Regulation of autophagy by the PI3K-AKT pathway in Astragalus membranaceus - Cornus officinalis to ameliorate diabetic nephropathy

Abstract

Aims and background: Autophagy plays an increasingly significant role in diabetic nephropathy (DN), but the mechanism by which autophagy participates in DN injury is not well understood. Our previous studies have shown that Astragalus membranaceus - Cornus officinalis (AM-CO) improves DN lipid metabolism disorders, however, the exact mechanism of which is also not well defined. The aim of this study was to investigate the therapeutic effects of AM-CO officinalis on DN and the mechanism of action on DN using lipidomic techniques and network pharmacological approaches.

Experimental methods: The in vivo experiments were carried out using the KKAy mice model with the intervention of AM-CO. Analysis of kidney and serum samples from KKAy mice treated with AM-CO using lipidomic technology to obtain biomarkers for the treatment of DN and to identify the main targets associated with DN; Analyse potential signalling pathways for the treatment of DN using network pharmacology methods. In vitro experiments were performed with PA-induced HK-2 cells and results verified by protein blotting and immunofluorescence.

Results: Lipidomic analysis revealed 363 differential metabolites in serum and 195 differential metabolites in kidney tissue, which were compared and analysed to find their common differential metabolites belonging to the phosphatidylethanolamine (PE) classes, respectively. In addition, PE plays a vital functiona in the process of autophagy. And the network analysis results speculated that Calycosin (Cal), a major component of AM-CO, could ameliorate DN injury by regulating autophagy through modulating the PI3K-AKT signaling pathway. In vivo experiments showed that AM-CO could induce autophagy, an increase in LC3II expression and a decrease in P62 expression. Meanwhile, in vitro experiments showed that Cal could also increase the expression of LC3II and inhibit the protein expression levels of p62, PI3K, P-AKT and AKT. The addition of a PI3K activator resulted in a reversal of protein expression.

Conclusion: In conclusion, Cal can ameliorate the injury in DN by regulating autophagy, and PI3K-AKT is the main pathway for its regulation of autophagy and a key pathway for the action of AM-CO.

Keywords: Astragalus membranaceus -Cornus officinalis; calycosin; diabetic nephropathy; lipidomic technology; network analysis.

FIGURE 1

Lipidomic approach to the analysis of differential metabolites. (A) Metabolite classification and proportion of donut plot in control and DN groups (In kidney tissue); (B) Metabolite classification and proportion of donut plot in DN and DN+AM-CO groups (In kidney tissue); (C) Score scatter plot 3D of PCA model in kidney tissue; (D) Metabolite classification and proportion of donut plot in control and DN groups (In serum); (E) Metabolite classification and proportion of donut plot in DN and DN+AM-CO groups (In serum); (F) Score scatter plot 3D of PCA model in serum; (G) Heatmap of hierarchical clustering analysis in kidney tissue; (H) Heatmap of hierarchical clustering analysis in serum; (I) Chrodplot analysis in kidney tissue; (J) Chrodplot analysis in serum.

FIGURE 2

Network pharmacological analysis of Cal-related proteins for DN treatment. (A) Relevant targets of DN predicted by the database; (B) Target-gene interaction PPI network; (C) PPI network plot after combining Cytoscape; (D) GO enrichment analysis of predicted targets of Cal for DN; (E) KEGG analysis of predicted proteins of Cal for DN; (F) Pathway map of Cal‘s potential therapeutic targets for DN.

FIGURE 3

AM-CO attenuates abnormal physiological parameters and renal function in DN mice. (A) AM-CO reduced the level of body weight. (B) AM-CO reduced the level of blood glucose. (C) AM-CO decreased TC in blood; (D) AM-CO decreased TG in blood; (E) AM-CO decreased LDL-C in blood; (F) AM-CO decreased HDL-C in blood; (G) AM-CO decreased the level of UACR; (H) AM-CO AM-CO reduced the level of Scr in blood; (I) MOR decreased the level of BUN; (J) Western blot analysis demonstrates the expression of P62 and LC3. Data are expressed as mean ± S.D.; N = 10; *P < 0.05; **P < 0.01; ***P < 0.001, and ****P < 0.0001 compared with the DN group.

FIGURE 4

Effects of AM-CO treatment on renal pathology and autophagy in DN mice. (A) HE staining reacts to the degree of tubular dilatation, PAS staining shows the degree of glycoprotein accumulation, Masson staining the degree of collagen fibre deposition (scale bars: 25/50 μm). (B) Immunofluorescence staining showed expression of P62 and LC3 (scale bars: 50 μm).

FIGURE 5

Effects of different concentrations of Cal on HK-2 cell viability and autophagy. (A) Effect of different concentrations of Cal on HK-2 cell viability; (B) Western blot showed the expression of P62, LC3, PAKT, PPI3K when different concentrations of Cal (5 μM, 10 μM, 20 μM) were given under PA stimulation; (C) Immunofluorescence results of P62, LC3, PAKT, PPI3K at different administration concentrations (scale bars: 50 μm). Compared to PA group, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

FIGURE 6

Addition of PI3K activators (740 Y-P) reverses Cal regulation of autophagy. (A) Protein expression levels of P62, LC3, PAKT, PPI3K after 740 Y-P treatment. Compared to PA group, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. (B) Immunofluorescence results of P62, LC3, PAKT, PPI3K after 740 Y-P treatment (scale bars: 50 μm). Compared to PA group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns:P > 0.05.

FIGURE 7

A mechanistic map of the formation and regulation of autophagy and the regulation of autophagy by Astragalus membranaceus -Cornus officinalis and Calycosin. The ULKI complex and PI3K III nucleation complex initiate autophagy, and the downstream Atg5-Atg12-Atg16 complex can turn PE-coupled LC3I into lipidated LC3II. P62 functions as an autophagy receptor and binds to LC3 to complete autophagy fusion and amplification. PI3K acts on AKT, which can directly act on ULKI complexes to regulate autophagy downstream. Astragalus membranaceus -Cornus officinalis as well as Calycosin can improve DN by modulating autophagy.