Intestinal epithelium, intraepithelial lymphocytes and the gut microbiota-Key players in the pathogenesis of celiac disease

Celiac disease(CD) is a chronic immune-mediated disorder triggered by the ingestion of gluten in genetically predisposed individuals. Before activating the immune system, gluten peptides are transferred by the epithelial barrier to the mucosal lamina propria, where they are deamidated by intestinal tissue transglutaminase 2. As a result, they strongly bind to human leucocyte antigens(HLAs), especially HLA-DQ2 and HLA-DQ8, expressed on antigen-presenting cells. This induces an inflammatory response, which results in small bowel enteropathy. Although gluten is the main external trigger activating both innate and adaptive(specific) immunity, its presence in the intestinal lumen does not fully explain CD pathogenesis. It has been hypothesized that an early disruption of the gut barrier in genetically susceptible individuals, which would result in an increased intestinal permeability, could precede the onset of gluten-induced immune events. The intestinal barrier is a complex functional structure, whose functioning is dependent on intestinal microbiotahomeostasis, epithelial layer integrity, and the gutassociated lymphoid tissue with its intraepithelial lymphocytes(IELs). The aim of this paper was to review the current literature and summarize the role of the gut microbiota, epithelial cells and their intercellular junctions, and IELs in CD development.

Effects of Estrogen on Mucosal Structure and Numbers and Distribution of Intraepithelial Lymphocytes and Goblet Cells in Small Intestine of Rats

To study the effects of estrogen on the structure of the intestinal mucosal barrier, 18 healthy female Wistar Rats underwent estrus synchronization. In diestrus, they were divided into three groups： one sham operated control group （SHAM） ; one ovariec- tomized group （OVX） ; and one ovariectomized plus estradiol benzoate group （ OVX ＋ E2 ）. Intestinal mu- cosal epithelial cells, intraepithelial lymphocytes （[EL）, and goblet cells （GCs） were observed by light microscope. The results showed that in the OVX group, the intestinal mucosa damaged obviously, the villus atrophied, the ratio of villus height to crypt depth reduced, and the number of IELs and GCs re- duced. The indicators of OVX ＋ Ez group were signif- icantly higher than OVX group, but some indicators were lower than SHAM. These indicated that the function of intestinal mucosal barrier was greatly dam- aged in ovariectomied rat, and proper dosage of estra- diol benzoate Would improve the function of small in- testinal mucosal barrier in ovariectomied rat to some degree.

Mesenteric adipose tissue B lymphocytes promote intestinal injury in severe acute pancreatitis by mediating enteric pyroptosis

Background:Visceral adipose tissue(VAT)has been linked to the severe acute pancreatitis(SAP)prognosis,although the underlying mechanism remains unclear.It has been reported that pyroptosis worsens SAP.The present study aimed to verify whether mesenteric adipose tissue(MAT,a component of VAT)can cause secondary intestinal injury through the pyroptotic pathway.Methods:Thirty-six male Sprague Dawley(SD)rats were divided into six different groups.Twelve rats were randomly divided into the SAP and control groups.We monitored the changes of MAT and B lymphocytes infiltration in MAT of SAP rats.Twelve SAP rats were injected with MAT B lymphocytes or phosphate buffer solution(PBS).The remaining twelve SAP rats were first injected with MAT B lymphocytes,and then with MCC950(NLRP3 inhibitor)or PBS.We collected blood and tissue samples from pancreas,gut and MAT for analysis.Results:Compared to the control rats,the SAP group showed inflammation in MAT,including higher expression of tumor necrosis factor(TNF-α)and interleukin-6(IL-6),lower expression of IL-10,and histological changes.Flow cytometry analysis revealed B lymphocytes infiltration in MAT but not T lymphocytes and macrophages.The SAP rats also exhibited intestinal injury,characterized by lower expression of zonula occludens-1(ZO-1)and occludin,higher levels of lipopolysaccharide and diamine oxidase,and pathological changes.The expression of NLRP3 and n-GSDMD,which are responsible for pyroptosis,was increased in the intestine of SAP rats.The injection of MAT B lymphocytes into SAP rats exacerbated the inflammation in MAT.The upregulation of pyroptosis reduced tight junction in the intestine,which contributed to the SAP progression,including higher inflammatory indicators and worse histological changes.The administration of MCC950 to SAP+MAT B rats downregulated pyroptosis,which subsequently improved the intestinal barrier and ameliorated inflammatory response of SAP.Conclusions:In SAP,MAT B lymphocytes aggravated local inflammation,and promoted the injury to the intestine through the enteric pyroptotic pathway.

