1. Introduction

Osteoarthritis (OA) is a chronic progressive disabling disorder of the elderly population that causes structural changes in the joints [1]. Over 595 million people are suffering from OA around the globe [2]. The quality of life of the affected individuals is greatly compromised by chronic pain, stiffness and physical impairment [3]. OA has a significant financial impact, from direct medical expenses to reduced productivity at work [4,5]. Treatment for this disease remains unsatisfactory as there are no disease-modifying drugs to date, non-steroidal anti-inflammatory drugs constitute the cornerstone of OA pain management, but they have substantial gastrointestinal, renal, and hepatic adverse effects and increased cardiovascular risks which restricted their usage to the shortest possible duration and have a little positive effect on joint structures [6,7]. Intra-articular corticosteroid therapy has been found to give short-term symptomatic relief but their long-term detrimental effects on articular cartilage remains a major concern [8]. Various nutrients and nutraceuticals were trialed in OA management with inconsistent outcomes [9]. Total knee replacement is considered as a last resort when nonsurgical treatment is ineffective which is associated with high complication and revision rate [10]. Hence the search for a safe and effective treatment option for slowing disease progression and improving symptoms continues.

Astaxanthin, commonly known as a "marine carotenoid," is a natural lipid-soluble and red-orange oxycarotenoid pigment that belongs to the xanthophyll group of carotenoids [11]. It is widely found in a variety of aquatic animals including shrimp, lobster, salmon, trout, red seabream and fish eggs [12]. The richest source of natural astaxanthin for human consumption is astaxanthin obtained from algae Haematococcus pluvialis [13]. Astaxanthin is a strong anti-oxidant with multiple health benefits for which it received significant attention and now it is a well-known neutraceutical [14]. Oxidative stress is regarded as a significant contributor to the etiology and disease progression in OA [15]. Anti-oxidant activity of astaxanthin is 10 fold higher than of other carotenoids and 100 times higher than α tocopherol [16].

The Matrix Metalloproteinases family (MMPs) is well established to have significant roles in cartilage breakdown [17]. It was reported that astaxanthin reduce MMP expression in chondrocytes and improve loss of cartilage [18]. which was confirmed by morphological and histological evaluation on the articular cartilage in experimental OA in rabbits [19]. Though once considered as a simple disease of wear and tear in recent years’ low grade chronic inflammation was linked with OA evidenced by increased levels of pro-inflammatory cytokines IL-1β, IL-6 and tumor necrosis factor-α (TNF-α) [20]. Astaxanthin can reduce critical mediators of inflammation including CRP, IL-1β, IL-6, NF-κB and nitric oxide (NO) [21].

All of this scientific evidence highlights astaxanthin’s enormous potential to improve disease progression and symptoms in OA patients, implying that it could be a promising therapeutic option for the treatment of OA. The goal of this trial was to evaluate the efficacy of 12 mg of commercially available astaxanthin supplement daily on pain, stiffness and physical function in patients with moderate to severe knee OA compared with a placebo over an 8-weeks period. Changes in serum inflammatory markers-high-sensitivity C-reactive protein (hsCRP) and IL-6 level was also evaluated.

2. Methods

This study was a randomized, double-blind, placebo-controlled trial conducted in the Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh in collaboration with the Department of Pharmacology, the Department of Physical Medicine and Rehabilitation and the Department of Microbiology and Immunology. Before the study’s conduct, the research protocol was submitted to the Institutional Review Board (IRB) of BSMMU to review the scientific and ethical issues related to the research to obtain the required approval. The protocol was reviewed and the IRB of BSMMU issued a Clearance Letter (Memo No. BSMMU/2022/5680). The trial was registered on ClinicalTrials.gov (trial ID number NCT05437601) and conducted in accordance with the International Council on Harmonization Good Clinical Practice (ICH GCP) guidelines. The intervention phase was executed from June 2022 to January 2023.

