Zisheng Shenqi Decoction Ameliorates Monosodium Urate-Mediated Gouty Arthritis in Rats via Promotion of Autophagy through the AMPK/mTOR Signaling Pathway

Abstract

Gouty arthritis (GA) is an inflammatory disease owing to the accumulation of monosodium urate (MSU) in joints, leading to redness and burning pain. In this study, the effect of Zisheng Shenqi Decoction (ZSD) on a rat model of MSU-induced GA was investigated. ZSD obviously diminished the right paw thickness, the degree of the swelling of the paw, and the infiltration of the inflammatory cell, as well as cartilage erosion, and widened the joint space in MSU-treated rats. Besides, MSU remarkably elevated the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-18; however, ZSD treatment dose dependently lowered these levels and resulted in a significant decrease in articular elastase activity. Also, ZSD administration increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) but declined malondialdehyde (MDA) and nitrogen monoxide (NO) contents. Importantly, western blotting analysis revealed that NOD-like receptor protein 3 (NLRP3), cleaved caspase-1, IL-1β, nuclear factor-E2-related factor 2 (Nrf2) in the cytoplasm, phosphorylated mammalian target of rapamyclin (p-mTOR), and p62 expressions were downregulated, whereas the levels of nuclear Nrf2, phosphorylated AMP-activated protein kinase (p-AMPK), Beclin-1, and LC3II/I were upregulated by ZSD. Immunofluorescence assay indicated that ZSD evidently promoted nuclear translocation of LC3. Taken together, ZSD inhibited inflammation and oxidative stress and facilitated autophagy through the activation of the AMPK pathway and suppression of the mTOR signaling pathway, demonstrating its potential for preventing and curing GA.

Figure 1

ZSD attenuated MSU-induced GA in rats. (a) Thickness of the right paw in each group was measured for 0, 24, 48, and 72 h after MSU injection, respectively. (b) After the injection of MSU for 72 h, the swelling of the right paw in each group was observed and photographed. Then, rats were anesthetized again and sacrificed. (c) The pathological changes in the ankle joint were detected by H&E staining. The scale bar = 200 μm. ((d)–(f)) Histopathological changes in the joint space, inflammatory cell infiltration, and cartilage erosion were scored. Results were expressed as means ± SD (n = 6). ^###P < 0.001 compared with the control group; ^∗∗P < 0.01 and ^∗∗∗P < 0.001 compared with the MSU group. ZSD: Zisheng Shenqi Decoction; MSU: monosodium urate; GA: gouty arthritis; H&E: hematoxylin and eosin.

Figure 2

ZSD inhibited inflammation mediated by MSU in rats. ((a)–(d)) The levels of TNF-α, IL-1β, IL-6, and IL-18 in rat serum were determined via ELISA. (e) The activity of articular elastase in the rat ankle joint was evaluated with the neutrophil elastase (ELA2) detection kit. Results were presented as means ± SD. ((f)–(i)) The mRNA levels of TNF-α, IL-1β, IL-6, and IL-18 in the rat ankle joint were assessed by qRT-PCR. β-Actin was used as an internal reference. (j) The expressions of NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1β, and IL-1β were detected by western blotting. β-Actin was used as an internal reference. Results were presented as means ± SD. ^###P < 0.001 compared with the control group; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 compared with the MSU group. TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; ELISA: enzyme-linked immunosorbent assay; qRT-PCR: quantitative real-time polymerase chain reaction; NLRP3: NOD-like receptor protein 3.

Figure 3

ZSD increased antioxidant status and suppressed oxidative stress in the GA rat model. ((a)–(e)) The activities of GSH-Px, SOD, and CAT, and contents of MDA and NO in serum of rats were determined using corresponding kits. Results were expressed as means ± SD. ^###P < 0.001 compared with the control group; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 compared with the MSU group. GSH-Px: glutathione peroxidase; SOD: superoxide dismutase; MDA: malondialdehyde; NO: nitrogen monoxide; CAT: catalase.

Figure 4

ZSD increased Nrf2 activation. (a) The expression of Nrf2 in the cytoplasm and nucleus of the rat ankle joint was evaluated utilizing western blotting, respectively. β-Actin and histone H3 were utilized as internal references, respectively. (b) The Nrf2 mRNA level in the rat ankle joint was detected by qRT-PCR. β-Actin was utilized as an internal control. Results were expressed as means ± SD. ^###P < 0.001 compared with the control group; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 compared with the MSU group. (c) The level of Nrf2 in the rat ankle joint was assessed via the immunofluorescence assay. The scale bar = 50 μm. Arrows represented Nrf2-positive cells. Nrf2: nuclear factor-E2-related factor 2.

Figure 5

ZSD promoted tissue autophagy in MSU-treated rats. ((a)–(c)) The levels of p-AMPK, AMPK, p-mTOR, mTOR, p62, Beclin-1, and LC3II/I in the rat ankle joint were detected by western blotting. β-Actin was used as an internal control. Results were presented as means ± SD. ^###P < 0.001 compared with the control group; ^∗P < 0.05 and ^∗∗∗P < 0.001 compared with the MSU group. (d) The level of LC3 in the rat ankle joint was evaluated by the immunofluorescence assay. The scale bar = 50 μm. Arrows represented LC3-positive cells. AMPK: AMP-activated protein kinase; p-: phosphorylated; mTOR: mammalian target of rapamyclin.