Involvement of the RhoA/ROCK signaling pathway in the pathogenesis of knee osteoarthritis

Involvement of the RhoA/ROCK signaling pathway in the pathogenesis of knee 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 Research Article Involvement of the RhoA/ROCK signaling pathway in the pathogenesis of knee osteoarthritis Tianxiang Yang, Yi Wang, Zige Liu, Bowen Zhang, Jinning Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4112556/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 Purpose This study compared the expression of the Ras homolog gene family member A (RhoA) and Rho-associated coiled-coil kinase (ROCK) signaling pathway members in knee joint synovial tissue from patients with primary knee osteoarthritis (KOA) who underwent lower limb disarticulation. Methods The specimens were separated into observation (synovial tissue removed during total knee arthroplasty of 30 patients with primary KOA) and control groups (synovial tissue removed during lower limb disarticulation of 25 patients). Results The observation group specimens showed high proliferation, congestion, edema, unorganized cellular arrangement, and presence of vacuolated cells. The control group specimens were white with a smooth surface and non-edematous texture, and showed organized and homogenous cell/stroma arrangement. The observation group showed higher expression of RhoA and ROCK transcripts and proteins than the control. Conclusion Patients with KOA expressed high levels of RhoA and ROCK transcripts and proteins in synovial tissues, suggesting a relationship between RhoA/ROCK and KOA. Our findings provide a potential therapeutic target for delaying synovial lesions in KOA. Lower limb disarticulation Knee osteoarthritis Synovium RhoA/ROCK signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Osteoarthritis (OA) mostly occurs in older adults. Its incidence rate in China is approximately 17% in the middle-aged population, with higher prevalence in women [ 1 , 2 ]. This disease commonly affects the weight-bearing joints, with knee osteoarthritis (KOA) accounting for > 80% of all OA cases [ 3 , 4 ]. KOA manifests as sclerosis of the subchondral bone and periarticular cartilage, in addition to the wear and tear, degeneration, softness, deformation, and disappearance of the articular cartilage. Sclerosis of the subchondral bone, periarticular synovial hyperplasia, degeneration and atrophy of the joint capsule, peripheral ligaments, and other tissues, destruction of the integrity of the joint surface, and narrowing of the joint space can lead to knee deformity [ 5 , 6 ]. Pathological changes mainly occur in the synovial membrane, cartilage, meniscus, and patella in the early stages of KOA [ 7 ], while knee joint pain, stiffness, swelling, limited activity, popping, and deformity appear gradually. KOA can involve the muscles, seriously affecting patients’ physical and mental health and daily activities [ 8 ]. Therefore, early diagnosis, intervention, and treatment have become the focus of joint surgeries. Cartilage wear and tear is the core pathological change in KOA; however, pathological cartilage changes occur in the middle and late stages [ 9 ]. Synovial inflammation, an important early pathological change in KOA, can result in knee pain and structural changes, and its incidence has been increasing continuously [ 10 ]. Normal synovial and cartilage tissues are loose connective tissues that secrete synovial fluid to nourish the cartilage, reduce friction, maintain intra-articular metabolism, and provide nutrition to the articular cartilage [ 11 , 12 ]. Intra-articular lesions lead to the loss of synovial nutritional function and production of many proteases and cytokines, damaging the chondrocytes. The pro-inflammatory factors released from synovial and cartilaginous tissues stimulate the production of protein hydrolyzing enzymes, thereby aggravating articular cartilage degeneration, which causes joint pain and deterioration [ 13 – 16 ]. Ras homolog gene family member A (RhoA) proteins are expressed in all tissues and affect cell growth, transformation, cytoskeletal regulation, actin stress fiber formation, and actin-myosin contraction [ 17 – 19 ]. Rho-associated protein kinase (ROCK), a downstream effector of RhoA, exists as two isoforms, ROCK1 and ROCK2. The RhoA/ROCK signaling pathway is involved in cell growth, differentiation, migration, and development [ 20 , 21 ], and is required for neuronal synaptic growth, bone formation, dorsal closure, and myogenesis [ 22 ]. Its aberrant activation is associated with various cancer types and diseases affecting the neurological, endocrine, and cardiovascular systems [ 23 – 28 ]. Previous studies have shown that the RhoA/ROCK signaling pathway is closely related to cartilage degeneration in KOA, but such association has not been studied in synovial lesions in KOA. The aim of this study was to investigate the correlation between the RhoA/ROCK signaling pathway and the pathogenesis of KOA by comparing the expression of RhoA and ROCK in the synovial tissue of patients with primary KOA as a way to potentially identify a therapeutic target for delaying synovial lesions in KOA. Methods Patients Observation group: The observation group comprised synovial tissue samples removed during total knee arthroplasty (TKA) from 30 patients with primary KOA between January 2020 and December 2022. The patients were 57–82 years of age (average age, 66.40 ± 6.05 years), and included 11 men and 19 women. The inclusion criteria were as follows: primary KOA and fulfillment of the diagnostic criteria of the Chinese Osteoarthritis Diagnostic and Treatment Guidelines (2018 edition) (Table 1 ); poor outcomes after receiving conservative treatment; unilateral knee arthroplasty for the first time; and provision of informed consent by patients or their family members. Table 1 Diagnostic criteria for knee osteoarthritis (2018 version). No. Symptoms or signs 1 Recurrent knee pain within the last 1 month 2 Radiography (standing or weight-bearing position) showing narrowing of the joint space, subchondral osteosclerosis and/or cystic degeneration, and formation of bone capillaries at the joint margins 3 Examination of the synovial fluid in the knee joint consistent with osteoarthritis (clear, viscous, white blood cell count < 2.0 × 10 9 /L) 4 Age ≥ 50 years 5 Morning stiffness ≤ 30 min 6 Sound of bone friction (sensation) during activity Knee osteoarthritis can be diagnosed by meeting at least diagnostic criteria 1 and 2; 1, 3, 5, and 6; 1, 4, 5, and 6. The exclusion criteria were as follows: serious blood-related diseases or receipt of prolonged anticoagulant drug treatment in preoperative manner; combined joint tuberculosis, tumor, and other serious diseases; serious knee deformity or trauma preventing cooperation while receiving treatment; history of drug injection into the joint cavity within half a year; and receipt of oral treatment for OA within the previous 2 weeks. Control group: Twenty-five control specimens