LC-MS-Based Metabolomics Analysis Revealed Deleterious Effect of Infrapatellar Fat Pad on Articular Chondrocytes of Osteoarthritis


 Objective: To investigate the interaction of infrapatellar fat pad/cartilage and related mechanisms in knee osteoarthritis (OA) using the metabolomics method.Method: Fat-conditioned media (FCM) of the infrapatellar fat pad from patients with OA were used to treat human OA chondrocytes. The extracellular metabolites of human OA chondrocytes were detected by nontargeted metabolic footprint analysis based on liquid chromatography and mass spectrometry (LC-MS). Then, the different metabolites were found, and the main metabolic pathways were explored, combined with bioinformatics methods.Results: After treatment with FCM for 48 h, the proliferation of human OA chondrocytes was slowed down, indicating that FCM had a certain inhibitory effect on the proliferation of human OA chondrocytes (P = 0.023). On the pattern diagram of principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA), after FCM treatment, the data sample areas were obviously separated, indicating that FCM can significantly affect the metabolic footprint of human OA chondrocytes. Through metabonomic identification, 131 different metabolites were screened after FCM treatment compared with before treatment. For 4 pathways in total, significantly different activity levels were discovered in pairwise comparisons: alanine, aspartate, and glutamate metabolism; citrate cycle (TCA cycle); arginine and proline metabolism; and phenylalanine metabolism.Conclusion: The infrapatellar fat pad aggravates OA chondrocyte injury and is involved in OA by disturbing the chondrocyte TCA cycle, amino acid metabolism, and glutamine metabolism, among others.


Introduction
Osteoarthritis (OA), a chronic and progressive disorder of the joints, is the most common form of arthritis [1,2]. Epidemiological analyses have suggested that approximately 10% of the population above 60 years of age have symptomatic OA worldwide [2]. However, its pathogenesis is not fully understood. OA is now widely viewed as a disease of the whole "joint organ," with direct and indirect interactions among multiple joint tissues (articular cartilage, subchondral bone, synovium, and periarticular adipose tissue) involved in both the maintenance and degeneration of joints [3].
As a periarticular adipose tissue located below the patella, the infrapatellar fat pad (IFP) has been con rmed to play an important role in the development of knee OA [4,5]. To understand how the IFP might be involved in this process, the behaviors of chondrocytes have been previously examined after being cultured with IFP-derived fat-conditioned media (FCM) from OA patients, but with inconsistent results. For example, IFP-derived FCM from OA patients induced an enhanced expression of matrix metalloproteinase (MMP)1 and MMP13, as well as an enhanced collagen release from bovine cartilage, implying a deleterious effect of the IFP on cartilage [6]. On the other hand, the opposite effect was also suggested. FCM was reported to increase collagen type II gene expression and inhibit the cell mediator in in ammatory response production (such as NO), as well as MMP1 and MMP3 gene expression [7].
In addition, these studies investigated the interaction between IFP and cartilage from the perspective of genes and protein. However, many pathway feedback mechanisms might not be re ected by the changes in protein and gene concentration. In ammation and metabolic disorders of chondrocytes are regarded as major pathological changes in OA [8]. Acting as a secretory organ, IFP releases in ammatory mediators that participate in the local in ammation state and metabolic state of the joint during OA development [3]. However, the fundamental mechanisms of the IFP in knee OA development are largely unknown, especially at the metabolite level.
Metabolomics is a quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modi cation. It is regarded as an e cient approach for probing signi cant biochemical alterations and exploring potential pathophysiological mechanisms [9,10]. It has been applied to nd biomarkers and elucidate the pathogenesis of OA, with samples mainly coming from serum and synovial uid [11,12]. Nevertheless, studies focusing on the cell metabolism of joint components and their interaction are lacking. Hence, to better understand the effects of IFP tissue on articular cartilage, a metabolomic analysis was applied in this study to explore the functional interaction between IFP and cartilage in a coculture model.

Preparation of FCM and chondrocytes
Thirteen IFPs obtained as anonymous waste material from human subjects with OA who underwent total knee arthroplasty were used to produce FCM. The protocol was approved by the ethical committee of First A liated Hospital of Fujian Medical University ([2019]254). The inner parts of the fat pads, where no synovium is present, were cut into small pieces of approximately 50 mg and cultured in suspension for 24 h in a concentration of 0.3 g tissue/ml in DMEM culture medium containing 5% fetal calf serum and antibiotics. Then, the medium was replaced with serum-free DMEM (0.3 g tissue/ml) and incubated for 72 h at 37°C. After harvesting, the media were lter sterilized and stored at −80°C before use.
Primary human articular chondrocytes were derived from articular cartilage obtained from joint replacement patients diagnosed with OA (the same as those used to produce FCM). Enzymatic digestion of tissue and maintenance and culture of cells were used as previously described [13].

