营养失调与治疗杂志

抽象的

临床营养学 2019：康普茶对小鼠肠道完整性的影响 - Elaheh Mahmoudi - 阿尔伯茨医科大学

埃拉赫·马哈茂迪

目的：氧化应激与高血压的发病机制有关，而高血压是心血管发病率和死亡率的危险因素。多项人体研究表明，番茄类胡萝卜素可影响人体健康的各个方面。在本次演讲中，作者将讨论两个问题：a) 平衡皮肤细胞对紫外线照射的反应；b) 减轻升高的生命体征。a) 多项人体研究表明，番茄类胡萝卜素可通过减少红斑和改善胶原蛋白生成与分解之间的平衡来减少紫外线引起的损伤。我们推测，番茄类胡萝卜素和多酚的混合物可能比它们活性总和所预期的保护皮肤的效果更好。事实上，我们了解到，番茄营养复合物（含番茄红素）与迷迭香提取物（含多酚鼠尾草酸）的混合物可协同降低炎症标志物并诱导皮肤细胞的抗氧化活性，首先是降低基质金属蛋白酶 (MMP)，从而可能减少胶原蛋白分解并延缓皮肤老化。 b) 高血压可能是心血管发病和死亡的危险因素。我们进行了剂量反应分析，以发现番茄营养复合补充剂在高血压患者中维持正常体征的最佳有效剂量。 材料和方法：

使用饮料中的葡聚糖硫酸钠连续七天在两组幼年和成年小鼠中诱导筛状肠。之前，对患有结肠炎的小鼠进行 fKT 治疗，并与年龄匹配的正常和未治疗的结肠炎动物进行比较。

康普茶 (KT) 制备

将红茶（伊朗德黑兰戈勒斯坦）加入沸水（1.2%w/v）中，混合，然后冲泡 10 分钟。然后通过无菌筛过滤茶，并将蔗糖（10%）溶解在茶中。为了制备 KT，将 200 毫升冷却的茶接种 3%w/v 茶菌和 10%v/v 预先发酵的 KT 液体。然后将收集物在 28°C 下培养 14 天进行发酵。为了形成过滤 KT（fKT），将所得发酵茶以 5000 rpm 的速度离心 20 分钟，并使用配备气泵的 0.45 µm 纤维素过滤器进行过滤。

实验组和研究设计

Male NMR mice were purchased from the Pasteur Institute Experimental Animal Center, Tehran, Iran. All animals were housed for one week before the experiments began in light- and temperature-regulated rooms at the traditional animal department of Alborz University of Medical Sciences.  All experimentations were permitted by the native ethical group (reference No Abzums.Rec.1395.51) and performed consistent with Animal Care and Use Protocol of Alborz University of Medical Sciences.

The animals were divided into two groups of young and old. Each group was then further subdivided into two groups (8 in each) including normal and colitis-induced. because the figure indicates, each of colitis-induced old or young groups was further subdivided into two subgroups, including colitis-induced with no treatment and colitis-induced treated with fKT. The study was performed in three phases. within the initiative , DSS-induced colitis was found out in young (2 months) and old (16 months) mice during a period of 21 days during which weight loss and therefore the clinical score were evaluated and compared with the age-matched healthy animals. within the second phase, the effect of fKT administration on survival analysis and therefore the clinical score were evaluated during a period of 21 days. After completion of phase I clinical trial and II of the study, molecular and histological evaluations were performed on young and old healthy controls, DSS-induced colitis, and DSS-induced colitis treated with fKT animals in phase III clinical trial . Considering the deathrate and clinical signs that occurred within the animals with colitis, animals at this phase of the study were sacrificed on day 14 after the start of the study.

Colitis induction

Colitis was induced on day 0 using administration of drinking water containing 3.5% (w/v) dextran sodium sulfate salt (DSS) (40000 kDa, MP Biomedical, Eschwege, Germany) per mouse per day. The animals and clinical signs of disease were daily monitored, indicated by weight loss, occurrence of blood in the stool or around the rectum, and diarrhea until day seven after the colitis induction. Weight loss was determined by comparing the body weight for each mouse to the baseline body weight and expressed as a percentage of weight loss. Other symptoms were scored according to the previously suggested system by Siegmund et al. Briefly, the different signs for stool consistency were scored as follows: score 0, well-formed pellets; score 2, pasty and semi-formed stools that did not adhere to the anus; score 4, liquid stools that did adhere to the anus. The different signs for bleeding were scored as follows: score 0, no blood measured using the Hemoccult system (Beckman Coulter); score 2, positive Hemoccult; score 4, gross bleeding. Animals with borderline scores were given a one-half score.

Histological and histopathological analysis

To perform histological evaluation of the colon, the animals were sacrificed under ether anesthesia after the last treatment with beverage or fKT. Colon was initially flushed with 1x ice-cold phosphate buffered saline (PBS) to get rid of feces completely. Tissue samples of the colon were then removed, fixed in 10% buffered formalin, and processed for paraffin sectioning. Sections of about 5 μm thickness were taken and stained with Hematoxylin and Eosin (H&E). The stained sections were examined with an Olympus cX41 microscope and photographed using an Olympus D330 camera . Damage score ranged from 0 to 4 scale was judged based on: inflammation represented by number and extent of leukocyte infiltration, epithelial defects represented by the severity of injury to the somatic cell layer, crypt atrophy estimated visually for the percent of atrophy within the crypts, edema, polymorphonuclear cells(PMNs) infiltration, and mucosal disruption.

