Pharmacological basis for use of madecassoside in gouty arthritis: anti-inflammatory,anti-hyperuricemic,and NLRP3 inhibition

Objectives: Gouty arthritis is caused by the deposition of monosodium urate (MSU) crystals in joints, which is associated with the rise of serum urate content. This study aims to investigate the therapeutic effect of Madecassoside on gouty arthritis and hyperuricemia.

Methods: DBA/1 mice were intradermally injected with MSU to stimulate joint inflammation or intraperitoneally injected with MSU to trigger peritonitis. Moreover, ICR mice were exposed to potassium oxonate to stimulate hyperuricemia.

Results: Madecassoside repressed MSU-triggered pad swelling, joint 99mTc uptake, and joint inflammation in DBA/1 mice with gouty arthritis. Neutrophil infiltration and IL-1β & IL-6 & MCP-1 secretion was also alleviated in lavage fluids from DBA/1 mice with peritonitis due to Madecassoside treatment. Furthermore, Madecassoside decreased MSU-induced neutrophil cytosolic factor 1, caspase-1 and NLRP3 expression in mice with peritoneal inflammation. In hyperuricemic mice, Madecassoside improved renal dysfunction. Serum uric acid, BUN, and creatinine were down-regulated by Madecassoside.

Conclusion: These findings indicate that Madecassoside has potential to ameliorate inflammation in both acute gouty arthritis model and peritonitis model, probably via regulating IL-1β and NLRP3 expression.

Practical point: Madecassoside also exhibited a urate-lowering effect and a renal protective effect in hyperuricemic mice.     

三金分散片的鉴别及含量测定

目的 建立三金分散片鉴别和含量测定的方法.方法 采用TLC法及HPLC法.结果 TLC鉴别金樱根和积雪草药材,色谱清晰,分离度好.HPLC测定羟基积雪草苷,线性范围为1.206～12.064 μg(r=0.9991);平均回收率为99.69%(RSD=1.49%).结论 鉴别重复性好,专属性强;含量测定方法灵敏、准确.     

羟基积雪草苷与积雪草苷对LPS诱导大鼠发热模型的影响

目的观察积雪草苷和羟基积雪草苷的解热作用,并比较两者在解热作用上的差异。方法将80只SD大鼠随机分为空白组、模型组(LPS 100μg/kg)、溶酶对照组(羧甲基纤维素钠)、积雪草苷5,15,45 mg/kg、羟基积雪草苷10,20,40mg/kg、对乙酰氨基酚50 mg/kg组。腹腔注射LPS(100μg/kg)建立SD大鼠发热模型,绘制各组平均体温变化曲线,比较测量体温与基础体温差值ΔT。结果羟基积雪草苷(10,20,40 mg/kg)与积雪草苷(5,15,45 mg/kg)对LPS诱导的SD大鼠发热模型体温有显著的降温作用(P<0.05)。结论羟基积雪草苷和积雪草苷对LPS诱导的SD大鼠发热模型的体温均有降温作用,积雪草苷在某些时间点(如LPS注射5 h后)降温效应优于羟基积雪草苷。     

羟基积雪草苷对胶原关节炎大鼠肠道黏膜免疫的影响

目的:考察胶原关节炎大鼠肠道黏膜免疫的改变及羟基积雪草苷的调节作用。方法:在Wistar大鼠复制胶原关节炎模型,其后将大鼠分为正常对照组、模型组和羟基积雪草苷组,灌胃给药,连续21 d。ELISA法测定大鼠肠内容物中s Ig A含量及小肠组织中IFN-γ含量。流式细胞术检测小肠上皮内及固有层细胞中CD4+和CD8+T细胞的比例,计算CD4+/CD8+比值。实时定量PCR法检测小肠组织中CD80、CD86、IL-6、IL-12及Foxp3 mRNA的表达水平。结果:与正常对照组比较,模型组大鼠肠内容物中s Ig A含量及小肠组织中IFN-γ含量显著升高,小肠上皮内及固有层T细胞中CD4+/CD8+比值上升,小肠组织中CD80、CD86、IL-6和IL-12 mRNA表达显著上调,而Foxp3 mRNA的表达变化无统计学意义。羟基积雪草苷灌胃给药可降低模型大鼠肠内容物s Ig A和小肠组织IFN-γ含量、CD4+/CD8+比值及CD80、CD86、IL-6和IL-12 mRNA的表达,显著上调Foxp3 mRNA的表达。结论:关节炎状态下大鼠肠道黏膜免疫应答上调,抗原呈递细胞异常活化且免疫耐受状态遭到破坏,羟基积雪草苷可下调关节炎大鼠肠道黏膜免疫应答,促进肠道黏膜免疫耐受的形成和维持。     

