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. 2022 Dec 7:13:1028994.
doi: 10.3389/fimmu.2022.1028994. eCollection 2022.

"VSports最新版本" Upregulation of leucine-rich alpha-2 glycoprotein: A key regulator of inflammation and joint fibrosis in patients with severe knee osteoarthritis

Ashish Sarkar  1   2 , Debolina Chakraborty  1   2 , Vijay Kumar  3 , Rajesh Malhotra  3 , Sagarika Biswas  1
Affiliations

Upregulation of leucine-rich alpha-2 glycoprotein: A key regulator of inflammation and joint fibrosis in patients with severe knee osteoarthritis

Ashish Sarkar et al. Front Immunol. .

Abstract

Introduction: Osteoarthritis (OA) is a degenerative disease of the joints mainly affecting older individuals. Since the etiology behind the progression of OA is not well understood, several associated consequences, such as synovial joint stiffness and its progression due to joint fibrosis, are still poorly understood. Although a lot of developments have been achieved in the diagnosis and management of OA, synovial fibrosis remains one of the major challenging consequences. The present study was therefore focused on understanding the mechanism of synovial fibrosis, which may further contribute to improving symptomatic treatments, leading to overall improvements in the treatment outcomes of patients with OA VSports手机版. .

Methods: We used advanced proteomic techniques including isobaric tag for relative and absolute quantitation and sequential window acquisition of all theoretical mass spectra for the identification of differentially expressed proteins in the plasma samples of patients with OA. An in silico study was carried out to evaluate the association of the identified proteins with their biological processes related to fibrosis and remodeling of the extracellular matrix (ECM). The most significantly upregulated protein was then validated by Western blot and enzyme-linked immunosorbent assay. The target protein was then further investigated for its role in inflammation and joint fibrosis using an in vitro study model V体育安卓版. .

Results: Leucine-rich alpha-2 glycoprotein (LRG1) was found to be the most highly differentially expressed upregulated (9. 4-fold) protein in the plasma samples of patients with OA compared to healthy controls. The knockdown of LRG1 followed by in vitro studies revealed that this protein promotes the secretion of the ECM in synovial cells and actively plays a role in wound healing and cell migration. The knockdown of LRG1 further confirmed the reduction of the inflammatory- and fibrosis-related markers in primary cells V体育ios版. .

Conclusion: LRG1 was identified as a highly significant upregulated protein in the plasma samples of patients with OA VSports最新版本. It was found to be associated with increased fibrosis and cell migration, leading to enhanced inflammation and joint stiffness in OA pathogenesis. .

Keywords: SWATH; biomarker; fibrosis; iTRAQ; inflammation; leucine-rich alpha-2 glycoprotein; osteoarthritis. V体育平台登录.

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Conflict of interest statement

