Transcriptome Analysis of the Anti-TGFß Effect of Schisandra chinensis Fruit Extract and Schisandrin B in A7r5 Vascular Smooth Muscle Cells.

Schisandra chinensis fruit extract (SCE) has been used as a traditional medicine for treating vascular diseases. However, little is known about how SCE and schisandrin B (SchB) affect transcriptional output-a crucial factor for shaping the fibrotic responses of the transforming growth factor ß (TGFß) signaling pathways in in vascular smooth muscle cells (VSMC). In this study, to assess the pharmacological effect of SCE and SchB on TGFß-induced transcriptional output, we performed DNA microarray experiments in A7r5 VSMCs. We found that TGFß induced distinctive changes in the gene expression profile and that these changes were considerably reversed by SCE and SchB. Gene Set Enrichment Analysis (GSEA) with Hallmark signature suggested that SCE or SchB inhibits a range of fibrosis-associated biological processes, including inflammation, cell proliferation and migration. With our VSMC-specific transcriptional interactome network, master regulator analysis identified crucial transcription factors that regulate the expression of SCE- and SchB-effective genes (i.e., TGFß-reactive genes whose expression are reversed by SCE and SchB). Our results provide novel perspective and insight into understanding the pharmacological action of SCE and SchB at the transcriptome level and will support further investigations to develop multitargeted strategies for the treatment of vascular fibrosis.

Additive effect of oral LDD175 to tamsulosin and finasteride in a benign prostate hyperplasia rat model.

OBJECTIVE: We investigated the benefits of the BKCa agonist 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid (LDD175) combined with tamsulosin and finasteride, in a benign prostatic hyperplasia (BPH) rat model. MATERIALS AND METHODS: Castration was performed by bilateral orchiectomy under ketamine anesthesia. A rat model of BPH was established by daily intramuscular administration of testosterone propionate plus 17ß-estradiol for 8 weeks. Model rats were administered combinations of 20 mg/kg LDD175, 0.01 mg/kg tamsulosin and 1 mg/kg finasteride once daily by oral gavage for 4 weeks from week 6 to 9 post-surgery. Intraurethral pressure induced by electrostimulation of the hypogastric nerve was measured at the end of administration. Body and genitourinary organ weights were recorded, serums were assayed for hormone concentrations, and tissues were subjected to histopathology, and analyses of α1-adrenoceptor mRNA and protein expression levels after treatment. RESULTS: Combined LDD175, tamsulosin, and finasteride significantly decreased prostatic index, serum hormone levels, epithelial thickness, and prostate expression of α1-adrenoceptors in BPH model rats. The 3-drug combination was more effective than any other combination or LDD175 alone. CONCLUSION: These results suggest that LDD175 addition to tamsulosin and finasteride may be beneficial for the treatment of BPH patients who do not respond to tamsulosin plus finasteride.

Schisandrol B and schisandrin B inhibit TGFß1-mediated NF-κB activation via a Smad-independent mechanism.

Aberrant transforming growth factor ß1 (TGFß1) signaling plays a pathogenic role in the development of vascular fibrosis. We have reported that Schisandra chinensis fruit extract (SCE), which has been used as a traditional oriental medicine, suppresses TGFß1-mediated phenotypes in vascular smooth muscle cells (VSMCs). However, it is still largely unknown about the pharmacologic effects of SCE on various TGFß1 signaling components. In this study, we found that SCE attenuated TGFß1-induced NF-κB activation and nuclear translocation in VSMCs. Among the five active ingredients of SCE that were examined, schisandrol B (SolB) and schisandrin B (SchB) most potently suppressed TGFß1-mediated NF-κB activation. In addition, SolB and SchB effectively inhibited IKKα/ß activation and IκBα phosphorylation in TGFß1-treated VSMCs. The pharmacologic effects of SolB and SchB on NF-κB activation were independent of the Smad-mediated canonical pathway. Therefore, our study demonstrates that SCE and its active constituents SolB and SchB suppress TGFß1-mediated NF-κB signaling pathway in a Smad-independent mechanism. Our results may help further investigations to develop novel multi-targeted therapeutic strategies that treat or prevent vascular fibrotic diseases.

Magnolia Officinalis Bark Extract Induces Depolarization of Pacemaker Potentials Through M2 and M3 Muscarinic Receptors in Cultured Murine Small Intestine Interstitial Cells of Cajal.

