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ORIGINAL ARTICLE
Year : 2022  |  Volume : 18  |  Issue : 79  |  Page : 783-789  

Angelica gigas nakai attenuated inflammatory response on monosodium iodoacetate-induced osteoarthritis


1 Department of Herbology, Korean Medicine of College, Daegu Haany University, Suseong-gu, Deagu, Korea
2 DHU Bio Convergence Testing Center, 1 Hanuidae-ro, Gyeongsan 38610, Korea

Date of Submission08-Oct-2020
Date of Decision23-Mar-2021
Date of Acceptance13-May-2021
Date of Web Publication19-Sep-2022

Correspondence Address:
Seong-Soo Roh
Department of Herbology, Korean Medicine of College, DaeguHaany University, 136, Shinchendong–ro, Suseong-gu, Deagu 42158
Korea
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_422_20

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   Abstract 


Background: Angelica gigas Nakai (AGN) is well-known for anti-inflammatory and anti-oxidative effects; however, the mechanism underlying the protective effects of AGN on monosodium iodoacetate (MIA)-induced osteoarthritis (OA) remains unknown. Objectives: Our objectives of this study were to evaluate the anti-inflammatory effect of AGN on MIA-induced OA and investigate the potential mechanism which underlies such effect. Materials and Methods: The dried herb AGN was extracted with distilled water. After the experiment was conducted for 4 weeks using 7-week-old male Sprague–Dawley rat. AGN (200 mg/kg body weight/day) was orally administered for 14 days from day 7 after intra-articular injection of MIA (50 μL with 80 mg/mL), and the effects were compared with those of MIA-treated control group. We examined the weight-bearing ability of hind paws, biomarkers related to oxidative stress in cartilaginous tissue. In addition, various factors associated with inflammatory and cartilage degeneration in cartilaginous tissue detected by the Western blot analysis. Results: Our results from an in vivo model of OA indicate that AGN inhibits inflammatory factors such as inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, interleukin-6 (IL-6), and IL-1β via nuclear factor kappa B signaling pathway. AGN suppresses the expression of matrix metalloproteinase, proteolytic enzyme, and also elevates tissue inhibitor of metalloproteinase which inhibited degradation of the extracellular matrix. Moreover, antioxidant defense was enhanced through the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Conclusion: Taken together, these results demonstrate the effectiveness of AGN at the inflammation associated with joint disorders. Our results also suggest that AGN could serve as a potential candidate for alternative therapeutic treatment of OA.

Keywords: Angelica gigas Nakai, anti-inflammatory, antioxidant, monosodium iodoacetate, osteoarthritis


How to cite this article:
Lee JA, Kim MJ, Shin MR, Park HJ, Roh SS. Angelica gigas nakai attenuated inflammatory response on monosodium iodoacetate-induced osteoarthritis. Phcog Mag 2022;18:783-9

How to cite this URL:
Lee JA, Kim MJ, Shin MR, Park HJ, Roh SS. Angelica gigas nakai attenuated inflammatory response on monosodium iodoacetate-induced osteoarthritis. Phcog Mag [serial online] 2022 [cited 2022 Sep 26];18:783-9. Available from: http://www.phcog.com/text.asp?2022/18/79/783/356394



SUMMARY

  • Angelica gigas Nakai (AGN) is well-known for anti-inflammatory and anti-oxidative effects. Our objectives of this study were to evaluate the anti-inflammatory effect of AGN on monosodium iodoacetate (MIA)-induced osteoarthritis (OA). As a result of the experiment, it was confirmed that AGN suppressed excessive ROS in osteoarthritis and increased the antioxidant effect through Nrf2/HO-1. Moreover, AGN could inhibit the expression of inflammation factors by NF-κB inactivation. Our results suggest that AGN could serve as a potential candidate for alternative therapeutic treatment of OA.




Abbreviations Used: AGN: Angelica gigas Nakai; MIA: Monosodium Iodoactate; OA: Osteoarthritis; Indo: Indomethacin; HWBD: Hindpaw weight bearing distribution; ROS: Reactive oxygen species; ONOO-: Peroxynitrite; Nrf2: Nuclear factor erythroid 2 related factor 2; HO-1: Heme oxygenase 1; MMPs: Matrix metalloproteinases; TIMPs: Tissue inhibitor of metalloproteinases; NF-κBp65: Nuclear factor kappa B p65; IκBɑ: Inhibitor of κB.


