Confirmation that Luobuma ameliorates the deterioration of antioxidant defense in senescence-accelerated mice

To determine whether Luobuma extract ameliorates the deterioration in antioxidant defense with aging, the effect of Luobuma extract was investigated in senescence-accelerated mice (SAM). In comparison with AKR/N Slc mice, a strain consistent with SAM but exhibiting normal aging, SAM treated with extract showed a lower glutathione (GSH) and glutathione/glutathione disulfide (GSH/GSSG) ratio in the liver and kidney, and increased levels of malondialdehyde (MDA), a lipid peroxidation product. Administration of Luobuma extract increased the GSH level and GSH/GSSG ratio, and suppressed MDA production. On the other hand, the reduced activities of hepatic superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase participating in the glutathione redox cycle were increased significantly by administration of Luobuma extract. A significant increase in renal SOD activity was also observed. In addition, the increased level of MDA in hepatic tissue was reduced in SAM given Luobuma extract. These findings indicate that Luobuma extract helps to ameliorate oxidative stress in SAM. 羅布麻エキスが老化における酸化防御機構にいかなる影響を及ぼしているかについて,老化促進マウス(SAM)を用い検討した。SAMと同じ系統ではあるが,通常の老化過程を辿るAKR/N Slcマウスに比べ,SAMでは肝,腎組織中のグルタチオンレベルとグルタチオン/グルタチオンジスルフィド比は低下し,脂質過酸化物のマロンジアルデヒドは逆に増加していた。一方,羅布麻エキスを投与したSAMでは,これらパラメーターがいずれも改善し,グルタチオン酸化還元サイクルに関係している肝組織中のスーパーオキシドジスムターゼ,グルタチオンペルオキシダーゼ,グルタチオンレダクターゼ活性と腎組織中のスーパーオキシドジスムターゼ活性が有意に上昇していた。また肝組織中のマロンジアルデヒドも低下し,SAMで認められた酸化的ストレス状態を羅布麻エキスが緩和していた

Molecular and Genetic Interactions between CCN2 and CCN3 behind Their Yin-Yang Collaboration

Cellular communication network factor (CCN) 2 and 3 are the members of the CCN family that conduct the harmonized development of a variety of tissues and organs under interaction with multiple biomolecules in the microenvironment. Despite their striking structural similarities, these two members show contrastive molecular functions as well as temporospatial emergence in living tissues. Typically, CCN2 promotes cell growth, whereas CCN3 restrains it. Where CCN2 is produced, CCN3 disappears. Nevertheless, these two proteins collaborate together to execute their mission in a yin-yang fashion. The apparent functional counteractions of CCN2 and CCN3 can be ascribed to their direct molecular interaction and interference over the cofactors that are shared by the two. Recent studies have revealed the mutual negative regulation systems between CCN2 and CCN3. Moreover, the simultaneous and bidirectional regulatory system of CCN2 and CCN3 is also being clarified. It is of particular note that these regulations were found to be closely associated with glycolysis, a fundamental procedure of energy metabolism. Here, the molecular interplay and metabolic gene regulation that enable the yin-yang collaboration of CCN2 and CCN3 typically found in cartilage development/regeneration and fibrosis are described

Effect of Luobuma leaves against oxidation of low-density lipoprotein : a cell culture assay

In a previous study, we observed an improvement in the atherosclerosis index, together with a decrease in blood cholesterol, in rats given Luobuma extract orally and fed a high-cholesterol diet. The present study was designed to examine the function of oxidized low-density lipoprotein (LDL) in atherosclerotic lesions, using cultured cells. When endothelial cells were cultured with LDL in the presence of Cu^, the release of thiobarbituric acid (TBA) -reactive substance and lactic dehydrogenase into the culture medium was increased, with a decrease in cell viability. However, when Luobuma extract was also present in the culture medium, changes in these parameters were more favorable. In another in vitro system using macrophages, the levels of TBA-reactive substance, total cholesterol and esterified cholesterol were all significantly lower in the presence of Luobuma extract than in its absence. There was also morphological evidence that foam cell formation through incorporation of oxidized LDL was suppressed. These findings indicate that Luobuma suppresses the progression of atherosclerosis, in which oxidized LDL is involved. 先に,高コレステロール食投与ラットに羅布麻エキスを経口投与した場合,高コレステロール血症の低下とともに動脈硬化指数の改善作用が認められたので,今回,粥状動脈硬化病変への酸化LDLの機能を細胞を用い検寸寸した。まず内皮細胞にLDLとCu^を添加して培養した場合,培地中へのチオバルビツール酸反応物質,総LDHの放出が増加して,細胞生存率の低下が観察された。しかし羅布麻エキス添加群ではこれらパラメータがいずれも改善し,またマクロファージを用いた系でもチオバルビツール酸反応物質,コレステロールエステル,コレステロールエステル/遊離コレステロール比がいずれも無添加群より有意に低下し,形態学的な変化も酸化LDLのとり込みに伴う泡沫化の形成を抑制する知見が得られた。このことから,羅布麻は酸化LDLが関与する動脈硬化の進展過程を抑制することが明らかとなった

