| Objectives: In recent years, more and more bisphosphonate-related osteonecrosis patients were reported. People gradually realized that bisphosphonate-related osteonecrosis was one of the severe side-effects after using long-term bisphosphonate(BP) drug. There may be special relations between systemic using of bisphosphonate(BP) drug and osteonecrosis. Zoledronic acid was one of the most widely used BP drug, and had the strongest action in inhibiting bone absorption. Now bisphosphonate-related osteonecrosis hadbeen paid wide attention in the whole the world, but its mechanism is not clear. This study aimed to explore the mechanism when zoledronic acid was used in SD rats by applying gene different expression.Methods: Twenty-four healthy female SD rats with age of three month and weights from 200 to 240 gram were selected in the study. All the rats were free access to food and water for one week. The rats were divided into three groups with each group included 8 samples. Group A(Simple zoledronate group): Peritoneal injection was done by zoledronate with 0.2mg/kg(concentration of 0.1 mg/ml) twice a day for 8 weeks and physiological saline 0.5ml per 100 g in weihgtwas given by gavage once a day.Group B(zoledronate combined calcium): Peritoneal injection was done by zoledronate with 0.2mg/kg(concentration of 0.1 mg/ml) twice a day for 8 weeks. Concentration of 0.84% of Calcuim carbonate suspension liquid 0.5ml per 100 g in weihgt was given by gavage once a day. Group C(control group): Peritoneal injection was done by zoledronate with physiological saline 2ml/kg for 8 weeks, and physiological saline 0.5ml per 100 g in weihgt was given by gavage once a day for 8 weeks. In 8 weeks after injection, all the each group(8 SD rats) wsa randomly divided into extraction group(2 rats) and non-extraction group(6 rats). Extraction group: The first molar on the mandibular left side was extracted under 10% chloral hydrate general anesthesia. The animals were sacrificed and their mandibulae were collected in 4 weeks after extraction.The non-extraction group: 2 SD rats were randomly sacrificed in 8,10,12 weeks after injection and mandibule specimens were collected. All mandiblar specimans were completely stored in liquid nitrogen and transferred to-80℃ refrigerator.Then total RNA was extracted from the mandible by TRIZOL, and the integrity and purity of DNA were determined. Gene of all the specimens was tested by Affymetrix whole genome expression microarray. Then gene expression was scanned and analyzed by the Transcriptome Analysis Console V3.0 to find out the differential gene.Results: 1 Identification of purity and integrity the of total RNAThe result of spectrophotometric analysis: the OD260/OD280 ratios of all total RNA were between 1.9~2.1. The result of gel electrophoresis showed that there were three bands appeared in 1% agarose gel electrophoresis. The first band(28s) was brighter than the second band, and the third band was the darkest among three bands. 2 The result of microarray detection 2.1 Comparison between Group A and Group CIn 8 weeks after injection, there were 1799 genes with differential expression according to 2 times filtering condition. In these genes, 698 genes were up-regulated and 1101 genes were down-regulated.In 10 weeks after injection, there were 361 genes with differential expression according to 2 times filtering condition. In these genes, 158 genes were up-regulated and 203 genes were down-regulated. In 12 weeks after injection, there were 208 genes with differential expression according to 2 times filtering condition. In these genes, 97 genes were up-regulated and 111 genes were down-regulated. In 12 weeks after injection in extraction group, there were 180 genes with differential expression according to 2 times filtering condition. In these genes, 98 genes were up-regulated and 82 genes were down-regulated.2.2 Comparison between Group B and Group CIn 8 weeks after injection, there were 1287 genes with differential expression according to 2 times filtering condition. In these genes,580 genes were up-regulated and 707 genes were down-regulated. In 10 weeks after injection, there were 310 genes with differential expression according to 2 times filtering condition. In these genes, 193 genes were up-regulated and 117 genes were down-regulated. In 12 weeks after injection, there were 635 genes with differential expression according to 2 times filtering condition. In these genes, 273 genes were up-regulated and 362 genes were down-regulated. In 12 weeks after injection in extraction group, there were 320 genes with differential expression according to 2 times filtering condition. In these genes, 161 genes were up-regulated and 159 genes were down-regulated.2.3 Comparison between Group A and Group BIn 8 weeks after injection, there were 677 genes with differential expression according to 2 times filtering condition. In these genes, 264 genes were up-regulated and 413 genes were down-regulated. In 10 weeks after injection, there were 303 genes with differential expression according to 2 times filtering condition. In these genes, 131 genes were up-regulated and 172 genes were down-regulated. In 12 weeks after injection, there were 453 genes with differential expression according to 2 times filtering condition. In these genes, 250 genes were up-regulated and 203 genes were down-regulated. In 12 weeks after injection in extraction group, there were 189 genes with differential expression according to 2 times filtering condition. In these genes, 115 genes were up-regulated and 74 genes were down-regulated.2.4 Analysis resultes of differential genes associated with bone metabolism anHigh expression of MMP-9, Ccl9 and c-Fos were found in all time points between Group A and Group C. High expression of MMP-9, Ccl9, c-Fos and FDC-SP were found in all time points between Group B and Group C. High expression of FDC-SP was found in all three time points between Group B and Group A. High expression of MMP9 was found in Group B and Group A after extraction. High expression of FDC-SP was found in Group C(after extraction).Through the analysis by Pathway, these genes are the important regulator genes in the MAPK signal pathway and PANK/RANKL signal pathway which was related to bone metabolism. FDC-SP gene participated in the periodontal defense reaction.Conclusions:1 After Systemic using of Zoledronic acid affected the gene expression of mandible of SD rats. Some genes were up-regulated and some genes were down-regulated, Mmp9, Ccl9, c-fos and FDC-SP was significantly up-regulated.2 The mechanism of Bisphosphonate-related osteonecrosis may be affact bone metabolism by regulation of MAPK signal pathway and RANK/RANKL signal pathway.3 The application of zoledronic acid、calcium and vitamin D could prevent bisphosphonate-related osteonecrosis by improving the ability of periodontal defence. |
