Various stimuli such as toxic pathogens, noxious chemical and mechanical agents play a significant role in the inflammatory reaction and create a situation of tissue damage and infection (Eng and Lee, 2003). Macrophages play a significant role in the expansion, resolution, and maintenance of inflammation (Xu et al., 2003). Various stimuli viz., bacterial endotoxin lipopolysaccharides (LPS), interferon gamma (IFN-γ), extracellular matrix proteins, pro-inflammatory (IL-1β, TNF-α, and IL-6) and other chemical mediators (Pahan et al., 1998). During the inflammatory reaction, macrophages become secreted and activate due to release of an excessive amount of inflammatory mediators viz., IL-1β, TNF-α, and IL-6; that have been linked to the numerous inflammatory disease including rheumatoid arthritis, Crohn’s disease, cancer, asthma, Alzheimer’s disease, auto-immune pathologies, and psoriasis to name few (Kmieć, 2001).

Activation of few inflammatory enzymes including inducible nitric oxide synthetase (iNOS), cyclooxygenase (COX-2) and secretion of prostaglandin E₂ (PGE₂) from arachidonic acid, and have played a significant role in the expansion of oncogenesis and inflammatory reaction (Gungor et al., 2014). Rahman (2002), suggest that the inhibition of overproduction of inflammatory mediators such as TNF-α, PGE₂, NO, IL-1β, and IL-6 (Park et al., 2013)with up-regulation of COX-2 and iNOS is an imperative target for the prevention and treatment of inflammation and its related complications (Esser et al., 2014).

Typically arthritis is considered as the common inflammatory disease of joint characterized by being restricted joint movement, pain, and inflammation of synovial membrane (Sellam and Berenbaum, 2010). Rheumatoid arthritis (RA) is one of the common chronic inflammatory conditions which persists over months or years in developed countries and affects the synovial joints, a common cause of deformity and disability (Scott et al., 2010; Walsh and McWilliams, 2012). Joints changes, which probably characterize the autoimmune reaction, consisting of inflammatory reaction, erosion of bone, cartilage, and proliferation of synovial. The epidemiology study of arthritis showed the arthritis ratio between male and female is 3:1 and prevalence is in 1% world population (Scutellari and Orzincolo, 1998; Jimenez-Boj et al., 2005).

Anderson hadclaimed that the enhancement of hind paw edema during the adjuvant-induced infection in rats is paralleled and its due to up-regulation of lysosomal enzymes activities in that particular area (Tekieh et al., 2011). The above-discussed enzymes are involved in the degradation of structural macromolecules in cartilage proteoglycans and connective tissue (Martel-Pelletier et al., 2008; Sindhu et al., 2012). They also induce the destruction of extracellular activity by enhancing the activity of lysosomal enzymes. Further, they destroy the cellular structures and may participate in tissue damage in RA (Kaur et al., 2012). Complete Freund’s adjuvant (CFA) is considered as the most reliable model for estimation of an anti-arthritic drug. CFA containing Mycobacterium tuberculosis per milliliter sterile paraffin oil (Bhalekar et al., 2015; Kumar et al., 2015). CFA induced the acute inflammation is linked with mast cell activation, leukocyte infiltration and secretion of free radicals and cytokines in circulation. CFA induced rheumatoid arthritis (RA) characterized by rapid activation of macrophage and secretion of various enzymes into the circulation playing a significant role in the vascular destruction, fibrosis and tissue destruction over a period of time (Harford et al., 2011; Kasimanickam et al., 2014).

Researchers are in the opinion that macrophage-derived cytokines like TNF-α, IL-6, and IL-1β are directly involved in every phase of inflammatory reactions linked with the pathogenesis of RA initiating from autoimmunity in peri-articular phase to upholding joining tissue destruction and chronic inflammation of synovitis (Scott et al., 2010). As on date available treatment of RA is steroidal, non-steroidal and immunosuppressive drugs which are used to control the inflammation symptoms and pain, they are associated with various undesirable side effects. With these obstructions, the researcher focuses on their research on alternative therapies, such as herbal and herbal based drugs. Now, 80% of the World population used the plant-based drug for the effective treatment of pain associated with the RA (Sahoo et al., 2010; Mori et al., 2011; Boissier et al., 2012).

