Abstract:

This study aims to investigate the effect of cryptotanshinone (CTS) on regulating macrophage polarization through the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) axis and its impact on gouty arthritis (GA) in rats. Rats were randomly divided into five groups: control group, GA group, CTS group (30 mg/kg), JAK2 activator coumermycin A1 (C-A1) group (5 mg/kg), and CTS+C-A1 group (30 mg/kg CTS+5 mg/kg C-A1), with 12 rats in each group. Except for the control group, the GA model was established in the other groups by injecting monosodium urate (MSU) crystals into the ankle joints. After the last administration, the degree of ankle joint swelling was observed in each group. Histopathological changes in the synovial tissue of the ankle joints were observed and scored using HE staining. ELISA kit for detecting interleukin (IL)-1β, tumour necrosis factor-α (TNF-α), IL-6, and IL-10 levels in rat serum. Flow cytometry was used to detect the proportions of M1-type (CD86⁺) and M2-type (CD206⁺) macrophages in the synovial tissue of the ankle joints. RT-qPCR was used to detect the mRNA expression levels of M1-polarised markers inducible nitric oxide synthase (iNOS) and IL-1β, as well as M2-polarised markers arginase-1 (ARG-1) and IL-10 in rat ankle joint synovial tissue. Western blot analysis of JAK2, p-JAK2, STAT3, and p-STAT3 protein expression levels in rat ankle joint synovial tissue. Macrophages were isolated and cultured from rat femurs and tibiae. Cell viability was assessed using the CCK-8 assay to determine the optimal concentration of CTS. Macrophages were randomly assigned to control, MSU, and CTS groups, with three replicates per group. Except for the control group, all other groups were stimulated with MSU crystals to establish an inflammatory model. RT-qPCR was used to assess the expression levels of M1 and M2 polarisation markers in the cells of each group. The results showed that compared with the GA group, CTS intervention reduced joint swelling and histological scores in GA rats, decreased serum IL-1β, TNF-α, and IL-6 levels, downregulated the proportion of CD86⁺ M1-type macrophages, iNOS, IL-1β mRNA expression, and p-JAK2/JAK2, p-STAT3/STAT3 expression in synovial tissue (P<0.05), increased serum IL-10 levels, and upregulated the proportion of CD206⁺ M2-type macrophages in synovial tissue, as well as ARG-1 and IL-10 mRNA expression levels (P<0.05). The experimental results demonstrated that CTS partially reversed the aforementioned effects of C-A1. In vitro findings revealed that, compared with the MSU group, the CTS group exhibited reduced expression of iNOS and IL-1β mRNA (P<0.05), alongside increased expression of ARG-1 and IL-10 mRNA (P<0.05). In summary, CTS can promote the polarization of macrophages towards the M2 phenotype in rats by inhibiting the JAK2/STAT3 axis, thereby alleviating joint inflammation in GA rats.

Key words:

cryptotanshinone; gouty arthritis, JAK2/STAT3 axis, macrophage polarization