BACKGROUND: Magnolia officinalis Rehder and EH Wilson (M. officinalis) are traditional Chinese medicines widely used for gastrointestinal (GI) tract motility disorder in Asian countries. We investigated the effects of an ethanol extract of M. officinalis (MOE) on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) in vitro and its effects on GI motor functions in vivo. METHODS: We isolated ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs in vitro. Both gastric emptying (GE) and intestinal transit rates (ITRs) were investigated in normal and GI motility dysfunction (GMD) mice models in vivo. RESULTS: MOE depolarized ICC pacemaker potentials dose-dependently. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) and 4-DAMP (a muscarinic M3 receptor antagonist) inhibited the effects of MOE on the pacemaker potential relative to treatment with MOE alone. In addition, MOE depolarized pacemaker potentials after pretreatment with Y25130 (a 5-HT3 receptor antagonist), GR113808 (a 5-HT4 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist). However, pretreatment with RS39604 (a 5-HT4 receptor antagonist) blocked MOE-induced pacemaker potential depolarizations. Intracellular GDPßS inhibited MOE-induced pacemaker potential depolarization, as did pretreatment with Ca2+ free solution or thapsigargin. In normal mice, the GE and ITR values were significantly and dose-dependently increased by MOE. In loperamide-and cisplatin-induced GE delay models, MOE administration reversed the GE deficits. The ITRs of the GMD mice were significantly reduced relative to those of normal mice, which were significantly and dose-dependently reversed by MOE. CONCLUSION: These results suggest that MOE dose-dependently depolarizes ICCs pacemaker potentials through M2 and M3 receptors via internal and external Ca2+ regulation through G protein pathways in vitro. Moreover, MOE increased GE and ITRs in vivo in normal and GMD mouse models. Taken together, the results of this study show that MOE have the potential for development as a gastroprokinetic agent in GI motility function.

High-Dose Radiation-Induced Changes in Murine Small Intestinal Motility: Are the Changes in the Interstitial Cells of Cajal or in the Enteric Nervous System?

Murine small intestinal motility consists of phasic contraction from interstitial cells of Cajal (ICC) and migrating motor complexes (MMCs) from the enteric nervous system. The number of ICC is reduced in various gastrointestinal disorders, and this effect can be reversed once the disorder is resolved through cellular and tissue remodelling. Exposure to high-dose radiation can induce inflammation and alter intestinal motility. In this study, we investigated the changes in the small intestinal motility of 8- to 10-week-old male C3H/HeN mice after high-dose (13 Gy) irradiation. The aim of this study was to determine whether those changes are caused by changes in the ICC or enteric nervous system. After irradiation, the small intestine was dissected and stored in oxygenated Krebs-Ringer bicarbonate solution. The tension of contractions and intracellular membrane potentials were recorded at day 0, 1, 3 and 5 after irradiation and compared with those of sham-irradiated mice. Histological evaluation was performed by immunohistochemistry and apoptosis was evaluated. Quantitative real-time polymerase chain reaction (qPCR) for c-kit mRNA was also performed. Phasic contractions were not changed at day 0, 1, 3 and 5 after irradiation and did not significantly differ from those in the control mice. Slow waves were also sustained after irradiation. However, the frequency of migrating motor complexes (MMCs) was significantly higher at day 0 and 1 after exposure and the amplitude and area under the curve were significantly lower at day 3 after exposure compared with control mice. MMCs were recovered at day 5 with no difference from those of the control mice. ICC were detected after irradiation by immunohistochemistry for c-kit, and c-kit mRNA levels did not differ between sham-irradiated and irradiated mice. Histological evaluation showed that the most severe inflammation was detected at day 3 after irradiation, and apoptosis was detected only in the mucosa. Acetylcholine increased the contractility after irradiation, and tetrodotoxin decreased the number of MMCs in sham-irradiated and irradiated mice. N(w)-oxide-l-arginine (L-NA) increased the number of MMCs. MMCs were recovered after L-NA treatment at day 3 after irradiation. Sodium nitroprusside decreased the MMCs in sham-irradiated and irradiated mice. Exposure to high-dose radiation did not alter phasic contractions and slow waves in the small intestine of mice, which suggests that ICC and their functions may be sustained after high-dose irradiation. Mucosal inflammation was severe after irradiation and there were some changes in MMCs related to the enteric nervous system.

