2 Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310000, China;
3 State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210000, China;
4 Key Laboratory for the Research of Aromatic Chinese Medicine in Yunnan, College of Pharmaceutical Science, Yunnan University of Traditional Chinese Medicine, Kunming 650000, China;
5 School of Pharmacy, Xinjiang Medical University, Urumqi 830001, China
Notopterygii Rhizoma Et Radix is obtained from the roots and rhizomes of Notopterygium incisum Ting ex H. T. Chang and has been historically used as "Qianghuo (QH)" in China. QH is considered as a traditional medicine with multiple efficacies of expelling wind, eliminating dampness and relieving pain. It is commonly used to treat a number of diseases and conditions, including cold, headache, rheumatoid arthritis, fever, sleepiness, and neck and back stiffness. Among those, the anti-rheumatic activity for the treatment of rheumatoid arthritis (RA) has been highlighted. QH also has potent effects in relieving joint pain and stiffness. Moreover, Notopterygium incisum Ting ex H. T. Chang - Clematis chinensis Osbeck combination has profound anti-rheumatic property in adjuvant-induced arthritis rats [1-2].
RA is a systemic, progressive, and inflammatory autoimmune disorder, affecting 0.5%-1% of the global population. It is pathologically characterized by inflammatory cell infiltration, synovial hyperplasia, angiogenesis, and subsequent destruction, and deformity of joints . Despite the unknown etiology of RA, the orchestrated interactions involved in these pathological processes play crucial roles in the initiation, development, and progression. For example, in inflammatory synovitis and angiogenesis, cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), are secreted by infiltrated inflammatory cells, These cytokines directly promote the chronic inflammation and also play irreplaceable roles in triggering the synovial angiogenesis. On the other hand, synovial angiogenesis is an important step in the initiation and maintenance of pannus, subsequently leading to cartilage and bone destruction and joint remodeling. Moreover, the new blood vessels formed during synovial angiogenesis enhance the local nutrients and oxygen supply, which then augment inflammatory cell mass and aggravates synovitis [4-5].
According to the phytochemical researches, the chemical constituents of the roots and rhizomes of N. incisum include coumarins, volatile oils, organic acids, phenolic compounds, sterols, fatty acids, polyacetylenes, glycosides, alkaloids, polyacetylenes, amino acids, and others [6-11]. Volatile compounds are the major components of N. incisum. Researchers have characterized and identified about 40 constituents from the water distilled volatile oils of N. incisum and determined the major components of Notopterygium volatile oils, including α-pinene, β-pinene, and limonene . The pharmacological activities of Notopterygium volatile oils have also been verified. For example, d-limonene possesses multiple properties, including anti-carcinogenic, antitussive, and anti-bacterial activities [13-14]. However, there are only few studies reported that are focused on the anti-inflammatory and anti-angiogenic properties of Notopterygium volatile oils.
Hence, in the present study, we utilized the gas chromatography-mass spectrometry (GC-MS) to determine the phytochemical ingredients and their proportions in the volatile oils distilled from the roots and rhizomes of N. incisum to obtain more detailed qualitative and quantitative characteristics. Meanwhile, on the basis of profound anti-rheumatic efficacy of N. incisum, the rats with adjuvant-induced arthritis (AIA) were used to evaluate the anti-rheumatic efficacy for the obtained Notopterygium volatile oils. Furthermore, in view of the importance of inflammatory synovitis and angiogenesis in human RA, we observed the anti-inflammatory and anti-angiogenic properties of Notopterygium volatile oils on two cell models of nitric oxide (NO) release and endothelial cell proliferation and tube formation, respectively.Materials and Methods Plant materials, chemicals and reagents
