Journal of Acupuncture Research 2023; 40(1): 35-43
Published online February 28, 2023
https://doi.org/10.13045/jar.2022.00269
© Korean Acupuncture & Moxibustion Medicine Society
Correspondence to : Ho-Sueb Song
Department of Acupuncture & Moxibustion Medicine, College of Korean Medicine, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam 13120, Korea
E-mail: hssong70@gachon.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: This study aimed to examine the effect of Scutellaria baicalensis extract (SBE) on ameliorating pain response and inflammation in an animal model.
Methods: The effects of SBE on joint inflammation-induced rats and pain writhing response were measured. In rats with monosodium iodoacetate (MIA)-induced knee osteoarthritis (OA), the weight-bearing distribution of the hind legs was measured, the actual joint condition was visually confirmed, and serum cytokines were extracted from whole blood and measured. In addition, the acetic acid-induced pain was measured by the number of abdominal wall contractions and writhing responses.
Results: 1. The weight-bearing distribution of the hind limbs of the SBE group was remarkably improved compared with that of the control group 7 days after MIA treatment, and the SBE 300 group was improved similarly to that of the indomethacin group. 2. Cartilage erosion was significantly recovered in the SBE and indomethacin groups, and the degree of healing of cartilage erosion by SBE was similar to that by indomethacin. 3. The serum levels of cytokines interleukin-1β, tumor necrosis factor-α, and interleukin-6 were significantly decreased in the SBE group compared with that in the control group, and the SBE 300 group had reduced levels of cytokines similar to the indomethacin group. 4. As regards acetic acid-induced writhing response, the number of writhes was significantly reduced in the SBE and ibuprofen groups, and the SBE 600 group had fewer writhes than the ibuprofen group.
Conclusion: SBE significantly improves knee OA and pain and is expected to show similar therapeutic effects to indomethacin and ibuprofen.
Keywords Baicalein; Inflammation; Knee osteoarthritis; Pain; Scutellaria baicalensis extract
Knee osteoarthritis (OA) is the most representative chronic joint disease and causes more dysfunctions and dependence than any other disease in older people [1]. The knee joint has high mobility and is the biggest synovial joint in the human body; however, it has an unstable structure and mainly undergoes degenerative changes under direct weight [2]. Knee OA refers to histological changes in soft tissues and joints, such as the overproduction of cartilage around and within joints, destruction of articular cartilage, and deformation of joints [3], causing inflammation, pain, swelling, and shortening, and stiffness of muscles. These cause movement disorders and lower the quality of life [4].
Knee OA is diagnosed based on clinical manifestation or a combination of radiology and clinical findings. OA has several classification systems. The European League Against Rheumatism recommends diagnosing knee OA if three symptoms (reduced function, persistent pain, and limited morning stiffness) and three signals (crepitus, restricted range of motion, and bony enlargement) are present [5]. The higher the number of elements present, the more likely that the diagnosis is OA, and when all six symptoms and signals are present, OA is confirmed in radiographs at 99% [6]. In addition, one of the most frequently used clinical categorization criteria was established by the American College of Rheumatology, in which OA diagnosis is based on laboratory criteria, clinical symptoms, and radiographs [7].
As a non-surgical treatment, nonsteroidal anti-inflammatory drugs (NSAIDs) are generally used for the medical care of knee arthritis, and a recent meta-analysis reported that diclofenac 150 mg/day is the most helpful NSAID available to improve both pain and function [8]. However, the excessive use of these drugs may cause cardiovascular and gastrointestinal disorders, requiring caution [9]. Although intra-articular knee injection provides potential pain reduction and function improvement and has a low risk of injury, long-term use is limited, and no studies have proved the objective effect [5].
