[Effect of Kanli Granule on Myocardial Mechanics in Pressure Overload Induced Diastolic Heart Failure Rats].

OBJECTIVE: To observe the effect of Kanli Granule (KG) on myocardial mechanics in pressure overload induced diastolic heart failure (DHF) rats. METHODS: Totally 60 male Wistar rats were divided into the sham-operation group, the model group, the KG group, and the Valsartan group according to random digit table, 15 in each group. The pressure overload induced DHF model was established in all groups except the sham-operation group using abdominal aortic constriction surgery. Totally 7 rats died after modeling (with the mortality of 10. 67%) , and the rest 53 finished the following test. Rats in the KG group were administered with KG extract (calculated as 6. 75 g crude drug/kg) by gastrogavage. Rats in the Valsartan group were administered with Valsartan (7.2 µg/g) by gastrogavage. Equal volume of double distilled water was administered to rats in the model group and the sham-operation group by gastrogavage. All rats were intervened for 32 weeks. The response of isolated heart papillary muscle tonus to isoprenaline (ISO) and adenylate cyclase (Forskolin) was respectively observed. The enhancement phenomenon after resting development force (DF) of isolated heart papillary muscle tonus, and changes of DF in different Ca²âº concentrations were observed. RESULTS: (1) In the ISO response test: Compared with the sham-operation group, the amplifications of DF, ±df/dt, -df/dt were obviously elevated in the model group (P < 0.05). Compared with the model group, the amplifications of DF and ±df/dt were obviously lowered in the KG group (P < 0.01), and the amplification of ±df/dt was also reduced in the Valsartan group (P < 0.01). (2) In the Forskolin response test: Compared with the sham-operation group, the amplifications of DF and ±df/dt obviously increased in the model group (P < 0.05). Compared with the model group, the amplifications of DF and ±df/dt were obviously reduced in the KG group (P < 0.01), and the amplification of DF was also reduced in the Valsartan group (P < 0.05). (3) In post-resting DF enhancement test: Compared with the sham-operation group, the amplification of DF showed gradually decreasing tendency along with prolonged resting time in the model group, and they were obviously lowered at all time points (P < 0.05). Compared with the model group, the amplification of DF was gradually increasing along with prolonged resting time in the KG group. The amplification of DF at post-resting 240 s was obviously larger in the KG group than in the model group (P < 0.05). The amplification of post-resting DF still showed gradually decreasing tendency along with prolonged resting time in the Valsartan group, with increased amplifications of DF at post-resting 60 s and 120 s (P < 0. 05) (4) The amplifications of DF in different Ca²âº concentrations: Compared with the sham-operation group, the amplifications of DF were significantly elevated in different Ca²âº concentrations (1.75, 3.5, 7.0 mmol/L ) (P < 0.05, P < 0.01). Compared with the model group, there was no statistical difference in amplification of DF in different Ca²âº concentrations in the KG group (P > 0.05). The amplifications of DF in different Ca²âº concentrations were significantly reduced in the Valsartan group (P < 0.05). CONCLUSIONS: The ISO response and the Forskolin response were enhanced in isolated heart papillary muscle tonus of pressure overload induced DHF rats; enhanced post-resting DF was reduced; DF in different supra-physiologic levels of Ca²âº was still enhanced. KG could significantly improve excessive enhancement of pressure overload induced DHF rats in ISO response and Forskolin response, and improve enhancement of post-resting myocardium.

Kurarinone Synergizes TRAIL-Induced Apoptosis in Gastric Cancer Cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been identified as a promising anti-tumor agent against in a variety of cancers. However, gastric cancer cells are less sensitive than other cancer cells to TRAIL-induced apoptosis. Here, we combined TRAIL with kurarinone, a natural compound, to induce apoptosis in gastric cancer cell lines SGC7901. After the cells were treated with TRAIL and/or kurarinone, the cell viability and apoptosis were examined by MTT and flow cytometry, respectively. The expression of apoptosis-associated proteins was determined by western blot and q-RT-PCR. Kurarinone at low concentration significantly potentiated the cytotoxic effect of TRAIL by enhancing apoptosis as well as cell cycle arrest at G2/Mphase. The enhancement of apoptosis TRAIL induced by kurarinone involved downregulation of anti-apoptotic proteins Mcl-1 and c-FLIP as well as inhibition of STAT3 signaling. Moreover, we found that STAT3 inhibitor could synergistically enhanced TRAIL-induced apoptosis, similar to kurarinone. Kurarinone synergizes TRAIL-induced apoptosis in human gastric cancer cells. The synergistic effect between these two drugs is associated with downregulation of Mcl-1 and c-FLIP via inhibiting STAT3 signaling. The combination of TRAIL and kurarinone might be an effective regimen for the treatment of advanced gastric cancer.

