Protective effect of Sanguisorbae Radix against peroxynitrite-mediated renal injury

3-Nitrotyrosine, an oxidative product of protein that is produced via peroxynitrite (ONOO^-) nitration, was detected by HPLC analysis in plasma obtained from rats injected with lipopolysaccharide (LPS) and subjected to renal ischemia followed by reperfusion (LPS+ischemia-reperfusion), but not in rats subjected to sham-treatment. Rats pretreated with Sanguisorbae Radix extract orally for 30 days before LPS+ischemia-reperfusion, had lower 3-nitrotyrosine levels than rats without the pretreatment. Plasma levels of urea nitrogen and creatinine, indicators of renal dysfunction, were markedly lower in the animals pretreated with Sanguisorbae Radix extract than in those without the pretreatment. In addition, DNA fragmentation in renal tissues was significantly inhibited by administration of Sanguisorbae Radix prior to LPS+ischemia-reperfusion. These results suggest that Sanguisorbae Radix extract ameliorates oxidative damage caused by ONOO^-. パーオキシナイトライトは蛋白中のチロシンをニトロ化して3-ニトロチロシンを生成するが,この3-ニトロチロシンをHPLCで測定した結果,リポポリサッカライドと虚血-再灌流を施したラット血漿で検出され,偽処理した場合には検出されなかった。一方,リポポリサッカライドと虚血-再灌流を施す前に30日間地楡エキスを経口投与したラットでは,非投与群より低い3-ニトロチロシン値を示し,腎機能の指標の血漿尿素窒素,クレアチニンレベルも著しく低下していた。また腎組織中のDNA断片化も抑制され,地楡エキスがパーオキシナイトライトによる酸化的損傷を軽減することが推測された

Radical-scavenging activity of Wen-Pi-Tang and its component crude drugs : with special reference to the effects on nitric oxide, superoxide and peroxynitrite

In renal diseases, active oxygen and free radicals play various roles in the development and progression of the pathological condition. Our previous studies have provided evidence that the Oriental medical prescription Wen-Pi-Tang normalizes the kidney under conditions of increased oxidative stress. In the present study, we examined the antioxidant capacity of Wen-Pi-Tang and its component crude drugs in a nitric oxide, superoxide and peroxynitrite generation system. It was found that the radical-scavenging effect of Wen-Pi-Tang is dose-dependent, and that three of its component crude drugs, i.e., Rhei Rhizoma, Zingiberis Rhizoma and Glycyrrhizae Radix, play important roles in the antioxidant action.腎疾患において活性酸素,フリーラジカルはさまざまな形でその病態の成立,進展に関与しているが,漢方方剤温脾湯が酸化的ストレス状態にある腎を是正する作用をこれまで報告してきた。本研究では温脾湯と構成和漢薬の抗酸化能をNO,O^-_2並ぴにONOO^-発生系を用い検討し,温脾湯によるこれらラジカル消去作用は用量依存的に認められ,また構成和漢薬では大黄,乾姜,甘草が重要な役割を担っていることが明らかとなった

Polysaccharide-Containing Macromolecules in a Kampo (Traditional Japanese Herbal) Medicine, Hochuekkito: Dual Active Ingredients for Modulation of Immune Functions on Intestinal Peyer's Patches and Epithelial cells

A traditional Japanese herbal (Kampo) medicine, Hochuekkito (Bu-Zhong-Yi-Qi-Tang in Chinese, TJ-41) is a well-known Kampo formula, and has been found to enhance antigen-specific antibody response in not only local mucosal immune system in upper respiratory tract, but also systemic immune system through upper respiratory mucosal immune system. Although this immunopharmacological effect has been proposed to express by modulation of intestinal immune system including Peyer's patches and intestinal epithelial cells, active ingredients are not known. TJ-41 directly affected the production of bone marrow cell-proliferative growth factors from murine Peyer's patch immunocompetent cells in vitro. Among low molecular, intermediate size and macromolecular weight fractions prepared from TJ-41, only fraction containing macromolecular weight ingredients showed Peyer's patch-mediated bone marrow cell-proliferation enhancing activity. Anion-exchange chromatography and gel filtration gave 17 subfractions comprising polysaccharides and lignins from the macromolecular weight fraction of TJ-41, and some of the subfractions showed significant enhancing activities having different degrees. Some of the subfractions also expressed stimulating activity on G-CSF-production from colonic epithelial cells, and statistically significant positive correlation was observed among enhancing activities of the subfractions against Peyer's patch immunocompetent cells and epithelial cells. Among the fractions from TJ-41 oral administration of macromolecular weight ingredient fraction to mice succeeded to enhance antigen-specific antibody response in systemic immune system through upper respiratory mucosal immune system, but all the separated fractions failed to enhance the in vivo antibody response in upper respiratory tract

Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity.

Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer\u27s disease (AD). β-amyloid (Aβ) lies upstream of abnormal tau behavior, including detachment from microtubules, phosphorylation at several disease-specific sites, and self-aggregation into toxic tau species in AD brains. To prevent the cascade of events leading to neurodegeneration in AD, it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. Currently, however, these mechanisms remain unclear. In this study, using transgenic Drosophila co-expressing human tau and Aβ, we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD

Loss of axonal mitochondria promotes tau-mediated neurodegeneration and Alzheimer\u27s disease-related tau phosphorylation via PAR-1.

Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer\u27s disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, we found that RNAi-mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD-related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD

Phenotypic analysis of a transgenic Drosophila model of Alzheimer’s amyloid-β toxicity

Summary: For decades, the fruit fly Drosophila melanogaster has been an efficient genetic model to investigate many aspects of human neurodegenerative diseases. Through genetic and pharmacologic approaches, these studies have revealed the molecular mechanisms underlying disease pathogenesis and provided therapeutic implications. Here, we describe a protocol for assessing Alzheimer's disease-related amyloid-β toxicity in a transgenic fly model through biochemical, histological, and behavioral analyses. We also discuss the advantages and limitations of our protocols.For complete details on the use and execution of this protocol, please refer to Wang et al. (2021)