祛浊通痹颗粒对高尿酸血症小鼠降尿酸作用的研究

Research on the Hypouricemic Effect of Quzhuo Tongbi Granules on Mice with Hyperuricemia

    摘要
  • 摘要:
    目的 探讨祛浊通痹颗粒(QZTB)对高尿酸血症(Hyperuricemia，HUA)小鼠降尿酸及抗炎的作用机制。
    方法 采用超高效液相色谱-质谱(UPLC-MS)方法鉴定祛浊通痹颗粒(QZTB)成分。随机将64只C57BL/6小鼠分成对照组、模型组、苯溴马隆组、QZTB低剂量组、QZTB中剂量组、QZTB高剂量组、MCC950组、MCC950+QZTB中剂量组，每组8只。腺嘌呤(100 mg·kg-1)联合氧嗪酸钾(500 mg·kg-1)建立HUA小鼠模型。除对照组外，其余各组均先造模2周后给药4周。造模2周后采用小鼠眼底静脉丛取血检测血尿酸(SUA)水平，以SUA水平作为造模成功依据，持续给药4周后处死取材。采用全自动生化分析仪检测SUA、尿素氮(BUN)、血肌酐(Cr)水平; 采用酶联免疫吸附测定法(ELISA)检测小鼠血清白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-18(IL-18)、肿瘤坏死因子-α(TNF-α)水平; 采用qPCR法检测肾脏组织中尿酸转运蛋白1(URAT1)、三磷酸腺苷结合转运蛋白G2(ABCG2)、葡萄糖转运蛋白9(GLUT9)、PDZ蛋白激酶1(PDZK1)mRNA表达; 采用蛋白免疫印迹法(Western blot)检测小鼠肾脏组织中尿酸转运体(URAT1、ABCG2、GLUT9、PDZK1)、核转录因子κB(NF-κB)总蛋白和磷酸化NF-κB(p-NF-κB)、Nod样受体蛋白3(NLRP3)、半胱氨酸蛋白酶-1(Cleaved Caspase-1)蛋白和半胱氨酸蛋白酶-1前体蛋白(Pro-Caspase-1)表达; 采用免疫组化测定肾脏组织中尿酸转运体(URAT1、ABCG2、PDZK1、GLUT9)表达水平。
    结果 在QZTB中鉴定了9种有代表性的活性成分。造模2周后，与对照组相比，模型组SUA显著上升(P < 0.000 1);给药4周后，模型组血清SUA、BUN、Cr显著升高(P < 0.000 1)，IL-1β、IL-6、IL-18和TNF-α水平升高(P < 0.01, P < 0.001)，肾组织ABCG2、PDZK1蛋白表达减少(P < 0.01, P < 0.001, P < 0.000 1)，URAT1、GLUT9、NLRP3、p-NF-κB p65/NF-κB p65和Cleaved Caspase-1/Pro-Caspase-1蛋白表达显著升高(P < 0.01, P < 0.001, P < 0.000 1)。与模型组相比，苯溴马隆组和QZTB低、中、高剂量干预组SUA、BUN、Cr存在不同程度的降低(P < 0.001, P < 0.000 1)，QZTB能有效降低血清炎症因子IL-1β、IL-6、IL-18和TNF-α水平(P < 0.05, P < 0.01)，提高肾组织ABCG2、PDZK1蛋白表达(P < 0.05, P < 0.01, P < 0.000 1)，下调URAT1、GLUT9、NLRP3、p-NF-κB p65/NF-κB p65和Cleaved Caspase-1/Pro-Caspase-1蛋白表达(P < 0.05, P < 0.01, P < 0.001, P < 0.000 1)。与模型组相比，MCC950组下调NLRP3、p- NF - κB p65 / NF - κB p65和Cleaved Caspase-1 / Pro-Caspase-1蛋白表达(P < 0.01)。与MCC950组或QZTB组相比, MCC950+QZTB组下调NLRP3、p - NF - κB p65/NF - κB p65、Cleaved Caspase-1/Pro-Caspase-1蛋白表达(P < 0.05, P < 0.01, P < 0.000 1)。
    结论 QZTB能够通过抑制NF-κB/NKRP3信号通路促进尿酸排泄，从而改善HUA症状。

     

    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.

     

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