Airway hyperresponsiveness induced by repeated esophageal infusion of HCl in guinea pigs.

Gastroesophageal reflux is a common disorder closely related to chronic airway diseases, such as chronic cough, asthma, chronic bronchitis, and chronic obstructive disease. Indeed, gastroesophageal acid reflux into the respiratory tract causes bronchoconstriction, but the underlying mechanisms have still not been clarified. This study aimed to elucidate functional changes of bronchial smooth muscles (BSMs) isolated from guinea pigs in an animal model of gastroesophageal reflux. The marked airway inflammation, hyperresponsiveness and remodeling were observed after guinea pigs were exposed to intraesophageal HCl infusion for 14 days. In addition, contractile responses to acetylcholine (ACh), KCl, electrical field stimulation, and extracellular Ca(2+) were greater in guinea pigs infused with HCl compared with control groups. The L-type voltage-dependent Ca(2+) channels (L-VDCC) blocker, nicardipine, significantly inhibited ACh- and Ca(2+)-enhanced BSM contractions in guinea pigs infused with HCl. The Rho-kinase inhibitor, Y27632, attenuated ACh-enhanced BSM contractions in guinea pigs infused with HCl. Moreover, mRNA and protein expressions for muscarinic M2 and M3 receptors, RhoA, and L-VDCC in BSM were detected by real-time PCR and Western blot. Expressions of mRNA and protein for muscarinic M3 receptors, RhoA, and L-VDCC were greater than in BSM of HCl-infused guinea pigs, whereas levels of muscarinic M2 receptors were unchanged. We demonstrate that acid infusion to the lower esophagus and, subsequently, microaspiration into the respiratory tract in guinea pigs leads to airway hyperresponsiveness and overactive BSM. Functional and molecular results indicate that overactive BSM is the reason for enhancement of extracellular Ca(2+) influx via L-VDCC and Ca(2+) sensitization through Rho-kinase signaling.

Tangeretin, a citrus polymethoxyflavonoid, induces apoptosis of human gastric cancer AGS cells through extrinsic and intrinsic signaling pathways.

Tangeretin, a natural polymethoxyflavone present in citrus peel oil, is known to have anticancer activities in breast cancer, colorectal carcinoma and lung carcinoma, yet, the underlying mechanisms of tangeretin in human gastric cancer AGS cells have not been investigated to date. In the present study, the apoptotic mechanisms of tangeretin in AGS cells were explored. It was observed that tangeretin increased the apoptotic rates of AGS cells following treatment with tangeretin for 48 h in a dose-dependent manner by Annexin V-FITC and PI double staining. In addition, characteristic apoptotic morphology such as nuclear shrinkage and apoptotic bodies was observed after Hoechst 33258 staining. Flow cytometric assay showed that treatment of AGS cells with tangeretin decreased the mitochondrial membrane potential (MMP) in a dose-dependent manner, which indicated that mitochondrial dysfunction was involved in the tangeretin-induced apoptosis. Caspase-3, -8 and -9 activities were increased by tangeretin in a dose-dependent manner. Western blotting showed that the protein levels of pro-apoptotic proteins including cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax, Bid, tBid, p53, p21/cip1, Fas and FasL were significantly upregulated by tangeretin. In addition, PFT-α (a p53 inhibitor) reduced the apoptotic rates and the expression of p53, p21, caspase-3 and caspase-9 induced by tangeretin, indicating that tangeretin-induced apoptosis was p53-dependent. In conclusion, these results suggest that tangeretin induces the apoptosis of AGS cells mainly through p53-dependent mitochondrial dysfunction and the Fas/FasL-mediated extrinsic pathway.

