This study explored the mechanism of Xiangsha Liujunzi Decoction(XSLJZD) in the treatment of functional dyspepsia(FD) based on inositol-requiring enzyme 1(IRE1)/apoptosis signal-regulating kinase 1(ASK1)/c-Jun N-terminal kinase(JNK) pathway-mediated autophagy in interstitial cells of Cajal(ICC). Forty-eight SPF-grade male SD suckling rats were randomly divided into a blank group and a modeling group, and the integrated modeling method(iodoacetamide gavage + disturbance of hunger and satiety + swimming exhaustion) was used to replicate the FD rat model. After the model replications were successfully completed, the rats were divided into a model group, high-dose, medium-dose, and low-dose groups of XSLJZD(12, 6, and 3 g·kg~(-1)·d~(-1)), and a positive drug group(mosapride of 1.35 mg·kg~(-1)·d~(-1)), and the intervention lasted for 14 days. The gastric emptying rate and intestinal propulsion rate of rats in each group were measured. The histopathological changes in the gastric sinus tissue of rats in each group were observed by hematoxylin-eosin(HE) staining. The ultrastructure of ICC was observed by transmission electron microscopy. The immunofluorescence double staining technique was used to detect the protein expression of phospho-IRE1(p-IRE1), TNF receptor associated factors 2(TRAF2), phospho-ASK1(p-ASK1), phospho-JNK(p-JNK), p62, and Beclin1 in ICC of gastric sinus tissue of rats in each group. Western blot was used to detect the related protein expression of gastric sinus tissue of rats in each group. Compared with those in the blank group, the rats in the model group showed decreased body weight, gastric emptying rate, and intestinal propulsion rate, and transmission electron microscopy revealed damage to the endoplasmic reticulum structure and increased autophagosomes in ICC. Immunofluorescence staining revealed that the ICC of gastric sinus tissue showed a significant elevation of p-IRE1, TRAF2, p-ASK1, p-JNK, and Beclin1 proteins and a significant reduction of p62 protein. Western blot revealed that the expression levels of relevant proteins in gastric sinus tissue were consistent with those of proteins in ICC. Compared with the model group, the body weight of rats in the high-dose and medium-dose groups of XSLJZD was increased, and the gastric emptying rate and intestinal propulsion rate were increased. Transmission electron microscopy observed amelioration of structural damage to the endoplasmic reticulum of ICC and reduction of autophagosomes, and the p-IRE1, TRAF2, p-ASK1, p-JNK, and Beclin1 proteins in the ICC of gastric sinus tissue were significantly decreased. The p62 protein was significantly increased. Western blot revealed that the expression levels of relevant proteins in gastric sinus tissue were consistent with those of proteins in ICC. XSLJZD can effectively treat FD, and its specific mechanism may be related to the inhibition of the expression of molecules related to the endoplasmic reticulum stress IRE1/ASK1/JNK pathway in ICC and the improvement of autophagy to promote gastric motility in ICC.
Animals ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Autophagy/drug effects* ; Rats ; Rats, Sprague-Dawley ; Interstitial Cells of Cajal/metabolism* ; Dyspepsia/physiopathology* ; Protein Serine-Threonine Kinases/genetics* ; MAP Kinase Kinase Kinase 5/genetics* ; MAP Kinase Signaling System/drug effects* ; Humans ; Endoribonucleases/genetics* ; Multienzyme Complexes

OBJECTIVETo explore the mechanism of acupuncture on promoting the restoration of interstitial cells of Cajal (ICCs).

METHODSThirty SD rats were randomly divided into a blank group, a model group and an acupuncture group, ten rats in each one. The rats in the model group and acupuncture group were treated with colonic anastomosis to establish the model. After successful establishment of the model, the rats in the acupuncture group were treated with acupuncture at bilateral "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Taichong" (LR 3) for 15 min, once a day for 10 days. Rats in the model group and blank group were put into the fixator for 15 min at the same time daily. The propulsive rate of small intestine was measured in each group. Colonic tissues were collected to detect c-kit expression by using immunohistochemistry. The nitricoxide (NO) content was measured by nitrate reductase method and nitric oxide synthase (NOS) activity was measured by method of L-arginine.

