Yuxin Zhang

Cycloastragenol, a triterpene aglycone derived from Radix astragali, suppresses the accumulation of cytoplasmic lipid droplet in 3T3-L1 adipocytes

Cycloastragenol (CAG), a bioactive triterpenoid sapogenin isolated from the Chinese herbal medicine Radix astragali, was reported to promote the phosphorylation of extracellular signal-regulated protein kinase (ERK). Here we investigated the effect of CAG on adipogenesis. The image-based Nile red staining analyses revealed that CAG dose dependently reduced cytoplasmic lipid droplet in 3T3-L1 adipocytes with the IC50 value of 13.0 μM. Meanwhile, cytotoxicity assay provided evidence that CAG was free of injury on HepG2 cells up to 60 μM. In addition, using calcium mobilization assay, we observed that CAG stimulated calcium influx in 3T3-L1 preadipocytes with a dose dependent trend, the EC50 value was determined as 21.9 μM. There were proofs that elevated intracellular calcium played a vital role in suppressing adipocyte differentiation. The current findings demonstrated that CAG was a potential therapeutic candidate for alleviating obesity and hyperlipidemia.

Identification of Natural Compound Carnosol as a Novel TRPA1 Receptor Agonist

The transient receptor potential ankyrin 1 (TRPA1) cation channel is one of the well-known targets for pain therapy. Herbal medicine is a rich source for new drugs and potentially useful therapeutic agents. To discover novel natural TRPA1 agonists, compounds isolated from Chinese herbs were screened using a cell-based calcium mobilization assay. Out of the 158 natural compounds derived from traditional Chinese herbal medicines, carnosol was identified as a novel agonist of TRPA1 with an EC50 value of 12.46 µM. And the agonistic effect of carnosol on TRPA1 could be blocked by A-967079, a selective TRPA1 antagonist. Furthermore, the specificity of carnosol was verified as it showed no significant effects on two other typical targets of TRP family member: TRPM8 and TRPV3. Carnosol exhibited anti-inflammatory and anti-nociceptive properties; the activation of TRPA1 might be responsible for the modulation of inflammatory nociceptive transmission. Collectively, our findings indicate that carnosol is a new anti-nociceptive agent targeting TRPA1 that can be used to explore further biological role in pain therapy.

Identification of berberine as a direct thrombin inhibitor from traditional Chinese medicine through structural, functional and binding studies

Thrombin acts as a key enzyme in the blood coagulation cascade and represents a potential drug target for the treatment of several cardiovascular diseases. The aim of this study was to identify small-molecule direct thrombin inhibitors from herbs used in traditional Chinese medicine (TCM). A pharmacophore model and molecular docking were utilized to virtually screen a library of chemicals contained in compositions of traditional Chinese herbs, and these analyses were followed by in vitro bioassay validation and binding studies. Berberine (BBR) was first confirmed as a thrombin inhibitor using an enzymatic assay. The BBR IC50 value for thrombin inhibition was 2.92 μM. Direct binding studies using surface plasmon resonance demonstrated that BBR directly interacted with thrombin with a KD value of 16.39 μM. Competitive binding assay indicated that BBR could bind to the same argartroban/thrombin interaction site. A platelet aggregation assay demonstrated that BBR had the ability to inhibit thrombin-induced platelet aggregation in washed platelets samples. This study proved that BBR is a direct thrombin inhibitor that has activity in inhibiting thrombin-induced platelet aggregation. BBR may be a potential candidate for the development of safe and effective thrombin-inhibiting drugs.