Curcumin induces apoptosis in human gastric carcinoma AGS cells and colon carcinoma HT-29 cells through mitochondrial dysfunction and endoplasmic reticulum stress.

In the present study, we investigate the effect of curcumin, a major active component isolated from rhizomes of Curcuma longa, on the cytotoxicity of three human carcinoma cell lines (AGS, HT-29 and MGC803) in gastrointestinal tract and a normal gastric epithelial cell line GES-1, and the mechanism of curcumin-induced apoptosis. The results indicated that curcumin inhibited the gastrointestinal carcinoma cell growth in a dose-dependent manner and cytotoxicity was more towards the gastric carcinoma cell AGS and colon carcinoma cell HT-29 compared to normal gastric cell GES-1, and increased externalization of phosphatidylserine residue was observed by Annexin V/PI staining in the two cell lines. Treatment of AGS and HT-29 cells with curcumin enhanced the cleavage of procaspase-3, -7, -8 and -9. Meanwhile, curcumin induced endoplasmic reticulum (ER) stress and mitochondrial dysfunction as evidenced by up-regulation of CCAAT/enhancer binding protein homologous protein (CHOP), phosphorylation of JNK and down-regulation of SERCA2ATPase, release of cytochrome c, decrease of Bcl-2 and reduction of mitochondrial membrane potential in both AGS and HT-29 cells. Overexpression of bax, total JNK, phospho-FADD and total FADD were also observed in curcumin-treated HT-29 cells. Moreover, curcumin decreased cytosolic and ER Ca(2+), but increased mitochondrial Ca(2+) in the two cell lines. 2-Aminoethoxydiphenyl borate, an antagonist of inositol 1, 4, 5-triphosphate receptor, partly blocked curcumin-induced cytosolic Ca(2+) decrease in AGS and HT-29 cells. Additionally, carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial Ca(2+) uptake, reversed curcumin-triggered AGS and HT-29 cells growth inhibition. siRNA to CHOP markedly reduced curcumin-induced apoptosis. These results suggest that curcumin can impact on ER stress and mitochondria functional pathways in AGS and HT-29 cells, death receptor pathway was also involved in curcumin-treated HT-29 cells, thus identifying specific well-defined molecular mechanisms that may be targeted by therapeutic strategies.

Targeted inhibition of KCa3.1 channel attenuates airway inflammation and remodeling in allergic asthma.

KCa3.1 has been suggested to be involved in regulating cell activation, proliferation, and migration in multiple cell types, including airway inflammatory and structural cells. However, the contributions of KCa3.1 to airway inflammation and remodeling and subsequent airway hyperresponsiveness (AHR) in allergic asthma remain to be explored. The main purpose of this study was to elucidate the roles of KCa3.1 and the potential therapeutic value of KCa3.1 blockers in chronic allergic asthma. Using real-time PCR, Western blotting, or immunohistochemical analyses, we explored the precise role of KCa3.1 in the bronchi of allergic mice and asthmatic human bronchial smooth muscle cells (BSMCs). We found that KCa3.1 mRNA and protein expression were elevated in the bronchi of allergic mice, and double labeling revealed that up-regulation occurred primarily in airway smooth muscle cells. Triarylmethane (TRAM)-34, a KCa3.1 blocker, dose-dependently inhibited the generation and maintenance of the ovalbumin-induced airway inflammation associated with increased Th2-type cytokines and decreased Th1-type cytokine, as well as subepithelial extracellular matrix deposition, goblet-cell hyperplasia, and AHR in a murine model of asthma. Moreover, the pharmacological blockade and gene silencing of KCa3.1, which was evidently elevated after mitogen stimulation, suppressed asthmatic human BSMC proliferation and migration, and arrested the cell cycle at the G0/G1 phase. In addition, the KCa3.1 activator 1-ethylbenzimidazolinone-induced membrane hyperpolarization and intracellular calcium increase in asthmatic human BSMCs were attenuated by TRAM-34. We demonstrate for the first time an important role for KCa3.1 in the pathogenesis of airway inflammation and remodeling in allergic asthma, and we suggest that KCa3.1 blockers may represent a promising therapeutic strategy for asthma.

