Arthritis is more prevalent autoimmune disease that affects the cartilage, bones, and synovial membranes (McInnes and Schett, 2007). The exact pathology of rheumatism is unknown, however, important validations have shown that its extensive destruction of bone and cartilage is caused by synovial neovascularization, proteinase-mediated suspension of cartilage articular matrix, elevated proinflammatory cytokine expression and osteoclast-mediated bone disintegration (Liu et al., 2013). The pathological similarity of the CFA-induced arthritis model to human arthritis makes it a highly recommended model for preclinical drug testing (Neng-Yu Lin et al., 2013). Inflammatory reactions start a few days after CFA injection inoculation, and secondary lesions appear two weeks after primary lesions (Alamgeer et al., 2017). CFA, which activates cell-arbitrated immunity and speeds up the production of antibodies, is made up of temperature-killed M. tuberculosis added in liquefied paraffin (Kim et al., 2016). There are three stages to mycobacterium-induced edema: starts with the induction phase without any sign of synovitis, early synovitis, and late synovitis. This showed the accumulation of protein debris and extracellular fluid at the site of inflammation (Jitta et al., 2019). The goal of the current study was to assess the effectiveness of Arbutin in CFA induced model of arthritis.
The administration of Arbutin, (25, 50 and 100 mg/kg, oral) has significantly decreased arthritic index and paw volume in treated rats as compared to the arthritic control group. Our compound Arbutin has an inhibitory effect on TNF-α, IL-1β, IL-6, PGE2, IL-8 and COX-2, which are the markers of inflammation (Qasim et al., 2023) as shown in Fig. 1a, 1c. Further, weight loss in treated groups was prevented through the reduction in IL-1β and expression level of TNF-α (Fu et al., 2019) as shown in Fig. 1b. Furthermore, the standard drug piroxicam also has reduced arthritic index and paw volume and there was increased in weight of CFA induced rats. Rheumatoid cachexia developed as a result of lysosomal protease-induced muscle proteolysis, which is triggered by decreased absorption of prostaglandin-E2, 14C-glucose, and 14C-leucine in the intestines of rats (Alamgeer et al., 2017). Intestinal absorption in rats was also resolved and reduced the expression of pro-inflammatory cytokines (Ahsan et al., 2021).
Hematological and biochemical tests in CFA-induced arthritis were conducted which show anemia in rats indicated by a decrease in the level of hemoglobin (Hb) and red blood cell (RBCs) count. Anemia may be caused by the destruction of premature reticuloendothelial cells, a decrease in erythropoietin, or a decrease in iron loading in the RES and synovial joints (Alamgeer et al., 2017). The significant increase in Hb and RBCs by Arbutin, most likely as a result of NF-κB reduced expression. Thus, it prevented the inflammatory cascade and restored the normalcy of inflammatory cell invasion in the synovial fluid (Iqubal et al., 2019). Furthermore, the production of cytokines like TNF-α and IL-6 leads to an increase in platelet and white blood cells (WBCs) counts in arthritic rats (Fig. 2h, 2e). Additionally, due to the greater secretion of IL-6 and TNF-alpha, which initiates the acute phase reaction in RA, arthritic rats showed increased levels of platelets and WBCs. Despite inhibiting the production of IL-6 and TNF-α, Arbutin has reduced the number of platelets and WBCs in rats; however, this may be because of its immunomodulatory action. Arbutin has normalized inflammatory cell invasion in synovial fluid and inhibited the inflammation cascade. It also increased hemoglobin and RBCs, possibly as a result of decreased NF-κB expression (Isaacs and Iqbal, 2019).
The two major markers for the early detection of systemic polyarthritis are elevated serum ferritin and C-reactive protein (CRP). An effective method for assessing the active inflammatory process is the CRP, which is a significant predictor of systemic inflammation (Razi et al., 2012). When compared to arthritic control rats, Arbutin treatment prevented the acute phase reactions. Increased levels in ESR and CRP in arthritic control rats indicated infection in the blood, whereas rats given piroxicam and Arbutin, the levels decreased which is in line with previous study (Prakash Babu et al., 2014). A possible parameter implicated in the pathobiology of RA could be abnormal changes in the values of RF and also in CRP in serum. Initiation of immune cascade progresses arthritis by the production of factor that is RF which is directed against the Fc portion of autoantibodies. There was a noteworthy reduction in Rheumatoid factor (RF) levels among treatment groups with Arbutin. Immunoglobulin molecules like rheumatoid factor (RF) have the ability to trigger an immune response because the body perceives them as "non-self" (Yildirim et al., 2004). Furthermore, increased bone erosion and lysosomal enzyme integrity may be the cause of the diseased control group's elevated serum ALP concentration (Chakraborty et al., 2010). Rats in the disease control group showed remarkable increased levels of the RF and CRP, indicating that these had been exposed to an inflammatory stimulus following CFA vaccination. Conversely, the groups treated with piroxicam and Arbutin (100 mg/kg) showed a marked reduction (p < 0.001) in the values of RF and C-reactive proteins as given in Table 2. Nonetheless, the ALP was markedly decreased when Arbutin at varying doses was given to the treatment groups. This also prevented the abnormal increase in SGPT and SGOT, which may have been brought on by a decrease in bone loss and an improvement in lysosome stability as given in Table 2. Renal safety of Arbutin was assessed through urea and creatinine levels in treated groups of rats (Silpavathi et al., 2023).