Chondrocyte culture
To test multiple FCM batches in one experiment, avoiding cartilage donor differences, we mixed the FCM collected several times, removed the bacteria again, and added the culture medium to prepare the conditioned medium containing 50% FCM. A total of 12 plates of chondrocytes in good condition were cultured in the FCM and divided into OA + FCM 0 h and OA + FCM 48 h groups. Culture media were collected at 0 and 48 h, frozen in liquid nitrogen, and stored in a refrigerator at −80℃.
Ckk-8 assay for cell proliferation Cells were kept in the incubator with 5% CO2 at 37°C after adding 10 ul of CCK-8. The microplate reader was used for measurement of optical density (OD) value, and the proliferative detection was carried out after culturing in the FCM for 0 and 48 h.

Metabolomic analysis
Culture media samples from the OA + FCM 0 h and OA + FCM 48 h groups were evaluated via ultra-high performance liquid chromatography-quadrupole/time of ght mass spectrometry (UHPLC/TOF-MS). A 100 lL aliquot of each culture media sample was added to 400 lL of methanol (containing 5 lg/mL 2chloro-L-phenylalanine as an internal standard) and vortex mixed for 60 s. The mixture was then centrifuged at 12,000 rpm, 4 °C for 10 min, and all the supernatant was transferred to a new 2 ml centrifuge tube, concentrated, and dried in a vacuum. Then, the methanol solution was reconstituted, and the supernatant was ltered with a 0.22 µm membrane to obtain samples for testing. From each sample, 20 µL was taken to be tested and mixed into quality control (QC) samples (used to correct the deviation of the analysis results of mixed samples and errors caused by the analysis instrument itself). The remaining samples were detected by liquid chromatography-mass spectrometry (LC-MS).

Data analysis
ProteoWizard software (v3.0.8789) was used to convert the obtained raw data into mzXML format (xcms input le format). The converted data were imported into the XCMS package of R (v3.3.2) for calculation of the normalized peak intensity, exact mass, and retention time. Subsequently, these data were employed in the nal dataset for multivariate statistical and pattern recognition analysis.

Effects of FCM on OA chondrocyte proliferation
The effects of FCM on the proliferation of OA chondrocytes was examined by performing a CCK-8 assay, and the results suggested that FCM could decrease the viability of OA chondrocytes (Fig. 1). After coculturing with FCM, the viability of OA chondrocytes decreased signi cantly when compared with the control (P = 0.023). It appears that FCM inhibited the proliferation of OA chondrocytes.

Effects of FCM on the metabolism of OA chondrocytes
Using the optimal chromatography condition described above, representative LC-MS total ion current (TIC) chromatograms from both groups in the positive and negative ion modes are shown in Fig. 2 The analytical stability of the system throughout the experiment was evaluated by analyzing QC samples. The PCA results (Fig. 3) show that the QC samples (red circle) were tightly clustered together, indicating good stability of the LC-MS system, which was su cient for metabolomic analysis.

Metabolomic analysis
To determine whether FCM would likely alter the metabolic pattern of OA chondrocytes and whether the metabolite concentration would change signi cantly, multivariate data analysis techniques were conducted, including PCA, PLS-DA, and OPLS-DA, based on the LC-MS data, to explore the tendency of groups in both ESI+ mode (Fig. 4A) and ESI− mode (Fig. 4B). A clear separation in the PCA and PLS-DA score plots between control and model groups was noted, which indicated that they had different metabolic pro les. In addition, the total variable of principle components (R2Y) of the PLS-DA model in positive and negative modes was 0.993 and 0.999, and Q2 was 0.954 and 0.955, respectively. These results indicated that the PLS-DA model exhibited goodness of t and good prediction. Furthermore, to maximize the discrimination between the two groups, OPLS-DA analysis was carried out. OPLS-DA loading plots revealed that this model was e cient and showed clear separation between the FCMtreated group and the control group (R2X = 0.829, R2Y = 0.979, and Q2 = 0.706). Moreover, a permutation test with 200 iterations con rmed that the constructed PLS-DA and OPLS-DA model was valid and not over-tted.