RESULTSCompared with the blank group, the propulsive rate of small intestine in the model group was decreased; NO content was increased; iNOS activity was elevated; cNOS activity was declined; total NOS (tNOS) activity was increased and the counting of c-kit positive ICCs was decreased (all P < 0.05). Compared with the model group, the propulsive rate of small intestine in the acupuncture group was increased; NO content was decreased; iNOS activity was reduced; cNOS activity was elevated; NOS activity was decreased and the counting of c-kit positive ICCs was increased (all P < 0.05).

CONCLUSIONAcupuncture can regulate NO content and NOS activity in postoperative restoration environment of ICCs, which may participate in the process of acupuncture promoting the restoration of ICCs.

Acupuncture Points ; Acupuncture Therapy ; Anastomosis, Surgical ; Animals ; Colon ; metabolism ; surgery ; Colonic Diseases ; enzymology ; metabolism ; surgery ; therapy ; Female ; Humans ; Interstitial Cells of Cajal ; enzymology ; metabolism ; Male ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe the effect of Banxia Xiexin Decoction (BXD) on expression of the interstitial cells of Cajal (ICCs) and stem cell factor (SCF) in the antrum of rats with diabetes mellitus (DM).

METHODSTotally sixty healthy male SD rats were randomly divided into the control group, the model group, the BXD group, and the Western medicine group (WM, treated by domperidone), 15 in each group. Diabetic rat models were established by a single intraperitoneal injection of streptozotocin (STZ, 55 mg/kg). Those in the BXD group were perfused with BXD at the daily dose of 5.4 g/kg. An equal volume of distilled water was given by gastrogavage to those in the WM group and the control group for 8 successive weeks. The body weight and blood glucose of all rats were detected, and the gastric residual rates were detected with semisolid nutrient paste by gastrogavage. The expression of positive ICCs and SCF were observed by immunohistochemical method and quantified image analyzer.

RESULTSCompared with the control group,the body weight reduced, blood glucose and gastric residual rates increased, and the mean optical density of positive ICCs and SCF significantly decreased in the model group (P < 0.05). Compared with the model group,symptoms such as polydipsia, polyphagia, polyuria were relieved, spirits improved, the body weight and mean optical densities of positive ICCs and SCF significantly increased (P < 0.05), and gastric residual rates significantly decreased in the BXD group and the WM group (P < 0.05). The blood glucose significantly decreased (P < 0.05) in the BXD group. The mean optical density of positive ICCs was higher in the BXD group than in the WM group (P < 0.05).

CONCLUSIONSBXD could promote the expression of positive ICCs and SCF. It could improve the gastric motility in DM rats by partially inverting abnormal changes of gastric antral ICCs and SCF.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Interstitial Cells of Cajal ; metabolism ; Male ; Pyloric Antrum ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cell Factor ; metabolism

OBJECTIVETo investigate the association of expression of c-kit (marker of interstitial cells of Cajal, ICC) in colon with slow transit constipation (STC) in rats.

METHODSSlow transit constipation (STC) rat model was induced by intragastric administration of compound diphenoxylate. Western blotting was used to measure the expression of c-kit in colon of STC rats (model group) and normal rats (control group). Gray scale ratio of c-kit to β-actin was used as the relative quantity of c-kit.

RESULTSFecal quantity per day of STC group was (1.3±0.7) g/100 g, significantly lower than that in normal rats [(1.6±0.9) g/100 g, t=10.798, P<0.05]. In model rats, the time of discharge of the first black fecal was (461.6±150.8) min, significantly longer than that in normal rats [(351.3±119.9) min, t=2.291, P<0.05]. Western blotting revealed that the average values of gray scale ratio of c-kit in proximal colon were 0.277±0.077 and 0.576±0.081 (t=10.719, P<0.05), in distal colon were 0.280±0.075 and 0.571±0.079 (t=10.700, P<0.05) in model group and control group respectively.

