A network pharmacology approach to decipher the mechanism of total flavonoids from Dracocephalum Moldavica L. in the treatment of cardiovascular diseases

Abstract

Aim of the study: Cardiovascular disease (CVD) seriously endangers human health and is characterized by high mortality and disability. The effectiveness of Dracocephalum moldavica L. in the treatment of CVD has been proven by clinical practice. However, the mechanism by which DML can treat CVD has not been systematically determined.

Materials and methods: The active compounds in DML were screened by literature mining and pharmacokinetic analysis. Cytoscape software was used to construct the target-disease interaction network of DML in the treatment of CVD. Gene ontology and signalling pathway enrichment analyses were performed. The key target pathway network of DML compounds was constructed and verified by pharmacological experiments in vitro. A hydrogen glucose deprivation/reoxygenation model was established in H9c2 cells using hypoxia and glucose deprivation for 9 h combined with reoxygenation for 2 h. The model simulated myocardial ischaemic reperfusion injury to investigate the effects of total flavonoids of Cymbidium on cell viability, myocardial injury markers, oxidative stress levels, and reactive oxygen radical levels. Western blot analysis was used to examine NOX-4, Bcl-2/Bax, and PGC-1α protein expression.

Results: Twenty-seven active components were screened, and 59 potential drug targets for the treatment of CVD were obtained. Through the compound-target interaction network and the target-disease interaction network, the key targets and key signalling pathways, such as NOX-4, Bcl-2/Bax and PGC-1α, were obtained. TFDM significantly decreased LDH and MDA levels and the production of ROS and increased SOD activity levels in the context of OGD/R injury. Further studies indicated that NOX-4 and Bax protein levels and the p-P38 MAPK/P38 MAPK andp-Erk1/2/Erk1/2 ratios were suppressed by TFDM. The protein expression of Bcl-2 and PGC-1α was increased by TFDM.

Conclusions: Our results showed that DML had multicomponent, multitarget and multichannel characteristics in the treatment of CVD. The mechanism may be associated with the following signalling pathways: 1) the NOX-4/ROS/p38 MAPK signalling pathway, which inhibits inflammation and reactive oxygen species (ROS) production, and 2) the Bcl-2/Bax and AMPK/SIRT1/PGC-1α signalling pathways, which inhibit apoptosis.

Keywords: Cardiovascular disease; Network pharmacology; Pharmacological evaluation; Recombinant NADPH oxidase 4; Total Flavonoids from Dracocephalum Moldavica L.

Fig. 1

The compound-target network of DML. The purple nodes represent candidate active compounds, and the red nodes represent potential protein targets. The edges represent the interactions between them

Fig. 2

GOBP enrichment analysis of the identified genes encoding proteins targeted by DML in biological processes

Fig. 3

Potential active ingredient target—disease interaction network diagram of the active ingredients of DML

Fig. 4

New pathways by which DML can treat CVD (the bioactive components in DML that treat CVD mainly target three signalling pathways and involve nine therapeutic modules)

Fig. 5

Screening the hypoxia/reoxygenation time in the OGD/R model of H9c2 cells. (n = 3, ^####P < 0.0001 versus the control group)

Fig. 6

Effect of TFDM on the viability of OGD/R-stimulated H9c2 cells. (n = 3, ^##P < 0.01 versus the control group, *P < 0.05 versus the model group, **P < 0.01 versus the model group)

Fig. 7

Effect of TFDM on the changes in LDH (A) and MDA (B) levels in OGD/R-stimulated H9c2 cells. A n = 3, ^##P < 0.01 versus the control group, *P < 0.05 versus the model group. B n = 3, ^###P < 0.001 versus the control group, ***P < 0.001 versus the model group, ****P < 0.0001 versus the model group)

Fig. 8

Effect of TFDM on the inhibition of oxidative stress in H9c2 cells exposed to OGD/R. A The activity of SOD. n = 3, ^####P < 0.0001 versus the control group, **P < 0.01 versus the model group, ****P < 0.0001 versus the model group. B The levels of ROS. n = 3, ^####P < 0.0001 versus the control group, ***P < 0.001 versus the model group, ****P < 0.0001 versus the model group

Fig. 9

TFDM pretreatment inhibited apoptosis in H9c2 cells after OGD/R stimulation. A Western blot analysis of the expression levels and quantification of Bax. n = 3, ^##P < 0.01 versus the control group, **P < 0.01 versus the model group, *P < 0.05 versus the model group. B Expression levels and quantification analysis of Bcl-2 were measured by western blotting. n = 3, ^##P < 0.01 versus the control group, **P < 0.01 versus the model group, *P < 0.05 versus the model group

Fig. 10

Effects of TFDM on the expression of NOX-4, PGC-1α, p-P38 MAPK/P38 MAPK and p-Erk1/2/Erk1/2. A Western blotting was performed to detect the expression levels of NOX-4. n = 3, ^##P < 0.01 versus the control group, **P < 0.01 versus the model group, *P < 0.05 versus the model group. B Western blotting was performed to detect the expression levels of PGC-1α. n = 3, ^####P < 0.0001 versus the control group, ****P < 0.0001 versus the model group, *P < 0.05 versus the model group. C Western blotting was performed to detect the expression levels of p-P38 MAPK and P38 MAPK. n = 3, ^##P < 0.01 versus the control group, **P < 0.01 versus the model group, *P < 0.05 versus the model group. D Western blotting was performed to detect the expression levels of p-Erk1/2 and Erk1/2. n = 3, ^#P < 0.05 versus the control group, ***P < 0.001 versus the model group, **P < 0.01 versus the model group