ω-(5-Phenyl-2H-tetrazol-2-yl)alkyl-substituted glycine amides and related compounds as inhibitors of the amine oxidase vascular adhesion protein-1 (VAP-1).

Vascular adhesion protein-1 (VAP-1), also known as plasma amine oxidase or semicarbazide-sensitive amine oxidase, is an enzyme that degrades primary amines to aldehydes with the formation of hydrogen peroxide and ammonia. Among others, it plays a role in inflammatory processes as it can mediate the migration of leukocytes from the blood to the inflamed tissue. We prepared a series of ω-(5-phenyl-2H-tetrazol-2-yl)alkyl-substituted glycine amides and related compounds and tested them for inhibition of purified bovine plasma VAP-1. Compounds with submicromolar activity were obtained. Studies on the mechanism of action revealed that the glycine amides are substrate inhibitors, i.e., they are also converted to an aldehyde derivative. However, the reaction proceeds much more slowly than that of the substrate used in the assay, whose conversion is thus blocked. Examination of the selectivity of the synthesized glycine amides with respect to other amine oxidases showed that they inhibited diamine oxidase, which is structurally related to VAP-1, but only to a much lesser extent. In contrast, the activity of monoamine oxidase A and B was not affected. Selected compounds also inhibited VAP-1 in human plasma. The IC50 values measured were higher than those determined with the bovine enzyme. However, the structure-activity relationships obtained with the glycine amides were similar for both enzymes.

Recombinant human diamine oxidase prevents hemodynamic effects of continuous histamine infusion in guinea pigs.

OBJECTIVE: To test whether recombinant human diamine oxidase (rhDAO) with a mutated heparin-binding motif (mHBM), which shows an increased alpha-distribution half-life, prevents histamine-induced hemodynamic effects. MATERIAL: Thirty-eight female guinea pigs were either pretreated with rhDOA_mHBM or buffer. TREATMENT AND METHODS: Guinea pigs received a continuous infusion of histamine. Heart rate (HR), body core temperature and mean arterial pressure (MAP) were measured and blood was collected. RESULTS: Continuous intravenous infusion of 8 µg/kg/min histamine increased mean peak plasma histamine levels from 5 (± 0.3 SEM) to 28 ng/mL (± 4.9 SEM) after 30 min but had no effect on oxygen saturation. Guinea pigs pretreated with 4 mg/kg rhDAO_mHBM showed lower mean HR (p = 0.008), histamine plasma concentrations (p = 0.002), and higher body core temperatures at the end of the histamine challenge (p = 0.02) compared to controls. Cessation of histamine infusion led to a rebound increase in MAP, but this hemodynamic instability was prevented by rhDAO_mHBM. Pretreatment with 4 mg/kg rhDAO_mHBM reduced urinary histamine (p = 0.004) and 1-Methylhistamine (p < 0.0001) concentrations compared to controls. CONCLUSIONS: Prophylactic infusion of rhDAO_mHBM prevents hemodynamic effects in a guinea pig model of continuous histamine infusion. These findings might help in the translation from animals to humans and in the selection of the optimal dosing of rhDAO_mHBM during human histamine challenge studies.

Amine oxidase copper-containing 3 aggravates cardiac remodelling by generating hydrogen peroxide after myocardial infarction.

Amine oxidase copper-containing 3 (AOC3) is a member of the semicarbazide-sensitive amine oxidase enzyme family. It acts as an ectoenzyme catalysing the oxidative deamination of primary amines and generating hydrogen peroxide (H2 O2 ). While AOC3 is implicated in cardiovascular diseases such as atherosclerosis, its role in cardiac remodelling after myocardial infarction (MI) is unclear. In this study, we first confirmed a long-term upregulation of AOC3 in both cardiac myofibroblasts after MI in vivo and angiotensin II (ANGII)-treated cardiac fibroblasts in vitro. AOC3 knockdown not only inhibited the activation of cardiac fibroblasts induced by ANGII but also alleviated cardiac fibrosis in mice after MI. Using sh-AOC3 lentiviruses, exogenous recombinant AOC3 (r-AOC3), semicarbazide (an AOC3 inhibitor), and catalase (a hydrogen peroxide scavenger) treatments, we also demonstrated that AOC3 promoted H2 O2 generation, increased oxidative stress, and enhanced ERK1/2 activation, which were responsible for the activation of cardiac fibroblasts. In particular, AOC3 knockdown also improved cardiac function and hypertrophy after MI. Through a coculture system, we confirmed that AOC3 expressed on cardiac myofibroblasts was able to enhance oxidative stress and induce hypertrophy of cardiomyocytes by promoting H2 O2 generation. Similarly, r-AOC3 promoted H2 O2 generation and resulted in oxidative stress and hypertrophy of cardiomyocytes, which were almost inhibited by both semicarbazide and catalase. In conclusion, AOC3 plays a critical role in cardiac fibrosis and hypertrophy after MI by promoting the generation of H2 O2 . AOC3 is a promising therapeutic target against cardiac remodelling. © 2023 The Pathological Society of Great Britain and Ireland.

