关键词: 计算机技术, 矢志方, 高尿酸血症, 尿酸转运蛋白, ERK1/2信号通路

Abstract: Object: To combine computer technology with molecular biology experiments to explore the effects of Shizhi Fang on uric acid-related transport proteins in hyperuricemia and the role of the ERK1/2 signaling pathway. Methods: After screening the active ingredients of Shizhi Fang obtained by ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-Q-TOF-MS) in the previous research of the research group, molecular docking was conducted with uric acid transport-related proteins (URAT1, ABCG2, OAT1, OAT3) to investigate their relationship with uric acid transport proteins. In vitro cultured human renal tubular epithelial cells (HK-2), after modeling and drug administration, Western blotting and immunofluorescence technology were used to detect the expression of uric acid transport-related proteins and the ERK1/2 signaling pathway. Twenty-five SD rats were randomly divided into a normal group, a model group, a Shizhi Fang group, a febuxostat group, and an ERK1/2 inhibitor group, with 5 rats in each group. In addition to the normal group, which was given normal saline by gavage, the other groups used oxonate (1500 mg/kg) and adenine (50 mg/kg) to prepare a hyperuricemia rat model by gavage, and the Shizhi Fang group, febuxostat group, and inhibitor group were given Shizhi Fang (6.75 g/kg), febuxostat (3.6 mg/kg), and inhibitor U0126 (30 mg/kg) by gavage for 4 weeks, respectively. HE staining was used to observe the pathological changes of kidney tissue, and toluidine silver staining was used to observe the formation of urate crystals. Western blotting and immunohistochemistry were used to detect the expression of uric acid transport-related proteins. Results: Ten active ingredients were screened, and molecular docking violin plots showed that, compared to uric acid reabsorption-related protein (URAT1), uric acid secretion-related proteins (OAT1, OAT3) could form stable docking with more active ingredients of Shizhi Fang, with lower docking energy for ABCG2. However, the heat map indicated that the binding energy of URAT1 with the active ingredients of Shizhi Fang was higher and more stable than the others. In vitro and in vivo experiments showed that, compared to the normal group, the model group had significantly increased URAT1 protein expression, and decreased ABCG2, OAT1, OAT3 protein expression. In comparison to the model group, the expression of URAT1 protein was significantly reduced in the various treatment groups, while OAT1 and OAT3 protein expression increased, with no statistically significant changes in ABCG2. Alcian blue staining revealed urate crystal formation in the model group, which was significantly reduced in the treatment groups. Pathological staining showed renal damage in the model group, which was attenuated in the treatment groups. Conclusion: Shizhi Fang can significantly reduce URAT1 expression in hyperuricemia and increase OAT1 and OAT3 expression to exert its therapeutic effect. This effect is closely related to the ERK1/2 signaling pathway, and the active ingredients of Shizhi Fang, including Deoxyadenosine, P-Hydroxybenzaldehyde, and Sinapic acid, may play a major role in this process.

Key words: Computer technology, Shizhi Fang, Hyperuricemia, Uric acid transport proteins, ERK1/2 signaling pathway