Panaxatriol exerts anti-senescence effects and alleviates osteoarthritis and cartilage repair fibrosis by targeting UFL1

Abstract

Introduction: Osteoarthritis (OA), the most common degenerative joint disease, can eventually lead to disability. However, no safe or effective intervention is currently available. Therefore, there is an urgent need to develop effective drugs that reduce cartilage damage and treat OA.

Objectives: This study aimed to ascertain the potential of panaxatriol, a natural small molecule, as a therapeutic drug for alleviating the progression of OA.

Methods: An in vitro culture of human cartilage explants and C28/I2 human chondrocytes and an in vivo surgically induced OA mouse model were used to evaluate the chondroprotective effect of panaxatriol. The Drug Affinity Responsive Target Stability assay, CRISPR-Cas9 assay, Whole-transcriptome RNA sequencing analysis and agonist or antagonist assays were used to identify the target and potential signaling pathways of panaxatriol. Poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) was used to construct the sustained-release system of panaxatriol.

Results: Panaxatriol protected against OA by regulating chondrocyte metabolism. Ubiquitin-fold modifier 1-specific E3 ligase 1 (UFL1) was identified as a novel target of panaxatriol. Whole transcriptome RNA sequencing showed that UFL1 was closely related to cell senescence. Panaxatriol inhibited chondrocyte senescence through UFL1/forkhead box O1 (FOXO1)/P21 and UFL1/NF-κB/SASPs signaling pathways. It also could inhibit fibrocartilage formation during cartilage repair via the UFL1/FOXO1/Collagen 1 signaling pathway. Finally, we constructed a sustained-release system for panaxatriol based on PLGA-PEG, which reduced the number of intra-articular injections, thereby alleviating joint swelling and injury.

Conclusions: Panaxatriol exerts anti-senescence effects and has the potential to delay OA progression and reduce cartilage repair fibrosis by targeting UFL1.

Keywords: Cell senescence; Fibrosis; Osteoarthritis; PLGA; Panaxatriol; UFL1.

Graphical abstract

Fig. 1

Panaxatriol promotes anabolism and reduces catabolism of human cartilage explants. Human cartilage explants were incubated in the absence or presence of 10 ng/mL IL-1β or a series concentration of panaxatriol for 7 days. (A) Structural formula of panaxatriol. (B) qRT-PCR was used to detect the mRNA level of anabolism markers (SOX9, COL2, ACAN) and catabolism markers (MMP13, ADAMTS-4, COL10). (C) Western blot was used to detect the protein level of COL2, ACAN and MMP13. (D) Quantification of (C). (E) Representative images of H&E stained, safranin O/Fast green stained and immunofluorescence stained sections of human cartilage explants. (F), Quantification of immunofluorescence staining of ACAN and MMP13 in (E). The values are mean ± SEM of at least three independent experiments. (*,P < 0.05; **,P < 0.01;***,P < 0.001; ****,P < 0.0001;ns, no significance).

Fig. 2

Panaxatriol reduces knee joint cartilage damage and pain caused by DMM surgery in mice. (A) Experimental flow chart, every group have at least 5 mice. (B) Representative images of safranin O/Fast green stained sections of knee joints from mice treated with DMSO or panaxatriol. (C) The severity of OA of mice was evaluated by OARSI score system, chondrocytes number and cartilage thickness based on safranin O/Fast green stained sections. P values were determined with two-sided Kruskal-Wallis test followed by Mann-Whitney U test. (D) Representative images of immunofluorescence staining of COL2 and MMP13 of mice joint sections. (E) Three-dimensional micro-CT images of subchondral bone in the groups. (F) Quantification of (E) including mean density of bone volume(BMD), bone volume/total volume(BV/TV), bone surface/bone volume(BS/BV), number of trabecular bone(Tb.N), separation of trabecular bone (Tb.Sp) and thickness of trabecular bone (Tb.Th). (G) Mechanical sensitivity was measured using von Frey filaments every week. (H) Representative images of immunofluorescence staining of the pain-related marker CGRP of mice joint sections. (I) Quantification of (I). (J) RT-PCR was used to detect the mRNA level of pain-related markers (Ngf and Mcp-1) of mice knee joint cartilage. (K) Representative images of footprint of mice hind legs, left hind leg(purple), right hind leg(green). (L) Quantification of mice gait analysis, all data were presented as right hind leg/left hind leg (RH/LH). (*,P < 0.05; **,P < 0.01;***,P < 0.001; ****,P < 0.0001;ns, no significance).