CYP450 1A2 and multiple UGT1A isoforms are responsible for jatrorrhizine metabolism in human liver microsomes.

Jatrorrhizine, one of the protoberberine alkaloids derived from the plant Coptis chinensis, is expected to be developed as a new gastric prokinetic drug, but its metabolic characteristics in humans remain unknown. This study characterized the phase I and phase II metabolites, metabolic kinetics, and cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of jatrorrhizine in human liver microsomes (HLMs). Chemical inhibition in HLMs and metabolism by recombinant human CYP or UGT enzymes were employed to determine the key metabolic enzyme subtypes. In HLMs, demethyleneberberine (demethylated product) and jatrorrhizine glucuronide were identified as the phase I and phase II metabolites, respectively. The enzyme kinetics for both demethylation and glucuronidation were fitted to the Michaelis-Menten equation. Demethylation was inhibited significantly by furafylline and predominantly catalysed by recombinant CYP1A2, whereas glucuronidation was inhibited by silibinin, quercetin, as well as 1-naphthol and catalysed by recombinant UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9 and UGT1A10. These results showed that jatrorrhizine is metabolized by human CYP1A2 and multiple UGT1A isoforms.

α-asarone from Acorus gramineus alleviates epilepsy by modulating A-type GABA receptors.

Alpha (α)-asarone is a major effective compound isolated from the Chinese medicinal herb Acorus gramineus, which is widely used in clinical practice as an antiepileptic drug; however, its mechanism of action remains unclear. In this study, we have characterized the action of α-asarone on the excitability of rat hippocampal neurons in culture and on the epileptic activity induced by pentylenetetrazole or kainate injection in vivo. Under cell-attached configuration, the firing rate of spontaneous spiking was inhibited by application of α-asarone, which was maintained in the Mg(2+)-free solution. Under whole-cell configuration, α-asarone induced inward currents in a concentration-dependent manner with an EC(50) of 248 ± 33 µM, which was inhibited by a GABA(A) receptor blocker picotoxin and a competitive GABA(A) receptor antagonist bicuculline but not a specific glycine receptor inhibitor strychnine. Measurement of tonic GABA currents and miniature spontaneous inhibitory postsynaptic currents indicated that α-asarone enhanced tonic GABAergic inhibition while left phasic GABAergic inhibition unaffected. In both pentylenetetrazole and kainate seizure models, α-asarone suppressed epileptic activity of mice by prolonging the latency to clonic and tonic seizures and reducing the mortality as well as the susceptibility to seizure in vivo presumably dependent on the activation of GABA(A) receptors. In summary, our results suggest that α-asarone inhibits the activity of hippocampal neurons and produces antiepileptic effect in central nervous system through enhancing tonic GABAergic inhibition.

Subunit-specific inhibition of glycine receptors by curcumol.

Emerging evidence has suggested that inhibitory glycine receptors (GlyRs) are an important molecular target in the treatment of numerous neurological disorders. Rhizoma curcumae is a medicinal plant with positive neurological effects. In this study, we showed that curcumol, a major bioactive component of R. curcumae, reversibly and concentration-dependently inhibited the glycine-activated current (IGly) in cultured rat hippocampal neurons. The inhibitory effect was neither voltage- nor agonist concentration-dependent. Moreover, curcumol selectively inhibited homomeric α2-containing, but not α1- or α3-containing, GlyRs. The addition of ß subunit conferred the curcumol sensitivity of α3-containing, but not α1-containing, GlyRs. Site-directed mutagenesis analysis revealed that a threonine at position 59 of the α2 subunit is critical for the susceptibility of GlyRs to curcumol-mediated inhibition. Furthermore, paralleling a decline of α2 subunit expression during spinal cord development, the degree of IGly inhibition by curcumol decreased with prolonged culture of rat spinal dorsal horn neurons. Taken together, our results suggest that the GlyRs are novel molecular targets of curcumol, which may underlie its pharmaceutical effects in the central nervous system.

Pharmacokinetics and metabolism of jatrorrhizine, a gastric prokinetic drug candidate.

