Gaining Insights To Understand The AntiInammatory Functions of Mesenchymal Stem Cell-Derived Conditioned Medium In Combination With Stigmasterol In IL-1β-Stimulated Rat Articular Chondrocytes – An In-Vitro Approach

Osteoarthritis (OA) is the most prevalent joint disease predominantly characterized by inammation which drives cartilage destruction. Mesenchymal stem cells-condition medium (MSC-CM) or the secretome is enriched with bioactive factors and possesses anti-inammatory and regenerative effects. The present study aimed at evaluating the effects of combining MSC-conditioned medium with stigmasterol compared with per se treatments in alleviating interleukin-1beta (IL-1β)-induced inammation in rat chondrocytes. Stigmasterol is a phytosterol exhibiting anti-inammatory effects. IL-1β (10ng/ml) was used to induce inammation and mimic OA in-vitro in primary rat articular chondrocytes. The IL-1β-stimulated chondrocytes were treated with MSC-CM, stigmasterol, and a combination of MSC-CM and stigmasterol for 24 hours. Cell viability was measured using MTT assay. Protein expression of inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), collagen II (COL2A1) and matrix metalloproteinase (MMP) ‐ 13 were evaluated by immunouorescence. Gene expression levels of MMP-3, MMP-13 and A Disintegrin-like and Metalloproteinases with Thrombospondin Motifs (ADAMTS)-5 were measured using qRT-PCR. NF-κB signaling pathway was studied using western blotting. A signicant reduction in the expression of iNOS, IL-6, MMP-3, MMP-13 and ADAMTS-5 and a signicant increase in COL2A1 expression was observed in the rat chondrocytes across all the treatment groups. However, the combination treatment of MSC-CM and stigmasterol remarkably reversed the IL-1β-induced pro-inammatory/pro-catabolic responses to near normal levels comparable to the control group. The combination treatment (MSC-CM+stigmasterol) elicited a superior anti-inammatory/anti-catabolic effect by inhibiting the IL-1β-induced NF-κB activation evidenced by the negligible phosphorylation of p65 and IκBα subunits, thereby emphasizing the benet of the combination therapy over the per se treatments. attenuated


Introduction
Osteoarthritis (OA) is the common joint disorder primarily affecting the diarthrodial joints and advances into severe degenerative arthritis. OA forms the leading cause of disability and joint pain impacting the socio-economic status across the globe with a prevalence of 16% (Cui et al., 2020). With a multifactorial etiology including age, obesity, trauma, sports injuries and altered biomechanics, OA progresses as an irreversible, often heterogeneous disease commonly ending up with complete joint destruction . In ammation triggered by mechanical trauma vis-à-vis initial cartilage breakdown has been identi ed to be a critical determinant in initiating and accelerating OA development(van der Kraan and van den Berg, 2012). Evidence(s) suggest for a prominent role for IL-1β, a key early-stage proin ammatory cytokine, in exacerbating the pathogenesis of OA by activating key signaling pathways including the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen-activated protein kinases (MAPK), and transcription factors (Daheshia and Yao, 2008). Consequently, this triggers a sequelae of intracellular events that culminate in the activation of cartilage-degrading proteinases (MMPs and ADAMTS), production of in ammatory molecules (iNOS), pro-in ammatory cytokines (IL-6), causing a decrease in the synthesis of cartilage extracellular matrix ultimately leading to cartilage degradation and joint damage (Ding et al., 2020). Till now there is no single therapeutic agent that has been deemed safe and effective for treating OA. Strategies aimed at targeting the IL-1β-induced catabolic metabolism and in ammatory responses have proven to offer immense promise(s) in curtailing OA progression. our earlier study re-instates the e cacy of combination line of approach in OA management (Pragasam et al., 2021). Phytosterols or plant sterols are a group of naturally occurring plant compounds that have been shown to elicit immense health bene ts across a wide range of disease conditions. Stigmasterol, also known as anti-stiffness factor, is the most common phytosterol found in severalnuts, seeds, legumes, banana, cabbage, and medicinal herbs including Akebia quinata, Gypsophila oldhamiana, Emilia sonchifolia, Eucalyptus globules, Aralia cordata, Emilia sonchifolia, Theobroma cacao L (Yadav et al., 2018). In addition to its proven anti-osteoarthritic effects (Chen et (Haque et al., 2021). Very recently, we demonstrated that intra-articular transplantation of mesenchymal stem cells combined with stigmasterol resulted in e cient cartilage repair/regeneration compared to the per se treatments in a monosodium-iodoacetate induced rat model of OA (Pragasam et al., 2021). Hence, in the present study we aimed to assess the potential of a combining rat bone marrow mesenchymal stem cells-derived conditioned medium (MSC-CM) with stigmasterol in ameliorating the IL-1β-induced in ammatory responses in rat primary chondrocytes. We hypothesize that combining MSC-CM with stigmasterol will elicit a superior anti-in ammatory vis-à-vis bene cial effect compared to the per se treatments in-vitro.