Effects of probiotics on intestinal flora, intestinal function and T lymphocyte level in patients with cervical cancer after radiotherapy / 中国基层医药

Objective:To investigate the effects of probiotics on intestinal flora, intestinal function, and T lymphocyte level in patients with cervical cancer after radiotherapy.Methods:A total of 92 patients with cervical cancer who underwent pelvic radiotherapy in The Second Affiliated Hospital of Zhengzhou University from September 2020 to February 2022 were included in this study. They were randomly divided into control and experimental groups ( n = 46/group). The patients in the experimental group took probiotics during radiotherapy, while the patients in the control group did not take probiotics during radiotherapy. The amount of intestinal flora, D-lactic acid, diamine oxidase, and T lymphocyte subset levels pre- and post-radiotherapy were compared between the two groups. Urinary lactulose (L) and mannitol (M) concentrations were determined in each group. Urinary excretion ratios of L to M were calculated. Results:After 10, 15, and 20 times of radiotherapy and after all radiotherapies, the amount of Escherichia coli and Enterococcus in the experimental group was significantly lower than that in the control group ( F = 128.60, 224.99, all P < 0.05). The amount of Bifidobacteria and Lactobacilli in the experimental group was significantly higher than that in the control group ( F = 2 065.46, 948.23, both P < 0.05). After 10, 15, and 20 times of radiotherapy and after all radiotherapies, plasma D-lactic acid level in the experimental group was (9.34 ± 1.63) μg/L, (9.15 ± 1.36) μg/L, (8.68 ± 1.06) μg/L, and (8.05 ± 0.82) μg/L, respectively. After 10, 15, and 20 times of radiotherapy and after all radiotherapies, plasma diamine oxidase level in the experimental group was (86.34 ± 20.25) μg/L, (84.28 ± 17.45) μg/L, (80.40 ± 13.35) μg/L, and (76.85 ± 10.87) μg/L, respectively, and urinary excretion ratio of L to M in the experimental group was (1.84 ± 0.16), (1.55 ± 0.12), (1.26 ± 0.09), (0.98 ± 0.06), respectively, all of which were significantly lower than those in the control group ( F = 121.60, 31.73, 417.84, all P < 0.05). After 10, 15, and 20 times of radiotherapy and after all radiotherapies, CD4 + level in the experimental group was (39.80 ± 4.90)%, (40.92 ± 5.30)%, (42.52 ± 6.14)%, (43.83 ± 6.55)%, respectively, CD4 +/CD8 + was (1.52 ± 0.25), (1.63 ± 0.22), (1.71 ± 0.39), (1.83 ± 0.22), respectively, all of which were significantly higher than those in the control group ( F = 58.69, 31.07, all P < 0.05). Conclusion:Probiotics can improve the status of intestinal flora and intestinal barrier function in patients with cervical cancer after radiotherapy, and simultaneously improve the cellular immune function of patients.

Semicarbazide-Sensitive Amine Oxidase (SSAO) and Lysyl Oxidase (LOX) Association in Rat Aortic Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. ßAPN abolished LOX activity (** p < 0.01 for 8 vs. 3 and * p < 0.05 for 5 vs. 8) and had no effect on LOX protein and mRNA levels. MDL72527 reduced LOX activity at passage 3 and 5 (## p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (**** p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). ßAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (#### p < 0.0001 for Si6 vs. Sictrl and ### p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (# p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.

