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Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis
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Osteoarthritis and Cartilage xxx (2015) 1e7
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Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis
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B. Bai y, Y. Li z * y Orthopedics Department, The First Affiliated Hospital, Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, China z School of Public Health, Health Science Center of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, China
a r t i c l e i n f o
s u m m a r y
Article history: Received 8 January 2015 Accepted 9 October 2015
Objective: To evaluate the efficacy of Danshen on histological parameters and antioxidative activity in the articular cartilage of rabbits with osteoarthritis (OA). Design: Twenty-four rabbits were randomly divided into three groups (control, OA, and Danshen OA; eight rabbits per group). Anterior cruciate ligament transection (ACLT) of the left hind knees was performed in all rabbits in the OA and Danshen OA group for induction of OA. The rabbits in the control group underwent a sham operation. After surgery, 3 g/kg body weight of Danshen granules dissolved in 5 mL distilled water was administered by gastric intubation once per day and over a 6-week period to the Danshen OA group. The same volume of distilled water was administered to the OA and control groups. After 6 weeks, the medial femoral condyles and synoviums of the left hind knees in all three groups were harvested and used for histological and biochemical analyses. Results: Severe articular cartilage degeneration as well as lower proteoglycan (PG) content were noted in the OA group compared to the Danshen OA group (P < 0.05). The glutathione (GSH) levels in the synovium and articular cartilage of the rabbits in the Danshen OA group were significantly higher compared to the OA group (P < 0.001). The malondialdehyde (MDA) levels of the synovium and articular cartilage in the Danshen OA group was markedly depleted compared to the OA group (P < 0.001). Conclusion: Danshen can prevent articular cartilage degeneration in OA though the defense against oxidative stress. © 2015 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.
Keywords: Danshen Osteoarthritis Articular cartilage Degeneration Oxidative stress
Introduction Osteoarthritis (OA) is the most common joint disease that leads to pain and disability. The prevalence of symptomatic OA in those 60 years old and above was 9.6% in men and 18% in women1. It will be the fourth leading cause of disability worldwide by 20202, resulting in a large worldwide socioeconomic burden. The etiology and pathogenesis of OA currently remains unclear. Nevertheless, increasing evidence from both experimental and clinical studies suggest that oxidative stress plays a pivotal role in the pathological process of OA3,4. Oxidative stress is generated from an imbalance in the production and elimination of reactive oxygen species (ROS)5. In
* Address correspondence and reprint requests to: Y. Li, School of Public Health, Health Science Center of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, China. Tel: 86-29-82655108. E-mail addresses: [email protected] (B. Bai), [email protected] (Y. Li).
order to maintain balance under physiological conditions, ROS are produced and removed in the human body by the cellular antioxidant defense system. If regulated improperly, the excess ROS can inhibit the activity of normal cells by damaging cellular lipid, protein, and DNA content4. Previous studies have demonstrated the critical role played by oxidative stress in directly promoting chondrocyte apoptosis, catabolic processes, and matrix degradation6. Furthermore, some studies suggest that the telomere shortening and reduced number and function of mitochondria seen in OA chondrocytes are due to oxidative stress4,7. According to these studies, the oxidative stress in OA patients is due to either increased lipid peroxidation or decreased antioxidant levels8e11. The excess ROS can result in oxidative damage to various components of the joint, including collagen, proteoglycans, and hyaluronan6,12. Malondialdehyde (MDA) is one of the most prevalent end byproducts of lipid peroxidation during oxidative stress. As an oxidative biomarker, MDA has been extensively detected in biological and clinical samples in previous studies on oxidative
http://dx.doi.org/10.1016/j.joca.2015.10.004 1063-4584/© 2015 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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stress13,14. Glutathione (GSH) is a naturally occurring tripeptide with nucleophilic and reducing properties that play a central role in the antioxidant system of most aerobic cells. Since GSH is a naturally occurring antioxidant with several functions including detoxification of xenobiotics and removal of hydroperoxides and free radicals, GSH concentration has been determined in many studies on oxidative stress15,16. Most medicines for OA are administered in order to alleviate the pain temporarily. Since all pain relievers may have gastrointestinal and cardiovascular adverse effects17, there is a clinical need for disease-modifying OA drugs (DMOADS) for OA patients. However, currently available drugs and therapies are unable to effectively delay the progression of OA. Joint replacement is the only treatment available for end-stage OA18,19. Danshen (Salvia miltiorrhiza), a traditional Chinese medicine with a number of physiological benefits, is widely used for the treatment of heart disease20. The pharmacokinetic and pharmacodynamic studies on the active components of Danshen indicate that Danshen is a compound that contains mainly two types of constituents, lipid soluble diterpenoid quinines (e.g., tanshinone and cryptotanshinone) and water soluble phenolics (e.g., danshensu, rosmarinic acid, salvianolic acids, protocatechuic acid, and protocatechuic aldehyde)21e23. Both components are responsible for the pharmacological activities of Danshen. It has been reported that Danshen has antioxidative, antiinflammatory, vasodilative, antihypertensive, anticoagulant, antibacterial, and anticancer effects24. Danshen has been widely used for the treatment of angina pectoris, myocardial infarction, and stroke20,21. Although the mechanism for antioxidant activity remains unclear, Danshen is believed to function as an active oxygen inhibitor25e27. It scavenges the oxygen free radicals generated by a myocardial ischemic reperfusion injury as effectively as superoxide dismutase28. Another study demonstrated the mechanisms by which Danshen protect endothelial cells against oxidative stress29. Additionally, it has been reported that Danshen could prevent the occurrence of oxidative stress in the eye and aorta of diabetic rats without affecting their hyperglycemic state30. OA has been experimentally induced in animals through a variety of techniques including joint immobilization, joint structure destabilization by surgery, and intra-articular injection of an agent. It has been determined that simple anterior cruciate ligament transection (ACLT) can lead to a progressively degenerative OA of the knee in the New Zealand rabbit31. Another study demonstrated that measured changes on the femoral condyle (specifically the medial femoral condyle) were good indicators of cartilage degeneration32. The ACLT model in rabbits is a reproducible and effective OA model. The cartilage lesions of this OA model and their response to therapy can be graded according to an adapted histological and histochemical grading system. Therefore, we hypothesized that Danshen supplementation at the initial stage of OA, may prevent the further degeneration of articular cartilage through its antioxidative activity. The purpose of this study was to evaluate the effects of Danshen on histological parameters and GSH and MDA concentrations in the articular cartilage of the medial femoral condyles of New Zealand rabbits with early OA induced by ACLT.
