Sema3a inhibits the differentiation of Raw264.7 cells to osteoclasts under 2Gy radiation by reducing inflammation

<div><p>Astronauts and cancer patients receive different types of radiation, and radiation decreases bone strength and leads to radiation-induced osteoporosis. This effect is attributed to the activation of osteoclasts. Our aim was to study the effect of Sema3a on the differentiation of the murine macrophage cell line Raw264.7 into osteoclasts upon irradiation. Raw264.7 cells were divided into four groups: A, receiving no radiation; B, receiving no radiation + 50ngng/ml Sema3a; C, receiving 2Gy radiation; and D, receiving 2Gy radiation +50ngng/ml Sema3a. After treatment, cells were subjected to a proliferation assay, migration assay, live and apoptosis assay, and an ROS assay, along with analyses of bone resorption activity, TRAP staining and RT-PCR to assess the effect of Sema3a on Raw264.7 cells under 2Gy radiation. Sema3a inhibited the proliferation of Raw264.7 cells and showed statistical significance at a concentration of 100ngng/ml (P<0.05). Under 2Gy radiation, cell migration was reduced (P<0.05). In addition, 2Gy radiation resulted in more apoptotic cells, a higher level of ROS, larger bone resorption lacunae and more Trap-positive cells (p<0.05), and radiation increased <i>CSTK</i>, <i>NFAT</i>, <i>TRAP-5b</i>, <i>Rankl/OPG</i>, <i>IL-1</i>, <i>IL-6</i>, <i>TNFa</i> and <i>P53</i> gene expression (P<0.05). Sema3a had an inhibitory effect on the differentiation of Raw264.7 cells and the migration and activity of osteoclasts upon irradiation but did not affect ROS. Sema3a also decreased the expression of <i>CSTK</i>, <i>NFAT</i>, <i>TRAP-5b</i>, <i>Rankl/OPG</i>, <i>IL-1</i>, <i>IL-6</i> and <i>TNFa</i> on the 3rd and 7th days after irradiation (p<0.05), whereas <i>P53</i> expression was increased (P<0.05). Sema3a reduced the inflammation induced by radiation and negatively regulated osteoclast differentiation. Sema3a promoted Raw264.7 cell apoptosis after irradiation, indicating that Sema3a could be a potential therapeutic target for radiation-induced osteoporosis.</p></div

ROS levels.

<p>At 2 h, the ROS level increased approximately 4-fold upon 2Gy radiation compared to 0Gy radiation (*P<0.05), and Sema3a showed no effect on ROS generation under 0 Gy or 2Gy radiation. At 8 h following 2Gy radiation, the ROS level decreased approximately 10% compared to 2 h. Although Sema3a also decreased the ROS level, the differences between the 0ng/ml of Sema3a group and the 50ng/ml Sema3a group were not statistically significant (P>0.05, N = 5).</p

Cell migration assay.

<p>Raw264.7 cells given 2Gy or 0Gy radiation were placed in the upper chamber with no FBS, and medium with 10% FBS containing 0 or 50ng/ml Sema3a was added to the bottom chamber; 24 h later, cells were fixed with methanol and stained with crystal violet for 10 min. The cells on the upper side of the membrane were scrubbed away gently. The white triangles indicate the cells that migrated from the upper chamber to the lower chamber (Fig. 4A). A: 0Gy + 0ng/ml Sema3a; B: 0Gy + 50ng/ml Sema3a; C: 2Gy + 0ng/ml Sema3a; D: 2Gy + 50ng/ml Sema3a (N = 5). Fig. 4B shows the number of migrated cells: 2Gy radiation significantly decreased cell migration (*P<0.05), and Sema3a also inhibited cell migration; however, only under 2Gy radiation did Sema3 show a statistically significant effect (*P<0.05, N = 5).</p

Gene expression.

