Semen cassiae Extract Inhibits Contraction of Airway Smooth Muscle

β2-adrenoceptor agonists are commonly used as bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), however, they induce severe side effects. Therefore, developing new bronchodilators is essential. Herbal plants were extracted and the extracts’ effect on airway smooth muscle (ASM) precontraction was assessed. The ethyl alcohol extract of semen cassiae (EESC) was extracted from Semen cassia. The effects of EESC on the ACh- and 80 mM K+-induced sustained precontraction in mouse and human ASM were evaluated. Ca2+ permeant ion channel currents and intracellular Ca2+ concentration were measured. HPLC analysis was employed to determine which compound was responsible for the EESC-induced relaxation. The EESC reversibly inhibited the ACh- and 80 mM K+-induced precontraction. The sustained precontraction depends on Ca2+ influx, and it was mediated by voltage-dependent L-type Ca2+ channels (LVDCCs), store-operated channels (SOCs), TRPC3/STIM/Orai channels. These channels were inhibited by aurantio-obtusin, one component of EESC. When aurantio-obtusin removed, EESC’s action disappeared. In addition, aurantio-obtusin inhibited the precontraction of mouse and human ASM and intracellular Ca2+ increases. These results indicate that Semen cassia-contained aurantio-obtusin inhibits sustained precontraction of ASM via inhibiting Ca2+-permeant ion channels, thereby, which could be used to develop new bronchodilators

Andrographolide Attenuates Established Pulmonary Hypertension via Rescue of Vascular Remodeling

Pulmonary hypertension (PH) is characterized by vascular remodeling caused by marked proliferation of pulmonary artery smooth muscle cells (PASMCs). Andrographolide (ANDRO) is a potent anti-inflammatory agent which possesses antioxidant, and has anticarcinogenic activity. The present study examined potential therapeutic effects of ANDRO on PH in both chronic hypoxia and Sugen5416/hypoxia mouse PH models. Effects of ANDRO were also studied in cultured human PASMCs isolated from either healthy donors or PH patients. In vivo, ANDRO decreased distal pulmonary arteries (PAs) remodeling, mean PA pressure and right ventricular hypertrophy in chronic hypoxia- and Sugen/hypoxia-induced PH in mice. ANDRO reduced cell viability, proliferation and migration, but increased cell apoptosis in the PASMCs isolated from PH patients. ANDRO also reversed the dysfunctional bone morphogenetic protein receptor type-2 (BMPR2) signaling, suppressed [Ca2+]i elevation, reactive oxygen species (ROS) generation, and the upregulated expression of IL-6 and IL-8, ET-1 and VEGF in PASMCs from PH patients. Moreover, ANDRO significantly attenuated the activation of TLR4/NF-κB, ERK- and JNK-MAPK signaling pathways and reversed the inhibition of p38-MAPK in PASMCs of PH patients. Further, ANDRO blocked hypoxia-triggered ROS generation by suppressing NADPH oxidase (NOX) activation and augmenting nuclear factor erythroid 2-related factor 2 (Nrf2) expression both in vitro and in vivo. Conventional pulmonary vasodilators have limited efficacy for the treatment of severe PH. We demonstrated that ANDRO may reverse pulmonary vascular remodeling through modulation of NOX/Nrf2-mediated oxidative stress and NF-κB-mediated inflammation. Our findings suggest that ANDRO may have therapeutic value in the treatment of PH

Polygonum aviculare L. extract and quercetin attenuate contraction in airway smooth muscle

Abstract Because of the serious side effects of the currently used bronchodilators, new compounds with similar functions must be developed. We screened several herbs and found that Polygonum aviculare L. contains ingredients that inhibit the precontraction of mouse and human airway smooth muscle (ASM). High K+-induced precontraction in ASM was completely inhibited by nifedipine, a selective blocker of L-type voltage-dependent Ca2+ channels (LVDCCs). However, nifedipine only partially reduced the precontraction induced by acetylcholine chloride (ACH). Additionally, the ACH-induced precontraction was partly reduced by pyrazole-3 (Pyr3), a selective blocker of TRPC3 and stromal interaction molecule (STIM)/Orai channels. These channel-mediated currents were inhibited by the compounds present in P. aviculare extracts, suggesting that this inhibition was mediated by LVDCCs, TRPC3 and/or STIM/Orai channels. Moreover, these channel-mediated currents were inhibited by quercetin, which is present in P. aviculare extracts. Furthermore, quercetin inhibited ACH-induced precontraction in ASM. Overall, our data indicate that the ethyl acetate fraction of P. aviculare and quercetin can inhibit Ca2+-permeant LVDCCs, TRPC3 and STIM/Orai channels, which inhibits the precontraction of ASM. These findings suggest that P. aviculare could be used to develop new bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease

The SH3BGR/STAT3 Pathway Regulates Cell Migration and Angiogenesis Induced by a Gammaherpesvirus MicroRNA