Effect of 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid on the intraurethral pressure in a rat model of benign prostatic hyperplasia.

OBJECTIVES: To investigate the effect of 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid, a new benzofuroindole derivative, on the intraurethral pressure in a rat model of benign prostatic hyperplasia. METHODS: Benign prostatic hyperplasia was induced by testosterone and 17ß-estradiol, which were administered intramuscularly once a day for 12 weeks. The effects of 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid and tamsulosin on the intraurethral pressure induced by the electrostimulation of hypogastric nerves after a single intravenous injection of 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid (10 mg/kg) or tamsulosin (10 µg/kg) were evaluated in a benign prostatic hyperplasia model. The electrostimulation-induced intraurethral pressure was measured just before and after the injection of 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid. Bodyweight and genitourinary organ weights were recorded, and serums and tissues were subjected to hormone assays and histopathology. In addition, the expression of α1-adrenoceptors in the prostate was measured by western blotting. RESULTS: The benign prostatic hyperplasia groups showed increased prostatic index, increased concentrations of testosterone, free testosterone and estradiol in serum, and increased epithelial thickness of the prostate. An injection of 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid or tamsulosin significantly inhibited the elevation of electrostimulation-induced intraurethral pressure. In addition, 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid did not cause a significant change in the blood pressure compared with tamsulosin. While the benign prostatic hyperplasia group showed increased the expression of α1-adrenoceptors, the 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid or tamsulosin injection into a rat model of benign prostatic hyperplasia decreased the expression of α1-adrenoceptors. CONCLUSIONS: These findings show that 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid might be beneficial for lowering the intraurethral pressure associated with benign prostatic hyperplasia, and it could represent a therapeutic option for benign prostatic hyperplasia patients.

Prospective investigation of change in the prostate-specific antigens after various urologic procedures.

PURPOSE: Prostate-specific antigen (PSA) is the most important marker in the diagnosis and follow-up of patients with prostate cancer. The primary objective of this study was to evaluate the effect of various urologic procedures in prostatic area on serum free and total PSA levels. SUBJECTS AND METHODS: A series of 62 patients (8 after digital rectal examination [DRE], 12 after transrectal ultrasonography [TRUS], 11 after rigid cystoscopy, 13 after prostatic massage, 8 after TRUS-guided prostate biopsy, and 10 after transurethral resection of prostate [TURP]) were enrolled in the study. Blood samples were taken from each patient before procedure and at 10, 30, 60, and 120 minutes after procedures. RESULTS: Prostate massage, rigid cystoscopy, TURP, and TRUS-guided prostate biopsy caused statistically significant rise in total and free PSA levels in the serum. There was no significant increase in total and free PSA levels in the serum after DRE and TRUS. The mean differences were greater for free PSA level in the serum for TURP, TRUS-guided prostate biopsy, prostate massage, and rigid cystoscopy. CONCLUSION: Total and free PSA levels in the serum are altered by prostate massage, rigid cystoscopy, TRUS-guided prostate biopsy, and TURP. The PSA rises were related to the stimulation strength of the procedures. The total and free PSA levels were increased significantly from 10 minutes after procedures, except DRE and TRUS, and were increased to maximal level at 60 minutes after procedures.