Jatrorrhizine, a protoberberine alkaloid derived from Coptis chinensis, is currently under investigation as a natural gastric prokinetic drug candidate. In vitro and in vivo studies were conducted to characterize its pharmacokinetics and metabolism. After intravenous administration, the plasma concentration kinetics and major metabolites in rats were investigated. The metabolic kinetics, key cytochrome P450 enzymes and UDP-glucuronosyltransferase isoforms (UGTs) of jatrorrhizine were studied in rat liver microsomes (RLMs). After intravenous administration, plasma jatrorrhizine concentrations showed a biphasic decline, dose-independent clearance and half-life of terminal elimination phase, and a relatively large distribution volume. The metabolic pathway for the conversion of jatrorrhizine was important for its elimination. In addition, the demethylated and glucuronidated products were found to be the major metabolites in rats. The enzyme kinetics for both demethylation and glucuronidation were fitted to the hyperbolic Michaelis-Menten equation in RLMs. CYP3A1/2 and CYP2D2 were mainly responsible for demethylation, and UGT 1A1 and 1A3 were responsible for glucuronidation in RLMs. The metabolic properties of jatrorrhizine suggest multiple metabolic pathways. These results will contribute to promote further research and development of jatrorrhizine.

Effect of jatrorrhizine on delayed gastrointestinal transit in rat postoperative ileus.

OBJECTIVES: Postoperative ileus is major cause of postoperative complication and prolonged hospitalization. Jatrorrhizine, which is a protoberberine alkaloid isolated from the medicinal plants Berberis aristata and Coptis chinensis, has been found to increase contractility of gastric antral and ileum smooth muscles of rat gastrointestinal tract. We have investigated whether jatrorrhizine could offset gastrointestinal transit in rat with postoperative ileus. METHODS: Postoperative ileus was induced by laparotomy with intestinal manipulation under anaesthesia. Gastrointestinal transit was evaluated by measurement of gastric emptying, geometric centre and the migration of Evans blue. KEY FINDINGS: Postoperative ileus significantly delayed gastric emptying and intestinal transit. Jatrorrhizine dose-dependently (0.1, 0.3 and 1 mg/kg) offset delayed gastric emptying and intestinal transit (geometric centre and the migration of Evans blue) in postoperative ileus. Pretreatment of animals with atropine inhibited the action of jatrorrhizine on gastric emptying and intestinal transit, but pretreatment of animals with SB204070 did not influence the effect of jatrorrhizine on gastric emptying and intestinal transit in postoperative ileus. CONCLUSIONS: Jatrorrhizine offset postoperative ileus-induced delayed gastric emptying and intestinal transit in rats, an action mediated via the cholinergic pathway, but not involving activation of 5-HT(4) receptors.

[Effects of sinensetin on proliferation and apoptosis of human gastric cancer AGS cells].

OBJECTIVE: To study the effects and mechanisms of sinensetin on proliferation and apoptosis of human AGS gastric cancer cells. METHOD: MTT assay was used to detect the growth inhibition rates of human AGS gastric cancer cells treated with sinsesectin in different concentrations and times. The cell cycle distribution was measured by flow cytometry. The apoptosis was examined by Annexin-FITC/PI staining and DNA fragment analysis. The apoptosis morphology was observed by inverted fluorescence microscope after Hoechst 33342 staining. The protein expressions of p21 and p53 were detected by western blot. RESULT: MTT assay showed that sinensetin inhibited the growth of AGS gastric cancer cells in a dose- and time-dependent manner. Sinensetin blocked AGS cells in G2/ M and increased the apoptosis rates of AGS cells in a dose-dependent manner. DNA ladder was observed in cells treated with 60 micromol x L(-1) sinensetin for 48 h. The typical apoptotic morphological changes including cell nucleus shrinkage, chromatin condensation and apoptotic bodies were observed when treated with different dose of sinensetin. Western blot showed that sinensetin increased expressions of p53 and p21 in a dose-dependent manner. CONCLUSION: Sinensetin could inhibit human AGS gastric cancer cells proliferation and induce cell cycle block in G2/M phase and apoptosis. The up regulation of p53 and p21 protein might be one of the mechanisms.