were obtained from the synovial tissue of knee joints removed during “lower limb disarticulation” during the same period at our hospital. The patients were 19–39 years of age (average age, 31.07 ± 6.10 years), including 12 men and 13 women. The inclusion criteria for the control group were as follows: age, 18–40 years; non-KOA; “lower limb disarticulation” because of malignant tumors of the lower limbs or lower limb disfigurement injuries; and voluntary study participation. The exclusion criteria were as follows: arthritis on preoperative imaging; rheumatoid arthritis; knee joint infection; autoimmune disease; tuberculosis and other serious systemic diseases; and serious blood-related diseases. This study was approved by the responsible ethics review board (No. 2020 − 974). Informed consent was obtained from the patients before enrollment in the study. Reagents The chemicals and reagents used included anti-ROCK1 (ab134181; Abcam, Cambridge, UK)/anti-ROCK2 (ab228000; Abcam) and anti-RhoA (ab54835; Abcam) antibodies, RNA solids (Wuhan Xavier Biotechnology Co., Ltd., Wuhan, China), and polymerase chain reaction (PCR) primers (Shanghai Sangong Bioengineering Service Co., Ltd., Shanghai, China). All the other reagents were obtained from Yinchuan Runyan Biotechnology Co., Ltd. (Yinchuan, China). Hematoxylin and eosin (HE) staining The tissue sections were soaked in xylene I and II for 15 min for dewaxing. Subsequently, they were immersed in the following gradients of alcohol for 3–5 min each: anhydrous ethanol Ⅰ and Ⅱ, 95% ethanol, 80% ethanol, and 70% ethanol; the sections were then removed and rinsed with distilled water for 5 min. Then, 1% hydrochloric acid in alcohol was used for differentiation for 3–5 s, followed by rinsing for 5 min. To dye them blue, the tissue sections were immersed in 0.2% ammonia for 1 min, followed by rinsing in distilled water for 5 min. Next, the tissue sections were stained with 0.6% eosin for 30 s to 1 min. After dehydration, the sections were soaked in xylene I and II for 10 min each and sealed with optical resin glue. Immunohistochemistry The slices were put in the oven at 60°C for > 6 h, followed by dewaxing and rehydration. The sections were rinsed thrice for 5 min. Then, they were boiled in citrate buffer (pH 6.0) for 15 min and placed in a mixture of ice and water at 0°C for 30 min. The residual citrate buffer was washed out thrice using distilled water for 5 min each. The washed tissue sections were soaked in 3% H 2 O 2 solution for 15 min and rinsed thrice for 5 min. The synovial tissue was blocked with goat serum at 20–25 ℃ for 15 min. A drop of one antigen was added to the synovial tissue, and the tissue sections were incubated overnight in the refrigerator in a humid box. The sections were rinsed thrice using phosphate buffered saline (PBS) for 10 min. In total, 2–3 drops of horseradish peroxidase-labeled goat anti-rabbit IgG antibody were added to cover the synovial tissue, and the tissue sections were incubated for 1 h at 37°C. The slices were rinsed thrice with PBS (for 5 min at a time). The slices were counterstained using the following sequence of reagents: hematoxylin staining solution for 2 min and 1% hydrochloric acid in alcohol solution for 30 s. The sections were dehydrated for 10 min. Western blotting Approximately 500 mg of each specimen was ground and transferred to an Eppendorf tube. A mixture of the whole protein lysate, protease inhibitor cocktail, and phenylmethylsulfonyl fluoride were added to each tube, mixed well, and placed on a vortex oscillator for 30 s; it was then placed on ice for tissue lysis, vortexed, and centrifuged again after 15 min; this procedure was repeated thrice. The tissue lysate was centrifuged (12,000 rpm) at 4 °C for 20 min. The supernatant and 1/4th of the supernatant were aspirated into 5× loading buffer, placed into a new Eppendorf tube for mixing, and boiled in hot water for 10 min. Equal amounts of protein from each sample were subjected to electrophoresis after adding loading buffer to achieve equal volumes across samples. Electrophoresis was initiated at 80 V and 400 mA, and after 30 min, the voltage and current were adjusted to 120 V and 400 mA, respectively. The gel was transferred to a methanol-soaked polyvinylidene fluoride (PVDF) membrane at 250 V and 260 mA for 90 min. The PVDF membrane was washed thrice with PBS-Tween 20 for 45 min. After sealing the membrane, the latter was washed with PBST) three times (10 min each time) and incubated overnight at 4°C on a refrigerator shaker with primary antibodies diluted at different ratios. Subsequently, the membrane was incubated with a secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG; 1:5,000 dilution) for 60 min at 20–25 ℃ on a shaking bed. The membrane was then washed thrice prior to development using a developing solution and visualization. Quantitative reverse transcription-PCR Approximately 30–40 mg synovial tissue was weighed and ground with liquid nitrogen to a fine powder, followed by the addition of 1 ml Trizol lysis buffer. The tissue was ground until liquification in the Trizol buffer. Two hundred microliters of chloroform was added, mixed to emulsify, placed at room temperature for 5–10 min, and centrifuged for 20 min at 12,000 rpm, 4°C. Five hundred microliters of the supernatant and an equal amount of isopropanol were mixed in a new tube. The tube was vortexed to separate the DNA, left standing for 5 min, and centrifuged for 20 min under the same conditions, following which the supernatant was discarded. One milliliter of 75% ethanol was used to wash the pellet and centrifuged in a low-temperature ultracentrifuge for 5 min at 4°C (7,500 rpm). The upper ethanol layer was discarded and the precipitate was put on the ultra-clean bench for 5 min. Diethyl pyrocarbonate-treated water was added to dissolve the precipitate, followed by heating at 65°C for 10 min. The cDNA was synthesized via reverse transcription of mRNA using an Applied Biosystems (Waltham, MA, USA) instrument under the following conditions: 37°C (15 min), 85°C (5 s), and 4°C (∞). Primers were designed and synthesized using the sequences listed in Table 2 . GAPDH was used as an internal reference, and the relative expression of the target mRNAs was calculated using the 2 −△△Ct method and a LightCyder480 instrument. Table 2. Primer names and sequences. Data analysis All data were analyzed using the SPSS software (v25.0; IBM Corp., Armonk, NY, USA) and were expressed as mean ± standard deviation; comparisons between groups were performed using the Student’s t -test for independent samples. Statistical results were plotted using the GraphPad Prism software (v7.0; GraphPad Software, La Jolla, CA, USA), with a significance level of α = 0.05. Results Appearance and morphology of the synovial tissue In the observation group, the synovial tissue showed high levels of proliferation and edema, with obvious congestion and a yellowish appearance (Fig. 1 a). The synovial membrane surface in the control