The traditional herbal medicine, Ge-Gen-Tang, inhibits pacemaker potentials by nitric oxide/cGMP dependent ATP-sensitive K(+) channels in cultured interstitial cells of Cajal from mouse small intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Ge-Gen-Tang (GGT) is a traditional Chinese medicinal formula composed of Puerariae radix (Pueraria lobata Ohwi), Ephedrae Herba (Ephedra sinica Stapf), Cinnamomi Ramulus (Cinnamomum cassia Blume), Paeoniae Radix (Paeonia lactiflora Pallas), Glycyrrhizae Radix preparata (Glycyrrhiza uralensis Fischer), Zingiberis Rhizoma (Zingiber officinale Roscoe), and Zizyphi Fructus (Ziziphus jujuba Mill. var. inermis Rehder) and is widely used to ameoliorate the symptoms of gastrointestinal (GI) disorders related to diarrhea and intestinal mucosal immunity and for anti-cold, antipyretic and analgesic in Eastern Asia. AIM OF THE STUDY: Interstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract that generate rhythmic oscillations in membrane potentials known as slow waves. We investigated the effects of GGT on pacemaker potentials in cultured ICCs from the mouse small intestine, and sought to identify the receptors and the action mechanisms involved. MATERIALS AND METHODS: Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. All experiments on ICCs were performed on within 12h after culture. A whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. Intracellular Ca(2+) ([Ca(2+)]i) increase was studied in cultured ICCs using fura-2AM. All of the experiments were performed at 30-32°C. RESULTS: Under the current clamping mode, GGT decreased the amplitude and frequency of pacemaker potentials; however, these effects were blocked by intracellular GDPßS, a G-protein inhibitor, and glibenclamide, a specific ATP-sensitive K(+) channels blocker. Prazosin (α1-adrenoceptor antagonist) and butoxamine (ß2-adrenoceptor antagonist) did not block the GGT-induced effects, whereas atenolol (ß1-adrenoceptor antagonist) blocked the GGT-induced effects. Also, yohimbine (α2-adrenoceptor antagonist) partially blocked the GGT-induced effects. Pretreatment with SQ-22536, an adenylate cyclase inhibitor, did not block the GGT-induced effects, whereas pretreatment with ODQ, a guanylate cyclase inhibitor, or L-NAME, an inhibitor of nitric oxide (NO) synthase, did. Additionally, [Ca(2+)]i analysis showed that GGT decreased [Ca(2+)]i. CONCLUSION: These results suggest that GGT inhibits pacemaker potentials in ICCs in a G protein-, cGMP- and NO-dependent manner through stimulation of α2 and ß1-adrenoceptors.

Effects of Schisandra chinensis extract on gastrointestinal motility in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra chinensis (Turcz.) Baill. (SC) continues to be used as a traditional folk medicine in Asia, especially for the treatment of gastrointestinal (GI) disorders related to gastritis, diarrhea, enterocolitis and abnormal GI motility. AIM OF THE STUDY: Because GI disorders, especially abnormal GI motility, are major lifelong problems, we investigated the effects of SC on the pacemaker activity of the interstitial cells of Cajal (ICCs) in murine small intestine and GI motility. MATERIALS AND METHODS: Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials generated by cultured ICCs. In vivo effects of SC on GI motility were investigated by measuring the intestinal transit rate (ITR) of Evans blue in normal and GI motility dysfunction mice. RESULTS: SC extracts depolarized the membrane potentials of ICCs in a dose dependent manner. Pretreatment with Ca(2+) free solution or thapsigargin (a Ca(2+)-ATPase inhibitor in the endoplasmic reticulum) abolished the generation of pacemaker potentials by ICCs, and under these conditions, SC extract did not depolarize the membrane potentials of ICCs. In addition, membrane depolarizations were inhibited by intracellular GDPßS and by U-73122 (an active phospholipase C (PLC) inhibitor). In normal mice, ITRs were significantly increased by SC extract (0.1-1g/kg, intragastrically (i.g.)) in a dose dependent manner. Also, SC extract significantly recovered the GI motility dysfunctions in acetic acid (AA)-injected and streptozotocin (STZ)-induced diabetic mice, which are the GI motility animal models. MATERIALS AND METHODS: SC extract modulates pacemaker potentials in ICCs in a dose dependent manner via external and internal Ca(2+) regulations, and via G protein and the PLC pathway. In addition, SC extract increased ITRs in normal and abnormal GI motility mice models. This study shows that SC extract offers a basis for the development of a prokinetic agent that prevents or alleviates GI motility dysfunctions.

Effects of Schisandra chinensis extract on the relaxation of isolated human prostate tissue and smooth muscle cell.

ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra chinensis has been commonly used as a traditional herbal medicine to treat various diseases including body weakness, dysentery, impotence, enuresis and frequent urination in many countries including Korea, China and Russia. Benign prostate hyperplasia is a common disease for the elderly men and it induces lower urinary tract symptoms which hinder general activity and quality of life. We evaluated the therapeutic potential of Schisandra chinensis extract (SCE) in benign prostate hyperplasia using human prostate tissue. MATERIALS AND METHODS: Schisandra chinensis fruit was collected and extracted with ethanol. Human prostate tissues were obtained from 14 prostate cancer patients. Macroscopically normal tissue was excised from the transition zone and the periurethral regions. Isolated prostate tissue strips were mounted in an organ-bath system, and the relaxation effect of SCE was evaluated by cumulative addition to prostate strips pre-contracted with 10(-5)M norepinephrine. The effect of tamsulosin was compared, and the additive effect was evaluated. Electrophysiological studies using cultured human prostate smooth muscle cells (HPrSMC) were conducted. RESULTS: Cumulative dosing of SCE induced concentration-dependent relaxation in contracted prostate tissue (n=18, P<0.05). Simultaneous dosing of SCE and tamsulosin showed an additive relaxation effect. The relaxation effect of SCE was abolished by inhibition of K+ channels by pre-treatment with tetraethylammonium. In HPrSMC, extracellular application of 100 µg/mL SCE significantly increased outward currents, and this effect was significantly attenuated by treatment with 100 nM Iberiotoxin. CONCLUSIONS: SCE showed a dose dependent relaxation effect on human prostate tissue as well as an additive effect with tamsulosin. The relaxation effects of SCE on HPrSMC were, in part, due to the activation of K+ channels.

The protective effects of Schisandra chinensis fruit extract and its lignans against cardiovascular disease: a review of the molecular mechanisms.

Schisandra chinensis fruit extract (SCE) has traditionally been used as an oriental medicine for the treatment of various human diseases, including cardiovascular disease. Advances in scientific knowledge and analytical technologies provide opportunities for translational research involving S. chinensis; such research may contribute to future drug discovery. To date, emerging experimental evidence supports the therapeutic effects of the SCE or its bioactive lignan ingredients in cardiovascular disease, unraveling the mechanistic basis for their pharmacological actions. In the present review, we highlight SCE and its lignans as promising resources for the development of safe, effective, and multi-targeted agents against cardiovascular disease. Moreover, we offer novel insight into future challenges and perspective on S. chinensis research to future clinical investigations and healthcare strategies.

Effects of San-Huang-Xie-Xin-tang, a traditional Chinese prescription for clearing away heat and toxin, on the pacemaker activities of interstitial cells of Cajal from the murine small intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: San-Huang-Xie-Xin-Tang (SHXXT) is a traditional Chinese medicinal formula composed of Coptidis rhizoma (Coptis chinesis Franch), Scutellariae radix (Scutellaria baicalensis Georgi), and Rhei rhizoma (Rheum officinale Baill) and is widely used in Eastern Asia, especially to ameliorate the symptoms of gastrointestinal (GI) disorders related to gastritis, gastric bleeding, peptic ulcers, and abnormal GI motility AIM OF THE STUDY: Interstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract that generate rhythmic oscillations in membrane potentials known as slow waves. Because GI disorders, especially abnormal GI motility, are major lifelong problems, the authors investigated the effects of SHXXT on mouse small intestine ICCs, and sought to identify the receptors and the action mechanisms involved. MATERIALS AND METHODS: Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials generated by cultured ICCs. RESULTS: SHXXT produced membrane depolarization in current-clamp mode, and Y25130 (a 5-HT3 receptor antagonist) and RS39604 (a 5-HT4 receptor antagonist) blocked SHXXT-induced membrane depolarizations, whereas SB269970 (a 5-HT7 receptor antagonist) did not. However, during external Ca2+ free conditions or in the presence of thapsigargin, SHXXT did not exhibit membrane depolarization. Furthermore, the application of flufenamic acid (a nonselective cation channel (NSCC) blocker) or DIDS (a chloride channel blocker) abolished pacemaker potential generation and blocked SHXXT-induced membrane depolarizations. In addition, SHXXT-induced membrane depolarizations, which are dependent on G-protein, in ICCs were blocked by PD 98059 (a p42/44 mitogen-activated protein kinase (MAPK) inhibitor), SB203580 (a p38 MAPK inhibitor), and by a c-jun NH2-terminal kinase (JNK) II inhibitor. Regarding the components of SHXXT, Coptidis rhizome and Rhei rhizoma modulated ICC pacemaking activity, whereas Scutellariae radix did not. CONCLUSION: SHXXT modulates pacemaker potentials via 5-HT3 and 5-HT4 receptor-mediated pathways, external Ca2+ influx, and Ca2+ release from internal stores. Furthermore, NSCCs and Cl- channels play important roles in the regulation of pacemaking activity in a MAPK dependent manner in ICCs. The regulation of pacemaking activity by SHXXT may be due to the activity of Coptidis rhizome and Rhei rhizome. The study shows SHXXT can modulate the pacemaking activity of ICCs in the GI tract, and thus, suggests SHXXT has potential pharmacological relevance for the treatment of GI motility disorders.