Increased atherosclerotic plaque in AOC3 knock-out in ApoE-/- mice and characterization of AOC3 in atherosclerotic human coronary arteries.

Introduction: Amine oxidase copper containing 3 (AOC3) displays adhesion between leukocytes and endothelial cells and enzymatic functions. Given its controversial role in atherogenesis, we proposed to investigate the involvement of AOC3 in the formation of atherosclerotic plaques in ApoE-/-AOC3-/- mice and human coronary arteries. Methods: Lesions, contractile markers, and AOC3 were studied in aortic tissues from 15- and 25-week-old mice and different stages of human coronary atherosclerotic arteries by immunohistochemistry (IHC) and/or western blot. Human VSMCs, treated or not with LJP1586, an AOC3 inhibitor, were used to measure differentiation markers by qPCR. AOC3 co-localization with specific cell markers was studied by using confocal microscopy in mice and human samples. Results: At 15 weeks old, the absence of AOC3 was associated with increased lesion size, α-SMA, and CD3 staining in the plaque independently of a cholesterol modification. At 25 weeks old, advanced plaques were larger with equivalent staining for α-SMA while CD3 increased in the media from ApoE-/-AOC3-/- mice. At both ages, the macrophage content of the lesion was not modified. Contractile markers decreased whereas MCP-1 appeared augmented only in the 15-week-old ApoE-/-AOC3. AOC3 is mainly expressed by mice and human VSMC is slightly expressed by endothelium but not by macrophages. Conclusion: AOC3 knock-out increased atherosclerotic plaques at an early stage related to a VSMC dedifferentiation associated with a higher T cells recruitment in plaques explained by the MCP-1 augmentation. This suggests that AOC3 may have an important role in atherosclerosis independent of its canonical inflammatory effect. The dual role of AOC3 impacts therapeutic strategies using pharmacological regulators of SSAO activity.

ω-(5-Phenyl-2H-tetrazol-2-yl)alkyl-substituted hydrazides and related compounds as inhibitors of amine oxidase copper containing 3 (AOC3).

Amine oxidase copper containing 3 (AOC3), also known as plasma amine oxidase, semicarbazide-sensitive amine oxidase, or vascular adhesion protein-1, catalyzes the oxidative deamination of primary amines to aldehydes using copper and a quinone as cofactors. Because it is involved in the transmigration of inflammatory cells through blood vessels into tissues, AOC3 is thought to play an important role in inflammatory diseases. Therefore, inhibitors of this enzyme could lead to new therapeutics for the treatment of inflammation-related diseases. Recently, 6-(5-phenyl-2H-tetrazol-2-yl)hexan-1-amine was found to be a tight-binding substrate of AOC3. To obtain novel inhibitors of the enzyme, the amino group of this substrate was replaced with functional groups that occur in known AOC3 inhibitors, such as hydrazide or glycine amide moieties. In addition, derivatives of the compounds obtained in this way were prepared. The obtained hydrazide 5, which proved to be the most effective, was subjected to further structural modifications. Selected hydrazides were evaluated for selectivity toward some other amine oxidases.

Diamine oxidase knockout mice are not hypersensitive to orally or subcutaneously administered histamine.

OBJECTIVE: To evaluate the contribution of endogenous diamine oxidase (DAO) in the inactivation of exogenous histamine, to find a mouse strain with increased histamine sensitivity and to test the efficacy of rhDAO in a histamine challenge model. METHODS: Diamine oxidase knockout (KO) mice were challenged with orally and subcutaneously administered histamine in combination with the ß-adrenergic blocker propranolol, with the two histamine-N-methyltransferase (HNMT) inhibitors metoprine and tacrine, with folic acid to mimic acute kidney injury and treated with recombinant human DAO. Core body temperature was measured using a subcutaneously implanted microchip and histamine plasma levels were quantified using a homogeneous time resolved fluorescence assay. RESULTS: Core body temperature and plasma histamine levels were not significantly different between wild type (WT) and DAO KO mice after oral and subcutaneous histamine challenge with and without acute kidney injury or administration of HNMT inhibitors. Treatment with recombinant human DAO reduced the mean area under the curve (AUC) for core body temperature loss by 63% (p = 0.002) and the clinical score by 88% (p < 0.001). The AUC of the histamine concentration was reduced by 81%. CONCLUSIONS: Inactivation of exogenous histamine is not driven by enzymatic degradation and kidney filtration. Treatment with recombinant human DAO strongly reduced histamine-induced core body temperature loss, histamine concentrations and prevented the development of severe clinical symptoms.

