Introduction: 1. Introduction:
OA is a persistent, progressive infectious joint illness, that causes cartilage, and bone destruction. In addition to progressive destruction of articular cartilage, the disease develops with symptoms such as calcium crystal deposition (calcification) on cartilage, subchondral bone thickening, osteophyte formation, synovial inflammation, and chondrocyte hypertrophic differentiation of chondrocytes (1,2). However, the most frequent symptom and reason of disability is the pain in patients with osteoarthritis. Inflammation can accelerate the process of cartilage and bone destruction. One of the factors that cause inflammation is cartilage fragmentation and their floating in the synovial fluid that stimulates the synovial membrane and eventually causes synovitis. This inflamed membrane subsequently releases other inflammatory mediators that further damage the cartilage (3).
We know that cytokines, along with growth factors, play a key role in the pathophysiology of osteoarthritis. They are intimately related to functional changes in the synovium as well as cartilage, followed by subcutaneous bone. Cytokines, along with growth factors, are secreted mainly by synovial membrane cells and propagated through cartilage by synovial fluid. In the process, they inactivate cartilage, resulting in the secretion of secondary metabolites such as pro-inflammatory cytokines as well as proteases. The major cytokines (anti-inflammatory) and antagonists - involved in the pathophysiology of OA such as the Interleukin 1 family, especially iL-1 α, iL-1β as well as interleukins include iL-4, iL-6, iL-8, iL-10, iL-11, iL-13, iL-17 besides TNF-α and iL-1Ra. Growth factors such as TGF-β, FGF, PDGF, and IGF are involved in OA, in which TGF-β has both a synthetic and a catabolic effect that depends on several factors such as concentration, target cell, and related tissue. (4,5). It is right that pro-inflammatory cytokines, especially iL-1β and TNF-α, play a major function in the onset and progression of the OA so that iL-1β causes cartilage destruction and TNF-α promoting the inflammatory process (6,7).
Cartilage and synovial cells are stimulated by iL-1 beta and TNF-alpha, and produce other cytokines such as LIF, il-6 and iL-8, as well as prostaglandin E2 or PGE2 (8). The cytokine iL-1β plays a potential role in OA. It can independently induce catabolic effects and inflammatory response and combine with other intermediaries. It is one of 11 members of the IL-1 (IL-1F) family (9). In patients with osteoarthritis, it is observed that the concentration of IL-1𝛽 in the joint fluid and synovial membrane, as well as cartilage, is significantly increased (10,11).
In the course of OA, Tumor necrosis factor-alpha (TNF-𝛼), together with IL-1𝛽 have been identified as two very important and fundamental factors occurring in the pathophysiological processes (12). TNF-𝛼 is made and splashed by the similar joint cells produced IL-1𝛽, and an increase in its concentration is also noticed in the similar parts, like SF, SM, cartilage, and SCB layer, where rose amounts of IL-1𝛽 are also noted (13, 14). Besides the FLS cells, tumor necrosis factor receptors 1 are rose (15).
Interleukin-6 is a complex distinguished by comprehensive interactions in actions taking place in human beings. It may be classified as anti-inflammatory interaction which powerfully operates the immune system and raises the inflammatory responses, even if several are considered of its results (16). IL-6 production in damaged joint tissues is normally in reaction to Interleukin -1𝛽, TNF-𝛼 also is mostly mediated by osteoblasts, chondrocytes, adipocytes, FLS, and macrophages (17). An increase in the concentration of interleukin-6 in serum and synovial fluid has been observed and this increase is emphatically associated with the severity of wounds on X-ray body scans (18, 19, and 20). The effect that Interleukin -6 has on articular cartilage is no different from other cytokines, but it reduces the production of type 2 collagen and MMP enzymes. (21, 22). Interleukin-6 is known as the crucial cytokine, brings about variations in the subchondral bone layer (23, 24). It is influenced by osteoclast structure or bone defect while manifesting its cooperation with Interleukin -1β, and TNF (25).
Another corporator of IL-18 is the Interleukin -1F family (26). At first, pro-interleukin-18 is like precursor form which has 192 amino acids but after activation of caspase-1 or proteinase-3, it reduces to 157 amino acids and switches to active forms. The rise of caspase-1 and articular cartilage or synovium cause to an active form of IL-18 and IL-1β (16). The creation of interleukin-18 is produced in osteoblasts, chondrocytes, macrophage, and FLS (27). There is a significant relationship between increasing gene expression of iL-18 in synovium, synovial fluid, blood serum, and cartilage and the severity of the disease seen in radiographic images (28, 29).