Identi cation of signi cant metabolites
According to the OPLS-DA analysis, signi cant differences in metabolites between the FCM-treated group and the control group were identi ed. Using a VIP > 1.0 in the OPLS-DA models and p < 0.05 using a twotailed Student's t-test, the metabolites with signi cant changes were screened out. There were 131 metabolites with VIP > 1.0 and p < 0.05 after FCM treatment (Fig. 5), of which 91 were mainly increased to various degrees, whereas the other 40 mainly showed a downward trend, indicating that FCM affected the metabolic secretion of substances in the OA chondrocytes and caused a signi cant change in their content. Fig. 6 shows the changes of amino acids with signi cant differences before and after coculturing. After coculturing with FCM for 48 h, we observed increased levels of 14 amino acids and declined levels of 3 amino acids in the OA chondrocytes culture medium.
Classi cation of metabolites between the OA + FCM 0 h group and OA + FCM 48 h group Using the HMDB for classi cation (Fig.6B)., of the 131 metabolites, 24% were sub-grouped as organic acids and derivatives, about 15% were amino acids (shown in Fig. 6A), peptides, and analogs, about 9% were lipids and lipid-like molecules, 8% were carbohydrates and carbohydrate conjugates, and 7% were pyrimidines and pyrimidine derivatives and purines and purine derivatives.
These metabolites were primarily located in the cytoplasm (53%), membrane (21%), extracellular (19%), and mitochondria (19%) cellular locations. In terms of biofunction distribution, these metabolites mainly participated in the biological processes of amino acid and energy metabolism and lipid metabolism, among others.

Effects of FCM on metabolic pathways of OA chondrocytes
Functional analysis showed that the differential metabolites identi ed are primarily involved in amino acid metabolism, energy metabolism, and lipid and oxidative stress metabolism. For 4 pathways in total, signi cantly different activity levels were discovered in pairwise comparisons. As illustrated in Fig. 7A, the analysis results showed that the main affected pathways were alanine, aspartate, and glutamate metabolism, the citrate cycle (TCA cycle), arginine and proline metabolism, and phenylalanine metabolism. Fig. 7B shows the correlation networks of the main potential affected pathways.

Discussion
It is evident that the IFP might interact with cartilage to participate in the development of OA, however, the underlying mechanism remains unclear. In this study, by coculturing FCM derived from human OA IFP and chondrocytes from OA patients, we found that FCM might inhibit the proliferation of OA chondrocytes. Furthermore, a total of 131 metabolites obtained in the culture media of OA chondrocytes were signi cantly changed. Pathway analysis revealed that those metabolic pathways signi cantly affected in OA chondrocytes included the TCA cycle, amino acid metabolism, and glutamine metabolism. These results suggest that IFP has a deleterious effect on cartilage, and the involvement of the IFP in the development of OA might be associated with the effect of induced metabolic disturbances of chondrocytes.

Effect of FCM on TCA cycle pathways of human OA chondrocytes
The TCA cycle is a key biochemical pathway that provides cellular energy in the form of ATP by oxidative conversion of carbohydrates [14]. The increased levels of citric acid and succinic acid and decreased levels of isocitrate in the culture medium OA chondrocytes after treatment of FCM indicate enhanced activity of the TCA cycle, possibly due to perturbed metabolism in the cartilage cells. Akhbari et al.'s [15] study on synovial uid (SF) showed that when compared with the metabolites of the SF in rheumatoid arthritis patients, OA patients had higher levels of metabolites involved in both TCA and glycolysis. Another study examined the metabolites of SFs between early-and late-stage OA and found that TCA intermediate metabolites including malate, citrate, succinate, and fumarate levels were signi cantly higher in the late-stage OA group than in the early-stage OA group, suggesting that the upregulation of the TCA pathway is greater in the late stage of OA [16]. In addition, a study on urine also indicated that the increased levels of TCA intermediate metabolites in the urine of in ammatory OA participants compared with non-OA controls suggested an enhanced activity of the TCA cycle [14]. Brie y, these data suggest that the TCA pathway is upregulated in OA, and our results agree with these studies, demonstrating that the upregulated TCA pathway of OA is most likely to increase the production of ATP needed to repair damaged cartilage. On the other hand, most of the enzymes involved in the TCA cycle are located inside the mitochondrial matrix of the cartilage cells, and the enhanced TCA cycle activity provides metabolic evidence of mitochondrial dysfunction of the cartilage cells in OA [17]. Back to our ndings, these results showed that FCM treatment accelerated the injury of OA chondrocytes by enhancing the TCA cycle activity.

