Alterations in serotonin, transient receptor potential channels and protease-activated receptors in rats with irritable bowel syndrome attenuated by Shugan decoction.

AIM: To determine the molecular mechanisms of Shugan decoction (SGD) in the regulation of colonic motility and visceral hyperalgesia (VHL) in irritable bowel syndrome (IBS). METHODS: The chemical compounds contained in SGD were measured by high-performance liquid chromatography. A rat model of IBS was induced by chronic water avoidance stress (WAS). The number of fecal pellets was counted after WAS and the pain pressure threshold was measured by colorectal distension. Morphological changes in colonic mucosa were detected by hematoxylin-eosin staining. The contents of tumor necrosis factor (TNF)-α in colonic tissue and calcitonin-gene-related peptide (CGRP) in serum were measured by ELISA. The protein expression of serotonin [5-hydroxytryptamide (5-HT)], serotonin transporter (SERT), chromogranin A (CgA) and CGRP in colon tissue was measured by immunohistochemistry. RESULTS: SGD inhibited colonic motility dysfunction and VHL in rats with IBS. Blockers of transient receptor potential (TRP) vanilloid 1 (TRPV1) (Ruthenium Red) and TRP ankyrin-1 (TRPA1) (HC-030031) and activator of protease-activated receptor (PAR)4 increased the pain pressure threshold, whereas activators of PAR2 and TRPV4 decreased the pain pressure threshold in rats with IBS. The effect of SGD on pain pressure threshold in these rats was abolished by activators of TRPV1 (capsaicin), TRPV4 (RN1747), TRPA1 (Polygodial) and PAR2 (AC55541). In addition, CGRP levels in serum and colonic tissue were both increased in these rats. TNF-α level in colonic tissue was also significantly upregulated. However, the levels of 5-HT, SERT and CgA in colonic tissue were decreased. All these pathological changes in rats with IBS were attenuated by SGD. CONCLUSION: SGD alleviated VHL and attenuated colon motility in IBS, partly by regulating TRPV1, TRPV4, TRPA1, PAR2, 5-HT, CgA and SERT, and reducing CGRP and TNF-α level.

The ethanol extract isolated from Weiqi Decoction induces G2/M arrest and apoptosis in AGS cells.

OBJECTIVE: To evaluate the effects of the ethanol extract isolated from Weiqi Decoction (WQD-EE) on AGS cell proliferation and apoptosis. METHODS: By using high-performance liquid chromatography with ultraviolet detectors (HPLC-UV) assay and MTT method, the main compounds in WQD-EE and cell viability were detected. And cell cycle distributions were determined by flow cytometry with propidium iodine (PI) staining while apoptosis was detected by flow cytometry with annexin V/PI double staining. Finally, caspase-3 activities were measured by colorimetric method and protein expression was determined by Western blotting. RESULTS: HPLC analysis showed that naringin (35.92 µg/mg), nobiletin (21.98 µg/mg), neohesperidin (17.98 µg/mg) and tangeretin (0.756 µg/mg) may be the main compounds in WQD-EE. WQD-EE not only inhibited AGS and MCF 7 cell proliferation in a dose-dependent manner, but also blocked cell cycle progression at G2/M stage as well as inducing cell apoptosis at concentrations triggering significant inhibition of proliferation and cell cycle arrest in AGS cells. While at 0.5 mg/mL, WQD-EE significantly increased caspase-3 activity by 2.75 and 7.47 times at 24 h and 48 h, respectively. Moreover, WQD-EE in one hand reduced protein expressions of p53 and cyclin B1, and in other hand enhanced protein expressions of cytochrome c and Bax. Protein levels of Bcl-2, Fas L and Fas were not significantly affected by WQD-EE. CONCLUSIONS: WQD-EE inhibits AGS cell proliferation through G2/M arrest due to down-regulation of cyclin B1 protein expression, and promotes apoptosis by caspase-3 and mitochondria-dependent pathways, but not by p53-dependent pathway.

Shugan-decoction relieves visceral hyperalgesia and reduces TRPV1 and SP colon expression.

