芍药苷对溃疡性结肠炎大鼠肠道屏障功能和ERK信号通路的影响

摘要 目的：探究芍药苷对溃疡性结肠炎（UC）发生过程中肠道屏障功能和ERK信号通路的影响。方法：将24只7-8周龄SPF级雄性SD大鼠随机分为4组：正常组（Normal组,未造模及给药的大鼠）、模型组（Model组,100 mg/kg TNBS给药造模）、低剂量芍药苷组（LPF组,100 mg/kg TNBS +10 mg/kg芍药苷给药处理）和高剂量芍药苷组（HPF组,100 mg/kg TNBS +100 mg/kg芍药苷给药处理）,每组6只大鼠。对大鼠推注5%三硝基苯磺酸（TNBS）进行UC大鼠造模,然后灌胃指定浓度的芍药苷,连续处理14 d。通过苏木精伊红（HE）染色进行组织病理学观察,通过阿尔辛蓝（AB）染色计算结肠粘液层厚度。通过ELISA法检测结肠组织中细胞因子（IL-6、IL-1β、TNF-α和IL-10）、髓过氧化物酶（MPO）和粘蛋白（MUC2和MUC5AC）的水平。通过免疫组化检测各组大鼠结肠组织中IL-6和IL-10的蛋白表达。通过Western blotting分析蛋白激酶Cα（PKCα）、p-PKCα、ERK1/2和p-ERK1/2的蛋白表达。结果：与Model组（8.38±0.42 cm）相比,LPF组（9.88±0.49 cm）和HPF组（10.92±0.55 cm）UC大鼠的结肠长度显著增加（P<0.05）。与Model组（22.54±1.13 μm）相比,LPF组（41.07±2.05 μm）和HPF组（50.33±2.52 μm）UC大鼠结肠粘液层厚度显著增加（P<0.05）。与Model组相比,LPF组和HPF组UC大鼠的结肠形态明显改善,结肠组织中IL-6、IL-1β、TNF-α和MPO的水平显著降低,而IL-10显著升高（P<0.05）。与Model组相比,LPF组和HPF组UC大鼠结肠组织中MUC2和MUC5AC水平均显著升高,p-PKCα和p-ERK1/2的磷酸化水平也显著升高（P<0.05）。结论：芍药苷抑制了TNBS诱导的UC大鼠结肠炎症并增加了结肠粘液层厚度,从而保护了肠道屏障功能,其机制可能与ERK信号通路的激活有关。     

Interaction between Shaoyao–Gancao–Tang and a laxative with respect to alteration of paeoniflorin metabolism by intestinal bacteria in rats

Shaoyao-Gancao-Tang (SGT), a traditional Chinese herbal medicine (Kampo formulation) containing Shaoyao (Paeoniae Radix) and Gancao (Glycyrrhizae Radix), is co-administered with laxative sodium picosulfate as a premedication for relieving the pain accompanying colonoscopy. Paeoniflorin (PF), an active glycoside of SGT, is metabolized into the antispasmodic agent paeonimetabolin-I (PM-I) by intestinal bacteria after oral administration. The objective of the present study was to investigate whether the co-administered laxative (sodium picosulfate) influences the metabolism of PF to PM-I by intestinal bacteria. We found that the PF-metabolizing activity of intestinal bacteria in rat feces was significantly reduced to approximately 34% of initial levels by a single sodium picosulfate pretreatment and took approximately 6 days to recover. Repeated administration of SGT after the sodium picosulfate pretreatment significantly shortened the recovery period to around 2 days. Similar results were also observed for plasma PM-I concentration. Since PM-I has muscle relaxant activity, the present results suggest that repetitive administration of SGT after sodium picosulfate pretreatment might be useful to relieve the pain associated with colonoscopy.     

Paeoniflorin diminishes ConA-induced IL-8 production in primary human hepatic sinusoidal endothelial cells in the involvement of ERK1/2 and Akt phosphorylation