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

VSports手机版 - Figures

Figure 1
Figure 1
Validation of LRG1 by Western blot (WB). WB images were taken by Chemidoc (Bio-Rad, Hercules, CA, USA), and densitometry analysis of all WB images was carried out using Image Lab v6 (Bio-Rad). All graphs were generated and the statistical analysis carried out using GraphPad Prism (version 8.4.686). Data are presented as the mean ± standard deviation (SD). Paired Student’s t-test was performed for group comparisons in (B, D, E) to show statistical significance. (A) WB image of the expression of LRG1 in pooled plasma samples from patients with OA and from HCs (upper panel) and Ponceau staining of blots showing total protein as the loading control (lower panel). (B) Graph of the densitometric analysis of the WB images using Image Lab software. LRG1 was found to be 2.3-fold upregulated in the plasma samples from OA patients compared to those from HCs. (C) WB images of the LRG1 expression and Ponceau staining of blots showing total protein in the SF samples from patients who had TKR compared to those who had UKR. (D) Bar graph showing the 1.6-fold upregulation of LRG1 in SF samples (p = 0.015). (E) ELISA results showing the concentrations of LRG1 in plasma samples from patients with OA compared to those from HCs. The fold change was 1.66 in OA compared to HC plasma. *p ≤ 0.05; ****p ≤ 0.0001. ns, non-significant; LRG1, leucine-rich alpha-2 glycoprotein; OA, osteoarthritis; SF, synovial fluid; HC, healthy control; TKR, total knee replacement; UKR, unicompartmental knee replacement.
Figure 2
Figure 2
In silico study of the DEPs involved in fibrosis-related disease. The proteins were converted to their corresponding gene ID and analyzed using the Cytoscape (3.8.2) online analysis tool. The protein–protein interaction in Cytoscape showed that LRG1 plays a central role in fibrosis (red). Closely associated proteins are shown in yellow, while the other distantly related proteins are shown as sky blue. LRG1, leucine-rich alpha-2 glycoprotein; DEPs, differentially expressed proteins.
Figure 3
Figure 3
In silico study and annotation. (A) The DEPs were used to map the pathways involved in the pathogenesis of osteoarthritis (OA). The pathways are depicted by the colored circle, where the size of the circle indicates the number of proteins and the color of the circle indicates the p-value. (B) The GO annotation tool was used to determine the molecular and functional roles of the identified DEPs. The number of proteins involved in biological processes, molecular functions, and cell components are shown in green, red, and black, respectively. The top 10 GO entries with p ≤ 0.05 are depicted. DEPs, differentially expressed proteins; GO, Gene Ontology; ECM, extracellular matrix.
Figure 4
Figure 4
LRG1 promotes fibrosis in primary synovial cells. Western Blot (WB) and agarose gel images were taken by Chemidoc (Bio-Rad, Hercules, CA, USA), and densitometry analysis of all the images was carried out using Image Lab V6 (Bio-Rad). All graphs were generated and the statistical analysis carried out using GraphPad Prism (version 8.4.686). Data are presented as the mean ± standard deviation (SD). Paired Student’s t-test was applied for groups comparisons in (B) to show statistical significance, while one-way ANOVA was used for group comparisons in (C, E, F). (A) Western blot image showing LRG1 knockdown using siRNA (50 nM) in primary fibroblast-like cells isolated from OA synovium. The upper panel shows the LRG1 expression level with the transfection reagent for 48 h, while the lower panel shows GAPDH as the loading control in the respective sample. (B) Bar graph plotted using the densitometric values after the analysis of bands depicting the 60% downregulation of the LRG1 expression after knockdown using siRNA. (C) Bar graph showing the mRNA level of COMP after the knockdown of LRG1 in OA primary cells compared to the vehicle control group. (D) PCR products run on 1% agarose gel for visualization of the mRNA expression levels of IL-6 and COMP after treatment with siRNA. (E) Bar graph showing the mRNA level of LRG1 after knockdown. (F) Bar graph showing the mRNA level of the inflammatory marker IL-6 after the knockdown of LRG1 compared to the untreated groups. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001. ns, non-significant; LRG1, leucine-rich alpha-2 glycoprotein; OA, osteoarthritis; COMP, cartilage oligomeric matrix protein; IL-6, interleukin 6; VC, vehicle group; HC, healthy control; GAPDG, glyceraldehyde-3-phosphate dehydrogenase.
Figure 5
Figure 5
Hydroxyproline assay after stimulation/knockdown of LRG1. Microscopy images were taken at ×10 magnification using Nikon Eclipse 650 (NIKON, Tokyo, Japan). The optical density of hydroxyproline was measured with a spectrophotometer (Molecular Devices, San Jose, CA, USA). Graphs were generated and the statistical analysis carried out using GraphPad Prism (version 8.4.686). Data are presented as the mean ± standard deviation (SD). One-way ANOVA was used to compare the data among groups to show statistical significance. (A) Increased extracellular matrix observed in primary synovial cells from patients with OA after stimulation using pure LRG1 (100 ng/ml); a reverse effect was observed morphologically after silencing the LRG1 gene using 50 nM siRNA compared to the non-treated group. (B) Level of collagen measured with the hydroxyproline assay. The level of hydroxyproline indicated a 1.3-fold increase in the stimulated (with 100 ng/ml LRG1) group compared to the control group, with 0.74 and 0.9-fold decreased levels of hydroxyproline in the knockdown (50 nm si-LRG1) group compared to the LRG1-treated and the non-treated group, respectively. *p ≤ 0.05; **p ≤ 0.01. LRG1, leucine-rich alpha-2 glycoprotein; OA, osteoarthritis.
Figure 6
Figure 6
Wound healing and scratch assay. Microscopy images taken at ×10 magnification using Nikon Eclipse 650 (NIKON, Tokyo, Japan). Analysis was carried out with ImageJ software using the freehand area selection tool. Graphs were generated and the statistical analysis carried out using GraphPad Prism (version 8.4.686). Data are presented as the mean ± standard deviation (SD). One-way ANOVA was used for comparisons of the data among groups to show statistical significance. (A) Wound healing and migration of primary synovial cells after stimulation with pure LRG1 (100 ng/ml) or knockdown with si-LRG1 (50 nM). (B) Graph showing wound healing of OA primary cells after knockdown with siRNA (50 nM) or stimulation with recombinant LRG1 as a function of reduction in the scratch surface area after 72 h of treatment. The results showed a 0.026-fold reduction in the wound healing capacity after knockdown, but a reverse effect was seen when the cells were stimulated with pure LRG1, which showed a 1.4-fold increased wound healing and migration compared to untreated cells. ****p ≤ 0.0001. ns, non-significant; LRG1, leucine-rich alpha-2 glycoprotein; OA, osteoarthritis.
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References

    1. Martel-Pelletier J, Barr AJ, Cicuttini FM, Conaghan PG, Cooper C, Goldring MB, et al. . Osteoarthritis. In: Nature reviews disease primers. United kingdom: Nature Publishing Group; (2016). 2:1–18. Available at: https://www.nature.com/articles/nrdp201672 (V体育官网入口). - PubMed
    1. Pal CP, Singh P, Chaturvedi S, Pruthi KK, Vij A. Epidemiology of knee osteoarthritis in India and related factors. Indian journal of orthopaedics (2016) 50(5):518–22. doi: 10.4103/0019-5413.18960 - DOI - PMC - PubMed
    1. Rim YA, Ju JH. The role of fibrosis in osteoarthritis progression. Life (2020) 11(1):3. doi: 10.3390/life11010003 - DOI - PMC - PubMed
    1. Watt FE. Osteoarthritis biomarkers: year in review. Osteoarthr Cartil [Internet] (2018) 26(3):312–8. doi: 10.1016/j.joca.2017.10.016 - "VSports" DOI - PubMed
    1. Braun HJ, Gold GE. Diagnosis of osteoarthritis: Imaging. Bone (2012) 51(2):278–88. doi: 10.1016/j.bone.2011.11.019 - DOI (VSports app下载) - PMC - PubMed

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