Fig. 3

Panaxatriol interacts with UFL1 as its specific target. A), Coomassie blue staining of SDS-PAGE gel of DARTS assay. The band with molecular weight between 75 and 100 kDa was protected by panaxatriol. The right panel is the UFL1 adapted image from mass spectrometry. B), The level of UFL1 was evaluated by Western blot following panaxatriol/C28I2 cells DARTS assay. C), CETSA assay, the protein from C28I2 cells incubated with DMSO or panaxatriol for 1 h, were denatured under various temperatures. The the protein level of UFL1 were assayed using Western blot. D), The densitometry analysis curve of (C). E), The visualized image of molecular docking between UFL1 and panaxatriol. UFL1 is showed as grey color and panaxatriol is green color. The amino acid residue is showed as red color. Hydrogen bonds are represented by dotted green lines. Pi-Alkyl bonds are represented by dotted purple lines. F), Schematic view of UFL1 domain and the design of truncations. The right panel is the DARTS assay for different truncation of UFL1. G), The sequencing results of deletions of UFL1 and the DARTS assay for the deletions.The values are mean ± SEM of at least three independent experiments. (*,P < 0.05; **,P < 0.01;***,P < 0.001; ****,P < 0.0001;ns, no significance).

Fig. 4

UFL1 is downregulated in osteoarthritis and Panaxatriol regulates chondrocyte metabolism dependent on UFL1. (A) Western blot was used to detect the expression of UFL1 in lesion area (OA) and non-lesion area (NC) of cartilage in OA patients. (B) Quantification of (A). (C) RT-PCR was used to detect the mRNA level of UFL1 in lesion area (OA) and non-lesion area (NC) of cartilage in OA patients. (D) Western blot was used to detect the protein level of UFL1 of human cartilage explants which treated with or without 10 ng/mL IL-1β and a series concentration of panaxatriol for 7 days. (E) Quantification of (D). (F) Representative images of immunofluorescence staining of UFL1 in the sections of human cartilage explants. (G) Representative images of immunofluorescence staining of UFL1 of knee joint sections of DMM mice treated with DMSO or panaxatriol. (H) Quantification of (F). (I) Quantification of (G). (J-K) UFL1 degradation were analyzed by cycloheximide chase assay. (L) Based on the UFL1 knock-out C28I2 cells(UFL-KO), western blot was used to detect the influence of UFL1 on the ability of panaxatriol to regulate COL2 and MMP13. OE-UFL1 means overexpression of UFL1 in UFL1-KO cells. (M) Quantification of (J).The values are mean ± SEM of at least three independent experiments. (*,P < 0.05; **,P < 0.01;***,P < 0.001; ****,P < 0.0001;ns, no significance).

Fig. 5

Normal C28I2 cells and UFL1-KO C28I2 cells were send to RNA sequencing analysis. (A) Venn diagram of the detected genes in normal C28I2 cells and UFL1-KO cells. (B) Volcano plot of RNA sequencing data. (C) Hot map of differentially expressed genes. (D) Gene Ontology (GO) enrichment analysis of differentially expressed genes. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes. (F) Gene Set Enrichment Analysis (GSEA) of cellular senescence. (G) GSEA of AGE-RAGE signaling pathway and NF-κB signaling pathway.

