MFE40-the active fraction of Mume Fructus alcohol extract-alleviates Crohn's disease and its complications.

ETHNOPHARMACOLOGICAL RELEVANCE: Mume Fructus (MF) is a well-known traditional Chinese medicine used to treat chronic cough, prolonged diarrhea, and other inflammation-related diseases. We previously confirmed the anti-colitis effect of its ethanol extract on a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced Crohn's disease (CD) rat model. However, the active ingredients and underlying mechanisms of MF remain unknown. AIM OF THE STUDY: To clarify the material basis and potential mechanism of the ethanol extract of MF (MFE) in alleviating CD and its complications, such as lung injury and intestinal obstruction. MATERIALS AND METHODS: MF was extracted with 80% ethanol aqueous solution and separated with 0, 40, and 100% ethanol aqueous solutions. MFE and its fractions were screened in a TNBS-induced CD rat model. For the bioactive fraction, the chemical composition was identified and quantified using ultrahigh-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight tandem mass spectrometry. Interleukin (IL)-1ß, IL-6, IL-17, transforming growth factor (TGF)-ß, and lipopolysaccharide (LPS) levels in the colon, lungs, and/or plasma were detected using enzyme-linked immunosorbent assays. The expression levels of zonula occludens-1 (ZO-1) and occludin in the colon were measured using immunohistochemical staining, and the intestinal microbiota and short-chain fatty acid (SCFA) levels were analyzed using 16S rRNA gene sequencing and gas chromatography/mass spectrometry. RESULTS: The 40% ethanol fraction of MF (MFE40), which mainly contained methyl citrate, ethyl citrate, and caffeoylquinic acid ethyl esters, was identified as the active fraction that could alleviate CD in rats. MFE40 could ameliorate inflammation and fibrosis in the colon and lung tissues by inhibiting the secretion of cytokines, such as IL-1ß, IL-6, IL-17, and TGF-ß, along with intestinal obstruction and lung injury in CD rats. The possible mechanisms of MFE40 were related to increased expression of ZO-1 and occludin in the colon, reduction in plasma LPS levels, and restoration of SCFAs via reduction in the relative abundance of Adlercreutzia, Clostridium_sensu_stricto_1, Erysipelatoclostridium, Faecalibaculum, norank_f_Erysipelotrichaceae, Phascolarctobacterium Coriobacteriaceae_UGG_002, and Allobaculum and increase in the relative abundance of Escherichia shigella, Christensenella, Acetivibrio_ethanolgignens, and Butyricicoccus. MFE40 had no significant influence on the inflammatory factors in healthy rats. CONCLUSIONS: Citrate esters and hydroxycinnamate esters are the main active constituents of MFE40. MFE40 exhibited a remission effect on CD rats and inhibited intestinal obstruction and lung injury via anti-inflammatory effects and regulation of the intestinal microbiota-gut-lung homeostasis.

Structural and molecular features of intestinal strictures in rats with Crohn's-like disease.

AIM: To develop a new rat model we wanted to gain a better understanding of stricture formation in Crohn's disease (CD). METHODS: Chronic colitis was induced locally by the administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The relapsing inflammation characteristic to CD was mimicked by repeated TNBS treatments. Animals were randomly divided into control, once, twice and three times TNBS-treated groups. Control animals received an enema of saline. Tissue samples were taken from the strictured colonic segments and also adjacent proximally and distally to its 60, 90 or 120 d after the last TNBS or saline administrations. The frequency and macroscopic extent of the strictures were measured on digital photographs. The structural features of strictured gut wall were studied by light- and electron microscopy. Inflammation related alterations in TGF-beta 2 and 3, matrix metalloproteinases 9 (MMP9) and TIMP1 mRNA and protein expression were determined by quantitative real-time PCR and western blot analysis. The quantitative distribution of caspase 9 was determined by post-embedding immunohistochemistry. RESULTS: Intestinal strictures first appeared 60 d after TNBS treatments and the frequency of them increased up to day 120. From day 90 an intact lamina epithelialis, reversible thickening of lamina muscularis mucosae and irreversible thickening of the muscularis externa were demonstrated in the strictured colonic segments. Nevertheless the morphological signs of apoptosis were frequently seen and excess extracellular matrix deposition was recorded between smooth muscle cells (SMCs). Enhanced caspase 9 expression on day 90 in the SMCs and on day 120 also in myenteric neurons indicated the induction of apoptosis. The mRNA expression profile of TGF-betas after repeated TNBS doses was characteristic to CD, TGF-beta 2, but not TGF-beta 3 was up-regulated. Overexpression of MMP9 and down-regulation of TIMP1 were demonstrated. The progressive increase in the amount of MMP9 protein in the strictures was also obvious between days 90 and 120 but TIMP1 protein was practically undetectable at this time. CONCLUSION: These findings indicate that aligned structural and molecular changes in the gut wall rather than neuronal cell death play the primary role in stricture formation.