The root of
Chromatographic analysis of SBE was performed by High-Performance Liquid Chromatography (HPLC) using a 1100 series HPLC system (Agilent, Santa Clara, CA, USA). A Zorbax EclipseXDB C18 column (4.6 × 250 mm, 5 µm; Agilent) at 30℃ was used for chromatic separation. A 1-mg sample was diluted in 10 mL of 50% methanol and sonicated for 10 minutes. Samples were filtered through a 0.45-μm syringe filter (Waters Corp., Milford, MA, USA). The mobile phase composition was 0.1% formic acid (A) and acetonitrile (B), and the column was eluted as follows: 0–3 minutes, 20%; 3–15 minutes, 20–45%; 15–20 minutes, 45–60%; 20–22 minutes, 60% solvent B with a flow rate of 1.0 mL/min. The outflow was indicated at 276 nm using an injection volume of 10 μL. The analysis was performed in triplicate.
Five-week-old male Sprague-Dawley (SD) rats and 6-week-old male ICR mice were provided by DBL Co., Ltd. (Eumseong, Korea). The animals were acclimatized to standard laboratory conditions (22 ± 2℃, 55 ± 10% humidity, and 12 hours light/dark cycle) for at least 7 days before the experiment. The animals were given feed and water freely. All experimental procedures described above were approved by the Gachon University Center of Animal Care and Use (GIACUC-R2020028).
SD rats were separated into five groups (n = 9 per group), namely, sham, control, indomethacin, SBE 100 mg/kg, and SBE 300 mg/kg groups. After being anesthetized using 2% isofluorane with O2 mixture, the rats were injected with 50 μL of 40 mg/mL Monosodium iodoacetate (MIA) (Sigma-Aldrich, St. Louis, MO, USA) intra-articularly into the knee joints to induce OA. The sham and control groups were provided basic diet (American Institute of Nutrition [AIN]-93G), the indomethacin group were provided AIN-93G diet with 0.003% indomethacin (final dose: 3 mg/kg), and each SBE group was provided AIN-93G diet with 0.11% and 0.33% SBE (final dose: 100 and 300 mg/kg). After the induction of OA by MIA, a diet of 18 g per 200 g body weight was provided every day for 24 days.
After inducing OA in SD rats, the weight-bearing of the hind limb was recorded on days 0, 3, 7, 10, 14, 17, 21, and 24 using an incapacitance Meter Tester 600 (IITC Life Science Inc., Woodland Hills, CA, USA), and the strength applied to each limb was averaged over 10 seconds. The percentage of weight distributed in the hind limb of the treated side (right) was calculated using the following equation: weight-bearing ratio (%) = (weight on the right hind limb / weight on the left and right hind limbs) × 100.
Whole blood was drawn from the abdominal vein and left for 30 minutes to clot. The samples were centrifuged for 10 minutes at 4,000 rpm, and the separated serum was stored at −70℃. The multiplex assay was conducted with interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α using the Premixed Multi-Analyte Kit (R&D Systems Inc., Minneapolis, MN, USA) for measurements of cytokines in the serum, and the results were analyzed with the Luminex MAGPIX Analyzer (Luminex Corp., Austin, TX, USA). The experimental procedure of all multiplex assays was performed according to the manufacturer’s protocols.
ICR mice were isolated into four groups (n = 8 per group) and administered with SBE (200 and 600 mg/kg), distilled water (control), and ibuprofen 200 mg/kg (Sigma-Aldrich). Thirty minutes after oral administration, 0.7% acetic acid was injected intraperitoneally at 10 mL/kg, and writhing responses were recorded 10 minutes later. A writhing response was composed of an abdominal wall contraction and a pelvic turn following the swelling of the hind limbs.
Statistical analysis was conducted using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) with one-way analysis of variance and Dunnett’s post hoc test. The significance was verified at
In this study, baicalein was obtained, and measured in the SBE with the HPLC-UV method. The SBE contained 0.37 mg/g of baicalein. An HPLC chromatogram of the analysis along with the chemical structure of the component is shown in Fig. 1.
The hind limb’s weight-bearing is an indicator of discomfort, and joint pain is generally used in animal models to evaluate the analgesic effects of herbal medicine in OA. The weight-bearing ratio between the right and left limbs was measured 24 days after OA induction. As shown in Fig. 2A, the weight-bearing distribution in the sham group was unchanged. However, in the control (MIA) group, it remarkably decreased on day 7 and was gradually lower than that in the sham group. In the SBE group, the weight-bearing of MIA rats notably increased. Specifically, the weight-bearing increase by SBE 300 was equivalent to that by indomethacin (Fig. 2B).