number: Z61021162 approved by the State; specification: 10 g 10 bags; one bag is equivalent to the original crude drugs 10 g). As a clinical drug, Danshen granules have been widely used for the treatment of angina pectoris in China. The Glutathione (GSH) Colorimetric Kit (ApoGSH™) was a product of BioVision Inc. (USA). The OxiSelect™ Thiobarbituric Acid Reactive Substance (TBARS) Assay Kit for Malondialdehyde (MDA) Quantitation was purchased from Cell Biolabs, Inc. (USA). Experimental animals Twenty-four healthy mature New Zealand White rabbits (weighing 2.2e2.8 kg; aged 9e10 weeks; 12 males and 12 females) were obtained from the Animal Experimental Center at Xi'an Jiaotong University. The rabbits were randomly divided into three groups (eight rabbits per group) designated as the control, OA, and Danshen OA groups, respectively and fed sterilized food and redistilled water. Each rabbit was housed in a single clean cage (measuring 60 60 45 cm). Environmental conditions were kept at a temperature of 20 ± 2 C and humidity of 60% ± 5%, with good ventilation, under 4 watts of light intensity per square meter and for 12 h per day. In the animal operation room, all the rabbits were anesthetized with sodium pentobarbital (at a dose of 30 mg/kg body weight through the ear edge vein). ACLT of the left hind knees was performed under an arthroscope in all the rabbits in OA and Danshen OA groups in order to induce OA31. The same surgical techniques were imitated under arthroscope with the exception of ACLT in all of the rabbits of the control group. After surgery, all rabbits were permitted activity in the cages without immobilization. The rabbits in the Danshen OA group were postoperatively treated with 3 g/kg body weight of Danshen granules dissolved in 5 mL distilled water by gastric intubation once per day over a 6-week period. The dose (3 g/kg body weight) was determined based on the dose used in humans, as well as on other literature involving rats30. The rabbits in both the control and OA groups were given the same volume of distilled water. The body weights of the rabbits were monitored once a week until sacrifice. The dose of Danshen administered was adjusted every week on the basis of their body weights. During housing, rabbits were monitored twice daily for their health status. No adverse events were observed. Handling and care of the animals were in accordance with the policies of the Animal Experimental Center of Xi'an Jiaotong University. The Committee on Animal Experimentation at the Xi'an Jiaotong University approved this study. All sections of this report adhere to the Animal Research Reporting in Vivo Experiments (ARRIVE) Guidelines for reporting animal research. Tissue collection Six weeks after surgery, all the rabbits were sacrificed by the intracardiac injection of T-6I euthanasia solution in three groups. The medial femoral condyles and the synovium from the infrapatellar fat pad were harvested (except for the synovium at the incision site) from all the rabbits. The trimmed tissues blocks were rinsed briefly with ice-cold saline solution and quickly blotted dry. The tissues were immediately frozen in liquid nitrogen and stored at 80 C until use.
Materials and methods Histological analysis Drugs and reagents Danshen granules are a type of pure Danshen product that are manufactured in compliance with good manufacturing practices (GMP) (Shaanxi Aoxing Pharmaceutical Co. Ltd. China; approval
In each group, the medial femoral condyle tissue was fixed in 10% buffered formalin at room temperature for 72 h and decalcified with 10% EDTA solution (pH ¼ 7.4). The EDTA was changed on a 48 h/48 h/72 h cycle for approximately 10 weeks until complete
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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decalcification was confirmed by X-ray. After decalcification, the medial femoral condylar tissues were dehydrated in a graded series of ethanol and xylol and embedded in paraffin. The paraffin tissue blocks were sagittally cut into serial sections (5 mm) for staining. Cartilage cell morphological changes were observed by hematoxylin and eosin staining (HE staining) of the slides. The cartilage histopathology grade, stage, and score were assessed in safranin-ostained sections according to the OARSI OA Cartilage Histopathology Assessment System33 which classifies OA severity into seven grades. Based on the extent of the cartilage surface involved, four OA stages were also defined33. The OA score was computed by multiplying the grade with the stage. Toluidine blue staining was also used to evaluate proteoglycan (PG) preservation in the cartilage matrix. The severity of toluidine blue staining loss was divided into seven grades according to Zur et al.34 The sections that were used respectively for HE, safranin-o and toluidine blue staining were stained in one batch. All the stained sections were evaluated by a pathologist blindly.
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Statistical analysis All statistical analyses were performed using the SPSS 13.0 statistical software. The Kruskal-Wallis test was utilized to test for differences in the grade of toluidine blue staining loss, the grade, stage, and score of the OARSI OA Cartilage Histopathology Assessment System evaluation (median-range) between the three groups (control, OA and Danshen OA). A P value < 0.05 was considered as statistically significant. The ManneWhitney for two independent samples was adopted for the comparison between two groups, and a P value < 0.017 was considered as statistically significant. The GSH and MDA concentrations were expressed as mean ± 95% confidence intervals (CIs), and one-way ANOVA with LSD post-hoc tests were performed for comparing GSH and MDA concentrations between the three groups. Results Histology
GSH determination The frozen tissues (100 mg) were homogenized using a mechanical homogenizer in 0.4 mL of glutathione buffer. The tissue homogenates were added to 100 mL of 5% SSA, mixed well and centrifuged at either 8000 rpm (for synovium) or at 12,000 rpm (for articular cartilage) for 10 min at 4 C. The supernatants were transferred to a fresh tube and used for glutathione assay. GSH was assayed by a colorimetric method using the ApoGSHTM Glutathione Colorimetric Assay Kit. The samples were assayed in a 96-well plate and beach well containing 20 mL NADPH Generating Mix, 20 mL Glutathione Reductase and 120 mL Glutathione Reaction Buffer. The reaction mix (160 mL) was added to each well and incubated at room temperature for 10 min in order to generate NADPH. The sample solution (20 mL) was subsequently added to each well and the plate was incubated at room temperature for 5e10 min. Finally, substrate solution (20 mL) was added to each well and the plate was incubated at room temperature for 5e10 min. The absorbance was read at 405 nm using a microplate reader. The concentration of GSH in the sample solutions was determined using the standard glutathione calibration curve. MDA determination The Thiobarbituric Acid Reactive Substances (TBARS) Assay Kit was used for the direct quantitative measurement of MDA in samples. The iced tissue (100 mg) was homogenized in 1 mL PBS containing 1BHT. The tissue homogenate mixture was centrifuged at 20,000 rpm for 5 min to collect the supernatant. The 100 mL supernatant was added to separate microcentrifuge tubes. The SDS lysis solutions (100 mL) were added to the supernatant and mixed thoroughly. The samples were subsequently incubated for 5 min at room temperature. After being incubated, the 250 mL TBA reagents were added to each sample. Each tube was subsequently closed and incubated at 95 C for 45e60 min. The tubes were removed and cooled to room temperature in an ice bath for 5 min. All sample tubes were centrifuged at 3000 rpm for 15 min. The supernatant was removed from samples for further analysis. The 200 mL of the samples were transferred to a 96-well microplate compatible with a spectrophotometric plate reader. The absorbance was read at 532 nm. Blank control was prepared and assessed in the same way to correct for the contribution of a 532 nm to the sample. The MDA concentration was calculated from the standard curve generated from known quantities of MDA.
