Loureirin B Exerts its Immunosuppressive Effects by Inhibiting STIM1/Orai1 and KV1.3 Channels

Loureirin B (LrB) is a constituent extracted from traditional Chinese medicine Resina Draconis. It has broad biological functions and an impressive immunosuppressive effect that has been supported by numerous studies. However, the molecular mechanisms underlying Loureirin B-induced immune suppression are not fully understood. We previously reported that Loureirin B inhibited KV1.3 channel, calcium ion (Ca2+) influx, and interleukin-2 (IL-2) secretion in Jurkat T cells. In this study, we applied CRISPR/Cas9 to edit KV1.3 coding gene KCNA3 and successfully generated a KV1.3 knockout (KO) cell model to determine whether KV1.3 KO was sufficient to block the Loureirin B-induced immunosuppressive effect. Surprisingly, we showed that Loureirin B could still inhibit Ca2+ influx and IL-2 secretion in the Jurkat T cells in the absence of KV1.3 although KO KV1.3 reduced about 50% of Ca2+ influx and 90% IL-2 secretion compared with that in the wild type cells. Further experiments showed that Loureirin B directly inhibited STIM1/Orai1 channel in a dose-dependent manner. Our results suggest that Loureirin B inhibits Ca2+ influx and IL-2 secretion in Jurkat T cells by inhibiting both KV1.3 and STIM1/Orai1 channels. These studies also revealed an additional molecular target for Loureirin B-induced immunosuppressive effect, which makes it a promising leading compound for treating autoimmune diseases.

Resina Draconis Reduces Acute Liver Injury and Promotes Liver Regeneration after 2/3 Partial Hepatectomy in Mice

Aim. To investigate the protective effects and possible mechanisms of action of resina draconis (RD) on acute liver injury and liver regeneration after 2/3 partial hepatectomy (PH) in mice. Methods. 2/3 PH was used to induce acute liver injury. Mice were divided into three groups: sham, vehicle + 2/3 PH, and RD + 2/3 PH. Resina draconis was administered intragastrically after 2/3 PH into the RD + 2/3 PH group, and the same volume of vehicle (1% sodium carboxymethyl cellulose) was injected into the vehicle + 2/3 PH group and sham group mice. The index of liver to body weight (ILBW) and proliferating cell nuclear antigen (PCNA) were assayed to evaluate liver regeneration. Blood and liver tissues were collected for serological and western blotting analysis. Results. Resina draconis protected against 2/3 PH-induced acute severe liver injury and promoted liver regeneration as shown by significantly increased ILBW compared with that of controls. 2/3 PH increased serum AST and ALT levels, which were significantly decreased by RD treatment, while 2/3 PH decreased serum TP and ALB, which were increased by RD treatment. In the RD + 2/3 PH group, PCNA expression was significantly increased compared with the 2/3 PH group. Further, hepatocyte growth factor (HGF), TNFα, and EGFR levels were increased in the RD group at postoperative days 2 and 4 compared with the those in the 2/3 PH group. Conclusion. Our results suggest that RD ameliorates acute hepatic injury and promotes liver cell proliferation, liver weight restoration, and liver function after 2/3 PH, probably via HGF, TNFα, and EGFR signaling.

Rapid Evaluation of Chemical Consistency of Artificially Induced and Natural Resina Draconis Using Ultra-Performance Liquid Chromatography Quadrupole-Time-of-Flight Mass Spectrometry-Based Chemical Profiling

Resina Draconis is a highly valued traditional medicine widely used in Arabia since ancient times, and it has been commonly used as an antidiarrheic, antimicrobial, antiulcer, blood circulation promoter as well as an anti-inflammatory agent. The tree source from which this medicine orignates grows extremely slowly, producing a very low yield of Resina Draconis. To meet the increasing market demand, artificial methods for stimulating Resina Draconis formation have been developed and applied. However, the chemical differences between artificially induced Resina Draconis (AIRD) and natural Resina Draconis (NRD) have been rarely studied. The aim of this research was to explore and identify the chemical constituents of AIRD and NRD using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS) based chemical profiling. A total of 56 chromatographic peaks were detected in AIRD, of these, 44 peaks have had their structures tentatively characterized based on high-resolution mass spectra (HRMS) data, fragmentation ions information, reference standards data and literature review. In total, 40 peaks were found both in AIRD and NRD. The potential chemical transformation mechanisms active in Resina Draconis during formation were explored. To the best of our knowledge, this is the first evaluation of the chemical profiles of both AIRD and NRD. Furthermore, these findings are expected to provide a rational basis for the quality assessment of AIRD and the use of AIRD as a substitute for NRD.

Bioactivity-Guided Fractionation of the Traditional Chinese Medicine Resina Draconis Reveals Loureirin B as a PAI-1 Inhibitor

Thrombotic diseases have become a global burden due to morbidity, mortality, and disability. Traditional Chinese medicine has been proven effective in removing blood stasis and promoting blood circulation, but the exact mechanisms remain unclear. Plasminogen activator inhibitor-1 (PAI-1) is a natural inhibitor of tissue-type and urokinase-type plasminogen activators. In this study, we screened four fractions of Resina Draconis (a traditional Chinese medicine) extract for PAI-1 inhibitory activity. Bioactivity-guided purification and chromogenic substrate-based assay led to the identification of loureirin B as the major PAI-1 inhibitor, with an IC50value of 26.10 μM. SDS-PAGE analysis showed that formation of the PAI-1/uPA complex was inhibited by loureirin B, and the inhibitory effect of loureirin B on PAI-1 was also confirmed by clot lysis assay. In vivo studies showed that loureirin B significantly prolonged the tail bleeding time and reduced the weight and size of arterial thrombus, reduced hydroxyproline level, and partly cured liver fibrosis in mice. Taken together, the results revealed loureirin B as a PAI-1 inhibitor, adding a new pharmacological target for loureirin B and uncovering a novel mechanism underlying the antithrombotic property of Resina Draconis, which might be useful in the treatment of cardiovascular diseases such as thrombosis and fibrosis.