2.1 Participants

All the patients (both male and female) with knee pain and above 40 years old attending the outpatient department and Osteoporosis, Osteoarthritis and Postmenopausal Muscle Bone Health Clinic of the Department of Physical Medicine and Rehabilitation, BSMMU were given X-ray of the affected knee joints in a standing position and were diagnosed according to the Kellgren-Lawrence radiographic grading scale. Those with Grade III and Grade IV knee OA were selected for the study by a competent Physiatrist. Patients with prior history of knee trauma or surgery, history of presence of systemic inflammatory conditions such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, current smokers, known allergy to seafood, shellfish or astaxanthin, taking immunosuppressant, pregnant and nursing mothers, history of taking intra-articular steroid within 6 months of the study were excluded from the study. Patients with major comorbidities or inability to cooperate with study requirements were precluded entry. The study objectives were explained to each participant and they were informed about the potential benefits and risks associated with the intervention. Participants were also informed that they can participate in this study of their free will and also, they had every right to refuse to participate or to withdraw at any time without compromising their medical care. Patients who were convinced and agreed willingly after adequate understanding were enrolled in the study with written consent. Participants’ confidentiality was rigorously protected. The participant’s personal information regarding name, age, sex and other information was not disclosed anywhere and were used for research purpose only.

2.2 Study design and procedure

Medicines and placebo were purchased from the manufacturer at the original market price so that there was no conflict of interest. Astaxanthin capsules (Astagen 4 mg) and placebo capsules were purchased from General Pharmaceuticals Limited, a leading Pharmaceutical company in Bangladesh. The capsule placebo was identical to the capsule astaxanthin 4mg in size, shape and color and was also supplied in an identical container.

Participants were randomly allocated in a double-blind manner in equal numbers to receive either astaxanthin 12 mg daily or placebo. Online GraphPad software was used to perform the randomization. The software automatically generated two distinct sets of random numbers after giving the necessary inputs (sample size, sets of numbers). Here every patient had an equal chance to be assigned to any one of the groups (Placebo and Intervention). The whole process of randomization was conducted by a competent third person who had no relationship with this research. Medicine was given to participants at the baseline after taking baseline blood sample and filling the WOMAC form. Compliance was assessed by returned capsule counts at the end of eight weeks (56±4 days) of treatment.

Patients were requested to avoid non-steroidal anti-inflammatory drugs (NSAIDs) for the next six weeks and any dietary supplement during the study period. Paracetamol was allowed for breakthrough pain <2000mg/day. If any patient required paracetamol after the initial two weeks of the treatment, the patients were instructed to stop medications 48 hours before the follow-up visit. They were allowed to withdraw from the study if their pain could not be relieved by paracetamol at a dose upto 2000 mg/day. Quadriceps strengthening exercises were advised to all the participants. Instruction for the activities of daily living (avoiding stairs, walking on flat surface, prohibiting weight carrying, using walking aid and kneecap during walking, avoiding kneeling and squatting, avoiding sitting for prolonged periods in with bent knees in one position, measures to reduce weight) was prescribed for all participants (both placebo and intervention group). They were in touch with the investigator over the phone during the study period.

2.3 Outcome measures

Knee pain, stiffness, and physical function of the index knee were assessed using the translated and validated Bangla version of WOMAC index version VA3.01 [22]. WOMAC is a 24 –item self-report questionnaire, that includes pain score (5 domain), stiffness score (2 domain) and physical function score (17 domain). Each domain in the visual analog scale VA3.01 is presented as a 0-100 mm line, where the patient will mark according to his/her severity perception. A higher score implies a deterioration in the condition.

Each domain score was converted into a scale of 0 to 100 for better representation. Finally, they were summed up for the total WOMAC score from 0 to 300. After ensuring that patients were free from any analgesic drugs in the last 48 hours they were assessed by the by the translated and validated Bangla version of WOMAC index version VA3.01 and after 8 weeks, the patients were again assessed by the WOMAC index. The total procedure took approximately 20-30 minutes for each patient in each visit.

