Integrating serum pharmacochemistry and network pharmacology to explore potential compounds and mechanisms of Alpiniae oxyphyllae fructus in the treatment of cellular senescence in diabetic kidney disease

Abstract

Background: Diabetic kidney disease (DKD), one of the microvascular complications in patients with diabetes mellitus, is a common cause of end-stage renal disease. Cellular senescence is believed to be an essential participant in the pathogenesis of DKD. Although there is evidence that Alpiniae oxyphyllae fructus (AOF) can ameliorate DKD progression and organismal senescence, its ability to ameliorate renal cellular senescence in DKD as well as active components and molecular mechanisms remain to be explored.

Purpose: This study aimed to investigate the role of AOF in the treatment of cellular senescence in DKD and to explore its active components and potential molecular mechanisms.

Methods: The pharmacological efficacy of AOF in ameliorating cellular senescence in DKD was assessed by establishing DKD mouse models and HK-2 cells under high glucose stress. UHPLC-QTOF-MS was used to screen the active compounds in AOF, which were used in conjunction with network pharmacology to predict the molecular mechanism of AOF in the treatment of cellular senescence in DKD.

Results: In vivo experiments showed that AOF reduced GLU, mAlb, Scr, BUN, MDA, SOD levels, and ameliorated renal pathological damage and renal cell senescence in DKD mice. In vitro experiments showed that AOF-containing serum improved the decline in HK-2 cell viability and alleviated cellular senescence under high glucose intervention. The results of the UHPLC-QTOF-MS screened 26 active compounds of AOF. The network pharmacological analyses revealed that Cubebin, 2',6'-dihydroxy-4'-methoxydihydrochalcone, Chalcone base + 3O,1Prenyl, Batatasin IV, and Lucidenolactone were the five core compounds and TP53, SRC, STAT3, PIK3CA, and AKT1 are the five core targets of AOF in the treatment of DKD. Molecular docking simulation results showed that the five core compounds had good binding ability to the five core targets. Western blot validated the network pharmacological prediction results and showed that AOF and AOF-containing serum down-regulate the expression of TP53, and phosphorylation of SRC, STAT3, PIK3CA, and AKT.

Conclusion: Our study shows that AOF may delay the development of cellular senescence in DKD by down-regulating the levels of TP53, and phosphorylation of SRC, STAT3, PIK3CA, and AKT.

Keywords: Alpiniae oxyphyllae fructus; UHPLC-QTOF-MS; cellular senescence; diabetic kidney disease; molecular docking; network pharmacology; traditional Chinese medicine.

Figure 1

The brief flow chart of this study. In this paper, the efficacy of AOF in ameliorating renal cellular senescence in DKD was firstly confirmed by in vitro and in vivo experiments, then the active compounds of AOF and their potential molecular mechanisms were identified by UHPLC-QTOF-MS analysis and network pharmacology analysis, and finally the predicted results of network pharmacology were verified by molecular docking and western blot.

Figure 2

AOF ameliorated biochemical indices and kidney histopathology in DKD mice. Effect of AOF on the GLU (A), mAlb (B), Scr (C), BUN (D), MDA (E), SOD (F) after treatment. Histopathological changes in kidney tissues evaluated by HE staining (original magnification × 40.0) (G). The scale bar represents 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Figure 3

AOF ameliorates cellular senescence in the kidney of DKD mice. Immunohistochemical staining images of P16, P21, P53 (A); P16, P21, P53 protein expression levels (B). P16, P21, P53 mRNA expression levels (C); IL-6, IL-1β, TGF-β, MMP3, MCP1 mRNA expression levels (D). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

AOF-containing serum ameliorates senescence of HK-2 cells under high glucose intervention. The effect of different doses of AOF on HK-2 cell viability (A); The representative images of SA-β-gal stained in HK-2 cells (B); P16, P21, P53 protein expression levels (C); P16, P21, P53 mRNA expression levels (D); Immunofluorescence staining images of P16, P21, P53 (E); IL-1β, TGF-β, MMP3, MCP1 mRNA expression levels (F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Positive and negative ion chromatograms of AOF-containing serum (A), blank serum (B), and AOF (C).

Figure 6

The result of network pharmacology analysis. Venn diagram of AOF, DKD, cellular senescence related targets (A); AOF active compounds-therapeutic targets network, and blue diamond represents AOF, orange round rectangle represents active compound, green ellipse represents therapeutic target, pink triangle represents cellular senescence in DKD (B). PPI network of targets for SQP treatment of DKD. The degree value of this network node is obtained based on the network topology analysis. The more centrally located the node is, the larger the circle is, the greener the color is, the greater the degree value is, indicating that the node is more critical in the overall network (C). The main pathways and biological processes in KEGG enrichment analysis (D). The main pathways and biological processes in GO enrichment analysis (E).

Figure 7

Molecular docking results. Minimum binding energy of each target to AOF bioactive compounds (A) and and diagrams of their interactions (B).

Figure 8

The effect of AOF on the regulation of core targets in kidneys of DKD mice. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9

The effect of AOF-containing serum on the regulation of core targets in HK-2 cells. *P < 0.05, **P < 0.01.