Abstract:
OBJECTIVE To investigate the mechanism of action of Quzhuo Tongbi Granules (QZTB) in reducing uric acid and anti-inflammation in mice with hyperuricemia (HUA).
METHODS The components of QZTB were identified by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Sixty-four C57BL/6 mice were randomly divided into control group, model group, benzbromarone group, QZTB low-dose group, QZTB medium-dose group, QZTB high-dose group, MCC950 group, and MCC950+QZTB medium-dose group, with 8 mice in each group. Adenine (100 mg ·kg-1) and potassium oxonate (500 mg ·kg-1) were used to establish the HUA mouse model. Except for the control group, all other groups underwent 2 weeks of modeling followed by 4 weeks of treatment. After 2 weeks of modeling, blood was collected from the orbital venous plexus to measure serum uric acid (SUA) levels as the criterion for successful model induction. Mice were sacrificed after 4 weeks of continuous treatment for sample collection.An automatic biochemical analyzer was used to measure serum levels of SUA, urea nitrogen (BUN), and creatinine (Cr). Enzyme-linked immunosorbent assay (ELISA) was employed to detect serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-18 (IL-18), and tumor necrosis factor-α (TNF-α). The qPCR was used to assess mRNA expression of urate transporter 1 (URAT1), ATP-binding cassette transporter G2 (ABCG2), glucose transporter 9 (GLUT9), and PDZ domain-containing protein kinase 1 (PDZK1) in kidney tissue. Western blot was performed to measure protein expression of urate transporters (URAT1, ABCG2, GLUT9, PDZK1), nuclear transcription factor κB (NF-κB) total protein, phosphorylated NF-κB (p-NF-κB), Nod-like receptor protein 3 (NLRP3), Cleaved Caspase-1 and Pro-Caspase-1 proteins in kidney tissue. Immunohistochemistry was used to determine the expression levels of urate transporters (URAT1, ABCG2, PDZK1, GLUT9) in kidney tissue.
RESULTS A total of 9 representative active ingredients were identified in QZTB. Two weeks after modeling, SUA in the model group was significantly increased compared with that in the control group (P < 0.000 1). Four weeks after administration, serum SUA, BUN and Cr in the model group were significantly increased (P < 0.000 1), IL-1β, IL-6, IL-18 and TNF-α levels were increased (P < 0.01, P < 0.001), the expression of ABCG2 and PDZK1 proteins in renal tissue was decreased (P < 0.01, P < 0.001, P < 0.000 1), and the expression of URAT1, GLUT9, NLRP3, p-NF-κB p65/NF-κB p65 and Cleaved Caspase-1/Pro-Caspase-1 proteins was significantly increased (P < 0.01, P < 0.001, P < 0.000 1). Compared with the model group, SUA, BUN and Cr in the benzbromarone group and the low-, medium- and high-dose QZTB intervention groups were reduced to varying degrees (P < 0.001, P < 0.000 1). QZTB could effectively reduce the levels of serum inflammatory factors IL-1β, IL-6, IL-18 and TNF-α (P < 0.05, P < 0.01), increase the expression of ABCG2 and PDZK1 proteins in renal tissue (P < 0.05, P < 0.01, P < 0.000 1), and downregulate the expression of URAT1, GLUT9, NLRP3, p-NF-κB p65/NF-κB p65 and Cleaved Caspase-1/Pro-Caspase-1 proteins (P < 0.05, P < 0.01, P < 0.001, P < 0.000 1). Compared with the model group, the MCC950 group downregulated the protein expressions of NLRP3, p-NF-κB p65/NF-κB p65, and Cleaved Caspase-1/Pro-Caspase-1 (P < 0.01). Compared with the MCC950 group or the QZTB group, the MCC950 + QZTB group downregulated the protein expressions of NLRP3, p-NF-κB p65/NF-κB p65, and Cleaved Caspase-1/Pro-Caspase-1 (P < 0.05, P < 0.01, P < 0.000 1).
CONCLUSION QZTB can promote uric acid excretion by inhibiting the NF-κB/NLRP3 signaling pathway, thereby improving the symptoms of HUA.