2.4 Estimation of Laboratory Parameters

4 ml blood was collected from all the participants at baseline and after 8 weeks into clot activator test tubes for estimation of serum hsCRP and IL-6. The tubes were placed in the sampling rack for at least 30 minutes. Then the tubes were centrifuged for 10 minutes at 7000-8000 rpm. Serum hsCRP was measured by the nephelometric system at BN ProSpec automated analyzer and serum IL-6 was measured by chemiluminescence immunoassay at Snibe Maglumi in the department of Microbiology and Immunology, BSMMU.

Adverse events: After every 15 days over the telephone each participant was asked in a no leading manner about occurrence of any adverse events or discomfort. For each adverse events detailed information regarding onset, duration, action taken and outcome was recorded. To facilitate the use of computers a special spreadsheet prepared by the researcher was used in this study.

2.5 Statistical analysis

Statistical analysis was done by Microsoft Office Excel 2007. A Chi-square test was done to see the association between the intervention and the placebo arm. An unpaired t-test was done to compare a score between the two arms. Spearman’s rank correlation test was done to see a correlation between changes in serum levels with changes in WOMAC scores. The significant p value is <0.05.

3. Result

A total of 300 participants were screened (Figure 1). Of these Eighty (80) patients were enrolled based on the study’s eligibility criteria. They were randomly assigned to allocated interventions (astaxanthin group: n=40; placebo: n=40). 7 participants (8.75%) discontinued intervention or were lost to follow-up (astaxanthin group: n=4; placebo: n=5).

The baseline characteristics summarized in Table 1 reflect males and females, with moderate to severe knee OA involving unilateral or both knees. The majority of participants had Grade III or moderate knee OA.

Table 1: Baseline characteristics of participants at the time of enrollment (n=71).

Figure 1: Flowchart of Consolidated Standards of Reporting Trials (CONSORT).

3.1 Knee pain, stiffness and physical function:

Table 2 shows WOMAC knee outcome changes from baseline to after 8 weeks of treatment. In the intervention arm, compared to the placebo arm, WOMAC pain, physical function, and overall WOMAC ratings all significantly decreased. All 3 domains of the WOMAC index and total WOMAC scores also reduced significantly in the intervention arm compared to the baseline. But the reduction of stiffness score in intervention arm in comparison to the placebo arm is not statistically significant.

*; p<0.05. Unpaired t-test was done between placebo and intervention arm

Table 2: Comparison of WOMAC Score between two arms (at baseline and after 8 weeks of treatment).

3.2 Serum inflammatory markers

Table 3 summarizes changes in inflammatory markers- serum hsCRP and IL-6 from baseline to the end of intervention. Serum hsCRP reduced from 3.61 ± 1.49 to 3.02 ± 1.44 mg/L in the intervention arm which was statistically significant compared to the placebo arm. Serum IL-6 level reduced in the intervention arm from 7.06 ± 2.09 to 6.21 ± 2.45 pg/ml which was statistically significant compared to the placebo arm. On the other hand, a rise of serum hsCRP and IL-6 was observed in the placebo arm after 8 weeks.

Statistically significant positive monotonic correlation was found when a correlation test was done between changes in serum hsCRP with total WOMAC scores changes (p=0.00) in Figure 2 and changes in serum IL-6 with total WOMAC scores changes (p=0.000) in Figure 3.

Figure 2: Scatter diagram showing positive monotonic correlation of serum hsCRP level changes with total WOMAC Score changes in intervention arm after 8 weeks. Spearman’s rank correlation coefficient after 8 weeks was r= 0.43.

*; p<0.05. Unpaired t-test was done between placebo and intervention arm

Table 3: Comparison of serum High-Sensitivity C-Reactive Protein (hsCRP) level (mg/L) and Interleukin-6 (IL-6) level (pg/ml) between two arms (at baseline and after 8 weeks of treatment).