Crocetin (carotenoid) extracted from the saffron flower and its medicinal properties have been tested in Chinese medicine (Bhandari, 2015). Multiple studies have suggested the role of spices in the reduction of chemical inflammation in the rodent. Very rare studies suggest that the saffron also inhibit the expansion of inflammation in the mice (Festuccia et al., 2014; Bhandari, 2015). Glycoprotein considers as the important compound which is involved in the cellular function and play an important role in the surface properties of cells and also involved in the tumorigenesis inflammation and mediator of immunological specificity (Gutheil et al., 2012). The available literature suggests that the carbohydrate moieties of glycoproteins have been implicated in the movement of metabolites inside the cell membrane and also create a direct relationship between the tumorigenesis, inflammation, and glycoprotein (Gutheil et al., 2012; Festuccia et al., 2014; Bhandari, 2015). To our knowledge, this is the first attempt to scrutinize the anti-inflammatory effect of crocetin in LPS induced RAW 264.7 macrophages and CFA induced chronic inflammation.

This study was an effort to scrutinize the anti-inflammatory effect of crocetin in in vitro and in vivo experimental model by LPS stimulated RAW 267.4 macrophages and CFA induced chronic arthritis model, respectively. Crocetin exhibited the inhibitory potential against the COX-2, iNOS, pro-inflammatory (IL-1β, IL-6, IL-10, and TNF-α) in in vitro assays, a potent in vivo potential in a rat model, suggested the protective and therapeutic effect on inflammatory tissue. Lipopolysaccharide (prototypical endotoxin) isolated from the Gram-negative bacterial membrane can directly activate the endothelial cells, macrophages and complement triggering generation of inflammatory mediators viz., COX-2, iNOS, pro-inflammatory (IL-1β, IL-6, IL-10, and TNF-α), respectively.

Excessive generation of NO, play a significant role in the provocation of chronic inflammatory and circulatory shock diseases, like inflammatory lung disease, septic shock, colitis and inflammatory hepatic dysfunctions (Zhang et al., 2017). Many of these disease conditions show the quick onset and expansion, frequently that result in the failure of conventional anti-inflammatory treatment with high mortality rates, a concurrent down-regulation of NO production pathways (Zhang et al., 2012), as exposed by crocetin may suggest managing the quick progression of inflammatory reactions. In vitro macrophages cell model or other cell lines model are very useful to study the high-level production of NO (Zhang et al., 2012). On the basis of the result, we can conclude that crocetin inhibits the LPS stimulate NO production via suppression of inflammatory expression including COX-2 and iNOS (Figure 1). On the other hand, it was also scrutinized the cytotoxic effect of crocetin in RAW 267.4 macrophages by MTT assay that suggest that its minimum dose did not influence the viability of RAW 267.4 cells. Therefore, the suppression of LPS induced NO production via crocetin was not the result of a probable cytotoxic upshot on these cells.

For confirmation of NO and PGE₂ inhibitory mechanism of Crocetin, it showed the significant inhibition of PGE₂ and slight reduction of NO production. The current finding suggests that Crocetin directly down-regulated the COX-2 and also affected the iNOS production in a different way (Hseu et al., 2005; Yoon et al., 2009; Heo et al., 2012).

Various researchers suggest that the excessive generation of PGE₂ and NO play a significant role in the provocation of chronic disease including pulmonary and hepatic dysfunction disease (Ohgami et al., 2003). Secretion of PGE₂ and NO play an important role in the alteration of molecular mechanisms that regulate the PGE₂ generation pathway (Heo et al., 2012). The available reports suggest that the generation of both PGE₂ and NO was blocked via NOS inhibitors in RAW 267.4 macrophages cells, this suppressive effect was reverted via co-incubation with NO synthesis precursor (L-arginine) (Zhang et al., 2012). Therefore, reduction of iNOS via non-selective NOS inhibitors calmed the generation of PGE₂ and NO simultaneously in LPS induced macrophages, suggesting the secretion of endogenous NO from the macrophages that exerted a stimulatory action on boosting the PGE₂production (Hseu et al., 2005; Yoon et al., 2009; Heo et al., 2012). On the contrary, activation of COX, turn into the alteration of the L-arginine-NO production pathway, whereas the suppression of COX down-regulate the NOS effect in human platelets. Our results are the outcome of cross-talk between PGE₂ and NO pathways.

