Study on the changes of FABP4 and PPARγ levels in knee tissues of patients with osteoarthritis

Study on the changes of FABP4 and PPARγ levels in knee tissues of patients with osteoarthritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Study on the changes of FABP4 and PPARγ levels in knee tissues of patients with osteoarthritis Shiqi Zhang, Ningning Liu, Yu Zhang, Xiaochun Yang, Xiaojie Wu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6446806/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: To investigate the expression and significance of fatty acid-binding protein4（FABP4）and peroxisome proliferator-activated receptor γ（PPARγ）in the tissues around the knee joint of patients with osteoarthritis (OA). Method: A total of 32 obese patients with OA（BMI>25 Kg/m 2 ）and 20 non-obese patients with OA were collected from Hospital affiliated to Shandong First Medical University who underwent surgical treatment. In addition, 25 patients with joint surgical injuries who underwent knee surgery at the same time were selected as the control group. The clinical data of the three groups (including age, height, blood routine indexes and biochemical routine indexes) were collected, and the statistics were collated. At the same time, specimens of periarticular tissues (including articular cartilage, synovium, suprapatellar fat pads, and infrapatellar fat pads) that had been discarded during the surgical procedure were collected from all three groups of patients. Western blot, qRT-PCR and immunofluorescence double staining were used to detect the expression and distribution of FABP4 and PPARγ in the tissues around the knee joint in obese patients with OA and non-obese patients with OA. Confirmation that informed consent was obtained from all subjects or their legal guardians. Result: In synovial tissue, the expression of FABP4 in OA obese patients was significantly higher than that in OA non-obese patients and control group ( P <0.01), and the expression of PPARγ in OA obese patients was significantly lower than that in non-obese OA patients and control group ( P <0.01). In the synovial tissues of OA patients, the green fluorescence intensity of FABP4 was significantly enhanced ( P <0.01), and the red fluorescence intensity of PPARγ was significantly decreased ( P <0.01). Conclusion: Aberrant expression of FABP4 and PPARγ may be a potential therapeutic target affecting the development of OA. Health sciences/Diseases/Rheumatic diseases Health sciences/Diseases/Rheumatic diseases/Rheumatoid arthritis Osteoarthritis synovial tissue FABP4 PPARγ Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Osteoarthritis (OA) is one of the most common types of arthritis, and it is also a chronic degenerative joint inflammatory disease. Its Key features are articular cartilage damage, sclerosis of subchondral bone, synovial hypertrophy, synovial cell inflammation and proliferation, and vascular hyperplasia [ 1 ] . OA is more common in middle-aged and elderly people and obese people, the clinical symptoms are joint pain, swelling, deformity, and mobility dysfunction, which are the main causes of pain and disability in patients [ 2 ] . According to statistics, about 16% of the world's population suffers from osteoarthritis [ 3 ] . The pathogenesis of OA is still unclear, and there is no effective way to prevent and treat OA. Recently, a growing number of studies have found that the occurrence of the disease may be related to a variety of factors such as age, gender, fat, and genetics [ 4 ] . At the same time, it was found that the incidence of OA increased significantly in obese people, but weight and weight may not be the direct cause of OA, but may affect the occurrence and development of OA by the deposition of adipose tissue [ 5 ] . Adipose tissue plays an important role in the secretion of adipokines, which is a key player in the regulation of inflammation, immunity, angiogenesis, fibrinolysis [ 6 ] , and is a highly active endocrine organ that secretes inflammatory factors, and low-grade chronic inflammatory state is very common in obese and overweight people, so obesity is considered to be one of the key factors in the pathogenesis of osteoarthritis [ 7 ] . Fatty acid-binding protein 4 (FABP4) is a small cytosolic lipid-binding protein mainly secreted by macrophages and adipocytes [ 8 – 9 ] , which can promote lipolysis, inhibit adipose tissue lipogenesis, reduce free fatty acid levels, and play an important role in inflammatory diseases. In the case of obesity, the excessive accumulation of fat increases the level of the small cytosolic lipid-binding protein FABP4 [ 10 – 11 ] , which may promote the development of osteoarthritis. Peroxisome proliferator-activated receptor γ (PPARγ) is fatty acid-activating transcription factor, plays an important role in adipogenesis and lipid synthesis [ 12 ] . PPARγ is a downstream factor of FABP4, which can transcriptionally regulate the expression of FABP4, and its activation can correct the disorder of lipid metabolism. At the same time, it acts with FABP4 to bind intracellular lipids to biological targets to complete signal transduction, which is the main regulator of adipocyte differentiation [ 13 ] . In related studies, it has been found that activation of PPARγ can enhance the phagocytosis and immunomodulatory functions of cells and reduce the occurrence and development of inflammation, but the overactivity of FABP4 affects the abnormal expression of PPARγ [ 14 ] , so it may affect the occurrence and development of osteoarthritis. At present, it has not been clarified whether the FABP4, PPARγ may affect the occurrence and development of OA. Therefore, the aim of this study was to investigate the changes in the levels of FABP4 and PPARγ in the periarticular tissues of obese patients with OA and non-obese patients with OA. Methods 1.Study population OA patients undergoing surgical treatment in Hospital affiliated to Shandong First Medical University were selected. Collection of periarticular tissue specimens abolished during patient surgery (including articular cartilage, articular synovium, suprapatellar fat pad and infrapatellar fat pad), clinical characteristics, clinical examination results and other relevant clinical data. The experiment has received ethical approval from the Research Ethics Committee of Binzhou is People's Hospital (Approval No. 2021. 266).in full compliance with Declaration of Helsinki, The Belmont Report, CIOMS Guidelines and the International Conference on Harmonisation-Good Clinical Practice (ICH-GCP). All methods were performed in accordance with these guidelines and regulations. All participants provided written informed consent. Inclusion Criteria: (1) meet the relevant diagnostic criteria for osteoarthritis; (2) The patient did not have serious organic lesions of the cardiovascular, digestive, respiratory and other systems; (3) Bone and joint surgery was performed during hospitalization, and some tissue samples around the knee bone and joint were taken for pathological examination; (4) The hospital ethics committee has approved it, and the patient and family members have informed consent; (5) Complete clinical data. Exclusion Criteria: (1) Patient has a history of previous joint surgery; (2) Patients with cancer, liver and kidney insufficiency, and hematologic diseases; (3) arthritis caused by autoimmune diseases; (4) Incomplete or missing clinical patient cases. 2. Clinical data and specimen collection The enrolled patients (all male patients) were divided into 32 cases of obese OA patients (BMI > 25 Kg/m 2 ) and 20 cases of non-obese OA patients (study group) and 25 cases of patients with arthro-surgical injuries who underwent knee surgery at the same time (control group), and the sample tissues abolished during their surgeries were collected; and the relevant clinical data of the patients as well as of normal subjects were also collected. 3. Western blot Radioimmunoprecipitation (RIPA) buffer homogenate 1ml was added to the cryopreserved tissue samples, the supernatant was obtained after centrifugation, and the protein concentration was determined by ultra-micro volumetric spectrophotometer. After adjusting the protein concentration of each sample, 5X protein loading buffer was added, and heated in a metal bath at 100℃ for 5min. Equal amounts of extracted proteins were separated by 12% SDS-PAGE electrophoresis, and wet transmembrane (PVDF membrane) was conducted for 1 h. After being blocked at room temperature for 20 min with Rapid blocking solution, and then primary antibodies including FABP4(Abcam; ab13979), PPARγ(Proteintech;16643-1-AP), BAX (Abcam;ab32503), Caspase3 (Proteintech;66470-2-Ig), Bcl-2 (Immunoway; YM3041), or GAPDH(Abcam; ab9485) were added, and the incubation were performed overnight at 4℃. The membranes were washed 4-time in TBST, and then incubated with the second antibody for 1 h at room temperature. After 4-time washing in TBST, the membrane was incubated with ECL solution and exposed using a G-Box scanner (Gene Company Limited). Finally, the densitometric analysis was performed using Image-J software. 4. Quantitative real-time PCR Total RNA extraction reagent was used to RNA extraction from tissue sample. RNA samples were then transcribed into cDNA using a cDNA Synthesis Kit (Monad;MR05101), added SYBR Green PCR Master Mix and corresponding primers Performing an amplification reaction. PCR reactions were carried out in circulations, pre-denaturation at 95℃for 5 min and denaturation at 95℃ for 30s, primer annealing at 58℃ for 30s, and extension at 74℃ for 5 min, followed by melt curve analysis. The GAPDH gene was used as the reference gene. The value of the corresponding target gene was calculated by the 2 −△△CT method. 