Figure 3: Scatter diagram showing positive monotonic correlation of serum IL-6 level changes with total WOMAC scores changes in intervention arm after 8 weeks. Spearman’s rank correlation coefficient after 8 weeks was r= 0.61.

3.3 Adverse events during treatment

Table 4 shows the adverse events in the placebo and intervention arms that occurred during the treatment period. There was no significant difference between adverse events of the placebo and intervention arm after 8 weeks of treatment (P=0.674). Adverse events in the intervention arm were - increased frequency of bowel movement (3) and anorexia (1). 3 participants had increase frequency with normal stool consistency around 3-5 times per day on first 2 days of treatment which resolved spontaneously on subsequent days. Whereas adverse events in the control arm were - insomnia (1) and nausea (1). All the events were mild in form and did not require discontinuation of treatment.

Fisher’s exact test was done between placebo and intervention arm

Table 4: Comparison of adverse events between two arms.

4. Discussion

This study is so far the first RCT conducted to explore the effects of astaxanthin on knee symptoms and serum inflammatory markers. There was no significant difference between placebo and intervention arms in baseline clinical characteristics. 

After 8 weeks of treatment, the WOMAC pain scores were significantly lower in the intervention arm (p<0.05) than in the placebo arm (p>0.05). It indicates that astaxanthin could effectively reduce pain in knee OA patients. Astaxanthin was found to be as effective as indomethacin in reducing inflammation-induced thermal and mechanical hyperalgesia in mice [23]. Anti-nociceptive function of astaxanthin was also found in experimentally induced pain in mice [24-26]. Intra-articular astaxanthin administration gave better outcomes than corticosteroid and hyaluronic acid in both dynamic gait parameters and histological examinations of articular structures in a rat osteoarthritis model [27]. This study further contributed to the evidence of the analgesic potential of astaxanthin.

The WOMAC stiffness scores decreased in the intervention arm after 8 weeks of treatment, however, this decrease was not statistically significant compared to the placebo arm (p>0.05). All of the study participants were given standard advice for symptomatic improvement including topical application of heat and quadriceps strengthening exercises, which might have contributed to the improvement of stiffness in both arms. As the exact mechanism of the effect of astaxanthin on joint stiffness is not yet known, further study warrants exploring the effect of astaxanthin on joint stiffness.

This study found, after 8 weeks of intervention, WOMAC physical function scores were significantly lower (p<0.05) in the intervention arm than the placebo arm (p>0.05). Reduction of pain might have contributed to the improvement of physical activities. Similar to this study, krill oil supplementation (a combination of astaxanthin and omega-3 fatty acids) resulted in significant improvement in the WOMAC index [28,29]. However, such a statistically significant improvement was not found in a study where 2g/day krill oil supplementation was given [30]. That study included participants with more severe disease (who had effusion synovitis and significant knee pain) whereas, such clinical severity was not in the inclusion criteria of others.

In this study, after 8 weeks of intervention, the total WOMAC scores were significantly lower (p<0.05) in the intervention arm than in the placebo arm (p>0.05). In this regard, we must mention that the WOMAC index is widely used in the clinical trials of knee and hip osteoarthritis and its improvement is considered as a significant therapeutic potential. Currently used NSAIDs and intra-articular steroids were also studied for their capacity in improving the WOMAC index [31-33]. So, the improvement in the WOMAC index indicates that astaxanthin has the potential to be an effective treatment option in OA and future studies should be directed to evaluate and include astaxanthin in OA treatment.