The HE staining findings of the articular cartilage were as follows. In the control group, the articular cartilage surface was smooth and intact with flattened chondrocytes in the superficial zone. In the mid and deep zones, the cell shape was rounded with organized chondrocytes and homogeneous matrix staining [Fig. 1(A)]. However, in the OA group, the surface of articular cartilage is uneven with disorganized chondrocytes in the superficial, mid, and deep zones. Proliferative chondrocyte clusters were also seen in the superficial and mid zone. Vertical clefts into the transitional zone were also observed in some specimens. The matrix demonstrated loss of staining [Fig. 1(B)]. In the Danshen OA group, chondrocytes in the superficial zone were disorganized with proliferative chondrocyte clusters with decreased matrix staining [Fig. 1(C)]. The grade of toluidine blue staining loss of the cartilage matrix was showed in Fig. 2. The median grade was 0, 5 (3e5) and 3 (3e4) in the control, OA and Danshen OA groups, respectively. There were significant differences in grade between the three groups (F ¼ 19.83, P < 0.001). The grade of toluidine blue staining loss of the cartilage matrix of both the OA and the Danshen OA groups was significantly higher compared to that of the control group (P < 0.001). The grade of Danshen OA group was significantly lower compared to that of the OA group (P ¼ 0.008). The grade, stage, and score of the articular cartilage histopathology are exhibited in Fig. 3. The median grade of the articular cartilage histopathology was 0, 3 (2e4) and 2 (2e3) in the control, OA and Danshen OA groups, respectively. The median stage was 0, 3 (2e3), and 2 (1e3) in the control, OA and Danshen OA groups, respectively. For the score, the median was 0, 9 (4e12), and 4 (3e9), for each of the three groups, respectively. There were significant differences in the grade, stage, and scores of the articular cartilage histopathology between the three groups (P < 0.001). The grade, stage, and score of articular cartilage histopathology of both the OA and the Danshen OA groups were significantly higher compared to that of the control group (P < 0.001). The grade and score of the Danshen OA group was significantly lower compared to that of OA group (P ¼ 0.013 and P ¼ 0.009). However, there were no significant differences in the stage of articular cartilage histopathology between the Danshen OA and OA groups (P ¼ 0.045). The GSH concentration of synovium and articular cartilage Fig. 4 showed that the GSH concentration of synovium differed significantly between the three groups (F ¼ 79.87, P < 0.001). The GSH concentration of the synovium in the OA group (0.033, 95% CI
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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Fig. 1. Histomorphological sections stained with HE staining (magnification: 1010) and illustrating articular cartilage lesions. A (control group): Normal articular cartilage with intact surface. B (OA group): The cartilage matrix demonstrated loss of straining and proliferative chondrocyte clusters in the superficial and middle zones. C (Danshen OA group): Unsmooth surface. The cartilage cells in the superficial zone appear disorganized.
(F ¼ 38.64, P < 0.001). The MDA level of the synovium in the OA group (0.163, 95% CI 0.141e0.185 nmol/mg protein) was significantly increased compared to the control group (0.065, 95% CI 0.058e0.073 nmol/mg protein, P < 0.001). However, with the 6-week Danshen treatment, the MDA level in the synovium of the Danshen OA group was markedly depleted (0.090, 95% CI 0.076e0.105 nmol/ mg protein) compared to the OA group (P < 0.001). Similarly, the MDA level in the articular cartilage of the OA group was significantly increased (0.206, 95% CI 0.181e0.243 nmol/mg protein) compared to the control group (0.085, 95% CI 0.079e0.091 nmol/mg protein, P < 0.001). With the 6-week Danshen treatment, the MDA level in the cartilage of the Danshen OA group was markedly depleted (0.131, 95% CI 0.106e0.155 nmol/mg protein) compared to the OA group (P < 0.001). Discussion Fig. 2. Comparison of grade for the toluidine blue staining loss of the cartilage matrix between the experimental groups.*P < 0.001 compared to control group; #P < 0.017 compared to OA group.
0.031e0.036 mmol/mg protein) was significantly lower compared to the control group (0.061, 95% CI 0.056e0.066 mmol/mg protein, P < 0.001). However, the GSH level in the Danshen OA group (0.055, 95% CI 0.052e0.058 mmol/mg protein) was significantly higher compared to that in the OA group (P < 0.001) after 6 weeks of Danshen treatment. A similar pattern was also observed in the articular cartilage. The GSH concentration of the articular cartilage also differed significantly between three groups (F ¼ 162.87, P < 0.001). The GSH level of the articular cartilage in the OA group (0.025, 95% CI 0.022e0.027 mmol/mg protein) was significantly lower than that in control group (0.055, 95% CI 0.051e0.059 mmol/ mg protein, P < 0.001). With 6-week Danshen treatment, the GSH level of the articular cartilage in the Danshen OA group (0.048, 95% CI 0.045e0.049 mmol/mg protein) was significantly higher compared to that in the OA group (P < 0.001). The MDA concentration of synovium and articular cartilage Fig. 5 demonstrates the statistically significant differences in that the MDA concentration of synovium between the three groups
The main pathological change in OA is the degeneration of articular cartilage. The pathological process of OA is slow and progressive. In the present study, the OA model was induced by ACLT under arthroscope in the rabbit left hind knee joint. Six weeks after ACLT, the early characteristic changes of articular cartilage degeneration were detectable in the OA and Danshen OA groups. This result indicated that the ACLT OA model in the rabbit was effective. In the present study, the OARSI OA Cartilage Histopathology Assessment System was used to evaluate the severity and extent of degeneration of articular cartilage in the three experimental groups. The score was 0 in control group, which indicates a normal articular cartilage. This differs from previous studies utilizing the ACLT model, which demonstrated abnormal sections of articular cartilage in the sham operation group31. In this study, the sham operation in the control group was performed under arthroscope, with slight surgical injury to the joint such that impact to the balance structure of the joint could be negligible. However, in other studies, the sham operation was a general open surgery where the surgical injury could impact the balance structure of the joint. In addition, the average score of the OA group was higher compared to that of the Danshen OA group. The results demonstrated a more severe degeneration of the articular cartilage in OA group compared to the Danshen OA group. This indicates that Danshen might protect the articular cartilage against degeneration.
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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Fig. 3. Comparison of grade, stage, and scores for articular cartilage histopathology between the experimental groups. (A) The grade evaluation of articular cartilage histopathology. (B) The stage evaluation of articular cartilage histopathology. (C) The score evaluation of articular cartilage histopathology. *P < 0.001 compared to control group; #P < 0.017 compared to OA group.