The typical image of the knee joints in each experimental group shows that SBE inhibited the cartilage damage induced by MIA. As illustrated in Fig. 3, the joint cartilage of the sham group was shiny and smooth, whereas the cartilage of the control group was less gleamed and irregular with damages in several areas. The MIA-induced cartilage erosion was remarkably recovered in the indomethacin and SBE groups. Remarkably, the recovery of cartilage erosion in the SBE group was similar to that in the indomethacin group.
The levels of TNF-α, IL-1β, and IL-6 in the rat serum were recorded after isolating the serum from blood samples collected from each experimental group. Compared with the control group, the SBE group demonstrated a notable dose-dependent decrease in the serum concentrations of TNF-α, IL-1β, and IL-6. Intriguingly, the SBE 300 group had reduced levels of cytokine close to that of the indomethacin group (Fig. 4).
The pain-reducing effects of SBE were evaluated by the writhing responses of acetic acid-induced mice. The average number of writhes in the control group for 10 minutes was set to 100. SBE decreased the number of writhes compared with the control. The SBE 600 group had a mean number of writhes of 43.86, which was lower than that of the ibuprofen group (49.65). This result suggests the pain-reducing effects of SBE (Fig. 5).
Knee OA management aims to reduce pain, improve function, and significantly improve joint damage. The primary goal of the clinician should be the reduction of risk factors [14]. Because the effectiveness of current therapy is negligible, combination therapy is commonly used for treatment, and safer treatments should be prioritized before considering potentially harmful opioids or NSAIDs. In addition to benefits, adverse event profiles, patient-specific disorders and preferences, intervention local availability, and budgets should be considered [15].
Non-pharmacological methods are the main treatment for knee OA management and should be tried before other treatments and, when necessary, with medications to relieve pain. Non-pharmacological treatments include weight management, exercise, education, and, if necessary, the use of assistive devices [16].
In the initial stage of drug treatment, additional drugs are used in patients who have not responded appropriately to non-pharmaceutical measures or whose symptoms have worsened. Pharmacological treatment should only be used during the symptomatic period, as no drug can improve OA itself. The main drugs used for knee OA include oral and topical NSAIDs, with limited use of duloxetine, topical capsaicin, and intra-articular glucocorticoids [17].
Total joint replacement is the most common surgical treatment, which is very successful in patients with knee OA when conservative treatment and drug therapy fail to provide appropriate pain relief [18]. However, a systematic review of 14 studies found that 9–20% of patients undergoing total hip and knee replacement reported moderate-to-severe long-term pain after surgery [19]. In addition, the patient may experience serious complications, especially in younger patients; thus, reoperation may be necessary [20].
Currently, treatments used in OA in traditional Korean medicine include herbal medicine, acupuncture, moxibustion, and pharmacopuncture. The recently used components of pharmacopuncture include Chinemys reevesii Gary, Cnidium officinale, Angelica, Salvia miltiorrhiza, Homnis placenta, sweet bee venom, etc. [21]. Especially, sweet bee venom acupuncture showed statistically significant results in reducing knee joint pain and improving function in randomized controlled trials [22]. Corni fructus, Achyranthis bidentatae radix, Aconiti radix, Curcuma longa, and Vernonia amygdalina, which are currently reported as the latest traditional Korean medicine treatments for OA, can suppress inflammatory stimulation, and reduce pain symptoms in OA. In addition, traditional Korean medicine treatment restores the function of the damaged joint, significantly increases the treatment effectiveness, and significantly reduces the recurrence rate [23-25].
In this study, OA, and pain were induced experimentally using MIA and acetic acid. OA was evaluated through the hind limb’s weight-bearing distribution; levels of inflammatory cytokines TNF-α, IL-1β, and IL-6; and actual cartilage dissection, and pain was evaluated through writhing response. Moreover, whether SBE reduces inflammation, pain, and discomfort in the joints was investigated. In addition, the effect of SBE was compared with those of indomethacin and ibuprofen, which are traditionally used drugs, and the difference according to the concentration of SBE was also investigated.