Downregulation of amine oxidase copper containing 1 inhibits tumor progression by suppressing IL-6/JAK/STAT3 pathway activation in hepatocellular carcinoma.

Amine oxidase copper containing 1 (AOC1) is a copper-containing amine oxidase that catalyzes the deamination of polyamines. AOC1 functions as an oncogene in human gastric cancer. There is little information available regarding the function of AOC1 in hepatocellular carcinoma (HCC). In the present study, reverse transcription-quantitative PCR was used to detect the expression levels of AOC1 in HCC tissues, and the role of AOC1 in HCC progression was determined using western blot, Cell Counting Kit 8, clone formation, wound-healing and Transwell assays. An AOC1 survival curve was generated with data downloaded from The Cancer Genome Atlas, and Gene Set Enrichment Analysis was performed to investigate the potential biological mechanisms of AOC1 in HCC. AOC1 was found to be upregulated in HCC tissues, which was associated with a poor prognosis. Furthermore, AOC1-knockdown inhibited HCC cell proliferation, migration and invasiveness, suppressed IL-6 expression, as well as decreasing JAK2 and STAT3 phosphorylation. Ultimately, the results of the present study illustrate that AOC1 promoted the proliferation, migration and invasiveness of HCC cells by regulating the IL-6/JAK/STAT3 pathway.

Amine oxidase copper-containing 3 (AOC3) inhibition: a potential novel target for the management of diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR), a microvascular complication of diabetes, is the leading cause of visual impairment in people aged 20-65 years and can go undetected until vision is irreversibly lost. There is a need for treatments for non-proliferative diabetic retinopathy (NPDR) which, in comparison with current intravitreal (IVT) injections, offer an improved risk-benefit ratio and are suitable for the treatment of early stages of disease, during which there is no major visual impairment. Efficacious systemic therapy for NPDR, including oral treatment, would be an important and convenient therapeutic approach for patients and physicians and would reduce treatment burden. In this article, we review the rationale for the investigation of amine oxidase copper-containing 3 (AOC3), also known as semicarbazide-sensitive amine oxidase and vascular adhesion protein 1 (VAP1), as a novel target for the early treatment of moderate to severe NPDR. AOC3 is a membrane-bound adhesion protein that facilitates the binding of leukocytes to the retinal endothelium. Adherent leukocytes reduce blood flow and in turn rupture blood vessels, leading to ischemia and edema. AOC3 inhibition reduces leukocyte recruitment and is predicted to decrease the production of reactive oxygen species, thereby correcting the underlying hypoxia, ischemia, and edema seen in DR, as well as improving vascular function. CONCLUSION: There is substantial unmet need for convenient, non-invasive treatments targeting moderately severe and severe NPDR to reduce progression and preserve vision. The existing pharmacotherapies (IVT corticosteroids and IVT anti-vascular endothelial growth factor-A) target inflammation and angiogenesis, respectively. Unlike these treatments, AOC3 inhibition is predicted to address the underlying hypoxia and ischemia seen in DR. AOC3 inhibitors represent a promising therapeutic strategy for treating patients with DR and could offer greater choice and reduce treatment burden, with the potential to improve patient compliance.

Effects of different doses of dexmedetomidine on levels of serum claudin-1 and diamine oxidase in patients undergoing laparoscopic radical resection of gynecological malignant tumors / 肿瘤研究与临床