PG is an essential part of the extracellular matrix of the articular cartilage. The excessive degradation (depolymerization) and loss of PG are the earliest metabolic changes in OA, and occur even earlier than pathologic changes35. Previous studies have indicated decreased PG content in early and advanced arthritic cartilages compared to normal cartilages36,37. A similar result was observed in
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this study. The grade of toluidine blue staining loss of both the OA and the Danshen OA groups was significantly higher compared to that of the control group. It indicated that the PG content of both the OA and the Danshen OA groups was lower than that of the control group. After 6 weeks of Danshen treatment, the grade of Danshen OA group was significantly lower compared to that of OA group. It indicated that PG content in the Danshen OA group was higher compared to the OA group suggesting that Danshen might prevent the degeneration of PG. In order to explore the mechanism of Danshen preventing articular cartilage degeneration, the GSH and MDA concentrations of the synovial and cartilage tissues were detected in the control, OA, and Danshen OA groups. The GSH concentration in the synovium and cartilage of the OA group were significantly lower compared to that of the control group. The results are in agreement with other studies, which reported that significantly lower GSH concentration in patients with OA compared to healthy persons38. The GSH level is maintained mainly by GSH oxidation and the regeneration of reduced GSH by GSHperoxidase and oxidized glutathione (GSSG) reductase, respectively. The decrease in GSH levels of the synovium and cartilage tissues represents an increased consumption and/or a reduced production of this antioxidant. Increased production of ROS under articular cartilage injury has been previously reported3. The reduced level of GSH in the synovium and articular cartilage of OA rabbits is probably attributed to increased oxidation of the GSH by GSH-peroxidase in order to combat the ROS generated by articular cartilage injury. However, with 6-week Danshen treatment, the GSH level in the synovium and articular cartilage of the Danshen OA group was significantly higher compared to the OA group. Similar results have not been reported in previous studies on OA. However, this result was consistent with previous study involving the 6-week Danshen treatment, which demonstrated significantly increased GSH levels in the eye and aorta of diabetic rats compared to the control group30. This suggests that Danshen may either preserve the endogenous GSH or induce GSH synthesis during articular cartilage injury. Some of the components in Danshen probably act as a ROS scavenger that prevents depletion of GSH25. It is also possible that some Danshen components upregulate the activity of GSH reductase, which catalyzes the reduction of GSSG to GSH26. According to Goranov NV, a statistically significant increase in MDA concentration was observed during the development of OA in a canine model39. Enhanced lipid peroxidation was observed in the synoviocytes of patients with OA compared to healthy controls40. A similar result was observed in this study. The MDA levels in the synovium and articular cartilage tissue of the OA group were significantly increased compared to the control group. However, with the 6-week Danshen treatment, the MDA levels in the synovium and articular cartilage tissue of the Danshen OA group were markedly depleted compared to the OA group. The results suggest that Danshen inhibited lipid peroxidation in the synovium and articular cartilage tissue of OA rabbits. A similar result was observed involving inhibition of lipid peroxidation in the eye and aortic tissues of diabetic rats30. According to Kuang et al., the inhibition of lipid peroxidation was conceivably due to the free radical scavenging activity of Danshen41. Moreover, a study results indicated that Danshen could elevate the activity of superoxide dismutase and reduce the MDA content in an animal model of chronic hepatic damage42. Furthermore, the vitro study indicated that Danshen could inhibit lipid peroxidation in cell microsomes and this effect might be mediated by the scavenging of superoxide anion radicals21. The present study is one of few investigations that assess the effects of Danshen, as an antioxidant on osteoarthritic joints in
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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GSH in synovium 0.08
*# 0.06
*
0.04 0.02 0.00
Control
OA
Danshen OA
GSH concentration (μmol/mg prote in)
B. Bai, Y. Li / Osteoarthritis and Cartilage xxx (2015) 1e7
GSH concentration (μmol/mg prote in)
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GSH in articular cartilage 0.08
*#
0.06 0.04
*
0.02 0.00
Control
OA
Danshen OA
MDA in synovium 0.3
*
0.2
*# 0.1
0.0
Control
OA
Danshen OA
M DA concentration (nmol/mg protein)
Fig. 4. A comparison of the GSH concentration levels in the synovium and articular cartilage tissue between the control, OA, and Danshen OA groups, respectively (mean ± 95% CI, n ¼ 8).*P < 0.001 compared to control group; #P < 0.001 compared to OA group.
MDA concentration (nmol/mg protein)
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MDA in articular cartilage 0.3
* *#
0.2
0.1
0.0
Control
OA
Danshen OA
Fig. 5. A comparison of the MDA concentration levels in the synovium and articular cartilage tissues between control, OA, and Danshen OA groups (mean ± 95% CI, n ¼ 8).*P < 0.001 compared to control group; #P < 0.001 compared to OA group.
rabbits. We acknowledge there are some limitations in this study. For example, Danshen was used immediately after surgery, which is earlier than the treatment period of OA patients in the clinical setting. The OARSI Assessment System is dependent on multiples of primes for individual scores, so it omits the OA scores 7, 11, 13, 14, 17, 21, 22, and 23, and is nonlinear33. However, these omitted scores mostly involved OA at a high grade and extensive stage, and these scores may be seen as averages of the individual scores. The ratio of GSH/GSSG was not calculated. Despite the above limitations, the results of this study are reliable because some measures were taken to ensure the reliability of the data including appropriate sample size, randomization, sham control, same treatment except of drug and blinding. The exact mechanism by which Danshen exerts its protective effect on oxidative damage remains unknown. Whether Danshen acts purely as a free radical scavenger, or through the stimulation of antioxidant enzyme activities, necessitates further investigations.
Conclusions In conclusion, this study demonstrates that Danshen could prevent the degeneration of articular cartilage by its antioxidation effects in rabbits with OA. It has been suggested that Danshen supplementation may be useful in the treatment of OA.
Author contributions All the authors of this study made substantial contributions in the conception and design of the study, data acquisition, analysis and interpretation of data, drafting the article or revising it critically for important intellectual content and final approval.
Conflict of interest The authors reported no conflicts of interests associated with this manuscript. Acknowledgments This research was supported by Shaanxi province government research grant (2008K14-01). References 1. Bedson J, Jordan K, Croft P. The prevalence and history of knee osteoarthritis in general practice: a case-control study. Fam Pract 2005;22(1):103e8. 2. Woolf AD, Pfleger B. Burden of major musculoskeletal conditions. Bull World Health Organ 2003;81(9):646e56. 3. Henrotin Y, Kurz B, Aigner T. Oxygen and reactive oxygen species in cartilage degradation: friends or foes? Osteoarthritis Cartilage 2005;13:643e54. 4. Yudoh K, Nguyen T, Nakamura H, Hongo-Masuko K, Kato T, Nishioka K. Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function. Arthritis Res Ther 2005;7: R380e91. 5. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature 2000;408:239e47. 6. Maneesh M, Jayalekshmi H, Suma T, Chatterjee S, Chakrabarti A, Singh TA. Evidence for oxidative stress in osteoarthritis. Indian J Clin Biochem 2005;20:129e30. 7. Afonso V, Champy R, Mitrovic D, Collin P, Lomri A. Reactive oxygen species and superoxide dismutases: role in joint diseases. Joint Bone Spine 2007;74:324e9.
