SC-514, a selective inhibitor of IKKβ attenuates RANKL-induced osteoclastogenesis and NF-κB activation

The RANKL-induced NF-κB signaling pathway is essential for osteoclastogenesis. This study aims to identify specific inhibitors targeting NF-κB signaling pathway, which might serve as useful small molecule inhibitors for the treatment and alleviation of osteoclast-mediated bone lytic diseases. By screening for compounds that selectively inhibit RANKL-induced NF-κB activation in RAW264.7 cells as monitored by luciferase reporter gene assay, we identified SC-514, a specific inhibitor of IKKβ, as a candidate compound targeting osteoclastogenesis. SC-514 dose-dependently inhibits RANKL-induced osteoclastogenesis with an IC50 of <5 μM. At high concentrations, SC-514 (≥12.5 μM) induced apoptosis and caspase 3 activation in RAW264.7 cells. Moreover, SC-514 specifically suppressed NF-κB activity owing to delayed RANKL-induced degradation of IκBα and inhibition of p65 nuclear translocation. Taken together, our results indicate that SC-514 impairs RANKL-induced osteoclastogenesis and NF-κB activation. Thus, targeting IKKβ by SC-514 presents as a potential treatment for osteoclast-related disorders such as osteoporosis and cancer-induced bone loss.     

经典骨质疏松症模型大鼠的肾虚证研究

目的:观察采用双侧卵巢切除术制备的骨质疏松症模型大鼠的一般情况、生理状态,脏器湿重及血清学指标,以验证绝经后骨质疏松模型大鼠的肾虚证表现,为中医药临床防治骨质疏松症采用补肾治本的原则提供一定的理论基础。方法:24只雌性SD大鼠分为3组:模型组、假手术组和正常组。模型组麻醉后行双侧卵巢切除术,假手术组麻醉后行假手术处理,正常组进行麻醉处理。饲养3个月进行股骨的病理组织观察及骨密度检测,确认造模成功后进行大鼠的一般情况观察及生理状态的量化检测。麻醉下处死大鼠,用电子天平称垂体、肾、肾上腺和子宫的湿重,采用ELISA法进行ACTH、E2、T3、T4的血清学检测。结果:骨质疏松模型组大鼠出现体毛稀疏无光泽、畏寒倦卧、群聚角落、精神萎靡、眼睛黯淡、眼球运动慢、反应迟钝、活动迟缓、食欲下降、身体肥胖、嗜睡、大便略稀、小便清长等一系列肾虚证的表现;活动度、肛温及进食量均低于假手术组与正常组;血清ACTH、E2、T3、T4水平均低于假手术组与正常组;肾上腺、肾、子宫的湿重均低于假手术组与正常组。结论:采用经典的骨质疏松症造模方法即双侧卵巢切除术制备的骨质疏松症模型大鼠具有肾虚的器质性及功能性变化表现,骨质疏松症模型大鼠存在肾虚病机,具有肾虚的临床表现。     

Development and assessment of a complete-detoxication strategy for Fuzi (lateral root of Aconitum carmichaeli) and its application in rheumatoid arthritis therapy

Ethnopharmacological relevance

Fuzi (lateral root of Aconitum carmichaeli) is a popular traditional Chinese medicine well known for its both therapeutic and high-toxic activities. Its toxic alkaloid ingredients, mainly aconitine, mesaconitine, and hypaconitine, are responsible for the high toxicity. However, to date, no detoxication strategy is available to completely eliminate Fuzi's toxicity, and, whether Fuzi's efficacy could be kept after detoxication, remain unknown and debatable.