group was smooth. There were no signs of edema or congestion, and the tissue appeared white (Fig. 1 b). HE staining RhoA expression in the synovial tissues RhoA protein was expressed widely in the synovial tissues of 30 patients with KOA compared with that in the control group. RhoA expression appeared as light yellow or brownish-yellow particles in the cell cytoplasm, cytosol, and interstitium (Fig. 3 a, c). RhoA expression was low in the control group (Fig. 3 b, d). ROCK1 expression in the synovium ROCK1 was expressed in the synovial tissues of 30 KOA joints, and the positively-stained particles were distributed in the cell membranes (Fig. 4 a, c). ROCK1 expression was low in the control group (Fig. 4 b, d). ROCK2 expression in the synovial membrane Anti-ROCK2 staining was observed in the synovial tissue of 30 KOA joints, and the positively-stained particles were distributed in the cytoplasm (Fig. 5 a, c). Anti-ROCK2 staining was low in the control group (Fig. 5 b, d). Compared with the control group, numerous samples in the observation group showed significant positive immunoreactivity for RhoA and ROCK, which were distributed widely ( P < 0.05; Table 3 ). The observation group showed higher RhoA and ROCK protein levels than the control group ( P < 0.05; Fig. 6 ). The synovial tissue in the observation group showed higher levels of RHOA and ROCK transcripts than that in the control group ( P < 0.05; Fig. 7 ). Table 3. Comparison between RhoA, ROCK1, and ROCK2 Expression Intensity in the Two Groups (Mean ± SD). Group Observation group Control group t -Value P RhoA 7.11 ± 1.61 2.43±0.78 14.328 <0.001 ROCK1 6.61 ± 1.45 2.92±0.54 13.043 <0.001 ROCK2 5.05 ± 1.14 3.20±0.53 8.063 <0.001 Discussion Surgical management using artificial joint arthroplasty is considered in clinical practice for patients with severe KOA, which affects their normal life activities. TKA can reduce the clinical symptoms, restore knee joint function, and improve the quality of daily life [ 29 ]. However, ensuring the survival of TKA prostheses after wear and tear, and rehabilitation of patients after knee arthroplasty are associated with high social, medical, and economic burdens. Early diagnosis and treatment can reduce the pain experienced by the patients and the economic burden. Hence, the study of early synovial inflammation pathogenesis in patients with KOA is crucial for its prevention and treatment [ 30 , 31 ]. In our study, the high expression of RhoA and ROCK transcripts and proteins during synovial inflammation was verified using immunohistochemistry, western blotting, and quantitative reverse transcription-PCR, confirming the relevance of the RhoA/ROCK signaling pathway with the development of KOA synovitis. Synovial inflammation in KOA is considered an early-stage lesion. The RhoA/ROCK signaling pathway plays a key role in lipopolysaccharide-induced inflammatory pain and has been associated with signaling pathway activation to cause nociceptive hypersensitivity and the concomitant release of tumor necrosis factor-α and interleukin-1β [ 32 , 33 ]. Suppression of ROCK expression and articular cartilage degeneration have been observed after the injection of RhoA/ROCK signaling inhibitors in rats, while the production of inflammatory factors in vivo and joint pain scores were reduced and chronic neuropathic pain was alleviated, consistent with our results [ 34 , 35 ]. Synovial inflammation can increase peripheral vascular permeability, lower pain thresholds, and increase nociceptive sensitivity. Activation of the RhoA/ROCK signaling pathway promotes the release of inflammatory factors and participates in sensory processing of spinal cord injury, which in turn aggravates inflammatory responses and increases knee pain. This study had certain limitations. We only verified the correlation between the RhoA/ROCK signaling pathway and KOA synovitis but did not use serology, cytology, and gene knockdown technology to further understand the mechanisms underlying the correlation. Moreover, the sample size was small, and the same group of specimens could not be used for all the testing techniques. Therefore, a larger sample size and comprehensive analysis are necessary to clarify the contribution of the pathway. Conclusions The expression of RhoA and ROCK transcripts and proteins was higher in the synovial tissue of patients with KOA than in those of the control, indicating that the RhoA/ROCK signaling pathway was involved in KOA. This study shed light on novel approaches for screening target genes for early diagnosis of KOA and development of preventive treatment strategies. In the future, it would be important to study the correlation between this signaling pathway and KOA synovitis in depth, and to develop drugs that slow down the progression of synovitis and improve synovitis-induced joint pain. Abbreviations KOA knee osteoarthritis TKA total knee arthroplasty RhoA Ras homolog gene family member A ROCK Rho-associated coiled-coil kinase Declarations Ethics approval and consent to participate This study was approved by the Medical Ethics Review Board of the People’s Hospital of Ningxia Hui Autonomous Region (No. 2020-974). Consent for publication Informed consent was obtained from the patients before enrollment in the study. Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests All authors declare that they have no conflicts of interest related to the research or writing of the manuscript. Funding This study was supported by the National Natural Science Foundation of China (82060408), the Key Research and Development Program of Ningxia Hui Autonomous Region (2021BEG03049), the Innovation and Entrepreneurship Project of Ningxia Overseas Students (2020-75), and the First-Class Discipline Construction Founded Project of Ningxia Medical University and the School of Clinical Medicine (NXYLXK2017A05). The financial support for the project did not influence the views stated in the article or the analysis of the results of the study and their reporting. Authors' contributions T.Y. was responsible for case data compilation and manuscript preparation. Y.W. was responsible for instructing the lab. J.Z. and B.Z. were responsible for statistical analysis and literature review. A.Y. and W.W. were responsible for case data collection. Z.L. and D.C. supervised the manuscript preparation. Acknowledgements Not applicable. References Li D, Li S, Chen Q, Xie X. 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Chin J Pain Med. 2016;22:493-500. Takeshita N, Yoshimi E, Hatori C, Kumakura F, Seki N, Shimizu Y. Alleviating effects of AS1892802, a Rho kinase inhibitor, on osteoarthritic disorders in rodents. J Pharmacol Sci. 2011;115:481-9. https://doi.org/10.1254/jphs.10319fp. Additional Declarations No competing interests reported. 