To explore how astaxanthin plays role in improving OA symptoms pathophysiology of OA was considered we observed astaxanthin’s effect on serum hsCRP and IL-6 levels. After 8 weeks of treatment, hsCRP levels decreased significantly in the intervention arm whereas, an increase was observed in the placebo arm. A similar decrease in hsCRP level was observed in studies using 4mg [34] and 2mg of astaxanthin [35]. On the contrary, a high dose of astaxanthin (12mg), not a low dose (6mg) was found to reduce hsCRP significantly [36]. In gouty arthritis, 8mg/day astaxanthin administration was as potent as 100 mg celecoxib in reducing CRP levels [37]. A significant inhibitory effect of astaxanthin on COX-II activity found in that study might have contributed to the reduction of hsCRP by astaxanthin. However, statistically non-significant changes in hsCRP levels with astaxanthin was also reported in several studies [38-40]. Among them 2 studies were conducted in healthy volunteers whose hsCRP levels were within the normal limits with not much scope to improve [38,40]. In the other study, research was conducted in renal transplant patients who had high CRP levels at baseline [39]. It indicates that there might be a certain range of baseline CRP within which the effect of astaxanthin is more prominent which needs to be confirmed in further studies.

In this study, serum IL-6 levels decreased significantly (p<0.05) in the intervention arm after 8 weeks of treatment, whereas, a non-significant increase was observed in the placebo arm (p>0.05). Astaxanthin reduces the expression of different cytokines including IL-6, IL-1β and TNF-ɑ [24,41,42], also downregulates IL-6 production by inhibiting the NF-κB signaling pathway [43] which might have contributed to the IL-6 reduction. This reduction of systemic IL-6 is supposed to be comparable and correspondingly reflect in synovial fluid as serum and synovial fluid IL-6 concentration correlates highly [44]. A similar significant reduction of serum IL-6 levels was found in other study also [36]. But an increase in serum IL-6 levels was found in another [35]. As the study included healthy and young females, the outcome might not be the same when pathology is present. A significant decrease in serum IL-6 levels with astaxanthin treatment was also showed in pre-clinical studies [45,46]. As IL-6 is known to decrease the production of type II collagen and increase the production of MMPs enzymes [47] the reduction of IL-6 seems to be beneficial in halting the OA progression.

A significant positive monotonic association was found between changes in serum hsCRP and serum IL-6 levels with changes in the total WOMAC scores. Changes in the hsCRP were noted to be positively and significantly associated with the change in knee pain [48]. A positive correlation was also observed between functional pain disability status and serum IL-6 levels [44]. A significant correlation between synovial fluid IL-6 with pain and total WOMAC sores was also found [49,50]. Regarding hsCRP, a significant association with WOMAC pain scores was also observed [50,51]. This indicates that the reduction of serum hsCRP and IL-6 might have contributed to the reduction of the WOMAC index in this study.

The incidence of treatment-related adverse events that were seen in the present trial was mild, did not require any treatment or discontinuation of treatment with astaxanthin and did not differ statistically between arms. This study contributes to the excellent safety profile of astaxanthin in knee OA patients.

To the best of our knowledge, this is the first study conducted on the effects of astaxanthin on adults with moderate to severe knee osteoarthritis. One of the strengths of the current study is its rigorous design, which hides the treatment assignment from all study participants and researchers and employs placebo capsules and containers that are identical to astaxanthin capsules. Another strength includes using a study population for whom the intervention is targeted, participants with appropriately diagnosed moderate to severe knee OA with room to improve knee pain and function were recruited. The use of a valid, reliable, and responsive standardized globalized measure (WOMAC Index) to assess knee pain, stiffness and function further contributed to the study's strength. Findings from this study could help millions of OA patients throughout the world by guiding future investigations into OA and possibly bolstering the usage of astaxanthin to treat it.

This study also has several limitations. It was a short duration study. Participants were given both naproxen/paracetamol and astaxanthin in the first 2 weeks of the study, so it is possible that there was a synergistic effect. The values of IL-6 and hsCRP measured after the intervention do not reflect the actual anti-inflammatory effect of astaxanthin as paracetamol was allowed as rescue medication for breakthrough pain. Number of days where rescue medication was needed was not possible to determine. Less than 10% of the study population had severe OA, so the effect of astaxanthin in severe knee OA patients was not clearly reflected.