羟基积雪草甙对慢性铝中毒痴呆小鼠的治疗作用

目的研究羟基积雪草甙(madecassoside,MC)对慢性铝中毒痴呆小鼠的保护作用。方法葡萄糖酸铝按铝400 mg.kg-1.d-1灌胃90 d建立慢性铝中毒痴呆小鼠模型。通过HE染色、Morris水迷宫试验和生化实验,观察MC三种剂量(30、60和120 mg.kg-1.d-1)同步给药90d对小鼠海马神经元损伤、空间学习记忆能力和脑组织单胺氧化酶B(MAO-B)活性的影响。结果与模型组比较,30和60 mg.kg-1.d-1MC明显减轻铝过负荷所致的海马神经元损伤,明显缩短小鼠寻找平台潜伏期(s)(35.9±10.9 vs 16.5±8.4和19.6±10.5)(P<0.05);同时三种剂量MC均明显降低小鼠脑组织中MAO-B活性(U.h-1.mg-1prot)(18.9±1.8 vs 14.6±1.7,13.7±2.3和13.6±1.4)(P<0.05)。结论MC对慢性铝中毒小鼠海马神经元有保护作用,改善痴呆小鼠学习记忆能力。     

羟基积雪草苷对LPS诱导小胶质细胞增殖的抑制作用及机制研究

目的:研究羟基积雪草苷对LPS刺激的小胶质细胞增殖的抑制作用及机制。方法:取SD大鼠的新生小鼠,进行小胶质细胞的原代培养,分离纯化小胶质细胞;MTT法筛选LPS刺激小胶质细胞增殖的最佳浓度,观察不同浓度羟基积雪草苷对LPS刺激小胶质细胞后的作用。ELISA检测细胞上清液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)的含量,流式细胞术检测细胞周期及细胞凋亡,Western blotting法检测Toll样受体4(TLR4)蛋白的表达,RT-PCR检测NF-κB mRNA的表达。结果:LPS可以明显诱导体外培养小胶质细胞的增殖和炎症因子释放。与LPS组比较,羟基积雪草苷对LPS诱导的小胶质细胞增殖具有显著抑制作用,且具有剂量依赖性,羟基积雪草苷处理小胶质细胞48 h的IC50为10.97 nmol/L。同时羟基积雪草苷使小胶质细胞TNF-α和IL-6的释放显著降低(P0.05);羟基积雪草苷使小胶质细胞的G2期细胞与细胞凋亡率增加,并降低小胶质细胞TLR4和NF-κB的表达。结论:羟基积雪草苷对LPS刺激的小胶质细胞的增殖和炎症因子的生成具有抑制作用,其作用机制可能与抑制TLR-4和NF-κB表达、改变细胞周期并诱导细胞凋亡有关。     

羟基积雪草苷对急性脊髓损伤大鼠的保护作用

目的 观察羟基积雪草苷(MC)对大鼠急性脊髓损伤(SCI)的保护作用.方法 制备SCI模型,成年大鼠150只随机分为假手术组、模型组、MC高、低剂量治疗组及甲基强的松龙(MP)对照组,每组30只,术后24 h、72 h、7 d、14 d、28 d各个时间点观察大鼠后肢运动功能及脊髓病理变化情况,检测脊髓中丙二醛(MDA)、神经元特异性烯醉化酶(NSE)含量及超氧化物歧化酶(SOD)活性.结果 高、低剂量MC及MP用药能明显提高SCI大鼠行为学评分,改善病理损伤,降低MDA含量,提高SOD活性,增加NSE表达,MC的作用呈剂量依赖性.结论 MC可减轻SCI大鼠的脊髓损伤,此作用可能与抑制损伤脊髓的脂质过氧化反应有关.     