QH was purchased from Simcere Drugstore (Nanjing, China) and identified as Notopterygium incisum Ting ex H. T. Chang by Professor LI Ping (Department of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing, China). Voucher specimens (No. CH20160515) were deposited in the State Key Laboratory of Natural Medicines in China Pharmaceutical University (Nanjing, China). Ethanol (Analytical grade) was purchased from Shanghai Titan Scientific Co., Ltd. (Shanghai, China). The deionized water was prepared with a Millipore Milli-Q water purification system provided by Merck Millipore (Bedford, MA, USA). Mycobacterium butyricum and Freund's incomplete adjuvant were purchased from Difco Laboratories Inc. (Detroit, MI, USA). Tween 80, 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and vascular endothelial growth factor (VEGF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI)-1640 medium and Dulbecco's modified eagle's medium (DMEM) were purchased from Gibco-BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from PAA Laboratories (Pasching, Austria). Matrigel was purchased from BD Biosciences (Franklin Lakes, New Jersey, USA). The kits for Nitrate/nitrite colorimetric assay and 0.4% paraformaldehyde were purchased from Jiancheng Chemical (Nanjing, China). Ibuprofen (IBU) was purchased from Zhongmei Tianjin Shike (Tianjin, China).Animals
Male Sprague-Dawley rats (weighing 220-240 g) were purchased from Shanghai BK Experimental Animal Center (Certificate No: SCXK2013-0016, Shanghai, China). All the rats were acclimatized under standard laboratory conditions with 12 h/12 h light/dark cycles. The room temperature was set at (22 ± 2) ℃ and room humidity was set at 50% ± 20%. The rats were allowed free access to food and tap water. All the experimental procedures were followed the guidelines of current ethical regulations for institutional animal care and use in China Pharmaceutical University (No. SYSK2016-0011, 1/27/2016-1/26/2021) and approved by China Pharmaceutical University Ethics Committee for Animal Laboratory Research.Apparatus and chromatographic conditions
GC-MS was performed with an Agilent 7890B gas chromatograph (Agilent Co., Palo Alto, CA, USA) coupled with a 5977A mass spectrometer (Varian Inc., Walnut Creek, CA, USA) and MassHunter GC/MS Acquisition Software (B.07.00). The column was initiated at 50 ℃ and lasted for 10 min, followed by 55 ℃ at the rate of 2 ℃·min-1 and lasted for 7 min, 80 ℃ at a rate of 5 ℃·min-1, 110 ℃ at a rate of 8 ℃·min-1 and lasted for 10 min, and then increased to 118 ℃ at a rate of 1.8 ℃·min-1 and sustained for 10 min. Later, the temperature was settled from 118 ℃ to 125 ℃ at a rate of 1.8 ℃·min-1 and lasted for 10 min. Finally, the temperature was programmed from 125 ℃ to 150 ℃ at 1.8℃·min-1. The injection volume was 1 μL and the split ratio was settled as 50 : 1. High-purity helium (99.99%) was used as the carrier gas at a flow rate of 1.2 mL·min-1 and the solvent delay was 3 min. The spectrometer was operated in the electron-impact mode, and the temperature of ion source and quadrupole were set at 230 ℃ and 150 ℃, respectively. The ionization voltage energy was set at 70 eV. The scan ranged from 30-600 amu at a rate of 3.8 scan/s.Sample preparation
150 g of properly dried and crushed QH sample (through a 40-mesh sieve) was weighed accurately, refluxed with 1500 mL of water for 5 h. Then the QH volatile oil was obtained by water-steam hydrodistillation, and the oil yield ratio was 2.3% (mL·g-1). The obtained essential oil was dried using sodium sulfate anhydrous (Na2SO4) and kept at 4 ℃ till use. Then, 1 μL of diluted volatile oil (diluted with acetic ether) was injected into the GC-MS system for analysis.Induction, assessment, and treatment AIA in rats
Freund's complete adjuvant was prepared one day ahead of AIA induction by dispersing lyophilized M. butyricum into Freund's incomplete adjuvant at a concentration of 10 mg·mL-1. On Day 0, 0.1 mL of Freund's complete adjuvant was intradermally injected into the plantar skin of the left hind-paw .
The rats were then monitored every day by examiners who were blinded to the experimental design. On Days 0, 7, 14, 17, 21, 24, and 27, body weight of each rat was measured as a general physical sign, and signs of arthritis were evaluated using two clinical parameters, paw swelling and arthritis index (AI). Paw swelling was determined by measuring the volume of hind paws using plethysmometer. AI scoring was performed on a 0-4 scale, where 0 = no swelling or erythema, 1 = slight swelling and/or erythema, 2 = low-to-moderate edema, 3 = pronounced edema with limited joint usage, and 4 = excess edema with joint rigidity.
