Tanshinone I Mitigates Steroid-Induced Osteonecrosis of the Femoral Head and Activates the Nrf2 Signaling Pathway in Rats

Steroid-induced osteonecrosis of the femoral head (SIONFH) is a frequent orthopedic disease caused by long-term or high-dose administration of corticosteroids. Tanshinone I (TsI), a flavonoid compound isolated from Salvia miltiorrhiza Bunge, has been reported to inhibit osteoclastic differentiation in vitro. This study aimed to investigate whether TsI can ameliorate SIONFH. Herein, SIONFH was induced by intraperitoneal injection of 20 μg/kg lipopolysaccharide every 24 h for 2 days, followed by an intramuscular injection of 40 mg/kg methylprednisolone every 24 h for 3 days. Four weeks after the final injection of methylprednisolone, the rats were intraperitoneally administrated with low-dose (5 mg/kg) and high-dose (10 mg/kg) TsI once daily for 4 weeks. Results showed that TsI significantly alleviated osteonecrotic lesions of the femoral heads as determined by micro-CT analysis. Furthermore, TsI increased alkaline phosphatase activity and expressions of osteoblastic markers including osteocalcin, type I collagen, osteopontin, and Runt-related transcription factor 2 and decreased tartrate-resistant acid phosphatase activity and expressions of osteoclastic markers including cathepsin K and acid phosphatase 5. TsI also reduced inflammatory response and oxidative stress and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in the femoral heads. Taken together, our findings show that TsI can relieve SIONFH, indicating that it may be a candidate for preventing SIONFH.

Screening of Potential Thrombin and Factor Xa Inhibitors from the Danshen–Chuanxiong Herbal Pair through a Spectrum–Effect Relationship Analysis

In this study; a spectrum–effect relationship analysis combined with a high-performance liquid chromatography–mass spectrometry (LC–MS) analysis was established to screen and identify active components that can inhibit thrombin and factor Xa (THR and FXa) in Salviae Miltiorrhizae Radix et Rhizoma–Chuanxiong Rhizoma (Danshen–Chuanxiong) herbal pair. Ten potential active compounds were predicted through a canonical correlation analysis (CCA), and eight of them were tentatively identified through an LC–MS analysis. Furthermore; the enzyme inhibitory activity of six available compounds; chlorogenic acid; Z-ligustilide; caffeic acid; ferulic acid; tanshinone I and tanshinone IIA; were tested to verify the feasibility of the method. Among them; chlorogenic acid was validated to possess a good THR inhibitory activity with IC50 of 185.08 µM. Tanshinone I and tanshinone IIA are potential FXa inhibitors with IC50 of 112.59 µM and 138.19 µM; respectively. Meanwhile; molecular docking results show that tanshinone I and tanshinone IIA; which both have binding energies of less than −7.0 kcal·mol−1; can interact with FXa by forming H-bonds with residues of SER214; GLY219 and GLN192. In short; the THR and FXa inhibitors in the Danshen–Chuanxiong herbal pair have been successfully characterized through a spectrum–effect relationship analysis and an LC–MS analysis.

Monoamine Oxidase Inhibition by Major Tanshinones from Salvia miltiorrhiza and Selective Muscarinic Acetylcholine M4 Receptor Antagonism by Tanshinone I

Monoamine oxidases (MAOs) and muscarinic acetylcholine receptors (mAChRs) are considered important therapeutic targets for Parkinson’s disease (PD). Lipophilic tanshinones are major phytoconstituents in the dried roots of Salvia miltiorrhiza that have demonstrated neuroprotective effects against dopaminergic neurotoxins and the inhibition of MAO-A. Since MAO-B inhibition is considered an effective therapeutic strategy for PD, we tested the inhibitory activities of three abundant tanshinone congeners against recombinant human MAO (hMAO) isoenzymes through in vitro experiments. In our study, tanshinone I (1) exhibited the highest potency against hMAO-A, followed by tanshinone IIA and cryptotanshinone, with an IC50 less than 10 µM. They also suppressed hMAO-B activity, with an IC50 below 25 µM. Although tanshinones are known to inhibit hMAO-A, their enzyme inhibition mechanism and binding sites have yet to be investigated. Enzyme kinetics and molecular docking studies have revealed the mode of inhibition and interactions of tanshinones during enzyme inhibition. Proteochemometric modeling predicted mAChRs as possible pharmacological targets of 1, and in vitro functional assays confirmed the selective M4 antagonist nature of 1 (56.1% ± 2.40% inhibition of control agonist response at 100 µM). These findings indicate that 1 is a potential therapeutic molecule for managing the motor dysfunction and depression associated with PD.