In the MIA-treated group, the right weight-bearing was significantly reduced compared with that of the Siamese group. In the drug and control groups, it decreased similarly until day 3. From day 7, the indomethacin group showed a significant increase in weight-bearing compared with the control group, and in the SBE group, the weight-bearing increased significantly from day 10. On day 24, the last day of evaluation, all experimental groups showed significantly improved results (
As a result of joint dissection, the sham group showed normal results; however, the MIA-treated group was precisely less shiny and irregular with damage in some areas. The MIA-induced erosion was notably recovered in the SBE and indomethacin groups. Remarkably, the healing of cartilage erosion by SBE 300 resembled that by indomethacin. As a result, SBE was found to help repair cartilage damage in arthritis.
Cytokines in the serum were extracted from the abdominal vein, left for 30 minutes, and then centrifuged at 4,000 rpm for 10 minutes. The SBE group showed a significant reduction in serum TNF-α, IL-1β, and IL-6 concentrations in a dose-dependent manner compared with the control group (IL-1β,
To confirm the analgesic effect of SBE, acetic acid-induced writhes were assessed. The analgesic effect of SBE was confirmed by comparing the number of writhes. In the control group, the average number of writhes for 10 minutes was 100. The number of writhes was remarkably decreased in the SBE group compared with that in the control group (
This study was conducted to confirm whether SBE improves OA and pain. Before drug administration, HPLC chromatogram was performed using baicalein as a major component in SBE as an index. SBE contained 0.37 mg/g of baicalein. In previous studies, SBE-related studies have focused on baicalin, baicalein, and wogonin, and among them, baicalein was the most studied. Baicalein has been reported to have antibacterial, antiviral, and antioxidant effects and inhibit inflammatory cytokines [11,13,14,26-29]. Therefore, the present study agrees with previous studies that have investigated whether inflammatory cytokines were suppressed. In addition, the present study investigated the recovery of OA-induced dysfunction and damaged cartilage. The results indicated that SBE inhibited inflammatory cytokines, similar to results of other studies, rehabilitate motor dysfunction caused by OA, and improved damaged cartilage. This is considered the effect of baicalein.
In conclusion, SBE improved pain, and discomfort in an animal model and showed anti-inflammatory effects similar to indomethacin by inhibiting inflammatory cytokines. In addition, it aided in the recovery of damaged joints and had a similar effect to ibuprofen on pain. Therefore, SBE shows potential as a new alternative to the treatment of knee OA and pain.
The SBE group had significantly improved hind-limb weight-bearing distribution in the MIA-induced knee OA group compared with the control group. As for the degree of recovery of knee cartilage damage, cartilage erosion was significantly recovered in the SBE and indomethacin groups, and the degree of healing of cartilage erosion by SBE was similar to that by indomethacin. The serum levels of cytokines TNF-α, IL-1β, and IL-6 dose-dependently decreased in the SBE group compared with those in the control group. The SBE 300 group had reduced levels of cytokines similar to the indomethacin group. In the acetic acid-induced writhing test, the number of writhes was significantly reduced in the SBE and ibuprofen groups, and the SBE 600 group had fewer writhes than the ibuprofen group. Therefore, SBE significantly improves knee OA and pain and is expected to show similar therapeutic effects to indomethacin and ibuprofen.
Conceptualization: HS, HSS. Data curation: HS, HSS. Formal analysis: HS, HSS. Investigation: HS, HSS. Methodology: HS, HSS. Project administration: HS, HSS. Resources: HS, HSS. Software: HS, HSS. Supervision: HS, HSS. Validation: HS, HSS. Visualization: HS, HSS. Writing – original draft: HS, HSS. Writing – review & editing: All authors.
The authors have no conflicts of interest to declare.
This study was approved by the Gachon University Center of Animal Care and Use (GIACUC-R2020028).