Immunofluorescence studies of ZO-1 and ZO-2 expression

Sections of 5 µm paraffin-embedded colon tissues were prepared from each sample and then dewaxed, hydrated, and incubated in a protein block solution. Subsequently, the sections were incubated with the primary rabbit monoclonal ZO-1 or ZO-2 antibody (diluted 1:100 in 0.01 mol /L PBS; Zo-1: ab214228, Zo-2: ab2273, UK) followed by incubation with a goat anti-rabbit Alexa flour 488 (ab150077, Abcam, Cambridge shire, UK). The images were captured using a DeltaPix fluorescent microscope (Smorum, Denmark) and evaluated independently by two expert pathologists.

Analysis of gene expression by real-time PCR

Total RNA was extracted from ~50 mg of frozen colon tissue using guanidine/phenol solution (reagent lysis Qiazol-USA) consistent with the manufacturer’s instruction. the standard and quantity of RNA concentrations were monitored employing a NanoDrop 2000c (Eppendorf, Germany). Then, 1 μg RNA was reversely transcribed to DNA using Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit (Munich, Germany), consistent with the manufacturer’s instructions. The relative expression of mRNA for GAPDH, ZO-1, and ZO-2 decided by preparing reaction mixer with PCR Master Mix (2X) (Amplicon-Denmark) and gene-specific primers with diluted cDNA and final volume made up to 10 μl with nuclease-free water. Quantification and analysis were administered in ABI real-time PCR. The sequences of primers, designed by Integrated DNA Technologies, were forward 5′-TGTCCCACTTGAATCCCC-3′ and reverse 5′-TGTTTCCTCCATTGCTGTG-3′ for ZO-1 and forward 5′-CTCCCTCTTCACATCTGCTTC-3′ and reverse R: 5′-CTGTTACTTGCTTTGGTCTGG-3′ for ZO-2. The efficiencies for primers utilized in the study varied between 95% and 105%. Primer pairs were validated to make sure the right size of the PCR product and therefore the absence of primer dimers. The GAPDH gene was chosen as an indoor control against which mRNA expression of the target gene was normalized. The resultant organic phenomenon level was presented as 2-ΔCt, during which ΔCt was the difference between Ct values of the target gene and GAPDH.

Statistical analysis

Statistical analysis was performed using Graph Pad Prism 7.01. Data are presented as means± SD. ANOVA was wont to indicate any significant difference between the groups. Survival rates were illustrated using Kaplan–Meier plots and compared using the log-rank test. Value of P was considered statistically significant when it had been but 0.05.

Results:

Characteristics and clinical course of DSS-induced colitis

DSS 诱发的小鼠结肠炎是研究结肠炎发病机制和评估治疗方法的常用动物模型。该模型由我们的实验室开发，并进行了为期 21 天（第一阶段）的监测。为此，雄性 NMR 小鼠连续七天在饮料中服用 3.5% DSS。在研究期间，每天检查动物的存活率、体重减轻和结肠炎临床症状（包括出血和腹泻），并与年龄匹配的健康动物进行比较。对接受 DSS 治疗的年轻动物的生存分析表明，第 7 天和第 14 天分别有 66% 和 33% 的动物存活，第 2 天所有动物均死亡。对于接受 DSS 治疗的老年动物，生存分析显示，第 7 天和第 14 天分别有 66% 和 50% 的动物存活，第 21 天所有动物均死亡。与年龄匹配的健康动物相比，接受 DSS 治疗的年轻组和老年组体重减轻幅度较大。接受 DSS 治疗的年轻小鼠在第 7 天和第 14 天分别减轻了约 10% 和 46% 的体重。接受 DSS 治疗的老年小鼠在第 7 天和第 14 天分别减轻了约 13.5% 和 15% 的体重。至于消化系统疾病症状，在接受 DSS 治疗后的第 2 天和第 3 天，接受 DSS 治疗的年轻小鼠和老年小鼠分别出现出血和腹泻。这些结果表明，接受 DSS 治疗的幼年小鼠比接受 DSS 治疗的老年小鼠表现出更严重的临床症状和更低的存活率。

组织学观察

对所有接受 DSS 治疗的年轻和老年小鼠的 H&E 染色组织切片进行组织学分析（图 6），结果显示与年龄匹配的健康小鼠相比，PMN 浸润、隐性丢失、上皮缺损、粘膜破坏、细胞凋亡、水肿和粘膜变薄的现象增加。用 fKT 治疗可减轻损伤程度，尽管在本研究期间实施的治疗方案下，它并没有完全逆转健康状态。值得注意的是，老年健康小鼠比年轻健康动物有更多的 PMN 浸润、隐性丢失和水肿。如图 6c 所示，由于 DSS 诱发的结肠炎的年轻和老年小鼠的临床评分均增加，因此 DSS 给药降低了粘膜厚度。 该研究部分在2019 年 3 月 4 日至 6 日于西班牙巴塞罗那举行的第 24 届^国际 临床营养会议上发表。