Correlation Study Between T Lymphocyte Subsets and Rheumatoid Arthritis

Objective:To study the effect of Helicobacter pylori infection on rheumatoid arthritis and T-lymphocyte subpopulations in patients with rheumatoid arthritis and to provide a new method for the treatment of rheumatoid arthritis by removing Helicobacter pylori from patients.Methods:60 patients with rheumatoid arthritis admitted to the hospital from May 2022 to May 2023 were selected for the study,and all patients underwent a 13-carbon urea breath test to detect gastric H.pylori and the test results showed that 20 cases were negative and 40 cases were positive.The 40 positive patients were divided into the treatment group(n=20)and non-treatment group(n=20)by random number table method and the treatment group was given anti-Helicobacter pylori treatment,and the non-treatment group was given maintenance rheumatoid basic treatment,comparing the anti-cyclic citrulline peptide(CCP),DS28 score,peripheral blood T-lymphocyte subsets(CD4^(+)T-lymphocytes,CD8^(+)T-lymphocytes,CD4^(+)/CD8^(+)ratio)before and after the treatment of patients by 13-carbon urea respiration test(pylori-negative group,20 patients)and those who were positive for the treatment of H pylori(pylori-positive group,40 patients).Besides,the correlation of peripheral blood T-lymphocyte subsets and disease activity between treatment and non-treatment groups in the pylori-positive group was identified together with the correlation of DS28 scores,TNF-αlevels,sedimentation and immunoglobulin,lymphocyte subsets in the pylori-positive treatment group and positive non-treatment group as well as the level of globulin,lymphocyte subsets,and peripheral blood lymphocytes before and after treatment.Results:Before treatment,CCP,DS28 score,CD8^(+)T lymphocyte level of the pylori-negative group were lower than that of the positive group,and CD4^(+)T lymphocyte and CD4^(+)/CD8^(+)ratio were higher than that of the positive group(P<0.05);after treatment,the indexes of the pylori-positive group improved,and there was no significant difference in the comparison of the indexes with those of the pylori-negative group(P>0.05);the positive treatment group had a DS28(3.19±1.02)points,positive non-treatment group DS28(5.36±1.85)points,non-treatment group DS28 score and CD4^(+)T lymphocytes,CD4^(+)/CD8^(+)negative correlation with CD8^(+)T lymphocytes showed a positive correlation(P<0.05);before the treatment,pylori-positive treatment group and non-treatment group DS28 scores,TNF-αlevels,peripheral blood T lymphocyte subpopulation levels were not significantly different(P>0.05);after treatment,DS28 score,TNF-αlevel,CD8^(+)T of the treatment group were lower than those of the non-treatment group,and CD4^(+)T lymphocytes and CD4^(+)/CD8^(+)ratio were higher than those of the non-treatment group(P<0.05).Conclusion:H.pylori affects the level of T lymphocyte subsets in patients with rheumatoid arthritis,and there is a certain correlation between the two.Removal of H.pylori can improve the level of T lymphocyte subsets,which is important for the treatment of patients with rheumatoid arthritis.

Effects of Two Drugs on Small Intestinal Epithelial Lymphocytes of Experimental Piglets with Spleen Deficiency

[ Objective] This study aimed to investigate the effects of Guchang Cuzhang powder and Smecta on small intestinal epithelial lymphocytes of piglets with spleen deficiency and diarrhea induced by Reserpine. [ Method] Eighteen piglets with reserpine-induced spleen deficiency and diarrhea were selected and randomly divided into three groups, six per group, including Guchang Cuzhang powder group, Smeeta group and spleen deficiency control group. Piglets in two drug adnfinistration groups were treated for 7 d using Guchang Cuzhang powder and Smecta, respectively. On the 2nd d and 8th d after drug withdrawal, three piglets in each group were euthanized by jugular vein bleeding to death. Intestine segments in different groups were collected for paraffin embedding, section preparation and HE staining, to conduct histological observation and count the number of small intestinal epithelial lymphocytes. [ Result ] Compared with the spleen deficiency control group, the number of lymphocytes in small intestinal epithelium of piglets in two drug administration groups increased significantly; in addition, the number of lymphocytes in small intestinal epithelium of piglets in Guchang Cuzhang powder group was significantly higher than that in Smecta group. [ Conclusion ] This study provided theoretical basis for the clinical applieation of Guchang Cuzhang powder and Smecta.