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-4112556","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281612740,"identity":"c6f623d7-92dd-4ef0-95c3-f0cbb63c256a","order_by":0,"name":"Tianxiang Yang","email":"","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Tianxiang","middleName":"","lastName":"Yang","suffix":""},{"id":281612741,"identity":"1546fb33-e01b-4651-806c-7fea9229bdb8","order_by":1,"name":"Yi Wang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Wang","suffix":""},{"id":281612742,"identity":"2c0f49a8-93b1-43b5-be8c-1b85c0ccf638","order_by":2,"name":"Zige Liu","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zige","middleName":"","lastName":"Liu","suffix":""},{"id":281612743,"identity":"8effc5fe-adf7-48df-94ae-4dad3edb9ac7","order_by":3,"name":"Bowen Zhang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Bowen","middleName":"","lastName":"Zhang","suffix":""},{"id":281612744,"identity":"f158e622-b109-403f-877a-c43a2826a80d","order_by":4,"name":"Jinning Zhang","email":"","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Jinning","middleName":"","lastName":"Zhang","suffix":""},{"id":281612745,"identity":"f7848907-8904-4b21-8a17-178475b3c585","order_by":5,"name":"Ang Yuan","email":"","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Ang","middleName":"","lastName":"Yuan","suffix":""},{"id":281612746,"identity":"3c0c7efa-5535-467d-a866-8d7e0f2d1f44","order_by":6,"name":"Wenpeng Wang","email":"","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Wenpeng","middleName":"","lastName":"Wang","suffix":""},{"id":281612747,"identity":"f6771f9c-b796-432c-b429-b04dfc5a0c61","order_by":7,"name":"Desheng Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYDACCSB+AGIcYD5w4MMPYrUkgLWwJR6c2UOaFh7jwxxsROjgn9387ENCRZ083/kzHw4z8DDI84sdIGDJnWPGMxLOHDaceSN3w+ECCwbDmbMT8GsxkEgwZkhsO5BgcIN3w+EZPAwJBrcJakn/zJD4ry7B4PyZB4d52IjSkgO0pYE5weBADgNxWiRu5BQzJBwD+SXNABjIEoT9wj8jfTPDhxpQiB1+/OHDDxt5fmkCWjBsJU35KBgFo2AUjALsAAAhOkk3pctpXQAAAABJRU5ErkJggg==","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":true,"prefix":"","firstName":"Desheng","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-03-16 10:14:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4112556/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4112556/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53253077,"identity":"23781967-6d6b-4bd0-b55d-1c2b66c8ce86","added_by":"auto","created_at":"2024-03-22 13:09:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":348456,"visible":true,"origin":"","legend":"\u003cp\u003eAppearance of synovial membrane specimens in the observation (a) and control (b) groups.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4112556/v1/e35640853c6718a495683c94.png"},{"id":53253074,"identity":"22c67993-63d2-4029-8100-9be569f631bc","added_by":"auto","created_at":"2024-03-22 13:09:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":542964,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin and eosin staining of the synovial tissue. (a) Observation and (b) control groups (10× magnification).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4112556/v1/209cd16b4e0c53405f33e9a4.png"},{"id":53253080,"identity":"e9bf46f2-df8c-4f2b-9c3f-e9c394a7e26f","added_by":"auto","created_at":"2024-03-22 13:10:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":921698,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry of RhoA in the synovial tissues of the observation and control groups. (a) Observation group (10× magnification); (b) control group (10× magnification); (c) observation group (40× magnification); (d) control group (40× magnification); “↑” indicates the colored RhoA-positive particles.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4112556/v1/e87a01c9e300023fd4da458d.png"},{"id":53253078,"identity":"0d9e7263-6cab-4ae1-a0c4-6f478934e40f","added_by":"auto","created_at":"2024-03-22 13:10:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":837278,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry of ROCK1 in the synovial tissues of the observation and control groups. (a) Observation group (10× magnification); (b) control group (10× magnification); (c) observation group (40× magnification); (d) control group (40× magnification); “↑” indicates the colored ROCK1-positive particles.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4112556/v1/f882ec162f15cfb002aaa5f2.png"},{"id":53253076,"identity":"0dbbbc1b-8639-49b8-8fbf-ebc13a65d169","added_by":"auto","created_at":"2024-03-22 13:09:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":758335,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry of ROCK2 in the synovial tissue of the observation and control groups. (a) Observation group (10× magnification); (b) control group (10× magnification); (c) observation group (40× magnification); (d) control group (40× magnification); “↑” indicates the colored ROCK2-positive particles.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4112556/v1/19a7f0dbd2c35a4b2ddb18ae.png"},{"id":53253075,"identity":"86c35d41-d73f-4178-a25b-9dab12e9f43d","added_by":"auto","created_at":"2024-03-22 13:09:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":95250,"visible":true,"origin":"","legend":"\u003cp\u003eRhoA and ROCK protein expression in the synovial tissues of the observation (D) and control (N) groups. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, and ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4112556/v1/d8b82408a1256648900a4053.png"},{"id":53253079,"identity":"d4cd456a-60f4-4bf0-a60b-c8523b115b13","added_by":"auto","created_at":"2024-03-22 13:10:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":47551,"visible":true,"origin":"","legend":"\u003cp\u003emRNA expression levels of \u003cem\u003eRHOA\u003c/em\u003e and \u003cem\u003eROCK\u003c/em\u003ein the synovial tissues of the observation (D) and control (N) groups. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4112556/v1/e3460af49948c31c8d7568e8.png"},{"id":53816065,"identity":"77142788-657f-44d4-abaf-32f76138750e","added_by":"auto","created_at":"2024-03-31 17:54:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3452454,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4112556/v1/7ff71351-4a84-4f54-ac5b-ad833c40d85a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Involvement of the RhoA/ROCK signaling pathway in the pathogenesis of knee osteoarthritis","fulltext":[{"header":"Background","content":"\u003cp\u003eOsteoarthritis (OA) mostly occurs in older adults. Its incidence rate in China is approximately 17% in the middle-aged population, with higher prevalence in women [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This disease commonly affects the weight-bearing joints, with knee osteoarthritis (KOA) accounting for \u0026gt;\u0026thinsp;80% of all OA cases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. KOA manifests as sclerosis of the subchondral bone and periarticular cartilage, in addition to the wear and tear, degeneration, softness, deformation, and disappearance of the articular cartilage. Sclerosis of the subchondral bone, periarticular synovial hyperplasia, degeneration and atrophy of the joint capsule, peripheral ligaments, and other tissues, destruction of the integrity of the joint surface, and narrowing of the joint space can lead to knee deformity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Pathological changes mainly occur in the synovial membrane, cartilage, meniscus, and patella in the early stages of KOA [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], while knee joint pain, stiffness, swelling, limited activity, popping, and deformity appear gradually. KOA can involve the muscles, seriously affecting patients\u0026rsquo; physical and mental health and daily activities [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, early diagnosis, intervention, and treatment have become the focus of joint surgeries.