   Introduction Top


Osteoarthritis (OA) is one of chronic arthritis characterized by inflammation and cartilage degradation. OA causes physical limitations, mental stress, socio-economic burden, and disability.[1],[2] It has been acknowledged through many studies that the cause of OA is related to inflammation. Therefore, anti-inflammatory drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) are used to relieve OA symptoms. However, these drugs cannot delay the progression of OA and it just relieves the pain. Hence, new strategies for its delay are urgently needed.[3]

Many studies have shown iinflammatory cytokines including interleukin (IL)-1 β and tumor necrosis factor (TNF)-α are important factors in OA pathogenesis,[4],[5],[6] which is intimately related to cartilage matrix degradation. Matrix metalloproteinases (MMPs), proteolytic enzyme, is another factor closely associated with the degradation of cartilage matrix, and both interleukin-1 (IL-1) β and TNF-α can up-regulate MMPs expression.[7],[8],[9],[10] Moreover, IL-1 β-induced nuclear factor kappa B (NF-κB) plays a central role in the expression of several inflammatory factors known as the pathogenesis of OA. Activated NF-κB can trigger extracellular matrix (ECM) degradation products, leading to OA.[11],[12] Hence, the inhibition of inflammation may be a valid approach for the improvement of OA.

Angelica gigas Nakai (AGN) (local name dang-gui), traditional herbal medicinal plant in Korea, is mainly used for the treatment of menopausal syndromes.[13] Its pyranocoumarin compounds, such as decursin, decursinol angelate (DA), nodakenin, and demethylsuberosin, exert varying pharmacological effects.[14],[15] Especially, both decursin and DA have been reported to exhibit anti-inflammatory effects.[16],[17],[18],[19]

The purpose of our study was to elucidate reveals the mechanism for AGN to reveal relief of inflammation and protection of ECM through inhibition of NF-κB activation in the monosodium iodoacetate (MIA)-induced OA model.


   Materials and Methods Top


Materials

MIA, phenylmethylsulfonyl fluoride (PMSF), and dithiothreitol (DTT), diethylenetriaminepenta-acetic acid (DTPA) and dihydrorhoda mine 123 (DHR 123) were purchased from Sigma Aldrich Co., Ltd (St. Louis, MO, USA). The protease inhibitor mixture solution and ethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries, Ltd., (Osaka, Japan). 2',7'-Dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular Probes (Eugene, OR, USA). ECL Western blotting detection reagents and pure nitrocellulose membranes were supplied by GE Healthcare (Piscataway, NJ, USA). 0.3 mL-insulin syringe was obtained from BD Medical-Diabetes Care (Holdrege, USA). Besides, all other chemicals and reagents were purchased from Sigma Aldrich Co., Ltd., (St Louis, MO, USA).

Preparation of Angelica gigas Nakai extract

AGN was purchased as a dried herb from Bonchowon (Yeoungcheon, Korea), in 2018. The plant materials were authenticated by Professor SS Roh at College of Korean Medicine at Daegu Haany University. The dried herb AGN (300 g) was extracted with distilled water (3 L) for 2 h (DW-790, Daewoongbio, Chungbuk, Korea). After filtration (285 mm), the solution was evaporated under reduced pressure (N-1100, Eyela, Tokyo, Japan) and then freeze dried (FD5508, IlShin, Seoul, Korea) to provide an extract with a yield of 27.1% by weight of the starting materials (number of the plant material; 2019-34). The concentrate was lyophilized and subsequently stored at −20°C until use.

Angelica gigas Nakai analysis by high-performance liquid chromatography chromatogram

The extract of AGN (10 mg) was dissolved in 1 mL of methanol. 10 μL of sample was injected into high-performance liquid chromatography (HPLC) using ZORBAX Eclipse XDB-C18 (4.6 mm × 150 mm; particle size 5 μm, [Agilent Technologies, Inc., USA]). The mobile phase was composed of MeOH (A) and water (B). The elution conditions were as follows: the initial mobile phase of 20% A and 80% B was held for 0 min; a gradient was the napplied to 70% A and 30% B for 30 min; 70% A and 30% B for 50 min wash with 100% B. The column temperature was kept at a constant 25°C throughout the analysis, with a flow rate of 2.0 mL/min and an injection volume of 10 mg/mL in 50% of Methanol. Identification was based on the comparisons of retention time sand UV spectra with those of commercial standards. Quantitation was performed based on external standards, with a mixture of standards of known concentration analyzed in duplicate before and after each batch of samples. The peak areas were used to calculate the sample contents of the compounds. The components of major compounds (Nodakenin, Demethylsuberosin, Decursin, and Decusinol angelate) were detected from AGN. The measurement was repeated three times for each sample. Representative HPLC results are illustrated in [Figure 1]
Figure 1: High performance liquid chromatography profile of Angelica gigas Nakai. (a) Chemical structure of Nodakenin, Demethylsuberosin, Decursin, and Decusinol angelate, (b) HPLC chromatogram of the extract of Angelica gigas Nakai

