Background: Tanshinone I (TI) is a primary component of Salvia miltiorrhiza Bunge (Danshen), which confers a favorable role in a variety of pharmacological activities including cardiovascular protection. However, the exact mechanism of the cardiovascular protection activity of TI remains to be illustrated. In this study, the cardiovascular protective effect and its mechanism of TI were investigated. Methods: In this study, tert-butyl hydroperoxide (t-BHP)-stimulated H9c2 cells model was employed to investigate the protective effect in vitro. The cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) kit. The reactive-oxygen-species (ROS) level and mitochondrial membrane potential (MMP) were investigated by the flow cytometry and JC-1 assay, respectively. While in vivo experiment, the cardiovascular protective effect of TI was determined by using myocardial ischemia–reperfusion (MI/R) model including hematoxylin–eosin (H&E) staining assay and determination of superoxide dismutase (SOD) and malondialdehyde (MDA). Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release were detected by Enzyme-linked immunosorbent assay (ELISA). Receptor interacting protein kinase 1 (RIP1), receptor interacting protein kinase 3 (RIP3), receptor interacting protein kinase 3 (MLKL), protein kinase B (Akt), Nuclear factor erythroid 2 related factor 2 (Nrf2), Heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO-1) were determined by western blotting. Results: Our data demonstrated that TI pretreatment attenuated t-BHP and MI/R injury-induced necroptosis by inhibiting the expression of p-RIP1, p-RIP3, and p-MLKL. TI activated the Akt/Nrf2 pathway to promote the expression of antioxidant-related proteins such as phosphorylation of Akt, nuclear factor erythroid 2 related factor 2 (Nrf2), quinone oxidoreductase-1 (NQO-1) and heme oxygenase-1 (HO-1) expression in t-BHP-stimulated H9c2 cells. TI relieved oxidative stress by mitigating ROS generation and reversing MMP loss. In vivo experiment, TI made electrocardiograph (ECG) recovery better and lessened the degree of myocardial tissue damage. The counts of white blood cell (WBC), neutrophil (Neu), lymphocyte (Lym), and the release of TNF-α and IL-6 were reversed by TI treatment. SOD level was increased, while MDA level was decreased by TI treatment. Conclusion: Collectively, our findings indicated that TI exerted cardiovascular protective activities in vitro and in vivo through suppressing RIP1/RIP3/MLKL and activating Akt/Nrf2 signaling pathways, which could be developed into a cardiovascular protective agent.

Background: Pneumonia refers to the inflammation of the terminal airway, alveoli and pulmonary interstitium, which can be caused by pathogenic microorganisms, physical and chemical factors, immune damage, and drugs. Anemoside B4, the major ingredient ofPulsatilla chinensis(Bunge) Regel, exhibited anti-inflammatory activity. However, the therapeutic effect of anemoside B4 on pneumonia has not been unraveled. This study aims to investigate that anemoside B4 attenuates the inflammatory responses inKlebsiella pneumonia(KP)- and influenza virus FM1 (FM1)-induced pneumonia mice model.Methods: The network pharmacology and molecular docking assays were employed to predict the targets of anemoside B4’s treatment of pneumonia. Two models (bacterial KP-infected mice and virus FM1-infected mice) were employed in our study. BALB/c mice were divided into six groups: control, model group (KP-induced pneumonia or FM1-induced pneumonia), anemoside B4 (B4)-treated group (2.5, 5, 10 mg/kg), and positive drug group (ribavirin or ceftriaxone sodium injection). Blood samples were collected for hematology analysis. The effects of B4 on inflammation-associated mediators were investigated by Enzyme-linked immunosorbent assay (ELISA) and hematoxylin and eosin staining (HE) staining. Proteins expression was quantified by western blotting.Results: The network results indicated that many pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) participated in anemoside B4’s anti-inflammatory activity. The counts of neutrophil (NEU) and white blood cell (WBC), the level of myeloperoxidase (MPO), and the release of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 increased by KP or FM1 infection, which were reversed by anemoside B4. In addition, anemoside B4 significantly suppressed the FM1-induced expression of toll-like receptor 4 (TLR4), myeloid differential protein-88 (MyD88), and myeloid differentiation protein-2 (MD-2), which were further validated by molecular docking data that anemoside B4 bound to bioactive sites of TLR4. Therefore, anemoside B4 exhibited a significant therapeutic effect on pneumonia via the TLR4/MyD88 pathway.Conclusion: Our findings demonstrated that anemoside B4 attenuates pneumonia via the TLR4/Myd88 signaling pathway, suggesting that anemoside B4 is a promising therapeutic candidate for bacterial-infected or viral-infected pneumonia.

