Effects of Oriental medicines on the production of advanced glycation endproducts

Advanced glycation endproducts (AGEs) are largely involved in the pathogenesis of diabetic nephropathy. It is apparent that inhibition of AGEs formation is important in preventing the occurrence and progression of nephropathy. Therefore, to seek possible AGEs inhibitors in Oriental medicines, we began our investigation with an in vitro evaluation system. Among the 12 Oriental medical prescriptions examined, Ompi-to inhibited AGEs formation to the greatest extent, followed by Tokaku-joki-to and Keishi-bukuryo-gan and Daio-botampi-to in that order. Among the 21 component galenicals examined, Rhei Rhizoma, Cinnamomi Cortex, Moutan Cortex and Paeoniae Radix all had a potent inhibitory action, indicating that Rhei Rhizoma, vascular system disturbance -eliminating drugs and tannin-containing crude drugs can all inhibit the formation of AGEs. These Oriental prescriptions and component galenicals proved to have more potent inhibitory activity than the positive control aminoguanidine. advanced glycation endproducts(AGE)は糖尿病性腎症の成因に大きく関与しており,AGE生成の抑制が腎症発症・進展防止に重要であることが知られている。このことから,本研究ではAGE阻害薬の可能性を和漢薬に求め,in vitroの評価系で検討した。使用した漢方方剤12種類のうち,最もAGE生成を抑制した漢方方剤は温脾湯で,次いで桃核承気湯,桂枝茯苓丸,大黄牡丹皮湯の順であった。実験に供した21種類の構成生薬では大黄,桂皮,牡丹皮,芍薬に強い抑制作用を認め,大黄剤,駆〓血剤ならびにタンニン生薬がAGEの生成を抑制しているものと考えられた。またこれらは陽性対照物質のアミノグアニジンより強い活性を示した

A study of Kampo medicines in a diabetic nephropathy model

The effects of four Kampo medicines, Ompi-to, Hachimi-jio-gan, Keishi-bukuryo-gan and Sairei-to, were investigated in rats with diabetic nephropathy induced by subtotal nephrectomy and injection of streptozotocin. To evaluate their effects on the glycation reaction, excessive activity of the polyol pathway and oxidative stress (abnormal bio-chemical processes induced by persistent hyperglycemia), we determined levels of the major endproducts of these processes: advanced glycation endproducts (AGEs) and sorbitol in the kidney and lipid peroxidation in the serum. These three processes were all enhanced in rats with untreated diabetic nephropathy. Oral administration of all four medicines significantly lowered AGEs levels. The renal sorbitol concentration was significantly lowered in the Hachimi-jio-gan-, Sairei-to- and Keishi-bukuryo-gan-treated groups compared with the untreated control group. Serum lipid peroxidation was significantly lowered in the Keishi-bukuryo-gan, Ompi-to and Sairei-to groups, while creatinine clearance and urinary protein excretion (parameters of renal function) were ameliorated by Keishi-bukuryo-gan and Hachimi-jio-gan, respectively, indicating retardation of the progression of diabetic nephropathy. These results suggest the potential therapeutic usefulness of Kampo medicines as a treatment for diabetic nephropathy. It is believed that their actions may occur through different mechanisms. 漢方方剤(温脾湯,八味地黄丸,桂枝茯苓丸,柴苓湯)の糖尿病性腎症に及ぼす影響を,モデルラットを用い検討した。慢性的な高血糖状態では糖化反応,ポリオール経路,酸化ストレスの亢進をひき起こすが,これらの指標の腎組織中のadvanced glycation endproducts(AGEs)とソルビトール,血中脂質過酸化量は,いずれも糖尿病性腎症ラットで有意に増加していた。これに対し,4種類の漢方方剤を投与した群では,いずれの場合も腎組織中のAGEsレベルが有意に低下していた。しかしソルビトール含量は八味地黄丸,柴苓湯,桂枝茯苓丸投与群で,血中脂質過酸化量は桂枝茯苓丸,温脾湯,柴苓湯投与群で有意に低下していた。また腎機能パラメーターのクレアチニンクリアランスと尿蛋白排泄量は桂枝茯苓丸,八味地黄丸投与群で有意に改善していた。このことから,4種類の漢方方剤はそれぞれ異なった作用機序で,糖尿病性腎症の進展を抑制している可能性が示唆された

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

Evaluation of Oriental medicines using a cultured renal epithelial cell line, LLC-PK_1 : Effects of Carthami Flos, Rhei Rhizoma and Astragali Radix