Objective:To explore the effect of different doses of dexmedetomidine (Dex) on levels of tight-junction protein claudin-1 and diamine oxidase (DAO) in patients undergoing laparoscopic radical resection of gynecological malignant tumors.Methods:A total of 60 patients with gynecological malignant tumors who were scheduled to undergo laparoscopic radical resection under general anesthesia from January 2019 to January 2020 in the Second Hospital of Shanxi Medical University were selected, including 43 cases of cervical cancer (stageⅠ-Ⅱ A), 9 cases of ovarian cancer (stageⅠ A-Ⅲ C), and 8 cases of endometrial carcinoma (stageⅠ). Accroding to the random number table method, the patients were divided into control group (group C), low-dose Dex group (group D 1) and high-dose Dex group (group D 2), with 20 cases in each group. Patients in group D 1 were given Dex 0.5 μg·kg -1·h -1 by constant rate intravenous infusion pump after induction until 30 min before the end of operation. Patients in group D 2 were given Dex 1.0 μg·kg -1·h -1 by constant rate intravenous infusion pump after induction until 30 min before the end of operation. Group C adopted the same calculation method and received the same amount of 0.9% sodium chloride solution by infusion pump. At 10 min before induction (T 1), 1 hour after pneumoperitoneum (T 2) and 12 hours after pneumoperitoneum (T 3), 5 ml of brachial vein blood was collected from the patients, and the levels of claudin-1 protein, DAO and blood glucose were measured. Results:At T 1, T 2 and T 3, the expression levels of claudin-1 in group C were (77.05±17.61) pg/ml, (66.76±12.97) pg/ml and (55.93±12.71) pg/ml, and the difference was statistically significant ( F = 10.449, P<0.05); the expression levels of DAO in group C were (4.83±0.93) ng/ml, (5.62±1.01) ng/ml and (5.98±1.21) ng/ml, and the difference was statistically significant ( F = 6.139, P < 0.05); the levels of blood glucose in group C were (4.82±0.66) mmol/L, (7.55±0.94) mmol/L and (6.51±0.54) mmol/L, and the difference was statistically significant ( F = 70.197, P < 0.05). At T 2, the expression level of claudin-1 in group D 1 was (69.12±13.02) pg/ml, which was not significantly different from group C ( t = -0.575, P > 0.05); the expression level of claudin-1 in group D 2 was (76.36±14.89) pg/ml, which was higher than that in group C, and the difference was statistically significant ( t = -2.175, P < 0.05). At T 3, the expression levels of claudin-1 in group D 1 and group D 2 were (66.14±14.36) pg/ml and (73.37±16.93) pg/ml, which were higher than that in group C, and the differences were statistically significant ( t values were -2.380 and -3.682, both P < 0.05). The expression levels of DAO in group D 1 and group D 2 were (5.02±0.84) ng/ml and (4.91±0.93) ng/ml at T 2, and (5.29±0.86) ng/ml and (5.20±0.98) ng/ml at T 3, which were lower than those in group C, and the differences were statistically significant ( t values were 2.051, 2.295, 2.079 and 2.285, all P < 0.05). The levels of blood glucose in group D 1 and group D 2 were (7.10±0.66) mmol/L and (6.77±0.97) mmol/L at T 2, and (5.95±0.94) mmol/L and (5.93±0.74) mmol/L at T 3, which were lower than those in group C, and the differences were statistically significant ( t values were 2.565, 5.374, 2.293 and 2.765, all P < 0.05). Conclusion:Continuous infusion of Dex can inhibit the stress response caused by long-term CO 2 pneumoperitoneum in laparoscopic radical resection of gynecological malignant tumors, and adjust the changes of expression levels of claudin-1 protein and DAO, reduce the damage of intestinal mucosal cells, facilitate the recovery of intestinal function, and the effect of high-dose Dex is better than low-dose Dex.

[Correlation between intestinal mucosal permeability and prognosis in patients with liver cirrhosis].