5. Conclusion

The present study provides scientific evidence on the symptomatic improvement of knee osteoarthritis patients and reinforces the potential mechanism of action. Astaxanthin can also be an effective alternative treatment for mild to moderate inflammatory diseases where long-term treatment is necessary to slow the disease progression. A large-scale study is warranted to explore the required dose, effect duration and cost-effectiveness of astaxanthin in the treatment of OA knee joints. Change in joint morphology and effect on joints other than knee need to be determined in future studies. In conclusion, astaxanthin is safe and effective as an adjunct therapy to reduce symptoms and inflammation in patients with moderate to severe knee osteoarthritis.

Acknowledgments

The authors gratefully thank Dr. Golam Rabbani, Assistant Professor, Rheumatology, Rangpur Medical College, for sharing his original research, the translated and validated Bangla Version of WOMAC VA3.01. The authors also express gratitude to Prof. Abul Khair Mohammad Salek, Chairman, Department of Physical Medicine & Rehabilitation, BSMMU for his permission and technical support to work in the respective department.

Conflicts of interest

There are no conflicts of interest declared by all authors.

Funding

This work is a self-funded research with a partial research grant from BSMMU.

References

1. Primorac D, Molnar V, Rod E, et al. Knee osteoarthritis: a review of pathogenesis and state-of-the-art non-operative therapeutic considerations. Genes 11 (2020): 854.
2. Steinmetz JD, Culbreth GT, Haile LM, et al. Global, regional, and national burden of osteoarthritis, 1990–2020 and projections to 2050: a systematic analysis for the Global Burden of Disease Study 2021. The Lancet Rheumatology 5 (2023): e508-22.
3. Milenović N, Hornjak M, Lukač S, et al. Assessment of the quality of life in patients with knee osteoarthritis. Medicinski pregled 75 (2022): 103-8.
4. Bitton R. The economic burden of osteoarthritis. The American journal of managed care 15 (2009): S230-5.
5. Leifer VP, Katz JN, Losina E. The burden of OA-health services and economics. Osteoarthritis and cartilage 30 (2022): 10-6.
6. Kolasinski SL, Neogi T, Hochberg MC, et al. 2019 American College of Rheumatology/Arthritis Foundation guideline for the management of osteoarthritis of the hand, hip, and knee. Arthritis & rheumatology 72 (2020): 220-33.
7. Hauser RA. The acceleration of articular cartilage degeneration in osteoarthritis by nonsteroidal anti-inflammatory drugs. Journal of Prolotherapy 2 (2010): 305-22.
8. Wernecke C, Braun HJ, Dragoo JL. The effect of intra-articular corticosteroids on articular cartilage: a systematic review. Orthopaedic Journal of Sports Medicine 3 (2015): 2325967115581163.
9. Lim YZ, Hussain SM, Cicuttini FM, et al. Nutrients and dietary supplements for osteoarthritis. Bioactive food as dietary interventions for arthritis and related inflammatory diseases 1 (2019): 97-137.
10. Kane RL, Saleh KJ, Wilt TJ, et al. Total knee replacement: summary. InAHRQ evidence report summaries 2003 Dec. Agency for Healthcare Research and Quality (US) (2003).
11. Zarneshan SN, Fakhri S, Farzaei MH, et al. Astaxanthin targets PI3K/Akt signaling pathway toward potential therapeutic applications. Food and Chemical Toxicology 145 (2020): 1-17.
12. Guerin M, Huntley ME, Olaizola M. Haematococcus astaxanthin: applications for human health and nutrition. Trends in Biotechnology 21 (2003): 210-216.
13. Olaizola M, Huntley ME. Recent advances in commercial production of astaxanthin from microalgae. Biomaterials and bioprocessing 9 (2003): 143-64.
