Zhilong Huoxue Tongyu Capsule regulates the macrophage polarization and inflammatory response via the let-7i/TLR9/MyD88 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhilong Huoxue Tongyu Capsule (ZL) is clinically prescribed for acute ischemic stroke (AIS). However, only a few studies have addressed the mechanisms of ZL in treating AIS. AIM OF THE STUDY: To explore the underlying mechanism of macrophage polarization and inflammation mediated by ZL, and to provide a reference for AIS treatment. MATERIALS AND METHODS: Sixteen SD rats were fed with different dose of ZL (0, 0.4, 0.8, and 1.6 g/kg/d) for 4 days to prepare ZL serum. After 500 ng/mL lipopolysaccharide (LPS) stimulation, RAW264.7 cells were administrated with ZL serum. Then, experiments including ELISA, flow cytometry, real-time quantitative PCR and Western blot were performed to verify the effects of ZL on macrophage polarization and inflammation. Next, let-7i inhibitor was transfected in RAW264.7 cells when treated with LPS and ZL serum to verify the regulation of ZL on the let-7i/TLR9/MyD88 signaling pathway. Moreover, the interaction between let-7i and TLR9 was confirmed by the dual-luciferase assay. RESULTS: ZL serum significantly decreased the expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), and increased the expression of IL-10 and transforming growth factor ß1 (TGF-ß1) of LPS stimulated-macrophages. Furthermore, ZL serum polarized macrophages toward M2, decreased the expressions of TLR9, MyD88, and iNOS, as well as increased the expressions of let-7i, CHIL3, and Arginase-1. It is worth mentioning that the effect of ZL serum is dose-dependent. However, let-7i inhibitor restored all the above effects in LPS stimulated-macrophages. In addition, TLR9 was the target of let-7i. CONCLUSIONS: ZL targeted let-7i to inhibit TLR9 expression, thereby inhibiting the activation of the TLR9/MyD88 pathway, promoting the M2 polarization, and inhibiting the development of inflammation in AIS.

Zhilong Huoxue Tongyu Capsule attenuates hemorrhagic transformation through the let-7f/TLR4 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hemorrhagic transformation after acute ischemic stroke is a life-threatening disease that currently has no effective chemotherapy. Zhilong Huoxue Tongyu Capsule (ZL) is an empirical prescription of traditional Chinese medicine that is used to prevent and treat cardiovascular and cerebrovascular diseases in China. However, only a few studies have addressed the mechanisms of ZL in treating hemorrhagic transformation. AIM OF THE STUDY: To evaluate the anti-inflammatory effects of ZL on hemorrhagic transformation model rats and lipopolysaccharide (LPS)-induced RAW264.7 macrophages and to explore the underlying molecular mechanisms. MATERIALS AND METHODS: Murine RAW264.7 cells were treated with ZL and LPS (1 µg/mL), and cell viability was detected by cell counting kit-8 assay. RT-qPCR was used to detect the expression of inflammatory chemokines, microRNA let-7a/e/i/f, toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa-B (NF-κB) p65. The protein expression levels of TLR4, MyD88, NF-κB p65, and apoptosis related molecules were determined by Western blotting. The apoptosis rate of RAW264.7 macrophages was detected by Annexin V-FITC/PI double staining. A hemorrhagic transformation model in rats was established by intraperitoneal injection of high glucose solution combined with thread embolization. Then, the model rats were observed behaviourally, pathologically, and molecularly. The gene expression of TLR4, MyD88, and NF-κB p65 was measured by RT-qPCR and used to evaluate the protective effect of ZL against hemorrhagic transformation in rats. RESULTS: ZL (5, 20, 40 µg/mL) was beneficial in cell proliferation. LPS (1 µg/mL) stimulated the production of inflammatory chemokines and inhibited the production of let-7a/e/i/f, with let-7f being influenced most strongly. Moreover, overexpression of let-7f decreased the gene and protein levels of TLR4, MyD88, and NF-κB p65, downregulated TLR4, and inhibited its transcriptional activity. ZL (5, 20, and 40 µg·mL-1) inhibited the production of TLR4, MyD88, and NF-κB p65 and promoted the production of let-7f in a concentration-dependent manner. Furthermore, the blockade of TLR4 antagonized the promoting effects of TLR4 pathway activation in cell inflammation and apoptosis by downregulating let-7f. Critically, it was confirmed in vivo and in vitro that ZL upregulated the expression of let-7f and inhibited the gene expression of TLR4, MyD88, and NF-κB p65 to reduce inflammatory cell infiltration, which determined the occurrence of hemorrhagic transformation. CONCLUSIONS: ZL can reduce inflammatory response by upregulating let-7f and subsequently inhibiting the TLR4 signaling pathway, thereby decreasing the occurrence of hemorrhagic transformation.

Antioxidant and anticancer constituents from the leaves of Liriodendron tulipifera.

Sixteen compounds were extracted and purified from the leaves of Liriodendron tulipifera. These compounds include aporphines, oxoaporphine, coumarin, sesquiterpene lactone, benzenoids, cyclitol and steroids. (+)-Norstephalagine (2) (an aporphine) and scopoletin (8) (a coumarin) were isolated from Liriodendron tulipifera leaves from the first time. The identified compounds were screened for their antiradical scavenging, metal chelating and ferric reducing power activities. The results have showed that these compounds have antioxidative activity. The study has also examined the chemopreventive property of the isolated compounds against human melanoma cells A375. The results shown that (-)-anonaine (1), (-)-liridinine (3), (+)-lirinidine (6), lysicamine (7) and epitulipinolide diepoxide (9) significantly inhibited the proliferation of melanoma cells. These results revealed that these compounds have antioxidative activity and chemopreventive activity in skin melanoma cells.

Thymol-evoked Ca+ mobilization and ion currents in pituitary GH3 cells.

In this study, an attempt was made to elucidate the effects of thymol, a monocyclic phenolic compound, on Ca2+ mobilization and ion currents in pituitary GH3 cells with the aid of fura-2 fluorimetry and the whole-cell voltage-clamp technique. Thymol increased intracellular Ca2+ concentrations ([Ca2+]i) in GH3 cells loaded with Ca2+-sensitive dye fura-2. Removing extracellular Ca2+ reduced the thymol-induced [Ca2+]i rise. In Ca2+ -free solution, thymol-evoked [Ca2+]i rise was unchanged by depleting the Ca2+ store with thapsigargin (1 microM), while the thapsigargin-induced [Ca2+]i rise was reduced by pretreatment with thymol. These results imply that the Ca2+ stores depleted by thymol comprise thapsigargin-sensitive and thapsigargin-insensitive pools. In addition, after depletion of the internal Ca2+ store with 100 microM thymol in Ca2+ -free solution, a subsequent application of Ca2+ greatly induced a [Ca2+]i increase. The results indicate that, similar to thapsigargin, 100 microM thymol may activate the capacitative calcium entry (CCE) channel. However, thymol (100 microM) had a slight depressant action in L-type calcium current (I(CaL)). The stimulatory actions of thymol on Ca2+ signaling may partly be responsible for the underlying cellular mechanisms through which it affects neuroendocrine functions.