Objective: To investigate the correlation between changes in intestinal mucosal permeability and prognosis of patients with liver cirrhosis. Methods: Data of 89 cases with liver cirrhosis who were hospitalized in the Hepatology Department of Shanxi Provincial Hospital of Traditional Chinese Medicine from January 2017 to August 2017 were collected as the liver cirrhosis experimental group, and 40 healthy subjects were randomly selected as the healthy control group. JY-DLT, the Intestinal Mucosal Barrier Biochemical Index Analysis System was used to measure the levels of serum diamine oxidase (DAO), D-lactic acid, and endotoxin (ETX) in two groups to evaluate intestinal mucosal barrier function. Spearman's rank correlation test was used to evaluate the correlation between liver cirrhosis prognosis and intestinal mucosal permeability. The results of the two groups were compared by Mann-Whitney H test of two independent samples. One-way Anova was used for intergroup comparison. The pairwise comparison between groups was performed using the LSD or SNK test. Results: The level of ETX in patients with decompensated cirrhosis was significantly higher than that in the compensated phase, and the difference was statistically significant (P < 0.05). The levels of DAO, D-lactic acid and ETX in the liver cirrhosis group were significantly higher than those in the healthy control group, and the differences were statistically significant (P < 0.01). The plasma levels of DAO, D-lactic acid and ETX in the Child-Pugh grade groups of patients with liver cirrhosis were significantly higher than those in the healthy control group, and the differences were statistically significant (P < 0.05). The results of intergroup comparison showed that there were statistically significant differences in DAO, D-lactic acid and ETX levels between Child-Pugh grade A and grade B groups (t = -4.255, 2.527, -2.179, P < 0.05). Furthermore, there were statistically significant differences in the levels of D-lactic acid and ETX between the Child-Pugh grade A and grade C groups (t = -2.693, -4.248, P < 0.01).The plasma levels of DAO, D-lactic acid and ETX levels were positively correlated (r = 0.205, 0.372, 0.342, P < 0.01). D-lactic acid and ETX levels were positively correlated with CTP score, Forns' index, RPR index, APRI score, FIB-4 index and FibroScan score(P < 0.01). Conclusion: The three indices (plasma DAO, D-lactic acid, and ETX) can accurately detect the changes in intestinal mucosal permeability. Moreover, the higher index of intestinal mucosal permeability causes the more severe degree of liver cirrhosis and the correlation between the intestinal mucosal permeability and the prognosis score of liver cirrhosis provides a reference for a new evaluation system and new ideas for the treatment of liver cirrhosis.

Correlation between intestinal mucosal permeability and prognosis in patients with liver cirrhosis / 中华肝脏病杂志

Objective@#To investigate the correlation between changes in intestinal mucosal permeability and prognosis of patients with liver cirrhosis.@*Methods@#Data of 89 cases with liver cirrhosis who were hospitalized in the Hepatology Department of Shanxi Provincial Hospital of Traditional Chinese Medicine from January 2017 to August 2017 were collected as the liver cirrhosis experimental group, and 40 healthy subjects were randomly selected as the healthy control group. JY-DLT, the Intestinal Mucosal Barrier Biochemical Index Analysis System was used to measure the levels of serum diamine oxidase (DAO), D-lactic acid, and endotoxin (ETX) in two groups to evaluate intestinal mucosal barrier function. Spearman’s rank correlation test was used to evaluate the correlation between liver cirrhosis prognosis and intestinal mucosal permeability. The results of the two groups were compared by Mann-Whitney H test of two independent samples. One-way Anova was used for intergroup comparison. The pairwise comparison between groups was performed using the LSD or SNK test.@*Results@#The level of ETX in patients with decompensated cirrhosis was significantly higher than that in the compensated phase, and the difference was statistically significant (P < 0.05). The levels of DAO, D-lactic acid and ETX in the liver cirrhosis group were significantly higher than those in the healthy control group, and the differences were statistically significant (P < 0.01). The plasma levels of DAO, D-lactic acid and ETX in the Child-Pugh grade groups of patients with liver cirrhosis were significantly higher than those in the healthy control group, and the differences were statistically significant (P < 0.05). The results of intergroup comparison showed that there were statistically significant differences in DAO, D-lactic acid and ETX levels between Child-Pugh grade A and grade B groups (t = -4.255, 2.527, -2.179, P < 0.05). Furthermore, there were statistically significant differences in the levels of D-lactic acid and ETX between the Child-Pugh grade A and grade C groups (t = -2.693, -4.248, P < 0.01).The plasma levels of DAO, D-lactic acid and ETX levels were positively correlated (r = 0.205, 0.372, 0.342, P < 0.01). D-lactic acid and ETX levels were positively correlated with CTP score, Forns’ index, RPR index, APRI score, FIB-4 index and FibroScan score(P < 0.01).@*Conclusion@#The three indices (plasma DAO, D-lactic acid, and ETX) can accurately detect the changes in intestinal mucosal permeability. Moreover, the higher index of intestinal mucosal permeability causes the more severe degree of liver cirrhosis and the correlation between the intestinal mucosal permeability and the prognosis score of liver cirrhosis provides a reference for a new evaluation system and new ideas for the treatment of liver cirrhosis.