\u003c/p\u003e \u003cp\u003eCartilage wear and tear is the core pathological change in KOA; however, pathological cartilage changes occur in the middle and late stages [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Synovial inflammation, an important early pathological change in KOA, can result in knee pain and structural changes, and its incidence has been increasing continuously [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Normal synovial and cartilage tissues are loose connective tissues that secrete synovial fluid to nourish the cartilage, reduce friction, maintain intra-articular metabolism, and provide nutrition to the articular cartilage [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Intra-articular lesions lead to the loss of synovial nutritional function and production of many proteases and cytokines, damaging the chondrocytes. The pro-inflammatory factors released from synovial and cartilaginous tissues stimulate the production of protein hydrolyzing enzymes, thereby aggravating articular cartilage degeneration, which causes joint pain and deterioration [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRas homolog gene family member A (RhoA) proteins are expressed in all tissues and affect cell growth, transformation, cytoskeletal regulation, actin stress fiber formation, and actin-myosin contraction [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Rho-associated protein kinase (ROCK), a downstream effector of RhoA, exists as two isoforms, ROCK1 and ROCK2. The RhoA/ROCK signaling pathway is involved in cell growth, differentiation, migration, and development [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and is required for neuronal synaptic growth, bone formation, dorsal closure, and myogenesis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Its aberrant activation is associated with various cancer types and diseases affecting the neurological, endocrine, and cardiovascular systems [\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Previous studies have shown that the RhoA/ROCK signaling pathway is closely related to cartilage degeneration in KOA, but such association has not been studied in synovial lesions in KOA.\u003c/p\u003e \u003cp\u003eThe aim of this study was to investigate the correlation between the RhoA/ROCK signaling pathway and the pathogenesis of KOA by comparing the expression of RhoA and ROCK in the synovial tissue of patients with primary KOA as a way to potentially identify a therapeutic target for delaying synovial lesions in KOA.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eObservation group: The observation group comprised synovial tissue samples removed during total knee arthroplasty (TKA) from 30 patients with primary KOA between January 2020 and December 2022. The patients were 57\u0026ndash;82 years of age (average age, 66.40\u0026thinsp;\u0026plusmn;\u0026thinsp;6.05 years), and included 11 men and 19 women.\u003c/p\u003e \u003cp\u003eThe inclusion criteria were as follows: primary KOA and fulfillment of the diagnostic criteria of the Chinese Osteoarthritis Diagnostic and Treatment Guidelines (2018 edition) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); poor outcomes after receiving conservative treatment; unilateral knee arthroplasty for the first time; and provision of informed consent by patients or their family members.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\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\u003eDiagnostic criteria for knee osteoarthritis (2018 version).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSymptoms or signs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRecurrent knee pain within the last 1 month\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRadiography (standing or weight-bearing position) showing narrowing of the joint space, subchondral osteosclerosis and/or cystic degeneration, and formation of bone capillaries at the joint margins\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExamination of the synovial fluid in the knee joint consistent with osteoarthritis (clear, viscous, white blood cell count\u0026thinsp;\u0026lt;\u0026thinsp;2.0 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u0026thinsp;\u0026ge;\u0026thinsp;50 years\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMorning stiffness\u0026thinsp;\u0026le;\u0026thinsp;30 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSound of bone friction (sensation) during activity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eKnee osteoarthritis can be diagnosed by meeting at least diagnostic criteria 1 and 2; 1, 3, 5, and 6; 1, 4, 5, and 6.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe exclusion criteria were as follows: serious blood-related diseases or receipt of prolonged anticoagulant drug treatment in preoperative manner; combined joint tuberculosis, tumor, and other serious diseases; serious knee deformity or trauma preventing cooperation while receiving treatment; history of drug injection into the joint cavity within half a year; and receipt of oral treatment for OA within the previous 2 weeks.\u003c/p\u003e \u003cp\u003eControl group: Twenty-five control specimens were obtained from the synovial tissue of knee joints removed during \u0026ldquo;lower limb disarticulation\u0026rdquo; during the same period at our hospital. The patients were 19\u0026ndash;39 years of age (average age, 31.07\u0026thinsp;\u0026plusmn;\u0026thinsp;6.10 years), including 12 men and 13 women.\u003c/p\u003e \u003cp\u003eThe inclusion criteria for the control group were as follows: age, 18\u0026ndash;40 years; non-KOA; \u0026ldquo;lower limb disarticulation\u0026rdquo; because of malignant tumors of the lower limbs or lower limb disfigurement injuries; and voluntary study participation.\u003c/p\u003e \u003cp\u003eThe exclusion criteria were as follows: arthritis on preoperative imaging; rheumatoid arthritis; knee joint infection; autoimmune disease; tuberculosis and other serious systemic diseases; and serious blood-related diseases.