Molecular Mechanism Of Prokinetic Effect Of Macrolides On Promoting Gastric Emptying

【Background&Aims】Ever since erythromycin was observed to mimic motilin, it has attracted attention of many investigators because it has been found to improve delayed gastric emptying of diabetic patients with gastroparesis. It has now been demonstrated that erythromycin , as well as its derivatives are nonpeptide motilin agonists in rabbits and human. Erythromycin has now been used to treat kinds of digestive diseases with hypomotility including diabetic gastroparesis. During clinical use of this prokinetics, however, we observed a quite different response varying from markedly effective to no effect at all.This observation agrees with that of published research. We aim to study molecular mechanism underlying this phenomenon.【Methods】1. Set up animal models of diabetic gastroparesis of S-D rats, observe prokinetic effect of erythromycin by scintigraphically scanning of 99m Tc-DTPA.2. Screen receptor-related genes that might be involved in prokinetic effect of erythromycin, using RT-PCR for identification.3. Synthesized peptide was used to raise rabbit anti-human antibody against N-terminal of motilin receptor; Elisa method was used to measure titer of the antiserum. After electro-transfect of 293 cells with GPR38 plasmid; RT-PCR was used to confirm successful transfection; Use Western Blot and Immunohistochemistry method to study protein expression of motilin receptor in transfected 293 cells and rat antrum tissues.4. PCR amplification of exons of GPR38 (motilin receptor) gene, then screen possiblely existed single nucleotide polymorphism (SNP) sites through direct sequencing of PCR product.5. Statistical analysis:t-test, paired t-test, ANOVA and Crosstab (SPSS 11.0).【Results】1. We successfully set up animal models of diabetic gastroparesis of S-D rats. Half time of gastric emptying (T1/2, min) was measured in every group. T1/2 of G group was longer than that of N group (62±6.8 vs 30±4.5), P＜0.01; But became shorter after treatment of erythromycin (62±6.8 vs 49±14.5), P＜0.05. According to effect of erythromycin gastroparetic rats were divided into E group (means effective), F group (means failure) and S group (blank control, use saline in stead of erythromycin). After treatment, T1/2 of E group became shorter (64±6.5 vs 36±7.3), P＜0.01; while the change of F and S group is insignificant (60±8.4 vs 52±9.5, 62±7.8 vs 60±7.6, respectively), P＞0.05. The difference of improvement value (expressed in form of difference of T1/2 by self-comparison before and after treatment) between E and F group is significant (28±7.5, vs 8.0±5.2), P＜0.01.2. Through microarray analysis we screened differetial expression of genes that might be involved in the effect of erythromycin. Among 10 genes screened out, dopamine D3 receptor (DRD3) and neuropeptide Y5 receptor (NPYY5) were submitted to RT-PCR identification, which showes consistant results: expression of D3 receptor is higher in E group than F group (0.26±0.04 vs 0.16±0.04), P＜0.01; There are no significant difference between E, F group and N, S group (0.21±0.06, 0.23±0.06, respectively), P＞0.05. expression of Y5 receptor is higher in E group than F group (0.94±0.10 vs 0.68±0.09), P＜0.01; There are no significant difference between E, F group and N, S group (0.80±0.18, 0.82±0.15, respectively), P＞0.05. Polyclonal antibody against motilin receptor was successfully raised using synthesized peptide of N-terminal with high titer;The antibody can combine with synthesized peptide and cloned GPR38. Motilin receptor was mainly expressed in glandular epithelial cells of antrum of stomach. Expression levels were higher in E group than F group (0.43±0.03 vs 0.30±0.04), P＜0.05. There are no significant difference between E, F group and N, S group (0.35±0.05 vs 0.36±0.04,3. respectively), P＞0.05.4. We successfully amplified 2 exons of GPR38 gene, direct DNA sequencing showes 2 SNP site in exon1, 117 A/C &799 C/T, neither changes codon of amino-acids.【Conclusions】1. Erythromycin may ac...