The development of arthritis is significantly influenced by proliferating macrophages, T lymphocytes, and synovial cells (Weyand et al., 2001). Macrophages and stimulated T cells release proinflammatory cytokines, which contribute to the development of arthritis (Vandooren et al., 2009). Immunity (T-cell mediated) triggers the release of pro-inflammatory cytokines and triggers antibody synthesis resulting in the degeneration of joints. Excessive expression of these cytokines results in apoptosis, bone loss, tissue eradication, irreversible tissue proliferation (Yang et al., 2013). Accordingly, possible counteragents for the cytokines have the power to shield cells from inflammatory processes (Shin et al., 2016). In the present study, Rt-qPCR was used to study the impact of Arbutin on the expression of pro-inflammatory cytokines panels (TNF-alpha, IL-6, IL-17 & IL-1β), inflammatory like NF-κB & COX-2, and anti-inflammatory gene pointers (IL-4 and IL-10). Osteoclast induction, bone erosion, and matrix metalloproteinase (MMP) production have all been related to IL-1β (Barksby et al., 2007). Moreover, the article refers to endogenous MMP inhibitors as tissue inhibitors of MMPs (TIMPs). Soluble IL-6R is characterized by the induction of TIMP formation in cultured chondrocytes and synovial fibroblasts by IL-6. Extracellular matrix turnover is aided by the protective catabolic response that tissues produce (Silacci et al., 1998).
Multiple studies have reported that infiltrating cells, monocytes, and macrophages secrete pro-inflammatory cytokines. Three main cytokines involved in initiation, progression and development of arthritis are TNF-α, IL-1β, and also IL-6 (Yap et al., 2018). As a result, pro-inflammatory cytokines have appeared as fascinating targets of arthritis. In our research, the administration of Arbutin to CFA-induced arthritis rats effectively decreased the TNF-α, IL-17, NF-κB, IL-6, COX-2 and also IL-1β levels which ultimately decreased proinflammatory cytokines as shown in Fig. 2. The present study found that rats given Arbutin and piroxicam showed a significant reduction in COX-2 levels, whereas animals with arthritis as control showed higher levels. As a result of the reduction in COX-2 production, this implies that piroxicam and Arbutin have partially prevented the harmful effects of CFA by blocking prostaglandin synthesis. Anti-arthritic actions of Arbutin may possibly be mediated through the inhibition of arachidonic acid metabolism in line with the already reported study (Uttra et al., 2018). Inflammation leads to increased release of TNF-α by phagocytes leading to leucocyte adhesion and penetration in the vascular endothelium. Additionally, TNF-α inhibits the synthesis of bone collagen mainly by causing increased bone deterioration and hyperplasia of the fibroblast (Singh et al., 2021). In our present study, Arbutin possesses anti-inflammatory potential in many mediators of inflammation (TNF, COX-2, IL-1 β and IL-17) and can used to treat arthritis. The main factors in the development of arthritic decay demonstrated that TNF-α buildup magnifies the appearance of PGE-2, IL-1 β, and also IL-6 leads to hyperplasia of the synovial joint, increased enzyme devastation accretion, collagenase activation, and osteoclast disease (Alunno et al., 2017).
In the current investigation, diseased control rats showed elevated expression of PGE-2 and COX-2; conversely, rats receiving Arbutin treatment (25, 50 and 100 mg/kg) have shown a significant decrease in PGE-2 and COX-2. Interleukin-10 alters arthritis synovitis by deactivating macrophages and suppressing the cellular defense mechanism (Bozkurt et al., 2006). As RA advances, IL-10 suppresses the immune response mediated by the Th1 and inhibits the functioning of antigen-presenting cells too, that provide protection to the integrity of the joints (Uttra et al., 2018). IL-17 has a crucial role in arthritis, affecting osteoclast genesis, granulopoiesis, and release of pro-inflammatory cytokines such as IL-1β, TNFα, IL-8, and IL-6 (Schinocca et al., 2021). In arthritic control rats, Arbutin decreased the raised IL-17, IL-1β, and IL-6 levels, suggesting its anti-inflammatory and anti-arthritic efficacy. IL-6 increases bone resorption and the production of autoantibodies. These mediators cause inflammatory tissues in the synovial membrane to release collagenase (Mima and Nishimoto, 2009).
