Additionally, this study showed the role of probiotics in preventing DSS/LPS-induced acute diverticulitis, so it can be employed as a promising treatment option for acute diverticulitis.\u003c/p\u003e","manuscriptTitle":"Protective Effects of Amoxicillin and Probiotics on Colon Disorders in an Experimental Model of Acute Diverticulitis Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-31 21:25:40","doi":"10.21203/rs.3.rs-1694157/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-05-27T15:08:45+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-05-26T16:00:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Inflammopharmacology","date":"2022-05-26T15:40:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-05-26T09:07:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammopharmacology","date":"2022-05-25T17:48:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a88e3759-5254-4c52-9456-895bb35e5adc","owner":[],"postedDate":"May 31st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-10-19T01:18:59+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-31 21:25:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1694157","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1694157","identity":"rs-1694157","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}