Aconiti Kusnezoffii Radix (Caowu) is often combined or processed with Chebulae Fructus (Hezi) to achieve attenuation purposes in Mongolian medicine. Mesaconitine (MA), a main bioactive ingredient of Caowu, is also famous for its high cardiotoxicity while exerting good anti-inflammatory and analgesic properties. Gallic acid (GA), one of the leading chemical components in Hezi, possesses cardiac protection. This study aimed to clarify the detoxification effects of GA from Hezi on MA-induced cardiotoxicity and whether the detoxification mechanism is related to the TRPV1 channel. Cell viability was determined by methyl thiazol tetrazolium (MTT), and lactate dehydrogenase (LDH) leakage rate was determined by ELISA. Hoechst 33258, JC-1, DCFH-DA, and Fluo-3 AM staining were conducted to detect apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS), and Ca2+ respectively; TRPV1 channel current was recorded by whole-cell patch-clamp technology to observe the effect of GA and MA alone or in combination on TRPV1 channel. The results showed that GA exhibited pronounced detoxification effects on MA-induced cardiotoxicity. GA significantly inhibited the MA-induced decrease in cell viability; suppressed the MA-induced LDH leakage rate, apoptosis, and the release of ROS and Ca2+; and alleviated the reduction of mitochondrial membrane potential. We found that MA-induced cardiotoxicity was significantly attenuated in H9c2 cells pretreated with the TRPV1 antagonist BCTC. In the whole-cell patch-clamp experiment, the TRPV1 channel current increase was caused by the GA and MA treatment, whereas it was reduced by the cotreatment of GA and MA. Our data demonstrate that GA in Hezi can reduce MA-induced cardiotoxicity by inhibiting intracellular Ca2+ influx, restoring mitochondrial membrane potential, and reducing apoptosis. The detoxification mechanism may be related to the desensitization of the TRPV1 channel by the combined application of MA and GA.

The pathogenesis of diabetic retinopathy (DR) is complicated, and there is no effective drug. Oxidative stress-induced human retinal microvascular endothelial cells (HRMECs) injury is one of the pathogenic factors for DR. Molecular switches are considered high-risk targets in disease progression. Identification of molecular switch is crucial to interpret the pathogenesis of disease and screen effective ingredients. In this study, a systematic process was executed to discover therapeutic candidates for DR based on HRMECs injury. First of all, the molecular mechanism of HRMECs oxidative stress injury was revealed by transcriptomics and network pharmacology. We found that oxidative stress was one of the pivotal pathogenic factors, which interfered with vascular system development, inflammation, cell adhesion, and cytoskeleton damaged HRMECs through crosstalk. Then, network topology analysis was used to recognize molecular switches. The results indicated that the Keap1-Nrf2-ARE signaling pathway was the molecular switch in HRMECs oxidative stress injury. On this basis, the HEK293-ARE overexpression cell line was applied to obtain 18 active traditional Chinese medicine (TCM) ingredients. Furthermore, andrographolide, one of the 18 candidates, was applied in the HRMECs oxidative stress model to evaluate the accuracy of the systematic process. The efficacy evaluation results showed that andrographolide could regulate oxidative stress, vascular system development, inflammation, adhesion, and skeleton tissue to inhibit HRMECs injury cooperatively. And its mechanism was related to the Nrf2 signaling pathway. Overall, our data suggest that the Nrf2 signaling pathway is the molecular switch in the HRMECs oxidative stress injury. 18 potential Nrf2 agonists are likely to be promising DR candidates.