Therapeutic effect and mechanism of electroacupuncture at Zusanli on plasticity of interstitial cells of Cajal: a study of rat ileum.

BACKGROUND: Electroacupuncture (EA) is one of the techniques of acupuncture and is believed to be an effective alternative and complementary treatment in many disorders. The aims of this study were to investigate the effects and mechanisms of EA at acupoint Zusanli (ST36) on the plasticity of interstitial cells of Cajal (ICCs) in partial bowel obstruction. METHODS: A Sprague Dawley rat model of partial bowel obstruction was established and EA was conducted at Zusanli (ST36) and Yinglingquan (SP9) in test and control groups, respectively. Experiments were performed to study the effects and mechanisms of EA at Zusanli on intestinal myoelectric activity, distribution and alteration of ICCs, expression of inflammatory mediators, and c-Kit expression. RESULTS: 1) EA at Zusanli somewhat improved slow wave amplitude and frequency in the partial obstruction rats. 2) EA at Zusanli significantly stimulated the recovery of ICC networks and numbers. 3) the pro-inflammatory mediator TNF-α and NO activity were significantly reduced after EA at Zusanli, However, no significant changes were observed in the anti-inflammatory mediator IL-10 activity. 4) EA at Zusanli re-expressed c-Kit protein. However, EA at the control acupoint, SP9, significantly improved slow wave frequency and amplitude, but had no effect on ICC or inflammatory mediators. CONCLUSIONS: We concluded that EA at Zusanli might have a therapeutic effect on ICC plasticity, and that this effect might be mediated via a decrease in pro-inflammatory mediators and through the c-Kit signaling pathway, but that the relationship between EA at different acupoints and myoelectric activity needs further study.

The role of L-arginine and inducible nitric oxide synthase in intestinal permeability and bacterial translocation.

BACKGROUND: Arginine has been shown to have several immunological and trophic properties in stressful diseases. Its metabolites, nitric oxide (NO) and polyamines, are related to arginine's effects. Thus, the aim of this study was to determine the effects of the NO donor L-arginine and the role of inducible NO synthase (iNOS) on intestinal permeability and bacterial translocation in a model of intestinal obstruction (IO) induced by a simple knot in the terminal ileum. MATERIAL AND METHODS: Male C57BL6/J wild-type (WT) and iNOS knockout (iNOS-/-) mice were randomized into 6 groups: Sham and Sham-/- (standard chow), IO and IO-/- (standard chow +IO), and Arg and Arg-/- (standard chow supplemented with arginine + IO). After 7 days of treatment with standard or supplemented chows, IO was induced and intestinal permeability and bacterial translocation were evaluated. The small intestine and its contents were harvested for histopathological and morphometric analysis and the determination of polyamine concentration. RESULTS: Pretreatment with arginine maintained intestinal permeability (P > .05; Arg and Arg-/- groups vs Sham and Sham-/- groups), increased polyamine concentration in intestinal content (P < .05; Arg vs IO group), and decreased bacterial translocation in WT animals (Arg group vs IO and IO-/- groups). Absence of iNOS also presented a protective effect on permeability but not on bacterial translocation. CONCLUSION: Arginine supplementation and synthesis of NO by iNOS are important factors in decreasing bacterial translocation. However, when intestinal permeability was considered, NO had a detrimental role.

