Soluble intercellular adhesion molecule-1,D-lactate and diamine oxidase in patients with inflammatory bowel disease

AIM： To study the levels of serum soluble intercellular adhesion molecule-1 （sICAM-1）, plasma D-lactate and diamine oxidase （DAO） in patients with inflammatory bowel disease （IBD）, and the potential clinical significance. METHODS： Sixty-nine patients with IBD and 30 healthy controls were included in this study. The concentration of sICAM-1 was detected with enzyme-linked immunosorbent assay, the level of D-lactate and DAO was measured by spectroscopic analysis, and the number of white blood cells （WBC） was determined by routine procedure. RESULTS： The levels of sICAM-I, DAO, and WBC in IBD patients were significantly higher than those in the control group （P 〈 0,01）, sICAM-I in IBD patients was found to be closely related to the levels of DAO and D-lactate （212.94 ± 69.89 vs 6.35 ± 2.35, P = 0.000）, DAO 212.94 ± 69.89 vs 8.65 ± 3.54, P = 0.000） and WBC （212.94 ± 69.89 vs 7.40 ± 2.61, P = 0.000）, but no significant difference was observed between patients with ulcerative colitis and patients with Crohn＇s disease. The post-treatment levels of sICAM-I, D-lactate and WBC were significantly lower than before treatment （sICAM-I 206.57 ± 79.21 vs 146.21 ± 64.43, P = 0.000）, （D-lactate 1.46 ± 0.94 vs 0.52± 0.32, P = 0.000） and （WBC 7.24 ± 0.2.33 vs 5.21 ± 3.21, P = 0.000）. CONCLUSION： sICAM-1, D-lactate and DAO are closely related to the specific conditions of IBD, and thus could be used as a major diagnostic index.

Biosensor-assisted CRISPRi high-throughput screening to identify genetic targets in Zymomonas mobilis for high d-lactate production

Lactate is an important monomer for the synthesis of poly-lactate(PLA),which is a substitute for the petrochemical plastics.To achieve the goal of high lactate titer,rate,and yield for commercial production,efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and tolerance.In this study,an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence,which could be the mutant strains with increased d-lactate production.Then,LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference(CRISPRi)library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole genome.Specifically,two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting(FACS),respectively.Two genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production,further knockout of ZMO1323 and ZMO1530 resulted in a 15%and 21%increase of d-lactate production,respectively.This work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in Z.mobilis,but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.

田七口服液对缺血性肠炎患者血清中I-FABP、D-LAC、Fg水平的影响观察

目的:探讨田七口服液在缺血性肠炎治疗中的效果。方法:选取本院2015年4月-2017年3月住院治疗的缺血性肠炎患者100例,按照随机数字表法将其分为治疗组和对照组,每组50例。治疗组给予田七口服液10 mL,3次/d;对照组静滴复方丹参注射液(5%葡萄糖注射液250 mL+复方丹参注射液20 mL),1次/d。两组均经药物加常规治疗2周,观察比较两组治疗前后血清中I-FABP、DLAC、Fg的水平变化。结果:治疗2周后,治疗组总有效率达96.0%,与治疗前比较,血清I-FABP、D-LAC及Fg水平均显著降低(P<0.01),且治疗组的血清Fg水平低于对照组(P<0.05);治疗组的血清I-FABP、D-LAC水平与对照组比较,差异均无统计学意义(P>0.05)。两组治疗均未出现相关不良反应(P>0.05)。结论:田七口服液治疗缺血性肠炎疗效显著。