Schisandrin B suppresses TGFß1-induced stress fiber formation by inhibiting myosin light chain phosphorylation.

ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra chinensis fruit extract (SCE) has been used as a traditional oriental medicine for treating vascular diseases. However, the pharmacologic effects and mechanisms of SCE on vascular fibrosis are still largely unknown. Transforming growth factor ß1 (TGFß1)-mediated cellular changes are closely associated with the pathogenesis of vascular fibrotic diseases. Particularly, TGFß1 induces actin stress fiber formation that is a crucial mechanism underlying vascular smooth muscle cell (VSMC) migration in response to vascular injury. In this study, we investigated the effect of SCE and its active ingredients on TGFß1-induced stress fiber assembly in A7r5 VSMCs. MATERIALS AND METHODS: To investigate pharmacological actions of SCE and its ingredients on TGFß1-treated VSMCs, we have employed molecular and cell biological technologies, such as confocal microscopy, fluorescence resonance energy transfer, western blotting, and radiometric enzyme analyses. RESULTS: We found that SCE inhibited TGFß1-induced stress fiber formation and cell migration. Schisandrin B (SchB) showed the most prominent effect among the active ingredients of SCE tested. SchB reduced TGFß1-mediated phosphorylation of myosin light chain, and this effect was independent of RhoA/Rho-associated kinase pathway. Fluorescence resonance energy transfer and radiometric enzyme assays confirmed that SchB inhibited myosin light chain kinase activity. We also showed that SchB decreased TGFß1-mediated induction of α-smooth muscle actin by inhibiting Smad signaling. CONCLUSIONS: The present study demonstrates that SCE and its active ingredient SchB suppressed TGFß1-induced stress fiber formation at the molecular level. Therefore, our findings may help future investigations to develop multi-targeted therapeutic strategies that attenuate VSMC migration and vascular fibrosis.

Poncirus trifoliate fruit modulates pacemaker activity in interstitial cells of Cajal from the murine small intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Poncirus fructus (PF) has been widely used as a traditional medicine in Eastern Asia, especially to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility. AIM OF THE STUDY: Poncirus fructus (PF), also known as Poncirus trifoliata (L.) Raf. (Rutaceae), is widely used as a traditional medicine in Eastern Asia mainly to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility. In a previous study, a methanol extract of PF was found to have particularly potent gastroprokinetic effects. Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal tract, but the action mechanisms of PF extract in mouse small intestinal ICCs have not been investigated. Therefore, in the present study, we investigated the effects of a methanol extract of PF (MPF) in mouse small intestinal ICCs. In addition, we sought to identify the receptors involved. MATERIALS AND METHODS: Enzymatic digestions were used to dissociate ICCs from small intestines. The whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. In addition, we analyzed intracellular Ca(2+) concentrations ([Ca(2+)]i). RESULTS: MPF decreased the amplitudes of pacemaker potentials in ICCs, and depolarized resting membrane potentials in a concentration dependent manner. Y25130 (a 5-HT3 receptor antagonist) and RS39604 (a 5-HT4 receptor antagonist) blocked MPF-induced membrane depolarizations, whereas SB269970 (a 5-HT7 receptor antagonist) did not. Pretreatment with Na(+) or Ca(2+)-free solution or thapsigargin (a Ca(2+)-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed MPF-induced activity. [Ca(2+)]i analysis showed that MPF increased [Ca(2+)]i. Furthermore, treatments with PD 98059, SB203580, or JNK II inhibitor blocked MPF-induced membrane depolarizations in ICCs. CONCLUSION: These results suggest that MPF modulates pacemaker potentials through 5-HT3 and 5-HT4 receptor-mediated pathways via external Na(+) and Ca(2+) influx, and via Ca(2+) release from internal stores in a mitogen-activated protein kinase dependent manner. The study shows MPF is a good candidate for the development of a gastroprokinetic agent. In view of the effects of MPF on ICCs, further research is required, particularly to identify the active compound(s) involved and to determine their action mechanisms.

Schisandrin B suppresses TGFß1 signaling by inhibiting Smad2/3 and MAPK pathways.