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. The amount was as follows: AGN: 8,902 μg/g Nodakenin, 6,650 μg/g Demethylsuberosin, 35,065 μg/g Decursin and Decusinol angelate.

Development of osteoarthritis with monosodium iodoacetate injections and Angelica gigas Nakai administration

Seven-week-old male Sprague–Dawley rat weighing 200–250 g at the start of the experiment was purchased from DBL Co. (Eumseong, South Korea). The animals were housed three per cage in a room with controlled temperature conditions (23°C ± 2°C), humidity (about 55 ± 5%), and lighting (12 h light/dark cycle) with access to freely food and water. All animal procedures were approved by the Animal Research Ethics Committee of the Daegu Haany University (Permit Number: DHU2019-047). After adaptation (1 week), rats are randomly arranged in the descending order of weight and assigned into four groups of equal number (n = 8) without statistical significance among the groups. Group 1 included the normal rats (Normal). Group 2 included the MIA control rats (Control). Group 3 included the Indomethacin 5 mg/kg-administered and MIA rats (Indo). Group 4 included the AGN 200 mg/kg-administered and MIA rats (AGN). The normal and MIA control rat groups were administrated water using a stomach tube, whereas the other groups were orally administered Indomethacin 5 mg/kg or AGN 200 mg/kg using a stomach tube for 2 weeks. OA was induced in SD rats employed the method of Wang et al. with minor modification.[20] After anesthetization with injection of zoletil mixture (Vibrac, France) 0.75 mg/kg intraperitoneally, rats were injected with MIA 80 mg/kg in a 50 μL volume using a 0.3 mL-insulin syringe (31G needle) inserted through the patellar ligament into the intra-articular space of the right knee; normal rats were injected with an equivalent volume of saline. The rats in all groups were sacrificed after the end of the experimental period. The rats were anesthetized using a mixture of zoletil and xylazine and euthanized by isoflurane overdose. The entire joint was removed immediately and frozen in liquid nitrogen. Blood was drawn from the abdominal vein using a 10 mL syringe, centrifuged at 3,000 rpm for 20 min at 4°C, and serum collected and stored at –80°C until analysis.

Measurements of hind paw weight-bearing distribution

Weight bearing was assessed using an in capacitance meter that measures the distribution of weight bearing across each hind limb. The balance in the weight-bearing capability of the hindpaws was disrupted after OA induction. A significant shift in weight from the arthritic site to the contralateral limb, i.e. a weight-bearing deficit, was considered to be an index of pain. Inflammatory pain was measured via the weight bearing of the paw load using Incapacitance Meter Apparatus (IITC Life Science Inc., CA, USA).[21] The weight distribution ratio was calculated using the following equation:



Measurement of reactive oxygen species level in cartilaginous tissue

The reactive oxygen species (ROS) levels were measured by employing the method of Ali et al.[22] 25 mM DCF-DA was added to cartilaginous tissue. After incubation for 30 min, the changes in fluorescence values were determined at an excitation wavelength of 486 nm and emission wavelength of 530 nm using a microplate fluorescence reader, model infinite M200 PRO (Tecan, Austria).

Measurement of ONOO Level in cartilaginous tissue

The peroxynitrite (ONOO) level was assessed by a modified Kooy's method with minor modifications,[23] which involves the monitoring of highly fluorescent rhodamine 123, which is rapidly produced from non-fluorescent dihydrorhodamine (DHR) 123 in the presence of ONOO. Moreover, its final fluorescent intensities remained unchanged over time. Cartilaginous tissue was added to the rhodamine buffer. In brief, the rhodamine buffer (pH 7.4) consisted of 50 mM sodium phosphate dibasic, 50 mM sodium phosphate monobasic, 90 mM sodium chloride, 5 mM potassium chloride and 5 mM diethylenetriamine penta-acetic acid. The final DHR 123 concentration was 5.0 μM. Five minutes after treating with or without the addition of authentic ONOO, the background and final fluorescent intensities of the samples were measured. The assay buffer was prepared prior to use and placed on ice. The fluorescence intensity of the oxidized DHR 123 was measured with a microplate fluorescence reader, model infinite M200 PRO (Tecan, Austria), at 485 nm excitation and 535 nm emission. The results were expressed as the inhibition level of oxidation of DHR 123 and calculated from the final fluorescence intensity minus background fluorescence intensity.