The effects of jatrorrhizine on contractile responses of rat ileum.

This study was designed to evaluate the effect of jatrorrhizine on smooth muscle contractions isolated from rat ileum longitudinal muscles. Jatrorrhizine increased the amplitude of spontaneous contractions of ileum longitudinal muscles in concentration-dependent manner with an EC(50) of 30.0±8.4µM. Preincubation of ileum strips with atropine (1µM), 4-diphenyllacetoxy-N (2-chloriethyl)-piperidine (4-DAMP, 1µM) or darifenacin (1µM) significantly inhibited acetylcholine (0.1µM)- and jatrorrhizine (100µM)-induced ileum longitudinal muscle contractions, whereas they were not affected by AF-DX116 (1µM) or hexamethonium (100µM). Pretreatment with SB204070 (1µM) rather than 3-tropanyl-indole-3-carboxyleat (tropisetron, 1µM) significantly inhibited 5-HT (10µM)-induced ileum longitudinal muscle contractions. In contrast, jatrorrhizine-induced ileum longitudinal muscle contractions were not inhibited by tropisetron or SB204070. Furthermore, jatrorrhizine-induced ileum longitudinal muscle contractions were strongly inhibited by nifedipine (1µM), and also attenuated by removal of extracellular Ca(2+), U73122 (1µM), ruthenium red (50µM) or 2-aminoethoxydiphenylborate (2-APB, 10µM). Taken together, jatrorrhizine-elicited spontaneous contractions in rat ileum longitudinal muscles are mediated by activation of acetylcholine receptors, mostly the M(3) receptor. Ca(2+) influx through L-type Ca(2+) channel is significantly contributed to jatrorrhizine-elicited spontaneous contractions, and Ca(2+) release via IP(3) and ryanodine pathways are also involved.

Traditional Chinese formula, lubricating gut pill, stimulates cAMP-dependent CI(−) secretion across rat distal colonic mucosa.

AIM OF THE STUDY: Lubricating gut pill (LGP), a traditional Chinese formula, had been conformed to improve the loperamide-induced rat constipation by stimulation of Cl(-) secretion, but its mechanism has not been fully explored. Thus, the purpose of this study was to identify the action sites of LGP-stimulated Cl(-) secretion across rat distal colonic mucosa. MATERIALS AND METHODS: Rat distal colonic mucosa was mounted in Ussing chambers and short circuit current (I(SC)), apical Cl(-) current and basolateral K(+) current were recorded. Intracellular cyclic adenosine monophosphate (cAMP) content and protein kinase A (PKA) activity were determined with ELISA kit and the non-radioactive PepTag test, respectively. RESULTS: LGP at 800µg/ml elicited a sustained increase in Cl(-) secretory response, which was inhibited by CFTR(inh)172, a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. Permeabilizing apical membrane with nystatin revealed that LGP-stimulated basolateral K(+) current was significantly inhibited by KCNQ1 K(+) channel inhibitor chromanol 293B. LGP-stimulated I(SC) was markedly reduced by pretreatment with cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2amine (MDL-12,330A) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), but not with inhibitors of Ca(2+)-dependent signaling pathway. Treatment of tissue with LGP resulted in an increase in intracellular cAMP level and the activation in protein kinase A. The E-prostanoid(4) (EP)(4) receptor antagonist L-161,982 completely eliminated LGP-induced response. CONCLUSIONS: The results showed that LGP enhances Cl(-) and fluid secretion via prostanoid receptor signaling and also cAMP and protein kinase A pathway, subsequently triggering the activation of apical Cl(-) channels mostly CFTR and basolateral cAMP-dependent K(+) channel.

[Effects of Coptis chinensis and Evodia rutaecarpa water extract on DMH-induced precancerous lesion of colon].