TGFß1 plays a crucial role in the pathogenesis of vascular fibrotic diseases. Schisandra chinensis (S. chinensis), which is used as an oriental herbal medicine, is effective in the treatment of vascular injuries that cause aberrant TGFß1 signaling. In this study, we investigated whether S. chinensis extract and its active ingredients inhibit TGFß1 signaling in A7r5 vascular smooth muscle cells. We found that S. chinensis extract suppressed TGFß1 signaling via inhibition of Smad2/3 phosphorylation and nuclear translocation. Among the active ingredients of S. chinensis extract, schisandrin B (SchB) most potently inhibited TGFß1 signaling. SchB inhibited sustained phosphorylation and nuclear translocation of Smad2/3. Moreover, SchB suppressed TGFß1-induced phosphorylation of p38 and JNK, which contributed to Smad2/3 inactivation. The present study is the first to demonstrate that S. chinensis extract and SchB inhibit TGFß1 signaling. Our results may help future investigations to understand vascular fibrosis pathogenesis and to develop novel therapeutic strategies for treatment of vascular fibrotic diseases.

Effects of Schisandra chinensis extract on the contractility of corpus cavernosal smooth muscle (CSM) and Ca2+ homeostasis in CSM cells.

UNLABELLED: What's known on the subject? And what does the study add? Schisandra chinensis extract (SCE) has been known to have relaxative effects on penile smooth muscle. A recent study showed that SCE could enhance slidenafil citrate-induced relaxation of penile corpus cavernosum. The current study investigated the mechanism of action of SCE and its constituents on corporal smooth muscle cells. And this study shows that SCE induced relaxation of CSM primarily through an endothelium independent pathway and the relaxation effects of SCE on corporal smooth muscle are, in part, due to the activation of K(+) channels and inhibition of TRPC6 channels, resulting in decreased [Ca(2+)]. OBJECTIVE: • To evaluate the relaxant effects of Schisandra chinensis extract (SCE) on corporal tissue in the penis and to investigate the mechanism of action of SCE and its constituents on corporal smooth muscle (CSM) cells. MATERIALS AND METHODS: • The fruit of SC was collected and extracted with ethanol. Six SC lignans (schisandrol A, schisandrol B, schisandrin A, schisandrin B, gomisin N, and schisandrin C) were isolated and purified, and the chemical structures were confirmed by (1)H-nuclear magnetic resonance (NMR) and (13)C-NMR data. • Isolated rabbit CSM strips were mounted in an organ-bath system, and the effects of SCE were evaluated. • To estimate the intracellular Ca(2+) level ([Ca(2+)](i)), we used a Fura-2 fluorescent technique, and a conventional whole-cell patch-clamp technique was used to measure the calcium-sensitive K(+) channels (K(Ca)), inward rectifier K(+) channels (K(IR)), and canonical transient receptor potential cation channel 6 (TRPC6) currents. RESULTS: • SCE induced concentration-dependent relaxation in contracted CSM tissue, and the removal of the endothelium did not significantly affect their relaxation potencies. • In CSM cells, extracellular application of SCE significantly increased whole-cell K(Ca) currents (117.4%) and K(IR) currents (110.0%). These effects were completely abolished by charybdotoxin or BaCl(2). • In contrast, carbachol-induced TRPC6 channel activity was significantly inhibited (87.3%) by SCE in green fluorescent protein-TRPC6 pcDNA transfected HEK 293 cells. [Ca(2+)](i) measurements showed that SCE effectively reduced basal [Ca(2+)](i) in both cell lines (CSM cells and A7r5 cells) and the [Arg8]-vasopressin (AVP)-induced [Ca(2+)](i) increase in A7r5 cells. • Among the six SC lignans, schisandrin A and schisandrin B most effectively attenuated the AVP-induced [Ca(2+)](i) increase. CONCLUSIONS: • SCE induced relaxation of CSM that occurred primarily via an endothelium-independent pathway. • The relaxation effects of SCE on CSM were, in part, due to the activation of K(+) channels and inhibition of TRPC6 channels, resulting in decreased [Ca(2+)](i).

The role of capillarisin from Artemisia capillaris on penile erection.