For studying the anti-arthritic capacity, the rats were treted with daily oral gavage of Notopterygium volatile oils (2, 4, and 8 g crude drug/kg, solubilized by Tween 80) and IBU (50 mg·kg-1) from Day 14 to 27, respectively. All the control and AIA rats were treated with equal volume of 0.1% Tween 80.Histopathological examination
The rats were euthanized by ether inhalation after 2 h of dosing on Day 27, and the right ankle joints were then removed and fixed in 0.4% paraformaldehyde. The fixed samples were decalcificated, embedded in paraffin, and sliced. Hematoxylin and eosin (H & E) staining was performed and histological changes in joints were determined by two pathologists blinded to experimental design, according to the reported scoring system .Cell culture
RAW264.7 mouse macrophages and human umbilical vein endothelial cell line EAhy926 were obtained from the Shanghai Cell Collection of China Academy of Science. The RAW264.7 cells were maintained in RPMI-1640 medium supplemented with 10% (V/V) fetal bovine serum, 100 U·mL-1 of penicillin, and 100 mg·mL-1 of streptomycin, at 37 ℃ in a 5% CO2 atmosphere. The EAhy926 cells were cultured in 10% FBS-DMEM with 100 U·mL-1 of penicillin, and 100 mg·mL-1 of streptomycin, at 37 ℃ in a 5% CO2 atmosphere. The cells in the exponential growth phase were used in further experiments.Cell viability assay
The cell viability was measured using the MTT assay. The RAW264.7 cells at a density of 3 × 104 cells/well were plated into 96-well plates and incubated at 37 ℃ and 5% CO2. The cells were then allocated to 6 groups: normal (cells in medium), control [cells in medium plus lipopolysaccharide (LPS, 1 μg·mL-1)], positive [cells in medium plus LPS (1 μg·mL-1) and IBU (50 μg·mL-1)], Notopterygium volatile oils [cells in medium plus LPS (1 μg·mL-1), and Notopterygium volatile oils (5, 15, and 45 μg·mL-1)]. After a 20-h incubation, 10 μL of MTT (5 mg·mL-1) was added to each well followed by incubation for additional 4 h. The supernatants were removed, and 150 μL of DMSO was added to each well. The formazan dye crystals were dissolved for 10 min, and absorbance at 490 nm was measured with a microplate reader. Cell viability was calculated using the formula below,
Cell viability = ODtreatment group/ODcontrol group × 100%.NO determination
The RAW264.7 cells at a density of 3 × 104 cells/well were cultured in 96-well plates and incubated at 37 ℃ and 5% CO2. The experimental group setting was the same with cell viability assay section. After 24-h treatment, 100 μL medium was collected and total NO was measured with a nitrate/nitrite colorimetric assay kit according to the manufacturer's instructions.Cell proliferation assay
The EAhy926 cells at a density of 1 × 104 cells/well were seeded into 96-well plate and incubated at 37 ℃ and 5% CO2. The cells were then allocated to 5 groups: normal (cells in medium), control [cells in medium plus VEGF (20 ng·mL-1)], Notopterygium volatile oils [cells in medium plus VEGF (20 ng·mL-1), and Notopterygium volatile oils (5, 15, and 45 μg·mL-1)]. The cells were pretreated with or without Notopterygium volatile oils for 4 h, and then VEGF was added into the medium. After a 72 h incubation, 10 μL of MTT (5 mg·mL-1) was added to each well followed by incubation for 4 h. The supernatants were then removed, and 150 μL of DMSO was added to each well. The formazan dye crystals were dissolved for 10 min, and absorbance at 490 nm was measured with a microplate reader. Percentage of proliferation ratio was calculated as ODtreatment group/ODcontrol group × 100%.Tube formation assay
The effect of Notopterygium volatile oils on in vitro angiogenesis was evaluated through tube formation assay. Matrigel was diluted in the medium and made up to 3 mg·mL-1 as the final concentration. Then 50 μL of liquid matrigel was added into a 96-well plate and incubated at 37 ℃ for 0.5 h to form a matrigel matrix. The EAhy926 cells (8 × 104 cells/well) in 1% FBS-DMEM were seeded into the coated 96-well plate in the presence/absence of VEGF (20 ng·mL-1) with or without various concentrations of Notopterygium volatile oils (5, 15, and 45 μg·mL-1). After a 12-h incubation at 37 ℃ and 5% CO2, the enclosed networks of tubes were photographed randomly under a microscope.Statistical analysis
All the data were expressed as means ± SD and all the experiments were carried out in triplicates. One-way analysis of variance (ANOVA) was used to determine the differences in assessment between the two groups. P < 0.05, < 0.01, and < 0.001 were considered to be statistically significant, very significant, and extremely significant, respectively.Results and Discussion Analysis of volatile compounds by GC-MS