5. Histopathological Analysis The periarticular tissue fixed with 4% paraformaldehyde was sectioned after paraffin embedding. Calcium in the knee joints was removed using a decalcifying solution for 28 days. The periarticular tissues were embedded in paraffin and coronally sectioned. The sections were stained with safranin O Staining or hematoxylin & eosin (H&E). 6. Immunofluorescence The tissue samples were fixed in 4% paraformaldehyde for 15 min, and washed with PBS three times. After added trypsin, incubated at room temperature at 37℃ for 30 min. The tissue samples were incubated with antibodies FABP4 and PPARγ at 4℃for 24 h. Then tissue samples were incubated with fluorescein-conjugated anti-rabbit IgG (Proteintech, 20001102) at 37℃for 1 h. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). The images were acquired by a fluorescence microscope. Fluorescence intensity was quantified by using Image-J software. 7. Statistical analysis Statistical Product and Service Solutions software version 26.0(SPSS 26.0) was used in the current study. Data were presented as means ± standard deviation ( \(\:\stackrel{-}{\text{x}}\) ±s); If the samples were normally distributed, one-way ANOVA was used, and the LSD-t test was used for comparison between the multi-sample mean and the single-group mean. If the samples did not conform to the normal distribution, the non-parametric test of two independent samples was used, and the Mann-Whitney U test was used for comparison between the means of multiple samples and the means of one group. P < 0.05 was considered to indicate a statistically significant difference. Result 1. Basic clinical information of the patient According to the inclusion and exclusion criteria, a total of 32 obese patients with OA, 20 non-obese patients with OA were included in the study group, and 25 patients with joint surgical injuries who underwent knee surgery during the same period were included in the control group. By collating, analyzing and statistically showing that there was no statistical difference in age and height between the three groups ( P > 0.05), and the weight and BMI of the obese OA group were significantly higher than those of the other two groups ( P < 0.05). Table 1 . Table 1 Comparison of general data of patients( \(\:\stackrel{-}{\text{x}}\) ±s) Control OA non-obesity OA obesity Patient number 25 20 32 Age(year) 68.00 ± 6.69 67.93 ± 6.68 66.21 ± 6.64 Weight(Kg) 59.67 ± 6.5 62.21 ± 7.26 75.66 ± 8.20* # Height(m) 1.59 ± 0.07 1.67 ± 0.07 1.63 ± 0.07 BMI(Kg/m 2 ) 23.51 ± 1.14 22.25 ± 1.97 28.37 ± 2.61* # * P < 0.05 vs. the control. # P < 0.05 vs. the non-obese OA. Through the collation, analysis and statistics of blood indicators, it was found that there were no statistically significant differences in TG (triglycerides), HDL-C (high-density lipoprotein), BUN (urea nitrogen), Cr (creatinine) and UA (uric acid) in the control group, OA non-obesity group and OA obesity group. The homocysteine, LDL-C and cysC (cystatin C) in the OA obesity group were significantly higher than those in the control group and the OA non-obesity group (P < 0.05). The level of erythrocyte sedimentation rate (ESR), FFA (free fatty acids) in the OA obesity group was higher than that in the control group ( P < 0.05). The level of TC (triglycerides) in the obese OA group was higher than that in the non-obese OA group ( P < 0.05). Table 2 . Table 2 Comparison of clinical blood indexes of patients( \(\:\stackrel{-}{\text{x}}\) ±s, n=45) Control OA non-obesity OA obesity ESR(mm/h) 6.67 ± 1.51 15.93 ± 5.62 22.84 ± 18.44* CysC(mg/L) 0. 80 ± 0.09 0.95 ± 0.17 1.11 ± 0.20* # HCY(umol/L) 5.74 ± 1.39 9.98 ± 4.11* 12.79 ± 3.56* # FFA(mmoL/L) 0.27 ± 0.13 0.41 ± 0.21 0.47 ± 0.27 TC(mmol/L) 4.22 ± 0.85 4.21 ± 0.91 4.90 ± 1.08 # TG(mmol/L) 1.30 ± 0.34 1.40 ± 0.77 1.71 ± 0.87 HDL-C(mmol/L) 1.40 ± 0.18 1.29 ± 0.30 1.19 ± 0.32 LDL-C(mmol/L) 1.95 ± 0.77 2.68 ± 0.72 3.17 ± 0.93* BUN(mmoL/L) 6.83 ± 0.48 5.31 ± 2.31 5.9 ± 1.92 Cr(umoL/L) 278.81 ± 67.24 324.50 ± 71.55 306.16 ± 93.41 UA(umoL/L) 74.70 ± 19.89 64.21 ± 10.53 63.08 ± 13.87 * P < 0.05 vs. the control. # P < 0.05 vs. the non-obese OA. 2. Expression of FABP4 and PPARγ in periarticular tissues of OA patients Western blot results showed that there were obvious individual differences in the protein expression of FABP4 and PPARγ in the suprapatellar fat tissue of the three groups, and there was no obvious trend (Fig. 1 A). In subpatellar fat, the obese OA group expressed more FABP4 than the other two groups, but the protein expression of PPARγ was unstable and there was an obvious outlier (Fig. 1 B). In cartilage tissue, the expression of FABP4 protein in the obese OA group was significantly higher than that in the control group and the non-obese OA group ( P < 0.05 or 0.01), while the expression of PPARγ protein in the obese OA group was significantly lower than that in the non-obese OA group and the control group ( P < 0.05 or 0.01) (Fig. 1 , C and E). In the synovial tissue, the expression of FABP4 protein in the control group, non-obese OA group and obese OA group showed a significant upward trend ( P < 0.01), while the expression of PPARγ protein in the control group was significantly higher than that in the non-obese OA group and the control group ( P < 0.05) (Fig. 1 , D and F). The results of qRT-PCR showed that there were obvious individual differences and free values in the mRNA expression of FABP4 and PPARγ in the suprapatellar adipose tissue of the three groups. In subpatellar fat, the expression of FABP4 in the obese OA group was more dispersed and there was an obvious free value (Fig. 2 A). In subpatellar fat, the mRNA expression of FABP4 was more in the obese OA group than in the other two groups, and the mRNA of PPARγ had an opposite trend, but there were also obvious free values (Fig. 2 B). In cartilage tissues, the mRNA expression of FABP4 in the obese OA group was significantly higher than that in the control group and the non-obese OA group ( P < 0.01), while the mRNA expression of PPARγ showed an opposite trend ( P < 0.01) (Fig. 2 C). In synovial tissues, the mRNA expression of FABP4 increased significantly in the control group, non-obese OA group and obese OA group ( P < 0.01), while the mRNA expression of PPARγ decreased ( P < 0.01) (Fig. 2 D). Therefore, by comparing the four parts of suprapatellar fat, lower fat, cartilage and synovium, it was found that the expression of FABP4 and PPARγ in suprapatellar and inferior fat was unstable and there were obvious individual differences, while synovium and cartilage were relatively stable. Therefore, cartilage and synovium were selected as the main research objects in the future. 3. Histopathological observation of synovial tissue and cartilage in OA patients Observation of the joint pathological sections of patients in the control group, OA non-obesity group and OA obesity group showed that in the synovial tissue, the synovial surface of the control group was smooth and there was no defect and protrusion, and the synovial cells lining the inner layer had clear structure and normal morphology. In the non-obese OA group, synovial fibroblasts proliferated in the synovial tissue, forming synovial dysmorphic protrusions, and inflammatory cell infiltration. A large number of fibroblastic synovial cells proliferate into the luminal tissues of the OA obese group, forming obvious abnormal protrusions and infiltrating a large number of inflammatory cells(Fig. 3A). In cartilage tissue, the perichondrium surface of the control group was smooth and the chondrocytes were intact. Necrosis and loss of chondrocytes were observed in the cartilage tissue of patients in the non-obese group of OA, and the perichondrium surface was not smooth. A large area of chondrocytes and loss of chondrocytes could be observed in the cartilage tissue of patients with OA obesity, and the perichondrium surface damage was an entophagocytic lesion(Fig. 3B). 4. Periarticular tissues of OA patients are stained with oil red O Observation of the joint sections of patients in the control group, OA non-obesity group and OA obesity group showed that no obvious lipid droplet formation was seen in the synovial tissue of the control group. A small number of red lipid droplets appeared in the synovial tissue of patients in the non-obese group of OA. A large number of red lipid droplets appeared in the synovial tissue of the obese OA group, and the intracellular lipid content was significantly higher than that in the non-obese group(Fig. 3A). In cartilage tissue, lipid droplet formation was not seen in the cartilage tissue of the control group; A small number of red lipid droplets appeared at the edge of the cartilage tissue in the patients in the OA non-obese group; A large number of red lipid droplets appeared at the edge of the cartilage tissue in the OA obese group(Fig. 3B). Therefore, by comparing the pathological changes of cartilage and synovium and oil red O staining, it can be found that the damage of articular cartilage in patients with OA mainly appeared at the margins, while synovial tissues appeared to have obvious inflammatory infiltration and high expression of lipid droplet content; therefore, we chose synovial tissues to further detect the expression of FABP4 and PPARγ as well as their distributions, and also to further validate them at the protein level and mRNA level. 