One of the most widely used dietary supplements containing medicinal plants is the extract of milk thistle. This supplement is one of the most popular products in the United States (30). Silybum marianum L, also known as wild artichoke is a kind of herbal plant. Silymarin is an active ingredient in milk thistle (47). Silymarin is a combination of flavonoliganes containing silibinin A, silibinin B, isosilibinin A, isosilibinin B, silydianin, silychristin, isosilychristin as well as taxifolin flavonoid (37,70). silymarin consists of approximately 50 to 60% of Silibinin, 20% of Silicristin, 10% of Silydianin and 5% of Isosilybin and silibinin has the highest biological activity. (31,32). Milk thistle is discovered throughout the milk thistle, nevertheless is more focused on its seeds and fruits. The seeds of milk thistle hold a relatively high level of oil, about 18-31 percent rich in unsaturated fatty acids, of which 42-54 percent is linoleic acid and 21-36 percent is oleic acid (43). Silymarin is a compound contains various properties like anty-inflammatory hepatoprotective, antioxidant (33,34), heart-protective (36,37), hypocholesterolemic (37), anti-diabetic (38,39), anticancer (40,41), and cardioprotective activities. Clinical studies have been demonstrated that silymarin has very rare side effects at high doses (>1500 mg/day), however it has quick metabolism and penurious absorption (42).
Although various treatment options are used to manage OA, these options do not affect on preventing the process of their degradation. Regularly used pharmacological agents include corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), and hyaluronan. Especially NSAIDs which are widely used and their long-term use is associated with serious side effects such as gastrointestinal lesions (48). The use of effective therapies with fewer side effects has led patients with OA to think of a way to control pain and confirm their function and quality of life. (43).
The intention of these experiments is to treat OA and acquiring a path to block the process of articular cartilage destruction we strived to demonstrate a new method of treatment based on herbal medicine to cure and diminish the symptoms of disease and pain by using the AESM (Alcoholic Extract Silybum marianum L), furthermore to protect patients from the severe side effects of common chemical drugs. In this study, we used dexamethasone and ibuprofen as steroids and non-steroidal anti-inflammatory drugs (NSAIDs), respectively. Meanwhile, the rabbits became infected using monosodium iodoacetate (MIA) (44).
Methods: TAK (TAK-242) or the TLR4 signaling pathway suppressor as well as anti-TLR4 production of Sigma- Aldrich (St. Louis, MO, USA), alkaline phosphates conjugate, Secondary antibody consist of Goat Anti-Rabbit IgG (H+L) (Sigma-Aldrich, Germany), MTT or 3-(4,5- dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide, CD90, CD68, CD14, and Vimentin, dimethylsulfoxide (DMSO) (Sigma-Aldrich, MO, USA). hematoxylin and eosin staining (H&E) (Bio-Optica, Italy). Bovine serum albumin (BSA), Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium, and 0.25% trypsin ethylenediaminetetraacetic acid (trypsin–EDTA). (Gibco, Life Technologies Corp, USA). TRIzol reagent (Carlsbad, Calif., USA). In addition (MIA, 4mg/50μl, Sigma-Aldrich, MO, USA) and RNA isolation kit (Cinna Pure RNA Kit Cat. No.: PR891620, IRAN Tehran).
Preparation of Silybum marianum L. Extract (ESM)
Silybum marianum L was obtained from Iran’s center of genetic resources. Then the plant was extracted under sterile conditions with a rotary evaporator vacuum device (Model 750, manufactured in West Germany). The ESM powder (1mg) needs to be soluble in DMSO and congested as aliquots (20 mM) at -20οC before reuse.
Ethics
In this research, the ethical ideology has been observed and acted by. We comply with international law on working with laboratory organisms. This paper was accepted by the Ethics Committee of the University of Payme Noor Tehran/Iran (IR.PNU.REC.1399.114).
Animals and Husbandry
Thirty healthy male New Zealand White rabbit sage 5 months, the average weight of 2.5 kg was used to investigate the role of AESM in osteoarthritis induced by MIA. The rabbits were maintained in an environment with precise temperature (25°C ± 1°C) and humidity settings(55% ± 15%), air conditioning, and regular light and dark cycles. All rabbits were retained in metal cages measuring (35×50×25 cm) and allowed to access standard food (Industrial Expansion Development Co Behparvar, Iran) and ad libitum water. at any time of the day or night.