CONCLUSIONDown-regulation of c-kit expression in proximal colon and distal colon is associated to the pathogenesis of slow transit constipation in rats.

Animals ; Chronic Disease ; Colon ; metabolism ; pathology ; Constipation ; metabolism ; pathology ; Disease Models, Animal ; Female ; Interstitial Cells of Cajal ; pathology ; Male ; Proto-Oncogene Proteins c-kit ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the location of telocytes in pulmonary tissues.

METHODSElectron microscopy, immunohistochemistry, primary cell culture and vital cell staining were used to identify the distribution of telocytes in mouse bronchial and pulmonary tissues.

RESULTSTelocytes were identified in the interstitial space between bronchial cartilage (cricoid) and smooth muscle by scanning and transmission electron microscope in mouse. By transmission electron micrscope and immunohistochemistry, telocytes were found in the interstitial spaces of lung parenchyma in connection with capillaries and bronchia. Telocytes expressed CD34, c-kit and vimetin by immunohistochemistry. After isolation, cultured telocytes demonstrated typical morphological feature, i.e. cells with telopode, which was seen as luminal structures with alternating thin and thick segments under electron microscope.

CONCLUSIONTelocytes are present in the interstitial space between cricoid cartilage and smooth muscle.

Animals ; Antigens, CD34 ; metabolism ; Bronchi ; cytology ; metabolism ; ultrastructure ; Cell Culture Techniques ; Immunohistochemistry ; Interstitial Cells of Cajal ; cytology ; metabolism ; ultrastructure ; Lung ; cytology ; metabolism ; ultrastructure ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Proto-Oncogene Proteins c-kit ; metabolism ; Vimentin ; metabolism

OBJECTIVETo observe the influence of Shenqing Recipe (SQR), a kind of Traditional Chinese Medicine, on the morphology and quantity of colonic interstitial cells of Cajal (ICC) in trinitrobenzene sulfonic acid (TNBS)-induced rat colitis, and to investigate the possible mechanism of SQR in regulating intestinal dynamics.

METHODSSixty rats were randomly divided into normal control, model 1, model 2, mesalazine, and high-dose, and low-dose SQR groups with 10 rats in each group. TNBS (10 mg) dissolved in 50% ethanol was instilled into the lumen of the rat colon of the latter five groups to induce colitis. On the 4th day after administration of TNBS, each treatment group was administered one of the following formulations by enteroclysis gavage once a day for 7 days: 600 mg•kg⁻¹•d⁻¹ mesalazine, 2.4 g•kg⁻¹•d⁻¹ SQR, and 1.2 g•kg⁻¹•d⁻¹ SQR. Model 2 rats received normal saline solution. After 7 days colonic samples were collected. While the colonic samples of model 1 group were collected on the 3rd day after TNBS administered. Ultrastructure of ICC in the damaged colonic tissues was observed with transmission electron microscope. Expression of c-kit protein in colonic tissue was determined by immunohistochemical staining and Western blot.

RESULTSThe ultrastructure of colonic ICC in the rat model of TNBS-induced colitis showed a severe injury, and administration of SQR or mesalazine reduced the severity of injury. Similarly, the expression of c-kit protein of TNBS-induced colitis rat model was significantly decreased compared with the normal control group (P < 0.05). Treatment with SQR or mesalazine significantly increased the expression of c-kit protein compared with the administration of control formulations (P < 0.05), especially the high-dose SQR group.

CONCLUSIONSQR could alleviate and repair the injured ICC, and improve its quantity, which might be involved in regulating intestinal motility.