5. The distribution and expression of FABP4 and PPARγ in the synovial tissues of OA patients were observed by immunofluorescence double staining The distribution and interaction of FABP4 and PPARγ were observed by immunofluorescence double staining of joint synovial tissues in the control group, OA non-obesity group and OA obesity group. The results showed that FABP4 and PPARγ were mainly distributed in the synovial lining cells of the synovial tissue of OA patients. However, in the control group, the green fluorescence of FABP4 was significantly darker, the red fluorescence of PPARγ was brighter, and the colocalization of FABP4 and PPARγ was clearly superimposed on the purple fluorescence, and the expression of FABP4 in the nucleus was reduced. Compared with the control group, the green fluorescence area of FABP4 in the synovial tissue of OA patients was significantly increased, the fluorescence intensity was significantly increased ( P < 0.01), and the red fluorescence area of PPARγ was decreased ( P < 0.01) (Fig. 4 . A, B, C and D). 6. mRNA and protein levels of apoptosis-related genes in synovial tissue of OA patients In order to verify the effects of FABP4 and PPARγ on osteoarthritis, the expression levels of apoptosis-related factors BAX, Bcl-2 and cleaved-Casepase3 were detected(Fig. 5 A). Compared with the control group and the non-obese group, the mRNA and protein levels of BAX and cleaved-Casepase3 in OA patients in the obese group were increased ( P < 0.01 or P < 0.05) (Fig. 5 , B and C), while the mRNA and protein expressions of Bcl-2 were decreased ( P < 0.01 or P < 0.05) (Fig. 5 , D). These results indicate that there is obvious apoptosis in the synovium of patients with osteoarthritis, which promotes the occurrence and development of osteoarthritis [ 15 ] . Discussion OA is a common chronic degenerative joint disease in middle-aged and elderly people, but obesity is one of the major risk factors for osteoarthritis. Studies have shown that as BMI increases, it leads to abnormalities in blood serum (ESR), resulting in an increase in joint pain symptoms and severity [ 16 ] . We detected the expression of blood indexes in OA obese patients, OA non-obese patients and control group. ESR, as an indicator of erythrocyte aggregation in blood, is a commonly used inflammatory marker, which can indicate and monitor the increase of inflammatory activity in vivo caused by one or more diseases. When the body is diseased, the erythrocyte sinks due to its high density, resulting in an increase in ESR. This study found that compared with the normal control group, the BMI of obese OA patients was significantly increased, and the contents of ESR, TC, LDL-C and FFA in blood indexes were also significantly increased. These data suggest that obesity is closely related to the occurrence and development of OA. In addition, the excess energy of the whole body caused by obesity not only changes the function of various cells in the tissue, but also releases a variety of adipokines, which may also be related to OA [ 17 ] . FABP4 is a novel adipokine expressed in adipocytes, macrophages, etc. It can preferentially bind to fatty acids, enter the cytoplasm for chemical modification such as oxidation and esterification, and transport to various parts of the body to participate in lipid synthesis, metabolism, inflammatory response and other signal transduction [ 18 ] . Recent studies have shown that overexpression of FABP4 gene promotes lipid deposition in transgenic mice and promotes the severity of atherosclerosis [ 19 ] . Gene knockout of FABP4 can protect mice from diseases such as cancer [ 10 ] and fatty liver [ 20 ] . On the other hand, PPARγ, as a lipid-sensitive nuclear receptor, is expressed in various cell tissues such as fat, muscle, and liver [ 21 ] . It has significant anti-inflammatory properties, which can regulate the immune inflammatory response. PPARγ activation reduced inflammatory response by negatively interfering with NF-κB and signal transducers and transcriptional activators [ 22 ] . The PPARγ signaling pathway has also been reported to be involved in the regulation of lipogenesis in C2C12 myoblast cells and C57BL/6 mouse meibocytes [ 23 ] . PPARγ gene knockout can promote cell apoptosis, resulting in cell damage, and aggravate the occurrence and development of various lipid metabolism and inflammatory diseases [ 24 ] . Therefore, in this study, we aimed to explore the potential role of FABP4 and PPARγ in OA. By detecting the protein and mRNA expression of FABP4 and PPARγ in the periarticular tissues of OA patients, we found that the expression of FABP4 and PPARγ in the suprapatellar fat and infrapatellar fat of OA patients was unstable and there were obvious individual differences. In synovium and cartilage, the expression of FABP4 in OA obese patients was significantly higher than that in control group and non-obese OA patients, while the expression of PPARγ showed an opposite trend. FABP4 has been confirmed to deliver specific ligands from the cytoplasm to the nuclear receptor PPARγ, which facilitates ligation and enhances the receptor transcriptional activity [ 25 ] . It has been found that FABP4 directly binds to and promotes the degradation of PPARγ, while PPARγ expression is significantly elevated in FABP4 knockout macrophages [ 26 ] . However, whether the interaction between FABP4 and PPARγ affects the occurrence and development of OA has not been reported. In this study, by immunofluorescence double staining of FABP4 and PPARγ in synovial membrane of three groups of patients, it can be found that the fluorescence expression of FABP4 in the OA obese group was significantly higher than that in the OA non-obese group and the control group, while the fluorescence expression of PPARγ was significantly suppressed, and at the same time, both of them appeared to have obvious co-localization, so there may be a relationship between the two interactions, and FABP4 can regulate the development of OA through negative feedback. PPARγ can promote cell proliferation, regulate lipid metabolism and thus alleviate OA. previous studies have also confirmed that FABP4 can reduce the gene and protein expression of PPARγ during adipose differentiation, inhibit adipogenesis through negative feedback regulation, improve the abnormal lipid accumulation that occurs in pleural hypertrophy, and also alleviate the fatty liver and other diseases [ 27 – 28 ] . FABP4 knockdown suppressed the inflammation, oxidative stress, apoptosis and extracellular matrix degradation of IL-1β-induced chondrocytes by activating PPARγ to inhibit the NF-κB signaling pathway [ 29 ] . In modern studies the development of OA is also affected by apoptosis, which induces cell lysis, leading to the massive release of pro-inflammatory factors and promoting the development of many inflammatory diseases [ 30 ] . Therefore, we also examined the expression of proliferation- and apoptosis-related indicators and analyzed the role of the FABP4/PPARγ signaling pathway in the proliferation and regulation of OA. BAX a protein located on the outer membrane of the mitochondria, is the key executor of mitochondria in the regulation of cell death, and in the process of apoptosis, BAX can change the permeability of the outer membrane of the mitochondria, form mitochondrial membrane channels, and release the cytochrome Bcl-2 is an anti-apoptotic protein that directly binds to pro-apoptotic proteins and inhibits BAX activity, thereby affecting the permeability of the outer mitochondrial membrane and exerting an anti-regulatory effect [ 31 ] .Caspase3 is a member of the cysteine protease family, known for its ability to mediate the cleavage of specific target proteins during the process of cellular death. Caspase3 can be a key executor in the regulation of cell death. process, Caspase3 cleaves a variety of downstream substrates, leading to morphological changes in the regulated cells and contributing to the onset of apoptosis [ 32 ] . The increased expression of BAX and Caspase3 and decreased expression of Bcl-2 in synovial tissues of OA patients indicated that apoptosis was abnormal in synovial tissues of OA patients, and thus increased apoptosis would also promote the development of OA. In summary, the expression level of FABP4 was significantly elevated and that of PPARγ was significantly reduced in synovial tissues of OA patients, so we hypothesized that FABP4 might inhibit the expression of PPARγ, and the abnormal expression of FABP4 and PPARγ might affect the development of OA. However, this study also has limitations such as the small number of clinical samples, which may make the results have a certain degree of error, so we will follow up with in-depth studies in animal and cellular experiments to investigate the detailed mechanism of FABP4 and PPARγ on the development of OA. Declarations Acknowledgements We would like to thank Binzhou Municipal People's Hospital for providing the tissue samples and the laboratory team who helped us in our routine experimental work. Author contributions S. Zhang: Conceptualization, Data curation, Writing-original draft N. Liu: Investigation, Validation Y. Zhang: Software, Validation Y. Zhao: Visualization X. Yang: Methodology, Resources X. Wu: Data curation, Formal analysis J. Yu: Supervision, Writing-review and editing S. Li: Data curation, J.Liu:Writing-review, Data curation, Conceptualization, Funding acquisition, Resources, Supervision H.Yu:Writing-review, Funding acquisition, Project administration, Resources, Supervision ICMJE COI statement The authors declare that they have no conflicts of interest. Ethical review statement Ethical approval was obtained from the Ethics Committee of Binzhou Municipal People's Hospital (Approval No. 2021. 