MicroRNA-200a Targets Cannabinoid Receptor 1 and Serotonin Transporter to Increase Visceral Hyperalgesia in Diarrhea-predominant Irritable Bowel Syndrome Rats

BACKGROUND/AIMS: MicroRNAs (miRNAs) were reported to be responsible for intestinal permeability in diarrhea-predominant irritable bowel syndrome (IBS-D) rats in our previous study. However, whether and how miRNAs regulate visceral hypersensitivity in IBS-D remains largely unknown. METHODS: We established the IBS-D rat model and evaluated it using the nociceptive visceral hypersensitivity test, myeloperoxidase activity assay, restraint stress-induced defecation, and electromyographic (EMG) activity. The distal colon was subjected to miRNA microarray analysis followed by isolation and culture of colonic epithelial cells (CECs). Bioinformatic analysis and further experiments, including dual luciferase assays, quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay, were used to detect the expression of miRNAs and how it regulates visceral hypersensitivity in IBS-D rats. RESULTS: The IBS-D rat model was successfully established. A total of 24 miRNAs were differentially expressed in the distal colon of IBS-D rats; 9 were upregulated and 15 were downregulated. Among them, the most significant upregulation was miR-200a, accompanied by downregulation of cannabinoid receptor 1 (CNR1) and serotonin transporter (SERT). MiR-200a mimic markedly inhibited the expression of CNR1/SERT. Bioinformatic analysis and luciferase assay confirmed that CNR1/SERT are direct targets of miR-200a. Rescue experiments that overexpressed CNR1/SERT significantly abolished the inhibitory effect of miR-200a on the IBS-D rats CECs. CONCLUSIONS: This study suggests that miR-200a could induce visceral hyperalgesia by targeting the downregulation of CNR1 and SERT, aggravating or leading to the development and progression of IBS-D. MiR-200a may be a regulator of visceral hypersensitivity, which provides potential targets for the treatment of IBS-D.

Osx and Satb2 regulate osteoblast differentiation, bone formation and repair / 中国组织工程研究

BACKGROUND:Osteoblasts occupy an important role in osteogenesis, which mainly come from bone marrow mesenchymal cels, and some transcription factors or local factors may promote the osteogenic differentiation of bone marrow stromal cels. OBJECTIVE: To study the role of Osx and Satb2 in C2C12cels in the repair process of osteoporosis. METHODS: Twenty wild-type Sprague-Dawley rats were assigned into normal control group (n=10), sham group (n=5) and osteoporosis group (model group,n=5). Another 10 Osx-KO rats were enroled in the study. Osteoporosis models were established by removal of both ovaries in the model group and Osx-KO group. In the sham group, bilateral ovaries were exposed but not removed. Changes in body mass and femoral bone density were detected in the four groups post operation. C2C12 cels were culturedin vitro, and siRNA-Satb2 and siRNA-Osx were designed. Expressions of Osx and Satb2 and their effects on osteoporosis were observed using cel experiments, gene silencing and western blot assay. RESULTS AND CONCLUSION:After 12 weeks, the body mass in the model and Osx-KO groups was significantly increased compared with the normal control and sham groups (P< 0.01); the bone density in the model and Osx-KO group was significantly decreased compared with the normal control and sham groups (P < 0.01). Satb2 and Osx were expressed in al the wild-type rats, but their expressions were decreased significantly in the Osx-KO rats (P < 0.001). Additionally, there was no difference in the Runx2 mRNA expression between the two kinds of rats. After silencing, the mRNA expressions of Satb2, Osx, Runx2 and ALP were al inhibited. These findings indicate that in the pathogenesis of osteoporosis, Osx and Satb2 may be protective molecules that have a regulatory role in the osteogenic differentiation, bone formation and repair.

Study of Extracts from Bushen Huoxue Decoction in Promoting Proliferation of Rat Bone Marrow Mesenchymal Stem Cells / 广州中医药大学学报

Objective To screen the active components of Bushen Huoxue Decoction ( BSHXD) involved in promoting the proliferation of bone marrow mesenchymal stem cells ( MSCs). Methods BSHXD and its subdivisions were extracted with petroleum ether, ethyl acetate, water-free ethanol and water respectively. MSCs were isolated and cultured by the bone marrow adherent method. At the third passage, MSCs were identified by the specific surface markers with immunofluorescence, and their osteogenic and adipogenic differentiation were tested by alizarin red staining and oil red “O” staining. After treated with the extracts of BSHXD and its subdivisions at gradient concentrations for 24 hours, cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay for the screening of active components and optimal concentration. MTT assay was used to describe the growth curve of MSCs treated with the most effective components, and cell cycle was analyzed by flow cytometry. Results Compared with the blank control group, the extracts of BSHXD and its subdivisions could protect MSCs from death to various degrees. Of all the extracts, the ethyl acetate extract of Bushen Division ( BSD) , ethyl acetate extract of BSHXD, ethyl acetate extract of Huoxue Division ( HXD) had the strongest effect, and the effect was dose-dependent, 100 μg/mL being the optimal active concentration while having no any cytotoxic reaction. The results of MTT assay revealed that BSD extracts promoted the proliferation of MSCs significantly and was the most effective component, and then came BSHXD. The results of flow cytometry indicated that BSD extract had the most strongest effect on increasing the amount of MSCs at proliferative phase, and then came BSHXD. Conclusion BSD ethyl acetate extract is the active component of BSHXD for promoting the proliferation of MSCs, showing an effect on increasing the proportion of MSCs at proliferative phase.