Ursolic acid inhibits NF-κB signaling and attenuates MMP-9/TIMP-1 in progressive osteoarthritis: a network pharmacology-based analysis

Abstract

Osteoarthritis (OA) is a degenerative joint disease, characterized by infiltration of monocytes into the synovial joint which promotes inflammation, stiffness, joint swelling, cartilage degradation and further bone destruction. The leaves of Ocimum forskolei have been used for inflammation-related disease management in traditional medicine. Additionally, the downregulation of NF-κB and the MMP/TIMP-1 ratio has been shown to protect against OA. The LC-HR-MS metabolic analysis of Ocimum yielded 19 putative compounds, among which ursolic acid (UA) was detected. Ursolic acid possesses significant anti-inflammatory effects and has been reported to downregulate oxidative stress and inflammatory biomarkers. It was tested on rats in a model of intra-articular carrageenan injection to investigate its efficacy on osteoarthritis progression. The UA emulgel exerted chondroprotective, analgesic and local anaesthetic efficacies confirmed via histopathological investigation and radiographical imaging. A network pharmacology followed by molecular docking highlighted TNF-α, TGF-β and NF-κB as the top filtered genes. Quantitative real-time PCR analysis showed that UA significantly attenuated serum levels of TNF-α, IL-1β, NF-κB, MMP-9/TIMP-1 and elevated levels of TGF-β. Taken together, these results suggest that UA could serve as a functional food-derived phytochemical with a multi-targeted efficacy on progression of OA, regulating the immune and inflammatory responses, particularly, attenuating chondrocytes degeneration via suppression of NF-κB and MMP-9/TIMP-1. Accordingly, UA might be a promising alternative to conventional therapy for safe, easily applicable and effective management of OA.

Fig. 1. Annotated compounds from HR-MS spectral data of n-hexane fraction of Ocimum forskolei.

Fig. 2. (a & b) SEM of O/W emulsion of UA-E2, (c) percent cumulative release of UA from suspension and UA-E2. Each value represents the mean ± SD (N = 6). Statistical analysis were done by one-way ANOVA followed by the Student's T-test (*P < 0.05).

Fig. 3. (a) Results of anti-inflammatory activity, (b) inhibition % of oedema, bars represent mean ± SD (N = 6). Statistical analysis were done by one-way ANOVA followed by the Student's T-test (*P < 0.05).

Fig. 4. [X-ray radiography of (a) untreated, (b) UA-emulgel, (c) Algason group], [H & E images of (d) & (e) normal group, (f) & (g) non-treated group, (h) Algason group and (i) UA-emulgel group]. Sc; stratum corneum, TEp; thickened epidermis, D; dermis, Gl.S; glandula sebacea, H.F.; hair follicle; yellow asterisks; cell infiltration, red asterisks; tissue necrosis, red arrows; congested blood vessels.

Fig. 5. (a) Results of analgesic activity, (b) maximum possible analgesia, (c) results of local anaesthetic activity. Each value represents the mean ± SD (N = 6). Statistical analysis were done by one-way ANOVA followed by the Student's T-test (*P < 0.05). Network pharmacology-based analysis.

Fig. 6. (a) Venn diagram for the integrated analysis of the related targets of ursolic acid and osteoarthritis, (b) network nodes represent suggested protein targets, and the edges represent protein–protein interactions. The size of nodes signifies the connectivity of each protein, the higher the node size the higher its connectivity to other nodes, (c) the top 10 hub genes: the darker the color, the higher the score and the stronger the connection.

Fig. 7. 2D & 3D views (a) & (b) of the ligand 307 re-docked in TNF-α active site, 2D & 3D views (c) & (d) of UA docked in TNF-α active site, 2D & 3D views (e) & (f) of naphthyridine re-docked in TGF-βR1 active site, 2D & 3D views (g) & (h) of ursolic acid docked in TGF-βR1 active site, 2D & 3D views (i) & (j) of UA docked in NF-κB active site, 2D & 3D views (k) & (l) of dexamethasone docked in NF-κB active site, top view (m) & (n) of 3D structure Nav 1.4-β 1 complex consisting of four VSD and Nav 1.4 pore presented in red circle, 2D & 3D views (o) & (p) of ursolic acid docked in Nav 1.4-β1 channel, 2D & 3D views (q) & (r) of Lidocaine docked in Nav 1.4-β1 channel, 2D & 3D views (s) & (t) of co-crystallized ligand (ID: N73) in MMP-9 active site, 2D & 3D views (u) & (v) of UA docked in MMP-9 active site.

Fig. 8. (a) Gene expression levels using quantitative RT-PCR. Data represent fold change relative to the normal control group. The relative fold change of mRNA was assessed using the 2^−ΔΔCt method and normalized to the house keeping gene GAPDH. Bars represent mean ± SD. Significant difference between groups is analysed by a one-way ANOVA test, where: *p < 0.05 compared with those of the non-treated OA group on the respective day and #p < 0.05 compared with those of the normal control group, (b) MMP-9/TIMP-1 ratios of different OA treatment groups.

Fig. 9. Proposed mechanism of ursolic acid for management of osteoarthritis.