Effects of Chinese Medicine on the Number of Intraepithelial Lymphocytes and Goblet Cells of Intestinal Villus of Heat Stress Layers

[ Objective] To study the effects of Chinese herbal additives on the number of intraepithelial lymphocyte and goblet cells of intestinal villus of heat stress layers. [Method] 180 healthy 88-day-old ISA brown egg roosters were selected and randomly divided into nine treatment groups, nor- mal temperature control group, high temperature control group, VC group, prescription one high-dose group, prescription one middle-dose group, prescription one low-dose group, prescription two high-dose group, prescription two middle-dose group, prescription two low-dose group, respec- tively. Prescription one and two groups were respectively fed with low, medium and high concentrations of the three doses of Chinese herbal ex- tracts, and VC group was fed with the VC in aqueous solution. Histological sections conventional technology and HE staining method were used to observe the number of intraepithelial lymphocytes and goblet cells in each sections of small intestine of chicken. [ Result] The number of chicken in- testinal epithelial lymphocytes and goblet cells showed a gradually decreasing trend in a high-temperature state. [ Conclusion] Prescription one and two groups could promote the cytopoiesis of goblet cells and lymphocytes, and the effect of prescription two was the best. Moreover, Adding the Chinese herbs had good effects on relieving the heat stress of layers.

Isolation of Lymphocytes and Their Innate Immune Characterizations from Liver,Intestine,Lung and Uterus

In steady-state conditions, the number and distribution of lymphocyte populations are under homeostatic control. New lymphocytes are continuously produced in primary and secondary lymphoid organs and then achieve immune-competence within different tissues, and they must challenge with resident cells for survival. The first step in the study of tissue lymphoid cells is their isolation in intact and viable form appropriate for establishment of in vitro culture systems. For reasons of simplicity, cell purity, cell yields and various purposes, lymphocytes obtained from different tissues in different labs were subjected to diverse protocols. To fully elucidate the nature of the local immune system as well as to adequately study the innate role of lymphocytes in liver, intestine, lung and uterus, we briefly reviewed the characterization of resident lymphocytes, and additional information on those cells from non-lymphoid tissues by using the recommended operation procedure was also presented.

Effect of Alemtuzumab on Intestinal Intraepithelial Lymphocytes and Intestinal Barrier Function in Cynomolgus Model

Background：Alemtuzumab has been used in organ transplantation and a variety of hematologic malignancies （especially for the treatment of B-cell chronic lymphocytic leukemia）.However,serious infectious complications frequently occur after treatment.The reason for increased infections postalemtuzumab treatment is unknown at this stage.We explore the effect ofalemtuzumab on intestinal intraepithelial lymphocytes （IELs） and intestinal barrier function in cynomolgus model to explain the reason of infection following alemtuzumab treatment.Methods：Twelve male cynomolguses were randomly assigned to either a treatment or control group.The treatment group received alemtuzumab （3 mg/kg,intravenous injection） while the control group received the same volume of physiological saline.Intestinal IELs were isolated from the control group and the treatment group （on day 9,35,and 70 after treatment） for counting and flow cytometric analysis.Moreover,intestinal permeability was monitored by enzymatic spectrophotometric technique and enzyme-linked immunosorbent assay.Results：The numbers of IELs were decreased significantly on day 9 after treatment compared with the control group （0.35 ± 0.07 × 10^8 and 1.35 ± 0.09 × 10^8,respectively; P 〈 0.05） and were not fully restored until day 70 after treatment.There were significant differences among four groups considering IELs subtypes.In addition,the proportion ofapoptotic IELs after alemtuzumab treatment was significantly higher than in the control group （22.01 ± 3.67 and 6.01 ± 1.42,respectively; P 〈 0.05）.Moreover,the concentration of D-lactate and endotoxin was also increased significantly on day 9 after treatment.Conclusions：Alemtuzumab treatment depletes lymphocytes in the peripheral blood and intestine of cynomolgus model.The induction of apoptosis is an important mechanism of lymphocyte depletion after alemtuzumab treatment.Notably,intestinal barrier function may be disrupted after alemtuzumab treatment.