14. Guerin M, Huntley ME, Olaizola M. Haematococcus astaxanthin: applications for human health and nutrition. TRENDS in Biotechnology 21 (2003): 210-6.
15. Altindag O, Erel O, Aksoy N, et al. Increased oxidative stress and its relation with collagen metabolism in knee osteoarthritis. Rheumatology international 27 (2007): 339-44.
16. Miki W. Biological functions and activities of animal carotenoids. Pure and applied chemistry 63 (1991): 141-6.
17. Mehana ES, Khafaga AF, El-Blehi SS. The role of matrix metalloproteinases in osteoarthritis pathogenesis: An updated review. Life sciences 234 (2019): 116786.
18. Chen WP, Xiong Y, Shi YX, et al. Astaxanthin reduces matrix metalloproteinase expression in human chondrocytes. International Immunopharmacology 19 (2014): 174-7.
19. Huang LJ, Chen WP. Astaxanthin ameliorates cartilage damage in experimental osteoarthritis. Modern Rheumatology 25 (2015): 768-71.
20. Lana JF, Rodrigues BL. Osteoarthritis as a chronic inflammatory disease: a review of the inflammatory markers. Osteoarthritis biomarkers and treatments (2019).
21. Chan KC, Pen PJ, Yin MC. Anticoagulatory and antiinflammatory effects of astaxanthin in diabetic rats. Journal of food science 77 (2012): H76-80.
22. Rabbani MG, Haq SA, Bellamy N, et al. Development, linguistic and clinimetric validation of the WOMAC® VA3. 01 Bangla for Bangladesh Index. Rheumatology international 35 (2015): 997-1003.
23. Kuedo Z, Sangsuriyawong A, Klaypradit W, et al. Effects of astaxanthin from Litopenaeus vannamei on carrageenan-induced edema and pain behavior in mice. Molecules 21 (2016): 382.
24. Jiang X, Yan Q, Liu F, et al. Chronic trans-astaxanthin treatment exerts antihyperalgesic effect and corrects co-morbid depressive like behaviors in mice with chronic pain. Neuroscience Letters 662 (2018): 36-43.
25. Sharma K, Sharma D, Sharma M, et al. Astaxanthin ameliorates behavioral and biochemical alterations in in-vitro and in-vivo model of neuropathic pain. Neuroscience letters 674 (2018): 162-70.
26. Zhao L, Tao X, Song T. Astaxanthin alleviates neuropathic pain by inhibiting the MAPKs and NF-κB pathways. European Journal of Pharmacology 912 (2021): 174575.
27. Çağlar C, Kara H, Ateş O, et al. Evaluation of different intraarticular injection therapies with gait analysis in a rat osteoarthritis model. Cartilage 13 (2021): 1134S-43S.
28. Deutsch L. Evaluation of the effect of Neptune Krill Oil on chronic inflammation and arthritic symptoms. Journal of the American college of nutrition 26 (2007): 39-48.
29. Stonehouse W, Benassi-Evans B, Bednarz J, et al. Krill oil improved osteoarthritic knee pain in adults with mild to moderate knee osteoarthritis: a 6-month multicenter, randomized, double-blind, placebo-controlled trial. The American journal of clinical nutrition 116 (2022): 672-85.
30. Laslett L, Scheepers L, Antony B, et al. POS0276 Efficacy of krill oil in the treatment of knee osteoarthritis: a 24-week multicentre randomised double-blind controlled trial (2021).
31. Raynauld JP, Buckland-Wright C, Ward R, et al. Safety and efficacy of long-term intraarticular steroid injections in osteoarthritis of the knee: A randomized, double-blind, placebo-controlled trial. Arthritis & Rheumatism 48 (2023): 370-377.
32. Essex MN, Bhadra P, Sands GH. Efficacy and tolerability of celecoxib versus naproxen in patients with osteoarthritis of the knee: a randomized, double-blind, double-dummy trial. Journal of International Medical Research 40 (2012): 1357-70.
33. Gallelli L, Galasso O, Falcone D, et al. The effects of nonsteroidal anti-inflammatory drugs on clinical outcomes, synovial fluid cytokine concentration and signal transduction pathways in knee osteoarthritis. A randomized open label trial. Osteoarthritis and cartilage 21 (2013): 1400-8.