\u003c/p\u003e \u003cp\u003eThis study was approved by the responsible ethics review board (No. 2020\u0026thinsp;\u0026minus;\u0026thinsp;974). Informed consent was obtained from the patients before enrollment in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eThe chemicals and reagents used included anti-ROCK1 (ab134181; Abcam, Cambridge, UK)/anti-ROCK2 (ab228000; Abcam) and anti-RhoA (ab54835; Abcam) antibodies, RNA solids (Wuhan Xavier Biotechnology Co., Ltd., Wuhan, China), and polymerase chain reaction (PCR) primers (Shanghai Sangong Bioengineering Service Co., Ltd., Shanghai, China). All the other reagents were obtained from Yinchuan Runyan Biotechnology Co., Ltd. (Yinchuan, China).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eHematoxylin and eosin (HE) staining\u003c/h2\u003e \u003cp\u003eThe tissue sections were soaked in xylene I and II for 15 min for dewaxing. Subsequently, they were immersed in the following gradients of alcohol for 3\u0026ndash;5 min each: anhydrous ethanol Ⅰ and Ⅱ, 95% ethanol, 80% ethanol, and 70% ethanol; the sections were then removed and rinsed with distilled water for 5 min. Then, 1% hydrochloric acid in alcohol was used for differentiation for 3\u0026ndash;5 s, followed by rinsing for 5 min. To dye them blue, the tissue sections were immersed in 0.2% ammonia for 1 min, followed by rinsing in distilled water for 5 min. Next, the tissue sections were stained with 0.6% eosin for 30 s to 1 min. After dehydration, the sections were soaked in xylene I and II for 10 min each and sealed with optical resin glue.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eThe slices were put in the oven at 60\u0026deg;C for \u0026gt;\u0026thinsp;6 h, followed by dewaxing and rehydration. The sections were rinsed thrice for 5 min. Then, they were boiled in citrate buffer (pH 6.0) for 15 min and placed in a mixture of ice and water at 0\u0026deg;C for 30 min. The residual citrate buffer was washed out thrice using distilled water for 5 min each. The washed tissue sections were soaked in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution for 15 min and rinsed thrice for 5 min. The synovial tissue was blocked with goat serum at 20\u0026ndash;25 ℃ for 15 min. A drop of one antigen was added to the synovial tissue, and the tissue sections were incubated overnight in the refrigerator in a humid box. The sections were rinsed thrice using phosphate buffered saline (PBS) for 10 min. In total, 2\u0026ndash;3 drops of horseradish peroxidase-labeled goat anti-rabbit IgG antibody were added to cover the synovial tissue, and the tissue sections were incubated for 1 h at 37\u0026deg;C. The slices were rinsed thrice with PBS (for 5 min at a time). The slices were counterstained using the following sequence of reagents: hematoxylin staining solution for 2 min and 1% hydrochloric acid in alcohol solution for 30 s. The sections were dehydrated for 10 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eApproximately 500 mg of each specimen was ground and transferred to an Eppendorf tube. A mixture of the whole protein lysate, protease inhibitor cocktail, and phenylmethylsulfonyl fluoride were added to each tube, mixed well, and placed on a vortex oscillator for 30 s; it was then placed on ice for tissue lysis, vortexed, and centrifuged again after 15 min; this procedure was repeated thrice. The tissue lysate was centrifuged (12,000 rpm) at 4 \u0026deg;C for 20 min. The supernatant and 1/4th of the supernatant were aspirated into 5\u0026times; loading buffer, placed into a new Eppendorf tube for mixing, and boiled in hot water for 10 min. Equal amounts of protein from each sample were subjected to electrophoresis after adding loading buffer to achieve equal volumes across samples. Electrophoresis was initiated at 80 V and 400 mA, and after 30 min, the voltage and current were adjusted to 120 V and 400 mA, respectively. The gel was transferred to a methanol-soaked polyvinylidene fluoride (PVDF) membrane at 250 V and 260 mA for 90 min. The PVDF membrane was washed thrice with PBS-Tween 20 for 45 min. After sealing the membrane, the latter was washed with PBST) three times (10 min each time) and incubated overnight at 4\u0026deg;C on a refrigerator shaker with primary antibodies diluted at different ratios. Subsequently, the membrane was incubated with a secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG; 1:5,000 dilution) for 60 min at 20\u0026ndash;25 ℃ on a shaking bed. The membrane was then washed thrice prior to development using a developing solution and visualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcription-PCR\u003c/h2\u003e \u003cp\u003eApproximately 30\u0026ndash;40 mg synovial tissue was weighed and ground with liquid nitrogen to a fine powder, followed by the addition of 1 ml Trizol lysis buffer. The tissue was ground until liquification in the Trizol buffer. Two hundred microliters of chloroform was added, mixed to emulsify, placed at room temperature for 5\u0026ndash;10 min, and centrifuged for 20 min at 12,000 rpm, 4\u0026deg;C. Five hundred microliters of the supernatant and an equal amount of isopropanol were mixed in a new tube. The tube was vortexed to separate the DNA, left standing for 5 min, and centrifuged for 20 min under the same conditions, following which the supernatant was discarded. One milliliter of 75% ethanol was used to wash the pellet and centrifuged in a low-temperature ultracentrifuge for 5 min at 4\u0026deg;C (7,500 rpm). The upper ethanol layer was discarded and the precipitate was put on the ultra-clean bench for 5 min. Diethyl pyrocarbonate-treated water was added to dissolve the precipitate, followed by heating at 65\u0026deg;C for 10 min. The cDNA was synthesized via reverse transcription of mRNA using an Applied Biosystems (Waltham, MA, USA) instrument under the following conditions: 37\u0026deg;C (15 min), 85\u0026deg;C (5 s), and 4\u0026deg;C (\u0026infin;). Primers were designed and synthesized using the sequences listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. \u003cem\u003eGAPDH\u003c/em\u003e was used as an internal reference, and the relative expression of the target mRNAs was calculated using the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method and a LightCyder480 instrument.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Primer names and sequences.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eAll data were analyzed using the SPSS software (v25.0; IBM Corp., Armonk, NY, USA) and were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation; comparisons between groups were performed using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test for independent samples. Statistical results were plotted using the GraphPad Prism software (v7.0; GraphPad Software, La Jolla, CA, USA), with a significance level of α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAppearance and morphology of the synovial tissue\u003c/h2\u003e \u003cp\u003eIn the observation group, the synovial tissue showed high levels of proliferation and edema, with obvious congestion and a yellowish appearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The synovial membrane surface in the control group was smooth. There were no signs of edema or congestion, and the tissue appeared white (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHE staining\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRhoA expression in the synovial tissues\u003c/h2\u003e \u003cp\u003eRhoA protein was expressed widely in the synovial tissues of 30 patients with KOA compared with that in the control group. RhoA expression appeared as light yellow or brownish-yellow particles in the cell cytoplasm, cytosol, and interstitium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, c). RhoA expression was low in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, d).