The effects of Carthami Flos，Rhei Rhizoma and Astragali Radix extract，which have been proved to ameliorate renal failure in rats with glycerol-induced renal failure，were examined in cell culture under three different culture conditions．Rhei Rhizoma　extract showed the most potent effect among the three crude drugs under any of the routine，hypoxia-reoxygenation and cisplatin exposure conditions employed，suggesting that its favorable effect on proximal tubule function is due to suppression　of lipid peroxidation via free radicals．In contrast，this anti-cytotoxic activity was low in Astragali Radix extract，and almost nil in Carthami Flos extract．These results indicate that Rhei Rhizoma，Astragali Radix and Carthami Flos exert their actions on different sites in the kidney

Chiyu extract stimulates antioxidant defense ability in senescence-accelerated mice

The effect of chiyu extract on antioxidant defense alteration in senescence-accelerated mice (SAM) was examined. Comparison with AKR/N Slc mice, a strain consistent with SAM but exhibiting normal aging, showed a lower glutathione (GSH) level and glutathione/glutathione disulfide (GSH/ GSSG) ratio in the kidney and liver of SAM, whereas malondialdehyde (MDA), a lipid peroxidation product, was increased significantly. Administration of chiyu extract increased the GSH level and GSH/ GSSG ratio, and markedly suppressed MDA production. On the other hand, detection of renal enzymes related to the glutathione redox cycle showed that catalase and glutathione peroxidase activities were largely decreased in SAM, whereas chiyu extract reversed this tendency. The reduced activities of hepatic catalase and glutathione reductase were increased significantly by the extract. These findings suggest that a decline of the antioxidant defense system occurs in SAM, and that chiyu extract may have a beneficial effect in ameliorating oxidative stress or damage. 老化促進マウス(SAM)の抗酸化防御機構に対し,地楡エキスがいかなる影響を及ぼしているかについて,SAMと同じ系統ではあるが,通常の老化過程を辿るAKR/N Slcマウスと比較検討した。その結果,SAMの腎,肝グルタチオンレベルとグルクチオン/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有意に上昇していたが,地楡エキスを投与したSAMでは,これらパラメーターがいずれも改善していた。一方,グルタチオン酸化還元サイクルに関係している酵素のうち,腎ではカタラーゼとグルクチオンペルオキシダーゼ活性がSAMで著しく低下し,地楡エキス投与によってこれら酵素の活性が回復していた。肝においても低下していたカタラーゼとグルタチオンレダクターゼ活性がエキス投与により著しく回復していた。これら所見は,SAMで認められる抗酸化防御系の低下が,地楡エキスによって是正されることを示唆するものである

Evaluation of Keishi-bukuryo-gan in a diabetic nephropathy model by comparison with aminoguanidine, butylated hydroxytoluene and captopril

桂枝茯苓丸の糖尿病性腎症に対する作用を,モデルラットを用い検討した。腎機能パラメーター,病理組織学的検討に加え,advanced glycation end products(AGEs)の蓄積,酸化ストレスに及ぼす影響を,アミノグアニジン(AGEs阻害薬),カプトプリル(アンジオテンシン変換酵素阻害薬),buthylated hydroxytoluene(BHT)(抗酸化剤)とで比較検討した。桂枝茯苓丸では腎機能(血清Cr,尿蛋白排泄量)と病理所見の有意な改善作用が認められ,糖尿病性腎症の進展を抑制することが実験的に明らかとなったが,このような腎保護作用はカプトプリルよりは弱く,アミノグアニジンと同程度であった。BHTには腎保護作用は認められなかった。腎組織中のAGEsの蓄積に対しては,桂枝茯苓丸,カプトプリル,BHTがいずれも有意に低下していたが,アミノグアニジンの作用よりは弱かった。腎組織中の脂質過酸化量はBHTで最も低下し,桂枝茯苓丸,カプトプリルでも有意に低下していた。一方,血中脂質過酸化に対しては,すべてにおいて有意な低下作用が認められたが,カプトプリルで最も強かった。このことから,桂枝茯苓丸はカプトプリルやアミノグアニジンとは異なった機序で糖尿病性腎症の進展を抑制している可能性が示された。 A study was done to investigate whether Keishi-bukuryo-gan can delay the progression of diabetic nephropathy in an experimentally induced diabetic nephropathy model. The efficacy of Keishi-bukuryo-gan against renal functional and structural changes and its influence on accumulation of advanced glycation end-products (AGEs) and oxidative stress were also examined by comparison with aminoguanidine (an AGEs inhibitor), butylated hydroxytoluene (BHT; an antioxidant) and captopril (an angiotensin converting enzyme inhibitor). Treatment with Keishi-bukuryo-gan for 10 weeks preserved renal function, as assessed in terms of proteinuria and serum creatinine, and prevented the morphological changes peculiar to diabetic nephropathy. However, its renoprotective activity was inferior to that of captopril and comparable to that of aminoguanidine. BHT lacked any of these effects. On the other hand, renal AGEs accumulation and oxidative stress were significantly enhanced in rats with untreated diabetic nephropathy compared with normal rats. Keishi-bukuryo-gan, captopril and BHT showed significant reduction of AGEs levels, but not to the extent shown by aminoguanidine. Renal lipid peroxidation levels were significantly lowered in the groups given Keishi-bukuryo-gan and captopril, but not to the extent shown in the rats given BHT. The reduction of serum lipid peroxidation levels by captopril was stronger than that by BHT. The effects of Keishi-bukuryo-gan and aminoguanidine on serum lipid peroxidation levels were similar to those of BHT. These results suggest that the pharmaceutical characteristics of Keishi-bukuryo-gan may differ from those of the other three medicines examined