<p>The expression levels of <i>NFAT</i>, <i>CTSK</i>, <i>TRAP-5b</i>, <i>Rankl/OPG</i>, <i>IL-6</i>, <i>TNFa</i>, <i>IL-1</i> and <i>P53</i> on the 3rd and 7th days are shown (Fig. 7A). Gene expression levels of NFAT and <i>Rankl/OGP</i> were higher on the 3rd day than on the 7th day, and 2Gy radiation significantly increased their expression at both time points. Sema3a decreased the expression of these genes in the 2Gy and 0Gy radiation groups. The expression of <i>CTSK</i> and <i>TRAP-5b</i> was higher under 2Gy radiation compared to 0Gy radiation at both time points, and Sema3a significantly reduced their expression at both time points. The expression of pro-inflammatory genes (<i>IL-1</i>, <i>IL-6 and TNFa</i>) increased with 2Gy radiation compared with 0Gy. The expression of the <i>IL-6</i> and <i>TNFa</i> genes was higher on the third day than on the 7th day, but expression of the <i>IL-1</i> gene was higher only on day 7. Sema3a significantly decreased <i>IL-1</i>, <i>IL-6</i>, <i>and TNFa</i> expression in the 2Gy group at both time points. With 2Gy radiation, the expression of the apoptosis-related gene P53 was higher on the 3rd day than on the 7th day. Sema3a increased <i>P53</i> expression at both time points. (*P<0.05, N = 5). Western blot analysis of <i>CTSK</i>, <i>NFAT</i>, <i>IL-1</i>, <i>IL-6</i>, <i>TNFa</i> and <i>P53</i> levels at 3 days post-radiation is shown in Fig 7B. The results indicate that 2Gy radiation increased levels of <i>CTSK</i>, <i>NFAT</i>, <i>P53</i>, <i>IL-1</i>, <i>IL-6</i> and <i>TNFa</i>. Sema3a showed a similar effect on the expression of these proteins, with the exception of <i>P53</i>, in both the 0Gy and 2Gy groups. (N = 5).</p

Bone resorption.

<p>3A shows the results of the bone resorption assay: Raw264.7 cells plated on bovine cortical bone slices were differentiated into osteoclasts by the addition of 50ng/ml of Rankl and 20ng/ml of M-CSF in the presence or absence of 50ng/ml Sema3a and/or 2Gy radiation. The cells were then lysed with 5% sodium hypochlorite for 15 min, and the bone slices were subsequently dried. The resorbed areas were photographed by SEM and were analyzed by the ImageJ program. Scale bars = 500 μm (N = 5). Fig. 3B shows the area of bone resorption assay: The resorbed areas were measured with LSM Image Browser software. The area of the bone resorption increased two-fold upon 2 Gy radiation compared to 0 Gy radiation; with Sema3a, bone resorption area decreased 4-fold and 6-fold in the 0 Gy and 2 Gy groups, respectively. (*P<0.05, N = 6).</p

table1.pdf

The primer pairs used in the stud

Cell proliferation.

<p>Sema3a at concentrations of 0ng/ml, 10ng/ml, 50ng/ml and 100ng/ml was used to analyze the effect on cell proliferation. On days 5 and 7, 100ng/ml significantly reduced cell proliferation compared to 0ng/ml (*P<0.05, N = 5).</p

Cell apoptosis assay.

<p>The cells were divided into four groups as described before (A: 0Gy + 0ng/ml Sema3a; B: 0Gy + 50ng/ml Sema3a; C: 2Gy + 0ng/ml Sema3a; D: 2Gy + 50ng/ml Sema3a). (A) Annexin V-FITC/PI double staining by flow cytometry. (B) Statistical analysis of the cell apoptosis assay. The number of apoptotic cells was the sum of Q2 and Q3 (P<0.05, N = 4). (Q1, PI positive and annexin negative; Q2, both annexin and PI positive; Q3, annexin positive and PI negative Q4, both annexin and PI negative).</p

Sema3a inhibits the differentiation of Raw 264.7 cells to osteoclasts under 2Gy radiation by reducing inflammation

the figures used in Sema3a inhibits the differentiation of Raw 264.7 cells to osteoclasts under 2Gy radiation by reducing inflammatio

Osteoclast differentiation.

<p>2A shows the Trap staining: After addition of Rankl to the four groups for 7 days, the cells were fixed and stained for Trap. The osteoclasts were stained red. The white-black arrows indicate the osteoclasts. A: 0Gy + 0ng/ml Sema3a; B: 0Gy + 50ng/ml Sema3a; C: 2Gy + 0ng/ml Sema3a; D: 2Gy + 50ng/ml Sema3a. A greater number of positive cells with more nuclei were seen in groups A and C. Fig. 2B shows the number of osteoclasts: Sema3a apparently inhibited osteoclast differentiation under 0Gy radiation and 2Gy radiation, and 2Gy radiation significantly promoted osteoclast differentiation without Sema3a (*P<0.05). With 50ng/ml of Sema3, the differences between 0Gy radiation and 2Gy radiation were not statistically significant (N = 5). Fig. 2C shows the number of osteoclast nuclei (P<0.05, N = 6).</p