Journal of Acupuncture Research 2023; 40(1): 35-43
Published online February 28, 2023 https://doi.org/10.13045/jar.2022.00269
Copyright © Korean Acupuncture & Moxibustion Medicine Society.
Department of Acupuncture & Moxibustion Medicine, College of Korean Medicine, Gachon University, Seongnam, Korea
Correspondence to:Ho-Sueb Song
Department of Acupuncture & Moxibustion Medicine, College of Korean Medicine, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam 13120, Korea
E-mail: hssong70@gachon.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: This study aimed to examine the effect of Scutellaria baicalensis extract (SBE) on ameliorating pain response and inflammation in an animal model.
Methods: The effects of SBE on joint inflammation-induced rats and pain writhing response were measured. In rats with monosodium iodoacetate (MIA)-induced knee osteoarthritis (OA), the weight-bearing distribution of the hind legs was measured, the actual joint condition was visually confirmed, and serum cytokines were extracted from whole blood and measured. In addition, the acetic acid-induced pain was measured by the number of abdominal wall contractions and writhing responses.
Results: 1. The weight-bearing distribution of the hind limbs of the SBE group was remarkably improved compared with that of the control group 7 days after MIA treatment, and the SBE 300 group was improved similarly to that of the indomethacin group. 2. Cartilage erosion was significantly recovered in the SBE and indomethacin groups, and the degree of healing of cartilage erosion by SBE was similar to that by indomethacin. 3. The serum levels of cytokines interleukin-1β, tumor necrosis factor-α, and interleukin-6 were significantly decreased in the SBE group compared with that in the control group, and the SBE 300 group had reduced levels of cytokines similar to the indomethacin group. 4. As regards acetic acid-induced writhing response, the number of writhes was significantly reduced in the SBE and ibuprofen groups, and the SBE 600 group had fewer writhes than the ibuprofen group.
Conclusion: SBE significantly improves knee OA and pain and is expected to show similar therapeutic effects to indomethacin and ibuprofen.
Keywords: Baicalein, Inflammation, Knee osteoarthritis, Pain, Scutellaria baicalensis extract
Knee osteoarthritis (OA) is the most representative chronic joint disease and causes more dysfunctions and dependence than any other disease in older people [1]. The knee joint has high mobility and is the biggest synovial joint in the human body; however, it has an unstable structure and mainly undergoes degenerative changes under direct weight [2]. Knee OA refers to histological changes in soft tissues and joints, such as the overproduction of cartilage around and within joints, destruction of articular cartilage, and deformation of joints [3], causing inflammation, pain, swelling, and shortening, and stiffness of muscles. These cause movement disorders and lower the quality of life [4].
Knee OA is diagnosed based on clinical manifestation or a combination of radiology and clinical findings. OA has several classification systems. The European League Against Rheumatism recommends diagnosing knee OA if three symptoms (reduced function, persistent pain, and limited morning stiffness) and three signals (crepitus, restricted range of motion, and bony enlargement) are present [5]. The higher the number of elements present, the more likely that the diagnosis is OA, and when all six symptoms and signals are present, OA is confirmed in radiographs at 99% [6]. In addition, one of the most frequently used clinical categorization criteria was established by the American College of Rheumatology, in which OA diagnosis is based on laboratory criteria, clinical symptoms, and radiographs [7].
As a non-surgical treatment, nonsteroidal anti-inflammatory drugs (NSAIDs) are generally used for the medical care of knee arthritis, and a recent meta-analysis reported that diclofenac 150 mg/day is the most helpful NSAID available to improve both pain and function [8]. However, the excessive use of these drugs may cause cardiovascular and gastrointestinal disorders, requiring caution [9]. Although intra-articular knee injection provides potential pain reduction and function improvement and has a low risk of injury, long-term use is limited, and no studies have proved the objective effect [5].