Identification of a specific agonist of human TAS2R14 from Radix Bupleuri through virtual screening, functional evaluation and binding studies

Bitter taste receptors (TAS2Rs) have attracted a great deal of interest because of their recently described bronchodilator and anti-inflammatory properties. The aim of this study was to identify natural direct TAS2R14 agonists from Radix Bupleuri that can inhibit mast cell degranulation. A ligand-based virtual screening was conducted on a library of chemicals contained in compositions of Radix Bupleuri, and these analyses were followed by cell-based functional validation through a HEK293-TAS2R14-G16gust44 cell line and IgE-induced mast cell degranulation assays, respectively. Saikosaponin b (SSb) was confirmed for the first time to be a specific agonist of TAS2R14 and had an EC50 value of 4.9 μM. A molecular docking study showed that SSb could directly bind to a TAS2R14 model through H-bond interactions with Arg160, Ser170 and Glu259. Moreover, SSb showed the ability to inhibit IgE-induced mast cell degranulation, as measured with a β-hexosaminidase release model and real-time cell analysis (RTCA). In a cytotoxicity bioassay, SSb showed no significant cytotoxicity to HEK293 cells within 24 hours. This study demonstrated that SSb is a direct TAS2R14 agonist that inhibit IgE-induced mast cell degranulation. Although the target and in vitro bioactivity of SSb were revealed in this study, it still need in vivo study to further verify the anti-asthma activity of SSb.

Salvianolic Acid A, as a Novel ETA Receptor Antagonist, Shows Inhibitory Effects on Tumor in Vitro

Endothelin-1 (ET-1) autocrine and paracrine signaling modulate cell proliferation of tumor cells by activating its receptors, endothelin A receptor (ETAR) and endothelin B receptor (ETBR). Dysregulation of ETAR activation promotes tumor development and progression. The potential of ETAR antagonists and the dual-ETAR and ETBR antagonists as therapeutic approaches are under preclinical and clinical studies. Salvianolic acid A (Sal A) is a hydrophilic polyphenolic derivative isolated from Salvia miltiorrhiza Bunge (Danshen), which has been reported as an anti-cancer and cardio-protective herbal medicine. In this study, we demonstrate that Sal A inhibits ETAR activation induced by ET-1 in both recombinant and endogenous ETAR expression cell lines. The IC50 values were determined as 5.7 µM in the HEK293/ETAR cell line and 3.14 µM in HeLa cells, respectively. Furthermore, our results showed that Sal A suppressed cell proliferation and extended the doubling times of multiple cancer cells, including HeLa, DU145, H1975, and A549 cell lines. In addition, Sal A inhibited proliferation of DU145 cell lines stimulated by exogenous ET-1 treatment. Moreover, the cytotoxicity and cardio-toxicity of Sal A were assessed in human umbilical vein endothelial cells (HUVEC) and Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which proved that Sal A demonstrates no cytotoxicity or cardiotoxicity. Collectively, our findings indicate that Sal A is a novel anti-cancer candidate through targeting ETAR.

Content decline of SERCA inhibitors saikosaponin a and d attenuates cardiotoxicity and hepatotoxicity of vinegar-baked Radix bupleuri

Improper usage of unprocessed Radix bupleuri root (chaihu) may cause cardiotoxicity and liver injury. Baking herb with vinegar is believed to attenuate the adverse responses. However, the chemical and molecular basis involved remained unclear. To this end, we investigated the in vitro toxicity of saikosaponin a, c, d, and their hydrolysates saikosaponin b1 and b2. Results showed that SSa and SSd possessed higher affinity with sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) by molecular docking, and exhibited stronger toxic responses on cardiomyocytes and hepatocytes than the other three saikosaponins in equivalent concentrations. Further, SSa and SSd induced LC3 puncta formation in U2OS-mCherry-EGFP-LC3 cells. Blockage of autophagy by 3-methyladenine did not abrogate the cytotoxicities induced by SSa and SSd. In parallel, none of SSc, SSb1, or SSb2 caused cell injury. Our study reveals how changes in chemical ingredients are connected to the toxicity of Chaihu during vinegar baking process and also provides a guidance for structure optimization to reduce drug induced toxicity.