Preparation of cytosol and nuclear fractions

Protein extraction was performed according to the method of Komatsu with minor modifications.[24] Cartilaginous tissue for cytosol fraction was homogenized with ice-cold lysis buffer A (250 mL) containing 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF and 1,250 μL protease inhibitor mixture solution. The homogenate incubated at 4°C for 20 min. And then 10% NP-40 was added and mixed well. After centrifugation (12,000 rpm for 2 min at 4°C) using Eppendorf 5415R (Hamburg, Germany), the supernatant liquid (cytosol fraction) was separated new e-tube. The left pellets were washed twice by buffer A and the supernatant was discarded. Next, the pellets were suspended with lysis buffer C (20 mL) containing 50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 1% (v/v) glycerol, and 100 μL protease inhibitor mixture solution suspended and incubated at 4°C for 30 min. After centrifugation (12,000 rpm for 10 min at 4°C), the nuclear fraction was prepared to collect the supernatant. The protein concentration was determined using BCA Protein Assays by Thermo Fisher Scientific (Waltham, USA). Both cytosol and nuclear fractions were kept at-80°C before the analysis.

Immunoblotting analysis

For the estimation of nuclear factor erythroid 2-related factor 2 (Nrf2), NF-κBp65 and histone (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) 10 μg of proteins from each nuclear fraction were electrophoresed through 8%–10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). Separated proteins were transferred to a nitrocellulose membrane, blocked with 5% (w/v) skim milk solution for 1 h, then incubated with primary antibodies to Nrf2, NF-κBp65 and histone, respectively, overnight at 4°C. After the blots were washed, they were incubated with anti-rabbit or anti-mouse immunoglobulin G HRP-conjugated secondary antibody (1:3000; Santa Cruz Biotechnology) for 1.5 h at the room temperature. In addition, 10–12 μg proteins of each cytosol fraction were electrophoresed 10%–14% sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) for immunodetection of p-IκBα/cyclooxygenase-2 (COX-2)/inducible nitric oxide synthase (iNOS)/TNF-α/IL-6/IL-1β/heme oxygenase-1 (HO-1)/MMP-1/tissue inhibitor of metalloproteinase-1 (TIMP-1)/β-actin (1:1000; Santa Cruz, USA). Each antigen-antibody complex was visualized using ECL Western blotting detection reagents and detected by chemiluminescence with Sensi-Q 2000 Chemidoc (Lugen Sci Co., Ltd., Gyeonggi-do, Korea). Band densities were measured using ATTO Densitograph Software (ATTO Corporation, Tokyo, Japan) and quantified as the ratio to histone or β-actin. The protein levels of the groups are expressed relative to those of the normal rat (represented as 1).

Statistical analysis

Data are expressed as mean ± standard error of the mean Statistical comparisons were assessed by one-way ANOVA followed by least-significant differences test (SPSS 22.0 for Windows, SPSS Inc., NY, USA). Values of P < 0.05 were considered significant.


   Results Top


Body weight changes

[Figure 2] shows the change in body weights during the experimental period. The groups, which were injected with MIA, displayed a lower body weight compared with normal group (P < 0.05). But there is no significance between each groups because of short experimental period.
Figure 2: Body weight changes for 2 weeks. Values are the mean ± standard error of the mean (n = 8). Normal; Normal rats, Control; MIA-induced osteoarthritis rats, Indo; monosodium iodoacetate -induced osteoarthritis rats administrated with indomethacin 5 mg/kg body weight, Angelica gigas Nakai; monosodium iodoacetate -induced osteoarthritis rats administrated with Angelica gigas Nakai 200 mg/kg body weight. Significance: #P < 0.05 versus normal rat values

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Change hindpaw weight-bearing distribution