超早期EN联合乌司他丁治疗SAP对免疫功能、D-lactate、endotoxin的影响

探讨分析急性重症胰腺炎(severe acute pancreatitis,SAP)患者采用超早期肠内营养(enteral nutrition, EN)联合乌司他丁治疗对免疫功能、D-lactate、endotoxin产生的影响效应。方法 本次研究时间节点为2022年1月—2022年12月,选择了在我院实施治疗的SAP患者80例,基于随机数字双盲法分组,对照组的治疗仅行超早期EN,观察组行超早期EN+乌司他丁治疗,对两组患者最后治疗结局进行对比和分析。结果 与对照组相比,观察组肠鸣音恢复所需时间、体温恢复正常时间、腹痛消失时间均更短(P<0.05);治疗前与治疗后第3天,两组APACHEⅡ评分存在的差异不明显(P>0.05),经过治疗,患者症状第 5 天发生了明显变化(P<0.05);治疗前,两组患者炎性因子指标差异不明显(P>0.05),经过治疗,观察组变化优于对照组(P<0.05);治疗前,两组患者肠道黏膜屏障指标差异不明显(P>0.05),经过治疗,观察组变化优于对照组(P<0.05);治疗前,两组患者CD3+、CD4+、CD8+水平差异不明显(P>0.05);经过治疗,观察组变化优于对照组(P<0.05);地比观察组与对照组并发症发生率,差异明显(5.00% VS 17.50%),P<0.05。结论 对于SAP患者的干预,采取超早期EN联合乌司他丁治疗,在促进患者症状缓解的同时,可以对患者的免疫功能、D-lactate、endotoxin指标起到改善作用,临床价值显著。

急性脑梗死及脑出血患者应激状态下肠黏膜屏障功能变化

目的 研究急性脑梗死、脑出血患者发病早期肠道屏障功能随病程进展的变化。方法 选取2013年9月-2015年12月徐州市中心医院神经内科,并确诊为急性脑梗死、脑出血的患者67例,分别于不同时间点取静脉血（12 h内、24 h、72 h、1周、3个月）。同时选取同期门诊健康体检者54例作为对照组,检测其血浆D-乳酸（D-LAC）、二胺氧化酶（DAO）水平。结果 （1）与正常对照组相比,急性脑梗死、脑出血患者在发病后均出现肠黏膜屏障功能改变,且脑梗死、脑出血组患者在发病24h后较发病初期（12 h内）D-LAC、DAO水平逐渐升高,72 h达到高峰,随后1周血清D-LAC及DAO持续高水平（P〈0.05）;（2）在发病12 h内、24 h、72 h、1周、3个月5个时间段内脑梗死、脑出血患者两组间的D-LAC、DAO水平对比,差异无统计学意义（P〉0.05）。结论 脑梗死及脑出血患者急性期应激状态下均会出现肠黏膜屏障功能的改变,且两者在相同发病时间段内无显著差异。

动态监测肠脂肪酸结合蛋白和D-乳酸对重度胃肠损伤患者早期肠内营养失败的预测价值

目的探讨动态监测肠脂肪酸结合蛋白(I-FABP)与D-乳酸(D-LAC)对重度胃肠损伤患者早期肠内营养(EN)失败的预测价值。方法①纳入2021年2月至2022年2月南京医科大学附属苏州医院不同监护室重度胃肠损伤患者76例,年龄27~90岁。②入院立即采静脉血作为基础对照(D1),24 h后再采血一次,记为D2。③24~48 h予以早期EN,观察7 d,EN未终止作为早期EN成功组;7 d内因持续性喂养不耐受或全身情况恶化而停止EN为早期EN失败组。④治疗过程不作干预,肠内喂养按照指南推荐意见实施;本研究所有患者入院第一个24 h内均未接受EN。⑤通过ELISA测定血清样本I-FABP和D-LAC。⑥比较两组一般资料、不同时间I-FABP和D-LAC,并定义患者入院第一个24 h内I-FABP、D-LAC的差值(ΔI-FABP、ΔD-LAC):ΔI-FABP=D2 I-FABP-D1 I-FABP;ΔD-LAC=D2 D-LAC-D1 D-LAC;探讨不同时间I-FABP、D-LAC、ΔI-FABP及ΔD-LAC对早期EN失败的预测价值。结果①早期EN成功组和失败组一般资料比较,年龄及入院APACHEⅡ评分差异有统计学意义(P<0.05)。②D2 I-FABP(AUC 0.783,95%CI 0.677~0.888,P=0.000)及ΔI-FABP(AUC 0.801,95%CI 0.701~0.902,P=0.000)对早期EN失败均表现出良好的预测作用,最佳预测值分别为D2 I-FABP 2838.8 pg/mL(敏感度83.3%,特异度72.5%),ΔI-FABP 175.0 pg/mL(敏感度80.6%,特异度75.0%)。③D2 D-LAC(AUC 0.776,95%CI 0.672~0.879,P=0.000)及ΔD-LAC(AUC 0.773,95%CI 0.664~0.881,P=0.000)对早期EN失败均有良好的预测作用,最佳预测值分别为D2 D-LAC 0.4015 mmol/L(敏感度75%,特异度70%),ΔD-LAC 0.1835 mmol/L(敏感度69.4%,特异度82%)。④年龄(AUC 0.656,95%CI 0.532~0.779,P=0.020)及APACHEⅡ评分(AUC 0.655,95%CI 0.532~0.778,P=0.020)对早期EN失败有一定的预测作用,但预测能力要明显低于D2 I-FABP、ΔI-FABP、D2 D-LAC及ΔD-LAC。⑤仅D2 D-LAC对28 d死亡有较低的预测作用(AUC 0.690,95%CI 0.562~0.818,P=0.006)。结论动态监测I-FABP和D-LAC对重度胃肠损伤患者早期EN失败具有预测价值。

