Role of the advanced glycation end products receptor in Crohn's disease inflammation

AIM:To investigate the level of mucosal expression and the involvement of the receptor for the advanced glycation end products(RAGE)in delayed apoptosis and tumor necrosis factor(TNF)-αproduction in Crohn’s disease(CD).METHODS:Surgical and endoscopic specimens from both inflamed and non-inflamed areas of the ileum and/or colon were collected from 20 and 14 adult CD patients,respectively,and used for the assessment of RAGE expression by means of immunohistochemistry and western blotting analysis.Normal tissues from 21 control subjects were used for comparison.The same polyclonal anti-human RAGE antibody(R and D System)was used in all experimental conditions.RAGE staining was quantized by a score including both the amount of positive cells and intensity of immunoreactivity;cellular pattern was also described.The effects of RAGE blocking on apoptotic rate and TNF-αproduction were investigated on immune cells freshly isolated from CD mucosa and incubated both with and without the muramyl dipeptide used as antigenic stimulus.Statistical analysis was performed via the test for trend,with regression models to account for intra-patient correlations.A 2-sided P<0.05 was considered significant.RESULTS:In inflamed areas,RAGE expression in both the epithelial and lamina propria compartments was higher than control tissues(P=0.001 and 0.021,respectively),and a cluster of positive cells were usually found in proximity of ulcerative lesions.Similar results were obtained in the lamina propria compartment of non-inflamed areas(P=0.025).The pattern of staining was membranous and granular cytosolic at the epithelial level,while in the lamina propria it was diffuse cytosolic.When evaluating the amount of protein expression by immunoblotting,a significant increase of both surface area and band intensity(P<0.0001 for both)was observed in CD inflamed areas compared to control tissue,while in non-inflamed areas a significant increase was found only for band intensity(P<0.005).Moreover,a significantly lower expression in noninflamed areas in comparison with inflamed areas was found for both surface area and band intensity(P<0.0006 for both).Finally,RAGE blocking largely affects both the apoptotic rate of mucosal cells(towards an increase in both non-inflamed and inflamed areas of P<0.001 and<0.0001,respectively)and TNF-αsecretion(towards a decrease in both non-inflamed and inflamed areas of P<0.05 and<0.01,respectively),mainly in the presence of antigenic stimulation.CONCLUSION:RAGE is up-regulated in CD,especially in inflamed areas,and it appears to play a role in the mechanisms involved in chronic inflammation.

肿瘤坏死因子-α对中性粒细胞凋亡的影响

细胞凋亡是生物界普遍的生命现象，是由基因控制的细胞自主的有序性的死亡，与临床疾病关系密切。中性粒细胞（ＰＭＮ）是一种主要的炎性细胞，本实验通过光镜、电镜、电泳、流式细胞仪等方法研究了重要的炎性介质肿瘤坏死因子－α（ＴＮＦ－α）对正常和炎症状态下外用血ＰＭＮ凋亡的影响。结果表明，ＴＮＦ－α可以诱导ＰＭＮ凋亡，且与作用时间和作用浓度相关。ＰＭＮ的凋亡在炎症微环境中被改变，ＰＭＮ凋亡延迟，对ＴＮＦ－α反应性下降。

中性粒细胞凋亡与凋亡中性粒细胞表面肿瘤坏死因子受体的研究

细胞凋亡是近年来的研究热点 ,中性粒细胞 (PMN)作为机体防御系统的重要组成部分 ,其凋亡情况倍受关注。为了了解PMN的自然凋亡 ,观察凋亡PMN表面肿瘤坏死因子受体(TNFR)数量的变化 ,本研究利用PMN自然凋亡模型 ,采用放射配基结合受体分析法 (RLBRA) ,检测凋亡PMNTNFR结合量。实验观察到PMN体外培养 0h ,6h ,2 0h ,TNFR结合量呈现增加趋势 ,而蛋白质合成抑制剂放线菌酮 (CHX)仅在一定程度上抑制TNFR与肿瘤坏死因子α(TNF -α)的结合。

Anti-inflammatory effect of Heliotropium indicum Linn on lipopolysaccharide-induced uveitis in New Zealand white rabbits

AIM： To investigate the anti-inflammatory effect of an aqueous whole plant extract of Heliotropium indicum（HIE） on endotoxin-induced uveitis in New Zealand white rabbits.·METHODS： Clinical signs of uveitis including flares,iris hyperemia and miosis, were sought for and scored in1.0 mg/kg lipopolysaccharide（LPS）-induced uveitic rabbits treated orally with HIE（30-300 mg/kg）,prednisolone（30 mg/kg）, or normal saline（10 m L/kg）. The number of polymorphonuclear neutrophils infiltrating, the protein concentration, as well as levels of tumor necrosis factor-α（TNF-α）, prostaglandin E2（PGE2）, and monocyte chemmoattrant protein-1（MCP-1） in the aqueous humor after the various treatments were also determined. A histopathological study of the anterior uveal was performed.· RESULTS： The extract and prednisolone-treatment significantly reduced（P ≤0.001） both the clinical scores of inflammation（1.0-1.8 compared to 4.40 ±0.40 in the normal saline-treated rabbits） and inflammatory cells infiltration. The level of protein, and the concentrationsof TNF-α, PGE2 and MCP-1 in the aqueous humor were also significantly reduced（P ≤0.001）. Histopathological studies showed normal uveal morphology in the HIE and prednisolone-treated rabbits while normal saline-treated rabbits showed marked infiltration of inflammatory cells.· CONCLUSION： The HIE exhibits anti-inflammatory effect on LPS-induced uveitis possibly by reducing the production of pro-inflammatory mediators.