CFA induced RA model used for the estimation of pharmacology and pathophysiological control of inflammatory processes, as well as the, scrutinize the anti-arthritic effect of drugs. CFA induced RA showed the chronic inflammation, in the first 2–4 days induced the local inflammation and after that induced the chronic inflammation which remains for next few weeks (Bernardi et al., 2009; Rayhana et al., 2014). CFA has been shown the number of clinical, immunological and chronic feature similar to human arthritis. The primary reaction of the CFA showed the expansion of inflammation and developed the arthritic nodules in the tail and ear and persist the swelling in contralateral and showed the delayed response (secondary reaction). CFA induced RA rats showed the enhancement of paw joint in from day 3 and remain increase until 28 days. CFA control group rats showed the increase joint diameter till 28 days and suggest the delayed immunological flare in the RA disease (Kuroki et al., 2011; Wang et al., 2012).

Currently, available treatment for the arthritis is NSAIDS (Non-Steroidal Anti-Inflammatory Drugs) viz., indomethacin, aceclofenac, phenylbutazone, ibuprofen etc., DMARDs (Disease Modifying Anti-Rheumatic Drugs) viz., cyclosporin A, methotrexate etc, Anticytokine therapy such as infliximab, adalimumab etc and immune suppressive drugs are commonly used to control the inflammation and pain during arthritis (Kumar et al., 2016). Indomethacin is the first choice of the drug due to its specific action on COX and PGE₂. But this drug has a limitation due to gastro intestinal irritation, diarrhea, rectal irritation, skin rash, hematologic toxicity and loss of response with chronic use (Kaithwas and Majumdar, 2010). It is moreover desirable that the more potent antiarthritic drugs be investigated for specific action on the inflammation and pain during arthritis.

During the arthritic condition, hematological parameters altered due to the expansion of disease. A similar result was observed in the CFA induced group rats. CFA group rats exhibited the increased level of ESR, WBC and reduced level of Hb RBC (Kumar et al., 2016). During the arthritic condition, the level of RBC and Hb decrease and create the anemia condition to the patient due to the deformability of erythrocyte (shorten the lifespan of erythrocyte). During the disease, the level of Hb was decreased due to the expansion of disease, resultant start the destruction of premature RBCs and reduction of erythropoietin (reduced the level of bone marrow erythropoietin) (Kumar et al., 2017). A similar condition was observed in our study. Another parameter such as WBC, its considers as the significant marker of the immune system which is linked with the induction of inflammation and its related other diseases. During the arthritic disease condition, IL-1β mediated WBC level increase and start the generation of colony stimulating factor, granulocyte and inflammatory macrophages (Jeyadevi et al., 2013). Crocetin significantly (P < 0.001) reduced the granulocytes and migration of inflammation macrophages at dose-dependent manner. Increased level of ESR start the generation of endogenous proteins and actively take part in the progression of the disease. Result suggests that the dose-dependent treatment of crocetin altered the hematological condition near to the normal level and suggest the anti-arthritic effect.

Various researchers suggest that the hepatic parameters such as ALP, SGPT, and SGOT are the tool for the estimation of the anti-arthritic effect of the tested drug (Kumar et al., 2016). Serum SGPT and SGOT level played an important role in the formation of biologically active chemical mediators such as bradykinins in the inflammatory process and suggest a positive relation between the enhanced activity of serum ALP and RA activity (Kaithwas and Majumdar, 2010). The aminotransferases enzymes were significantly boosted in disease rats since there is the marker of kidney and liver impairment, which are also considered as the features of adjuvant arthritis (Gautam et al., 2016). During arthritis, the level of ALP increased in the bone fraction and liver or may be due to increasing the level of both isoenzymes. This in turn implicates a localized bone loss in the form of bone erosion and periarticularosteopenia, as the enzyme is released into circulation in the course of bone formation and resorption (Kaithwas and Majumdar, 2010). Concentration-dependent treatment of crocetin significantly decreased the activity of hepatic enzymes such as SGOT, SGPT, ALP and suggest the reduced the bone loss and provide the protection to the organ against adjuvant-induced arthritis in the rats. From the result, we can say that crocetin provided the anti-arthritic effect against the CFA induced arthritic rats due to alteration of biochemical parameters.

In the current experimental study, we scrutinized the macrophage-derived pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β. Various researchers suggested that the level of pro-inflammatory cytokines increase during the CFA induced RA (Qureshi et al., 2011). These cytokines boosted the local systemic inflammatory response and induced the cartilage, infiltration of immune cells and start the degradation of joint via degrading the enzymes such as metalloproteinases (MMPs) (Impellizzeri et al., 2012). For confirmation of the exact mechanism of action, in the current experimental study, we evaluated the pro-inflammatory cytokines in synovium and serum (Hübner et al., 1996; Fernandes et al., 2002; Kim et al., 2007; Neurath, 2014). The level of TNF- α was up-regulated in the arthritis patient. The up-regulated level of TNF- α start the damage of gastrointestinal at indomethacin group rats. CFA induced group rats showed the up-regulation of TNF- α and dose-dependent treatment of crocetin showed the down-regulation of pro-inflammatory cytokines level.