Animals
The animal study was approved by the Institutional Animal Ethical Committee, National Institute of Nutrition (NIN), Hyderabad (P29F/III-IAEC/NIN/12/2016/SSJ/WNIN(CG)-6F/WNIN-Gr-Ob-42F). Four-weeks old female Wistar rats were obtained from the Animal Facility, NIN and were housed in standard polypropylene cages, maintained at 22 ± 1°C with 12 h dark/light cycles, and humidity of 50-60%, and were fed standard laboratory rat chow prepared at our animal facility with free access to water. All the experiments were performed in accordance with the regulations and guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Isolation of rat primary chondrocytes
Primary articular chondrocytes were isolated from four-weeks old WNIN Wistar rats according to a previously published method (Oseni et al., 2013) with slight modi cations (Pragasam and Venkatesan, 2020). Brie y, the rats were euthanized by CO 2 asphyxiation and the hind limbs were collected in sterile phosphate buffered saline (PBS, pH 7.4). The femur and tibia were separated from the hind limbs under sterile conditions. The cartilage at the ends of the femur and tibia were harvested using sterile scalpels, washed in sterile PBS, digested in 0.15% collagenase-II for 4 hours at 37℃, 5% CO 2, followed by the addition of culture medium (Dulbecco's Modi ed Eagle Medium/Ham's F12 (1:1) (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), P/S (penicillin (100 IU/ml) and streptomycin (100 IU/ml)). The digested cell suspension was then centrifuged at 300xg for 10 min using a Sigma 3-18KS centrifuge. The cell pellet was washed twice in culture medium and seeded in a T25 culture ask at a density of 5 x 10 3 cells/cm 2 . The culture medium was changed every 2 days and upon reaching con uency, the cells were trypsinized using 0.25% trypin-EDTA and sub-cultured subsequently. The cells at passage 2 were used for all the further experiments.

Isolation of rat bone marrow mesenchymal stem cells (BM-MSCs)
Post euthanization by CO 2 asphyxiation, the femur bones were harvested from four weeks old female WNIN Wistar rats under aseptic conditions in sterile PBS containing antibiotics. The BM-MSCs were isolated according to an earlier published protocol (Madhira et al., 2012). Brie y, the femurs were cut open at the metaphyseal ends and the bone marrow was ushed out using a 2 ml syringe containing DMEM/F12 containing 10% FBS and P/S. The ushed out bone marrow was adequately dispensed to get a uniform cell suspension which was washed thrice at 1800 rpm for 10 min. The resulting cell pellet was resuspended in the cell culture medium (DMEM/F12 containing 10% FBS and P/S and seeded in T25 asks at a seeding density of 5 x 10 3 cells/cm 2 . The culture medium was changed every 2 days and upon reaching con uency, the cells were trypsinized using 0.25% trypsin-EDTA. The BM-MSCs at passage 3 to 5 were used for the subsequent experiments. Brie y, the BM-MSCs were seeded in T75 asks at a density of 1.5 x 10 6 cells/ ask in DMEM/F12 containing 10% FBS and P/S. Upon reaching 80-90% con uency, the cells were washed with PBS and added with serum free DMEM/F12 and maintained at 37 ℃, 5% CO 2 . The asks were incubated for 48h after which the medium was removed and centrifuged at 1500 rpm for 5 min at 4 ℃ to remove any cell debris. The resulting supernatant was termed the MSC-CM and used for the subsequent experiments.