Effect of Dachengqi decoction on NF-kappaB p65 expression in lung of rats with partial intestinal obstruction and the underlying mechanism.

To investigate the effect of Dachengqi decoction on NF-kappaB p65 expression in lung of rats with partial intestinal obstruction and the underlying mechanism, 30 SD rats were randomly divided into three groups: sham-operation group, model group and Dachengqi decoction treatment group (Dachengqi group), with 10 animals in each group. The models were made by partially ligating their large intestines outside the body. The pathological changes were analyzed by HE staining. The expression of NF-kappaB p65 in rats lung were measured by using real-time polymerase chain reaction and immunohistochemistry respectively. Moreover, the expression of caveolin-1 in rats lung was also measured to. Increased edema, interstitial thickening, hemorrhage, and infiltration of inflammatory cells were found in the model group. In contrast, this change was significantly reduced in Dachengqi group as compared with model group. In addition, the up-regulated caveolin-1 and NF-kappaB p65 were also suppressed by Dachengqi decoction in lung of rats with partial intestinal obstruction. We are led to concluded that the caveolin-1-NF-kappaB pathway plays an important role in the development of lung injury of rats with partial intestinal obstruction and Dachengqi decoction could down-regulate the expression of caveolin-1 and NF-kappaB p65 in lung of rats with partial intestinal obstruction.

Involvement of superoxide anion in the pathogenesis of simple mechanical intestinal obstruction.

BACKGROUND: Mechanism underlying the pathogenesis of mechanical intestinal obstruction has been suggested to be closely associated with bowel inflammatory response in which reactive oxygen metabolites might play an important role. This study was designed to examine the involvement of superoxide anion in the obstruction-induced intestinal injury. MATERIALS AND METHODS: Rats were randomly aasigned to four groups: sham, obstruction, obstruction with polyethylene glycol (PEG), and obstruction with polyethylene glycol-superoxide dismutase (PEG-SOD) groups. A ligation at the ileum 20 cm proximal to the cecum was created under anesthesia. The superoxide anion production and the pathological manifestations in the obstructed intestine were measured after 24 h of ligation. RESULTS: There were significant intestinal shortening, distension, fluid accumulation and mucosal damage in the segment proximal to the ligation site. Pronounced generation of superoxide anion was found in the obstructed intestinal segment. Supplement of SOD, a superoxide free radicals scavenger, ameliorated obstruction-induced bowel distension, fluid accumulation and mucosal damage. CONCLUSION: These data suggest superoxide anion is one of the important mediators in the pathophysiologic changes of simple mechanical intestinal obstruction.

Functional ileus in neonates: Hirschsprung's disease-allied disorders versus meconium-related ileus.

Sixty-eight neonates with functional ileus were reviewed. Twelve required laparotomy; in seven, histological studies revealed decreased ganglia and ganglion cells of the myenteric plexus (MP) (Group A), and in five, MP was normal (Group B). In the remaining 56 cases, obstructive symptoms were relieved following conservative therapy (Group C). All Group A cases except one had normal birth weight, while Group B and C cases showed significantly lower birth weights. A marked caliber change of the small intestine and/or small-caliber distal intestine with meconium stagnation in the proximal intestine was commonly demonstrated at operation in Group A and B, or on contrast enema in Group C. Four Group A cases died of enteritis, and three survivors suffered from prolonged obstructive symptoms. The grade of histological abnormality of MP correlated with the clinical outcome. In Group B, three died of sepsis shortly after surgery, but two survivors have been free from symptoms. Group A can be categorized as Hirschsprung's disease-allied disorders (HAD). Group B and C can be categorized as meconium-related ileus (MRI). The similarity of the macroscopic findings of HAD and MRI, and the occurrence of MRI exclusively in low birth weight neonates, strongly suggest that functional immaturity of MP plays a role in the etiology of MRI.

Effect of glutamine or glucagon-insulin enriched total parenteral nutrition on liver and gut in 70% hepatectomized rats with colon stenosis.