Intraepithelial lymphocytes: bystanders or causative factors in functional gastrointestinal disorders?

Intraepithelial lymphocytes(IELs)are immune cells located in the epithelium of the mucosal layer of the gastrointestinal tract that typically show a CD3-positive phenotype.They are T lymphocytes that are involved in the primary immune response to several luminal antigens,such as food proteins and infectious agents.For this reason,their increase may be pathogenetic in several diseases,the most important being celiac disease.However,an increase above 25 IELs/100 enterocytes in the duodenum,which is considered the cutoff value for celiac disease,may occur in several conditions,such as nonceliac gluten sensitivity,food allergy,Helicobacter pylori(H.pylori)infection,gastroenteritis,and even irritable bowel syndrome(IBS).Therefore,the generic term“microscopic enteritis”or“duodenal lymphocytosis”has been proposed for this condition.

Isolation of γδT cells from mouse small intestine

Gamma/delta T cells (γδТ cells) are among the first lymphoid cells appearing in the ontogenesis. Many of γδТ cells are located in the small intestine/lamina propria of mice and human, where they cooperate with different cells (B-1 cells, in particular) and influence their activity. However, such interactions are studied rather scanty, and the functional role of γδТ cells in the intestine?is not yet fully elucidated. To study the interactions of mouse γδТ cells with other cells it is necessary to have purified cell populations. Unfortunately, most approaches used for isolation of human γδТ cells are not suitable for isolation of mouse γδТ cells. The aim of the present study is the modification of the method of mouse intestinal γδТ cell isolation in the quantities sufficient for the in vivo and in vitro experiments.

Mechanism of exogenous nucleic acids and their precursors improving the repair of intestinal epithelium after 7-irradiation in mice

AIM To clone expressed genes associated withrepair of irradiation-damaged mice intestinalgland cells treated by small intestinal RNA,andto explore the molecular mechanism ofexogenous nucleic acids improving repair ofintestinal crypt.METHODS The animal mode of test group andcontrol group was established,forty-five micebeing irradiated by γ ray were treated with smallintestinal RNA as test group,forty mice beingirradiated by γ ray were treated withphysiological saline as control group,five micewithout irradiation were used as normal control,their jejunal specimens were collectedrespectively at 6h,12h,24h,4d and 8d afterirradiation.Then by using LD-PCR based onsubtractive hybridization,these gene fragmentsdifferentially expressed between test group andcontrol group were obtained,and then werecloned into T vectors as well as beingsequenced.Obtained sequences were screenedagainst.GeneBank,if being new sequences,they were submitted to GeneBank.RESULTS Ninety clones were associated withrepair of irradiation-damaged intestinal glandcells treated by intestinal RNA.These clonesfrom test group of 6h,12h,24h,4d and 8dwere respectively 18,22,25,13,12.By screening against GeneBank,18 of which werenew sequences,the others were dramaticallysimilar to the known sequences,mainly similarto hsp,Nmi,Dutt1,alkaline phosphatase,homeobox,anti-CEA ScFv antibody,arginine/serine kinase and BMP-4,repA.Eighteen genefragments were new sequences,their acceptnumbers in GeneBank were respectivelyAF240164-AF240181.CONCLUSION Ninety clones were obtained tobe associated with repair of irradiation-damagedmice intestinal gland cells treated by smallintestinal RNA,which may be related toabnormal expression of genes and matchedproteins of hsp,Nmi,Duttl,Na,K-ATPase,alkalineph-osphatase,glkA,single strandedreplicative centromeric gene as well as 18 newsequences.