Hindpaw weight bearing distribution (HWBD), as a measure of OA progression, was measured as the difference between MIA-injected and contralateral hind limbs. The pain of knee joint leads to increase of HWBD, which has been proved for several study.[25] The HWBD on 0, 7 and 14 days is summarized in [Figure 3]. On day 0, MIA-treated group and AGN-treated group showed higher HWBD compared with normal group (P < 0.001). After 2 weeks, the increased HWBD was decreased at MIA-treated all experimental groups. Especially, when AGN-treated group improved significantly compared with MIA control group (P < 0.001). These results demonstrate a balance and relief of joint discomfort in the AGN-treated group.
Figure 3: Change in hind paw weight distribution. Values are the mean ± standard error of the mean (n = 8). Normal; normal rats, control; monosodium iodoacetate-induced osteoarthritis rats, Indo; monosodium iodoacetate -induced osteoarthritis rats administrated with indomethacin 5 mg/kg body weight, Angelica gigas Nakai; monosodium iodoacetate -induced osteoarthritis rats administrated with Angelica gigas Nakai 200 mg/kg body weight. Significance: ###P < 0.001 versus Normal rat values and ***P < 0.001 versus monosodium iodoacetate control rat values

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Tumor necrosis factor-α, interleukin-6, and interleukin-1β in cartilaginous tissue

[Figure 4] displayed the effect of AGN on the expression of inflammation-related cytokines, such as TNF-α, IL-6, and IL-1β. The MIA treatment significantly augmented the expressions of these cytokines (P < 0.05, P < 0.001). On the other hand, those expressions in Indo and AGN-treated groups were significantly decreased (P < 0.05, P < 0.01).
Figure 4: Tumor necrosis factor-α, interleukin-6 and interleukin-1β protein expressions in cartilaginous tissue. (a) Tumor necrosis factor-α, (b) interleukin-6 and (c) interleukin-1β. Values are the mean ± standard error of the mean (n = 8). Normal; normal rats, Control; monosodium iodoacetate induced osteoarthritis rats, Indo; monosodium iodoacetate -induced osteoarthritis rats administrated with indomethacin 5 mg/kg body weight, Angelica gigas Nakai; monosodium iodoacetate -induced osteoarthritis rats administrated with Angelica gigas Nakai 200 mg/kg body weight. Significance: #P < 0.05, ###P < 0.001 versus Normal rat values and *P < 0.05, **P < 0.01 versus monosodium iodoacetate control rat values

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Matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in cartilaginous tissue

Western bolt revealed that MIA treatment increased MMP-1 protein expression (P < 0.01). By contrast, TIMP-1 protein expression decreased (P < 0.05). On the other hand, AGN decreases MMP and increases TIMP-1 expression levels in cartilaginous tissue (P < 0.05, P < 0.01) [Figure 5].
Figure 5: Matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 protein expressions in cartilaginous tissue. (a) Matrix metalloproteinase-1 and (b) tissue inhibitor of metalloproteinase-1. Values are the mean ± standard error of the mean (n = 8). Normal; normal rats, Control; monosodium iodoacetate-induced osteoarthritis rats, Indo; monosodium iodoacetate -induced osteoarthritis rats administrated with indomethacin 5 mg/kg body weight, Angelica gigas Nakai; monosodium iodoacetate -induced osteoarthritis rats administrated with Angelica gigas Nakai 200 mg/kg body weight. Significance: #P < 0.05, ##P < 0.01 versus normal rat values and *P < 0.05, **P < 0.01 versus monosodium iodoacetate control rat values

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p-IκBα and nuclear factor kappa B p65 in cartilaginous tissue

NF-κB activity of nuclear fraction and p-IκBα of cytosol fraction from cartilaginous tissue were analyzed by western blotting. [Figure 6] indicated the effect of AGN. As expected, a significant elevation of NF-κB and p-IκBα activity were observed in MIA control group (P < 0.05, P < 0.001). Both NF-κB and p-IκBα activity were inhibited by AGN (P < 0.05, P < 0.01).
Figure 6: p-IκBα and nuclear factor kappa B p65 protein expressions in cartilaginous tissue. (a) p-IκBα and (b) nuclear factor kappa B p65. Values are the mean ± standard error of the mean (n = 8). Normal; normal rats, Control; monosodium iodoacetate-induced osteoarthritis rats, Indo; monosodium iodoacetate -induced osteoarthritis rats administrated with indomethacin 5 mg/kg body weight, Angelica gigas Nakai; monosodium iodoacetate -induced osteoarthritis rats administrated with Angelica gigas Nakai 200 mg/kg body weight. Significance: #P < 0.05, ###P < 0.001 versus normal rat values and *P < 0.05, **P < 0.01 versus monosodium iodoacetate control rat values