<div><p>Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is a gammaherpesvirus etiologically associated with KS, a highly disseminated angiogenic tumor of hyperproliferative spindle endothelial cells. KSHV encodes 25 mature microRNAs but their roles in KSHV-induced tumor dissemination and angiogenesis remain unknown. Here, we investigated KSHV-encoded miR-K12-6-3p (miR-K6-3p) promotion of endothelial cell migration and angiogenesis, which are the underlying mechanisms of tumor dissemination and angiogenesis. We found that ectopic expression of miR-K6-3p promoted endothelial cell migration and angiogenesis. Mass spectrometry, bioinformatics and luciferase reporter analyses revealed that miR-K6-3p directly targeted sequence in the 3’ untranslated region (UTR) of SH3 domain binding glutamate-rich protein (SH3BGR). Overexpression of SH3BGR reversed miR-K6-3p induction of cell migration and angiogenesis. Mechanistically, miR-K6-3p downregulated SH3BGR, hence relieved STAT3 from SH3BGR direct binding and inhibition, which was required for miR-K6-3p maximum activation of STAT3 and induction of cell migration and angiogenesis. Finally, deletion of miR-K6 from the KSHV genome abrogated its effect on the SH3BGR/STAT3 pathway, and KSHV-induced migration and angiogenesis. Our results illustrated that, by inhibiting SH3BGR, miR-K6-3p enhances cell migration and angiogenesis by activating the STAT3 pathway, and thus contributes to the dissemination and angiogenesis of KSHV-induced malignancies.</p></div

Cellular proteins downregulated >2.0 folds in HUVEC infected with miR-K6-3p.

<p>Cellular proteins downregulated >2.0 folds in HUVEC infected with miR-K6-3p.</p

Overexpression of SH3BGR inhibits miR-K6-3p-induced endothelial cell migration and angiogenesis <i>in vitro</i> and <i>in vivo</i>.

<p><b>(A)</b>. Transwell migration assay for HUVEC transduced with lentivirus-mediated miR-K6-3p (<b>miR-K6-3p</b>) or empty vector (<b>mpCDH</b>), which were subsequently co-transduced with lentivirus-SH3BGR (<b>SH3BGR</b>) or its control pHAGE (<b>pHAGE</b>), respectively. The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). <b>(B)</b>. Quantification of results in <b>(A)</b>. The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing five technical replicates. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. <b>(C)</b>. Microtubule formation assay for HUVEC treated as in (<b>A</b>). The representative images were captured at 2 and 5 h post seeding (original magnification, ×100). <b>(D)</b>. Quantification of results in (<b>C</b>). The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing five technical replicates. ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. <b>(E)</b>. The mRNA expression of MMP1, MMP13, VEGFA and VEGFR2 in HUVEC treated as in (<b>A</b>) were determined by qPCR. The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing four technical replicates. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. <i>n</i>.<i>s</i>., not significant. <b>(F)</b>. Western blotting was performed in HUVEC treated as in (<b>A</b>) with the indicated antibodies. The antibody against Flag-tag was used to detect the exogenous expression of SH3BGR. Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown. <b>(G)</b>. HUVEC treated as in (<b>A</b>) were examined for their proangiogenic effects in Matrigel plug assay in nude mice as described in the “Materials and Methods” section. Representative photographs of angiogenesis in the nude mice are shown. <b>(H)</b>. The hemoglobin level of the Matrigel plugs treated as in (<b>G</b>) was determined with hemoglobin content calculated based on the standard curve. Data represent mean ± SD, each group with five tumors (n = 5). Three independent experiments were performed and similar results were obtained. <b>(I)</b>. The mRNA expression of MMP1, MMP13, VEGFA and VEGFR2 in the Matrigel plugs treated as in (G) were determined by qPCR. The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing four technical replicates. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. <i>n</i>.<i>s</i>., not significant.</p

SH3BGR is directly targeted by miR-K6-3p.

<p><b>(A).</b> Luciferase assay of HEK293T cells co-transfected with pGL3-SH3BGR 3′UTR reporter together with an increasing amount (10, 20, and 50 nM) of negative control nucleotide of miRNA (<b>Neg. Ctrl.</b>) or a mimic of miR-K6-3p (<b>miR-K6-3p</b>) for 24 h. <b>(B).</b> HUVEC transfected with an increasing amount (20 and 50 nM) mimic of miR-K6-3p or negative control for 48 h. The transfected cells were collected and Western blotting was performed with the indicated antibodies. Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown. <b>(C).</b> Schematic illustration of the putative seed sequences of miR-K6-3p complementary with SH3BGR 3’UTR and mutagenesis of binding sites in the 3’UTR of SH3BGR. <b>(D).</b> Effect of mutation of the putative binding site on the SH3BGR 3’UTR reporter. After co-transfection of SH3BGR wild type 3’UTR (<b>WT SH3BGR</b>) or the mutant SH3BGR 3’UTR construct (<b>mut SH3BGR</b>) together with a negative control nucleotide of miRNA (<b>Neg. Ctrl.</b>), a mimic of miR-K6-3p (<b>miR-K6-3p</b>) or a mutant mimic of miR-K6-3p (<b>mut miR-K6-3p</b>) for 24 h in HEK293T cells, cells were collected and assayed for luciferase activity. * <i>P</i> < 0.05 for Student’s <i>t</i>-test versus Neg. Ctrl. group. <b>(E).</b> Mutant miR-K6-3p failed to target endogenous SH3BGR in HUVEC. A miRNA negative control nucleotide (<b>Neg. Ctrl.</b>), a mimic of miR-K6-3p (<b>miR-K6-3p mimic</b>; 10 nM) or a mutant mimic of miR-K6-3p (<b>mut miR-K6-3p</b>) lacking the seed sequences were transfected into HUVEC for 48 h, respectively. The transfected cells were collected and Western blotting was performed with the indicated antibodies. Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown. <b>(F).</b> Transfection of miR-K6-3p mimic (20 nM) has the same inhibition level on SH3BGR expression as that of KSHV infection. Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown.</p