Effect of sialyllactose on growth performance and intestinal epithelium functions in weaned pigs challenged by enterotoxigenic Escherichia Coli

Background:Sialyllactose(SL)is one of the most abundant oligosaccharides present in porcine breast milk.However,little is known about its effect on growth performance and intestinal health in weaned pigs.This study was conducted to explore the protective effect of SL on intestinal epithelium in weaned pigs upon enterotoxigenic Escherichia coli(ETEC)challenge.Methods:Thirty-two pigs were randomly divided into four treatments.Pigs fed with a basal diet or basal diet containing SL(5.0 g/kg)were orally infused with ETEC or culture medium.Results:SL supplementation elevated the average daily gain(ADG)and feed efficiency in the ETEC-challenged pigs(P<0.05).SL also improved the digestibilities of dry matter(DM),gross energy(GE),and ash in non-challenged pigs(P<0.05).Moreover,SL not only elevated serum concentrations of immunoglobulins(IgA,IgG,and IgM),but also significantly decreased the serum concentrations of inflammatory cytokines(TNF-α,IL-1β,and IL-6)upon ETEC challenge(P<0.05).Interestingly,SL increased the villus height,the ratio of villus height to crypt depth(V:C),and the activities of mucosal sucrase and maltase in the jejunum and ileum(P<0.05).SL also elevated the concentrations of microbial metabolites(e.g.acetic acid,propanoic acid,and butyric acid)and the abundance of Lactobacillus,Bifidobacterium,and Bacillus in the cecum(P<0.05).Importantly,SL significantly elevated the expression levels of jejunal zonula occludins-1(ZO-1),occluding,and fatty acid transport protein-4(FATP4)in the ETEC-challenged pigs(P<0.05).Conclusions:SL can alleviate inflammation and intestinal injury in weaned pigs upon ETEC challenge,which was associated with suppressed secretion of inflammatory cytokines and elevated serum immunoglobulins,as well as improved intestinal epithelium functions and microbiota.

富含萝卜硫素的西兰花提取物对小鼠外周血CD8^(+)T淋巴细胞活化及肠道菌群的影响

目的:探讨富含萝卜硫素(SFN)的西兰花提取物对小鼠肠道菌群及外周血CD8^(+)T淋巴细胞活化的影响。方法:将15只C57BL/6N小鼠随机分为空白对照(CON)组、阴性对照(NC)组和SFN组,每组5只小鼠,分别给予蒸馏水、不含SFN的西兰花提取物、含0.3 g/L SFN的西兰花提取物125μL灌胃,连续4周。每2 d测量1次体重。实验结束前采集小鼠外周血检测活化的CD8^(+)T淋巴细胞百分比以及CD8^(+)T淋巴细胞中IL-2、TNF-α的含量。收集小鼠粪便,进行肠道菌群分析。结果:3组小鼠不同时间点体重差异无统计学意义(P>0.05)。SNF组小鼠外周血活化的CD8^(+)T淋巴细胞百分比、CD8^(+)T淋巴细胞中IL-2和TNF-α含量高于CON组和NC组(P<0.05)。3组小鼠肠道菌群组成存在差异,SFN组小鼠肠道中疣微菌门及阿克曼菌属的相对丰度高于CON组和NC组(P<0.05)。肠道中阿克曼菌属的相对丰度与CD8^(+)T淋巴细胞中IL-2和TNF-α含量呈正相关(r=0.934和0.664,P<0.05)。结论:长期连续服用富含SFN的西兰花提取物能增加小鼠肠道中阿克曼菌属等有益菌的丰度,同时可增强外周血CD8^(+)T淋巴细胞的功能。

樱黄素抑制TLR4/MyD88通路减轻肠上皮炎症反应改善小鼠克罗恩病样结肠炎

目的探讨天然植物化合物樱黄素(PRU)对肠上皮细胞炎症和屏障结构的作用及其对小鼠克罗恩病样结肠炎的影响。方法建立脂多糖(LPS)诱导的结肠类器官炎症损伤模型和2,4,6-三硝基苯磺酸溶液(TNBS)诱导的小鼠结肠炎模型,用于评估PRU对肠上皮炎症反应和肠屏障的影响。另外,使用网络药理学结合体内外研究分析PRU调控肠上皮炎症影响肠炎的分子机制。结果PRU干预可抑制LPS诱导的结肠类器官中促炎介质肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-1β的释放以及改善TNBS诱导的小鼠结肠炎症状(体质量下降、疾病活动指数和炎症评分升高);同时,PRU促进LPS诱导的小鼠结肠类器官TNBS小鼠肠上皮细胞间紧密连接蛋白闭锁小带蛋白1(ZO-1),密封蛋白1(claudin-1)的表达和改善移位,同时保护肠屏障结构。PRU可靶向结合Toll样受体4(TLR4)并抑制TLR4/髓样分化因子88(MyD88)信号通路活化。结论PRU可通过靶向拮抗TLR4/MyD88信号的激活,抑制肠上皮细胞炎症和保护肠屏障损伤从而改善克罗病样肠炎。

