Guizhi-Shaoyao-Zhimu decoction attenuates rheumatoid arthritis partially by reversing inflammation-immune system imbalance

Abstract

Background: Guizhi-Shaoyao-Zhimu decoction (GSZD) has been extensively used for rheumatoid arthritis (RA) therapy. Marked therapeutic efficacy of GSZD acting on RA has been demonstrated in several long-term clinical trials without any significant side effects. However, its pharmacological mechanisms remain unclear due to a lack of appropriate scientific methodology.

Methods: GSZD's mechanisms of action were investigated using an integrative approach that combined drug target prediction, network analysis, and experimental validation.

Results: A total of 77 putative targets were identified for 165 assessed chemical components of GSZD. After calculating the topological features of the nodes and edges in the created drug-target network, we identified a candidate GSZD-targeted signal axis that contained interactions between two putative GSZD targets [histone deacetylase 1 (HDAC1) and heat shock protein 90 kDa alpha, class A member 1 (HSP90AA1)] and three known RA-related targets [NFKB2; inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB); and tumor necrosis factor-alpha (TNF-α)]. This signal axis could connect different functional modules that are significantly associated with various RA-related signaling pathways, including T/B cell receptor, Toll-like receptor, NF-kappa B and TNF pathways, as well as osteoclast differentiation. Furthermore, the therapeutic effects and putative molecular mechanisms of GSZD's actions on RA were experimentally validated in vitro and in vivo.

Conclusions: GSZD may partially attenuate RA by reversing inflammation-immune system imbalance and regulating the HDAC1-HSP90AA1-NFKB2-IKBKB-TNF-α signaling axis.

Keywords: Experimental validation; Guizhi-Shaoyao-Zhimu decoction; Network pharmacology; Rheumatoid arthritis; TCM herbal formula.

Fig. 1

A schematic diagram of the systematic strategies for unraveling the pharmacological mechanisms of herbal formula GSZD acting on RA

Fig. 2

The co-module underlying GSZD formula and pathways/pathological changes involved into RA progression. Co-module analysis was performed by mapping the herbs into shared modules if the distance between two herbs was very close (shared more putative targets or their putative targets had more interactions with each other) in the network. As a result, the herb-putative target network was divided into three modules, which were centered on Ramulus Cinnamomi, Paeonia lactiflora and Rhizoma Anemarrhenae

Fig. 3

Hub-pathway network of GSZD. The pathway enrichment analysis showed that the hubs, identified from the interaction network of putative targets of GSZD and known RA-related targets, were more frequently implicated into T/B cell receptor signaling pathway, Toll-like receptor signaling pathway, Osteoclast differentiation, NF-kappa B signaling pathway, TNF signaling pathway, Chemokine signaling pathway, VEGF signaling pathway and Neuroactive ligand-receptor interaction, which all play crucial roles in the main pathological events during the RA progression, such as inflammation, synovial pannus formation, inflammatory cell infiltration, angiogenesis, joint destruction and pain. Yellow nodes refer to the putative targets of GSZD; Blue nodes refer to the known RA-related targets; Green nodes refer to other human genes interacted with the putative targets of GSZD or the known RA-related targets

Fig. 4

Network of interactions among herbs of GSZD and hubs obtained from the network of putative targets of GSZD and known RA-related targets. Yellow nodes refer to the putative targets of GSZD; Blue nodes refer to the known RA-related targets; Green nodes refer to other human genes interacted with the putative targets of GSZD or the known RA-related targets

Fig. 5

The herbal formula GSZD. a Photos of nine Chinese herbs in GSZD, including Ramulus Cinnamomi (Guizhi, GZ), Paeonia lactiflora (Shaoyao, SY), Rhizoma Atractylodis Macrocephalae (Baizhu, BZ), Raidix Saposhnikoviae (Fangfeng, FF), RadixAconiti Lateralis Preparata (Fuzi, FZ), Herba Ephedrae (Mahuang, MH), RhizomaZingiberis Recens (Shengjiang, SJ), Rhizoma Anemarrhenae (Zhimu, ZM) and RadixGlycytthizae (Gancao, GC) in order. b HPLC graphs of GSZD. HPLC was performed to identify the phytochemical profiles of GSZD. c The main chemicals in GSZD

Fig. 6

Effects of GSZD on the severity of arthritis in AIA rats. a Macroscopic evidence of arthritis such as redness or swelling was obviously observed in AIA rats of the model group, while doses of 18.6 g/(kg day) GSZD and 0.2 mg/(kg day) MTX significantly reduced the severity of arthritis in AIA rats; b Doses of 4.65–18.6 g/(kg day) GSZD and 0.2 mg/(kg day) MTX significantly reduced the mean arthritis score of AIA rats; c Doses of 4.65–18.6 g/(kg day) GSZD and 0.2 mg/(kg day) MTX significantly reduced the arthritis incidence of AIA rats; d Doses of 4.65–18.6 g/(kg day) GSZD and 0.2 mg/(kg day) MTX significantly reduced the percentage of arthritis limbs of AIA rats; e Doses of 9.3 g/(kg day) and 18.6 g/(kg day) GSZD, and 0.2 mg/(kg day) MTX effectively extended the time of arthritis first appeared of AIA rats. Data are represented as the mean ± SD. ^#P < 0.05, comparison with the normal control (Con). *, **, and ***, P < 0.05, P < 0.01, and P < 0.001, respectively, comparison with the model control (Mod)

Fig. 7

Effect of GSZD on histologic lesions of AIA rats. a Inflammatory changes observed in different groups using H & E staining (×200); b Articular cartilage changes observed in different groups using H & E staining (×200); c Cartilage changes observed in different groups using toluidine blue staining; (×200); d Bone destruction changes observed in different groups using H & E staining (×200); e–g showed the inflammation score, the bone destruction score and the loss of toluidine blue staining in joints respectively, calculated as described in “Methods” section. Data are represented as the mean ± SD. *, **, and ***, P < 0.05, P < 0.01, and P < 0.001, respectively, in contrast with the model control (Mod)

Fig. 8

Effect of GSZD on the expression of HDAC1 (a), HSP90AA1 (b), NFKB2 (c), IKBKB (d) and TNF-α (e) proteins in the joint of AIA rats detected by Western blot analysis. Data are represented as the mean ± SD. ^# and ^##, P < 0.05 and P < 0.01, respectively comparison with the normal control (Con). * and **, P < 0.05 and P < 0.01, respectively, comparison with the model control (Mod)

Fig. 9

Effect of GSZD on the expression of HDAC1 (a), HSP90AA1 (b), NFKB2 (c), IKBKB (d) and TNF-α (e) proteins in HFLS-RA. Data are represented as the mean ± SD. ^# and ^##, P < 0.05 and P < 0.01, respectively, comparison with the control cells (Con). * and **, P < 0.05 and P < 0.01, respectively, comparison with the IL-1β-induced model control (Mod)