Uncovering the potential mechanism and bioactive compounds of Salviae Miltiorrhizae Radix et Rhizoma in attenuating diabetic retinopathy

Abstract

Background

Diabetic retinopathy (DR) is a serious microangiopathy resulting from diabetes. Salviae Miltiorrhizae Radix et Rhizoma (Danshen) is commonly used to treat cardiovascular diseases in clinics in China. However, whether it can also be used for DR treatment, along with its primary active compounds and underlying mechanisms of action, remains unclear.

Purpose

To evaluate the alleviation of water extract of Salvia miltiorrhiza Radix et Rhizoma (SWE) on DR, elucidate the underlying mechanisms, and identify the primary active compounds.

Methods

Mice were intraperitoneally injected with streptozotocin (STZ) to induce diabetes. Blood-retina barrier (BRB) breakdown was detected. The potential underlying mechanisms were predicted by network pharmacology and further validated by Western blot, leukostasis assay and real-time polymerase chain reaction (PCR). The primary active compounds in SWE were identified by integrating in vitro activity analysis and molecular docking.

Results

SWE attenuated BRB breakdown in STZ-induced DR mice. Results of network pharmacology and further experimental validation implied that inhibiting retinal inflammation and angiogenesis, and reversing endothelial barrier dysfunction were involved in the SWE-provided alleviation of DR, and the key involved signaling pathways were PI3K-AKT, VEGF, TNF, and NFκB pathways. Further results in vitro demonstrated that salvianolic acid A (SalA), salvianolic acid B (SalB), salvianolic acid C (SalC), and Tanshinone IIA (TanIIA) not only reduced the expression of pro-inflammatory cytokines but also inhibited the adhesion of inflammatory cells. However, danshensu (DSS), cryptotanshinone (CTS), and tanshinone I (TanI) only downregulated the expression of pro-inflammatory cytokines. SalA, SalB, and CTS reversed endothelial barrier dysfunction in vitro. SalA, SalB, SalC, CTS, DSS, and TanIIA decreased VEGF mRNA expression, and TanIIA also inhibited VEGF-induced angiogenesis in vitro. Molecular docking predicted potential interactions between these active compounds and several key molecules involved in regulating inflammation, angiogenesis, and cell-cell junctions. These compounds abrogated hyperglycemia-induced phosphorylation of AKT1 and PI3 K in vitro. Furthermore, the interactions of SalA, SalB, SalC, and TanIIA with TNFR1 were further validated using cellular thermal shift assay (CETSA).

Conclusion

SWE alleviated DR via reversing BRB breakdown and suppressing retinal inflammation and angiogenesis. SalA, SalB, SalC, TanIIA, and CTS might be primary active compounds in SWE, and they contributed greatly to the improvement of SWE against DR via reversing endothelial barrier injury, inhibiting inflammation and angiogenesis.