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis
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B. Bai y, Y. Li z * y Orthopedics Department, The First Affiliated Hospital, Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, China z School of Public Health, Health Science Center of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, China
a r t i c l e i n f o
s u m m a r y
Article history: Received 8 January 2015 Accepted 9 October 2015
Objective: To evaluate the efficacy of Danshen on histological parameters and antioxidative activity in the articular cartilage of rabbits with osteoarthritis (OA). Design: Twenty-four rabbits were randomly divided into three groups (control, OA, and Danshen OA; eight rabbits per group). Anterior cruciate ligament transection (ACLT) of the left hind knees was performed in all rabbits in the OA and Danshen OA group for induction of OA. The rabbits in the control group underwent a sham operation. After surgery, 3 g/kg body weight of Danshen granules dissolved in 5 mL distilled water was administered by gastric intubation once per day and over a 6-week period to the Danshen OA group. The same volume of distilled water was administered to the OA and control groups. After 6 weeks, the medial femoral condyles and synoviums of the left hind knees in all three groups were harvested and used for histological and biochemical analyses. Results: Severe articular cartilage degeneration as well as lower proteoglycan (PG) content were noted in the OA group compared to the Danshen OA group (P < 0.05). The glutathione (GSH) levels in the synovium and articular cartilage of the rabbits in the Danshen OA group were significantly higher compared to the OA group (P < 0.001). The malondialdehyde (MDA) levels of the synovium and articular cartilage in the Danshen OA group was markedly depleted compared to the OA group (P < 0.001). Conclusion: Danshen can prevent articular cartilage degeneration in OA though the defense against oxidative stress. © 2015 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.
Keywords: Danshen Osteoarthritis Articular cartilage Degeneration Oxidative stress
Introduction Osteoarthritis (OA) is the most common joint disease that leads to pain and disability. The prevalence of symptomatic OA in those 60 years old and above was 9.6% in men and 18% in women1. It will be the fourth leading cause of disability worldwide by 20202, resulting in a large worldwide socioeconomic burden. The etiology and pathogenesis of OA currently remains unclear. Nevertheless, increasing evidence from both experimental and clinical studies suggest that oxidative stress plays a pivotal role in the pathological process of OA3,4. Oxidative stress is generated from an imbalance in the production and elimination of reactive oxygen species (ROS)5. In
* Address correspondence and reprint requests to: Y. Li, School of Public Health, Health Science Center of Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, China. Tel: 86-29-82655108. E-mail addresses: [email protected] (B. Bai), [email protected] (Y. Li).
order to maintain balance under physiological conditions, ROS are produced and removed in the human body by the cellular antioxidant defense system. If regulated improperly, the excess ROS can inhibit the activity of normal cells by damaging cellular lipid, protein, and DNA content4. Previous studies have demonstrated the critical role played by oxidative stress in directly promoting chondrocyte apoptosis, catabolic processes, and matrix degradation6. Furthermore, some studies suggest that the telomere shortening and reduced number and function of mitochondria seen in OA chondrocytes are due to oxidative stress4,7. According to these studies, the oxidative stress in OA patients is due to either increased lipid peroxidation or decreased antioxidant levels8e11. The excess ROS can result in oxidative damage to various components of the joint, including collagen, proteoglycans, and hyaluronan6,12. Malondialdehyde (MDA) is one of the most prevalent end byproducts of lipid peroxidation during oxidative stress. As an oxidative biomarker, MDA has been extensively detected in biological and clinical samples in previous studies on oxidative
http://dx.doi.org/10.1016/j.joca.2015.10.004 1063-4584/© 2015 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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stress13,14. Glutathione (GSH) is a naturally occurring tripeptide with nucleophilic and reducing properties that play a central role in the antioxidant system of most aerobic cells. Since GSH is a naturally occurring antioxidant with several functions including detoxification of xenobiotics and removal of hydroperoxides and free radicals, GSH concentration has been determined in many studies on oxidative stress15,16. Most medicines for OA are administered in order to alleviate the pain temporarily. Since all pain relievers may have gastrointestinal and cardiovascular adverse effects17, there is a clinical need for disease-modifying OA drugs (DMOADS) for OA patients. However, currently available drugs and therapies are unable to effectively delay the progression of OA. Joint replacement is the only treatment available for end-stage OA18,19. Danshen (Salvia miltiorrhiza), a traditional Chinese medicine with a number of physiological benefits, is widely used for the treatment of heart disease20. The pharmacokinetic and pharmacodynamic studies on the active components of Danshen indicate that Danshen is a compound that contains mainly two types of constituents, lipid soluble diterpenoid quinines (e.g., tanshinone and cryptotanshinone) and water soluble phenolics (e.g., danshensu, rosmarinic acid, salvianolic acids, protocatechuic acid, and protocatechuic aldehyde)21e23. Both components are responsible for the pharmacological activities of Danshen. It has been reported that Danshen has antioxidative, antiinflammatory, vasodilative, antihypertensive, anticoagulant, antibacterial, and anticancer effects24. Danshen has been widely used for the treatment of angina pectoris, myocardial infarction, and stroke20,21. Although the mechanism for antioxidant activity remains unclear, Danshen is believed to function as an active oxygen inhibitor25e27. It scavenges the oxygen free radicals generated by a myocardial ischemic reperfusion injury as effectively as superoxide dismutase28. Another study demonstrated the mechanisms by which Danshen protect endothelial cells against oxidative stress29. Additionally, it has been reported that Danshen could prevent the occurrence of oxidative stress in the eye and aorta of diabetic rats without affecting their hyperglycemic state30. OA has been experimentally induced in animals through a variety of techniques including joint immobilization, joint structure destabilization by surgery, and intra-articular injection of an agent. It has been determined that simple anterior cruciate ligament transection (ACLT) can lead to a progressively degenerative OA of the knee in the New Zealand rabbit31. Another study demonstrated that measured changes on the femoral condyle (specifically the medial femoral condyle) were good indicators of cartilage degeneration32. The ACLT model in rabbits is a reproducible and effective OA model. The cartilage lesions of this OA model and their response to therapy can be graded according to an adapted histological and histochemical grading system. Therefore, we hypothesized that Danshen supplementation at the initial stage of OA, may prevent the further degeneration of articular cartilage through its antioxidative activity. The purpose of this study was to evaluate the effects of Danshen on histological parameters and GSH and MDA concentrations in the articular cartilage of the medial femoral condyles of New Zealand rabbits with early OA induced by ACLT.