OBJECTIVE: To investigate the effects of Coptis chinensis and Evodia rutaecarpa water extract on precancerous lesion of colon induced by DMH and proliferation and apoptosis changes of colon mucosa crypts. METHOD: Precancerous lesion of colon was induced by DMH. The changes of proliferation and apoptosis of colon mucosa crypts were detected by morphological analysis. The numbers of aberrant crypt foci (ACF) were measured by feulgen staining. RESULT: C. chinensis and E. rutaecarpa water extract could significantly inhibit the formation of ACF in model animals. The proliferative crypts were increased obviously in middle and distal colon, and decreased by C. chinensis and E. rutaecarpa water extract. The apoptosis crypts were increased in distal colon but not middle colon. C. chinensis and E. rutaecarpa water extract could promote apoptosis of both middle and distal colon. CONCLUSION: C. chinensis and E. rutaecarpa water extract could significantly inhibit the formation of ACF in model animals. These results indicated that C. chinensis and E. rutaecarpa water extract maybe have an inhibitory and clinically therapeutic effect on colon cancer, which were partly resulted from inhibiting proliferation and promoting apoptosis of crypts in middle and distal colon.

Transient receptor potential A1 is involved in cold-induced contraction in the isolated rat colon smooth muscle.

Transient receptor potential (TRP) A1, a member of TRP channel family, is activated by noxious cold. The aims of this study were to determine if TRPA1 contributed to cold-induced contractions in the isolated rat colon preparations and explore the potential mechanisms. The colon smooth muscle layers were surgically isolated from the male Wistar rats and changes in isotonic tension of longitudinal muscle under various treatments were recorded as colonic motilities. Cold stimuli were obtained by the reperfusion with Krebs-Henseleit solution at given temperature using Constant Flow Pump. The mRNA expressions of TRPA1, TRPV1 and TRPM8 in rat colon smooth muscle layer were examined by using reverse transcription-polymerase chain reaction (RT-PCR) techniques. The results showed that the contractions induced by cold stimuli (from 37 degrees C to 12 degrees C stepwise) were inversely proportional to the temperature with a maximum contraction at 17 degrees C in both proximal and distal colons (P<0.01). RT-PCR analysis revealed the expression of TRPA1, but not TRPM8 and TRPV1, in the rat proximal and distal colon smooth muscle layers. Cold-induced colonic contractions were specially inhibited by TRPA1 blocker, ruthenium red (30 µmol/L), in the proximal and distal colon (P<0.05). The cold-induced contractions of proximal (P<0.01, P<0.05) and distal colons (both P<0.001) were almost abolished or inhibited by the pretreatments of TRPA1 agonists, Allyl isothiocyanate (AITC, 300 µmol/L) and cinnamaldehyde (CA, 1 mmol/L). Extracellular calcium removal (EGTA, 1 mmol/L), PLC blocker (U73122, 10 µmol/L) and IP(3) receptor blocker (2-aminoethoxydiphenyl borate, 2-APB, 30 µmol/L) all decreased the contractions evoked by the cooling at 17 degrees C in the proximal and distal colon (P<0.001, P<0.05, P<0.001). Atropine (1 µmol/L) had no effects on these contractions. L-type Ca(2+) channels blocker nifedipine (1 µmol/L) and neurotoxin tetrodotoxin (TTX, 2 µmol/L) decreased the contractile response in the distal colon (P<0.01, P<0.05), but not in the proximal colon. In conclusion, TRPA1 contributes to cold-induced contractions of the rat colon smooth muscle, and the mechanism of TRPA1 activation involves PLC/IP(3)/Ca(2+) pathway. L-type Ca(2+) channel and neurogenic mechanism other than muscarinic receptor might be partially involved in cold-induced contraction of the distal colon, which probably resulted in higher contraction of distal colon compared with that of proximal colon.

Structural shifts of gut microbiota as surrogate endpoints for monitoring host health changes induced by carcinogen exposure.

This study monitored structural shifts of gut microbiota of rats developing precancerous mucosal lesions induced by carcinogen 1,2-dimethyl hydrazine (DMH) treatment using PCR-denaturing gradient gel electrophoresis (DGGE) and 454 pyrosequencing on the 16S rRNA gene V3 region. Partial least square discriminant analysis of DGGE fingerprints showed that the gut microbiota structure of treated animals was similar to that of the controls 1 and 3 weeks after DMH treatments, but significantly different 7 weeks after DMH treatments, when a large number of aberrant crypt foci (ACF) developed in their colons. Martens' uncertainty test, followed by anova test (P<0.05) identified Ruminococcus-like and Allobaculum-like bacteria as key variables for discrimination of DMH-treated rats from controls. Real-time PCR confirmed the significant increase of the Ruminococcus obeum and the Allobaculum-like bacteria in DMH-treated rats. UniFrac analysis based on V3 pyrosequencing further validated that the gut microbiota structures of treated and control animals were similar at an early stage, but segregated after ACF formation. Thirteen operational taxonomic units including Ruminococcus-like and Allobaculum-like bacteria were identified as key variables for the discrimination of DMH-treated rats from controls. Dynamic analysis of gut microbiota may become a noninvasive strategy for monitoring host health changes induced by carcinogen exposure.