It was reported that PGE-2 levels in CFA-induced rats were closely linked to cartilage erosion, blood vessel dilatation, joint swelling, redness and pain. Prostaglandin E synthase (PGES) produces PGE2 from COX-II. When arthritis is active, pro-inflammatory cytokines up-regulate COX-II, which results in an indirect increase in PGE-2 (Uttra et al., 2019). Angiogenesis, vasodilation, fluid extravasation and vascularization in the synovial membrane mediated by the production of PGE-2, are upregulated at the injection site due to increase in COX-2 levels (Shabbir et al., 2016). Increased PGE-2 expression results from an excess of pro-inflammatory cytokines. Increased PGE-2 levels spur vasodilation, bone destruction and also migration of leukocytes to the site of inflammation by speeding up osteoclast differentiation and producing degrading enzymes as shown in Fig. 3a.
Lipoxygenases are crucial in the production of leukotrienes (B4, C4, D4, and E4), which are important in inflammatory disorders (Gheorghe et al., 2009). The CFA-induced rise in PGE-2 and 5-LOX levels in arthritic rats was significantly reduced by administering Arbutin. Hence, the outcomes of the present investigation demonstrated that Arbutin caused a remarkable decrease in PGE-2 levels in rats with arthritis induced by CFA. In the current investigation, increased levels of anti-CCP were detected early on the 7th day using the CFA model followed by arthritis. The levels of Anti-CCP reduced significantly in Arbutin (25, 50 and 100 mg/kg) treated rodent groups. A decrease in Anti-CCP concentration explained the inhibition of TNF-α (El-Ashmawy et al., 2019) as shown in Fig. 3. ROS leads to alterations in cell organelles including DNA damage, protein oxidation, inactivation of enzymes, and peroxidation of lipids (Sghaier et al., 2011). Consequently, the injured joints are destroyed because superoxide anions produced during phagocytosis cause the second messenger system-dependent, NF-κB-dependent expression of the pro-inflammatory cytokines (Prakash Babu et al., 2014). These are shielded against tissue damage by reactive species by endogenous antioxidant enzymes such as catalase. The enzyme catalase contributes to the reduction of hydrogen peroxide (H2O2) in synovial tissue spaces. SOD catalyzes the conversion of superoxide to hydrogen peroxide and oxygen free radicals, which are then further catalyzed into molecules of oxygen and water by catalase enzymes. In comparison to healthy rats, the arthritic control rats showed decreased levels of anti-oxidant parameters (SOD, CAT and GSH) and raised levels of pro-oxidant marker (MDA). Arbutin has shown a potential role in mitigating the deterioration caused by free radicals. Oxidative enzymes like GSH, CAT and also SOD were overexpressed, MDA production decreased and lipid peroxidation was less prominent in rats with arthritis treated with Arbutin, indicating antioxidant activity which is given in Table 3. In vitro free radical scavenging assay through DPPH radical, is used for assessing the antioxidant potential of Arbutin which has shown strong antioxidant properties (Baliyan et al., 2022) .
In the current study, histopathological assessment showed that animal groups that received treatment with Arbutin inhibited the leukocyte migration, thereby protecting joints from joint deformity, cartilage destruction and formation of pannus as depicted in Fig. 4. Furthermore, radiological analysis demonstrated that, in comparison to arthritic control rats, Arbutin demonstrated a significant protective effect against the growth of secondary lesions and cartilage damage as illustrated in Fig. 5. As mediators in the pathophysiology of RA, free radicals and reactive oxygen species (ROS) have been supposed to cause tissue damage. Rheumatism causes various cells such as neutrophils, macrophages and dendritic cells to infiltrate the diseased articulations, and then produce ROS, which breaks through the antioxidant defense system and release more of them at the site of inflammation (Nich and Goodman, 2014).
We also analyzed appropriate poses for protein–ligand interactions, the dependable method of molecular docking, which is one of the most important docking subtypes in the pharmaceutical industry is protein-ligand docking (Azam and Abbasi, 2013). Strong interactions were indicated by the highest negative bond energy values in the docking (Herawati et al., 2021). In this way, the most suitable binding site for the ligand can be predicted along with the molecular interactions that are explained. Furthermore, interactions between the ligand atoms and amino acids could be identified according to their distance from one another. The objective of the current investigation was to study how Arbutin interacted with IL-17, iNOS, TNF-α, COX-2, NFκB, 5-LOX, IL-4, and IL-10. Together with pharmacological evidence supporting its anti-arthritic efficacy, Arbutin demonstrated strong binding interactions with each of these chosen targets. The in silico and in vivo analysis of the present study yielded very consistent results.