Materials and methods

The purpose of this study was to establish and validate a complete-detoxication strategy for Fuzi via acute toxicity test, to clarify the detoxication mechanism by HPLC and titrimetric analyses, and to evaluate the therapeutic effect of detoxicated Fuzi on adjuvant arthritis (AA). Three processed Fuzi (Bai-fu-pian) with 30-min, 60-min, and 120-min decoctions, respectively, named dBfp-30, dBfp-60, and dBfp-120, were prepared for this study. For the acute toxicity test, their oral doses to male and female Kunming mice were up to 70–190 g/kg body weight, and their toxicological profiles were evaluated by median lethal dose (LD₅₀), maximal tolerance dose (MTD), minimal lethal dose (MLD), no-observed-adverse-effect-level (NOAEL), and time–concentration–mortality (TCM) modeling methods using a 14-day schedule with up to five doses. The HPLC analysis was performed to determine the detoxication-induced changes in composition and amount of aconitine, mesaconitine and hypaconitine in Fuzi, whilst the titrimetric method was adopted to estimate the amount changes of Fuzi's total alkaloids. AA model was established by incomplete Freund's adjuvant injection in Wistar rats, and the animal's physiological (body weight, food intake, etc.), clinical (hind paw volume), and immunological (IL-1 and TNF-α) parameters were assessed as markers of inflammation and arthritis.

Results

With increasing decoction time, the acute toxicity of detoxicated Fuzi became decreased in the following order: dBfp-30 (LD₅₀ of 145.1 g/kg; MTD of 70 g/kg; MLD of 100 g/kg; NOAEL of 70 g/kg) >dBfp-60 (too large LD₅₀; MTD of 160 g/kg; MLD of 190 g/kg; NOAEL of 100 g/kg) >dBfp-120 (no LD₅₀; unlimited MTD; unlimited MLD; NOAEL of 130 g/kg). dBfp-30 and dBfp-60 displayed the toxicity at a dose-dependent manner with maximum mortalities reaching 100% and 50% respectively, whereas no mortality or signs of intoxication was induced by dBfp-120. The chemical analyses revealed a dramatic reduction of the toxic alkaloids as well as total alkaloids in Fuzi after the detoxication, from which no level of aconitine and only minimum residual of mesaconitine (0.56±0.02 μg/g) and hypaconitine (8.73±0.13 μg/g) were detected in dBfp-120. However, no significant difference of total alkaloid amount was found among dBfp-30, dBfp-60, and dBfp-120 (P>0.05), suggesting an equivalent conversion from toxic alkaloids to its non-toxic derivants in dBfp-120. Further, also no significant differences were seen among dBfp-30, dBfp-60, and dBfp-120 for the therapeutic effects on physiological, clinical, and immunological parameters in AA rat, indicating that dBfp-120 is of non-toxicity and efficacy.

Conclusions

A complete-detoxication strategy has been developed successfully for ensuring the safe and effective use of Fuzi. The detoxication mechanism associated with elimination of toxic alkaloids has kept Fuzi's efficacy, indicating a non-interdependent relationship between its efficacy and toxicity. This is the first report on such an optimal detoxication strategy and on the application of detoxicated Fuzi in AA. It may provide in depth understanding to the toxicological and pharmacological profiles of Fuzi and further benefit the herbal drug development with safety and efficacy for disease especially RA therapy.     

慢性骨髓炎的临床诊断与治疗

慢性骨髓炎是临床常见的病症之一.近些年来由于糖尿病、外周血管病以及创伤等诱发疾病增多,其患病率也有所上升.随着磁共振成像和CT成像技术的发展,大大提高了慢性骨髓炎临床诊断的准确性和描述感染特征的能力,为临床治疗提供可靠依据.目前对慢性骨髓炎的研究主要集中在影像学应用价值方面和抗生素优化使用途径控制炎症、缺损重建恢复血供等治疗方面.但其抗生素治疗的最佳时间以及运用方案仍不确定,对于清创后植骨的时机以及缺损修复后功能的快速恢复等还需进一步的研究.     