Animals ; Colitis ; chemically induced ; drug therapy ; pathology ; Colon ; cytology ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Interstitial Cells of Cajal ; drug effects ; pathology ; ultrastructure ; Male ; Medicine, Chinese Traditional ; Mesalamine ; therapeutic use ; Peroxidase ; metabolism ; Proto-Oncogene Proteins c-kit ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Trinitrobenzenesulfonic Acid ; adverse effects

OBJECTIVETo study the clinical significance of interstitial cell of Cajal (ICC) in spontaneous neonatal gastric perforation by examining the expression of c-kit and Cx43 in neonates with this disorder.

METHODSThe gastric specimens of 19 cases of neonatal gastric perforation from 2001 to 2010 and 8 cases of accidental death without digestive tract malformations (control) were collected. Immunohistochemical staining was employed to examine the expression of c-kit and Cx43 (immunomarkers of ICCs) in gastric tissues.

RESULTSThe muscular layer of the stomach wall became thinner or deficient in the gastric perforation group. C-kit and Cx43 positive cells in gastric tissues decreased significantly in the gastric perforation group compared with those in the control group (P＜0.01).

CONCLUSIONSThe development of spontaneous neonatal gastric perforation is associated with the decreased quantity of ICCs and damaged gap junction structure of the stomach wall.

Connexin 43 ; analysis ; Female ; Humans ; Infant, Newborn ; Interstitial Cells of Cajal ; pathology ; Male ; Proto-Oncogene Proteins c-kit ; analysis ; Rupture, Spontaneous ; Stomach ; chemistry ; Stomach Rupture ; congenital ; metabolism ; pathology

OBJECTIVETo look for the evidences of motilin receptor expression on interstitial cells of Cajal (ICC) of the rabbit.

METHODSmooth muscle segments with ICC were isolated from the small intestine of 10-day old rabbits. The tissue segments equilibrated in Ca(2+)-free Hanks' solution were dispersed with an enzyme solution containing collagenase type II and then Ficoll density centrifugation was used to dissociate ICC. The cells were suspended and cultured in the M199 medium. The c-kit antibody was applied to distinguish the cultured ICC. The motilin receptor was identified by immunocytochemical assay with GPR38 antibody, c-kit antibody and hoechst 33342 combined to label ICC. Cells cultured for a few days were sorted for ICC with c-kit stained green fluorescent through flow cytometry. The total RNA and proteins extracted from the sorted ICC were respectively used to verify motilin receptor on the ICC by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting.

RESULTWe had successfully dissociated and cultured ICC of rabbit small intestine in vitro. Fluorescent staining with c-kit antibody confirmed that the culture ICC was successful. Triple-labeled immunofluorescent staining had detected the motilin receptor on membrane of ICC. Flow cytometry analysis showed that the ratio of c-kit positive cell in the cultured cells was 64.3%. The number of sorted ICC was 6.7 × 10(5) and 5.6 × 10(6). The results of RT-PCR and Western blot confirmed that the ICC had motilin receptor expression.

CONCLUSIONOur study demonstrated presence of motilin receptor on ICC of the rabbit. The present results may suggest that ICC play an important role in gastrointestinal movement induced by motilin.

Animals ; Cells, Cultured ; Interstitial Cells of Cajal ; metabolism ; Intestine, Small ; cytology ; Rabbits ; Receptors, Gastrointestinal Hormone ; metabolism ; Receptors, Neuropeptide ; metabolism

This study sought to probe into the mechanism of spontaneous contraction of portal vein. The morphological and electrophysiological characteristics of the freshly isolated interstitial cells (ICs) of rabbit portal vein were investigated by using immunohistochemical and conventional whole-cell patch clamp techniques. The isolated interstitial cells exhibited stellate-shaped or spindle-shaped bodies with a variable number of thin processes projecting from cell bodies, and these cells were noted to be c-Kit immunopositive. Under conventional whole-cell patch clamp configuration, the membrane potential was held at -60 mV, the spontaneous rhythmic inward currents were recorded in ICs, and the frequencies of which were similar to those of spontaneous contraction of portal vein. The inward currents were insensitive to nicardipine (an L-type calcium channel blocker) but could be abolished by gadolinium (a non-selective cation channel blocker). The results suggested that the spontaneous rhythmic inward currents recorded in freshly isolated ICs may be pacemaker currents which elicit the spontaneous contraction of portal vein.
Action Potentials ; Animals ; Electrophysiology ; Female ; Interstitial Cells of Cajal ; physiology ; Male ; Muscle, Smooth, Vascular ; physiology ; Periodicity ; Portal Vein ; cytology ; physiology ; Rabbits ; Transient Receptor Potential Channels ; metabolism