AIM: To evaluate the therapeutic effect of Shugan-decoction (SGD) on visceral hyperalgesia and colon gene expressions using a rat model. METHODS: Ninety-six adult male Wistar rats were randomized into six equal groups for assessment of SGD effects on psychological stress-induced changes using the classic water avoidance stress (WAS) test. Untreated model rats were exposed to chronic (1 h/d for 10 d consecutive) WAS conditions; experimental treatment model rats were administered with intragastric SGD at 1 h before WAS on consecutive days 4-10 (low-dose: 0.1 g/mL; mid-dose: 0.2 g/mL; high-dose: 0.4 g/mL); control treatment model rats were similarly administered with the irritable bowel syndrome drug, dicetel (0.0042 g/mL); untreated normal control rats received no drug and were not subjected to the WAS test. At the end of the 10-d WAS testing period, a semi-quantitative measurement of visceral sensitivity was made by assessing the abdominal withdrawal reflex (AWR) to colorectal balloon-induced distension (at 5 mmHg increments) to determine the pain pressure threshold (PPT, evidenced by pain behavior). Subsequently, the animals were sacrificed and colonic tissues collected for assessment of changes in expressions of proteins related to visceral hypersensitivity (transient receptor potential vanilloid 1, TRPV1) and sustained visceral hyperalgesia (substance P, SP) by immunohistochemistry and real-time polymerase chain reaction. Inter-group differences were assessed by paired t test or repeated measures analysis of variance. RESULTS: The WAS test successfully induced visceral hypersensitivity, as evidenced by a significantly reduced AWR pressure in the untreated model group as compared to the untreated normal control group (190.4 ± 3.48 mmHg vs 224.0 ± 4.99 mmHg, P < 0.001). SGD treatments at mid-dose and high-dose and the dicetel treatment significantly increased the WAS-reduced PPT (212.5 ± 2.54, 216.5 ± 3.50 and 217.7 ± 2.83 mmHg respectively, all P < 0.001); however, the low-dose SGD treatment produced no significant effect on the WAS-reduced PPT (198.3 ± 1.78 mmHg, P > 0.05). These trends corresponded to the differential expressions observed for both TRPV1 protein (mid-dose: 1.64 ± 0.08 and high-dose: 1.69 ± 0.12 vs untreated model: 3.65 ± 0.32, P < 0.001) and mRNA (0.44 ± 0.16 and 0.15 ± 0.03 vs 1.39 ± 0.15, P < 0.001) and SP protein (0.99 ± 0.20 and 1.03 ± 0.23 vs 2.03 ± 0.12, P < 0.01) and mRNA (1.64 ± 0.19 and 1.32 ± 0.14 vs 2.60 ± 0.33, P < 0.05). These differential expressions of TRPV1 and SP related to mid- and high-dose SGD treatments were statistically similar to the changes induced by dicetel treatment. No signs of overt damage to the rat system were observed for any of the SGD dosages. CONCLUSION: Shugan-decoction can reduce chronic stress-induced visceral hypersensitivity in rats, and the regulatory mechanism may involve mediating the expressions of TRPV1 and SP in colon tissues.

[Effects of taraxerol and taraxerol acetate on cell cycle and apoptosis of human gastric epithelial cell line AGS].

OBJECTIVE: To investigate the effects of taraxerol and taraxerol acetate on cell cycle and apoptosis of human gastric epithelial cell line AGS cells. METHODS: The inhibitory effects of taraxerol and taraxerol acetate at different concentrations on AGS cell growth were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method and the concentrations of taraxerol and taraxerol acetate to be used in following experiments were decided. Then, cell cycle analysis was performed by FACScan flow cytometry after culture with taraxerol or taraxerol acetate. Annexin V-fluorescein isothiocyanate/propidium iodide staining was used to measure cell apoptosis. RESULTS: Taraxerol significantly inhibited AGS cell proliferation in a dose- and time-dependent manner. Taraxerol arrested the AGS cells at G(2)/M stage. 110 µmol/L taraxerol elevated the population of AGS cells arrested in G(2)/M phase compared with solvent (P<0.05). Taraxerol also promoted early cell apoptosis in AGS cells. 110 µmol/L taraxerol increased the early cell apoptosis rate from 4.45% to 10.29%, which was 1.31 times higher than that of the untreated cells. However, taraxerol acetate had a lower inhibitory effect than taraxerol, and it showed a tendency of G(2)/M arrest and apoptosis promotion but with no statistical significance (P>0.05). CONCLUSION: Taraxerol has inhibitory effects on AGS cell growth through inducing G(2)/M arrest and promotion of cell apoptosis. Taraxerol acetate has less effect on cell cycle arrest and apoptosis of AGS cells than taraxerol.