266). Open access funding This study was supported by the Science Foundation of the Ministry of Education of China（2023HT048） and the Natural Science Foundation of Shandong Province（ZR2021MH084）. Data availability statement The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Motta F, Barone E, Sica A, Selmi C. Inflammaging and Osteoarthritis. Clin Rev Allergy Immunol. 2023 Apr;64(2):222-238. Jin W，Wu Z，Dai N. 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Wu W, Sun Y, Zhao C, Chen X, Wang G, et al. Lipogenesis in myoblasts and its regulation of CTRP6 by AdipoR1/Erk/PPARγ signaling pathway. Acta Biochim Biophys Sin (Shanghai). 2016 Jun;48(6):509-19. Carrasco AG, Izquierdo-Lahuerta A, Valverde ÁM, Ni L, Flores-Salguero E, et al.The protective role of peroxisome proliferator-activated receptor gamma in lipotoxic podocytes. Biochim Biophys Acta Mol Cell Biol Lipids. 2023 Jul;1868(7):159329. Wang XW, Sun YJ, Chen X, Zhang WZ. Interleukin-4-induced FABP4 promotes lipogenesis in human skeletal muscle cells by activating the PPAR γ signaling pathway. Cell Biochem Biophys. 2022 Jun;80(2):355-366. Moseti D, Regassa A, Kim WK. Molecular Regulation of Adipogenesis and Potential Anti-Adipogenic Bioactive Molecules. Int J Mol Sci. 2016 Jan 19;17(1):124. Garin-Shkolnik T, Rudich A, Hotamisligil GS, Rubinstein M. FABP4 attenuates PPARγ and adipogenesis and is inversely correlated with PPARγ in adipose tissues. Diabetes. 2014 Mar;63(3):900-11. Cao Y, Li J, Qiu S, Ni S, Duan Y. ACSM5 inhibits ligamentum flavum hypertrophy by regulating lipid accumulation mediated by FABP4/PPAR signaling pathway. Biol Direct. 2023 Nov 14;18(1):75. Mao H, Han B, Li H, Tao Y, Wu W. FABP4 knockdown suppresses inflammation, apoptosis and extracellular matrix degradation in IL-1β-induced chondrocytes by activating PPARγ to regulate the NF-κB signaling pathway. Mol Med Rep. 2021 Dec;24(6):855. Chen Y, Liu Y, Jiang K, Wen Z, Cao X, et al. Linear ubiquitination of LKB1 activates AMPK pathway to inhibit NLRP3 inflammasome response and reduce chondrocyte pyroptosis in osteoarthritis. J Orthop Translat. 2022 Dec 1;39:1-11. Spitz AZ, Gavathiotis E. Physiological and pharmacological modulation of BAX. Trends Pharmacol Sci. 2022 Mar;43(3):206-220. Eskandari E, Eaves CJ. Paradoxical roles of caspase-3 in regulating cell survival, proliferation, and tumorigenesis. J Cell Biol. 2022 Jun 6;221(6):e202201159. Additional Declarations No competing interests reported. Supplementary Files Westernblot.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6446806","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":452108386,"identity":"58591b93-8373-4895-8cc6-ffd2cdf7b608","order_by":0,"name":"Shiqi Zhang","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shiqi","middleName":"","lastName":"Zhang","suffix":""},{"id":452108387,"identity":"efe928da-8ae9-4729-a03a-a23a45158b49","order_by":1,"name":"Ningning Liu","email":"","orcid":"","institution":"Binzhou people's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ningning","middleName":"","lastName":"Liu","suffix":""},{"id":452108388,"identity":"3451f845-2679-4c5a-bf64-7a0db748e06b","order_by":2,"name":"Yu Zhang","email":"","orcid":"","institution":"Binzhou people's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhang","suffix":""},{"id":452108389,"identity":"bad337e9-2c97-4025-9c5f-0269671ddeb0","order_by":3,"name":"Xiaochun Yang","email":"","orcid":"","institution":"Binzhou people's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaochun","middleName":"","lastName":"Yang","suffix":""},{"id":452108390,"identity":"521ef3a7-db1c-4175-8854-e6e08dc0fd29","order_by":4,"name":"Xiaojie Wu","email":"","orcid":"","institution":"Binzhou people's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaojie","middleName":"","lastName":"Wu","suffix":""},{"id":452108391,"identity":"2bc69dab-b8d2-4811-ac4e-10ec55c9ef52","order_by":5,"name":"Yifei Zhao","email":"","orcid":"","institution":"Binzhou people's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yifei","middleName":"","lastName":"Zhao","suffix":""},{"id":452108392,"identity":"a17ec5f9-4c89-4dae-9279-4244d221779e","order_by":6,"name":"Jianmin Yu","email":"","orcid":"","institution":"Binzhou people's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jianmin","middleName":"","lastName":"Yu","suffix":""},{"id":452108393,"identity":"534b0461-bed4-4062-bc36-ea4f6e0ce791","order_by":7,"name":"Shengguang Li","email":"","orcid":"","institution":"Binzhou people's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shengguang","middleName":"","lastName":"Li","suffix":""},{"id":452108394,"identity":"a82da377-29e2-45fd-a0be-998249402d00","order_by":8,"name":"Jiao Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBAC+wOMDWAGH8PhAwc+/CBCiwEDVAsbw7HEgzN7iNICBWwMPMaHOdiI0cJ+uO0z7w47BjbGMx8OM/AwyPOLHcCvxZ4nsXk275lkoC1nNxwusGAwnDk7gZDDEpuZedsOQLTM4GFIMLhNSAv/Q5iWMw8O87ARo0UCbssZBmK1PGxmnAv2yzEDYCBLEOEX/vTHDG+BIcYvcfjxhw8/bOT5pQloAQEm3gaG+gaJAyC2BGHlIMD4swFI8jcQp3oUjIJRMApGHgAA5vZDnAKvdv8AAAAASUVORK5CYII=","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Liu","suffix":""},{"id":452108395,"identity":"527d43ed-4bd3-4c54-8d85-3cb59ce55ca4","order_by":9,"name":"Hongjin Yu","email":"","orcid":"","institution":"Binzhou people's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongjin","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2025-04-14 14:08:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6446806/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6446806/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82176955,"identity":"ef4acfd6-2c44-42fe-9563-9cffaba1705f","added_by":"auto","created_at":"2025-05-07 11:15:47","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":95150,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of FABP4 and PPARγ proteins in periarticular tissues of OA patients. (A-B) Expression of FABP4 and PPARγ proteins in suprapatellar fat, subpatellar fat, n=7. (C-D) The protein expression of FABP4 and PPARγ were determined by western blot, n=3. (E-F) Expression of FABP4 and PPARγ proteins in cartilage, synovium, n=3. Values are expressed as x̄±s. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the control. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the non-obese OA.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6446806/v1/a6450df647e9d2ce8631078c.jpeg"},{"id":82176963,"identity":"84f97323-8e5b-4dec-8fc3-ec86a9853c80","added_by":"auto","created_at":"2025-05-07 11:15:47","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":143482,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of FABP4 and PPARγ mRNA in periarticular tissues of OA patients. (A-B) Expression of FABP4 and PPARγ mRNA in suprapatellar fat, subpatellar fat, n=7. (C-D) Expression of FABP4 and PPARγ mRNA in cartilage, synovium, n=3. Values are expressed as x̄±s. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the control. #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the non-obese OA.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6446806/v1/a68de4db0c3d3e64d155c804.jpeg"},{"id":82176953,"identity":"e4acb08f-15dd-41ac-94ef-c56a7b367cce","added_by":"auto","created_at":"2025-05-07 11:15:47","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":558755,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological observation of periarticular patients with OA. (A) H\u0026amp;E (×100,×400) staining and oil red O (×100) staining of synovium in the control group, OA non-obesity group and OA obesity group. (B) H\u0026amp;E (×100,×400) staining and oil red O (×100) staining of cartilage in the control group, OA non-obese group and OA obese group.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6446806/v1/55d453377f211214d783ed09.jpeg"},{"id":82176964,"identity":"c881691f-2dec-419b-8ba0-042bab609abf","added_by":"auto","created_at":"2025-05-07 11:15:47","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":727149,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution and expression of FABP4 and PPARγ in the synovium of OA patients. (A-B) Immunofluorescence of FABP4 and PPARγ were expressed in the synovium of the control group, OA non-obese group and OA obese group (×100,×400). (C) Immunofluorescence of FABP4 was quantified in the synovial membrane of the control group, OA non-obese group, and OA obese group, n=3. (D) Immunofluorescence of PPARγ was quantified in the synovial membrane of the control group, OA non-obese group, and OA obese group, n=3. Values are expressed as x̄±s. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the control. #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the non-obese OA.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6446806/v1/8a803599bb88d74d05e801f2.jpeg"},{"id":82178311,"identity":"1265bf84-ddf3-4321-b636-ae384d958fd0","added_by":"auto","created_at":"2025-05-07 11:23:47","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":88030,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of apoptosis in synovial tissue in OA patients. (A) The protein expression of cleaved-Casepase3、BAX and Bcl-2 were determined by western blot, n=3. (B-D) Expression of Caspase3、BAX and Bcl-2 proteins in synovium, n=3. (E-H) Expression of Caspase3、BAX and Bcl-2 mRNA in synovium, n=3. Values are expressed as x̄±s. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the control. #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the non-obese OA.