肠道菌群失调与RRTI患儿T淋巴细胞亚群、免疫球蛋白水平的关系

目的探讨肠道菌群失调与反复呼吸道感染(RRTI)患儿T淋巴细胞亚群、免疫球蛋白水平的关系。方法选取2020年5月至2023年5月在郑州大学第一附属医院治疗的130例RRTI患儿作为观察组,选取同期60例健康体检儿童作为对照组,取两组儿童粪便标本检测肠道菌群分布,根据检测结果进一步将观察组患儿分为菌群失调组和非菌群失调组,同时取空腹静脉血检测T淋巴细胞亚群和免疫球蛋白(Ig)水平。比较各组儿童肠道菌群失调情况、T淋巴细胞亚群和Ig水平,采用Pearson相关系数分析肠道菌群失调与RRTI患儿免疫功能之间的相关性。结果与健康对照组儿童相比,观察组患儿的双歧杆菌、乳酸杆菌总数及双歧杆菌/大肠杆菌总数比值(B/E)均明显减少,而大肠杆菌、肠球菌总数均明显增加,观察组患儿CD3^(+)、CD4^(+)百分率和CD4^(+)/CD8^(+)均降低,而CD8^(+)百分率升高,其IgA和IgG也均低于健康对照组,差异均有统计学意义(P<0.05);菌群失调组患儿CD3^(+)、CD4^(+)百分率和CD4^(+)/CD8^(+)分别为(58.31±10.65)%、(28.19±8.02)%和1.16±0.22,低于非菌群失调组的(62.13±9.80)%、(34.12±7.93)%和1.32±0.37,CD8^(+)百分率为(35.97±9.49)%,高于非菌群失调组的(29.14±9.11)%,IgA和IgG分别为(0.33±0.10)g/L和(7.52±2.12)g/L,低于非菌群失调组的(0.50±0.16)g/L和(8.29±2.07)g/L,差异均有统计学意义(P<0.05);Pearson法分析结果显示,RRTI患儿粪便标本中双歧杆菌、乳酸杆菌计数及B/E与CD3^(+)、CD4^(+)T淋巴细胞比例、CD4^(+)/CD8^(+)比值、IgA和IgG均呈正相关(P<0.05),与CD8^(+)呈负相关(P<0.05);而大肠杆菌和肠球菌计数与T淋巴细胞亚群及IgA、IgG均呈负相关(P<0.05)。结论RRTI患儿肠道菌群失调明显,这可能是影响其发病和病情加重的主要因素之一,同时患儿存在的肠道菌群紊乱与其外周血T淋巴细胞和免疫球蛋白水平异常有关,可能是造成患儿免疫功能降低的原因之一,因此对RRTI的防治还应重视肠道菌群紊乱的监测和肠道微生态环境的改善。

锌剂通过NF-κB信号通路修复肠黏膜屏障损伤的研究进展

肠黏膜屏障是机体抵御肠腔内有害抗原的第一道防线,缺血缺氧、理化免疫、肠道菌群变化、氧自由基等多种因素的参与可导致肠黏膜屏障损伤。锌是一种至关重要的微量元素,可通过减少肠道损伤、抗炎和肠道微生物群重建机制来调节黏膜完整性。锌可通过调节NF-κB信号通路来减少肠道损伤、对抗炎症、重构肠道微生物群来调节黏膜完整性,以达到修复肠道黏膜损伤的目的。这为研究锌剂修复肠黏膜屏障损伤的机制提供了参考。