BACKGROUND: Malnutrition of enterocytes is believed to facilitate the breakdown of the intestinal mucosal barrier, furthering a translocation of enteric bacteria with subsequent severe infection, which has been described after extensive hepatectomy. Glutamine and glucagon insulin are said to attenuate the malnutrition of enterocytes. To determine whether this was true, the effects on the remnant liver and the gut of total parenteral nutrition supplemented by admixtures of glutamine and/or glucagon insulin were investigated in rats subjected to massive hepatectomy and transient intestinal stasis. STUDY DESIGN: Rats underwent a permanent cannulation of the superior caval vein without restraining their mobility, a 70% hepatectomy, and a 24 hour string-ligation stenosis of the colon. A standard total parenteral nutrition solution was infused without or with 2% glutamine and without or with glucagon-insulin supplementation, respectively. RESULTS: Glutamine and glucagon-insulin supplemented total parenteral nutrition increased ileal mucosal DNA concentrations during and after intestinal stasis. Glutamine or glucagon-insulin alone had less pronounced effects. In the liver, the combined supplementation resulted in reduced adenosine triphosphate concentrations and increased mitochondrial adenosine triphosphate synthesis as well as in an early increase in DNA concentrations. CONCLUSION: Glutamine and glucagon-insulin enriched total parenteral nutrition attenuates malnutrition of enterocytes after massive abdominal stress and promotes liver regeneration after extensive hepatectomy.

Polyunsaturated phosphatidylcholine prevents stricture formation in a rat model of colitis.

BACKGROUND & AIMS: Polyunsaturated phosphatidylcholine stimulates collagen breakdown in experimental models of liver cirrhosis. Bowel strictures are characterized by excess deposition of collagen in the intestinal wall. The aim of this study was to investigate the effect of polyunsaturated phosphatidylcholine in the prevention of bowel strictures. METHODS: Colitis was induced by trinitrobenzenesulfonic acid. On day 21, the presence of strictures was assessed in control rats, rats with colitis, and phosphatidylcholine-fed (100 mg/day) rats with colitis. Furthermore, serum transforming growth factor beta1, collagen deposition, and collagenase activity in colonic tissue were measured in all groups. RESULTS: None of the control rats but 12 of 16 rats with colitis developed colonic strictures. In contrast, only 2 of 15 phosphatidylcholine-fed rats with colitis showed strictures. Collagen content was much higher in rats with colitis than in phosphatidylcholine-fed rats with colitis and control rats. Phosphatidylcholine-fed rats showed significantly higher collagenase activity in colonic tissue than rats with colitis and control rats. In an ancillary study, free linoleic acid-fed rats showed no differences when compared with rats with colitis. Stimulation of transforming growth factor beta1 was similar in all rats with colitis. CONCLUSIONS: Oral supplementation with polyunsaturated phosphatidylcholine prevents the accumulation of collagen in inflamed intestinal tissue and the formation of strictures. This effect is associated with an enhanced collagen catabolism.

Gut inflammation in children with cystic fibrosis on high-dose enzyme supplements.

We used a whole-gut perfusion technique to study subclinical gut inflammation in children with cystic fibrosis (18 elective tests, three lavages to treat distal intestinal obstruction syndrome); and in 12 control children with constipation or pre-colonoscopy. We assayed for haemoglobin, IgG, albumin, alpha-1-antitrypsin, granulocyte elastase, interleukin-1 beta (IL-1 beta) and IL-8 concentrations in whole-gut lavage fluid. Results for two children with distal intestinal obstruction syndrome, the only children in the series taking Nutrizym 22, were strikingly abnormal. This new test has revealed subclinical gut mucosal inflammation in a minority of CF children, for which distal intestinal obstruction syndrome, Nutrizym 22 treatment, or both, may be risk factors.

[Bioenergetic disorders of the intestinal wall in acute intestinal obstruction and the means for their correction]. / Narusheniia bioénergetiki kishechnoi stenki pri ostroi kishechnoi neprokhodimosti i sposoby ikh korrektsii.

Results of an investigation of bioenergetics of the mucous and intestinal layers of the adducting, strangulated and abducting parts of the small intestine at different terms after the creation and liquidation of acute occlusive and strangulated intestinal experimental ileus in 173 dogs are presented. The use of the method of correcting therapy described in the article for complex treatment of 326 patients with acute ileus has shown their advantage over traditional methods.