Effect of FCM on amino acid metabolism of human OA chondrocytes
Amino acids, small molecule metabolites, are considered the most important biological compounds, being involved in the synthesis of proteins and hormones and playing prominent metabolic and physiological roles (such as immune function, cytokine secretion) within organisms [18]. The current literature data suggest that the metabolic pathogenesis of OA might be signi cantly related to perturbations of amino acid metabolism [19]. In our study, after coculturing with FCM for 48 h, we observed increased levels of two main amino acids (threonine and glutamate) and declined levels of four main amino acids (cysteine, proline, arginine, and tyrosine) in the OA chondrocyte culture medium. Previous studies have found increased levels of glutamate and aspartate in dialysates from the injured joint of rats with anterior cruciate ligament transection (ACLT) [20]. Furthermore, the contents of glutamate and aspartate in the SF of patients with synovitis are 54 times and 28 times higher than those of healthy controls, respectively [21]. In addition, sheep with ACLT exhibited decreases in serum concentrations of tyrosine, valine, and isoleucine at week 4 after surgery, compared with the sham control [22]. Moreover, a study evaluated the short-term metabolic response of human chondrocytes to mechanical loading and suggested that rates of serine and threonine metabolism were increased. In rabbits with an ACLT injury, there were negative correlations between histological assessments and changes in plasma concentrations of arginine [23]. These ndings indicate that the decrease of arginine plasma concentration was due to the process and progress of OA. In summary, the increase of glutamate and threonine levels and the decrease of tyrosine and arginine levels could induce the occurrence and development of OA. Furthermore, prolines are thought to inhibit cell oxidative damage and have antiin ammatory effects [24]. In our study, after coculturing with FCM, the levels of arginine, proline, and tyrosine decreased, and the levels of glutamate and threonine increased in the culture medium of OA chondrocytes, indicating that FCM promotes the in ammatory response of OA chondrocytes and IFP has a deleterious effect on cartilage.

Effect of FCM on glutamine metabolism of human OA chondrocytes
Glutamine is a key source of carbon secondary only to glucose for energy production and anabolic processes [25]. Recently, a study found that glutamine suppressed OA progression [26]. Glutamine is a precursor of glucosamine and can upregulate glucosamine synthesis in chondrocytes, and glucosamine could inhibit cartilage destruction and promote biosynthetic activity of chondrocytes [27]. A study observed that the concentration of glutamine was lower in the SF from OA patients than in normal SF [15]. In addition, 2-keto-glutaramic acid (a deaminated metabolite of glutamine under abnormal conditions) has been found to be increased in the urine of in ammatory OA participants, indicating that glutamine metabolism might be disturbed in the chondrocytes [28]. Furthermore, Wang et al. [29] indicated that growth plate (GP) and articular cartilage chondrocytes exhibited metabolic plasticity that allowed these cells to switch to glutamine oxidation to support energy production when glucose metabolism was compromised. Glutamine oxidation enables GP chondrocytes to survive in glucoselimited conditions, however, normal proliferation and matrix production by these cells is impaired. In a word, in OA progression, glutamine metabolism could be disturbed in the chondrocytes, and lower glutamine might indicate the cartilage destruction of the joint. Our observations suggested that after coculturing with FCM, the level of glutamine declined. This result supports that IFP has a deleterious effect on cartilage. However, the mechanisms of glutamine involvement in OA pathogenesis are unclear.
During stress loading, exogenous glutamine supplementation has been shown to increase the concentration of glutathione (an essential intracellular antioxidant) that protects cells from injury. Moreover, glutamine has been shown to be vital for fueling mitochondrial metabolism [30], and the IFT might disrupt glutamine metabolism, resulting in mitochondrial dysfunction, and nally lead to the development of OA. Further research is needed to determine the mechanisms of the crosstalk between the IFP and cartilage.
There are a couple of potential limitations of the current study that must be acknowledged. First, we only evaluated the changes to OA chondrocyte culture medium metabolites after FCM treatment. The underlying mechanism of the interaction between cartilage and IFP could be better understood together with the intracellular metabolites, which should be evaluated in the future. Furthermore, our results indicated that IFP could disturb the TCA cycle, amino acid metabolism, and glutamine metabolism of OA chondrocytes, however, further investigations are warranted to verify the results of metabolomics.

Conclusion
In conclusion, we have shown that IFP has a deleterious effect on cartilage, and IFP involvement in the development of OA could be associated with disturbances to the chondrocyte TCA cycle, amino acid metabolism, and glutamine metabolism, among others. These metabolic disturbances could provide insight into the mechanisms underlying OA, and beyond that, to develop new strategies to slow down the degenerative process and explore an effective and timely diagnosis of the disease.   The PCA scores plot-obtained QC samples (red circle) and experimental samples (green circle). The QC samples were tightly clustered together, indicating good stability of the LC-MS system