Cell viability assay
The effect of stigmasterol on cell viability in the isolated rat primary chondrocytes was measured using the MTT assay. The chondrocytes were seeded at a density of 5x10 3 cells/well in a 96-well plate. After allowing the cells to adhere for 24h, the cells were treated with different concentrations of stigmasterol (0-100 μM) in culture medium. After 24 hours of the treatment, the media was removed, and the cells were incubated with MTT solution (5mg/ml) for 4 h at 37°C. The supernatant was removed and the formazan crystals were dissolved by adding DMSO to the wells. The absorbance was measured at 570 nm using a microplate reader (BioTek, US). The number of viable cells in the treatment groups was expressed as a percentage of the number in the control group.

Experimental approach
The chondrocytes were grown in 6-well plates at a seeding density of 2 x10 5 cells/well and cultured in DMEM/F12 containing 10% FBS and P/S until con uency. The study comprised of the following ve groups: (1)

Western Blot analysis
The total protein was extracted from the chondrocytes cultured in 6-well plates using ice-cold radio immunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors. The lysates were sonicated, kept on ice for 10 min followed by centrifugation at 12000 rpm for 15 min at 4°C. The protein concentration in the supernatants was measured using the bicinchoninic acid (BCA) protein assay kit (G-Biosciences, MO, USA). Equal amounts of protein (40 μg) were separated by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene di uoride membrane (Bio-Rad, USA). After blocking with 5% nonfat milk for 2h, the membranes were incubated with the primary antibodies against NF-κB p65 (1:1000, Cell Signaling Technology), phospho-NF-κB p65 (1:1000, Cell Signaling Technology), IκBα (1:1000, Novus Biologicals), phospho-IκBα (1:1000, Novus Biologicals) and β-actin (1:1000, Cell Signaling Technology) overnight at 4℃ with gentle rocking. The membranes were washed with TBST and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000, Southern Biotech) at room temperature for 1 hr. After washing with TBST, the membranes were visualized with an enhanced chemiluminescence reagent (G-Biosciences, MO, USA) and the images were captured using an iBrightFL1500 Imaging System (ThermoFisher Scienti c, USA).

Statistical analysis:
The values given represent average of three independent experiments, carried out in duplicates. All data have been expressed as mean ± standard deviation (SD). Signi cant difference between the groups was measured by using one way analysis of variance (ANOVA) followed by Dunnett's test using GraphPad Prism 8.0.2 software. p < 0.05 implied signi cance.

Results
Effect of stigmasterol on cell viability: The rat primary articular chondrocytes were isolated and cultured in DMEM/F12 containing 10% FBS and P/S. The cultured chondrocytes were identi ed by their typical polygonal shape when viewed under a bright eld inverted microscope (Fig. 1A). The effect of stigmasterol on the cell viability/cytotoxicity of the cultured rat articular chondrocytes was evaluated by the MTT assay. The cells were treated with stigmasterol at varying concentrations ranging from 0-100 µM for 24h. There was no signi cant cytotoxicity in the chondrocytes upon treatment with stigmasterol across all the tested concentrations (Fig. 1B). Stigmasterol at a concentration of 50 μM was used for all the further experiments of the study. This dosage was selected also considering a previous study by Gabay et al. (2010). Figure 1 Characterization of rat BM-MSCs The cultured rat BM-MSCs were identi ed by their typical broblast-like spindle shaped morphology when viewed under the microscope (Fig. 2A). The ability of the isolated BM-MSCs to differentiate into the chondrogenic, osteogenic and adipogenic lineages was assessed using commercially available kits following the manufacturer's instructions. The BM-MSCs differentiated into the chondrogenic lineage as identi ed by the Alcian Blue staining for proteoglycans (Fig. 2B). The osteogenic differentiation potential of the BM-MSCs was con rmed by the Alizarin Red staining for calcium phosphate deposits (Fig. 2C) and the adipogenic differentiation potential of the BM-MSCs was con rmed by the Oil-Red-O staining of the lipid droplets (Fig. 2D). The BM-MSCs also stained positive for the expression of MSC-speci c markers CD29 (Fig. 2E), CD73 (Fig. 2F). CD90 (Fig. 2G) and CD106 (Fig. 2H) as con rmed by immuno uorescence using a Leica SP5 confocal laser scanning microscope using the LAS software. Figure 2.
Effects of MSC-CM, stigmasterol, and combination of MSC-CM and stigmasterol on the expression of iNOS, IL-6, MMP-13 and COL2A1in IL-1β stimulated rat chondrocytes by immuno uorescence IL-β is the most critical pro-in ammatory cytokine implicated in OA pathogenesis which upregulates several in ammatory/catabolic mediators involved in cartilage degradation. The effects of MSC-CM and stigmasterol per se or in combination on the expression of key OA-related in ammatory markers such as iNOS (Fig. 3), IL-6 ( Fig. 4) and MMP-13 (Fig. 5), and cartilage turnover marker COL2A1 (Fig. 6)  The mRNA expression levels of MMP-3, MMP-13 and ADAMTS5 genes in IL-β-stimulated rat chondrocytes treated with MSC-CM and/or stigmasterol was evaluated using qRT-PCR. As depicted in Fig.7, IL-β stimulation in chondrocytes (IL-β group) resulted in a signi cant increase in the mRNA expression levels speci c for MMP-3 (Fig. 7A), MMP-13 (Fig. 7B) and ADAMTS5 (Fig. 7C). Per se treatments with MSC-CM (IL-β +CM) or stigmasterol (IL-β +S) resulted in a signi cant reduction in the gene expression levels of MMP-3, MMP-13 and ADAMTS5 compared to the IL-β group. Co-treatment of MSC-CM and stigmasterol (IL-1β + CM + S) also resulted in a signi cant reduction in the expression of MMP-3, MMP-13 and ADAMTS5 genes when compared to the IL-β group. However, the combination therapy (IL-1β + CM + S) markedly reduced the gene expression of these OA-speci c tissue degrading proteases to near normal levels as comparable to that of the control group underscoring the bene t of the combination therapy when compared to the per se treatments. Figure 7 Effects of MSC-CM, stigmasterol, and combination of MSC-CM and stigmasterol on the inhibition of IL-1β-mediated activation of NF-κB in rat chondrocytes by western blot analysis IL-1β exerts its downstream catabolic/in ammatory effects chie y mediated through the NF-κB signaling pathway. The effects of MSC-CM, stigmasterol, and the combination of MSC-CM and stigmasterol on the IL-1β-induced NF-κB activation in rat primary chondrocytes was evaluated using western blot analysis. As shown in Fig. 8, the results indicated that IL-1β treatment triggered NF-κB activation as seen by increased expression of phosphorylated-p65 (Fig. 8B) and phosphorylated-IκBα (Fig. 8C). In the presence of MSC-CM (IL-1β + CM) or stigmasterol (IL-1β +S), the phosphorylation of both p65 (Fig. 8B) and IκBα (Fig. 8C) decreased signi cantly. Nonetheless, the results revealed that the inhibition of IL-1βinduced NF-κB activation was the greatest in combination therapy group (MSC-CM + stigmasterol) evidenced by the negligible levels of phosphorylation of both p65 and IκBα comparable to control levels. The effects of the treatments in inhibiting IL-1β-induced NF-κB activation was found to be as follows: (IL-1β + CM + S) > (IL-1β + CM) > (IL-1β +S) when compared to the IL-1β group. Figure 8.

Discussion
Chronic low-grade in ammation driven by pro-in ammatory mediators has been identi ed to have a pivotal role in propelling OA pathogenesis characterized by cartilage destruction, subchondral bone remodeling, pain, joint failure and deformity (Robinson et al., 2016). Apparently, therapeutic strategies targeting the in ammatory responses in OA could hold immense clinical value in treating OA. In the current study, we evaluated for the rst time the bene cial effects of combining MSC-CM with stigmasterol compared with the per se treatments in negating the IL-1β-induced in ammatory responses in rat primary chondrocytes in-vitro.
Chondrocytes form the sole cellular components found in the cartilage tissue; they synthesize the extracellular matrix components and provide matrix turnover which are indispensable for maintaining the functional and structural integrity of the cartilage (Goldring and Marcu, 2009). However, under OA, this dynamic balance between the cartilage matrix synthesis and degradation is disrupted by the induction of stress-induced in ammatory mediators. Notably, IL-1β exerts its detrimental effects on the chondrocytes by compromising the chondrocyte viability, inhibiting the anabolic processes critical to cartilage homeostasis vis-à-vis production of ECM components, impairing their fate of differentiation and dysregulating their functions to induce pro-in ammatory and pro-catabolic responses (Charlier et al., 2016). Hence IL-1β induction in chondrocytes has been employed as a conventional way to generate an OA model in vitro (Li et al., 2019).
In our present study, we assessed the effects of IL-1β stimulation on the expression of in ammationassociated markers including iNOS, IL-6, and the cartilage turn-over markers COL2A1, MMP-3, MMP-13 and ADAMTS5 in rat chondrocytes. The inducible nitric oxide synthase (iNOS) contributes to OA pathogenesis by increasing the production of nitric oxide which in turn inhibits the synthesis of collagen type II and proteoglycan (Sasaki et al., 1998). IL-6 is another key pro-in ammatory cytokine and a well known player in amplifying OA by its inherent ability to work in synergy with IL-1β (Flannery et al., 2000) and through its role in transsignaling (Scheller et al., 2011). The cartilage matrix degrading enzymes including those that belong to the matrix metalloproteinase (MMP) family and the a disintegrin and metalloproteinase with thrombospondin type-1 motifs (ADAMTS) family have been identi ed as potent diagnostic/therapeutic targets for early OA. Of the many MMPs involved in OA pathogenesis, MMP-13 has been identi ed as the central node in the cartilage degradation network in OA and is highly e cient in degrading collagen II, in addition to degrading proteoglycan, types IV and type IX collagen, osteonectin and perlecan in cartilage (Shiomi et al., 2010). MMP-3 also plays a crucial role in cartilage degradation capable of degrading a broad range of substrates including collagen types II, III, and IV, laminin, proteoglycans, and bronectin, and also activates other MMPs including MMP-1, MMP-2, MMP-9 and MMP-13 (Tong et al., 2017). ADAMTS-5 is the primary aggrecanase responsible for aggrecan degradation in OA with evidence demonstrating for ADAMTS-5 knockout mice to exhibit signi cant resistance to cartilage erosion in a surgical model of OA (Glasson et al., 2005;Stanton et al., 2005). In our current study, we found thatIL-1β stimulation signi cantly up-regulated the protein expression of iNOS, IL-6, MMP-13 with a concomitant down-regulated protein expression of COL2A1 in the rat chondrocytes. IL-1β treatment also signi cantly increased the gene expression levels of MMP-3 and ADAMTS-5 in the chondrocytes. These altered gene/protein expression levels of key OA-speci c markers were signi cantly reversed by the MSC-CM/stigmasterol per se treatments. However, the combination therapy of MSC-CM and stigmasterol demonstrated a remarkably better anti-in ammatory and anti-catabolic effect by signi cantly restoring the levels of these OA speci c markers (iNOS, IL-6, COL2A1, MMP-3, MMP-13 and ADAMTS5) to near normal levels comparable to that of the control group accentuating the bene ts of the combination therapy over the individual treatments.
In OA, the overexpression of pro-in ammatory and catabolic factors in response to in ammatory stimuli is regulated by a set of pro-in ammatory signaling pathways. Chie y, the transcription factor nuclearfactor kappa B (NF-κB) mediated signaling is a critical and master regulator orchestrating the expression of various genes involved in in ammation, immune response, adhesion, tissue degradation and apoptosis implicated in OA pathophysiology (Choi et al., 2019). In chondrocytes, the IL-1β-mediated production of the downstream in ammatory/catabolic mediators such as the iNOS, IL-6 and MMPs is essentially regulated by the NF-κB pathway (Chow and Chin, 2020) and hence strategies interfering with the activation of the NF-κB pathway hold immense potential in curtailing OA. Under normal conditions, the p65 subunit of NF-κB is sequestered into the cytoplasm and kept blocked by the IκBα proteins.
However, during in ammation, NF-κB is activated where IκBα proteins are phosphorylated, followed by the subsequent phoshphorylation and translocation of the NF-κB p65 subunit into the nucleus, where it binds to the promoter regions of the DNA and activates the transcription of target genes . In our present study, western blot studies revealed that IL-1β stimulation resulted in NF-κB activation which was evidenced by a signi cant increase in phosphorylation of p65 and IκBα. Our results also indicated that the combination therapy (MSC-CM + stigmasterol) was the most effective in negating the IL-1β induced-NF-κB activation demonstrated by the negligible phosphorylation of p65 and IκBα which was comparable to that of the control group. These results reiterate the superiority of the combination therapy over the per se treatments in inhibiting iNOS, IL-6, MMP-3, MMP-13, ADAMTS5 and improved collagen-II synthesis which could apparently be attributed to the upstream suppression of NF-κB activation. The ndings from our current study using stigmasterol or MSC-CM per se treatments in IL-1β-stimuated rat chondrocytes are in line with the evidences from these earlier published reports. However, it is noteworthy that the anti-in ammatory/anti-catabolic effects exhibited by the combination therapy (MSC-CM and stigmasterol) clearly shows potent promise(s) as compared to per se treatments, underscoring for possible synergistic effect augmented by the combination treatments.
Substantiating these ndings, we had recently reported (Pragasam et al., 2021) in vivo that intraarticularly transplanted MSCs in combination with stigmasterol facilitated for signi cant cartilage repair and regeneration in a monosodium-iodoacetate induced rat model of OA. We attribute the bene cial effects to the paracrine, autocrine and immunomodulatory effects of MSCs vis-à-vis stigmasterol for its anti-in ammatory and antioxidant functions. Accumulating evidence(s) from several studies merit the distinctive advantages of employing MSC-CM/secretome over the use of stem cells in regenerative research/tissue engineering (Vizoso et al., 2017). The MSC-CM offers optimal platform for therapy in retaining all the bioactive factors, can be freeze-dried, packaged, and transported whilst avoiding the operational and logistic challenges observe d with stem cells, in addition to its ability as cell-free system to enhance the shelf life (Pawitan, 2014).