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6446806/v1/4d65e93b33a09b0427c8e438.jpeg"},{"id":82554927,"identity":"35662641-4b7a-4983-a9a6-9b78f01ad52c","added_by":"auto","created_at":"2025-05-12 22:46:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2170943,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6446806/v1/b63b3bfe-6037-45f9-acbd-006d5cfb71fd.pdf"},{"id":82179060,"identity":"be6c331b-fae3-4f5c-b340-340f0d8b0662","added_by":"auto","created_at":"2025-05-07 11:31:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":168472,"visible":true,"origin":"","legend":"","description":"","filename":"Westernblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6446806/v1/6670d1bd8ef5df3058d76bec.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Study on the changes of FABP4 and PPARγ levels in knee tissues of patients with osteoarthritis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is one of the most common types of arthritis, and it is also a chronic degenerative joint inflammatory disease. Its Key features are articular cartilage damage, sclerosis of subchondral bone, synovial hypertrophy, synovial cell inflammation and proliferation, and vascular hyperplasia\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. OA is more common in middle-aged and elderly people and obese people, the clinical symptoms are joint pain, swelling, deformity, and mobility dysfunction, which are the main causes of pain and disability in patients\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. According to statistics, about 16% of the world's population suffers from osteoarthritis\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The pathogenesis of OA is still unclear, and there is no effective way to prevent and treat OA.\u003c/p\u003e \u003cp\u003eRecently, a growing number of studies have found that the occurrence of the disease may be related to a variety of factors such as age, gender, fat, and genetics\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. At the same time, it was found that the incidence of OA increased significantly in obese people, but weight and weight may not be the direct cause of OA, but may affect the occurrence and development of OA by the deposition of adipose tissue\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Adipose tissue plays an important role in the secretion of adipokines, which is a key player in the regulation of inflammation, immunity, angiogenesis, fibrinolysis\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, and is a highly active endocrine organ that secretes inflammatory factors, and low-grade chronic inflammatory state is very common in obese and overweight people, so obesity is considered to be one of the key factors in the pathogenesis of osteoarthritis\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFatty acid-binding protein 4 (FABP4) is a small cytosolic lipid-binding protein mainly secreted by macrophages and adipocytes\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, which can promote lipolysis, inhibit adipose tissue lipogenesis, reduce free fatty acid levels, and play an important role in inflammatory diseases. In the case of obesity, the excessive accumulation of fat increases the level of the small cytosolic lipid-binding protein FABP4\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, which may promote the development of osteoarthritis.\u003c/p\u003e \u003cp\u003ePeroxisome proliferator-activated receptor γ (PPARγ) is fatty acid-activating transcription factor, plays an important role in adipogenesis and lipid synthesis\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. PPARγ is a downstream factor of FABP4, which can transcriptionally regulate the expression of FABP4, and its activation can correct the disorder of lipid metabolism. At the same time, it acts with FABP4 to bind intracellular lipids to biological targets to complete signal transduction, which is the main regulator of adipocyte differentiation\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In related studies, it has been found that activation of PPARγ can enhance the phagocytosis and immunomodulatory functions of cells and reduce the occurrence and development of inflammation, but the overactivity of FABP4 affects the abnormal expression of PPARγ\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, so it may affect the occurrence and development of osteoarthritis.\u003c/p\u003e \u003cp\u003eAt present, it has not been clarified whether the FABP4, PPARγ may affect the occurrence and development of OA. Therefore, the aim of this study was to investigate the changes in the levels of FABP4 and PPARγ in the periarticular tissues of obese patients with OA and non-obese patients with OA.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e1.Study population\u003c/p\u003e \u003cp\u003eOA patients undergoing surgical treatment in Hospital affiliated to Shandong First Medical University were selected. Collection of periarticular tissue specimens abolished during patient surgery (including articular cartilage, articular synovium, suprapatellar fat pad and infrapatellar fat pad), clinical characteristics, clinical examination results and other relevant clinical data. The experiment has received ethical approval from the Research Ethics Committee of Binzhou is People's Hospital (Approval No. 2021. 266).in full compliance with Declaration of Helsinki, The Belmont Report, CIOMS Guidelines and the International Conference on Harmonisation-Good Clinical Practice (ICH-GCP). All methods were performed in accordance with these guidelines and regulations. All participants provided written informed consent.\u003c/p\u003e \u003cp\u003eInclusion Criteria: (1) meet the relevant diagnostic criteria for osteoarthritis; (2) The patient did not have serious organic lesions of the cardiovascular, digestive, respiratory and other systems; (3) Bone and joint surgery was performed during hospitalization, and some tissue samples around the knee bone and joint were taken for pathological examination; (4) The hospital ethics committee has approved it, and the patient and family members have informed consent; (5) Complete clinical data.\u003c/p\u003e \u003cp\u003eExclusion Criteria: (1) Patient has a history of previous joint surgery; (2) Patients with cancer, liver and kidney insufficiency, and hematologic diseases; (3) arthritis caused by autoimmune diseases; (4) Incomplete or missing clinical patient cases.\u003c/p\u003e \u003cp\u003e2. Clinical data and specimen collection\u003c/p\u003e \u003cp\u003eThe enrolled patients (all male patients) were divided into 32 cases of obese OA patients (BMI \u0026gt; 25 Kg/m\u003csup\u003e2\u003c/sup\u003e) and 20 cases of non-obese OA patients (study group) and 25 cases of patients with arthro-surgical injuries who underwent knee surgery at the same time (control group), and the sample tissues abolished during their surgeries were collected; and the relevant clinical data of the patients as well as of normal subjects were also collected.\u003c/p\u003e \u003cp\u003e3. Western blot\u003c/p\u003e \u003cp\u003e Radioimmunoprecipitation (RIPA) buffer homogenate 1ml was added to the cryopreserved tissue samples, the supernatant was obtained after centrifugation, and the protein concentration was determined by ultra-micro volumetric spectrophotometer. After adjusting the protein concentration of each sample, 5X protein loading buffer was added, and heated in a metal bath at 100℃ for 5min. Equal amounts of extracted proteins were separated by 12% SDS-PAGE electrophoresis, and wet transmembrane (PVDF membrane) was conducted for 1 h. After being blocked at room temperature for 20 min with Rapid blocking solution, and then primary antibodies including FABP4(Abcam; ab13979), PPARγ(Proteintech;16643-1-AP), BAX (Abcam;ab32503), Caspase3 (Proteintech;66470-2-Ig), Bcl-2 (Immunoway; YM3041), or GAPDH(Abcam; ab9485) were added, and the incubation were performed overnight at 4℃. The membranes were washed 4-time in TBST, and then incubated with the second antibody for 1 h at room temperature. After 4-time washing in TBST, the membrane was incubated with ECL solution and exposed using a G-Box scanner (Gene Company Limited). Finally, the densitometric analysis was performed using Image-J software.\u003c/p\u003e \u003cp\u003e4. Quantitative real-time PCR\u003c/p\u003e \u003cp\u003eTotal RNA extraction reagent was used to RNA extraction from tissue sample. RNA samples were then transcribed into cDNA using a cDNA Synthesis Kit (Monad;MR05101), added SYBR Green PCR Master Mix and corresponding primers Performing an amplification reaction. PCR reactions were carried out in circulations, pre-denaturation at 95℃for 5 min and denaturation at 95℃ for 30s, primer annealing at 58℃ for 30s, and extension at 74℃ for 5 min, followed by melt curve analysis. The GAPDH gene was used as the reference gene. The value of the corresponding target gene was calculated by the 2\u003csup\u003e−△△CT\u003c/sup\u003e method.\u003c/p\u003e \u003cp\u003e5. Histopathological Analysis\u003c/p\u003e \u003cp\u003eThe periarticular tissue fixed with 4% paraformaldehyde was sectioned after paraffin embedding. Calcium in the knee joints was removed using a decalcifying solution for 28 days. The periarticular tissues were embedded in paraffin and coronally sectioned. The sections were stained with safranin O Staining or hematoxylin \u0026amp; eosin (H\u0026amp;E).\u003c/p\u003e \u003cp\u003e6. Immunofluorescence\u003c/p\u003e \u003cp\u003eThe tissue samples were fixed in 4% paraformaldehyde for 15 min, and washed with PBS three times. After added trypsin, incubated at room temperature at 37℃ for 30 min. The tissue samples were incubated with antibodies FABP4 and PPARγ at 4℃for 24 h. Then tissue samples were incubated with fluorescein-conjugated anti-rabbit IgG (Proteintech, 20001102) at 37℃for 1 h. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). The images were acquired by a fluorescence microscope. Fluorescence intensity was quantified by using Image-J software.\u003c/p\u003e \u003cp\u003e7. Statistical analysis\u003c/p\u003e \u003cp\u003eStatistical Product and Service Solutions software version 26.0(SPSS 26.0) was used in the current study. Data were presented as means ± standard deviation (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{\\text{x}}\\)\u003c/span\u003e\u003c/span\u003e±s); If the samples were normally distributed, one-way ANOVA was used, and the LSD-t test was used for comparison between the multi-sample mean and the single-group mean. If the samples did not conform to the normal distribution, the non-parametric test of two independent samples was used, and the Mann-Whitney U test was used for comparison between the means of multiple samples and the means of one group. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e1. Basic clinical information of the patient\u003c/p\u003e\u003cp\u003eAccording to the inclusion and exclusion criteria, a total of 32 obese patients with OA, 20 non-obese patients with OA were included in the study group, and 25 patients with joint surgical injuries who underwent knee surgery during the same period were included in the control group. By collating, analyzing and statistically showing that there was no statistical difference in age and height between the three groups (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05), and the weight and BMI of the obese OA group were significantly higher than those of the other two groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of general data of patients(\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{\\text{x}}\\)\u003c/span\u003e\u003c/span\u003e±s)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOA non-obesity\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOA obesity\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient number\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge(year)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.00 ± 6.69\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.93 ± 6.68\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.21 ± 6.64\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight(Kg)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.67 ± 6.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.21 ± 7.26\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75.66 ± 8.20*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeight(m)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.59 ± 0.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.67 ± 0.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.63 ± 0.07\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI(Kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.51 ± 1.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.25 ± 1.97\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.37 ± 2.61*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. the control. #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. the non-obese OA.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003eThrough the collation, analysis and statistics of blood indicators, it was found that there were no statistically significant differences in TG (triglycerides), HDL-C (high-density lipoprotein), BUN (urea nitrogen), Cr (creatinine) and UA (uric acid) in the control group, OA non-obesity group and OA obesity group. The homocysteine, LDL-C and cysC (cystatin C) in the OA obesity group were significantly higher than those in the control group and the OA non-obesity group (P \u0026lt; 0.05). The level of erythrocyte sedimentation rate (ESR), FFA (free fatty acids) in the OA obesity group was higher than that in the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). The level of TC (triglycerides) in the obese OA group was higher than that in the non-obese OA group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of clinical blood indexes of patients(\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{\\text{x}}\\)\u003c/span\u003e\u003c/span\u003e±s, n=45)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOA non-obesity\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOA obesity\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eESR(mm/h)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e6.67 ± 1.51\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e15.93 ± 5.62\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e22.84 ± 18.44*\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCysC(mg/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e0. 80 ± 0.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e0.95 ± 0.17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e1.11 ± 0.20*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCY(umol/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e5.74 ± 1.39\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e9.98 ± 4.11*\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e12.79 ± 3.56*\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFFA(mmoL/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e0.27 ± 0.13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e0.41 ± 0.21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e0.47 ± 0.27\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTC(mmol/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e4.22 ± 0.85\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e4.21 ± 0.91\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e4.90 ± 1.08\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTG(mmol/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e1.30 ± 0.34\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e1.40 ± 0.77\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e1.71 ± 0.87\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHDL-C(mmol/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e1.40 ± 0.18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e1.29 ± 0.30\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e1.19 ± 0.32\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLDL-C(mmol/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e1.95 ± 0.77\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e2.68 ± 0.72\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e3.17 ± 0.93*\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBUN(mmoL/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e6.83 ± 0.48\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e5.31 ± 2.31\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e5.9 ± 1.92\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCr(umoL/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e278.81 ± 67.24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e324.50 ± 71.55\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e306.16 ± 93.41\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUA(umoL/L)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e74.70 ± 19.89\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e64.21 ± 10.53\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e63.08 ± 13.87\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. the control. #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. the non-obese OA.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e2. Expression of FABP4 and PPARγ in periarticular tissues of OA patients\u003c/p\u003e\u003cp\u003eWestern blot results showed that there were obvious individual differences in the protein expression of FABP4 and PPARγ in the suprapatellar fat tissue of the three groups, and there was no obvious trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In subpatellar fat, the obese OA group expressed more FABP4 than the other two groups, but the protein expression of PPARγ was unstable and there was an obvious outlier (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In cartilage tissue, the expression of FABP4 protein in the obese OA group was significantly higher than that in the control group and the non-obese OA group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 or 0.01), while the expression of PPARγ protein in the obese OA group was significantly lower than that in the non-obese OA group and the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 or 0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, C and E). In the synovial tissue, the expression of FABP4 protein in the control group, non-obese OA group and obese OA group showed a significant upward trend (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01), while the expression of PPARγ protein in the control group was significantly higher than that in the non-obese OA group and the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, D and F).\u003c/p\u003e\u003cp\u003eThe results of qRT-PCR showed that there were obvious individual differences and free values in the mRNA expression of FABP4 and PPARγ in the suprapatellar adipose tissue of the three groups. In subpatellar fat, the expression of FABP4 in the obese OA group was more dispersed and there was an obvious free value (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In subpatellar fat, the mRNA expression of FABP4 was more in the obese OA group than in the other two groups, and the mRNA of PPARγ had an opposite trend, but there were also obvious free values (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In cartilage tissues, the mRNA expression of FABP4 in the obese OA group was significantly higher than that in the control group and the non-obese OA group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01), while the mRNA expression of PPARγ showed an opposite trend (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In synovial tissues, the mRNA expression of FABP4 increased significantly in the control group, non-obese OA group and obese OA group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01), while the mRNA expression of PPARγ decreased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eTherefore, by comparing the four parts of suprapatellar fat, lower fat, cartilage and synovium, it was found that the expression of FABP4 and PPARγ in suprapatellar and inferior fat was unstable and there were obvious individual differences, while synovium and cartilage were relatively stable. Therefore, cartilage and synovium were selected as the main research objects in the future.