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

Retrotransposons Manipulating Mammalian Skeletal Development in Chondrocytes

Retrotransposons are genetic elements that copy and paste themselves in the host genome through transcription, reverse-transcription, and integration processes. Along with their proliferation in the genome, retrotransposons inevitably modify host genes around the integration sites, and occasionally create novel genes. Even now, a number of retrotransposons are still actively editing our genomes. As such, their profound role in the evolution of mammalian genomes is obvious; thus, their contribution to mammalian skeletal evolution and development is also unquestionable. In mammals, most of the skeletal parts are formed and grown through a process entitled endochondral ossification, in which chondrocytes play central roles. In this review, current knowledge on the evolutional, physiological, and pathological roles of retrotransposons in mammalian chondrocyte differentiation and cartilage development is summarized. The possible biological impact of these mobile genetic elements in the future is also discussed

Effect of Subcultivation of Human Bone Marrow Mesenchymal Stem on their Capacities for Chondrogenesis, Supporting Hematopoiesis, and Telomea Length

Effects of subcultivation of human bone marrow mesenchymal stem cells on their capacities for chondrogenesis and supporting hematopoiesis, and telomea length were investigated. Mesenchymal stem cells were isolated from human bone marrow aspirates and subcultivated several times at 37℃ under a 5% CO2 atmosphere employing DMEM medium containing 10% FCS up to the 20th population doubling level (PDL). The ratio of CD45- CD105+ cells among these cells slightly increased as PDL increased. However, there was no marked change in the chondrogenic capacity of these cells, which was confirmed by expression assay of aggrecan mRNA and Safranin O staining after pellet cell cultivation. The change in capacity to support hematopoiesis of cord blood cells was not observed among cells with various PDLs. On the other hand, telomere length markedly decreased as PDL increased at a higher rate than that at which telomere length of primary mesenchymal stem cells decreased as the age of donor increased

Novel intracellular effects of human connective tissue growth factor expressed in Cos-7 cells

AbstractTo clarify the multiple functionality of connective tissue growth factor (CTGF), we examined the effects of nascent CTGF within the cell by transient expression. In Cos-7 cells, expression of human CTGF induced an altered cell morphology. It was associated with an increased cellular DNA content and loose attachment, indicating the cells were in G2/M phase. Overexpression of CTGF did not induce cell growth, whereas recombinant CTGF efficiently stimulated the proliferation extracellularly. These results indicate that intracellular CTGF may act as an antimitotic agent, thus it should also be noted that nascent CTGF was found to accumulate around the central mitotic machinery

Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Chondrocytes for Construction of Three-dimensional Cartilage Tissue

A differentiation method of human bone marrow mesenchymal stem cells (MSCs) to chondrocytes was developed for the construction of a three-dimensional (3D) cartilage tissue. The adhesive cells, which were isolated from a human bone marrow aspirate were embedded in type I collagen in a poly-l-lactate-glycolic acid copolymer (PLGA) mesh and cultivated for 4 week together with growth factors. The degree of cellular differentiation was estimated by quantitative RT-PCR of aggrecan and type II collagen mRNAs and by staining with Safranin O. The 3D culture showed a higher degree of differentiation even without growth factors than the conventional pellet culture with growth factors, namely, dexamethasone and transforming growth factor (TGF)-β 3. The 3D culture for 2 week with the combined addition of dexamethasone, TGF-β 3, and insulin-like growth factor (IGF)-I reached a 30% expression of aggrecan mRNA compared with that in primary human chondrocytes, while the aggrecan mRNA expression in the conventional pellet culture was less than 2%. The sequential two-step differentiation cultivation, during which the cells were cultivated in 3D for 1 week after the conventional two-dimensional (2D) culture for 1 week, could markedly accelerate the expression of aggrecan mRNA compared with the 3D cultivation for 2 week

Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes

To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes