Electroacupuncture alleviates stress-induced visceral hypersensitivity through an opioid system in rats

AIM:To investigate whether stress-induced visceral hypersensitivity could be alleviated by electroacupuncture(EA) and whether EA effect was mediated by endogenous opiates.METHODS:Six to nine week-old male SpragueDawley rats were used in this study.Visceral hypersensitivity was induced by a 9-d heterotypic intermittent stress(HIS) protocol composed of 3 randomly stressors,which included cold restraint stress at 4?℃ for 45 min,water avoidance stress for 60 min,and forced swimming stress for 20 min,in adult male rats.The extent of visceral hypersensitivity was quantified by electromyography or by abdominal withdrawal reflex(AWR) scores of colorectal distension at different distention pressures(20 mmHg,40 mmHg,60 mmHg and 80 mmHg).AWR scores either 0,1,2,3 or 4 were obtained by a blinded observer.EA or sham EA was performed at classical acupoint ST-36(Zu-San-Li) or BL-43(Gao-Huang) in both hindlimbs of rats for 30 min.Naloxone(NLX) or NLX methiodide(m-NLX) was administered intraperitoneally to HIS rats in some experiments.RESULTS:HIS rats displayed an increased sensitivity to colorectal distention,which started from 6 h(the first measurement),maintained for 24 h,and AWR scores returned to basal levels at 48 h and 7 d after HIS compared to pre-HIS baseline at different distention pressures.The AWR scores before HIS were 0.6 ± 0.2,1.3 ± 0.2,1.9 ± 0.2 and 2.3 ± 0.2 for 20 mmHg,40 mmHg,60 mmHg and 80 mmHg distention pressures,respectively.Six hours after termination of the last stressor,the AWR scores were 2.0 ± 0.1,2.5 ± 0.1,2.8 ± 0.2 and 3.5 ± 0.2 for 20 mmHg,40 mmHg,60 mmHg and 80 mmHg distention pressures,respectively.EA given at classical acupoint ST-36 in both hindlimbs for 30 min significantly attenuated the hypersensitive responses to colorectal distention in HIS rats compared with sham EA treatment [AWRs at 20 mmHg:2.0 ± 0.2 vs 0.7 ± 0.1,P = 4.23 711 E-4;AWRs at 40 mmHg:2.6 ± 0.2 vs 1.5 ± 0.2,P = 0.00 163;AWRs at 60 mmHg:3.1 ± 0.2 vs 1.9 ± 0.1,P = 0.003;AWRs at 80 mmHg:3.6 ± 0.1 vs 2.4 ± 0.2,P = 0.0023;electromyographic(EMG) at 20 mmHg:24 ± 4.7 vs 13.8 ± 3.5;EMG at 40 mmHg:60.2 ± 6.6 vs 30 ± 4.9,P = 0.00 523;EMG at 60 mmHg:83 ± 10 vs 39.8 ± 5.9,P = 0.00 029;EMG at 80 mmHg:94.3 ± 10.8 vs 49.6 ± 5.9,P = 0.00 021].In addition,EA at the acupuncture point BL-43 with same parameters did not alleviate visceral hypersensitivity in HIS rats.EA in healthy rats also did not have any effect on AWR scores to colorectal distention at distention pressuresof 20 and 40 mmHg.The EA-mediated analgesic effect was blocked by pretreatment with NLX in HIS rats [AWR scores pretreated with NLX vs normal saline(NS) were 2.0 vs 0.70 ± 0.20,2.80 ± 0.12 vs 1.50 ± 0.27,3 vs 2.00 ± 0.15 and 3.60 ± 0.18 vs 2.60 ± 0.18 for 20 mmHg,40 mmHg,60 mmHg and 80 mmHg;P = 0.0087,0.0104,0.0117 and 0.0188 for 20,40,60 and 80 mmHg,respectively].Furthermore,EA-mediated analgesic effect was completely reversed by administration of m-NLX,a peripherally restricted opioid antagonist(EMG pretreated with m-NLX vs NS were 30.84 ± 4.39 vs 13.33 ± 3.88,74.16 ± 9.04 vs 36.28 ± 8.01,96.45 ± 11.80 vs 50.19 ± 8.28,and 111.59 ± 13.79 vs 56.42 ± 8.43 for 20 mmHg,40 mmHg,60 mmHg and 80 mmHg;P = 0.05 026,0.00 034,0.00 005,0.000 007 for 20 mmHg,40 mmHg,60 mmHg and 80 mmHg,respectively).CONCLUSION:EA given at classical acupoint ST-36 alleviates stress-induced visceral pain,which is most likely mediated by opioid pathways in the periphery.     

Electroacupuncture at Acupoint ST-36 Promotes Contractility of Distal Colon via a Cholinergic Pathway in Conscious Rats

Purpose To investigate the effects and possible mechanism of electroacupuncture (EA) at acupoint ST-36 (Zusanli) on the contractility of the distal colon in conscious rats. Methods Rats were randomized into four groups, including the ST-36 group, the sham group, the saline-plus-ST-36 and the atropine-plus-ST-36 group. Needles were inserted into the acupoints for EA. The distal colonic contractility was measured with a manometric catheter inserted into the distal colon. The colonic contractility was recorded for 1 h before EA, 20 min during EA, and 3 h after EA. Saline or atropine was administrated in the saline-plus-ST-36 or atropine-plus-ST-36 group. Results (1) EA at ST-36 significantly increased the contractility of the distal colon. The mean AUC during and after EA were significantly higher than that before EA in the ST-36 group, a 27% increase during EA and 26% after EA. (2) Atropine abolished the accelerating effect of EA at ST-36. The mean AUC remained unchanged during or after EA compared with that before EA in the atropine-plus-ST-36 group. Conclusions EA at ST-36 has a stimulatory effect on the contractility of the distal colon in conscious rats, and the stimulatory effect may be mediated via the cholinergic pathway.     

Increased Expression of 5-HT3 and NK1 Receptors in 5-Fluorouracil-Induced Mucositis in Mouse Jejunum

Background and Objective

Although 5-fluorouracil (5-FU) is a widely used as chemotherapy agent, severe mucositis develops in approximately 80 % of patients. 5-FU-induced small intestinal mucositis can cause nausea and vomiting. The current study was designed to investigate peripheral alterations due to the 5-FU-induced mucositis of neuronal and non-neuronal 5-HT₃ and NK₁ receptor expression by immunohistochemical analysis.

Methods

5-FU was administered by i.p. injection to C57BL/6 mice. After 4 days, segments of the jejunum were removed. The specimens were analyzed by immunohistochemistry, real-time PCR, and enzyme immunoassay.

Results

The numbers of 5-HT₃ receptor immunopositive cells and nerve fibers in mucosa were increased by 5-FU treatment. The 5-HT₃ receptor immunopositive cell bodies were found only in jejunal submucosa and myenteric plexus in the 5-FU-treated mice. The numbers of NK₁ receptor cells in mucosa and immunopositive expression of NK₁ receptors in deep muscular plexus were dramatically increased in 5-FU-treated mice. Real-time PCR demonstrated that 5-FU treatment significantly increased mRNA levels of 5-HT_3A, 5-HT_3B, and NK₁ receptors. The amounts of 5-HT and substance P increased after 5-FU treatment. The 5-HT₃ or NK₁ receptor immunopositive cells colocalized with both 5-HT and substance P. Furthermore, 5-HT₃ and NK₁ receptors colocalized with CD11b.

Conclusions

The 5-HT₃ and NK₁ immunopositive macrophages and mucosal mast cells in lamina propria release 5-HT and substance P, which in turn activate their corresponding receptors on mucosal cells in autocrine and paracrine manners. It is assumed to result in the release of 5-HT and substance P in mucosa.     

Effects of electroacupuncture on corticotropin-releasing hormone in rats with chronic visceral hypersensitivity

AIM: To investigate the effect of electroacupuncture on corticotropin-releasing hormone (CRH) in the colon, spinal cord, and hypothalamus of rats with chronic visceral hypersensitivity.METHODS: A rat model of chronic visceral hypersensitivity was generated according to the internationally accepted method of colorectal balloon dilatation. In the 7^th week after the procedure, rats were randomly divided into a model group (MG), electroacupuncture group (EA), and sham electroacupuncture group (S-EA). After treatment, the abdominal withdrawal reflex (AWR) score was used to assess the behavioral response of visceral hyperalgesia. Immunohistochemistry (EnVision method), ELISA, and fluorescence quantitative PCR methods were applied to detect the expression of CRH protein and mRNA in the colon, spinal cord, and hypothalamus.RESULTS: The sensitivity of the rats to the colorectal distension stimulus applied at different strengths (20-80 mmHg) increased with increasing stimulus strength, resulting in increasing AWR scores in each group. Compared with NG, the AWR score of MG was significantly increased (P < 0.01). After conducting EA, the AWR scores of the rats were decreased compared with MG rats. The relative expression of CRH mRNA in the colon, spinal cord, and hypothalamus of MG rats was significantly increased compared with NG rats (P < 0.01). CRH mRNA in the colon and spinal cord of EA and S-EA rats was decreased to varying degrees (P > 0.05) compared with normal rats (NG). However, the decrease in EA compared with MG rats was statistically significant (P < 0.01). The average optical density of CRH expression in the colon of the MG rats was significantly enhanced compared with NG (P < 0.05), while the average optical density of CRH expression in the EA and S-EA rats was significantly decreased compared with MG rats (P < 0.01, P < 0.05, respectively). Compared with MG rats, the CRH concentration in the spinal cord of EA rats was significantly reduced (P < 0.01), but there was no significant change in S-EA rats (P > 0.05).CONCLUSION: Electroacupuncture at the Shangjuxu acupoint was able to significantly reduce the visceral hypersensitivity in rats, and regulated the expression of CRH protein and mRNA in the colon, spinal cord and hypothalamus at different levels, playing a therapeutic role in this model of irritable bowel syndrome.     

Host immune response determines visceral hyperalgesia in a rat model of post-inflammatory irritable bowel syndrome

Background

Irritable bowel syndrome (IBS) is associated with visceral hyperalgesia and frequently occurs after a transient gastrointestinal infection. Only a proportion of patients with acute gastroenteritis develop post-infectious IBS suggesting differences in host response to inflammatory stimuli. We aimed to investigate this concept by characterizing visceral sensitivity in two rat strains, following a chemically induced colitis.

Methods

Colorectal instillation of trinitrobenzenesulfonic acid (TNBS) in aqueous ethanol was used to induce a transient colitis in Lewis and F344 rats. The colitis was characterized semiquantitatively by histology, as well as by quantitative methods using ^99mTc-leukocytes (radioactive organ assay) and plasma IL-2 and IL-6 levels. Visceromotor response to colorectal distensions was assessed after 2 h and, 5, 14, and 28 days.

Results

The colitis peaked on day 5 and dissipated to no visible mucosal damage on day 14. Cytokines were significantly increased in TNBS-treated rats at 2 h and on day 5. On day 14 cytokines were still significantly enhanced in Lewis but not Fisher rats. Both strains had a highly inflamed to non-inflamed tissue ratio at 3 h after TNBS instillation with increased uptake in Lewis compared to F344 rats. No ^99mTc-tin-colloid-leukocytes were detected in colon samples on day 28. Visceromotor response was significantly elevated in both strains during the acute colitis (day 5), whereas only Lewis rats developed a post-inflammatory (day 28) visceral hyperalgesia.

Conclusion

Genetically determined host factors account for prolonged immune activation in response to a standardized inflammatory stimulus and are linked to susceptibility for a post-inflammatory visceral hyperalgesia.     

CD36 mRNA in the Gastrointestinal Tract Is Differentially Regulated by Dietary Fat Intake in Obesity-Prone and Obesity-Resistant Rats

Background

The gastrointestinal tract (GI) is important for detection and transport of consumed nutrients and has been implicated in susceptibility to diet-induced obesity in various rat strains.

Aims

The current studies investigated the regulation of CD36, a receptor which facilitates uptake of long-chain fatty acids, in the GI tract of obesity-prone Osborne–Mendel and obesity-resistant S5B rats fed a high-fat diet.

Methods

Osborne–Mendel and S5B rats consumed a high-fat diet (HFD, 55 % kcal from fat) or a low-fat diet (10 % kcal from fat) for either 3 or 14 days. CD36 messenger RNA (mRNA) levels were measured from circumvallate papillae of the tongue and from duodenal enterocytes.

Results

In Osborne–Mendel rats, consumption of HFD for 3 and 14 days led to an increase in CD36 mRNA on circumvallate papillae and in duodenal enterocytes. CD36 mRNA levels were positively correlated with body weight gain and kilocalories consumed at 3 days. In S5B rats, consumption of HFD for 3 days did not alter CD36 mRNA levels on circumvallate papillae or in the duodenum. Duodenal CD36 levels were elevated in S5B rats following 14 days of HFD consumption. CD36 mRNA levels in the duodenum were positively correlated with body weight gain and kilocalories consumed at 14 days.

Conclusions

These data support the differential sensing of nutrients by two regions of the GI tract of obesity-prone and obesity-resistant rats consuming HFD and suggest a role for CD36 in the strain-specific susceptibility to obesity.     

γ-Aminobutyric Acid B Receptor Improves Carbon Tetrachloride-Induced Liver Fibrosis in Rats

Background

It was well known that angiotension II can inhibit hepatic stellate cell activation. The GABA_B receptor was upregulated when the hepatic stellate cell line was stimulated by angiotension II in our previous study. But the role of the GABA_B receptor in liver fibrosis has never been reported.

Aim

In the present study, we investigated the effects of this receptor on carbon tetrachloride-induced liver fibrosis in rats.

Methods

The rats were divided into four groups including GABA_B receptor agonist, antangonist, model and control group. α-smooth muscle actin (α-SMA) and GABA_B receptor expression levels were detected by immunohistochemistry and real-time polymerase chain reaction. Liver function tests were performed once blood samples was taken; Western blot analysis was used to detect protein expression level of α-SMA and TGF-β1.

Results

We found baclofen ameliorated the CCl₄-induced rats’s liver fibrosis. The highest liver enzymes and α-SMA protein levels were found in the CGP35348 group.

Conclusion

The GABA_B receptor may have a protective role in the liver.     

Exogenous Interleukin-6 Facilitated the Contraction of the Colon in a Depression Rat Model

Background

Gut dysmotility is closely associated with proinflammatory cytokines both in irritable bowel syndrome and inflammatory bowel disease. There is a dose–response relationship between depression and these inflammatory cytokines.

Aims

In the present study, we aimed to investigate the effect of Interleukin-6 (IL-6) on colon motility in a rat model of depression induced by chronic unpredictable mild stress (CUMS).

Methods

The contraction of the circular muscle strips of proximal colon was monitored by a polygraph. IL-6 and IL-6 receptor (IL-6R) mRNA was assayed by real-time quantitative PCR. Immunohistochemistry staining was used to locate the IL-6 and IL-6R in the rat colon.

Results

IL-6 and IL-6R were expressed in the mucosal layer, smooth muscle cells, and myenteric plexus of the colon. Exogenous IL-6 (20 ng/ml) increased the contraction of the circular muscle strip. Pretreatment of tetrodotoxin (blocker of voltage-dependent Na⁺ channel on nerve fiber) blocks the excitatory effect of IL-6 on the contraction of the colon in non-stressed rats, but partially inhibited IL-6-induced excitatory effect on the muscle strips in CUMS-treated rats.

Conclusions

These results suggest that IL-6-induced the contraction of the colonic strip by acting on the gut’s nervous system and acting directly on the smooth muscle in rats with depression.     

Injection of l-glutamate into the insular cortex produces sleep apnea and serotonin reduction in rats

Purpose

Obstructive sleep apnea (OSA) is primarily characterized by repetitive episodes of complete or partial obstruction of airflow during sleep. The neuronal and cellular mechanisms underlying this process are not fully understood, although the focus of some studies is on putative serotonin (5-HT) mechanisms, and serotonergic therapy may be beneficial to OSA patients. This study aimed to demonstrate possible changes in 5-HT associated with induction of OSA in a rat model.

Methods

Apnea was induced in rats by injection of l-glutamate (l-Glu) into the insular cortex. We examined changes in: (1) simultaneous genioglossus and diaphragm EMG activity; and (2) peripheral and cerebral levels of 5-HT, by histology.

Results

Injection of l-glutamate (l-Glu) into the insular cortex induced apnea in the rats. l-Glu stimulation of the insular cortex also produced significant reductions in plasma 5-HT levels and the expression of 5-HT in the brainstem. In addition, lower activity was observed in the GG and a higher activity was observed in the diaphragm, as compared to controls.

Conclusion

Data indicate that l-Glu stimulation of the insular cortex simulates the electrical activity of the genioglossus muscle and diaphragm in sleep apnea, and contributes to reduced peripheral and cerebral 5-HT levels in rats. The results of our study suggest that 5-HT may play a role in the pathogenesis of OSA.     

A quantitative and qualitative analysis of the virtual unipolar electrograms from non-contact mapping of right or left-sided outflow tract premature ventricular contractions/ventricular tachycardia origins

Objective

This study was conducted to examine the virtual unipolar electrogram configuration of right/left outflow tract (OT) premature ventricular contraction (PVC)/ventricular tachycardia (VT) origins obtained from a non-contact mapping system (NCMS).

Methods

The subjects consisted of 30 patients with OT-PVCs/VT who underwent NCMS-guided ablation. We evaluated the virtual unipolar electrograms of the origin on 3D right ventricular (RV)-OT isochronal maps.

Results

Successful ablation was achieved from the RV in 20 patients (RVOT group), and it failed in 10 (non-RVOT group: including left-sided/pulmonary artery/deep RVOT foci). On the virtual unipolar electrograms, the earliest activation (EA) preceded the QRS onset by 11.2?±?2.6 ms in the RVOT group and by 7.4?±?10.5 ms in the non-RVOT group (P?=?0.138). The negative slope of the electrogram at the EA site (EA slope₅), quantified by the virtual unipolar voltage amplitude 5 ms after the EA onset, was significantly steeper in the RVOT group than in the non-RVOT group (0.66?±?0.52 mV vs. 0.14?±?0.17 mV, P?=?0.005). Cutoff values for the EA-to-QRS onset time and EA slope₅ of ??8 ms and >0.3 mV, respectively, completely differentiated the RVOT group from the non-RVOT group. A lesser EA slope₅ was associated with a greater radiofrequency energy delivery required to terminate RVOT-PVCs/VT.

Conclusions

These demonstrate the importance of the virtual unipolar electrograms from OT-PVC/VT origins obtained with the NCMS. The virtual EA predicts both successful and potentially difficult ablation sites from the RV side.     

Adjuvant radiotherapy and anastomosis in rectal cancer—disturbing evidence from animal studies

PURPOSE: Adjuvant radiotherapy is used increasingly in the management of rectal cancer. However, ionizing radiation is mutagenic, and, superimposed on a background of increased cellular proliferation as seen around anastomoses and in colorectal cancer patients, there is the potential for enhanced metachronous cancer risk. METHOD: The influence of preoperative irradiation on both carcinogenesis and cellular proliferation at colonic anastomoses was explored in 180 adult male F344 rats. Orthovoltage x-rays were delivered to the distal descending colon by parallel opposed fields. Ninety rats received 16 Gy in one fraction; the remainder received 36 Gy in two fractions one week apart. In each irradiation group 18 rats either acted as controls or one week after radiotherapy underwent distal colotomy and repair. RESULTS: We found the descending colon susceptible to radiation carcinogenesis; 26 colorectal tumors developed in the low-dose irradiation group and 47 in the highdose group (P=0.0008; Mann-Whitney U test, 16 vs. 36 Gy). Preferential tumor development was seen in the anastomotic region. In those animals that underwent surgery and irradiation, among the low-dose irradiation group only 3 of 72 had tumors within the descending colon compared with 21 of 72 at the anastomotic site (McNemar's test chi-squared=40.9; P<0.001), and in the high-dose irradiation group 5 of 72 had tumors within the descending colon compared with 36 of 72 at the anastomotic site (McNemar's test chi-squared=22.0; P<0.001). Anastomotic crypt cell production rates were increased for at least three months following exposure to irradiation (16 Gy: f=15.1,P<0.005; 32 Gy: f=9.4,P<0.005). CONCLUSIONS: Radiation carcinogenesis is greatly enhanced at colonic anastomoses and may result from altered anastomotic proliferation. This has potentially disturbing implications in view of the increasing use of adjuvant radiotherapy for rectal carcinoma.     

Role of nesfatin-1 in a rat model of visceral hypersensitivity

AIM: To explore the role of nesfatin-1 on irritable bowel syndrome (IBS)-like visceral hypersensitivity. METHODS: The animal model of IBS-like visceral hypersensitivity was induced by intracolonic infusion of 0.5% acetic acid (AA) in saline once daily from postnatal days 8-21. Experiments were performed when rats became adults. The visceral sensitivity of rats was evaluated by abdominal withdrawal reflex (AWR) and electromyographic (EMG) activity of the external oblique muscle to graded colorectal distension. The content of nesfatin-1 in serum was determined using enzyme-linked immunosorbent assay. After implantation of an intracerebroventricular (ICV) cannula and two electrodes into the external oblique muscle, model rats were randomly divided into four groups. Animals then received ICV injection of 8 μg of anti-nesfatin-1/ nucleobindin-2 (NUCB2), 50 μg of α-helical cortico-tropin releasing factor (CRF) 9-41 (non-selective CRF receptor antagonist), 50 μg of NBI-27914 (selective CRF1 receptor antagonist) or 5 μL of vehicle. After 1 h of ICV administration, visceral sensitivity of each group was measured again, and comparisons between groups were made. RESULTS: Rats treated with AA showed higher mean AWR scores and EMG activity at all distension pressures compared with controls (P < 0.05). On histopathologic examination, no evidence of inflammation or abnormalities in structure were noted in the colon of either control or AA-treated groups. Myeloperoxidase values were not significantly different between the two groups. The level of nesfatin-1 in serum was significantly higher in the AA-treated group than in the control group (5.34 ± 0.37 ng/mL vs 4.81 ± 0.42 ng/mL, P < 0.01). Compared with rats injected with vehicle, rats which received ICV anti-nesfatin-1/NUCB2, α-helical CRF9-41 or NBI-27914 showed decreased mean AWR scores and EMG activity at all distension pressures (P < 0.05). CONCLUSION: Nesfatin-1 may be associated with IBS-like visceral hypersensitivity, which may be implicated in brain C     

Functional 5-HT receptor subtypes in the isolated canine common carotid artery

Summary The vascular responses to 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT, a selective 5-HT₁-like receptor agonist), alphamethyl-5-HT (-M-5-HT, a relatively selective 5-HT₂ receptor agonist), noradrenaline (NA), and KCl were examined in isolated, cannulated, and perfused canine common carotid arterial preparations. They caused strong vasoconstrictions. The rank order of vasoconstrictive potency was 5-HT > -M-5-HT NA > 5-CT >> KCl. The 5-HT-induced vasoconstriction was significantly depressed by methysergide (a 5-HT₁ and 5-HT₂ receptor antagonist), ketanserin (a selective 5-HT₂ receptor antagonist), and spiperone (a selective 5-HT₂ receptor antagonist). The 5-CT- and -M-5-HT-induced vasoconstrictions were also significantly inhibited by methysergide, spiperone, and ketanserin. The NA-induced vasoconstriction was readily inhibited by bunazosin (an -adrenoceptor antagonist) and ketanserin but not significantly inhibited by spiperone and methysergide. KCl has a weak potency for producing a vasoconstriction of the canine common carotid artery. A relatively large dose of diltiazem (a calcium channel blocker) did not modify 5-HT-induced vasoconstrictions. From these results, we conclude that (a) the canine common carotid artery contains abundant 5-HT receptors; (b) there are no functional 5-HT₁ receptors, but 5-HT₂ receptors are prominent; (c) 5-CT-induced vasoconstrictions might be due to activation of 5-HT₂ but not to 5-HT₁ receptors; (d) 5-HT-induced vasoconstriction might not involve -adrenoceptors; and (e) the vasoconstriction related to 5-HT in the common carotid artery is not significantly mediated via activation of calcium ion channels of smooth muscle cells, but may be induced by calcium ions from intracellular stores.     

Conjugated linoleic acid suppresses colon carcinogenesis in azoxymethane-pretreated rats with long-term feeding of diet containing beef tallow

Background

We have indicated previously that long-term feeding of beef tallow increases colorectal cancer in rats. In this study, we investigated the effects of conjugated linoleic acid (CLA) on colon carcinogenesis in rats under long-term feeding of beef tallow diets, pretreated with azoxymethane (AOM).

Methods

Six-week-old male Sprague–Dawley rats were fed with 10% beef tallow diet only, 10% beef tallow with 1% CLA in triglyceride form (CLA-TG), or 10% beef tallow with 1% CLA in free fatty acid form (CLA-FFA). Colon carcinogenesis was induced by two intraperitoneal injections of AOM. Aberrant crypt foci (ACFs) were examined at 12 weeks. Cancer, cell proliferation, apoptosis, Wnt signaling, and the arachidonic acid cascade were examined at 44 weeks.

Results

At 12 weeks, CLA-TG and CLA-FFA attenuated the increase in ACFs induced by 10% beef tallow and AOM pretreatment. At 44 weeks, both forms of CLA attenuated multiple colon cancers, and CLA-FFA reduced the incidence of colon cancer to 50% of that seen with CLA-TG. CLA-TG and CLA-FFA decreased the number of 5-bromo-2′-deoxyuridine-positive cells in AOM-pretreated rats fed with 10% beef tallow. CLA-FFA increased the number of apoptotic cells and the activity of caspase-3 in the colon mucosa, and CLA-TG enhanced the activity of caspase-3. Both forms of CLA suppressed Wnt signaling and the arachidonic acid cascade in rats treated with beef tallow and AOM.

Conclusion

These results suggested that CLA-TG and CLA-FFA suppressed colon carcinogenesis in rats with long-term feeding of a 10% beef tallow diet, through several mechanisms. The results of the present study with rats might be applicable to humans.     

Serodolin,a β-arrestin–biased ligand of 5-HT7 receptor,attenuates pain-related behaviors

G protein–coupled receptors (GPCRs) are involved in regulation of manifold physiological processes through coupling to heterotrimeric G proteins upon ligand stimulation. Classical therapeutically active drugs simultaneously initiate several downstream signaling pathways, whereas biased ligands, which stabilize subsets of receptor conformations, elicit more selective signaling. This concept of functional selectivity of a ligand has emerged as an interesting property for the development of new therapeutic molecules. Biased ligands are expected to have superior efficacy and/or reduced side effects by regulating biological functions of GPCRs in a more precise way. In the last decade, 5-HT₇ receptor (5-HT₇R) has become a promising target for the treatment of neuropsychiatric disorders, sleep and circadian rhythm disorders, and pathological pain. In this study, we showed that Serodolin is unique among a number of agonists and antagonists tested: it behaves as an antagonist/inverse agonist on G_s signaling while inducing ERK activation through a β-arrestin–dependent signaling mechanism that requires c-SRC activation. Moreover, we showed that Serodolin clearly decreases hyperalgesia and pain sensation in response to inflammatory, thermal, and mechanical stimulation. This antinociceptive effect could not be observed in 5-HT₇R knockout (KO) mice and was fully blocked by administration of SB269-970, a specific 5-HT₇R antagonist, demonstrating the specificity of action of Serodolin. Physiological effects of 5-HT₇R stimulation have been classically shown to result from G_s-dependent adenylyl cyclase activation. In this study, using a β-arrestin–biased agonist, we provided insight into the molecular mechanism triggered by 5-HT₇R and revealed its therapeutic potential in the modulation of pain response.

Among 14 serotonin receptor subtypes, 5-hydroxytryptamine 7receptors (5-HT₇Rs) belong to the G protein–coupled receptor (GPCR) family or the so-called seven transmembrane-spanning receptor. It is the last identified member and has been cloned from several animal species, including human (1). 5-HT₇R couples to the heterotrimeric G protein G_s, which in turn stimulates adenylate cyclase (AC), leading to an increase of 3′-5′-cyclic adenosine monophosphate (cAMP) production in both recombinant and native systems (1). This allows the activation of cAMP-dependent protein kinase (PKA), which acts on the MAPK cascade in a cell type–specific manner (2–4).In HEK-293 cells, the observed agonist-induced ERK1/2 activation requires PKA, Ras, and Raf activation independently of Rap-1 (4). In neurons, the 5-HT₇R–induced ERK activation is mediated through a PKA-independent pathway that utilizes cAMP-guanine nucleotide exchange factor (cAMP-GEF) (3). It was shown that 5-HT₇R signaling also depends on the activation of Gα₁₂ protein that in turn triggers activation of multiple signaling pathways through the family of small Rho GTPases, Cdc42 and RhoA (5).The 5-HT₇R is expressed in the peripheral and central nervous system with highest densities in thalamus, hypothalamus, cerebral cortex, amygdala, and striatal complex (1). Numerous data have established 5-HT₇R implication in the control of circadian rhythms and thermoregulation, learning, and memory as well as in central nervous system (CNS) disorders such as depression, Alzheimer’s disease, and schizophrenia (6). There is mounting evidence that 5-HT₇R is an important modulator of nociceptive transmission (7). It was also reported that 5-HT₇R is involved in the antinociceptive effects of morphine (8), antidepressants, and nonopioid analgesics (9). Collectively, these observations underscore the interest of developing new 5-HT₇R ligands for the treatment of pain. To date, many 5-HT₇R potent agonists (5-CT, E55888, AS-19, and LP-211) and antagonists (SB269-970 and DR4004) against 5-HT₇R have been described (10); these are all ligands that commit the receptor to a G protein–dependent pathway, although few 5-HT₇R–biased ligands have been described (11, 12).In contrast to standard agonists and antagonists, which activate or inactivate the entirety of a receptor’s signaling network, some ligands termed biased ligands are capable of stabilizing subsets of receptor conformations, hence eliciting selective modulation within the network. From data obtained in the last two decades, the concept of functional selectivity of a ligand has emerged as an interesting property in modern drug discovery. Increasing preclinical data highlight the value of using such ligands, which exhibit a unique spectrum of pharmacological responses, for instance, by specifically targeting G protein– or β-arrestin–dependent signaling. Biased ligands, by selectively modulating a subset of receptor functions, may optimize therapeutic action and generate less pronounced side effects than compounds globally affecting receptor activity. Although binding of β-arrestins to GPCR has primarily been involved in the termination of G protein signaling by inducing desensitization and internalization of the receptor, numerous studies have indicated that β-arrestins can be intimately involved in additional signaling events through mechanisms dependent on or independent of G protein coupling (13, 14). Several β-arrestin–biased ligands have been identified and show therapeutic interest in other receptor classes (15). In particular, several studies demonstrate the role of β-arrestin signaling on opioid analgesia and tolerance (16, 17). In the present study, we used a combination of pharmacological, genetic, and behavioral approaches to identify a β-arrestin–biased 5-HT₇R ligand and evaluate its therapeutical potential for the treatment of pain.     

Gut Bacterial Translocation Contributes to Microinflammation in Experimental Uremia

Background

Microinflammation frequently develops in chronic uremia with pathological intestinal changes. However, the relationship between gut bacterial translocation and microinflammation in uremia has not been widely investigated.

Aim

This study aimed to investigate whether gut microbiome dysbiosis and translocation occurred in experimental uremia, and whether they consequently contributed to microinflammation.

Methods

Forty rats underwent surgical renal mass 5/6 ablation. The surviving (uremic group, n?=?21) and healthy (sham group, n?=?20) rats were used in the experiment. Postoperative blood, livers, spleens, and mesenteric lymph nodes (MLNs) were subjected to bacterial 16S ribosomal DNA amplification to determine if bacteria were present. Bacterial genomic DNA samples from the MLNs and colon were amplified with specific primers designed by the 16S rRNA sequence of the species obtained from blood, livers, and spleens. Pyrosequencing was used to analyze the colonic microbiome of each subject. Intestinal permeability to ^99mTc-DTPA, plasma hs-CRP, and IL-6 were measured.

Results

Bacterial DNA in extraintestinal sites and altered colonic microbiomes were detected in some rats in the uremic group. Bacterial genomic DNA in MLNs and colon were obtained by primers specific for bacterial species observed from blood, livers, and spleens of identical individuals. Intestinal permeability, plasma hs-CRP, and IL-6 levels were statistically higher in the uremic group compared with the sham group. Plasma hs-CRP and IL-6 were significantly higher in uremic rats with bacterial DNA in their blood than in those without.