GC-MS is the combination of high-resolution gas chromatography and high-sensitivity mass spectrometry. It is suitable for analyzing low molecular weight compounds, especially for the quantitative and qualitative analysis of volatile components extracted from traditional Chinese medicines. In the present study, volatile compounds from QH exerted different efficacies, which were then analyzed and identified by GC-MS using Mass Hunter. The compounds were identified according to the NIST online spectrum library developed by the National Institute of Standards. The information included the name, formula, structure, molecular weight, and other information of the unknown compound at the same time. The first one shown in the results was usually the most accurate, but the match should be larger than 850. Generally, the higher match score, the more reliable the result. If necessary, it was compared with other related literatures to further confirm these components. Moreover, the peak area percentage can be used as a reference for the relative content of these compounds by peak area normalization method. The results in Fig. 1 (chromatography profiling) and Table 1 (relative contents) showed that 70 compounds were determined in QH. According to the percentage of different compounds, the contents of 14 compounds were more than 1%, and 5 compounds were more than 5%. Moreover, the contents of 3 compounds exceeded 10%, and they are 16.48% (γ-Terpinene), 21.61% (α-Pinene), and 24.19% (β-Pinene), respectively. β-Pinene and α-Pinene has been shown to have several pharmacological effects including anti-inflammatory [17-18], anti-hypertensive , anti- viral , anti-depressant-like , anti-tumor , anti-angiogenic activities [23-24]. In addition, many studies have reported that γ-Terpinene also has anti-inflammatory properties [23-26]. Therefore, β-Pinene, α-Pinene, and γ-Terpinene might be the main effective composition of QH that exert anti-inflammatory and anti-angiogenic activities.
AIA, which appears in rats immunized with Freund's complete adjuvant, shows suggestive similarities in clinical onset and course, as well as the pattern of lesions in joints, synovitis, periostitis, and tendonitis, to those observed in human rheumatoid arthritis . This reproducible model has been commonly used in the screening and development of anti-arthritic agents. In our study, Freund's complete adjuvant inoculated in the paw of rats showed an acute inflammation on Day 0 with acute swelling and signs of AIA developed around two weeks after injection and perpetuated for more than two weeks. On Day 17, the mean body weight of rats in model group was significantly less than that of normal group during the autoimmune phase. The AI scores and paw swelling in immune rats indicated that the secondary arthritis began from about Day 14 and reached a plateau on Day 21.
Notopterygium volatile oils, IBU, and dissolvent were orally administered after the onset of the secondary arthritis. Notopterygium volatile oils (2, 4, and 8 g crude drugs/kg) exerted a clear therapeutic effect on ongoing AIA in a dose- dependent manner. Both Notopterygium volatile oils and IBU (50 mg·kg-1), anon-steroidal anti-inflammatory drug (NSAID), significantly attenuated the clinical signs of arthritis, as determined by improved loss of body weight, decreased swelling of maximum paw, enhanced resolution of swelling, and reduced maximum polyarthritis index (Fig. 2). Meanwhile, Notopterygium volatile oils showed a slightly higher potency than the positive drug IBU, especially in the improvement of loss of body weight (Fig. 2A).
The rat ankles were removed after euthanization and then fixed in 0.4% paraformaldehyde on Day 27 for histological examination of tissue sections. As shown in Fig. 3A, the normal rat ankles appeared clear and complete histological architecture under the microscope, whereas the AIA rat ankle joints appeared to have abnormal histological architecture including profound synovial hyperplasia, massive inflammatory cell infiltration, angiogenesis (increased microvessel density), and extensive erosion in the cartilage and bone (Figs. 3B, G).
IBU (50 mg·kg-1) and high dose of Notopterygium volatile oils (8 g crude drugs/kg) preserved almost normal histological architecture of the ankle joints with mild synovial hyperplasia. The pathohistological scores were remarkably decreased in these two groups compared with model group, suggesting their pronounced anti-arthritic efficacy (Figs. 3E, F, and G). Moreover, the medium dose of Notopterygium volatile oils (4 g crude drugs/kg) also showed that therapeutic capacity on rat AIA was somewhat milder than the high dose of the volatile oils, while low dose (2 g crude drugs/kg) of volatile oils exerted little improvement on the pathohistological changes (Figs. 3C, D, and G). Results gained in the histological assessment are in accordance with those obtained in general and observation of arthritis sign, indicating that Notopterygium volatile oils possessed substantial anti-arthritic efficacy in a dose-dependent manner.Downregulation of NO production by RAW264.7 macrophages
RA is the most common inflammatory arthritis with clinical manifestations of multi-small-joint damage, which in turn lead to chronic pain, joint deformity and functional disability. It is well known that abnormal inflammatory response in synovium is one of the key events of this disease, despite the etiology and pathogenesis still remains to be elucidated. Under the inflammatory conditions associated with massive infiltrated inflammatory cells and proinflammatory cytokines, the macrophage-like and fibroblast-like synoviocytes undergo uncontrolled proliferation, eventually forming the pannus and destroying the articular . During the abnormal inflammatory response, pro-inflammatory cells, mainly activated macrophages, produce inflammatory cytokines (i.e., TNF-α, IL-1β, and IL-6) and other inflammatory mediators, including NO, during abnormal inflammatory response. NO is produced by inducible NO synthase in activated macrophages and other immune cells that are exposed to stimulants such as LPS and interferon-gamma (IFN-γ). Therefore, inhibition of the production of NO is a major strategy in the treatment of inflammatory diseases [27-28].
