ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of estradiol on the expression of antioxidant enzymes in osteoblasts and its role in postmenopausal osteoporosis.</p><p><b>METHODS</b>Rat models of osteoporosis established by ovariectomy were treated with estradiol for 3 months, and the changes in serum levels of reactive oxygen species (HO) and antioxidant enzymes (γ -GCS, GSH-ST and GSH-px) were detected. The effects of estradiol on the expression of γ -GCS mRNA and protein in osteoblast-like cells MC3T3-E1, MG63 and OB were examined with PCR and Western blotting. Using a mRNA microarray, we analyzed the changes in the expressions of 84 antioxidant enzymes in the osteoblast cell line MC3T3-E1 following estradiol treatment, and the enzymes with significant changes were verified by PCR. CCK-8 kit was used to evaluate the effect of estradiol and antioxidant NAC on the proliferation of MC3T3-E1 cells.</p><p><b>RESULTS</b>Rat models of osteoporosis were successfully established with ovariectomy. The osteoporotic rats showed significantly increased serum level of reactive oxygen species (H2O2) and decreased levels of antioxidant enzymes. Estrogen treatment of the osteoporotic rats obviously reversed the phenotype of osteoporosis, lowered serum level of reactive oxygen species, and increased the level of γ -GCS. In MC3T3-E1, MG63 and OB cells, estradiol treatment significantly upregulated the expression levels of γ -GCS mRNA and protein. In MC3T3-E1 cells treated with estrogen, the mRNA chip identified 6 upregulated antioxidant enzymes (Gpx6, Gstk1, Nos2, Prdx2, Ngb and Ccs), and the results of PCR verified that estradiol upregulated Ccs and Ngb mRNAs in MC3T3-E1, MG63 and OB cells. Estradiol and antioxidant NAC obviously promoted the proliferation of MC3T3-E1 cells.</p><p><b>CONCLUSION</b>Estradiol significantly increases the expression of antioxidase γ -Gcs, Ccs and Ngb in osteoblasts in vitro. Postmenopausal osteoporosis is closely related with the increase of reactive oxygen species and the decrease of antioxidant levels. In osteoblasts, estrogen deficiency may increase the level of reactive oxygen species, decrease the level of antioxidant enzymes, activate the oxidative stress cascade, and consequently inhibit the proliferation of osteoblasts to aggravate the condition of osteoporosis.</p>
ABSTRACT
<p><b>OBJECTIVE</b>To establish a method for gene delivery in murine renal tissue using lentivirus vector encoding miR-483-5p.</p><p><b>METHODS</b>Thirty-five C57BL/6J mice were randomly divided into control group, low-dose treatment group (5 µL each kidney) , and high?dose treatment group (20 µL each kidney), and in the latter two groups, the lentivirus vector encoding miR-483-5p were injected in the renal cortex. The tissue samples were collected at 7 and 21 days after the injection. A transgenic mouse model with inducible systemic overexpression of miR-483-5p was established in TG483 mice. The Cre-loxp system was used to create a mouse model with renal tubule-specific expression of miR-483-5p. The levels of BUN in the mice were detected and HE staining and fluorometric TUNEL assay were used to observe the morphological changes of the kidneys; real-time qPCR was used to detect miR-483-5p expression in the renal cortex.</p><p><b>RESULTS</b>The mice with overexpression of miR-483-5p had normal renal function without obvious pathological changes or apoptosis in the renal tissue. Renal cortex injection of 20 µL lentivirus resulted in obviously increased level of miR-483-5p at 21 days (1.2∓0.43 vs 8.6∓1.09, P<0.001). miR-483-5p showed a low expression (0.9∓0.09 vs 1.7∓0.19, P<0.05) in TG483 mice and a high expression in the kidney of the transgenic mice established using the Cre-loxp system (1.6∓1.13 vs 12.36∓3.89, P<0.05).</p><p><b>CONCLUSION</b>The transgenic mice with renal tubule-specific expression of miR-483-5p show normal renal function, and this model facilitates further study of the role of miR-483-5p in the kidney.</p>