The sequences of the shRNAs.

<p>The sequences of the shRNAs.</p

Deletion of miR-K6 from KSHV genome attenuates KSHV induction of endothelial cell migration and angiogenesis.

<p><b>(A)</b>. Transwell migration assay for HUVEC were treated with PBS (PBS), infected with BAC16 KSHV wide type virus (KSHV_WT) or BAC16 KSHV miR-K6 deletion mutant virus (miR-K6_Mut). The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). <b>(B)</b>. Quantification of the results in (<b>A</b>). The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing five technical replicates. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. (<b>C</b>). Microtubule formation assay for HUVEC treated as in (<b>A</b>). The representative images were captured at 3 and 6 h post seeding (original magnification, ×100). <b>(D)</b>. Quantification of the results in (<b>C</b>). The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing five technical replicates. ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. <b>(E)</b>. The mRNA expression of MMP1, MMP13, VEGFA and VEGFR2 in HUVEC treated as in (<b>A</b>) were determined by RT-qPCR. The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing four technical replicates. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. <b>(F)</b>. Western blotting analysis of expression of SH3BGR, phosphorylated STAT3, STAT3 and VEGFA in HUVEC treated as in (<b>A</b>) with the indicated antibodies. Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown.</p

Ectopic expression of miR-K6-3p promotes endothelial cell migration and angiogenesis.

<p><b>(A)</b>. Transwell migration for HUVEC transduced with 1 MOI lentivirus empty vector (<b>mpCDH</b>) or lentivirus-miR-K6-3p (<b>miR-K6-3p</b>). The representative images were captured at 6 and 12 h post seeding (original magnification, ×100). <b>(B)</b>. The quantification results of Transwell migration assay in (<b>A</b>). The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing at least four technical replicates. <b>(C)</b>. Microtubule formation assay for HUVEC transduced with lentivirus empty vector (<b>mpCDH</b>; top) or lentivirus-miR-K6-3p (<b>miR-K6-3p</b>; bottom). The representative images were captured under the light microscope (<b>Phase</b>) and fluorescent microscope (<b>RFP</b>) at 2 and 5 h post seeding (original magnification, ×100). <b>(D)</b>. The quantification results of microtubule formation assay in (<b>C</b>). The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing four technical replicates. <b>(E)</b>. The mRNA expression of MMP1, MMP13, VEGFA and VEGFR2 in HUVEC treated as in (<b>A</b>) was determined by RT-qPCR. The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing four technical replicates. ** <i>P</i> < 0.01 for Student’s <i>t</i>-test versus mpCDH group. <b>(F)</b>. Western blotting analysis of the expression of VEGFA protein in HUVEC treated as in (<b>A</b>). Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown. <b>(G)</b>. HUVEC treated as in (<b>A</b>) were examined for their proangiogenic effects in Matrigel plug assay in nude mice as described in the “Materials and Methods” section. Representative photographs of angiogenesis in the nude mice are shown. <b>(H)</b>. The hemoglobin level of the Matrigel plugs treated as in (<b>G</b>) was determined with hemoglobin content calculated based on the standard curve. Data represent mean ± SD, each group with five tumors (n = 5). Three independent experiments were performed and similar results were obtained. ** <i>P</i> < 0.01 for Student’s <i>t</i>-test versus mpCDH group. <b>(I)</b>. Hematoxylin and eosin staining analysis of histologic features (<b>left</b>; ×400) and immunohistochemical (IHC) staining analysis of the expression of SMA (<b>middle</b>; ×400) and VEGFA (<b>right</b>; ×400) in plugs induced by HUVEC transduced with mpCDH or miR-K6-3p. Black arrows point to neovascularization and hemorrihagic foci in H&E staining sections and the SMA in IHC staining sections, respectively. <b>(J)</b>. Quantification of results in (<b>I</b>). ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test versus mpCDH group. <b>(K)</b>. The mRNA expression of MMP1, MMP13, VEGFA and VEGFR2 in the Matrigel plugs treated as in <b>(G)</b> were determined by RT-qPCR. The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing four technical replicates. ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test versus mpCDH group.</p