Cardamonin, a Novel Antagonist of hTRPA1 Cation Channel, Reveals Therapeutic Mechanism of Pathological Pain

The increasing demand for safe and effective treatments of chronic pain has promoted the investigation of novel analgesic drugs. Some herbals have been known to be able to relieve pain, while the chemical basis and target involved in this process remained to be clarified. The current study aimed to find anti-nociceptive candidates targeting transient receptor potential ankyrin 1 (TRPA1), a receptor that implicates in hyperalgesia and neurogenic inflammation. In the current study, 156 chemicals were tested for blocking HEK293/TRPA1 ion channel by calcium-influx assay. Docking study was conducted to predict the binding modes of hit compound with TRPA1 using Discovery Studio. Cytotoxicity in HEK293 was conducted by Cell Titer-Glo assay. Additionally, cardiotoxicity was assessed via xCELLigence RTCA system. We uncovered that cardamonin selectively blocked TRPA1 activation while did not interact with TRPV1 nor TRPV4 channel. A concentration-dependent inhibitory effect was observed with IC50 of 454 nM. Docking analysis of cardamonin demonstrated a compatible interaction with A-967079-binding site of TRPA1. Meanwhile, cardamonin did not significantly reduce HEK293 cell viability, nor did it impair cardiomyocyte constriction. Our data suggest that cardamonin is a selective TRPA1 antagonist, providing novel insight into the target of its anti-nociceptive activity.

Discovery of TAS2R14 Agonists from Platycodon grandiflorum Using Virtual Screening and Affinity Screening Based on a Novel TAS2R14-Functionalized HEMT Sensor Combined with UPLC–MS Analysis

TAS2R14 is of great potential as a therapeutic target against asthma, and the discovery of TAS2R14 agonists can be very valuable for treating this disease. Herein, we developed a strategy using virtual screening and affinity screening based on a fabricated biosensor combined with UPLC-MS analysis to screen TAS2R14 agonists from Platycodon grandiflorum. By ligand-based virtual screening, 16 best-fit candidates were yielded. A novel TAS2R14-functionalized high-electron-mobility transistor (HEMT) sensor was applied to detect and fish out the potential TAS2R14 agonists from P. grandiflorum extracts. Those components captured by the immobilized TAS2R14 were eluted and characterized on UPLC-QTOF MS. As a result, six potential TAS2R14 agonists were screened out and identified. Among them, platycodin L was confirmed to be a special agonist of TAS2R14 for the first time and had an EC50 of 15.03 ± 1.15 μM via intracellular calcium mobilization assay ( n = 6). The results indicated that the proposed strategy was efficient to discover TAS2R14 agonists from the herb directly.

DanHong injection targets endothelin receptor type B and angiotensin II receptor type 1 in protection against cardiac hypertrophy

Cardiac hypertrophy (CH) is an independent risk factor for cardiovascular diseases (CVDs). Mitigating or preventing CH is the most effective strategy for the treatment of CVDs. DanHong injection (DH) is a Chinese herbal medicine preparation (CHMP) widely used in clinical treatment of several CVDs in China. However, the direct targets and cellular mechanisms for these protective effects remain unclear. This study was designed to illustrate the direct targets of DH in protecting against CH and investigate CH molecular pathogenesis. A hypertrophic cell model was induced by endothelin-1 (ET-1) on human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). Real time cellular analysis (RTCA) cardio system and high content analysis (HCA) were used to detect the changes in contractile function, morphology and protein level of hypertrophic hiPS-CMs. Agonist and antagonist assay on receptors were performed using calcium mobilization high-throughput screening (HTS). DH significantly attenuated CH by modulating myocardial contractility, suppressing cell area enlargement and down-regulating ET-1-induced brain natriuretic peptide (BNP), actinin alpha 2 (ACTN2) and cardiac muscle troponin T (TNNT2) protein expression (P < 0.05). Endothelin receptor type B (ETBR) and angiotensin II receptor type 1 (AT1R) were DH direct targets, with IC50 value of 25.67 μL/mL and 1.10 μL/mL, respectively. Proteomics analysis showed that proteins involved in cell cycle inhibition, RNA processing, mitochondrial translation and cytoskeleton are significant regulated by DH treatment. These data revealed that ETBR and AT1R are DH direct targets on protecting against CH, providing a strategy to explore direct targets of CHMPs.