| Background:Slow transit constipation (STC) is a colonic motor disorder, which is characterized by measurably delayed movement of materials through the colon. Although abnormalities in the neuronal networks of the colon have been demonstrated in patients with STC, the aetiology of it remains unclear. The severity of its symptoms and the failure of the conservative therapy ultimately led to colectomy for some patients with STC.Health surveys revealed that chronic constipation caused by slow transit is common in women with an F/M ratio of 9:1. The cause and mechanism responsible for this syndrome are unknown. Among the female hormones, estrogen is one of the most important female homones,it can have an indispensable effect on body with estrogen receptor (ER), ER have two kinds of recptors, ie, ERαand ERβ. Studies reported ERβis the major ER in colon.That is to say, ERβcan determine the effect of estrogen and its antagonist on colon. Previous studies in our laboratories have shown that the expression of ERβprotein decreased greatly in mucous layer of sigmoid colon of patients with STC though immunohistochemistry method, which indicated ERβwas related to the pathogenesis of STC.Interstitial cells of Cajal (ICC) have been shown to be the pacemaker cells of the intestine and have been implied in the pathogenesis of a number of gastrointestinal motility dysfunction including idiopathic slow transit constipation. A major breakthrough in this field was the discovery that the tyrosine kinase receptor c-kit are critical in the normal development, maturation, and maintenance of phenotype of ICC, and ICC can be reliably identified by c-kit immunohistochemical technique. Blockade of c-kit with neutralized antibody could induce transdifferentiation of ICC to a smooth muscle phenotype, and intestinal slow waves disappear. A loss-of-function mutation of c-kit results in depletion of stem cells and ICC, while its gain-of-function mutation results in their oncogenesis. Several studies indicate that ICC decreased in the colon of patients with STC, and the expression of c-kit mRNA was also reduced. So, abnormal expression of c-kit gene may disturb the SCF/c-kit signal pathway, and contribute to the alteration of ICC in STC. However, the mechanism is unknown.Recent study suggested the blockade of ER signaling can lead to the decreased expression of c-kit mRNA, and ER may regulate c-kit signal pathway. So, we want to investigate further the expression of ERβin patients and rats with STC in transcription or translation phase. we study the changes of c-kit and ICC though blocking the ERβsignal pathway, and explore whether ERβhas any effects on ICC.Objective: The aims of this study were:(1) To investigate the sexual homones in patients and rats with STC, and to determine if sexual homones are abnormal.(1) To detect the expression of ERβprotein and its isoform mRNA within the colon of patients and rats with STC, and to determine if the expression of ERβare abnormal.(2) To explore the motility of rat colon though blocking the ERβsignal pathway, and further to investigate the expression of c-kit and ICC in the colon of experimental group, and to determine whether ERβhas any effects on ICC.Methods:Twenty patients with STC and Twenty age-matched controls were studied. The sexual homones were detected by Chemiluminescence. The distribution and expression of ERβwere observed with immunohistochemistry. Western blotting and Revers-transcriptional polymerase chain reaction (RT-PCR) technique were employed to determine the expression of ERβprotein and ERβisoform mRNA. An experimental rat model of STC was established with Rhubarb. Chemiluminescence was used to detect sexual homones. Frequency and amplitude of colonic slow waves were examined though electrophysiology. Western blotting and RT-PCR technique were employed to investigate the expression of ERβprotein and ERβisoform mRNA. Though blocking ERβpathway with tamoxifen (TAM), frequency and amplitude of colonic slow waves were examined though electrophysiology in both groups. Western blotting and RT-PCR technique were employed to determine the expression of c-kit protein and c-kit mRNA. The distribution and configuration of ICC were observed with immunohistochemistry. With an immunofluorescence staining, ICC were examined with fluorescence microscope and the area occupied by ICC were calculated with an image analysis system.Results:(1) Chemiluminescence displayed that FSH, LH, E2, PRL, P and Testo in patients with STC had no differences with that of control group (P>0.05).(2) Immunohistochemistry displayed that ERβwas detected in mucous layer, myenteric nerve plexus and submucous nerve plexus, but not detected in muscular layer in the sigmoid colon of patients with STC and the controls. ERβwas mainly expressed in cytoplasm. The expression of ERβin each of the three regions in STC group decreased significantly compared with the control group (P<0.05).(3) The RT-PCR results indicated that the ERβ1, ERβ2 and ERβ5 mRNA were detected in the sigmoid colon of patients with STC and the controls, but ERβ3 and ERβ4 mRNA were not detected. In comparison with the control group, the expression of ERβprotein and ERβ1, ERβ2 and ERβ5 mRNA of STC group significantly declined (P<0.01).(4) Western blotting technique showed that ERβprotein reduced markedly in the sigmoid colon of patients with STC compared with the controls (P<0.01).(5) An experimental rat model of STC was established with Rhubarb for 3 months. The frequency and amplitude of colonic slow waves were decreased in vitro (P<0.05).(6) Chemiluminescence displayed that FSH, E2, LH, P and Testo in rats with STC had no differences with that of control group (P>0.05), and PRL was too little to be detected.(7) The RT-PCR results indicated that the rERβ1 and rERβ2 mRNA were detected in the two groups,but rERβ1δ3, rERβ2δ3 and rERβ1δ4 mRNA were not detected. In comparison with the control group, the expression of rERβ1 and rERβ2 mRNA in the colon of rats with STC significantly declined (P<0.01).(8) Western blotting technique showed that ERβprotein in the colon of rats with STC reduced markedly compared with the controls (P<0.01).(9) Though blocking the ERβsignal pathway with TAM, the frequency and amplitude of slow waves of the experimental group were reduced significantly in vitro and in vivo compared with the control group(P<0.05). The RT-PCR and Western blot results indicated that the expression of c-kit mRNA and c-kit protein also decreased significantly (P<0.01). Immunohistochemistry displayed that ICC were located in the four regions (ICC-LM, ICC-MP, ICC-CM, ICC-SMB), and ICC in the experimental group appeared blunted and shorter compared with the controls. Immunofluorescence staining showed that the percentage of the area occupied by ICC in SMB, MP regions were significantly decreased while compared with the controls (p<0.01), and ICC in CM and LM regions almost can not be find.Conclusions:(1) Our studies revealed the sexual homones in patients and rats with STC had no differences with that in controls, which indicated that the serum sexual homones level had no direct relationship with STC.(2) The reduction of ERβin myenteric nerve plexus and submucous nerve plexus may relate to the pathogenesis of STC.(3) Abnormal expression of ERβgene in the colon of patients and rats with STC at transcription or translation phase may relate to the pathogenesis of STC.(4) Abnormal expression of ERβgene may disturb the c-kit signal pathway, and contribute to the alteration of ICC.
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