The root of
Chromatographic analysis of SBE was performed by High-Performance Liquid Chromatography (HPLC) using a 1100 series HPLC system (Agilent, Santa Clara, CA, USA). A Zorbax EclipseXDB C18 column (4.6 × 250 mm, 5 µm; Agilent) at 30℃ was used for chromatic separation. A 1-mg sample was diluted in 10 mL of 50% methanol and sonicated for 10 minutes. Samples were filtered through a 0.45-μm syringe filter (Waters Corp., Milford, MA, USA). The mobile phase composition was 0.1% formic acid (A) and acetonitrile (B), and the column was eluted as follows: 0–3 minutes, 20%; 3–15 minutes, 20–45%; 15–20 minutes, 45–60%; 20–22 minutes, 60% solvent B with a flow rate of 1.0 mL/min. The outflow was indicated at 276 nm using an injection volume of 10 μL. The analysis was performed in triplicate.
Five-week-old male Sprague-Dawley (SD) rats and 6-week-old male ICR mice were provided by DBL Co., Ltd. (Eumseong, Korea). The animals were acclimatized to standard laboratory conditions (22 ± 2℃, 55 ± 10% humidity, and 12 hours light/dark cycle) for at least 7 days before the experiment. The animals were given feed and water freely. All experimental procedures described above were approved by the Gachon University Center of Animal Care and Use (GIACUC-R2020028).
SD rats were separated into five groups (n = 9 per group), namely, sham, control, indomethacin, SBE 100 mg/kg, and SBE 300 mg/kg groups. After being anesthetized using 2% isofluorane with O2 mixture, the rats were injected with 50 μL of 40 mg/mL Monosodium iodoacetate (MIA) (Sigma-Aldrich, St. Louis, MO, USA) intra-articularly into the knee joints to induce OA. The sham and control groups were provided basic diet (American Institute of Nutrition [AIN]-93G), the indomethacin group were provided AIN-93G diet with 0.003% indomethacin (final dose: 3 mg/kg), and each SBE group was provided AIN-93G diet with 0.11% and 0.33% SBE (final dose: 100 and 300 mg/kg). After the induction of OA by MIA, a diet of 18 g per 200 g body weight was provided every day for 24 days.
After inducing OA in SD rats, the weight-bearing of the hind limb was recorded on days 0, 3, 7, 10, 14, 17, 21, and 24 using an incapacitance Meter Tester 600 (IITC Life Science Inc., Woodland Hills, CA, USA), and the strength applied to each limb was averaged over 10 seconds. The percentage of weight distributed in the hind limb of the treated side (right) was calculated using the following equation: weight-bearing ratio (%) = (weight on the right hind limb / weight on the left and right hind limbs) × 100.
Whole blood was drawn from the abdominal vein and left for 30 minutes to clot. The samples were centrifuged for 10 minutes at 4,000 rpm, and the separated serum was stored at −70℃. The multiplex assay was conducted with interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α using the Premixed Multi-Analyte Kit (R&D Systems Inc., Minneapolis, MN, USA) for measurements of cytokines in the serum, and the results were analyzed with the Luminex MAGPIX Analyzer (Luminex Corp., Austin, TX, USA). The experimental procedure of all multiplex assays was performed according to the manufacturer’s protocols.
ICR mice were isolated into four groups (n = 8 per group) and administered with SBE (200 and 600 mg/kg), distilled water (control), and ibuprofen 200 mg/kg (Sigma-Aldrich). Thirty minutes after oral administration, 0.7% acetic acid was injected intraperitoneally at 10 mL/kg, and writhing responses were recorded 10 minutes later. A writhing response was composed of an abdominal wall contraction and a pelvic turn following the swelling of the hind limbs.
Statistical analysis was conducted using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) with one-way analysis of variance and Dunnett’s post hoc test. The significance was verified at
In this study, baicalein was obtained, and measured in the SBE with the HPLC-UV method. The SBE contained 0.37 mg/g of baicalein. An HPLC chromatogram of the analysis along with the chemical structure of the component is shown in Fig. 1.
The hind limb’s weight-bearing is an indicator of discomfort, and joint pain is generally used in animal models to evaluate the analgesic effects of herbal medicine in OA. The weight-bearing ratio between the right and left limbs was measured 24 days after OA induction. As shown in Fig. 2A, the weight-bearing distribution in the sham group was unchanged. However, in the control (MIA) group, it remarkably decreased on day 7 and was gradually lower than that in the sham group. In the SBE group, the weight-bearing of MIA rats notably increased. Specifically, the weight-bearing increase by SBE 300 was equivalent to that by indomethacin (Fig. 2B).