In the present study, we proved that the inflammatory cell infiltration in the synovial tissue was weakened by Notopterygium volatile oils in AIA rats. Then, the test of the inhibitive capacity of the volatile oils on inflammatory cells was conducted in vitro. The cell viability assay was initially conducted and the results demonstrated that the Notopterygium volatile oils exerted no cytotoxicity against mouse RAW264.7 macrophages (Fig. 4A). Then, we measured its effect on NO release to evaluate its anti-inflammatory property. As shown in Fig. 4B, the NO levels in the mouse RAW264.7 macrophages culture supernatants were decreased by more than 50% in each group pretreated by different concentrations of Notopterygium volatile oils. These results indicated that the potent anti-arthritic efficacy of the volatile oils was related to the inhibition of NO release from the inflammatory cells. Meanwhile, Ibuprofen, a type of NSAIDs, has been chosen as a positive control because of its definite effect of relieving pain, fever and inflammation in RA .
Synovial angiogenesis in RA showes abnormal and unregulated growth of blood vessels, which results in increased leucocyte migration into the synovial tissue, forming the pannus. The pannus then invades the cartilage and bone, eventually leading to articular damage through destroying the subtle balance between the action of osteoclasts and osteoblasts. Thus, targeting angiogenesis remains an attractive strategy for the treatment of RA . Angiogenesis is a tightly orchestrated process that involves a complex cascade of proinflammatory cytokines and growth factors. VEGF, an endothelial cell-specific mitogenic and chemotactic agent, plays a key regulator role in angiogenesis. In experimental arthritis, VEGF expression level was correlated with the degree of angiogenesis and arthritis severity. Furthermore, it has been reported that at early stage of vascular morphogenesis, VEGF triggers endothelial cell proliferation and migration [30-31].
In the present study, a decreased effect on synovial blood vessels by Notopterygium volatile oils was observed under the microscope with H & E staining, indicating potent anti- angiogenic capacity of the volatile oils. Therefore, we further validated the anti-angiogenic effect of Notopterygium volatile oils through VEGF stimulation of endothelial cell proliferation and tube formation assays. As shown in Fig. 5A, the volatile oils exerted a concentration-dependent downregulation on VEGF stimulation of endothelial cell proliferation. The stimulation indices were decreased by 24% and 30% at the concentrations of 15 and 45 μg·mL-1, respectively. Meanwhile, an angiogenic model was used to test the capacity of Notopterygium volatile oils to interfere endothelial cell capillary tube formation. Fig. 5C showed the normal tube formation of EAhy926 cells by VEGF stimulation. Notopterygium volatile oils effectively delayed endothelial tube formation in a concentration-dependent manner (Figs. 5D-F) compared with the VEGF treated group (Figs. 5C). The tube structure was obviously disrupted when exposed to Notopterygium volatile oils at concentrations above 5 μg·mL-1, resulting in the incomplete tube network formation in the field of vision. To sum up, the significant inhibition on these two events suggested that volatile oils might target angiogenesis.
Our GC-MS analyses revealed that, among the determined 70 compounds in volatile oils distilled from QH, the contents of 14 compounds were more than 1%, 5 compounds were more than 5%, and 3 compounds were more than 10%, showing γ-terpinene of 16.48%, α-pinene of 21.61%, and β-pinene of 24.19%, respectively. Animal experiments demonstrated substantial anti-arthritis efficacy of Notopterygium volatile oils, as gauged by the obvious alleviation of synovial hyperplasia, angiogenises, inflammatory cell infiltration, and cartilage and bone destruction. Further in vitro studies via NO release, endothelial cell proliferation, and tube formation assays demonstrated potent anti-inflammatory and anti-angiogenesis activities of Notopterygium volatile oils, which might contribute to the anti-arthritic efficacy of QH in vivo.
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