CFA induced control group rats showed that the rat’s hind paw increased in diameter till the end of the experimental study. The increase in joint diameters increase is a result of arthritis disease (Hussein et al., 2012; Kumar et al., 2017). CFA induced group rats showed the reduced body weight and crocetin treatment showed the increased body weight and suggests the anti-arthritic effect. CFA induced showed the decreased body weight due to deficient absorption of nutrient via intestine and the treatment showed the increased body weight suggest the normalizing process of the intestine (Jalalpure et al., 2011; Nair et al., 2011; Suke et al., 2013; Jiang et al., 2014). The possible mechanism may be improved the level of nutrient from intestine absorption and down-regulation of distress induced through the severity of arthritis.

In the current investigation, the joint inflammation of crocetin treated group rats decreases at the end of the experimental study. CFA control rats showed the increased level of TNF- α and dose-dependent treatment of crocetin exhibited the reduction of TNF-α, however, there was a higher TNF- α level was observed in the indomethacin group (Gautam et al., 2016). The increased level of TNF- α found in the indomethacin group due to gastrointestinal damage, which further caused the gastrointestinal ulcer. Inflammatory mediators such as IL-6, IL-1β, VEGF, and TNF-R1 are correlated with our finding in the animal investigation, as the expression of IL-6, IL-1β, VEGF, and TNF-R1 were observed to be attenuated in the synovial membrane of crocetin and indomethacin group (De Groote et al., 1992; Stevens et al., 2008; Nadeau et al., 2011; Studer et al., 2011). Indomethacin was more effectual in reducing the joint inflammation via inhibiting the inflammatory mediators. The same results were observed in the crocetin group rats, which showed the reduction of joint inflammation and pro-inflammatory cytokines in a dose-dependent manner. The result suggests that crocetin preventing the destruction of joint and synovial membrane via the different/additional mechanism.

Various researchers suggest that pathogenesis of RA having various inflammatory reactions such as transcription factor, NF-κB, which play a significant role in the joint degradation and inflammation via increased the expression of pro0inflammatory genes viz., IL-6, IL-1β, TNF-α, MMPs, and chemokines (Bonizzi and Karin, 2004; Shakibaei et al., 2007; Choi and Lee, 2010; Ali et al., 2011). The increased level of NF-κB in the synovial joint of animal and human during the RA is well observed. Now, the researcher focuses their research on the RA via inhibiting the NF-κB expression for the treatment of it. In the current investigation, we scrutinized the effect of NF-κB in the control and treated group rats (Makó et al., 2010; Mori et al., 2011). Dose-dependent treatment of crocetin significantly (P < 0.001) decreased the expression of NF-κB as compared to CFA induced rats. The current finding suggests that crocetin could be responsible for anti-arthritic activity via inhibition of NF-κB expression.

Nrf2 is considered as the significant transcription factor which was found to numerous set of enzymes including GPx, glutathione S-transferase and HO-1 via binding of DNA sequence called antioxidant response element (ARE), which has the ability to reduced the pro-inflammatory pathway and antioxidant response (Itoh et al., 1999; Li et al., 2004; Jung and Kwak, 2010; Vomhof-DeKrey and Picklo, 2012). Various researchers suggest that cross relationship between NF-κB/IL-1β and Nrf2 and serve the joint destruction and oxidative injury in animals (Lee and Surh, 2005; Vomhof-DeKrey and Picklo, 2012). In the current experimental study, we have found that the crocetin treatment increased the mRNA expression of HO-1 and Nrf2 in CFA induced rats as compared to CFA control group rats and suggesting the suppression of oxidative stress and pro-inflammatory cytokines via altering the oxidative stress and pro-inflammatory cytokines level.

On the basis of the result, we can conclude that crocetin was an effective agent in attenuating the adjuvant-induced arthritis rats in a concentration-dependent manner, and therefore it could be scrutinized as a probable treatment for human chronic arthritis condition. The results suggest that crocetin is beneficial in the treatment of painful inflammatory and chronic arthritic condition. Additionally, particular identify the gene responsible for the anti-arthritic effect.

YL and RK performed the experimental study. JW, YL, and RK interpreted the biochemical data. All the authors equally contributed for the proofreading.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.