Liver diseases are closely associated with elevated levels of interleukin-8 (IL-8), suggesting the ability to inhibit IL-8 production could enhance the treatment of liver diseases. Paeoniflorin is a major active constituent of dried Paeoniae Radix Alba root (Baishao in Chinese) which is widely used in China to treat liver diseases. We examined the effects and underlying mechanisms of paeoniflorin on IL-8 production in primary human hepatic sinusoidal endothelial cells (HHSECs). Concanavalin A (ConA) at 20 μg/mL produced a 5.2-fold increase in IL-8 mRNA by 8 h, and a 14.2-fold rise in IL-8 levels by 16 h. Inhibition of MEK (ERK kinase) and extracellular signal-regulated kinase (ERK) by PD98059 and U0126, or inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 blocked both ConA-induced IL-8 mRNA expression and IL-8 secretion. Paeoniflorin reduced ConA-induced IL-8 mRNA expression and IL-8 release by 57.9% and 52.8%, respectively, and also decreased ConA-stimulated phosphorylation of ERK1/2 and Akt, suggesting paeoniflorin inhibits IL-8 expression and release by inhibiting the ERK1/2 and Akt pathways. Combining paeoniflorin with U0126 or LY294002 at low doses showed supra-additive inhibition of not only phospho-ERK1/2 and phospho-Akt by 46.4% and 35.0%, but also IL-8 release by 42.4% and 36.1% and IL-8 mRNA expression by 43.5% and 31.8%, respectively. In conclusion, paeoniflorin most likely contributes to the therapy for liver disease by exerting anti-inflammatory effects on HHSECs through blocking IL-8 secretion via downregulation of ERK1/2 and Akt phosphorylation.     

薄层层析-紫外分光光度法测定十全大补胶囊中芍药苷含量

目的 建立薄层层析-紫外分光光度法测定十全大补胶囊中芍药苷含量的方法。方法 使用硅胶GF254薄层板,醋酸乙酯：甲酸：冰醋酸：水(30：2：2：4)为展开剂,在紫外光灯(254nm)下定位,230nm波长处检测。结果 点样量在25～125μg范围内呈现良好的线性关系,其回归方程为Y=0．004658C-0．01959,r=0．9994,平均回收率为99．20％,RSD=1．19％。结论 本法准确,适用于该药的质量控制。     

芍药甘草汤及其活性成分的脑保护作用机制研究概述

芍药甘草汤由东汉张仲景创立,仅有芍药和甘草两味药物组成,具有调和肝脾,缓急止痛的功效,是经典的止痛方剂,临床主要用于血虚津伤所导致的各种内脏疼痛,腓肠肌痉挛的治疗中。已经展开的基础研究主要集中解痉、止痛和抗炎方面的机理研究。近年逐步认识到芍药甘草汤及其活性成分在神经保护领域的作用,在脑卒中、帕金森病、阿尔海默氏症、癫痫等中枢神经系统疾病基础研究中,取得成果。文章研读近年来国内外相关文献,分别从芍药、甘草的活性成份以及芍药甘草汤方剂在脑保护领域进行的基础研究的进展做一综述,显示了芍药甘草汤及其活性成分可以抑制脑组织免疫炎症反应、抑制氧化反应、抗谷氨酸毒性,改善脑血流、抗细胞凋亡和保护神经元的作用,阐述了芍药甘草汤的多途径、多靶点的药理优势及在脑保护领域的应用前景。     

A Preliminary Study of the Chemistry and Systematics of Paeoniaceae

Abstract From the root of Paeonia suffruticosa and P. lactiflora seven compounds have been isolated. They are identified as paeoniflorin I, benzoylpaeoniflorin II, oxypaeoniflorin III, paeonol IV, paeonoside V, paeonolide VI and apiopaeonoside VII. The contents of the seven constituents in the root of 23 Paeonia taxa, including 19 species and 6 varieties, are determined by means of the quantitative HPTLC scanning method. The results show that paeoniflorin occurs ubiquitously in all the species examined and is the characteristic constituent of the family Paeoniaceae; The paeonol compounds (IV, V. VI, VII) are found to be restricted to the woody Sect. Moutan and notably absent in the herbaceous Sect. Paeon. This differentiation gives a support to the division of sections in Paeoniaceae. The chemical comparison between Paeoniaceae and 15 possibly related families shows that Paeoniaceae is different from Ranunculaceae, Ranunculales of Magnoliales and is similar to Dilleniaceae, Theaceae and Rosaceae in some respects. The chemical data support the suggestion that Paeoniaceae be elevated to the order rank, Paeoniales.     