肠黏膜屏障损伤与脑动脉硬化相关性研究

脑动脉硬化至缺血性脑卒中对机体来说是一个持续强刺激，可以引起全身应激的播散效应，引发远隔脏器的病理生理甚至病理解剖的改变，肠道是应激反应的中心器官，同时也是重要的靶器官之一，

Functional changes of intestinal mucosal barrier insurgically critical patients

BACKGROUND： The gut is capable of inducing multiple organ dysfunction syndrome （MODS）. In the diagnosis and treatment of critical ill patients, doctors should pay particular attention to the protection or recovery of intestinal barrier function. However, no reliable diagnostic criteria are available clinically. This study aimed to assess the changes of intestinal mucosal barrier function in surgically critical ill patients as well as their signi？ cance.METHODS： Thirty-eight surgically critical ill patients were enrolled as a study group （APACHE II〉8 scores）, and 15 non-critical ill patients without intestinal dysfunction were selected as a control group （APACHE II〈6）. General information, symptoms, physical signs, and APACHE II scores of the patients were recorded. The patients in the study group were subdivided into an intestinal dysfunction group （n=26） and a non-intestinal dysfunction group （n=12）. Three ml venous blood was collected from the control group on admission and the same volume of plasma was collected from the study group both on admission and in the period of recovery. The plasma concentrations of endotoxin, diamine oxidase （DAO）, D-lactate, and intestinal fatty-acid binding protein （iFABP） were detected respectively. The data collected were analyzed by the SPSS 17.0 software for Windows. RESULTS： The levels of variables were significantly higher in the study group than in the control group （P〈0.01）. They were higher in the intestinal dysfunction group than in the non-intestinal dysfunction group （DAO P〈0.05, endotoxin, D-lactate, iFABP P〈0.01）. In the non-intestinal dysfunction group compared with the control group, the level of endotoxin was not significant （P〉0.05）, but the levels of DAO, D-lactate and iFABP were statistically significant （P〈0.05）. The levels of variables in acute stage were higher than those in recovery stage （P〈0.01）.The death group showed higher levels of variables than the survival group （endotoxin and D-lactate P〈0.01, DAO and iFABP P〈0.05）.CONCLUSION： The plasma concentrations of endotoxin, DAO, D-lactate, and intestinal fatty-acid binding protein （iFABP） could re？ ect a better function of the intestinal mucosa barrier in surgically critical ill patients.