Results: 3. Results
3.1. Effects of Treatment on Body Weight
At 35 days after surgery, the average weight of rabbits in each group was 2.39 ± 0.15, respectively. No significant difference in body weight was observed between the groups.
3.2. Effects of Treatment on Knee Thickness
We accurately measured the diameter of rabbit joints before OA and then one week after OA and also after treatment. The mean joint diameter of rabbits was measured before the onset of the disease at 15.8 mm and this value increased to an average of 19.8 mm after MIA. Finally, after treatment, the rabbit joint diameter, although generally reduced, did not reach the initial mean and the average was recorded at 16.6 mm (Table 7).
3.3. Cytotoxicity assays
Although cell cultures were served 24 hour with AESM, it did not bring about any changes in cell numbers at the concentrations of (1x10-3, 9x10-3, 1x10-2, 9x10-2, 1x10-1, 9x10-1, 1, 9,18, 36, 45, 54, 63, 72, 81, 90 and 100μg/ml). There is no sign of side effects higher than 1x-10 μg/ml. Enhancement of concentration 9 μg/ml leads to a particular diminished in the cell viability (LC50 was calculated 45μg/ml and its average was 12.38 μg/ml) (56)(Fig.6).
3.4. The result of AESM in RFLS
RFLS cells natured for 3 days in the medium lonely, AESM leads to decrease the level of genes such as iL-1, iL-6, iL-18, and TNF-α regarding to LPS-induced chondrocytes (Table 4 and Fig.3). Operated of cells by 20 ng/mL of LPS caused a remarkable rise in the articulation of the proinflammatory cytokines. As expected, dexamethasone and NSAID had a significant effect on reducing the expression of cytokines in stimulated cells, but DMSO did not affect. The AESM almost caused a decrease in the percentage of cells stimulated by 50% which is a significant decrease compared to Dexamethasone and NSAID (Table 4,6 and Fig. 3).
3.5. The Effect of AESM on Cytokine Gene Expression in Cartilages
The plant extract was able to compete with chemical drugs and also had a significant effect on reducing inflammation and decreasing the process of joint cartilage destruction. The highest impact effect of AESM after the iL-1 at 29.85 is related to iL-18 at 49.48(Table 5 and Fig.4). Similar results were obtained in the articulation of the Interleukin-6 and Tumor Necrosis Factor-α genes which was acceptable.
3.6. The Change in serum level of proinflammatory cytokines
Examination of blood samples and analysis of its data indicated that consumption of AESM not only significantly reduced the expression of pro-inflammatory genes in the blood, especially iL-6 but also proved its anti-inflammatory properties and confirmed the results of former tests. Therefore, it can be a worthy therapeutic purpose for OA patients in the future. We believe that the effects of the AESM on reducing pro-inflammatory genes expression will be more severe with increasing concentration (Fig.5).
3.7. Rabbits histological evaluation
Thickening of the middle part of cartilage in rabbits was done by hematoxylin and eosin staining (H&E) (Bio-Optica, Italy) and the results are shown on Figure 7. The concentration of proteoglycans and elastin fibers in the matrix of cartilage in the group of rabbits with OA treated with AESM was obviously increased compared to the control group(P<0.05). According to the results, it can be mentioned that the plant extract has increased the thickness of the middle part of cartilage in rabbits with OA. Due to the high amount of essential fatty acids, AESM has caused the accumulation of indispensable proteoglycans of cartilage. Increasing cartilage thickness and accelerating the healing process of cartilage wounds and accumulation of proteoglycans in the group treated with AESM indicate that AESM has been able to improve articular cartilage and help treat OA (Fig 7).
