Salivary flow rate (SFR) was calculated at 30^th, 60^th, and 90^th day after irradiation.

<p>Sham1: Pre-sterilized water group; sham2: Pre+Post sterilized water group; sham3: Post-sterilized water group. Data is presented as means ± SEM. *: P<0.05 compared with normal group; **: P<0.05 between two individual groups; ***: P>0.05 compared with normal group.</p

Restoring the Secretory Function of Irradiation-Damaged Salivary Gland by Administrating Deferoxamine in Mice

<div><p>Objectives</p><p>One of the major side effects of radiotherapy for treatments of the head and neck cancer is the radiation-induced dysfunction of salivary glands. The aim of the present study is to investigate the efficacy of deferoxamine (DFO) to restore the secretory function of radiation-damaged salivary glands in mice.</p><p>Methods</p><p>DFO (50 mg/kg/d) was administered intraperitoneally in C₅₇BL/6 mice for 3 days before and/or after point-fixed irradiation (18 Gy) of submandibular glands. The total 55 mice were randomly divided into: (1) Normal group: mice received no treatment (n = 5); (2) Irradiation group (IR): mice only received irradiation (n = 5); (3) Pre-DFO group (D+IR) (n = 10); (4) Pre+Post DFO group (D+IR+D) (n = 10); (5) Post-DFO group (IR+D) (n = 10); (6) For each DFO-treated group, the mice were intraperitoneally injected with 0.1 ml sterilized water alone (by which DFO was dissolved) for 3 days before and/or after irradiation, and served as control. Sham1: Pre-sterilized water group (n = 5); sham2: Pre+Post sterilized water group (n = 5); sham3: Post-sterilized water group (n = 5). The salivary flow rate (SFR) was assessed at 30^th, 60^th and 90^th day after irradiation, respectively. After 90 days, all mice were sacrificed and their submandibular glands were removed for further examinations.</p><p>Results</p><p>The salivary glands showed remarkable dysfunction and tissue damage after irradiation. DFO restored SFR in the irradiated glands to a level comparable to that in normal glands and angiogenesis in damaged tissue was greatly increased. DFO also increased the expression levels of HIF-1α and VEGF while reduced apoptotic cells. Furthermore, Sca-1⁺cells were preserved in the salivary glands treated with DFO before IR.</p><p>Conclusions</p><p>Our results indicate DFO could prevent the radiation-induced dysfunction of salivary glands in mice. The mechanism of this protective effect may involve increased angiogenesis, reduced apoptosis of acinar cells and more preserved stem cells.</p></div

The weight of salivary gland harvested at 90^th day post irradiation.

<p>The weight of salivary gland shows no significant difference among individual groups. Sham1: Pre-sterilized water group; sham2: Pre+Post sterilized water group; sham3: Post-sterilized water group. Data is presented as means ± SEM. *: P<0.05 compared with normal group.</p

Detection of proliferating cells in submandibular gland tissue at 90^th day after irradiation.

<p>A: Immunohistochemical staining of PCNA from each group. Images of lower panel represent the higher magnification of the boxed area in corresponding images of upper panel. B: Surface area occupied by PCNA-positive cells (% per gland) between different groups. Sham1: Pre-sterilized water group; sham2: Pre+Post sterilized water group; sham3: Post-sterilized water group. Data is presented as means ± SEM. *: P<0.05 compared with normal group; **: P<0.05 between two individual groups.</p

DFO administration improved survival of salivary stem/progenitor cells.

<p>Immunohistochemical staining of Sca-1 in different groups. Images of lower panel represent the higher magnification of the boxed area in corresponding images of upper panel. Arrows indicate Sca-1⁺ cells.</p

Analysis of apoptotic cells by TUNEL determination at 90^th day after irradiation.

<p>TUNEL assay of apoptotic cells in different groups. A: TUNEL-positive cells of submandibular gland tissue from each group. B: Ratio of TUNEL-positive cells to total nuclei (% per gland) between different groups. Sham1: Pre-sterilized water group; sham2: Pre+Post sterilized water group; sham3: Post-sterilized water group. Data is presented as means ± SEM. *: P<0.05 compared with normal group; **: P<0.05 between two individual groups.</p

DFO administration improved angiogenesis in irradiated tissue via activating HIF-1α-VEGF Pathway.