34. Baralic I, Andjelkovic M, Djordjevic B, et al. Effect of astaxanthin supplementation on salivary IgA, oxidative stress, and inflammation in young soccer players. Evidence-based complementary and alternative medicine 2015 (2015): 783761.
35. Park JS, Chyun JH, Kim YK, et al. Astaxanthin decreased oxidative stress and inflammation and enhanced immune response in humans. Nutrition & Metabolism 7 (2010): 1-10.
36. Chan KC, Chen SC, Chen PC. Astaxanthin attenuated thrombotic risk factors in type 2 diabetic patients. Journal of functional foods 53 (2019): 22-7.
37. Zhang L, Chen H, Ding K, et al. Astaxanthin intake alleviates gouty arthritis in patients and rats by modulating the levels of various inflammatory markers. Journal of Functional Foods 87 (2021): 104823.
38. Karppi J, Rissanen TH, Nyyssönen K, et al. Effects of astaxanthin supplementation on lipid peroxidation. International journal for vitamin and nutrition research 77 (2007): 3-11.
39. Coombes JS, Sharman JE, Fassett RG. Astaxanthin has no effect on arterial stiffness, oxidative stress, or inflammation in renal transplant recipients: a randomized controlled trial (the XANTHIN trial). The American journal of clinical nutrition 103 (2016): 283-9.
40. Chen JT, Kotani K. Effects of astaxanthin on liver and leukocyte parameters in healthy climacteric women: Preliminary data. Journal of Medicinal Food 20 (2007): 724-725.
41. Sakai S, Nishida A, Ohno M, et al. Astaxanthin, a xanthophyll carotenoid, prevents development of dextran sulphate sodium-induced murine colitis. Journal of clinical biochemistry and nutrition 64 (2019): 66-72.
42. Kochi T, Shimizu M, Sumi T, et al. Inhibitory effects of astaxanthin on azoxymethane-induced colonic preneoplastic lesions in C57/BL/KsJ-db/db mice. BMC gastroenterology 14 (2014): 1-0.
43. Kim YH, Koh HK, Kim DS. Down-regulation of IL-6 production by astaxanthin via ERK-, MSK-, and NF-κB-mediated signals in activated microglia. International immunopharmacology 10 (2010): 1560-72.
44. Azim S, Nicholson J, Rebecchi MJ, et al. Interleukin-6 and leptin levels are associated with preoperative pain severity in patients with osteoarthritis but not with acute pain after total knee arthroplasty. The Knee 25 (2018): 25-33.
45. Park JH, Yeo IJ, Han JH, et al. Anti-inflammatory effect of astaxanthin in phthalic anhydride-induced atopic dermatitis animal model. Experimental dermatology 27 (2018): 378-85.
46. Qiu X, Fu K, Zhao X, et al. Protective effects of astaxanthin against ischemia/reperfusion induced renal injury in mice. Journal of translational medicine 13 (2015): 1-9.
47. Wojdasiewicz P, Poniatowski ŁA, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis. Mediators of inflammation 2014 (2014): 561459.
48. Zhu Z, Otahal P, Wang B, et al. Longitudinal associations between serum inflammatory cytokines and knee bone marrow lesions in patients with knee osteoarthritis. Osteoarthritis and Cartilage 24 (2016): S332.
49. Radojčić MR, Thudium CS, Henriksen K, et al. Biomarker of extracellular matrix remodelling C1M and proinflammatory cytokine interleukin 6 are related to synovitis and pain in end-stage knee osteoarthritis patients. Pain 158 (2017): 1254-63.
50. Calvet J, Orellana C, Gimenez NA, et al. Differential involvement of synovial adipokines in pain and physical function in female patients with knee osteoarthritis. A cross-sectional study. Osteoarthritis and cartilage 26 (2018): 276-84.
51. Stürmer T, Brenner H, Koenig W, et al. Severity and extent of osteoarthritis and low-grade systemic inflammation as assessed by high sensitivity C reactive protein. Annals of the rheumatic diseases 63 (2004): 200-5.