Fig. 6

UFL1 is associated with celluar senescence and Panaxatriol modulates chondrocyte senescence through UFL1. (A) Western blot was used to detect the protein level of UFL1, P21 and SASPs (MMP9,MMP13,ADAMTS-4,IL-6,TNF-α) in UFL1-KO cells. (B) Representative images of immunofluorescence staining of P21 and γ-H2AX of knee joint sections of DMM mice treated with DMSO or panaxatriol and the quantification results. (C) Representative images of immunofluorescence staining of P21 and γ-H2AX in the sections of human cartilage explants treated with 10 ng/mL IL-1β or a series concentration of panaxatriol for 7 days. (D) Representative images of SA-beta-gal staining C28/I2 cells treated with 10 ng/mL IL-1β or a series concentration of panaxatriol for 2 days. (E) Based on the UFL1 knock-out C28I2 cells (UFL1-KO), western blot was used to detect the influence of UFL1 on the ability of panaxatriol to regulate P21 and SASPs. OE-UFL1 means overexpression of UFL1 in UFL1-KO cells. (F) Based on UFL1-KO cells, western blot was used to detect the influence of UFL1 on the ability of panaxatriol to regulate P65,p-P65 and FOXO1.The values are mean ± SEM of at least three independent experiments. (*,P < 0.05; **,P < 0.01;***,P < 0.001; ****,P < 0.0001;ns, no significance).

Fig. 7

Panaxatriol reduces fibrosis during cartilage repairing. (A) Representative images of immunofluorescence staining of COL1 of knee joint sections of DMM mice treated with DMSO or panaxatriol. (B) Quantification of (A). (C) RT-PCR was used to detect the mRNA level of COL1 in human cartilage explants. (D) Western blot and quantification. Based on the UFL1 knock-out C28I2 cells (UFL1-KO), western blot was used to detect the influence of UFL1 on the ability of panaxatriol to regulate COL1. OE-UFL1 means overexpression of UFL1 in UFL1-KO cells. (E) Based on over expression of FOXO1, Western blot was used to detect the influence of FOXO1 on the ability of panaxatriol to regulate COL1. (F) Based on the FOXO1 inhibitor (AS1842856) and UFL1-KO cells, Western blot was used to detect the influence of FOXO1 on the ability of UFL1 to regulate COL1. (G) Experimental flow chart of cartilage defect model, every group have at least 5 mice. (H) Representative images of macro view, safranin O/Fast green staining and immunofluorescence staining (COL2,COL10,COL1,UFL1 and FOXO1) of femoral trochlear groove of cartilage defect mice treated with DMSO or panaxatriol. (I) International Cartilage Repair Society (ICRS) macroscopic evaluation score assessment and histologic Wakitani cartilage repairing score assessment were performed. P values for ICRS macroscopic score and histologic Wakitani cartilage repairing score were determined with two-sided Kruskal-Wallis test followed by Mann-Whitney U test. (*,P < 0.05; **,P < 0.01;***,P < 0.001; ****,P < 0.0001;ns, no significance).

Fig. 8

PLGA-PEG@Panaxatriol enables sustained release of Panaxatriol and reduces joint damage. (A) Schematic view of PLGA-PEG@Panaxatriol. (B) Representative images of transmission electron microscope (TEM) image of PLGA-PEG@Panaxatriol. (C) The particle size of PLGA-PEG@Panaxatriol and PLGA-PEG@blank. (D) The Zeta potential of PLGA-PEG@Panaxatriol and PLGA-PEG@blank. (E) Representative images of small animal living imaging system, photos were taken after PLGA-PEG@Panaxatriol had been injected into mice knee joint for 0,1,3,5,9,12,15 days. (F) Quantification of (E). (G) Experimental flow chart of cartilage defect model or DMM model treated with PLGA-PEG@Panaxatriol or PLGA-PEG@blank or panaxatriol, every group have at least 5 mice. (H) Representative images of Safranin O/Fast green staining of joint sections and three-dimensional mirco-CT images of subchondral bone in the DMM mice model groups. (I) Representative images of macro view, safranin O/Fast green staining and immunofluorescence staining (COL2,COL10,COL1) of femoral trochlear groove of cartilage defect mice model groups. The values are mean ± SEM of at least three independent experiments. (*, P < 0.05; **,P < 0.01;***,P < 0.001; ****,P < 0.0001;ns, no significance).

Fig. 9

A proposed model for explaining the signaling pathways by which panaxatriol binds to UFL1, leading to the inhibition of celluar senescence and fibrosis and protection against OA.