\u003c/p\u003e\u003cp\u003e3. Histopathological observation of synovial tissue and cartilage in OA patients\u003c/p\u003e\u003cp\u003eObservation of the joint pathological sections of patients in the control group, OA non-obesity group and OA obesity group showed that in the synovial tissue, the synovial surface of the control group was smooth and there was no defect and protrusion, and the synovial cells lining the inner layer had clear structure and normal morphology. In the non-obese OA group, synovial fibroblasts proliferated in the synovial tissue, forming synovial dysmorphic protrusions, and inflammatory cell infiltration. A large number of fibroblastic synovial cells proliferate into the luminal tissues of the OA obese group, forming obvious abnormal protrusions and infiltrating a large number of inflammatory cells(Fig.\u0026nbsp;3A).\u003c/p\u003e\u003cp\u003eIn cartilage tissue, the perichondrium surface of the control group was smooth and the chondrocytes were intact. Necrosis and loss of chondrocytes were observed in the cartilage tissue of patients in the non-obese group of OA, and the perichondrium surface was not smooth. A large area of chondrocytes and loss of chondrocytes could be observed in the cartilage tissue of patients with OA obesity, and the perichondrium surface damage was an entophagocytic lesion(Fig.\u0026nbsp;3B).\u003c/p\u003e\u003cp\u003e4. Periarticular tissues of OA patients are stained with oil red O Observation of the joint sections of patients in the control group, OA non-obesity group and OA obesity group showed that no obvious lipid droplet formation was seen in the synovial tissue of the control group. A small number of red lipid droplets appeared in the synovial tissue of patients in the non-obese group of OA. A large number of red lipid droplets appeared in the synovial tissue of the obese OA group, and the intracellular lipid content was significantly higher than that in the non-obese group(Fig.\u0026nbsp;3A).\u003c/p\u003e\u003cp\u003eIn cartilage tissue, lipid droplet formation was not seen in the cartilage tissue of the control group; A small number of red lipid droplets appeared at the edge of the cartilage tissue in the patients in the OA non-obese group; A large number of red lipid droplets appeared at the edge of the cartilage tissue in the OA obese group(Fig.\u0026nbsp;3B).\u003c/p\u003e\u003cp\u003eTherefore, by comparing the pathological changes of cartilage and synovium and oil red O staining, it can be found that the damage of articular cartilage in patients with OA mainly appeared at the margins, while synovial tissues appeared to have obvious inflammatory infiltration and high expression of lipid droplet content; therefore, we chose synovial tissues to further detect the expression of FABP4 and PPARγ as well as their distributions, and also to further validate them at the protein level and mRNA level.\u003c/p\u003e\u003cp\u003e5. The distribution and expression of FABP4 and PPARγ in the synovial tissues of OA patients were observed by immunofluorescence double staining\u003c/p\u003e\u003cp\u003eThe distribution and interaction of FABP4 and PPARγ were observed by immunofluorescence double staining of joint synovial tissues in the control group, OA non-obesity group and OA obesity group. The results showed that FABP4 and PPARγ were mainly distributed in the synovial lining cells of the synovial tissue of OA patients. However, in the control group, the green fluorescence of FABP4 was significantly darker, the red fluorescence of PPARγ was brighter, and the colocalization of FABP4 and PPARγ was clearly superimposed on the purple fluorescence, and the expression of FABP4 in the nucleus was reduced. Compared with the control group, the green fluorescence area of FABP4 in the synovial tissue of OA patients was significantly increased, the fluorescence intensity was significantly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01), and the red fluorescence area of PPARγ was decreased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A, B, C and D).\u003c/p\u003e\u003cp\u003e6. mRNA and protein levels of apoptosis-related genes in synovial tissue of OA patients\u003c/p\u003e\u003cp\u003eIn order to verify the effects of FABP4 and PPARγ on osteoarthritis, the expression levels of apoptosis-related factors BAX, Bcl-2 and cleaved-Casepase3 were detected(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Compared with the control group and the non-obese group, the mRNA and protein levels of BAX and cleaved-Casepase3 in OA patients in the obese group were increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 or \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e, B and C), while the mRNA and protein expressions of Bcl-2 were decreased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 or \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e, D). These results indicate that there is obvious apoptosis in the synovium of patients with osteoarthritis, which promotes the occurrence and development of osteoarthritis \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOA is a common chronic degenerative joint disease in middle-aged and elderly people, but obesity is one of the major risk factors for osteoarthritis. Studies have shown that as BMI increases, it leads to abnormalities in blood serum (ESR), resulting in an increase in joint pain symptoms and severity\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. We detected the expression of blood indexes in OA obese patients, OA non-obese patients and control group. ESR, as an indicator of erythrocyte aggregation in blood, is a commonly used inflammatory marker, which can indicate and monitor the increase of inflammatory activity in vivo caused by one or more diseases. When the body is diseased, the erythrocyte sinks due to its high density, resulting in an increase in ESR. This study found that compared with the normal control group, the BMI of obese OA patients was significantly increased, and the contents of ESR, TC, LDL-C and FFA in blood indexes were also significantly increased. These data suggest that obesity is closely related to the occurrence and development of OA. In addition, the excess energy of the whole body caused by obesity not only changes the function of various cells in the tissue, but also releases a variety of adipokines, which may also be related to OA\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFABP4 is a novel adipokine expressed in adipocytes, macrophages, etc. It can preferentially bind to fatty acids, enter the cytoplasm for chemical modification such as oxidation and esterification, and transport to various parts of the body to participate in lipid synthesis, metabolism, inflammatory response and other signal transduction\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Recent studies have shown that overexpression of FABP4 gene promotes lipid deposition in transgenic mice and promotes the severity of atherosclerosis\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Gene knockout of FABP4 can protect mice from diseases such as cancer\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e and fatty liver\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. On the other hand, PPARγ, as a lipid-sensitive nuclear receptor, is expressed in various cell tissues such as fat, muscle, and liver\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. It has significant anti-inflammatory properties, which can regulate the immune inflammatory response. PPARγ activation reduced inflammatory response by negatively interfering with NF-κB and signal transducers and transcriptional activators\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. The PPARγ signaling pathway has also been reported to be involved in the regulation of lipogenesis in C2C12 myoblast cells and C57BL/6 mouse meibocytes\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. PPARγ gene knockout can promote cell apoptosis, resulting in cell damage, and aggravate the occurrence and development of various lipid metabolism and inflammatory diseases\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Therefore, in this study, we aimed to explore the potential role of FABP4 and PPARγ in OA. By detecting the protein and mRNA expression of FABP4 and PPARγ in the periarticular tissues of OA patients, we found that the expression of FABP4 and PPARγ in the suprapatellar fat and infrapatellar fat of OA patients was unstable and there were obvious individual differences. In synovium and cartilage, the expression of FABP4 in OA obese patients was significantly higher than that in control group and non-obese OA patients, while the expression of PPARγ showed an opposite trend.