number: Z61021162 approved by the State; specification: 10 g 10 bags; one bag is equivalent to the original crude drugs 10 g). As a clinical drug, Danshen granules have been widely used for the treatment of angina pectoris in China. The Glutathione (GSH) Colorimetric Kit (ApoGSH™) was a product of BioVision Inc. (USA). The OxiSelect™ Thiobarbituric Acid Reactive Substance (TBARS) Assay Kit for Malondialdehyde (MDA) Quantitation was purchased from Cell Biolabs, Inc. (USA). Experimental animals Twenty-four healthy mature New Zealand White rabbits (weighing 2.2e2.8 kg; aged 9e10 weeks; 12 males and 12 females) were obtained from the Animal Experimental Center at Xi'an Jiaotong University. The rabbits were randomly divided into three groups (eight rabbits per group) designated as the control, OA, and Danshen OA groups, respectively and fed sterilized food and redistilled water. Each rabbit was housed in a single clean cage (measuring 60 60 45 cm). Environmental conditions were kept at a temperature of 20 ± 2 C and humidity of 60% ± 5%, with good ventilation, under 4 watts of light intensity per square meter and for 12 h per day. In the animal operation room, all the rabbits were anesthetized with sodium pentobarbital (at a dose of 30 mg/kg body weight through the ear edge vein). ACLT of the left hind knees was performed under an arthroscope in all the rabbits in OA and Danshen OA groups in order to induce OA31. The same surgical techniques were imitated under arthroscope with the exception of ACLT in all of the rabbits of the control group. After surgery, all rabbits were permitted activity in the cages without immobilization. The rabbits in the Danshen OA group were postoperatively treated with 3 g/kg body weight of Danshen granules dissolved in 5 mL distilled water by gastric intubation once per day over a 6-week period. The dose (3 g/kg body weight) was determined based on the dose used in humans, as well as on other literature involving rats30. The rabbits in both the control and OA groups were given the same volume of distilled water. The body weights of the rabbits were monitored once a week until sacrifice. The dose of Danshen administered was adjusted every week on the basis of their body weights. During housing, rabbits were monitored twice daily for their health status. No adverse events were observed. Handling and care of the animals were in accordance with the policies of the Animal Experimental Center of Xi'an Jiaotong University. The Committee on Animal Experimentation at the Xi'an Jiaotong University approved this study. All sections of this report adhere to the Animal Research Reporting in Vivo Experiments (ARRIVE) Guidelines for reporting animal research. Tissue collection Six weeks after surgery, all the rabbits were sacrificed by the intracardiac injection of T-6I euthanasia solution in three groups. The medial femoral condyles and the synovium from the infrapatellar fat pad were harvested (except for the synovium at the incision site) from all the rabbits. The trimmed tissues blocks were rinsed briefly with ice-cold saline solution and quickly blotted dry. The tissues were immediately frozen in liquid nitrogen and stored at 80 C until use.
Materials and methods Histological analysis Drugs and reagents Danshen granules are a type of pure Danshen product that are manufactured in compliance with good manufacturing practices (GMP) (Shaanxi Aoxing Pharmaceutical Co. Ltd. China; approval
In each group, the medial femoral condyle tissue was fixed in 10% buffered formalin at room temperature for 72 h and decalcified with 10% EDTA solution (pH ¼ 7.4). The EDTA was changed on a 48 h/48 h/72 h cycle for approximately 10 weeks until complete
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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decalcification was confirmed by X-ray. After decalcification, the medial femoral condylar tissues were dehydrated in a graded series of ethanol and xylol and embedded in paraffin. The paraffin tissue blocks were sagittally cut into serial sections (5 mm) for staining. Cartilage cell morphological changes were observed by hematoxylin and eosin staining (HE staining) of the slides. The cartilage histopathology grade, stage, and score were assessed in safranin-ostained sections according to the OARSI OA Cartilage Histopathology Assessment System33 which classifies OA severity into seven grades. Based on the extent of the cartilage surface involved, four OA stages were also defined33. The OA score was computed by multiplying the grade with the stage. Toluidine blue staining was also used to evaluate proteoglycan (PG) preservation in the cartilage matrix. The severity of toluidine blue staining loss was divided into seven grades according to Zur et al.34 The sections that were used respectively for HE, safranin-o and toluidine blue staining were stained in one batch. All the stained sections were evaluated by a pathologist blindly.
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Statistical analysis All statistical analyses were performed using the SPSS 13.0 statistical software. The Kruskal-Wallis test was utilized to test for differences in the grade of toluidine blue staining loss, the grade, stage, and score of the OARSI OA Cartilage Histopathology Assessment System evaluation (median-range) between the three groups (control, OA and Danshen OA). A P value < 0.05 was considered as statistically significant. The ManneWhitney for two independent samples was adopted for the comparison between two groups, and a P value < 0.017 was considered as statistically significant. The GSH and MDA concentrations were expressed as mean ± 95% confidence intervals (CIs), and one-way ANOVA with LSD post-hoc tests were performed for comparing GSH and MDA concentrations between the three groups. Results Histology
GSH determination The frozen tissues (100 mg) were homogenized using a mechanical homogenizer in 0.4 mL of glutathione buffer. The tissue homogenates were added to 100 mL of 5% SSA, mixed well and centrifuged at either 8000 rpm (for synovium) or at 12,000 rpm (for articular cartilage) for 10 min at 4 C. The supernatants were transferred to a fresh tube and used for glutathione assay. GSH was assayed by a colorimetric method using the ApoGSHTM Glutathione Colorimetric Assay Kit. The samples were assayed in a 96-well plate and beach well containing 20 mL NADPH Generating Mix, 20 mL Glutathione Reductase and 120 mL Glutathione Reaction Buffer. The reaction mix (160 mL) was added to each well and incubated at room temperature for 10 min in order to generate NADPH. The sample solution (20 mL) was subsequently added to each well and the plate was incubated at room temperature for 5e10 min. Finally, substrate solution (20 mL) was added to each well and the plate was incubated at room temperature for 5e10 min. The absorbance was read at 405 nm using a microplate reader. The concentration of GSH in the sample solutions was determined using the standard glutathione calibration curve. MDA determination The Thiobarbituric Acid Reactive Substances (TBARS) Assay Kit was used for the direct quantitative measurement of MDA in samples. The iced tissue (100 mg) was homogenized in 1 mL PBS containing 1BHT. The tissue homogenate mixture was centrifuged at 20,000 rpm for 5 min to collect the supernatant. The 100 mL supernatant was added to separate microcentrifuge tubes. The SDS lysis solutions (100 mL) were added to the supernatant and mixed thoroughly. The samples were subsequently incubated for 5 min at room temperature. After being incubated, the 250 mL TBA reagents were added to each sample. Each tube was subsequently closed and incubated at 95 C for 45e60 min. The tubes were removed and cooled to room temperature in an ice bath for 5 min. All sample tubes were centrifuged at 3000 rpm for 15 min. The supernatant was removed from samples for further analysis. The 200 mL of the samples were transferred to a 96-well microplate compatible with a spectrophotometric plate reader. The absorbance was read at 532 nm. Blank control was prepared and assessed in the same way to correct for the contribution of a 532 nm to the sample. The MDA concentration was calculated from the standard curve generated from known quantities of MDA.