The objective of this study was to evaluate the effect and mechanism of capillarisin from Artemisia capillaris (A. capillaris) on rabbit penile corpus cavernosum (PCC). The pre-contracted New Zealand White rabbit (2.5-3.0 kg) penis with phenylephrine (Phe; 10⁻5 M) was treated with various concentrations of ethanol extract of A. capillaris (0.1, 0.5, 1, and 2 mg/mL) and capillarisin, the active component of A. capillaris (10⁻7, 10⁻6, 10⁻5 and 10⁻4 M). Capillarisin was also applied to PCC tissues contracted with Phe, which were pre-incubated with phosphodiesterase type 5 inhibitors (PDE5 Is). Cyclic nucleotides in the perfusate were measured by radioimmunoassay. The tissues were pre-incubated with Nω nitro-l-arginine-methyl ester (L-NAME, 10⁻³ M) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10⁻5 M) to block nitric oxide (NO) synthase and guanylate cyclase, respectively. Capillarisin induced penile relaxation and enhanced PDE5 Is-induced relaxation. Capillarisin increased cGMP and cAMP in the perfusate. The application of capillarisin on PCC pre-treated with L-NAME and ODQ significantly inhibited the relaxation. Capillarisin exerts the relaxing effect on PCC by activating the NO-cGMP and adenylyl cAMP signaling pathways and may become an alternative medicine for patients who want to use natural products to improve erectile function or do not completely respond to PDE5 Is.

Effects of ginseng total saponins on pacemaker currents of interstitial cells of Cajal from the small intestine of mice.

Although ginsenosides have a variety of physiologic or pharmacologic functions in various regions, there are only a few reports on the effects of ginsenosides on gastrointestinal (GI) motility. We studied the modulation of pacemaker activities by ginseng total saponins in the interstitial cells of Cajal (ICC) using the whole cell patch-clamp technique. Externally applied ginseng total saponins (GTS) produced membrane depolarization in the current-clamp mode and increased tonic inward pacemaker currents in the voltage-clamp mode. The application of flufenamic acid or niflumic acid abolished the generation of pacemaker currents, but only treatment with flufenamic acid inhibited the GTS-induced tonic inward currents. The tonic inward currents induced by GTS were not inhibited by the intracellular application of guanosine 5'-[beta-thio]diphosphate trilithium salt. Pretreatment with a Ca(2+)-free solution, with U-73122, an active phospholipase C inhibitor, and with thapsigargin, a Ca(2')-ATPase inhibitor of the endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the GTS-induced action. However, treatment with chelerythrine and calphostin C, protein kinase C inhibitors, did not block the GTS-induced effects on the pacemaker currents. These results suggest that ginsenosides modulate the pacemaker activities of the ICC, and the ICC can be targets for ginsenosides, and their interaction can affect intestinal motility.

Effects of pine needle extract on pacemaker currents in interstitial cells of Cajal from the murine small intestine.

Extracts of pine needles (Pinus densiflora Sieb. et Zucc.) have diverse physiological and pharmacological actions. In this study we show that pine needle extract alters pacemaker currents in interstitial cells of Cajal (ICC) by modulating ATP-sensitive K+ channels and that this effect is mediated by prostaglandins. In whole cell patches at 30 degrees , ICC generated spontaneous pacemaker potentials in the current clamp mode (I = 0), and inward currents (pacemaker currents) in the voltage clamp mode at a holding potential of -70 mV. Pine needle extract hyperpolarized the membrane potential, and in voltage clamp mode decreased both the frequency and amplitude of the pacemaker currents, and increased the resting currents in the outward direction. It also inhibited the pacemaker currents in a dose-dependent manner. Because the effects of pine needle extract on pacemaker currents were the same as those of pinacidil (an ATP-sensitive K+ channel opener) we tested the effect of glibenclamide (an ATP-sensitive K+ channels blocker) on ICC exposed to pine needle extract. The effects of pine needle extract on pacemaker currents were blocked by glibenclamide. To see whether production of prostaglandins (PGs) is involved in the inhibitory effect of pine needle extract on pacemaker currents, we tested the effects of naproxen, a non-selective cyclooxygenase (COX-1 and COX-2) inhibitor, and AH6809, a prostaglandin EP1 and EP2 receptor antagonist. Naproxen and AH6809 blocked the inhibitory effects of pine needle extract on ICC. These results indicate that pine needle extract inhibits the pacemaker currents of ICC by activating ATP-sensitive K+ channels via the production of PGs.