\u003c/p\u003e \u003cp\u003eFABP4 has been confirmed to deliver specific ligands from the cytoplasm to the nuclear receptor PPARγ, which facilitates ligation and enhances the receptor transcriptional activity\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. It has been found that FABP4 directly binds to and promotes the degradation of PPARγ, while PPARγ expression is significantly elevated in FABP4 knockout macrophages\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. However, whether the interaction between FABP4 and PPARγ affects the occurrence and development of OA has not been reported. In this study, by immunofluorescence double staining of FABP4 and PPARγ in synovial membrane of three groups of patients, it can be found that the fluorescence expression of FABP4 in the OA obese group was significantly higher than that in the OA non-obese group and the control group, while the fluorescence expression of PPARγ was significantly suppressed, and at the same time, both of them appeared to have obvious co-localization, so there may be a relationship between the two interactions, and FABP4 can regulate the development of OA through negative feedback. PPARγ can promote cell proliferation, regulate lipid metabolism and thus alleviate OA. previous studies have also confirmed that FABP4 can reduce the gene and protein expression of PPARγ during adipose differentiation, inhibit adipogenesis through negative feedback regulation, improve the abnormal lipid accumulation that occurs in pleural hypertrophy, and also alleviate the fatty liver and other diseases\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. FABP4 knockdown suppressed the inflammation, oxidative stress, apoptosis and extracellular matrix degradation of IL-1β-induced chondrocytes by activating PPARγ to inhibit the NF-κB signaling pathway\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn modern studies the development of OA is also affected by apoptosis, which induces cell lysis, leading to the massive release of pro-inflammatory factors and promoting the development of many inflammatory diseases\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Therefore, we also examined the expression of proliferation- and apoptosis-related indicators and analyzed the role of the FABP4/PPARγ signaling pathway in the proliferation and regulation of OA. BAX a protein located on the outer membrane of the mitochondria, is the key executor of mitochondria in the regulation of cell death, and in the process of apoptosis, BAX can change the permeability of the outer membrane of the mitochondria, form mitochondrial membrane channels, and release the cytochrome Bcl-2 is an anti-apoptotic protein that directly binds to pro-apoptotic proteins and inhibits BAX activity, thereby affecting the permeability of the outer mitochondrial membrane and exerting an anti-regulatory effect\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e.Caspase3 is a member of the cysteine protease family, known for its ability to mediate the cleavage of specific target proteins during the process of cellular death. Caspase3 can be a key executor in the regulation of cell death. process, Caspase3 cleaves a variety of downstream substrates, leading to morphological changes in the regulated cells and contributing to the onset of apoptosis\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. The increased expression of BAX and Caspase3 and decreased expression of Bcl-2 in synovial tissues of OA patients indicated that apoptosis was abnormal in synovial tissues of OA patients, and thus increased apoptosis would also promote the development of OA.\u003c/p\u003e \u003cp\u003eIn summary, the expression level of FABP4 was significantly elevated and that of PPARγ was significantly reduced in synovial tissues of OA patients, so we hypothesized that FABP4 might inhibit the expression of PPARγ, and the abnormal expression of FABP4 and PPARγ might affect the development of OA. However, this study also has limitations such as the small number of clinical samples, which may make the results have a certain degree of error, so we will follow up with in-depth studies in animal and cellular experiments to investigate the detailed mechanism of FABP4 and PPARγ on the development of OA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Binzhou Municipal People\u0026apos;s Hospital for providing the tissue samples and the laboratory team who helped us in our routine experimental work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS. Zhang: Conceptualization, Data curation, Writing-original draft\u003c/p\u003e\n\u003cp\u003eN. Liu: Investigation, Validation Y. Zhang: Software, Validation \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eY. Zhao: Visualization\u003c/p\u003e\n\u003cp\u003eX. Yang: Methodology, Resources\u003c/p\u003e\n\u003cp\u003eX. Wu: Data curation, Formal analysis\u003c/p\u003e\n\u003cp\u003eJ. Yu: Supervision, Writing-review and editing\u003c/p\u003e\n\u003cp\u003eS. Li: Data curation,\u003c/p\u003e\n\u003cp\u003eJ.Liu:Writing-review, Data curation, Conceptualization, Funding acquisition, Resources, Supervision\u003c/p\u003e\n\u003cp\u003eH.Yu:Writing-review, Funding acquisition, Project administration, Resources, Supervision\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eICMJE COI statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical review statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical \u0026nbsp; approval \u0026nbsp;was \u0026nbsp;obtained \u0026nbsp; from \u0026nbsp;the \u0026nbsp;Ethics \u0026nbsp; Committee \u0026nbsp;of Binzhou Municipal People\u0026apos;s Hospital (Approval No. 2021. 266).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen access funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Science Foundation of the Ministry of Education of China（2023HT048）\u0026nbsp;and the Natural Science Foundation of Shandong Province（ZR2021MH084）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMotta F, Barone E, Sica A, Selmi C. 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Linear ubiquitination of LKB1 activates AMPK pathway to inhibit NLRP3 inflammasome response and reduce chondrocyte pyroptosis in osteoarthritis. J Orthop Translat. 2022 Dec 1;39:1-11. \u003c/li\u003e\n\u003cli\u003eSpitz AZ, Gavathiotis E. Physiological and pharmacological modulation of BAX. Trends Pharmacol Sci. 2022 Mar;43(3):206-220. \u003c/li\u003e\n\u003cli\u003eEskandari E, Eaves CJ. Paradoxical roles of caspase-3 in regulating cell survival, proliferation, and tumorigenesis. J Cell Biol. 2022 Jun 6;221(6):e202201159. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteoarthritis, synovial tissue, FABP4, PPARγ","lastPublishedDoi":"10.21203/rs.3.rs-6446806/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6446806/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To investigate the expression and significance of fatty acid-binding protein4（FABP4）and peroxisome proliferator-activated receptor γ（PPARγ）in the tissues around the knee joint of patients with osteoarthritis (OA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod: \u003c/strong\u003eA total of 32 obese patients with OA（BMI\u0026gt;25 Kg/m\u003csup\u003e2\u003c/sup\u003e）and 20 non-obese patients with OA were collected from Hospital affiliated to Shandong First Medical University who underwent surgical treatment. In addition, 25 patients with joint surgical injuries who underwent knee surgery at the same time were selected as the control group. The clinical data of the three groups (including age, height, blood routine indexes and biochemical routine indexes) were collected, and the statistics were collated. At the same time, specimens of periarticular tissues (including articular cartilage, synovium, suprapatellar fat pads, and infrapatellar fat pads) that had been discarded during the surgical procedure were collected from all three groups of patients. Western blot, qRT-PCR and immunofluorescence double staining were used to detect the expression and distribution of FABP4 and PPARγ in the tissues around the knee joint in obese patients with OA and non-obese patients with OA. Confirmation that informed consent was obtained from all subjects or their legal guardians.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult: \u003c/strong\u003eIn synovial tissue, the expression of FABP4 in OA obese patients was significantly higher than that in OA non-obese patients and control group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01), and the expression of PPARγ in OA obese patients was significantly lower than that in non-obese OA patients and control group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). In the synovial tissues of OA patients, the green fluorescence intensity of FABP4 was significantly enhanced (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01), and the red fluorescence intensity of PPARγ was significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eAberrant expression of FABP4 and PPARγ may be a potential therapeutic target affecting the development of OA.\u003c/p\u003e","manuscriptTitle":"Study on the changes of FABP4 and PPARγ levels in knee tissues of patients with osteoarthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 11:15:42","doi":"10.21203/rs.3.rs-6446806/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fa9d968f-cbf8-42a8-8386-1181e49142be","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48081937,"name":"Health sciences/Diseases/Rheumatic diseases"},{"id":48081938,"name":"Health sciences/Diseases/Rheumatic diseases/Rheumatoid arthritis"}],"tags":[],"updatedAt":"2025-05-12T22:38:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 11:15:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6446806","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6446806","identity":"rs-6446806","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}