[The effects of dachengqi decoction, shaogan decoction, emodin and sennoside on the histamine level of intestinal mucosa in intestinally obstructed rats].

The experimental intestinal obstruction model was made by partly ligating the ileum in rat. The histamine levels of obstruction groups (8.14 +/- 2.28 micrograms/g) were obviously lower than those of control groups (P < 0.01). After administration of Dachengqi decoction, Shaogan decoction, emodin and sennoside, the levels of histamine were significantly higher than those of obstruction groups, but there were no significant differences as compared with control groups.

[Da cheng qi tang on 45Ca content of the isolated colon smooth muscle from experimental colon obstruction rats].

Da Cheng Qi Tang (DCQT) is a classical prescription of Chinese medicine for treatment of acute intestinal obstruction. In this paper, the isolated colon smooth muscle from normal and experimental colon obstruction (CO) rats were used to study the effect of DCQT on 45Ca content. The results showed that the 45Ca content on isolated colon smooth muscle (microns/g wet tissue, mean +/- S) was 0.043 +/- 0.009 in the normal and 0.057 +/- 0.012 in those treated by DCQT respectively. The content of 45Ca in CO was higher than the normal, DCQT can reduce the content of 45Ca in CO. It is known that the higher level of intracellular CA++ is related to the formation and the development of acute intestinal obstruction. The inhibitory effect of DCQT on 45Ca content may play an important role in the treatment of acute colon obstruction.

[Urinary excretion rate of methylguanidine as a new maker of active oxygen in vivo: demonstration in hyperbaric oxygen therapy].

Methylguanidine (MG) which is known as a uremic toxin, is synthesized from creatinine (Cre). We have clarified that active oxygen plays an important role on MG synthesis in vitro and in rat hepatocytes. On the other hand, hyperoxia is very injurious in various tissues, and it has been reported that active oxygen produced in hyperoxia plays an important role on the tissue injury. This study was performed to investigate the effect of hyperoxia on MG synthesis in vivo. The subjects in this study were patients who were treated by hyperbaric oxygen therapy (HBO). Serum Cre, MG, and urinary Cre, MG before and after HBO were measured in these subjects. The subjects were classified into four groups. Group I-III were undergone HBO with condition of 100% O2, 2 atmosphere absolute (ATA), 1 hour, (I: Ccr less than 10 ml/min, II: 10 less than or equal to Ccr less than 50 ml/min, III: Ccr greater than or equal to 50 ml/min) and group IV (Ccr greater than or equal to 50 ml/min) with 100%O2, 3ATA, 1 hour. Urinary excretion rate of MG (urine MG/urine Cre) significantly increased after HBO therapy in every group. Urine MG/urine Cre/serum Cre ratio which was used as a index of MG synthesis rate also increased. In this study, it is clarified that MG excretion rate increases in hyperoxic condition. These results suggest that active oxygen plays an important role on MG synthesis in vivo, and that the urine MG/urine Cre/serum Cre ratio can be a useful maker of the active oxygen products in vivo.

Effects of intraluminal hydrostatic pressure on L-methionine absorption in the obstructed small intestine of the rat.

The effects of elevated intraluminal hydrostatic pressure on the active absorption of the amino acid selenium 75 L-methionine has been analyzed in the normal and obstructed small intestine. An intestinal loop of defined position and length was included in a recircling perfusion system from which the elimination rate of the radiolabeled amino acid was measured. Preset pressure levels within the system were maintained by a servo-controlled unit, which added or subtracted volume to keep the pressure constant. The rate of amino acid elimination increased when the nonobstructed loop was subjected to a pressure of 10 cm H2O but decreased when exposed to 20cm H2O. Using a loop of intestine subjected to 48 hours of obstruction, amino acid elimination was greatly retarded compared with that of the nonobstructed loop. By increasing the intraluminal pressure to 10 and 20 cm H2O, the elimination rate increased, equalling that of the nonobstructed gut. The results suggest that intestinal obstruction per se decreases active absorption secondary to impaired intestinal viability. Moderately increased intraluminal pressure adds a driving force to L-methionine absorption, the mechanism of which is obscure.