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eROCK1 expression in the synovium\u003c/h2\u003e \u003cp\u003eROCK1 was expressed in the synovial tissues of 30 KOA joints, and the positively-stained particles were distributed in the cell membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, c). ROCK1 expression was low in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, d).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eROCK2 expression in the synovial membrane\u003c/h2\u003e \u003cp\u003eAnti-ROCK2 staining was observed in the synovial tissue of 30 KOA joints, and the positively-stained particles were distributed in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, c). Anti-ROCK2 staining was low in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, d). Compared with the control group, numerous samples in the observation group showed significant positive immunoreactivity for RhoA and ROCK, which were distributed widely (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The observation group showed higher RhoA and ROCK protein levels than the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The synovial tissue in the observation group showed higher levels of \u003cem\u003eRHOA\u003c/em\u003e and \u003cem\u003eROCK\u003c/em\u003e transcripts than that in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Comparison between RhoA, ROCK1, and ROCK2 Expression Intensity in the Two Groups (Mean \u0026plusmn; SD).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.974358974358974%\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.299145299145298%\"\u003e\n \u003cp\u003eObservation group\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.974358974358974%\"\u003e\n \u003cp\u003eControl group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.11111111111111%\"\u003e\n \u003cp\u003e\u003cem\u003et\u003c/em\u003e-Value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.641025641025642%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.974358974358974%\"\u003e\n \u003cp\u003eRhoA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.299145299145298%\"\u003e\n \u003cp\u003e7.11 \u0026plusmn; 1.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.974358974358974%\"\u003e\n \u003cp\u003e2.43\u0026plusmn;0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.11111111111111%\"\u003e\n \u003cp\u003e14.328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.641025641025642%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.974358974358974%\"\u003e\n \u003cp\u003eROCK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.299145299145298%\"\u003e\n \u003cp\u003e6.61 \u0026plusmn; 1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.974358974358974%\"\u003e\n \u003cp\u003e2.92\u0026plusmn;0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.11111111111111%\"\u003e\n \u003cp\u003e13.043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.641025641025642%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.974358974358974%\"\u003e\n \u003cp\u003eROCK2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.299145299145298%\"\u003e\n \u003cp\u003e5.05 \u0026plusmn; 1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.974358974358974%\"\u003e\n \u003cp\u003e3.20\u0026plusmn;0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.11111111111111%\"\u003e\n \u003cp\u003e8.063\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.641025641025642%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eSurgical management using artificial joint arthroplasty is considered in clinical practice for patients with severe KOA, which affects their normal life activities. TKA can reduce the clinical symptoms, restore knee joint function, and improve the quality of daily life [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, ensuring the survival of TKA prostheses after wear and tear, and rehabilitation of patients after knee arthroplasty are associated with high social, medical, and economic burdens. Early diagnosis and treatment can reduce the pain experienced by the patients and the economic burden. Hence, the study of early synovial inflammation pathogenesis in patients with KOA is crucial for its prevention and treatment [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, the high expression of RhoA and ROCK transcripts and proteins during synovial inflammation was verified using immunohistochemistry, western blotting, and quantitative reverse transcription-PCR, confirming the relevance of the RhoA/ROCK signaling pathway with the development of KOA synovitis. Synovial inflammation in KOA is considered an early-stage lesion. The RhoA/ROCK signaling pathway plays a key role in lipopolysaccharide-induced inflammatory pain and has been associated with signaling pathway activation to cause nociceptive hypersensitivity and the concomitant release of tumor necrosis factor-α and interleukin-1β [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Suppression of ROCK expression and articular cartilage degeneration have been observed after the injection of RhoA/ROCK signaling inhibitors in rats, while the production of inflammatory factors \u003cem\u003ein vivo\u003c/em\u003e and joint pain scores were reduced and chronic neuropathic pain was alleviated, consistent with our results [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Synovial inflammation can increase peripheral vascular permeability, lower pain thresholds, and increase nociceptive sensitivity. Activation of the RhoA/ROCK signaling pathway promotes the release of inflammatory factors and participates in sensory processing of spinal cord injury, which in turn aggravates inflammatory responses and increases knee pain.\u003c/p\u003e \u003cp\u003eThis study had certain limitations. We only verified the correlation between the RhoA/ROCK signaling pathway and KOA synovitis but did not use serology, cytology, and gene knockdown technology to further understand the mechanisms underlying the correlation. Moreover, the sample size was small, and the same group of specimens could not be used for all the testing techniques. Therefore, a larger sample size and comprehensive analysis are necessary to clarify the contribution of the pathway.