OBJECTIVE: To evaluate the roles of CG in visceral analgesia, intestinal inflammation, barrier function, and to develop computational molecular models.
METHODS: Visceral pain was recorded through colorectal distension (CRD) in a model of long-lasting colon hypersensitivity induced by an intra-rectal administration of TNBS [15 milligrams (mg)/kilogram (kg)] in 33 Sprague-Dawley rats. Intracolonic pressure was regularly assessed during the 9 wk-experiment (weeks 0, 3, 5, and 7) in animals receiving CG (n = 14) at a human equivalent dose (HED) of 1.5 g/d or 3.0 g/d and compared to negative control (tap water, n = 11) and positive control (phloroglucinol at 1.5 g/d HED, n = 8) groups. The anti-inflammatory effect of CG was evaluated using clinical and histological scores in 30 C57bl6 male mice with colitis induced by dextran sodium sulfate (DSS) administered in their drinking water during 14 d. HT-29 cells under basal conditions and after stimulation with lipopolysaccharide (LPS) were treated with CG to evaluate changes in pathways related to analgesia (µ-opioid receptor (MOR), cannabinoid receptor 2 (CB2), peroxisome proliferator-activated receptor alpha, inflammation [interleukin (IL)-10, IL-1b, and IL-8] and barrier function [mucin 2-5AC, claudin-2, zonula occludens (ZO)-1, ZO-2] using the real-time PCR method. Molecular modelling of CG, LPS, lipoteichoic acid (LTA), and phospholipomannan (PLM) was developed, and the ability of CG to chelate microbial pathogenic lipids was evaluated by docking and molecular dynamics simulations. Data were expressed as the mean ± SEM.
RESULTS: Daily CG orally-administered to rats or mice was well tolerated without including diarrhea, visceral hypersensitivity, or inflammation, as evaluated at histological and molecular levels. In a model of CRD, CG at a dosage of 3 g/d HED significantly decreased visceral pain perception by 14% after 2 wk of administration (P < 0.01) and reduced inflammation intensity by 50%, resulting in complete regeneration of the colonic mucosa in mice with DSS-induced colitis. To better reproduce the characteristics of visceral pain in patients with IBS, we then measured the therapeutic impact of CG in rats with TNBS-induced inflammation to long-lasting visceral hypersensitivity. CG at a dosage of 1.5 g/d HED decreased visceral pain perception by 20% five weeks after colitis induction (P < 0.01). When the CG dosage was increased to 3.0 g/d HED, this analgesic effect surpassed that of the spasmolytic agent phloroglucinol, manifesting more rapidly within 3 wk and leading to a 50% inhibition of pain perception (P < 0.0001). The underlying molecular mechanisms contributing to these analgesic and anti-inflammatory effects of CG involved, at least in part, a significant induction of MOR, CB2 receptor, and IL-10, as well as a significant decrease in pro-inflammatory cytokines IL-1b and IL-8. CG also significantly upregulated barrier-related genes including muc5AC, claudin-2, and ZO-2. Molecular modelling of CG revealed a new property of the molecule as a chelator of microbial pathogenic lipids, sequestering gram-negative LPS and gram-positive LTA bacterial toxins, as well as PLM in fungi at the lowesr energy conformations.
CONCLUSIONS: CG decreased visceral perception and intestinal inflammation through master gene regulation and direct binding of microbial products, suggesting that CG may constitute a new therapeutic strategy for patients with IBS or IBS-like symptoms.
目的:评价CG在内脏镇痛中的作用,肠道炎症,屏障功能,并开发计算分子模型。
方法:在33只Sprague-Dawley大鼠直肠内给药TNBS[15毫克(mg)/千克(kg)]诱导的长期结肠超敏反应模型中,通过结肠直肠扩张(CRD)记录内脏疼痛。在9周实验期间(第0、3、5和7周),在以1.5g/d或3.0g/d的人类等效剂量(HED)接受CG(n=14)的动物中定期评估结肠内压力,并与阴性对照(自来水,n=11)和阳性对照(间苯三酚在1.5g/dHED,n=8)组。用临床和组织学评分对30只饮用水中给予葡聚糖硫酸钠(DSS)诱导的结肠炎的C57bl6雄性小鼠进行了14天的临床和组织学评分,评估了CG的抗炎作用。在基础条件下和脂多糖(LPS)刺激后,用CG处理HT-29细胞,以评估与镇痛相关的途径的变化(µ阿片受体(MOR),大麻素受体2(CB2),过氧化物酶体增殖物激活受体α,炎症[白细胞介素(IL)-10,IL-1b,和IL-8]和屏障功能[粘蛋白2-5AC,claudin-2,小带闭塞(ZO)-1,ZO-2]使用实时PCR方法。CG的分子建模,LPS,脂磷壁酸(LTA),并开发了磷脂甘露聚糖(PLM),通过对接和分子动力学模拟评估了CG螯合微生物病原脂质的能力。数据表示为平均值±SEM。
结果:每天口服给大鼠或小鼠的CG耐受性良好,不包括腹泻,内脏过敏,或者炎症,在组织学和分子水平上评估。在CRD的模型中,CG在3g/d的HED剂量下,在给药2周后,内脏疼痛感知显着降低了14%(P<0.01),炎症强度降低了50%,导致DSS诱导的结肠炎小鼠的结肠粘膜完全再生。为了更好地再现IBS患者内脏痛的特点,然后,我们测量了CG对TNBS诱导的炎症大鼠对长期内脏高敏感性的治疗作用.1.5g/d剂量的CG在结肠炎诱导后5周降低了20%的内脏疼痛感知(P<0.01)。当CG剂量增加到3.0g/dHED时,这种镇痛作用超过了解痉剂间苯三酚,在3周内表现得更快,并导致疼痛感知抑制50%(P<0.0001)。导致CG的这些镇痛和抗炎作用的潜在分子机制涉及,至少在某种程度上,MOR的显著诱导,CB2受体,和IL-10,以及促炎细胞因子IL-1b和IL-8的显着减少。CG也显著上调屏障相关基因,包括muc5AC,Claudin-2和ZO-2.CG的分子建模揭示了该分子作为微生物病原脂质螯合剂的新特性,隔离革兰氏阴性LPS和革兰氏阳性LTA细菌毒素,以及真菌在低能量构象下的PLM。
结论:CG通过主基因调控和微生物产物的直接结合降低了内脏知觉和肠道炎症,提示CG可能构成IBS或IBS样症状患者的新治疗策略。