Traditional Chinese formula, lubricating gut pill, improves loperamide-induced rat constipation involved in enhance of Cl- secretion across distal colonic epithelium.

AIM OF THE STUDY: Lubricating gut pill (LGP), a traditional Chinese formula, was widely used for the treatment of chronic constipation, especially in the elderly, in China. However, it is unclear whether LGP-induced laxative and/or lubricating effect is involved in water and electrolytes transport in distal colonic epithelium. MATERIALS AND METHODS: The present study was designed to evaluate the effect of LGP on Cl(-) secretion across rat distal colonic epithelium mounted in Ussing chambers, and on a rat constipation model induced by loperamide, respectively. RESULTS: Application of LGP in the apical side elicited a sustained increase in short circuit current (I(SC)) response in a concentration-dependent manner. Evidence that LGP-stimulated I(SC) was due to Cl(-) secretion is based on inhibition of current by (a) a Na(+)-K(+)-2Cl(-) cotransporter inhibitor bumetanide, (b) removal of Cl(-) ions in bath solution, and (c) the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel blocker DPC, suggesting that a apical cAMP-dependent Cl(-) channel was activated. LGP-stimulated I(SC) was also strongly inhibited by pretreatment with clotrimazole, indicating that the basolateral K(+) channel was also involved in maintaining this cAMP-dependent Cl(-) secretion. Pretreatment of tissues with indomethacin, but not atropine, tetrodotoxin or hexamethonium, inhibited LGP-induced response. In a rat constipation model, oral administration with LGP was significantly restored number of fecal pellets, water content and mucus secretion compared with loperamide-treated group alone. CONCLUSIONS: LGP enhances Cl(-) secretion that is mostly mediated through the release of cyclooxygenase metabolites, by which provided an osmotic force for the subsequent laxative action observed in the rat constipation model.

[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.

Effects of Puerariae radix extract on the increasing intestinal permeability in rat with alcohol-induced liver injury.

AIM OF THE STUDY: Puerariae radix, as an edible plant, has been used for centuries in China to treat alcohol-related problems, including alcoholic liver disease (ALD). However, the mechanisms of Puerariae radix on the liver-protective effect have not been fully explored. Because an increased intestinal permeability is a major factor for ALD, the present study investigates whether Puerariae radix extract (PRE) inhibits ALD through prevention of alterations in intestinal permeability. MATERIALS AND METHODS: We used an animal model of chronic alcohol-induced liver injury that is associated with increased intestinal permeability. Male Wistar rats were given increasing alcohol doses from 2 g/kg/d to 8 g/kg/d and alcohol plus PRE via intragastric feeding for 10 weeks. Chronic alcohol exposure caused an elevation in serum alanine aminotransferase (ALT) as well as aspartate aminotransferase (AST) levels and a decrease in superoxide dismutase (SOD) activity, and hepatic damages including steatosis, inflammation, and necrosis, determined by serum enzymatic analysis and morphological analysis, respectively. The damage to small intestine induced by chronic alcohol treatment was examined by intestinal histological, immunohistochemical analysis, and permeability assays. RESULTS: Alcohol-induced hepatic pathological changes, elevations in ALT and AST, and a decrease in SOD activity were significantly inhibited in PRE treated animals. The inhibitory effect of PRE on alcohol-induced liver injury was associated with suppression of alcohol induced the increase of intestinal permeability. CONCLUSIONS: The results showed that this beneficial effect of PRE on ALD could be partly explained by improving intestinal barrier dysfunction induced by alcohol.