Antiproliferative effects of essential oil of a compound Chinese herbal medicine Weiqi Decoction on AGS cells.

OBJECTIVE: The main ingredients and the inhibitory effects of essential oil of a compound Chinese herbal medicine Weiqi Decoction (WQD) on AGS cell proliferation were to be investigated. METHODS: Chemical compounds of WQD essential oil were detected by gas chromatography and mass spectrometry analysis. Cell viability was measured by methyl thiazolyl tetrazolium method. Cell cycle distribution was detected by flow cytometry. Apoptosis and necrosis of AGS cells were determined by Hoechst 33342/propidium iodine staining. RESULTS: Chemical analysis showed that the main ingredients of WQD essential oil were bornylene and 3-n-butylphthalide. Ligustilide, which is the effective compound of Danggui (Radix Angelicae Sinensis), was not detected in WQD essential oil. The essential oil inhibited cell proliferation in a dose- and time-dependent manner, and blocked cell cycle progression at G(2)/M stage. At the concentrations that resulted in significant inhibition of cell proliferation and cell cycle arrest, essential oil induced both apoptosis and necrosis. CONCLUSION: The results suggest that WQD essential oil contains some effective ingredients for treating chronic atrophic gastritis and functional dyspepsia, and also has an antiproliferative effect on AGS cells through cell cycle arrest and apoptosis promotion in vitro. Therefore, essential oil should be retained as much as possible during stewing this decoction.

[Effect of Qingchang Suppository on intestinal permeability in rats with ulcerative colitis].

OBJECTIVE: To observe the effect of Qingchang Suppository (QCS, a Chinese herbal preparation) on intestinal permeability in rat ulcerative colitis (UC) model induced by trinitrobenzene sulforic acid, and to explore the mechanism of QCS for healing the ulceration. METHODS: Labelled by FITC-dextran 4 000 (FD-4), the permeability of colonic membrane in UC rat and effect of QCS on it were observed in vitro and in vivo. RESULTS: In vivo study showed that the colonic FD-4 permeability of UC rat was increased significantly, being 6-fold of normal in 30 min. After treated with QCS of high/moderate dosage, it significantly attenuated to different degrees (P < 0.05). FD-4 permeability coefficient (Papp) determination in vitro showed that Papp in model rats increased to (5.001 +/- 1.316) x10(-8) cm/s in 120 min, being 2.5-fold of control; and which could be decreased by high/moderate dose QCS effectively (P < 0.05). CONCLUSION: QCS could suppress the high colonic permeability in UC model rats, improve the barrier function of intestinal membrane and promote the healing of ulceration. Qingchang Suppository; ulcerative colitis; intestinal permeability in UC model rats, improve the barrier function of intestinal membrane and promote the healing of ulceration.

[Tongxie Yaofang inhibits the contraction of colonic smooth muscle isolated from rats through a mechanism related to calcium mobilization].

OBJECTIVE: To study the relationship between the inhibitory effects of Tongxie Yaofang, a compound traditional Chinese herbal medicine, on the contraction of the colonic smooth muscle isolated from rats and calcium mobilization. METHODS: By measuring the tension of the isolated colonic smooth muscle strips, the inhibitory effects of Tongxie Yaofang on the contraction induced by acetylcholine (ACh), KCl and exhausting Ca(2+) of internal calcium store were assessed respectively. RESULTS: Tongxie Yaofang could concentration-dependently inhibit the contraction of isolated rat colonic smooth muscle strips induced by KCl and exhausting the Ca(2+) of internal calcium store. Tongxie Yaofang could also inhibit the tension of the second contractile phase induced by ACh (P<0.01, vs control), but had no influence on the first contractile phase. CONCLUSION: Tongxie Yaofang can inhibit the contraction of isolated rat colonic smooth muscle strips mainly by preventing the influx of extracellular Ca(2+), which may be associated with blocking voltage-dependent channel, store-operated channel and receptor-operated channel, but not by preventing the release of internal Ca(2+) from calcium store.