大鼠“曲泽”,“大陵”和“中冲”穴区之间神经血管显微结构的比较

ObjectiveThe aim of this study was to compare the microstructure of blood vessels and nerve fibers between different acupoints in the rat skin from the perspective of histology.MethodsThe skin tissue at “Qūzé” (曲泽 PC3)” “Dàlíng (大陵 PC7)” and “Zhōngchōng (中冲 PC9)” regions was taken from the rat forelimb along the pericardium meridian, and cut sagittally with a freezing microtome. After that, the skin sections were stained by fluorescent histochemistry and immunohistochemistry with phalloidin and calcitonin gene-related peptide (CGRP) to reveal the microstructure of blood vessels and nerve fibers respectively, and then examined under a laser scanning confocal microscope.ResultsThe microstructure of blood vessels and nerve fibers was clearly labeled with phalloidin and CGRP, respectively. The blood vessels and nerve fibers were observed in the dermis of “PC3” “PC7” and “PC9” regions, where the bundle of nerve fibers distribute in parallel to the blood vessels, and sent out thin branches to surround the wall of the blood vessels. In contrast, the density of blood vessels and nerve fibers in “PC3” “PC7” and “PC9” were presented orderly in an increased tendency.ConclusionThe results of present study demonstrate the differences of the neurovascular microstructure among “PC3” “PC7” and “PC9” in the rat, providing a histological view to insight into the specificity of different acupoints.     

Byakangelicin inhibits IL-1β–induced mouse chondrocyte inflammation in vitro and ameliorates murine osteoarthritis in vivo

Osteoarthritis (OA) is a chronic musculoskeletal degeneration disease, resulting in severe consequences such as chronic pain and functional disability. Owing to the complex pathology, there are currently available preventative clinical therapies for OA. Several studies have reported the potential anti-inflammatory effects of byakangelicin (BYA), a component of the Angelica dahurica root extract; however, the effects of BYA in OA remain unknown. In this study, we investigated the protective effects of BYA in interleukin (IL)-1β–induced mouse chondrocytes in vitro and on surgical destabilization in a medial meniscus (DMM) mouse OA model in vivo. In vitro, BYA treatment inhibited IL-1β–mediated inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-alpha, and IL-6 expression. Moreover, BYA promoted the expression of type two collagen and aggrecan but inhibited the expression of thrombospondin motifs 5 and matrix metalloproteinases, leading to degradation of the extracellular matrix. In addition, BYA mechanistically suppressed nuclear factor-kappa B signaling in the IL-1β–induced chondrocytes. The protective effects of BYA in OA development were also observed in vivo using the DMM model. In conclusion, our results highlight BYA as a candidate for OA treatment and prevention.     

Chondroprotective activity of a detoxicated traditional Chinese medicine (Fuzi) of Aconitum carmichaeli Debx against severe-stage osteoarthritis model induced by mono-iodoacetate

Ethnopharmacological relevance: Fuzi is an effective but toxic traditional Chinese medicine (TCM) derived from Aconitum carmichaeli. In our previous study, detoxicated Fuzi (d-Fuzi) has been originally developed with no toxicity but significant efficacy. However, whether d-Fuzi can be used for therapy of osteoarthritis (OA), remain unknown.Materials and methods: Severe OA model was established by intra-articular mono-iodoacetate (MIA) injection (1.25 mg) into rats and orally treated with 2 g/ml d-Fuzi at a dosage of 7 ml/kg body weight for 28 days. In vivo, the articular radiographic and histopathologic analyses were performed to qualitatively assess the chondroprotective effect of d-Fuzi, followed by quantitative measurements of bone density and Mankin scores. In vitro, such effect on chondrocyte viability after MIA attack was evaluated. Hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS) was performed for chemical analysis of d-Fuzi.Results: d-Fuzi was demonstrated to possess chondroprotective activity on MIA-induced OA model by in vivo preventing the articular degeneration and the reducing of bone density and Mankin score, as well as by in vitro promoting the chondrocyte proliferation and inhibiting the MIA-induced chondrocyte damage. A total of 23 compounds were identified in d-Fuzi, most of which were deduced as the non-toxic derivatives of aconite alkaloids.Conclusions: This is the first report regarding chondroprotective effect and chemical profile of d-Fuzi, originally revealing its great anti-OA potential and thereby providing a promising TCM candidate for OA therapy.