HIF对乙醇诱导肠上皮细胞屏障功能损伤的影响

目的 探究低氧诱导因子(hypoxia-inducible factor, HIF)-1α(HIF-1α)和2α(HIF-2α)对乙醇诱导的结肠腺癌细胞(colorectal adenocarcinoma cell, Caco-2)单层膜肠上皮屏障功能损伤的作用及机制。方法 利用Caco-2细胞建立单层膜肠上皮细胞屏障模型,用不同浓度乙醇处理,MTT法检测细胞的增殖活性。选取合适的乙醇作用浓度和时间刺激Caco-2细胞,ELISA检测炎性细胞因子白介素-1β(interleukin 1β,IL-1β)和白介素-6(interleukin 6,IL-6)水平,Western blot法和RT-PCR检测HIF-1α、HIF-2α、人质膜型唾液酸酶(Neu3)蛋白及mRNA的表达,采用跨上皮电阻(transepithelial electrical resistance, TEER)评估细胞单层膜的通透性。转染小分子干扰RNA(small interfering RNA, siRNA)敲低HIF-1α、HIF-2α水平后,乙醇处理Caco-2细胞,检测细胞的增殖活性、炎性因子水平、TEER数值和Neu3的表达。最后,敲低Neu3的表达,乙醇处理细胞,检测细胞单层膜的TEER。结果当乙醇浓度>5%时,对Caco-2细胞增殖活性的抑制呈现浓度依赖性。用5%浓度的乙醇刺激Caco-2细胞1h,与对照组比较,发现其炎性细胞因子IL-1β和IL-6的分泌增加,HIF-1α、HIF-2α、Neu3的表达升高,TEER水平下降(P<0.05)。分别敲低Caco-2细胞的HIF-1α和HIF-2α表达后,与阴性序列对照组比较,其细胞增殖活性减弱,均促进了IL-1β和IL-6的分泌,两者TEER数值也进一步降低(P<0.05)。并且,敲低HIF-2α抑制了细胞中Neu3的表达(P<0.05)。最后,敲低Neu3使乙醇诱导的细胞单层膜TEER值下降(P<0.05)。结论 一定浓度和时间的乙醇暴露可引起肠上皮细胞增殖活性减弱和炎性细胞因子释放,使肠上皮细胞屏障功能受损,HIF-1α和HIF-2α参与了该过程的调节。敲低HIF-1α或HIF-2α可进一步加重乙醇诱导的肠上皮细胞屏障功能的损伤,具体机制可能是肠上皮细胞中HIF-2α,而不是HIF-1α通过调控Neu3来实现。

CD48在溃疡性结肠炎小鼠肠道免疫细胞中的表达及意义

目的探讨信号传导淋巴细胞活化分子家族2(signaltransduction lymphocyte activation molecule family 2,SLAMF2/CD48)在溃疡性结肠炎(ulcerative colitis,UC)小鼠肠道免疫细胞中的表达及意义。方法选择6~8周龄的C57BL/6J雄性小鼠5只,分离其结肠上皮细胞(intestinal epithelial cells,IECs)及固有层淋巴细胞(lamina propria lymphocytes,LPLs),提取细胞总RNA,RT-PCR、RT-qPCR和流式细胞术检测不同细胞中CD48的表达情况。将10只6~8周龄的C57BL/6J雄性小鼠随机分为对照组和UC组,每组5只。UC组小鼠以含2.5%DSS的饮用水喂养5 d诱导急性UC模型,对照组小鼠以正常饮用水喂养;采用流式细胞术检测小鼠结肠上皮细胞中EPCAM^(+)上皮细胞及CD45^(+)免疫细胞中CD48的表达及两组小鼠结肠LPLs中CD4^(+)T淋巴细胞、CD8^(+)T淋巴细胞及CD19^(+)B淋巴细胞和CD11b^(+)Ly6G^(+)中性粒细胞、CD11b^(+)F4/80^(+)巨噬细胞及CD11c^(+)MHCⅡ^(+)树突状细胞中CD48的表达情况。结果CD48在C57BL/6J雄性小鼠结肠LPLs中的表达高于IECs(P<0.001)。在结肠IECs内,CD45^(+)免疫细胞中CD48的表达高于EPCAM^(+)上皮细胞。与对照组相比,UC组CD11b^(+)F4/80^(+)巨噬细胞中CD48的表达降低(P=0.0065)。结论CD48在结肠免疫细胞中的表达高于上皮细胞,说明CD48可能通过免疫细胞而不是上皮细胞参与UC的发生发展过程。进一步研究发现,DSS诱导UC模型后,CD48在巨噬细胞中的表达下降,提示其可能通过巨噬细胞在急性UC的发生发展中发挥了一定作用。