Conclusion
Taken together, the ndings of our study clearly demonstrate the advantage of employing a combination therapy of MSC-CM and stigmasterol over the per se treatments in mitigating the IL-1β induced in ammatory response in a rat chondrocyte model of OA in vitro. The combination therapy signi cantly suppressed the IL-1β induced iNOS, IL-6, MMP3, MMP13 and ADAMTS5 expression chie y by inhibiting the NF-κB signaling pathway. The leads obtained from our previous and current studies have shown promise for the possible application and evaluation of this combination therapy in clinical settings at least in OA patients presenting with early OA-like changes.   treated with the combination of MSC-CM and stigmasterol. B. Quantitative uorescence measurements for IL-6 expression in the chondrocytes from three independent experiments and values represented as mean ± SD. Signi cant differences between the groups de ned as: **p < 0.01; ***p < 0.001 compared with control group; ## p < 0.01 ###p < 0.001 compared with IL-1β group.  Immuno uorescence studies for COL2A1 expression in rat chondrocytes. MSC-CM, stigmasterol, and combination of MSC-CM and stigmasterol treatment signi cantly increased COL2A1 expression in IL-1β stimulated rat chondrocytes. The combination of MSC-CM and stigmasterol treatment signi cantly restored COL2A1 expression to near normal levels comparable to the control group. B. Quantitative uorescence measurements for COL2A1 expression in the chondrocytes from three independent experiments and values represented as mean ± SD. Signi cant differences between the groups de ned as: **p < 0.01; ***p < 0.001 compared with control group; ###p < 0.001 compared with IL-1β group. Signi cant differences between the groups de ned as: * p < 0.05 compared with control; #p < 0.05 compared with IL-1β group.