The HE staining findings of the articular cartilage were as follows. In the control group, the articular cartilage surface was smooth and intact with flattened chondrocytes in the superficial zone. In the mid and deep zones, the cell shape was rounded with organized chondrocytes and homogeneous matrix staining [Fig. 1(A)]. However, in the OA group, the surface of articular cartilage is uneven with disorganized chondrocytes in the superficial, mid, and deep zones. Proliferative chondrocyte clusters were also seen in the superficial and mid zone. Vertical clefts into the transitional zone were also observed in some specimens. The matrix demonstrated loss of staining [Fig. 1(B)]. In the Danshen OA group, chondrocytes in the superficial zone were disorganized with proliferative chondrocyte clusters with decreased matrix staining [Fig. 1(C)]. The grade of toluidine blue staining loss of the cartilage matrix was showed in Fig. 2. The median grade was 0, 5 (3e5) and 3 (3e4) in the control, OA and Danshen OA groups, respectively. There were significant differences in grade between the three groups (F ¼ 19.83, P < 0.001). The grade of toluidine blue staining loss of the cartilage matrix of both the OA and the Danshen OA groups was significantly higher compared to that of the control group (P < 0.001). The grade of Danshen OA group was significantly lower compared to that of the OA group (P ¼ 0.008). The grade, stage, and score of the articular cartilage histopathology are exhibited in Fig. 3. The median grade of the articular cartilage histopathology was 0, 3 (2e4) and 2 (2e3) in the control, OA and Danshen OA groups, respectively. The median stage was 0, 3 (2e3), and 2 (1e3) in the control, OA and Danshen OA groups, respectively. For the score, the median was 0, 9 (4e12), and 4 (3e9), for each of the three groups, respectively. There were significant differences in the grade, stage, and scores of the articular cartilage histopathology between the three groups (P < 0.001). The grade, stage, and score of articular cartilage histopathology of both the OA and the Danshen OA groups were significantly higher compared to that of the control group (P < 0.001). The grade and score of the Danshen OA group was significantly lower compared to that of OA group (P ¼ 0.013 and P ¼ 0.009). However, there were no significant differences in the stage of articular cartilage histopathology between the Danshen OA and OA groups (P ¼ 0.045). The GSH concentration of synovium and articular cartilage Fig. 4 showed that the GSH concentration of synovium differed significantly between the three groups (F ¼ 79.87, P < 0.001). The GSH concentration of the synovium in the OA group (0.033, 95% CI
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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Fig. 1. Histomorphological sections stained with HE staining (magnification: 1010) and illustrating articular cartilage lesions. A (control group): Normal articular cartilage with intact surface. B (OA group): The cartilage matrix demonstrated loss of straining and proliferative chondrocyte clusters in the superficial and middle zones. C (Danshen OA group): Unsmooth surface. The cartilage cells in the superficial zone appear disorganized.
(F ¼ 38.64, P < 0.001). The MDA level of the synovium in the OA group (0.163, 95% CI 0.141e0.185 nmol/mg protein) was significantly increased compared to the control group (0.065, 95% CI 0.058e0.073 nmol/mg protein, P < 0.001). However, with the 6-week Danshen treatment, the MDA level in the synovium of the Danshen OA group was markedly depleted (0.090, 95% CI 0.076e0.105 nmol/ mg protein) compared to the OA group (P < 0.001). Similarly, the MDA level in the articular cartilage of the OA group was significantly increased (0.206, 95% CI 0.181e0.243 nmol/mg protein) compared to the control group (0.085, 95% CI 0.079e0.091 nmol/mg protein, P < 0.001). With the 6-week Danshen treatment, the MDA level in the cartilage of the Danshen OA group was markedly depleted (0.131, 95% CI 0.106e0.155 nmol/mg protein) compared to the OA group (P < 0.001). Discussion Fig. 2. Comparison of grade for the toluidine blue staining loss of the cartilage matrix between the experimental groups.*P < 0.001 compared to control group; #P < 0.017 compared to OA group.
0.031e0.036 mmol/mg protein) was significantly lower compared to the control group (0.061, 95% CI 0.056e0.066 mmol/mg protein, P < 0.001). However, the GSH level in the Danshen OA group (0.055, 95% CI 0.052e0.058 mmol/mg protein) was significantly higher compared to that in the OA group (P < 0.001) after 6 weeks of Danshen treatment. A similar pattern was also observed in the articular cartilage. The GSH concentration of the articular cartilage also differed significantly between three groups (F ¼ 162.87, P < 0.001). The GSH level of the articular cartilage in the OA group (0.025, 95% CI 0.022e0.027 mmol/mg protein) was significantly lower than that in control group (0.055, 95% CI 0.051e0.059 mmol/ mg protein, P < 0.001). With 6-week Danshen treatment, the GSH level of the articular cartilage in the Danshen OA group (0.048, 95% CI 0.045e0.049 mmol/mg protein) was significantly higher compared to that in the OA group (P < 0.001). The MDA concentration of synovium and articular cartilage Fig. 5 demonstrates the statistically significant differences in that the MDA concentration of synovium between the three groups
The main pathological change in OA is the degeneration of articular cartilage. The pathological process of OA is slow and progressive. In the present study, the OA model was induced by ACLT under arthroscope in the rabbit left hind knee joint. Six weeks after ACLT, the early characteristic changes of articular cartilage degeneration were detectable in the OA and Danshen OA groups. This result indicated that the ACLT OA model in the rabbit was effective. In the present study, the OARSI OA Cartilage Histopathology Assessment System was used to evaluate the severity and extent of degeneration of articular cartilage in the three experimental groups. The score was 0 in control group, which indicates a normal articular cartilage. This differs from previous studies utilizing the ACLT model, which demonstrated abnormal sections of articular cartilage in the sham operation group31. In this study, the sham operation in the control group was performed under arthroscope, with slight surgical injury to the joint such that impact to the balance structure of the joint could be negligible. However, in other studies, the sham operation was a general open surgery where the surgical injury could impact the balance structure of the joint. In addition, the average score of the OA group was higher compared to that of the Danshen OA group. The results demonstrated a more severe degeneration of the articular cartilage in OA group compared to the Danshen OA group. This indicates that Danshen might protect the articular cartilage against degeneration.