Tanshinone I Inhibits Oxidative Stress–Induced Cardiomyocyte Injury by Modulating Nrf2 Signaling

Cardiovascular disease, a disease caused by many pathogenic factors, is one of the most common causes of death worldwide, and oxidative stress plays a major role in its pathophysiology. Tanshinone I (Tan I), a natural compound with cardiovascular protective effects, is one of the main active compounds extracted from Salvia miltiorrhiza. Here, we investigated whether Tan I could attenuate oxidative stress and oxidative stress–induced cardiomyocyte apoptosis through Nrf2/MAPK signaling in vivo and in vitro. We found that Tan I treatment protected cardiomyocytes against oxidative stress and oxidative stress–induced apoptosis, based on the detection of relevant oxidation indexes such as reactive oxygen species, superoxide dismutase, malondialdehyde, and apoptosis, including cell viability and apoptosis-related protein expression. We further examined the mechanisms underlying these effects, determining that Tan I activated nuclear factor erythroid 2 (NFE2)–related factor 2 (Nrf2) transcription into the nucleus and dose-dependently promoted the expression of Nrf2, while inhibiting MAPK signaling activation, including P38 MAPK, SAPK/JNK, and ERK1/2. Nrf2 inhibitors in H9C2 cells and Nrf2 knockout mice demonstrated aggravated oxidative stress and oxidative stress–induced cardiomyocyte injury; Tan I treatment suppressed these effects in H9C2 cells; however, its protective effect was inhibited in Nrf2 knockout mice. Additionally, the analysis of surface plasmon resonance demonstrated that Tan I could directly target Nrf2 and act as a potential Nrf2 agonist. Collectively, these data strongly indicated that Tan I might inhibit oxidative stress and oxidative stress–induced cardiomyocyte injury through modulation of Nrf2 signaling, thus supporting the potential therapeutic application of Tan I for oxidative stress–induced CVDs.

Detection-confirmation-standardization-quantification: a novel method for the quantitative analysis of active components in traditional Chinese medicines

Background Quantitative analysis of the active ingredients of Traditional Chinese Medicine is a research tendency. The objective of this study was to build a novel method, namely, Detection-confirmation-standardization-quantification (DCSQ), for the quantitative analysis of active components in traditional Chinese medicines, without individual reference standard. Methods Danshen (the dried root of Radix Salvia miltiorrhiza) was used as the matrix. The “extraction” function in high-performance liquid chromatography-mass (HPLC-MS) instrument was used to find the peaks corresponding to cryptotanshinone, tanshinone I, and tanshinone IIA in the total ion current (TIC) chromatogram of Danshen. The multicomponent reference standard (MCRS) containing the three tanshinones mainly was prepared by preparative HPLC. The contents of them in the resulting MCRS were determined by NMR; moreover, the constituents of the MCRS were confirmed. The MCRS containing known content of the three tanshinones was used as the reference standard for the quantitative analysis of cryptotanshinone, tanshinone I and tanshinone IIA in Danshen Samples by analytical HPLC. Results The optimized HPLC conditions for the quantitative analysis of the active components in Danshen were established, and the assignments of the extracted peaks were confirmed by analyzing the characteristic fragments in their MS/MS product ion spectra and UV spectra. Then, the MCRS containing the three tanshinones were successfully prepared. The results of determination about the contents by NMR showed linearity fitted with high likelihood and calibration curves possessed high linearity. The results of determination on Danshen Samples obtained through DCSQ exhibited minimal deviations, in contrast to those obtained through individual reference standards. Conclusion The establishing DCSQ is independent and convenient for the quantitative analysis of the active components in TCMs by MCRS, without individual reference standard. This method is a great advance in quantitative analysis for complex composition, especially TCMs.

Monoamine Oxidase Inhibition by Major Tanshinones From Salvia Miltiorrhiza and Selective Muscarinic Acetylcholine M4 Receptor Antagonism by Tanshinone I

Monoamine oxidases (MAOs) and muscarinic acetylcholine receptors (mAChRs) are considered important therapeutic targets for Parkinson's disease (PD). Lipophilic tanshinones are major phytoconstituents in dried roots of Salvia miltiorrhiza that have demonstrated neuroprotective anti-Parkinson effects against dopaminergic neurotoxins and the inhibition of human monoamine oxidase (hMAO)-A. Since MAO-B inhibition is also considered an important therapeutic approach for PD, we accessed the potential of three abundant tanshinone congeners in hMAO-A and hMAO-B inhibition in vitro. In our study, tanshinone I (1) exhibited the highest potency against hMAO-A, followed by tanshinone IIA and cryptotanshinone with IC50 values less than 10 μM. They also suppressed hMAO-B activity with IC50 values lower than 25 μM. Although the hMAO-A inhibitory activity of tanshinones has been reported, the mechanism of enzyme inhibition and binding sites are yet to be studied. Thus, we conducted enzyme kinetics and molecular docking studies to evaluate the mode of enzyme inhibition and interactions. Proteochemometric modeling further predicted mAChRs as potential targets of 1 and an in vitro functional G-protein coupled receptor assay confirmed the selective M4 antagonist nature of 1. These findings indicate that 1 is a potential therapeutic molecule for managing the motor dysfunction and depression associated with PD.