Study on effect of imbalance of TRAF-6, IRAK-1 and NALP3 inflammatory factors in patients with gouty arthritis

Objective:To explore the effect of imbalance of tumor necrosis factor receptor related factor-6(TRAF-6),interleukin 1 receptor associated kinase-1(IRAK-1)and neutrophil alkaline phosphatase-3(NALP3)in patients with gouty arthritis.Methods:The retrospective experiment was conducted on 105 patients with gouty arthritis admitted to our hospital(47 patients with acute onset and 58 patients with remission,namely group A and group B);meanwhile,another 61 healthy volunteers were selected for control,namely group C.The enrolling of the three groups was dated from May 2017 to May 2018,and TRAF-6,IRAK-1 and NALP3 of all subjects were tested through real-time fluorescence quantification(RT-PCR),and the correlation between the three inflammatory factors and gouty arthritis was compared.Results:1)Through treatment,ESR,BUA and total addiment in group A and B were higher than those in group C,among which the three indicators in group A were higher than those in group B(P<0.05),while CRP was lower than that of group C,and the two indicators in group A were lower than those in group B(P<0.05).2)There was no significant difference in the relative expression of TRAF-6 mRNA between group A and group B before treatment(P>0.05),significantly lower than group C(P<0.05);the above indicators of group A and group B were improved to some extent after treatment,but group A was still lower than group B(P<0.05),and the degree of improvement of group A was also lower than that of group C(P<0.05),while the degree of improvement of group B was not significantly different from that of group C(P>0.05).3)The relative expression level of IRAK-1mRNA in group A and group B before treatment showed no significant difference(P>0.05),but was also lower than that in group C(P<0.05).The relative expression level of IRAK-1mRNA in group A and group B increased to some extent after treatment,with group A significantly lower than group C(P<0.05),and group B showed no significant difference compared with group C(P>0.05).4)The relative expression level of NALP-3 mRNA in group A and group B showed no significant difference(P>0.05)before treatment,significantly higher than that in group C(P<0.05);the relative expression of NALP-3 mRNA in group A was not significantly decreased(P>0.05)after treatment,while that in group B was significantly decreased after treatment(P<0.05),indicating significant different compared with group A and group C.5)There was no correlation between)TRAF-6,ESR,CRP and total addiment(P>0.05);IRAK-1 was negatively correlated with CRP,BUA and total addiment(P<0.05);NALP-3 was negatively correlated with ESR and CRP(P<0.05).Conclusion:TRAF-6,IRAK-1 and NALP-3 are all under abnormal expression in the developing of new gouty arthritis,acting as important participants in promoting the occurrence,development and outcome of illness states,so the intervening measures should be taken.

过敏性紫癜患者中性粒细胞及其IgA Fc受体对血管内皮细胞凋亡的影响及机制

目的 研究中性粒细胞及其IgA Fc受体（CD89）在过敏性紫癜（HSP）发病中的作用和对血管内皮细胞的损伤效应及调控机制。方法 入组30例急性期HSP患儿及9例健康对照儿童，分离外周血中性粒细胞；Jacalin亲和层析分离HSP患者血清IgA后，采用聚丙烯葡聚糖凝胶纯化IgA。实时定量PCR（qPCR）、Western印迹分别检测中性粒细胞CD89 mRNA和蛋白表达，流式细胞仪分析中性粒细胞活化标志分子CD11b的表达。分别将HSP患儿中性粒细胞、健康对照中性粒细胞与血管内皮细胞HUVEC共培养，以 HSP患儿血清分离的IgA（HSP IgA）、单体IgA（mIgA）、磷酸盐缓冲液（空白对照组）分别处理，流式细胞仪检测HUVEC细胞凋亡，ELISA检测上清液中白细胞介素8（IL-8）、肿瘤坏死因子α（TNF-α）水平。结果 HSP患儿组中性粒细胞CD89 mRNA表达与健康对照组差异无统计学意义（P = 0.98），但蛋白表达（0.60 ± 0.16）低于健康对照组（0.83 ± 0.24，P = 0.03）。HSP患者中性粒细胞CD11b表达（1 880.25 ± 388.29）显著高于健康对照组（1 109.25 ± 364.25，P 〈 0.01）。与HSP患者中性粒细胞共培养组HUVEC凋亡率（37.44% ± 5.49%）高于与健康对照中性粒细胞共培养组（17.14% ± 4.45%，P 〈 0.01）。HSP IgA处理组HUVEC凋亡率明显高于mIgA处理组（均P 〈 0.01）和空白对照组（P 〈 0.01），且上清液中IL-8、TNF-α浓度明显高于mIgA处理组（均P 〈 0.01）和空白对照组（P值分别为0.01、0.02）。结论 HSP患者外周血中性粒细胞活化，并可诱导血管内皮细胞凋亡。而且HSP IgA能促进中性粒细胞介导的血管内皮细胞凋亡和炎症因子IL-8、TNF-α分泌，而mIgA则可能具有一定抑制效应。