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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Fig. 3. Comparison of grade, stage, and scores for articular cartilage histopathology between the experimental groups. (A) The grade evaluation of articular cartilage histopathology. (B) The stage evaluation of articular cartilage histopathology. (C) The score evaluation of articular cartilage histopathology. *P < 0.001 compared to control group; #P < 0.017 compared to OA group.
PG is an essential part of the extracellular matrix of the articular cartilage. The excessive degradation (depolymerization) and loss of PG are the earliest metabolic changes in OA, and occur even earlier than pathologic changes35. Previous studies have indicated decreased PG content in early and advanced arthritic cartilages compared to normal cartilages36,37. A similar result was observed in
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this study. The grade of toluidine blue staining loss of both the OA and the Danshen OA groups was significantly higher compared to that of the control group. It indicated that the PG content of both the OA and the Danshen OA groups was lower than that of the control group. After 6 weeks of Danshen treatment, the grade of Danshen OA group was significantly lower compared to that of OA group. It indicated that PG content in the Danshen OA group was higher compared to the OA group suggesting that Danshen might prevent the degeneration of PG. In order to explore the mechanism of Danshen preventing articular cartilage degeneration, the GSH and MDA concentrations of the synovial and cartilage tissues were detected in the control, OA, and Danshen OA groups. The GSH concentration in the synovium and cartilage of the OA group were significantly lower compared to that of the control group. The results are in agreement with other studies, which reported that significantly lower GSH concentration in patients with OA compared to healthy persons38. The GSH level is maintained mainly by GSH oxidation and the regeneration of reduced GSH by GSHperoxidase and oxidized glutathione (GSSG) reductase, respectively. The decrease in GSH levels of the synovium and cartilage tissues represents an increased consumption and/or a reduced production of this antioxidant. Increased production of ROS under articular cartilage injury has been previously reported3. The reduced level of GSH in the synovium and articular cartilage of OA rabbits is probably attributed to increased oxidation of the GSH by GSH-peroxidase in order to combat the ROS generated by articular cartilage injury. However, with 6-week Danshen treatment, the GSH level in the synovium and articular cartilage of the Danshen OA group was significantly higher compared to the OA group. Similar results have not been reported in previous studies on OA. However, this result was consistent with previous study involving the 6-week Danshen treatment, which demonstrated significantly increased GSH levels in the eye and aorta of diabetic rats compared to the control group30. This suggests that Danshen may either preserve the endogenous GSH or induce GSH synthesis during articular cartilage injury. Some of the components in Danshen probably act as a ROS scavenger that prevents depletion of GSH25. It is also possible that some Danshen components upregulate the activity of GSH reductase, which catalyzes the reduction of GSSG to GSH26. According to Goranov NV, a statistically significant increase in MDA concentration was observed during the development of OA in a canine model39. Enhanced lipid peroxidation was observed in the synoviocytes of patients with OA compared to healthy controls40. A similar result was observed in this study. The MDA levels in the synovium and articular cartilage tissue of the OA group were significantly increased compared to the control group. However, with the 6-week Danshen treatment, the MDA levels in the synovium and articular cartilage tissue of the Danshen OA group were markedly depleted compared to the OA group. The results suggest that Danshen inhibited lipid peroxidation in the synovium and articular cartilage tissue of OA rabbits. A similar result was observed involving inhibition of lipid peroxidation in the eye and aortic tissues of diabetic rats30. According to Kuang et al., the inhibition of lipid peroxidation was conceivably due to the free radical scavenging activity of Danshen41. Moreover, a study results indicated that Danshen could elevate the activity of superoxide dismutase and reduce the MDA content in an animal model of chronic hepatic damage42. Furthermore, the vitro study indicated that Danshen could inhibit lipid peroxidation in cell microsomes and this effect might be mediated by the scavenging of superoxide anion radicals21. The present study is one of few investigations that assess the effects of Danshen, as an antioxidant on osteoarthritic joints in
Please cite this article in press as: Bai B, Li Y, Danshen prevents articular cartilage degeneration via antioxidation in rabbits with osteoarthritis, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.10.004
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GSH in synovium 0.08
*# 0.06
*
0.04 0.02 0.00
Control
OA
Danshen OA
GSH concentration (μmol/mg prote in)
B. Bai, Y. Li / Osteoarthritis and Cartilage xxx (2015) 1e7
GSH concentration (μmol/mg prote in)
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GSH in articular cartilage 0.08
*#
0.06 0.04
*
0.02 0.00
Control
OA
Danshen OA
MDA in synovium 0.3
*
0.2
*# 0.1
0.0
Control
OA
Danshen OA
M DA concentration (nmol/mg protein)
Fig. 4. A comparison of the GSH concentration levels in the synovium and articular cartilage tissue between the control, OA, and Danshen OA groups, respectively (mean ± 95% CI, n ¼ 8).*P < 0.001 compared to control group; #P < 0.001 compared to OA group.
MDA concentration (nmol/mg protein)
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MDA in articular cartilage 0.3
* *#
0.2
0.1
0.0
Control
OA
Danshen OA
Fig. 5. A comparison of the MDA concentration levels in the synovium and articular cartilage tissues between control, OA, and Danshen OA groups (mean ± 95% CI, n ¼ 8).*P < 0.001 compared to control group; #P < 0.001 compared to OA group.
rabbits. We acknowledge there are some limitations in this study. For example, Danshen was used immediately after surgery, which is earlier than the treatment period of OA patients in the clinical setting. The OARSI Assessment System is dependent on multiples of primes for individual scores, so it omits the OA scores 7, 11, 13, 14, 17, 21, 22, and 23, and is nonlinear33. However, these omitted scores mostly involved OA at a high grade and extensive stage, and these scores may be seen as averages of the individual scores. The ratio of GSH/GSSG was not calculated. Despite the above limitations, the results of this study are reliable because some measures were taken to ensure the reliability of the data including appropriate sample size, randomization, sham control, same treatment except of drug and blinding. The exact mechanism by which Danshen exerts its protective effect on oxidative damage remains unknown. Whether Danshen acts purely as a free radical scavenger, or through the stimulation of antioxidant enzyme activities, necessitates further investigations.
Conclusions In conclusion, this study demonstrates that Danshen could prevent the degeneration of articular cartilage by its antioxidation effects in rabbits with OA. It has been suggested that Danshen supplementation may be useful in the treatment of OA.
Author contributions All the authors of this study made substantial contributions in the conception and design of the study, data acquisition, analysis and interpretation of data, drafting the article or revising it critically for important intellectual content and final approval.
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