Co(II) is not oxidized during turnover in the copper amine oxidase from Hansenula polymorpha.

Co(II) substitution into the copper amine oxidases (CAOs) has been an effective tool for evaluating the mechanism of oxygen reduction in these enzymes. However, formation of hydrogen peroxide during turnover raises questions about the relevant oxidation state of the cobalt in these enzymes and, therefore, the interpretation of the activity of the metal-substituted enzyme with respect to its mechanism of action. In this study, Co(II) was incorporated into the CAO from Hansenula polymorpha (HPAO). The effect of hydrogen peroxide on the catalytic activity of cobalt-substituted HPAO was evaluated. Hydrogen peroxide, either generated during turnover or added exogenously, caused a decrease in the activity of the enzyme but did not oxidize Co(II) to Co(III). These results are in strong contrast with results from the CAO from Arthrobacter globiformis (AGAO), where hydrogen peroxide causes an increase in the activity of the enzyme as the Co(II) is oxidized to Co(III). The results of this study with HPAO support previous reports that have shown that this enzyme acts by transferring an electron directly from the reduced TPQ cofactor to dioxygen rather than passing the electron through the bound metal ion. Furthermore, these results provide additional evidence to support the idea that different CAOs use different mechanisms for catalysis.

Perivascular Adipose Tissue's Impact on Norepinephrine-Induced Contraction of Mesenteric Resistance Arteries.

Background: Perivascular adipose tissue (PVAT) can decrease vascular contraction to NE. We tested the hypothesis that metabolism and/or uptake of vasoactive amines by mesenteric PVAT (MPVAT) could affect NE-induced contraction of the mesenteric resistance arteries. Methods: Mesenteric resistance vessels (MRV) and MPVAT from male Sprague-Dawley rats were used. RT-PCR and Western blots were performed to detect amine metabolizing enzymes. The Amplex® Red Assay was used to quantify oxidase activity by detecting the oxidase reaction product H2O2 and the contribution of PVAT on the mesenteric arteries' contraction to NE was measured by myography. Results: Semicarbazide sensitive amine oxidase (SSAO) and monoamine oxidase A (MAO-A) were detected in MRV and MPVAT by Western blot. Addition of the amine oxidase substrates tyramine or benzylamine (1 mM) resulted in higher amine oxidase activity in the MRV, MPVAT, MPVAT's adipocyte fraction (AF), and the stromal vascular fraction (SVF). Inhibiting SSAO with semicarbazide (1 mM) decreased amine oxidase activity in the MPVAT and AF. Benzylamine-driven, but not tyramine-driven, oxidase activity in the MRV was reduced by semicarbazide. By contrast, no reduction in oxidase activity in all sample types was observed with use of the monoamine oxidase inhibitors clorgyline (1 µM) or pargyline (1 µM). Inhibition of MAO-A/B or SSAO individually did not alter contraction to NE. However, inhibition of both MAO and SSAO increased the potency of NE at mesenteric arteries with PVAT. Addition of MAO and SSAO inhibitors along with the H2O2 scavenger catalase reduced PVAT's anti-contractile effect to NE. Inhibition of the norepinephrine transporter (NET) with nisoxetine also reduced PVAT's anti-contractile effect to NE. Conclusions: PVAT's uptake and metabolism of NE may contribute to the anti-contractile effect of PVAT. MPVAT and adipocytes within MPVAT are a source of SSAO.

Expression of vascular adhesion protein-1 in severe hemorrhagic shock and resuscitation in rats / 第二军医大学学报