响应面法优选牡丹果荚中丹皮酚和芍药苷提取工艺的研究

以牡丹果荚为原料,采用响应面分析法对影响微波辅助提取牡丹果荚中芍药苷和丹皮酚的主要因素(料液比、微波功率、微波时间)进行优化。结果表明:微波辅助提取牡丹果荚中的芍药苷及丹皮酚的最佳提取工艺条件为:液料比10∶1、微波功率253 W、微波时间10 min,牡丹果荚芍药苷和丹皮酚的得率分别为2.92、0.91 mg·g-1。与传统提取法相比,微波辅助提取方法不仅提取时间短,原料使用量少,而且提取率高,是一个高效的提取方法。     

Effects of veratrine and paeoniflorin on isolated mouse vas deferens

In this study, we attempted to identify the interactions and mechanisms between veratrine and paeoniflorin on isolated mouse vas deferens. Paeoniflorin had no effect on isolated mouse vas deferens. Veratrine (1 x 10(-5) approximately 1 x 10(-3) g/ml) could directly induce contraction of isolated rat and mouse vas deferens. The concentration induced by veratrine (1 x 10(-5) g/ml) was completely inhibited by Ca2+-free solution and verapamil (1 x 10(-5) M), in both the epididymal and the prostatic portions of isolated mouse vas deferens. Naloxone (1 x 10(-5) M) did not alter the contraction induced by veratrine (1 x 10(-5) g/ml) in either the epididymal or the prostatic portions of isolated mouse vas deferens. Paeoniflorin (4.8 x 10(-5) g/ml) inhibited the contraction induced by veratrine (1 x 10(-5) g/ml) in both the epididymal and the prostatic portions of isolated mouse vas deferens. Paeoniflorin (4.8 x 10(-5) g/ml) potentiated norepinephrine (1 x 10(-5) M)-induced phasic contraction in the epididymal portion, but decreased contractions in the prostatic portion. Paeoniflorin (4.8 x 10(-5) g/ml) increased KCI (56 mM)-induced phasic contraction in the epididymal portion, but decreased the tonic contraction in either the epididymal or the prostatic portion. Veratrine (1 x 10(-5) g/ml)-induced contractions could be decreased by pretreatment with ryanodine (1 x 10(-5) M) in both the epididymal and the prostatic portions. Pretreatment with the combination of paeoniflorin (4.8 x 10(-5) g/ml) and ryanodine (1 x 10(-5) M) did not potentiate the inhibition of paeoniflorin in the veratrine-induced contraction in both the epididymal and the prostatic portions of isolated mouse vas deferens.     

Inhibitory effect of paeoniflorin on methylglyoxal-mediated oxidative stress in osteoblastic MC3T3-E1 cells

PurposeMethylglyoxal (MG) has been suggested to be one major source of intracellular reactive carbonyl compounds. In the present study, the effect of paeoniflorin on MG-induced cytotoxicity was investigated using osteoblastic MC3T3-E1 cells.MethodsOsteoblastic MC3T3-E1 cells were pre-incubated with paeoniflorin before treatment with MG, and markers of oxidative damage and mitochondrial function were examined.ResultsPretreatment of MC3T3-E1 cells with paeoniflorin prevented the MG-induced cell death and formation of intracellular reactive oxygen species, cardiolipin peroxidation, and protein adduct in osteoblastic MC3T3-E1 cells. In addition, paeoniflorin increased glutathione level and restored the activity of glyoxalase I to almost the control level. These findings suggest that paeoniflorin provide a protective action against MG-induced cell damage by reducing oxidative stress and by increasing MG detoxification system. Pretreatment with paeoniflorin prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss. Additionally, the nitric oxide and nuclear respiratory factor 1 levels were significantly increased by paeoniflorin, suggesting that paeoniflorin may induce mitochondrial biogenesis. Paeoniflorin treatment decreased the levels of proinflammatory cytokines such as TNF-α and IL-6.ConclusionsThese findings indicate that paeoniflorin might exert its therapeutic effects via upregulation of glyoxalase system and mitochondrial function. Taken together, paeoniflorin may prove to be an effective treatment for diabeteic osteopathy.     

Monoterpene glycosides from Paeonia hybrida

Monoterpene glycosides (1–5, and 7), together with 14 known compounds, were isolated from the methanol extract of the roots of Paeonia hybrida. These compounds included a paeoniflorin-related glycoside with a hybrid structure of paeoniflorin and paeonovicinoside (1), a monoterpene glucoside biogenetically related to lactiflorin (2), a paeoniflorin-related monoterpene (3), arbiflorin-related monoterpenes (4 and 5), and a tymol-related monoterpene glycoside (7). Their structures were elucidated by extensive spectroscopic examinations.