The typical image of the knee joints in each experimental group shows that SBE inhibited the cartilage damage induced by MIA. As illustrated in Fig. 3, the joint cartilage of the sham group was shiny and smooth, whereas the cartilage of the control group was less gleamed and irregular with damages in several areas. The MIA-induced cartilage erosion was remarkably recovered in the indomethacin and SBE groups. Remarkably, the recovery of cartilage erosion in the SBE group was similar to that in the indomethacin group.
The levels of TNF-α, IL-1β, and IL-6 in the rat serum were recorded after isolating the serum from blood samples collected from each experimental group. Compared with the control group, the SBE group demonstrated a notable dose-dependent decrease in the serum concentrations of TNF-α, IL-1β, and IL-6. Intriguingly, the SBE 300 group had reduced levels of cytokine close to that of the indomethacin group (Fig. 4).
The pain-reducing effects of SBE were evaluated by the writhing responses of acetic acid-induced mice. The average number of writhes in the control group for 10 minutes was set to 100. SBE decreased the number of writhes compared with the control. The SBE 600 group had a mean number of writhes of 43.86, which was lower than that of the ibuprofen group (49.65). This result suggests the pain-reducing effects of SBE (Fig. 5).
Knee OA management aims to reduce pain, improve function, and significantly improve joint damage. The primary goal of the clinician should be the reduction of risk factors [14]. Because the effectiveness of current therapy is negligible, combination therapy is commonly used for treatment, and safer treatments should be prioritized before considering potentially harmful opioids or NSAIDs. In addition to benefits, adverse event profiles, patient-specific disorders and preferences, intervention local availability, and budgets should be considered [15].
Non-pharmacological methods are the main treatment for knee OA management and should be tried before other treatments and, when necessary, with medications to relieve pain. Non-pharmacological treatments include weight management, exercise, education, and, if necessary, the use of assistive devices [16].
In the initial stage of drug treatment, additional drugs are used in patients who have not responded appropriately to non-pharmaceutical measures or whose symptoms have worsened. Pharmacological treatment should only be used during the symptomatic period, as no drug can improve OA itself. The main drugs used for knee OA include oral and topical NSAIDs, with limited use of duloxetine, topical capsaicin, and intra-articular glucocorticoids [17].
Total joint replacement is the most common surgical treatment, which is very successful in patients with knee OA when conservative treatment and drug therapy fail to provide appropriate pain relief [18]. However, a systematic review of 14 studies found that 9–20% of patients undergoing total hip and knee replacement reported moderate-to-severe long-term pain after surgery [19]. In addition, the patient may experience serious complications, especially in younger patients; thus, reoperation may be necessary [20].
Currently, treatments used in OA in traditional Korean medicine include herbal medicine, acupuncture, moxibustion, and pharmacopuncture. The recently used components of pharmacopuncture include Chinemys reevesii Gary, Cnidium officinale, Angelica, Salvia miltiorrhiza, Homnis placenta, sweet bee venom, etc. [21]. Especially, sweet bee venom acupuncture showed statistically significant results in reducing knee joint pain and improving function in randomized controlled trials [22]. Corni fructus, Achyranthis bidentatae radix, Aconiti radix, Curcuma longa, and Vernonia amygdalina, which are currently reported as the latest traditional Korean medicine treatments for OA, can suppress inflammatory stimulation, and reduce pain symptoms in OA. In addition, traditional Korean medicine treatment restores the function of the damaged joint, significantly increases the treatment effectiveness, and significantly reduces the recurrence rate [23-25].
In this study, OA, and pain were induced experimentally using MIA and acetic acid. OA was evaluated through the hind limb’s weight-bearing distribution; levels of inflammatory cytokines TNF-α, IL-1β, and IL-6; and actual cartilage dissection, and pain was evaluated through writhing response. Moreover, whether SBE reduces inflammation, pain, and discomfort in the joints was investigated. In addition, the effect of SBE was compared with those of indomethacin and ibuprofen, which are traditionally used drugs, and the difference according to the concentration of SBE was also investigated.