Conclusion: 4. Discussion
OA is known as a joint illness distinguished by inflammation of the synovial membrane and progressive loss of extracellular matrix proteins and gradual destruction of articular cartilage and is the best source of physical disease (58,59). The biochemical cause of OA is still unknown, but the onset of the disease usually begins with the formation of abnormal structures in the joint or abnormal pressure on the joint surface (60). Nowadays, no satisfactory and definitive treatment has been developed for OA (61). Common treatments for OA include pharmacotherapy, non-pharmacotherapy, supplementation, and surgery (62). Non-specific cyclooxygenase inhibitors (COX-ІІ) Non-steroidal anti-inflammatory drugs (NSAIDs) and oral analgesics such as acetaminophen (63), antidepressants, and glucosamine are used in combination with intra-articular injections of steroids and hyaluronic acid derivatives (64,65). Unfortunately, the use of these chemical drugs has many side effects such as severe gastrointestinal bleeding and digestive disorders, and has severely limited the use of this treatment by patients (66). Therefore, in this study, we are in pursuit of an appropriate therapeutic method to reduce the symptoms of the disease and pain with the least side effects in patients. Our method is a complementary, and alternative treatment method. We used the medicinal plant Silybum marianum L,(AESM) to treat sick rabbits and evaluated the effect of injecting administration of AESM in comparison with NSAIDs and steroid drugs in vitro or in vivo. Accurate identification of the components of AESM is possible using HPLC-UV (67,68). Milk thistle seeds contain 4 to 6% silymarin and its extract contains 65 to 80% silymarin (69) and about 25 to 35% of the oil full of fatty acids. The highest concentration of fatty acids is 48.88% unsaturated fatty acid Linoleic acid (70). The compounds are divided into two main groups, flavonolignans and non-flavonolignans, and Non- flavonolignans group itself is divided into two smaller groups, polyphenolic compounds, and flavonoids. Finally, the flavonoids group can be divided into taxifolin and quercetin. Silibinin (or Silybin) has the highest concentration and maximum biological properties among other components of silymarin (69-71). In OA, immune cells potentiate the inflammatory process in the joint by secreting proinflammatory cytokines such as Interleukin -6, Interleukin1-α, Tumor Necrosis Factor-α and Interleukin -1β (72). Interleukin -1β is not expressed by tissues under normal and physiological conditions of the body (73) and is first synthesized as pro-iL-1β, which is biologically inactive and converted to active iL-1β after caspase-1-dependent proteolysis (74,75). In addition, interleukin-1β is activated by neutrophils-derived elastase and cathepsin-G and mast cell-derived proteolysis (76,77). Unlike iL-1β, iL-1α does not require primary processing for biological activity and activation. Activated iL-1β along with iL-1α both persuade many cytokines and play an important role in the body's irritant response (78,79). Interleukin -6 is known as a proinflammatory cytokine required in many incurable inflammatory illnesses secreted by joint tissue and, after binding to its soluble receptor (iL-6R), transsignals and activates the immune system by calling mononuclear cells (such as monocytes) to the inflamed area of the joint (80). TLR4 activates the MyD88 pathway in the plasma mambrane. Then it enters the cytoplasm through CD14 dependent endocytosis and initiates the TRIF-dependent cascade. Also, TRIF activates TRAF3, TRIF3 which causes the release of type 1 interferons and CCL5. TRAF6 activates three important signaling pathways after TAK1 including AP-1, CREB and NF-κB. They increase the expression of proinflammatory cytokine genes such as iL-6, TNF-α , pro-iL-1β and pro-iL18. Our researches revealed that AESM has an ability to suppress the TLR4 signaling pathway and also the reduction of genes expression such as iL-1α, iL-6, iL-18 and TNF-α on plasma and after the induction of RFLS cells induced by MIA and AESM. Staining of cartilage cells by H&E method showed that AESM can also inhibiting inflammation accelerates of the healing process of articular cartilage (81).
5. Conclusion:
As we know, during OA disease, the immune system is activated by increasing the expression of proinflammatory cytokine genes such as iL-1α, iL-18, TNF-α, and iL-6 by immune cells and cartilage cells at the joint surface, the destruction of articular cartilage begins, intensifies progressively and eventually leading to cell death. Today, the drugs used to treat OA are not very effective and also have dangerous side effects. Therefore, patients are less inclined to take these drugs. This study aimed to find and propose a drug to reduce the symptoms of OA and treat it with minimal side effects and increase the community's hope for definitive treatment of osteoarthritis. In this paper, we examined the ESM effectiveness, on rabbits with OA. The rabbits became ill by injecting MIA into the wrist joint. After 60 days of treatment of rabbits with AESM, we measured the gene expression of proinflammatory cytokines including TNF-α, iL-6, iL-1α and iL-18. Also, we compared the effect of AESM with Dexamethasone and Ibuprofen(NSAID), on the expression of those genes related before. Our experiments indicated that consumption administration of AESM reduces the expression of TNF-α, iL-6, iL-1α and iL-18 genes and can compete well with common drugs in the treatment of OA. Reduction of expression of these cytokines in articular cartilage cells as well as immune cells (such as monocytes and macrophages) cause reduce joint inflammation and symptoms such as pain, swelling, and inflammation gradually disappear. We suggest that with further research on the medicinal plant milk thistle and its seeds extract, this plant can be introduced as a very effective and efficient drug to reduce joint inflammation, pain, and decrease the expression of proinflammatory cytokine genes and a new treatment for OA with minimal side effects.