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. 2022 Dec 19:2022:5851412.
doi: 10.1155/2022/5851412. eCollection 2022.

Tanshinone IIA Improves Acute Gouty Arthritis in Rats through Regulating Neutrophil Activation and the NLRP3 Inflammasome

Lianjie Xu  1 , Xiao Liu  2 , Yurong Zhang  1 , Tao Jia  3 , Limei Li  1 , Yan Du  1 , Wenhui Chen  1   4 , Shan Zhang  1   4
Affiliations

Tanshinone IIA Improves Acute Gouty Arthritis in Rats through Regulating Neutrophil Activation and the NLRP3 Inflammasome

Lianjie Xu et al. Dis Markers. .

Abstract

Objectives: To investigate the prevention and treatment effect of tanshinone IIA (TIIA) on acute gouty arthritis (AGA) and its mechanism.

Methods: The anti-AGA effect of TIIA was observed in vivo and in vitro. Neutrophils were isolated from the abdominal cavity of mice, and the anti-AGA effect of TIIA was investigated in a rat model of MSU-induced AGA. The pathological changes of the ankle joint tissues were assessed by H&E. Cytokine and chemokine expression were determined by ELISA and RT-qPCR. The NLRP3 inflammasome pathway protein levels in the ankle joint tissues were evaluated via western blotting. Neutrophil migration was evaluated in air pouch and transwell assays. Immunohistochemistry and immunofluorescence analysis evaluate the release of myeloperoxidase (MPO), neutrophil elastase (NE), and citrullination of histone H3 (CitH3). Beclin-1 and LC3B expressions were determined using western blotting and immunofluorescence. Key Findings. Treatment with TIIA alleviated synovial hyperplasia and neutrophil infiltration, regulated cytokine and chemokine expressions, and inhibited NLRP3 activation in AGA rats, neutrophil migration, MPO, NE, and CitH3 expression, and LC3B and Beclin-1 protein expression.

Conclusions: These results demonstrate that TIIA can effectively enhance AGA by focusing on the neutrophils and NLRP3 inflammasome, demonstrating that TIIA may act as a potential helpful agent for AGA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TIIA treatment alleviates MSU-induced AGA in rats. (a) The degree of swelling of right ankle joints was observed at 48 h after MSU induction. (b) The circumference of the ankle joints of rats. ##P < 0.01 when comparing the AGA group with the control (n = 8). (c) H&E staining of the ankle joint sections, the arrow indicates pannus formation, original magnification: ×400.
Figure 2
Figure 2
TIIA decreases UA, cytokine, or chemokine production in serum and neutrophils in vitro. (a–g) ELISA analyses of serum UA, XOD, IL-6, TNF-α, IL-8, CXCL1, CXCL2, S100A8A9, IL-1β, IL-18, and IL-1RA expressions in the MSU-induced AGA rats (n = 6). (h, i) QRT-PCR analyses of IL-1β, IL-6, TNF-α, CXCL1, and CXCL2 mRNA levels in neutrophils in vitro (n =6). #P < 0.05 and ##P < 0.01, when compared to the control; ∗P < 0.05 and ∗∗P < 0.01, when compared to the AGA.
Figure 3
Figure 3
TIIA inhibited protein expression in the NLRP3 inflammasome pathway. (a) Western blotting analysis of NLRP3 and caspase-1 in ankle joints. (b) Density analyses of NLRP3 and caspase-1 expression in (a). (c) Western blotting analysis of ASC and IL-1β in ankle joints. (d) Density analyses of ASC and IL-1β expression in (c) (n = 3). AGA vs. control, #P < 0.05; TIIA vs. AGA, ∗P < 0.05.
Figure 4
Figure 4
TIIA inhibited MSU-induced neutrophil migration. We administer MSU or TIIA into the air pouches. Exudates were collected after 6 h, and neutrophils were numbered and identified via cytology as described in Section 2.6. (a) The number of neutrophils in mouse air pouches (n = 6), AGA vs. control, ##P < 0.01; AGA+TIIA vs. AGA, ∗∗P < 0.01. (b) Transwell analysis results of in vitro neutrophil mobility (n = 6), AGA vs. control, #P < 0.05; MSU+TIIA vs. AGA, ∗P < 0.05.
Figure 5
Figure 5
TIIA inhibited the formation of MPO, NE, and CitH3. (a–c) Release of MPO, NE, and CitH3 by immunohistochemistry analysis in vivo, original magnification: ×400. Arrows indicate positive signals. (d–f) Immunofluorescence analyses of MSU-induced MPO, NE, and CitH3 expression in vitro.
Figure 6
Figure 6
TIIA inhibits autophagy in MSU-induced neutrophils. (a) The neutrophils of different groups were analysed for Beclin-1 and LC3B protein expression via western blotting (n = 3). (b) Quantification of Beclin-1 and LC3BII shown in (a). (c) Ratio of LC3BII over LC3BI expression. AGA vs. control, #P < 0.05; TIIA vs. AGA, ∗P < 0.05. (d, e) Immunofluorescence analyses of MSU-induced LC3B and Beclin-1 levels in vitro.
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