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Inducible nitric oxide synthase and cyclooxygenase-2 in cartilaginous tissue

[Figure 7] displayed the effect of AGN on the expression of inflammation-related mediators such as iNOS and COX-2. The MIA treatment significantly augmented the expressions of iNOS and COX-2 (P < 0.01). On the other hand, iNOS expression shows a decreasing tendency without a significance. Besides, COX-2 expression in AGN-treated group was significantly decreased (P < 0.05).
Figure 7: Inducible nitric oxide synthase and cyclooxygenase-2 protein expressions in cartilaginous tissue. (a) Inducible nitric oxide synthase and (b) cyclooxygenase-2 Values are the mean ± standard error of the mean (n = 8). Normal; normal rats, Control; monosodium iodoacetate -induced osteoarthritis rats, Indo; monosodium iodoacetate -induced osteoarthritis rats administrated with indomethacin 5 mg/kg body weight, Angelica gigas Nakai; monosodium iodoacetate -induced osteoarthritis rats administrated with Angelica gigas Nakai 200 mg/kg body weight. Significance: ##P < 0.01 versus normal rat values and *P < 0.05, **P < 0.01 versus monosodium iodoacetate control rat values

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ROS and ONOO in cartilaginous tissue

[Figure 8] shows the effect of AGN on ROS and ONOO level in cartilaginous tissue. The ROS level of MIA control group was significantly elevated to 197141 fluorescence/min/mg in comparison with that of normal group, at 94644 fluorescence/min/mg [Figure 3]a. On the other hand, it was significantly ameliorated by the administration of AGN, at 86737 fluorescence/min/mg (P < 0.01). The level of ONOO was also increased by MIA treatment, whereas it was markedly reduced by AGN administration [[Figure 3]b, P < 0.01].
Figure 8: Effect of Angelica gigas Nakai on monosodium iodoacetate induced ROS and ONOO formation in cartilaginous tissue. (a) ROS and (b) ONOO. Values are the mean ± standard error of the mean (n = 8). Normal; normal rats, Control; monosodium iodoacetate -induced osteoarthritis rats, Indo; monosodium iodoacetate -induced osteoarthritis rats administrated with indomethacin 5 mg/kg body weight, Angelica gigas Nakai; monosodium iodoacetate -induced osteoarthritis rats administrated with Angelica gigas Nakai 200 mg/kg body weight. Significance: #P < 0.05, ###P < 0.001 versus normal rat values and **P < 0.01 versus monosodium iodoacetate control rat values

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Nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway in cartilaginous tissue

As shown in [Figure 9], the Nrf2 protein expression was decreased in MIA control group as compared with normal group, whereas AGN treatment significantly upregulated (P < 0.05). Moreover, decreased HO-1 level by MIA treatment was significantly increased by AGN administration (P < 0.05, P < 0.01).
Figure 9: Nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 protein expressions in cartilaginous tissue. (a) Nuclear factor erythroid 2-related factor 2 and (b) Heme oxygenase-1. Values are the mean ± standard error of the mean (n = 8). Normal; Normal rats, Control; monosodium iodoacetate -induced osteoarthritis rats, Indo; monosodium iodoacetate -induced osteoarthritis rats administrated with indomethacin 5 mg/kg body weight, Angelica gigas Nakai; monosodium iodoacetate -induced osteoarthritis rats administrated with Angelica gigas Nakai 200 mg/kg body weight. Significance: #P < 0.05, ##P < 0.01 versus normal rat values and *P < 0.05 versus monosodium iodoacetate control rat values