OBJECTIVETo observe expressions of glucose-regulated protein (GRP78) and caspase-S12 in nervous system-interstitial cells of Cajal (ICC) in smooth muscle in colonic wall in rats with scald injury, as well as their relevant ultrastructural changes, so as to probe the possible mechanisms of dynamic damage in murine colon after a scald injury.

METHODSFifty healthy Sprague-Dawley rats were randomly divided into scald (n = 40) and control (n = 10) groups. Rats in scald group were inflicted with 30% TBSA full-thickness scald, and received an intraperitoneally injection of Ringer lactate solution (50 mg/kg) for resuscitation, while those in control group had similar treatment with the exception of scald. Rats in control group and scald group were sacrificed at 3, 6, 12, 24 post scald hour (PSH, 10 rats at each time point) for collection of 4 cm of colonic tissue, 5 cm proximal to the cecum. A segment of colonic wall, 1 cm in length, was obtained from the middle of the harvested segment of colon, and it was fixed with 3% glutaraldehyde or 10% formaldehyde. The samples fixed with glutaraldehyde were used to observe ultrastructural alterations under transmission electron microscope, while that with formaldehyde were used to observe expressions of GRP78 and caspase-12 in colonic wall by immunohistochemical assay.

RESULTSThe colonic smooth muscle cells of rats in control group showed regular arrangement, their organelles were abundant, nucleus centrally located, euchromatin distributed evenly with more abundant mitochondrial cristae and less smooth endoplasmic reticulum, neuronal organelles were abundant in intermuscular plexus, and ICC could be seen in the neighborhood of neurons. The colonic smooth muscle cells appeared in irregular and disordered manner in scald group, perinuclear space was widened, intercellular vacuoles were observed, mitochondria showed vacuolation degeneration with dissolved and condensed cristae, rough endoplasmic reticula were dilated with partial dissolution, and perinuclear cytoplasm of ICC was obviously decreased. The expression of GRP78 was increased in scald group at 3, 6, 12 PSH (4.3 +/- 0.9, 6.0 +/- 0.7, 4.8 +/- 1.1 score) as compared with that in control group (2.4 +/- 0.7 score, P < 0.05). The expression of caspase-12 in scald group at 6, 12, 24 PSH was higher than that in control group (P < 0.05). GRP78 was consistently expressed in cytoplasm in control group, while in scald group, it mainly appeared in mucosa, myenteric plexus, and stromal cells, but only moderately or lightly expressed in smooth muscle cells. The expression of GRP78 was positive in scald group at 3, 6, 12 PSH, strongly positive at 6 PSH, and it was also expressed in cytoplasm in control group. The expression of caspase-12 in scald group was not obviously positive at 3 PSH, and weakly positive at 6, 24 PSH, but strongly positive at 12 PSH, while no expression was shown in control group.

CONCLUSIONSMarked pathological changes are observed in enteric nervous system-interstitial cells of Cajal-smooth muscle in rats with severe scald injury. It may be related with cellular injuries induced by caspase-12 apoptotic pathways through activated endoplasmic reticulum stress.

Animals ; Apoptosis ; Burns ; metabolism ; pathology ; physiopathology ; Caspase 12 ; metabolism ; Colon ; cytology ; Endoplasmic Reticulum ; metabolism ; Female ; Heat-Shock Proteins ; metabolism ; Interstitial Cells of Cajal ; cytology ; pathology ; ultrastructure ; Male ; Rats ; Rats, Sprague-Dawley