Three new unsaturated fatty acids, (Z)-9,10,11-trihydroxy-12-octadecenoic acid (1), (Z)-7,8,9-trihydroxy-10-hexadecenoic acid (2) and (Z)-12-keto-7,8,9-trihydroxy-10-hexadecenoic acid (3) were isolated from the roots of Boehmeria nivea (L.) Gaudich, along with a known unsaturated fatty acid, (E)-8,11,12-trihydroxy-9-octadecenoic acid (4). The structures of the new compounds were established by HR ESI-MS, ¹H, ¹³C, 2D (¹H–¹H COSY, HSQC, HMBC) NMR experiments. The known compound was identified by a comparison of its spectral data with published references. The three new compounds showed some antifungal activities by agar assay.

Background: Myocardial ischemia/reperfusion injury (MIRI) is an important complication of reperfusion therapy, and has a lack of effective prevention and treatment methods. Oleuropein (OP) is a natural strong antioxidant with many protective effects on cardiovascular diseases, but its protective effect on MIRI has not yet been studied in depth. Methods: Tert-Butyl hydroperoxide (tBHP) was used to establish an in vitro oxidative stress model. Cell viability was detected by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and lactate dehydrogenase (LDH). Flow cytometry and fluorescence assays were performed for evaluating the ROS levels and mitochondrial membrane potential (MMP). Immunofluorescence analysis detected the NRF2 nuclear translocation and autophagy indicators. Further, Western blotting and quantitative real-time PCR were performed to evaluate the expression levels of proteins and mRNAs. Molecular docking, CETSA, and molecular interaction analysis explored the binding between OP and TLR4. The protective effects of OP in vivo were determined using a preclinical MIRI rat model. Results: OP protected against tBHP-treated injury, reduced ROS levels and reversed the damaged MMP. Mechanistically, OP activated NRF2-related antioxidant pathways, inhibited autophagy and attenuated the TLR4/MAPK signaling pathway in tBHP-treated H9C2 cells with a high binding affinity to TLR4 (K_D = 37.5 µM). The TLR4 inhibitor TAK242 showed a similar effect as OP. In vivo, OP could alleviate cardiac ischemia/reperfusion injury and it ameliorated adverse cardiac remodeling. Consistent with in vitro studies, OP inhibited TLR4/MAPK and autophagy pathway and activated NRF2-dependent antioxidant pathways in vivo. Conclusion: This study shows that OP binds to TLR4 to regulate oxidative stress and autophagy for protecting damaged cardiomyocytes, supporting that OP can be a potential therapeutic agent for MIRI.

Inflammation is a common pathogenesis in many diseases. Salvia miltiorrhiza Bunge (Danshen), a traditional Chinese medicine, has been considered to have good anti-inflammatory effects. In the present study, we investigated the anti-inflammatory effect of diethyl blechnic (DB), a novel compound isolated from Danshen, and its possible mechanisms in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. The results showed that DB can inhibit the LPS-induced pro-inflammatory cytokines release of prostaglandin E2 (PGE2) and mRNA expression of TNF-α, IL-6, and IL-1β. In addition, the results of the flow cytometry assay and the fluorometric intracellular ROS kit assay indicated that DB reduced the generation of ROS in LPS-stimualted RAW264.7 cells. DB reversed the LPS-induced loss of the mitochondrial membrane potential (MMP). Furthermore, DB suppressed the LPS-stimulated increased expression of Toll-like receptor 4 (TLR4), myeloid differential protein-88 (MyD88) and phosphorylation of TAK1, PI3K, and AKT. DB promoted NF-E2-related factor 2 (Nrf2) into the nucleus, increased the expression of heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1) and reduced the expression of Keap1. In summary, DB may inhibit LPS-induced inflammation, which mainly occurs through TLR4/MyD88 and oxidative stress signaling pathways in RAW264.7 cells.