<p>A: Real-time PCR detection of VEGF expression 90 days after irradiation. B: Western-blot analysis of HIF-1α in tissues received DFO. Sham1: Pre-sterilized water group; sham2: Pre+Post sterilized water group; sham3: Post-sterilized water group. Data is presented as means ± SEM. *: P<0.05 compared with normal group; **: P<0.05 between two individual groups. C: HIF-1α in each group. Annotate: 1A represents D+IR group; 1B represents sham1; 2A represents IR+D group; 2B represents sham3; 3A represents D+IR+D group; 3B represents sham2; 4 represents IR; 5 represents normal group; β-Actin represents reference.</p

Schematic representation of the experimental design.

<p>Local 18 Gy irradiation of salivary glands was given before or/and after 3-day DFO treatment (50 mg/kg/d). Normal: no treatment; IR: only Irradiation group; D+IR: Pre-DFO group; sham1: Pre-sterilized water group; IR+D: Post-DFO group; sham3: Post-sterilized water group; D+IR+D: Pre+Post DFO group; sham2: Pre+Post sterilized water group; DFO: deferoxamine.</p

CRH triggers the activation of primordial follicles in the cultured ovaries of newborn mice <i>in vitro</i>.

<p>(A) The mRNA expression level of CRH receptor CRHR1 in the ovaries of the CRS group was significantly higher compared with that in the control group (<i>n</i> = 6). (B) The cell viability of mice granulosa cells (GCs) increased with the treatment of increasing concentration of CRH using theCCK-8 assay (<i>n</i> = 12). (C) The mRNA expression level of Kitl in mice GCs after culturing in serial concentration of CRH for 48 h (<i>n</i> = 6). (D) H&E staining of cultured mice ovaries from the control or CRH groups. Ovaries were isolated from the 3-day postnatal mice and cultured <i>in vitro</i> with or without CRH (100nM) for 7 days (scale bar, 100 μm). (E) CRH treatment increased the proportion of developing follicles in cultured mice ovaries (<i>n</i> = 6). (F) CRH treatment increased the diameter of oocytes in developing follicles in cultured mice ovaries (<i>n</i> = 6). (G) The mRNA levels of Kitl, Kit, AMH, and Gdf9 in cultured mice ovaries increased after CRH treatment (<i>n</i> = 6). (*<i>P</i><0.05,**<i>P</i>< 0.01,***<i>P</i>< 0.001,****<i>P</i>< 0.0001).</p

Chronic restraint stress induces excessive activation of primordial follicles in mice ovaries

<div><p>Chronic stress is an important factor influencing people’s health. It usually causes endocrinal disorders and a decline in reproduction in females. Although studies of both human and animals suggest a detrimental effect of stress on reproduction, the influence of chronic stress on the ovarian reservation and follicular development is still not clear. In this study, a chronic restraint stress (CRS) mouse model was used to investigate the effect of stress on ovarian reservation and follicular development and explore the underlying mechanism. In this study, after 8 weeks of CRS, primordial follicles were excessively activated in the ovaries of the CRS group compared with the control group. Further results showed that the activation of primordial follicles induced by CRS was involved in the increasing expression level of Kit ligand and its receptor Kit and the activation of phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/protein kinase B (Akt) pathway. The corticotropin-releasing hormone (CRH) is a neuropeptide released due to stress, which plays an important role in regulating follicle development. A high level of serum CRH was detected in the CRS mouse model, and the real-time polymerase chain reaction assay showed that the mRNA level of its main receptor CRHR1increased in the ovaries of the CRS mouse group. Moreover, 100nM CRH significantly improved the activation of primordial follicles in newborn mouse ovaries <i>in vitro</i>. These results demonstrated that CRS could induce immoderate activation of primordial follicles accompanied by the activation of Kit-PI3K signaling, in which CRH might be an important endocrine factor.</p></div