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe expression of RhoA and ROCK transcripts and proteins was higher in the synovial tissue of patients with KOA than in those of the control, indicating that the RhoA/ROCK signaling pathway was involved in KOA. This study shed light on novel approaches for screening target genes for early diagnosis of KOA and development of preventive treatment strategies. In the future, it would be important to study the correlation between this signaling pathway and KOA synovitis in depth, and to develop drugs that slow down the progression of synovitis and improve synovitis-induced joint pain.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKOA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eknee osteoarthritis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTKA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etotal knee arthroplasty\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRhoA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRas homolog gene family member A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROCK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRho-associated coiled-coil kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethics Review Board of the People’s Hospital of Ningxia Hui Autonomous Region (No. 2020-974).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from the patients before enrollment in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflicts of interest related to the research or writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (82060408), the Key Research and Development Program of Ningxia Hui Autonomous Region (2021BEG03049), the Innovation and Entrepreneurship Project of Ningxia Overseas Students (2020-75), and the First-Class Discipline Construction Founded Project of Ningxia Medical University and the School of Clinical Medicine (NXYLXK2017A05). The financial support for the project did not influence the views stated in the article or the analysis of the results of the study and their reporting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.Y. was responsible for case data compilation and manuscript preparation. Y.W. was responsible for instructing the lab. J.Z. and B.Z. were responsible for statistical analysis and literature review. A.Y. and W.W. were responsible for case data collection. Z.L. and D.C. supervised the manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi D, Li S, Chen Q, Xie X. The prevalence of symptomatic knee osteoarthritis in relation to age, sex, area, region, and body mass index in China: A systematic review and meta-analysis. 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J Orthop Res. 2011; 29:369-74. https://doi.org/10.1002/jor.21257.\u003c/li\u003e\n\u003cli\u003eAspenstr\u0026ouml;m P. Activated Rho GTPases in cancer-the beginning of a new paradigm. Int J Mol Sci. 2018;19:3949. https://doi.org/10.3390/ijms19123949.\u003c/li\u003e\n\u003cli\u003eSantiago-Lopez AJ, Gutekunst CA, Gross RE. C3 Transferase gene therapy for continuous RhoA inhibition. Methods Mol Biol. 2018;1821:267-81. https://doi.org/10.1007/978-1-4939-8612-5_19.\u003c/li\u003e\n\u003cli\u003eUeyama T. Rho-Family Small GTPases: From highly polarized sensory neurons to cancer cells. Cells. 2019;8:92. https://doi.org/10.3390/cells8020092.\u003c/li\u003e\n\u003cli\u003eLv HS. Progress and status of artificial knee arthroplasty. Chin J Surg. 2004;1:26-9.\u003c/li\u003e\n\u003cli\u003eMa CH, Lv Q, Cao Y, Wang Q, Zhou XK, Ye BW, et al. Genes relevant with osteoarthritis by comparison gene expression profiles of synovial membrane of osteoarthritis patients at different stages. Eur Rev Med Pharmacol Sci. 2014;18:431-9.\u003c/li\u003e\n\u003cli\u003eKozhanova TG, Poliakova VS, Mkhitarian EE, Meshcheriakov KN. Morphological peculiarities of the synovial membrane of human knee joint in mature age. Morfologiia 2013;144:50-3. Russian.\u003c/li\u003e\n\u003cli\u003eWang C, Song S, Zhang Y, Ge Y, Fang X, Huang T, et al. Inhibition of the Rho/Rho kinase pathway prevents lipopolysaccharide-induced hyperalgesia and the release of TNF-\u0026alpha; and IL-1\u0026beta; in the mouse spinal cord. Sci Rep. 2015;5:14553. https://doi.org/10.1038/srep14553.\u003c/li\u003e\n\u003cli\u003eWong SSC, Lee UM, Wang XM, Chung SK, Cheung CW. Role of DLC2 and RhoA/ROCK pathway in formalin induced inflammatory pain in mice. Neurosci Lett. 2019;709:134379. https://doi.org/10.1016/j.neulet.2019.134379. \u003c/li\u003e\n\u003cli\u003eQiu Y,\u003cspan dir=\"RTL\"\u003e \u003c/span\u003eChen WY,\u003cspan dir=\"RTL\"\u003e \u003c/span\u003eWang ZY, Liu F, Ma C, Huang GY. Effects of simvastatin on neuropathic pain behavior and RhoA pathway expression in rats. Chin J Pain Med. 2016;22:493-500.\u003c/li\u003e\n\u003cli\u003eTakeshita N, Yoshimi E, Hatori C, Kumakura F, Seki N, Shimizu Y. Alleviating effects of AS1892802, a Rho kinase inhibitor, on osteoarthritic disorders in rodents. J Pharmacol Sci. 2011;115:481-9. https://doi.org/10.1254/jphs.10319fp.\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":"Lower limb disarticulation, Knee osteoarthritis, Synovium, RhoA/ROCK signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-4112556/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4112556/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThis study compared the expression of the Ras homolog gene family member A (RhoA) and Rho-associated coiled-coil kinase (ROCK) signaling pathway members in knee joint synovial tissue from patients with primary knee osteoarthritis (KOA) who underwent lower limb disarticulation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe specimens were separated into observation (synovial tissue removed during total knee arthroplasty of 30 patients with primary KOA) and control groups (synovial tissue removed during lower limb disarticulation of 25 patients).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe observation group specimens showed high proliferation, congestion, edema, unorganized cellular arrangement, and presence of vacuolated cells. The control group specimens were white with a smooth surface and non-edematous texture, and showed organized and homogenous cell/stroma arrangement. The observation group showed higher expression of RhoA and ROCK transcripts and proteins than the control.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003ePatients with KOA expressed high levels of RhoA and ROCK transcripts and proteins in synovial tissues, suggesting a relationship between RhoA/ROCK and KOA. Our findings provide a potential therapeutic target for delaying synovial lesions in KOA.\u003c/p\u003e","manuscriptTitle":"Involvement of the RhoA/ROCK signaling pathway in the pathogenesis of knee osteoarthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 13:09:51","doi":"10.21203/rs.3.rs-4112556/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":"5ef3e2e7-a12c-4e81-8393-ed8aa6dab695","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-31T17:46:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-22 13:09:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4112556","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4112556","identity":"rs-4112556","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}