Conclusions

Gut microbiome dysbiosis occurs and bacteria translocate to the systemic and lymph circulation, thereby contributing to microinflammation in experimental uremia.     

STW 5 is effective in dextran sulfate sodium-induced colitis in rats

Purpose

An herbal preparation, STW 5, used clinically in functional dyspepsia and irritable bowel syndrome, has been shown to possess properties that may render it useful in inflammatory bowel disease (IBD). The present work was conducted to study its effectiveness in a rat model of IBD.

Methods

An experimental model reflecting ulcerative colitis in man was adopted, whereby colitis was induced in Wistar rats by feeding them 5?% dextran sulfate sodium (DSS) in drinking water for one week. STW 5 and sulfasalazine (as a reference standard) were administered orally daily for 1?week before colitis induction and continued during DSS feeding. The animals were then sacrificed, and the severity of colitis was evaluated macroscopically and microscopically. Colon samples were homogenized for determination of reduced glutathione, tumor necrosis factor-α, and cytokine-induced neutrophil chemoattractant-3 as well as myeloperoxidase, glutathione peroxidase, and superoxide dismutase. In addition, colon segments were suspended in an organ bath to test their reactivity towards carbachol, KCl, and trypsin.

Results

STW 5 and sulfasalazine were both effective in preventing the shortening of colon length and the increase in both colon mass index and total histology score as well as the changes in biochemical parameters measured except changes in dismutase activity. DSS-induced colitis led to marked depression in colonic responsiveness to the agents tested ex vivo, an effect which was normalized by both drugs.

Conclusions

The findings point to a potential usefulness of STW 5 in the clinical setting of ulcerative colitis.     

High Fat Diet Differentially Regulates the Expression of Olfactory Receptors in the Duodenum of Obesity-Prone and Obesity-Resistant Rats

Background

The gastrointestinal tract is important in the regulation of food intake, nutrient sensing and nutrient absorption. Obesity-prone Osborne-Mendel (OM) rats are less sensitive to the satiating effects of a duodenal infusion of fatty acids than obesity-resistant S5B/Pl (S5B) rats, suggesting that the gastrointestinal tract differentially senses the presence of fat in these two strains. A microarray analysis was conducted to identify genes that were differentially expressed in the duodenal enterocytes of OM and S5B rats.

Aims

The present experiment evaluated the expression of olfactory receptors in the duodenal enterocytes of OM and S5B rats. It was hypothesized that olfactory receptors present in the duodenum of OM and S5B rats would be differentially regulated by the intake of a high fat diet.

Methods

The mRNA levels of four olfactory receptors (Olr1744, Olr50, Olr124, Olr1507) were assessed from the duodenal enterocytes of OM and S5B rats consuming a high fat diet for 14 days.

Results

The duodenal mRNA levels of Olr1744, Olr124 and Olr1507 were significantly elevated in OM rats fed the high fat diet, but not S5B rats. No differences in the expression of Olr50 receptor mRNA were detected.

Conclusions

These data suggest that several olfactory receptors present in the duodenum are selectively regulated by high fat diet intake in obesity-prone OM rats. Therefore, these receptors may play a role in the sensing and regulation of dietary fat, and may be important for the individual susceptibility to obesity in these two strains.     

Adenine Induces Differentiation of Rat Hepatic Stellate Cells

Background and Aims

Adenine is a uric acid pathway metabolite of no known function, and has recently been identified as a ligand for a rat G protein-coupled receptor. Due to the known role of other uric acid pathway metabolites in HSC biology, we tested the ability of adenine to induce HSC differentiation.

Methods

RT-PCR was performed for adenine receptor expression in T-6 and primary rat HSC. T-6 and primary rats HSC were cultured with and without adenine, and stellation examined. Next, we examined inhibition of calcium signaling using caged IP₃. To test if adenine inhibits HSC chemotaxis T-6 cells and rat HSCs were cultured with or without adenine for 24?h in a transwell assay with PDGF as the chemoattractant. cDNA was prepared from T-6 and primary HSC for quantification of collagen 1 mRNA using real-time PCR.

Results

We found that mRNA for the adenine receptor is expressed in T-6 cells and primary rat HSC. Also, adenine induces HSC stellation and adenine inhibits IP₃ mediated increase in cytosolic [Ca²⁺]_i and inhibits chemotaxis in T-6 cells and primary rat HSC. Adenine was also shown to up-regulate α-SMA and collagen 1, and this effect is lost by using specific si-RNA for the adenine receptor. Finally, adenine inhibits endothelin-1-induced gel contraction.

Conclusions

The adenine receptor is present in T-6 cells and primary rats HSC. Adenine, via the adenine receptor, induces morphological change, and cytosolic calcium signaling, inhibits chemotaxis, and up-regulates collagen 1 mRNA in HSCs.