Anxiolytic effects of standardized extract of Centella asiatica (ECa 233) after chronic immobilization stress in mice

Ethnopharmacological relevance

Centella asiatica has long been used for various neurological disturbances in Southeast Asian countries. The present study aims to demonstrate the anxiolytic effect of ECa 233, a standardized extract of C. asiatica containing triterpenoids not less than 80%, in comparison to diazepam.

Materials and methods

The test compound was given orally to non-stressed mice and mice subjected to chronic immobilization stress. Anxiolytic effect was assessed by an elevated plus maze (EPM), a dark-light box and an open-field tests.

Results

Anxiolytic effect of ECa 233 was clearly demonstrated in non-stressed mice subjected to acute stress in all behavioral tests employed. In the EPM test, chronically stressed mice showed significant decrease in the number of open arm entries, shortening the time spent in open arms and an increase of the latency to leave the central area, suggesting their release from the stress. In addition, ameliorating effect of ECa 233 was observed on the body weight and serum corticosterone which were adversely affected by immobilization stress. Madecassoside and asiaticoside, equal to their respective contents of the effective doses of ECa 233, exclusively presented anxiolytic effects in EPM, while no distinct effect was observed on the body weight and serum corticosterone.

Conclusions

The present study demonstrated anxiolytic effect of ECa 233 in both acutely and chronically stressed animals. These effects could be mainly accounted by madecassoside and asiaticoside, suggesting a possible use of ECa 233 for the treatment of both acute and chronic anxiety in the pathological state.     

HPLC法测定积雪草总苷中羟基积雪草苷和积雪草苷的含量

目的采用高效液相色谱法测定积雪草总苷中羟基积雪草苷和积雪草苷的含量.方法采用岛津Shimpack VP-ODSC18色谱柱(250 mm×4.6 mm,5 μm),以乙腈一水(2971)为流动相,流速1.0 ml·min-1 ,于205 nm测定,柱温室温.结果羟基积雪草苷和积雪草苷的线性范围分别为0.12～0.61 mg·ml-1和0.12～0.61 mg·ml-1,平均回收率分别为101.3%和98.4%,RSD分别为0.8%和0.6%(n=6).结论本法简便、快速,结果准确,重现性好,适用于积雪草总苷的质量控制.     

Madecassoside suppresses migration of fibroblasts from keloids: involvement of p38 kinase and PI3K signaling pathways

Keloid is a specific skin scar that expands beyond the boundaries of the original injury as it heals. The invasive nature of keloid and notable migratory activity of fibroblasts are a hallmark, which distinguishes keloids from other common scars. Madecassoside, a triterpenoid saponin occurring in Centella asiatica herbs, possesses unique pharmacological properties to enhance wound-healing and diminish keloid formation. However, the effects of madecassoside on the formation of keloid scars have been poorly understood. Here, we focused on the potential of madecassoside on the migration of keloid-derived fibroblasts (KFs) and its mechanism. Primary KF, originating from human earlobe keloids, were purified and cultured, and then treated with madecassoside (10, 30, and 100μM). In both transwell migration assays and scratch-wound-closure assays, KF migration was considerably suppressed by madecassoside pretreatment. Furthermore, KFs treated with madecassoside showed decreased F-actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. By Western blot analysis, madecassoside was shown to remarkably attenuate the phosphorylation of cofilin, p38 MAPK and phosphatidylinositol-3-kinase (PI3K)/AKT signaling, but only exhibited a minor effect on MMP-13 and little effect on ERK1/2 phosphorylation. It was concluded that madecassoside could be of great use in the treatment and/or prevention of hypertrophic scars and keloids.