Effect of Lianshu preparation on lipopolysaccharide-induced diarrhea in rats

AIM： To investigate the effect of Lianshu preparation on lipopolysaccharide （LPS）-induced diarrhea in rats. METHODS： A diarrhea model was established in Sprague Dawley rats via injection of 1 mL of 30 mg/kg LPS. A total of 40 rats were randomly divided into normal group, LPS group, LPS ＋ Lianshu group, LPS ＋ berberine group （n = 10 in each group）. Their intestinal mucosal barrier and frequency of diarrhea were observed. Levels of glucose, serum Na^＋, K^＋, Cl and hematocrit, plasma nitrogen monoxide （NO）, diamine oxidase （DAO）, and D （-）-lactate were measured. The number of IgA＋ plasma cells in small intestine was detected and SIgA levels in the intestinal fluid were measured. The antipyretic activity of Lianshu preparation in rats was evaluated using Brewer＇s yeast-induced pyrexia （10 mL/kg of 20% aqueous suspension）. Acetaminophen （250 mg/kg, intragastric administration, bid） was comparison. Temperature used as a standard drug for was recorded 1 h before and 6 h after Brewer＇s yeast injection. Finally, small intestina transmission in mice treated with Lianshu was detected after intraperitoneal injection of methyl prostigmin （2 mg/kg）. Atropine （10 g/kg） was used as a control. The ink content in intestine was determined and the total length of intestine was measured. RESULTS： The frequency of diarrhea was higher in LPS group than in LPS ＋ Lianshu group and LPS ＋ berberine group （36.70± 5.23 vs 28.50 ±4.06 and 32.70±9.30 respectively, P 〈 0.01）, and lower in LP5 ＋ Lianshu group than in LPS ＋ berberine group （P = 0.03）. The levels of Na＋, glucose, Cl, K^＋ were significantly lower in LPS ＋ Lianshu group than in LPS ＋ berberine group （140.35±3.19 mmol/L vs 131.99±4.86 mmol/L, 8.49 ±1.84 mmol/L vs 6.54±2.30 mmol/L, 106.29± 4.41 mmol/L vs 102.5±1.39 mmol/L, 5.08±0.66 mmol/L vs 4.32 ± 0.62 mmol/L respectively, P 〈 0.05）. The level of hematocrit was lower in LPS ＋ Lianshu group than in LPS ＋ berberine group （0.50% ±0.07% vs 0.59%± 0.10% respectively, P 〈 0.05）. The plasma levels of NO, DAO and D （-）-lactate were higher in LPS group than in normal group （79.74 ± 7.39μmol/L vs 24.94 ± 3.38μmol/L, 2.48 ±0.42μ/mL vs 0.82 ±0.33 p/mL, 5.63± 0.85μg/mL vs 2.01 ±0.32 μg/mL respectively, P 〈 0.01）, and lower in LPS ＋ Lianshu group than in LP5 ＋ berberine group （48.59±4.70μmol/L vs 51.56 ±8.38 μmol/L, 1.43± 0.53μmol/mL vs 1.81 ±0.42 μmol/mL, 4.00± 0.54 μg/mL vs 4.88 ± 0.77 pg/mL respectively, P 〈 0.05）. The morphology of the intestinal mucosa showed destroyed villi in LPS group and atrophied intestinal mucosa in other groups. The pathological intestinal mucosal changes were less in LPS ＋ Lianshu group than in LPS group. The number of IgA＋ plasma cells and amount of SIgA were higher in LPS ＋ Lianshu group than in LPS group （1.16±0.19/μm^2 vs 1.09±0.28/μm^2, P = 0.026; 0.59 ±0.12 mg/L vs 0.15± 0.19 mg/L respectively, P = 0.000）. Lianshu had counteractive effects on yeast-induced pyrexia and enterokinesia in rats. CONCLUSION： Lianshu preparation has therapeutic effects on LPS-induced diarrhea and enterokinesia in rats.

Controversies about the use of serological markers indiagnosis of in flammatory bowel disease

The serological markers are increasingly used in diagnosis of inflammatory bowel disease(IBD).D-lactate and diamine oxidase are new indicators that can be used to reveal the damage to intestinal mucosa and permeability alteration in IBD.Although the two biological markers seem more sensitive,recent clinical trials and animal experiments have shown controversies about the use of them in diagnosis of IBD.Therefore, these markers should be interpreted cautiously and further prospective studies are needed to establish their clinical role in diagnosis of IBD.