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   Discussion Top


AGN as herbal medicine has long been utilized and contains bioactive substances such as decursin (D), decursinol angelate (DA), nodakenin, demethylsuberosine, and decursinol (DOH). It is a well-studied pyranocoumarins with various pharmacological activities in cancer and inflammation.[26],[27] Among pyranocoumarins, major compounds are D and DA. In addition, they are abundantly present in AGN.[28],[29] After, the dried herb AGN (300 g) was extracted with distilled water (3 L) for 2 h, and it was named AGN. Our HPLC analysis also revealed a similar result [Figure 1]. AGN including these effective compounds may exert potential protective effects against OA. OA, which is characterized by chronic and degenerative joint disease, is the most common and prevalent type of arthritis.[30] The typical progression of OA causes pain and a substantial disability. The standard drugs available for the management of OA, such as NSAIDs, intraarticular corticosteroids, and cartilage protective agents can only relieve the clinical symptoms.[3] Another field of recent research is the efficacy of TNF-α blockers but anti-TNF-α agents are too expensive and also have various side effects.[31] So, new therapeutic approaches are needed. The present study was designed to explore the protective effect and possible underlying mechanism of AGN on a MIA-induced model of OA. The intra-articular injection of MIA, the chondrocyte glycolysis inhibitor is the common way to induce the OA chemically.[25],[32] Specifically, we observed changes in HWBD and levels of antioxidant and inflammatory factors in serum or cartilaginous tissue. Both inflammatory factors such as cytokines and mediators have been implicated in OA pathogenesis.[33]

Many studies have been demonstrated inflammatory cytokines included IL-1 β, IL-6, and TNF-α are critical roles in OA pathogenesis,[34],[35] which is intimately related to degradation of the ECM. Higher levels of these cytokines in the initiation and progression of articular cartilage destruction have been shown to correlate with pain and physical function of patients with OA.[36],[37],[38] In this study, the authors measured the IL-1 β, IL-6, and TNF-α expression levels within cartilage tissues in the MIA-induced OA model. The AGN treatment group showed a reduction of three cytokines production, especially, IL-6, inducing the production of acute-phase response proteins, reduced significantly. Pro-inflammatory cytokines are believed to cause cartilage damage including ECM degradation and several enzymes play important roles in this course; of these enzymes, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) have been diversely studied.[39] MMPs have been reported to degrade ECM alone or in combination with factors to facilitate the migration of inflammatory cells, whereas TIMPs inhibit MMP-induced ECM degradation to maintain normal homeostasis.[40] Therefore, excessive activation of MMPs increases tissue degradation. Hence, the balance between MMPs and TIMPs has to be well regulated to optimize ECM remodeling.[11] Our results in this study revealed that AGN suppresses the expression of MMP-1 and elevates TIMP-1, on MIA-induced OA.

A crucial cytokine-induced initiation of transcription is seen in the NF-κB signaling pathway. Inactivated NF-κB pathway, phosphorylation of IKKα/β and NF-κBp65 mediates the regulation of promoter activity of both anabolic and catabolic genes. NF-κB can influence the expression of inflammatory mediators such as iNOS and COX-2.[41] COX is a key pro-inflammatory enzyme that converts arachidonic acid into prostaglandins. Inhibition of NF-κB activation is associated with the down-regulation of COX-2 expression and synthesis. Moreover, the excessive iNOS induces the overproduction of NO. NO generates ONOO through reacting with O2- and this can accelerate OA.[42] The results of the present study show that AGN promotes the suppression of NF-κB activation in cartilaginous tissue. As the result, the elevated expressions of COX-2 and iNOS were markedly attenuated by AGN treatment.

Oxidative stress (OS) is correlated with a reduction of antioxidant defenses and an increase in the production of ROS. Several studies have reported antioxidant activities of AGN.[13],[43] Through this property, AGN might also protect against tissue damage in a cartilaginous tissue by regulating free radicals/ROS. The Nrf2 antioxidant pathway has been suggested as an important therapeutic target against OS. HO-1, a stress-responsive protein shortly activated by OA, has been known as the cytoprotective effect resistant to OA.[44] Herein, ROS were suppressed in AGN-treated group. In addition, our results revealed that AGN enhanced the ability of Nrf2 to migrate into the nucleus and activated the up-regulation of HO-1 expression through stimulation of Nrf2. As the result, AGN protected cartilaginous tissue from OS.


   Conclusion Top


Taken together, it suggests that AGN could suppress excessive production of ROS and elevate antioxidant effect through Nrf2/HO-1 pathway. Moreover, AGN could inhibit the expression of inflammation factors by NF-κB inactivation. As the result, AGN treatment may have the potential therapeutic ability in the treatment of OA. However, the complete molecular mechanism of AGN has not been fully elucidated. Further research on AGN in humans is required before treatments after that AGN can be used safely in the clinic.

Financial support and sponsorship

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2018R1A5A2025272) and '2019 Development of Technology on Bio MediFood Using Medicinal Crop' funded by Gyeongsangbuk-do and Gyeongsan-si.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]



 

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