Objective To observe the expression and activity changes of vascular adhesion protein-1 (VAP-1) in the small intestine and serum of rats during severe hemorrhagic shock and resuscitation, and to study its influence on shock prognosis. Methods Fifty rats were evenly randomized into sham group, hemorrhagic shock group, shock resuscitation group, control recovery group and the experimental recovery group. Rat models of severe hemorrhagic shock and resuscitation were established. Before shock, 1 hour after shock and 1 hour after resuscitation, the expressions of VAP-1 protein and mRNA in the intestinal tissues of rats were examined by Western blotting analysis and real-time RT-PCR, respectively; and the serum levels of VAP-1 and its activities were determined by ELISA kit. Rats in the experimental recovery group was resuscitated by injection of 20 mg/kg 2-bromoethylamine and those in the control recovery group were given 1 mL/kg normal saline, and then the blood pressure, intestinal mucosa injury (Chiu’s score), small intestinal epithelial cell apoptosis (TUNEL detection) and 24-hour survival rates were compared between the two recovery groups. Results The intestinal VAP-1 protein and mRNA expressions and the serum VAP-1 and its activities in the severe hemorrhagic shock group were significantly higher than those in the sham shock group (P<0.05). Compared with the shock group, the above parameters were decreased in the recovery group, but were still higher than those in the sham group. Compared with the saline control group, 20 mg/kg 2-bromoethylamine significantly increased the blood pressure of animals 1 h and 24 h after recovery (P = 0.010, 0.039), significantly improved the Chiu’s score and apoptosis index of small intestinal epithelial cells (P = 0.022, P = 0.002), and improved the 24-hour survival rates of rats(90% to 60%). Conclusion The levels of VAP-1 and its activities are increased in severe hemorrhagic shock rats, and fluid resuscitation can inhibit this increase. Inhibition of VAP-1 activities can improve the low blood pressure, intestinal mucosa injury and apoptosis of small intestinal mucosa cells after the severe hemorrhagic shock and resuscitation, improving the 24-hour survival rates of rats.

Protective effect of ulinastatin on intestinal barrier function of septic rats / 中华临床感染病杂志

Objective To observe the protective effect of ulinastatin (UTI) on the intestinal barrier function of septic rats.Methods Septic rat model was established using Sprague-Dawley rats by cecal ligation and puncture (CLP) method.Thirty Sprague-Dawley rats were randomly divided into 3 groups (n =10 for each grop) : sham group, septic group and UTI group.All rats received intraperitoneal injections of 0.9％ saline (10 mL/kg) after and 8 h after surgery.In UTI group, UTI (10 × 104 U/kg in 10 mL/kg saline) was injected after and 8 h after surgery.Collect blood samples after 0, 8, 12 h after surgery to examine levels of procalcitonin (PCT), intestinal fatty acid binding protein (iFABP) and diamine oxidase (DAO) by enzyme-linked immunosorbent assay (ELISA) method.Rats were killed 12 h after surgery to collect intestine tissue samples.Pathological changes of intestine were observed under microscopy, and the expression of tight junction protein-1 (ZO-1) and occludin were analyzed by Western blot.Results In sham group, the mucosa structure was complete and the shape was normal, and villi stood neatly.In septic group, intestinal was expanded, intertinal mucosal was atrophic, villi were scanty.An inflammatory infiltrate with numerous nuetrophils was found in the mucosal.In UTI group, the level of severity was relatively slight.The relative optical density of Western blot images were decreased on ZO-1 and occludin in CLP and UTI groups, and decreased more in CLP group (F =43.15 and 52.23, P ＜ 0.05).At 0h after surgery, the plasma values of PCT, iFABP and DAO were similar in three groups (F =11.17, 22.45 and 13.58, P ＞ 0.05).At 8h and 12h after surgery, values of PCT, iFABP and DAO in septic and UTI groups were much higher than those in the sham group, and those in UTI group were also significantly higher than those in septic group (F8h=85.26, 44.59 and 101.47, F12h =59.44, 49.26 and 69.57, all P＜0.05).PCT, iFABP and DAO levels were first increased and then fell down in sham group, those in septic group were keeping increasing, and those in UTI group were first increased and then kept stable.Conclusion UTI shows protective effect to intestinal barrier function in rats with sepsis.

The change of intestinal mucosal barrier in severe acute pancreatitis / 中国现代普通外科进展

Objective:To investigate the mechanism of the changes of the intestinal mucosal barrier in Severe acute pancreatitis.Methods:Twenty patients with Severe acute pancreatitis were admitted and 10 volunteers as the control group.The levels of serum tumor necrosis factor alpha(TNF_?)、nitric oxide(NO)、diamine oxidase(DAO) and the concentration of plasma endotoxin(ET) were measured.The ratio of lactulose to mannitol in urine was detected by HPLC with Pulsed Electrochemical Detection.Results:Compared with the control group,significantly increasing parameters could be seen in all the patients with Severe acute pancreatitis,including the ratio of lactulose to mannitol in urine (P