Protective effect of Sanguisorbae Radix against apoptosis and function of renal tissues subjected to ischemia-reperfusion

DNA ladders were detected by gel electrophoresis of DNA obtained from rat kidney subjected to 60 min of ischemia followed by 24 h of reperfusion, indicating the involvement of apoptosis in ischemia-reperfusion injury. This ladder formation was significantly inhibited by oral administration of Sanguisorbae Radix extract to rats for 30 days prior to ischemia-reperfusion. In addition, blood levels of urea nitrogen and creatinine, two parameters of renal function, were markedly lower in the Sanguisorbae Radix-treated animals than in the untreated controls. These results suggest that Sanguisorbae Radix has potential for attenuating renal injury and accelerating the recovery of renal function after ischemia-reperfusion injury, which might involve inhibition of apoptosis. 60分間虚血後,24時間再灌流したラット腎のDNAにラダーが出現し,腎虚血一再灌流障害にアポトーシスが関与していることが示唆された。このようなDNAの断片化は,地楡エキスを前もって30日間経口投与した場合,有意に抑制され,腎機能の指標の血中尿素窒素とクレアチニンレベルも地楡エキス処理群で著しく低下していた。このことから,地楡はアポトーシスが関与した腎虚血一再灌流障害を軽滅して,腎機能の回復をはかっているものと推測された

Increase of radical in rats with adenine-induced renal failure is suppressed by Wen-Pi-Tang

We analyzed the free radical reaction in the body in vivo under the conditions of renal failure, using an L-band electron spin resonance apparatus. In rats with adenine-induced renal failure, the attenuation velocity of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl was lowered in comparison with normal rats, indicating that they were in a state of augmented oxidation. In contrast, the attenuation velocity was higher in rats given Wen-Pi-Tang, showing a shift toward reduction. In the kidney of rats given Wen-Pi-Tang, we also found that a significant decrease in glutathione disulfide (GSSG) level caused an increase in the glutathione/GSSG ratio. In addition, there were significant reductions of increased thiobarbituric acid-reactive substance level and decreased superoxide dismutase and increased glutathione peroxidase activities, suggesting a decreased hydrogen peroxide production, which presumably drove the glutathione redox cycle toward reduction. The results of the present study suggest the possibility that Wen-Pi-Tang exerts an antioxidant effect through regulation of the redox cycle. L-バンドESR(生体計測用ESR)装置を用い,腎不全における生体内フリーラジカル反応を解析した。アデニン誘発腎不全ラット腎では,正常ラットより3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxylの減衰速度が遅延し,酸化亢進状態にあった。これに対し,温脾湯投与群では減衰速度が速やかとなり,還元方向に変換していた。一方,温脾湯投与群の腎臓では増加したグルタチオンジスルフィドレベルの有意な低下からグルタチオン/グルタチオンジスルフィド比の上昇が認められ,また増加したチオバルビツール酸反応物質レベル,低下したスーパーオキシドジスムターゼと上昇したグルタチオンレダクターゼ活性の有意な低下から,過酸化水素の産生低下が示唆され,グルタチオン酸化還元サイクルを還元方向に作動しているものと考えられた。以上より,温脾湯は酸化還元サイクル調節を介して抗酸化作用を発揮する可能性が示唆された

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