In the MIA-treated group, the right weight-bearing was significantly reduced compared with that of the Siamese group. In the drug and control groups, it decreased similarly until day 3. From day 7, the indomethacin group showed a significant increase in weight-bearing compared with the control group, and in the SBE group, the weight-bearing increased significantly from day 10. On day 24, the last day of evaluation, all experimental groups showed significantly improved results (
As a result of joint dissection, the sham group showed normal results; however, the MIA-treated group was precisely less shiny and irregular with damage in some areas. The MIA-induced erosion was notably recovered in the SBE and indomethacin groups. Remarkably, the healing of cartilage erosion by SBE 300 resembled that by indomethacin. As a result, SBE was found to help repair cartilage damage in arthritis.
Cytokines in the serum were extracted from the abdominal vein, left for 30 minutes, and then centrifuged at 4,000 rpm for 10 minutes. The SBE group showed a significant reduction in serum TNF-α, IL-1β, and IL-6 concentrations in a dose-dependent manner compared with the control group (IL-1β,
To confirm the analgesic effect of SBE, acetic acid-induced writhes were assessed. The analgesic effect of SBE was confirmed by comparing the number of writhes. In the control group, the average number of writhes for 10 minutes was 100. The number of writhes was remarkably decreased in the SBE group compared with that in the control group (
This study was conducted to confirm whether SBE improves OA and pain. Before drug administration, HPLC chromatogram was performed using baicalein as a major component in SBE as an index. SBE contained 0.37 mg/g of baicalein. In previous studies, SBE-related studies have focused on baicalin, baicalein, and wogonin, and among them, baicalein was the most studied. Baicalein has been reported to have antibacterial, antiviral, and antioxidant effects and inhibit inflammatory cytokines [11,13,14,26-29]. Therefore, the present study agrees with previous studies that have investigated whether inflammatory cytokines were suppressed. In addition, the present study investigated the recovery of OA-induced dysfunction and damaged cartilage. The results indicated that SBE inhibited inflammatory cytokines, similar to results of other studies, rehabilitate motor dysfunction caused by OA, and improved damaged cartilage. This is considered the effect of baicalein.
In conclusion, SBE improved pain, and discomfort in an animal model and showed anti-inflammatory effects similar to indomethacin by inhibiting inflammatory cytokines. In addition, it aided in the recovery of damaged joints and had a similar effect to ibuprofen on pain. Therefore, SBE shows potential as a new alternative to the treatment of knee OA and pain.
The SBE group had significantly improved hind-limb weight-bearing distribution in the MIA-induced knee OA group compared with the control group. As for the degree of recovery of knee cartilage damage, cartilage erosion was significantly recovered in the SBE and indomethacin groups, and the degree of healing of cartilage erosion by SBE was similar to that by indomethacin. The serum levels of cytokines TNF-α, IL-1β, and IL-6 dose-dependently decreased in the SBE group compared with those in the control group. The SBE 300 group had reduced levels of cytokines similar to the indomethacin group. In the acetic acid-induced writhing test, the number of writhes was significantly reduced in the SBE and ibuprofen groups, and the SBE 600 group had fewer writhes than the ibuprofen group. Therefore, SBE significantly improves knee OA and pain and is expected to show similar therapeutic effects to indomethacin and ibuprofen.
Conceptualization: HS, HSS. Data curation: HS, HSS. Formal analysis: HS, HSS. Investigation: HS, HSS. Methodology: HS, HSS. Project administration: HS, HSS. Resources: HS, HSS. Software: HS, HSS. Supervision: HS, HSS. Validation: HS, HSS. Visualization: HS, HSS. Writing – original draft: HS, HSS. Writing – review & editing: All authors.
The authors have no conflicts of interest to declare.
This study was approved by the Gachon University Center of Animal Care and Use (GIACUC-R2020028).
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