黑樹莓改善幽門螺旋桿菌感染及神經退化之研究

腸道菌群或腸道微生物菌叢是一個由細菌、真菌和其他微生物組成的動態群落，它們居住在胃腸道中，腸道中的改變（稱為腸道菌群失調）會影響中樞神經系統並引發損害，從而加重神經退化性疾病，例如阿茲海默症 (AD)。已知幽門螺旋桿菌感染會誘發腸道菌叢失調，一些研究顯示，幽門螺旋桿菌會透過幾種機制與 AD 的進展相關，由於多年來 AD 病例的盛行率增加，可能成為全球性的健康問題。健康的生活方式，例如高多酚飲食，可能有助於減少幽門螺旋桿菌感染並緩解 AD 症狀。黑樹莓 (Rubus occidentalis) 是一種漿果家族的水果，富含多酚和其他生物活性化合物，具有抗菌和神經保護功效。本研究透過AGS細胞模式和人體研究評估黑樹莓補充對幽門螺旋桿菌感染的影響，並使用HT-22細胞模式評估其對緩解β-澱粉樣蛋白（Aβ）誘發的神經傷害及突觸功能的影響。在AGS細胞模型中,測定H. pylori抗黏附活性、轉運細胞毒素相關基因A（CagA）和空泡毒素A（VacA）以及白細胞介素8（CXCL8）的分泌，而在人體研究中測定尿素呼氣試驗（UBT）C13（指示H. pylori感染）、糞便微生物菌叢、發炎、氧化指標以及臨床失智症值評估。在HT-22細胞中，檢測了細胞凋亡（caspase-3、cleaved caspase-3、細胞核碎裂）和粒線體功能標記（PGC-1α、Sirt1、Nrf2、粒線體膜電位和活性氧（ROS）生成）。也檢測了幾種與突觸功能相關的mRNA（neurexin-2、neurexin-3、neuroligin-2、PSD-95、BDNF和IGF-1）以及IGF-1訊號傳導標記（PI3K、Akt、GSK3β、tau、mTOR、細胞內酸性空泡）。結果顯示，黑樹莓粗萃物可有效抑制AGS細胞模式中的H. pylori黏附，並減少CagA、VacA的釋放並減少CXCL8的生成。從粗萃物中去除原花青素並未降低H. pylori的黏附性，黑樹莓中的原花青素可能不是抑制H. pylori黏附的主要貢獻者。人體的研究結果也有類似的發現，服用乾燥粉末(每天50公克)可能有減少幽門螺旋桿菌感染，改善腸道微環境，並在介入2 個月後顯著降低臨床失智數值(CDR)。黑樹莓及其乙醇冲堤物1可保護 HT-22 細胞免受 Aβ 毒性並改善粒線體功能。此外,也可以有效改善突觸功能。 Aβ 誘導的胰島素訊號阻斷和自噬損傷也可透過黑樹莓粗萃物及其乙醇冲堤物1予以改善。由此研究可知，黑樹莓可以有效抑制幽門螺旋桿菌感染並減輕 Aβ 誘導的神經及突觸損傷，黃酮類化合物是可能的貢獻成分, 但仍需要進一步確認主要貢獻成分或是相加或相乘效果所致。

Intestinal microbiota or gut microbiome is a dynamic community of bacteria, fungi, and other microbiomes that resides in gastrointestinal and alteration in the gut known as gut dysbiosis can affect the central nervous system and induce damage which worsens neurodegenerative diseases such as Alzheimer’s disease (AD). H. pylori infection is known to induce gut dysbiosis and several studies in the past show that H. pylori is also correlated with the progression of AD by several mechanisms and this might become a global health concern due to the increased prevalence of AD cases over the years. Healthy lifestyles such as high polyphenol diet might help to reduce H. pylori infection as well as alleviate AD symptoms. Black raspberry (Rubus occidentalis) is a fruit from the berries family that contains high polyphenols and other bioactive compounds those could act as antimicrobial and neuroprotective agents. This study aims to assess the effect of black raspberry supplementation on H. pylori infection by AGS cell model and human study and also to assess its effect on alleviating β-amyloid(Aβ)-induced neuron and synapse dysfunction by using HT-22 cell model. H. pylori antiadhesion activity, transported cytotoxin associated gene A (CagA) and vacuolating toxin A (VacA) and secretion of interleukin-8 (CXCL8) were measured in AGS cell model while urea breath test (UBT) C13 which indicated H. pylori infection, stool microbiome, inflammatory and oxidative indexes and also clinical dementia rating (CDR) value were measured in human study. Reduction of apoptosis (caspase-3, cleaved caspase-3, nuclear fragmentation) and mitochondrial markers (PGC-1α, Sirt1, Nrf2, mitochondria membrane potential and production of ROS) were measured in HT-22 cells. Several mRNA for synapse function (neurexin-2, neurexin-3, neuroligin-2, PSD-95, BDNF and IGF-1) were also measured together with IGF-1 signaling marker (PI3K, Akt, GSK3β, Tau, mTOR, intracellular acidic vacuole). Results showed that treatment of black raspberry crude extract suppressed H. pylori adhesion in AGS cell model and reduced the release of CagA, VacA, and also secretion of CXCL8. Removal of proanthocyanidin from the crude extract didn’t diminish the antiadhesion activity of black raspberry suggesting that proanthocyanidin could not the main contributor for this activity. Result from human study also showed similar finding where treatment of black raspberry powder (50 grams daily) also reduced H. pylori infection, improved gut microenvironment and also reduced CDR value after 2 months of intervention. Black raspberry and its ethanolic elution fraction 1 showed to protect HT-22 cells from Aβ toxicity and improved mitochondrial function. Crude extract and its ethanol elution fraction 1 also improved synapse function shown by normalization of mRNA expression after treatment. Aβ-induced insulin signaling blockade and autophagy impairment also reversed by the treatment of black raspberry crude extract and its ethanolic fraction 1. In conclusion, black raspberry treatment could suppress H. pylori infection and alleviate Aβ-induced damage and the main contributor might come from ethanolic fraction. Further study is needed to find the major contributor or recognize the additive or synergistic effect for the functions.