Effects of cardiopulmonary bypass on tight junction protein expressions in intestinal mucosa of rats

AIM： To investigate the tight junction protein expressions of intestinal mucosa in an experimental model of cardiopulmonary bypass （CPB） in rats. METHODS： Thirty anesthetized rats were randomly divided into two groups： Group S （n = 10） served as sham operation and group C （n = 20） served as CPB which underwent CPB for 1 h. Expression of occludin and zonula occludens-1 （ZO-1） were determined by Western blotting and immunotochemistry, respectively. Plasma levels of diamine oxidase （DAO） and d-lactate were determined using an enzymatic spectrophotometry. RESULTS： Immunohistochemical localization of occludin and ZO-1 showed disruption of the tight junctions in enterocytes lining villi at the end of CPB and 2 h after CPB. The intensities of the occludin and ZO-i at the end of CPB were lower than those of control group （76.4% ± 22.5% vs 96.5% ± 28.5% and 62.4% ± 10.1% vs 85.5% ±25.6%, P 〈 0.05） and were further lower at 2 h after CPB （50.5% ± 10.5% and 45.3% ± 9.5%, P 〈 0.05）. Plasma d-lactate and DAO levels increased significantly （8.688 ± 0.704 vs 5.745 ± 0.364 and 0.898 ± 0.062 vs 0.562 ± 0.035, P 〈 0.05） at the end of CPB compared with control group and were significantly higher at 2 h after CPB than those at the end of CPB （9.377 ± 0.769 and 1.038 ± 0.252, P 〈 0.05）. There were significant negative correlations between occludin or ZO-1 expression and DAO （r^2 = 0.5629,r^2 = 0.5424, P 〈 0.05） or d-lactate levels （r^2 = 0.6512,r^2 = 0.7073, P 〈 0.05） both at the end of CPB and 2 h after CPB. CONCLUSION： CPB markedly down-regulates the expression of occludin and ZO-1 proteins in intestinal mucosa of rats. The close correlation between expression of tight junctions （TJs） and plasma levels of DAO or d-lactate supports the hypothesis that intestinal permeability increases during and after CPB because of decreases in the expressions of TJs.

Increased intestinal permeability in pathogenesis and progress of nonalcoholic steatohepatitis in rats

AIM： To investigate whether increased intestinal permeability contributes to the pathogenesis and progress of nonalcoholic steatohepatitis by observing its dynamic change in rat models. METHODS： Rat models of nonalcoholic steatohepatitis were established by giving a fat-rich diet. The rats were sacrificed at wk 8, 12 and 16 during the study. Rats fed with normal diet were taken as control. Plasma D-lactate, plasma diarnine oxidase, serum lipids and liver transarninases were measured in blood of the femoral artery. Hepatic steatosis and inflammation were assessed by haematoxylin-eosin staining. RESULTS： A rat model of nonalcoholic steatohepatitis was established successfully. Plasma D-lactate level in model group at wk 8, 12 and 16 and diarnine oxidase level in model group at wk 12, 16 increased significantly compared with those in control group. There were notable differences of D-lactate and diarnine oxidase level in model group between wk 8 and 12 as well as between wk 12 and 16. Serum lipids, liver transaminases and liver injury also increased with disease development CONCLUSION： Increased intestinal permeability caused by intestinal bacterial overgrowth and endotoxin-induced intestinal destruction exists in rats with nonalcoholic steatohepatitis, which may partially explain the pathogenesis and progress of this disease.

Synthesis and molecular recognition characteristics of a tetrapodal benzene cage

In this contribution,we describe the preparation and recognition characteristics of a novel tetrapodal benzene cage(1).The cage can express a wide recognition range without losing selectivity for the object of appropriate size and functional groups.The key to obtaining the desired structural isomer of 1 is the synthesis and isolation of the o-bis(bromomethyl)benzene precursor(5).Three distinct guests,F^(−)(ex-tremely small size),d-lactate(appropriate size)and l-Asp(branched shape),were selected as examples to demonstrate the recognition characteristics of 1.By NMR titration studies,they all expressed good binding affinity(K>10^(5) L/mol)in competitive medium(10%DMSO/THF),indicating that 1 has a wide recognition scope.The highest binding constant was observed for d-lactate,revealing that 1 has good selectivity for d-lactate versus F^(−)and L-Asp.Moreover,the NMR titration study of F^(−)in DMSO indicates 1 can achieve different binding modes(1:1 and 2:1 guest-host)for small-sized guests,which allows for the further development of binary binding properties and thereafter applications in the field of catalysis.