Inhibitory Effect Of Sanguinarine On The Smooth Muscle Contractions In Isolated Rat Small Intestine And The Signal Pathway

Gastrointestinal spasms and cramps can result from gastrointestinal dysfunction and organic lesion. Atropine, a nonselective muscarinic receptors blocker, and the Ca2+channel blocker nifedipine can be used in the treatment of gastrointestinal diseases. As is true for all antimuscarinic agents, the clinical uses are limited due to severe side-effects. However, nifedipine has a potent negative dromotropic effect with dose increase after long-term administration. It's an important to develop the safe, efficient and no residue of anticholinergic or calcium channel antagonistic of natural medicine. Sanguinarine (SA) is a benzophenanthridine alkaloid which is derived mainly from the roots of Sauguinaria Canadensis L., Chelidonium majus L. and the seeds of Argemone mexicana L., and has been revealed for the treatment of gastrointestinal (GI) diseases by immediate the motility of the gastrointestinal (GI), including abdominal pain, diarrhea, and intestinal inflammation for recent of years. In our previous study, supplement of SA into the animal feed could improve the feed conversion and growth rate, and decrease the piglet diarrhea (Cai et al.,2010). However, the underlying mechanisms of action of SA on GI motility are unclear. Therefore, inhibitory effect of SA on smooth muscle cells contractions and the signal pathway in isolated rat small intestine were demonstrated by using the methods of cytobiology, molecular biology, and immunofluorescence.1. Duodenum tissue was isolated from rat for in vitro intestine movement experiment and biologic function analysis system was used to record the frequency and amplitude of the spontaneous intestinal muscle contractions induced by ACh/Atropine, HA/Proazamine and CaCl2/Nifedipine. SA dose dependently inhibited the frequency and amplitude of spontaneous contractions with a50%inhibition (IC50). The frequency and amplitude were28.31and11.37μM, respectively. In addition, SA inhibited the contractions of intestinal smooth muscle which were induced by acetylcholine (ACh), histamine (HA) and calcium chloride (CaCl2). The IC50of Ach's frequency and amplitude were25.14and13.77μM, respectively. The IC50of HA's frequency and amplitude were28.33and14.72μM, respectively. The IC50of CaCl2's frequency and amplitude were27.90and3.35μM, respectively. Also, SA significantly strengthened the relaxant effect caused by atropine, promethazine. and nifedipine. Therefore, the inhibitory effects of SA might be involved in Muscarinic acetylcholine receptors. H1receptor and an L-type Ca2+channel that may mediate the relaxation in intestinal muscle. 2. The smooth muscle cells were isolated from rat intestines using collagenase digestion, and then cells were treated with SA or agonists (ACh, HA, or KCl) after immunohistochemistry identification. Cell lengths were measured using a phase-contrast microscope. The result showed that SA dose-dependently inhibited spontaneous cell contractions and induced by ACh, HA, or KC1. Meanwhile, the inhibitory effect of atropine in cells was enhanced by SA.3. ISMC were incubated with different concentrations of SA for24h. The mRNA expression of M2and M3receptors were measured using Real-Time PCR analysis. The mRNA of M2and M3receptors in ISMC was expressed by RT-PCR, especially M2receptor. RT-PCR analysis showed that mRNA expression of M2and M3receptors were significantly inhibited in smooth muscle cells isolated from rat intestines exposed to SA.4. ISMC were respectively treated with SA or agonists (CCh or KCl), and then intracellular Ca2+concentration was measured using a Ca2+-sensitive fluorescent indicator fura-2/AM by spectrofluorophotometer. The result showed that SA dose-dependently inhibited the concentration of intracellular [Ca2+]i. Meanwhile, SA inhibited KCl or CCh mediated increase in resting [Ca2+]i in1.8mM Ca2+-Hepes buffer, but SA did not inhibited CCh or KCl induced raise [Ca2+]i under Ca2+-free conditions. These results demonstrated that SA was mainly inhibited extracellular Ca2+influx through the Ca2+channel, also known as receptor-operative Ca2+channel.5. We examined the effect of SA on the mRNA and protein expression of PKC isoforms and CPI-17using RT-PCR and Western blot analysis in ISMC. PKC isoforms (δ,ε,η) and CPI-17were detected in rat ISMC. CCh(10μM,10min) rapidly caused an increase in PKCs and PKCη mRNA, and at the concentration of100μM for60min significantly increased mRNA levels of PKCS, PKCη and CPI17. RT-PCR analysis showed that treatment of ISMC for30min with SA (1or3μM) significantly decreased mRNA expression of PKCδ, PKCε, PKCη and CPI17. SA (1μM) markedly inhibited CCh-mediated increase in PKCδ, PKCη and CPI17mRNA. Treatment of ISMC with SA for a short period (30min) caused a decrease in PKCδ protein expression; however, the amount of CPI-17was significantly inhibited for24h by Western bolts analysis. SA markedly inhibited CCh-mediated increase in PKCδ and CPI17protein. This result demonstrated that the inhibitory effect of SA by down regulates PKC and CPI-17mRNA and protein expression in rat ISMC.In conclusion, the GI smooth muscle excitation-contraction coupling is mediated by G protein regulation of PKC-CPI-17. The underlying mechanisms of the inhibitory effect of SA on intestinal smooth muscle contracture were determined by fluorescence labeling, real-time RT-PCR and Western bolt analysis. The results demonstrated that the inhibitory effect of SA is associated with alteration of M receptors, intracellular Ca2+concentration and PKC-CPI-17signal transduction that regulate smooth muscle contraction.