复合乳酸菌胶囊配合营养支持对重症肺炎患者营养生化指标及肠道微生态的影响

目的探讨复合乳酸菌胶囊配合营养支持对重症肺炎患者营养生化指标及肠道微生态的影响。方法选择2017年10月至2019年11月郑州市中心医院收治的117例重症肺炎患者为研究对象。根据治疗方案将患者分为对照组(n=58)和观察组(n=59)。2组患者均给予基础对症治疗,在此基础上,对照组患者给予肠内营养支持,观察组患者给予肠内营养支持和复合乳酸菌胶囊,2组患者均治疗2周。记录2组患者机械通气时间、住院时间、重症监护时间、抗生素使用时间、不良事件发生情况。分别于治疗前、治疗2周后,采用全自动生化分析仪检测2组患者血清白蛋白(ALB)、前白蛋白(PA)水平及淋巴细胞计数(TLC);流式细胞仪测2组患者辅助性T细胞(Th)1、Th2占比,并计算Th1/Th2比值;血气分析仪检测2组患者二氧化碳分压(PaCO_(2))、氧合指数(PaO_(2)/FiO_(2))、血氧饱和度(SaO_(2))。分别于治疗前及治疗2周后,采用急性生理与慢性健康状况评分Ⅱ(APACHEⅡ)评估患者疾病严重程度。分别于治疗前、治疗2周后,取2组患者新鲜粪便检测肠道微生态指标(乳酸杆菌、肠球菌、双歧杆菌计数)。结果观察组患者机械通气时间、住院时间、重症监护时间、抗生素使用时间均显著短于对照组(P<0.05)。治疗前2组患者的PA、TLC、ALB水平比较差异无统计学意义(P>0.05)。2组患者治疗2周后的PA、TLC、ALB水平均显著高于治疗前(P<0.05)。治疗2周后,观察组患者PA、TLC、ALB水平显著高于对照组(P<0.05)。治疗前2组患者PaO_(2)/FiO_(2)、PaCO_(2)、SaO_(2)及APACHEⅡ评分比较差异无统计学意义(P>0.05);2组患者治疗2周后的PaO_(2)/FiO_(2)、SaO_(2)均显著高于治疗前,PaCO_(2)及APACHEⅡ评分均显著低于治疗前(P<0.05);治疗2周后,观察组患者PaO_(2)/FiO_(2)、SaO_(2)显著高于对照组,PaCO_(2)、APACHEⅡ评分显著低于对照组(P<0.05)。治疗前2组患者的Th1、Th2占比及Th1/Th2比值比较差异无统计学意义(P>0.05)。2组患者治疗2周后的Th1占比、Th1/Th2比值显著高于治疗前,Th2占比显著低于治疗前(P<0.05)。治疗2周后,观察组患者的Th1占比、Th1/Th2比值显著高于对照组,Th2占比显著低于对照组(P<0.05)。治疗前、治疗2周后,2组患者粪便中乳酸杆菌、肠球菌、双歧杆菌计数比较差异无统计学意义(P>0.05)。2组患者治疗前与治疗后粪便中乳酸杆菌、肠球菌、双歧杆菌计数比较差异无统计学意义(P>0.05)。对照组患者呼吸机相关肺炎(VAP)、呼吸衰竭、腹泻发生率及28 d病死率分别为18.97%(11/58)、17.24%(10/58)、20.70%(12/58)、5.17%(3/58),观察组患者VAP、呼吸衰竭、腹泻发生率及28 d病死率分别为5.08%(3/59)、3.40%(2/59)、3.40%(2/59)、1.70%(1/59);观察组患者VAP、呼吸衰竭、腹泻发生率显著低于对照组(χ^(2)=5.350、6.0960x09、8.310,P<0.05),2组患者28 d病死率比较差异无统计学意义(χ^(2)=0.277,P>0.05)。结论复合乳酸菌胶囊配合营养支持有助于改善重症肺炎患者营养生化指标及血气指标,降低APACHEⅡ评分,减少不良事件发生。