A Comparison of the Effects of Estrogen and <i>Cimicifuga racemosa</i> on the Lacrimal Gland and Submandibular Gland in Ovariectomized Rats

<div><p>This study aims to observe the effects of estradiol and <i>Cimicifuga racemosa</i> on the lacrimal gland and submandibular gland of ovariectomized rats. We randomly divided 20 adult female SD rats into four groups—a sham-operated group (SHAM), ovariectomized (OVX) group, ovariectomized group treated with estradiol (OVX+ E), and ovariectomized group treated with the isopropanolic extract of <i>Cimicifuga racemosa</i> (OVX+ iCR). The SHAM group and OVX group used distilled water to instead the drugs. Two weeks after ovariectomy, the estradiol and iCR were administered for 4 weeks. Next, we used H&E staining and electron microscopy to observe any histological changes in the lacrimal and submandibular glands and immunohistochemical staining to observe the expressions of cleaved caspase-3 (Casp-3) and Cu-Zn SOD (superoxide dismutase). The H&E staining find that both drugs can prevent the cells of area from shrinkage in the two kinds of gland. But under the electron microscopy, estradiol and iCR have different efficacy. Estradiol is more effective at protecting mitochondria in lacrimal gland acinar cells than iCR, and iCR is more effective at suppressing endoplasmic reticulum expansion than estradiol. Both estradiol and iCR have a similar protective function on mitochondria in the submandibular gland. The protective function of the two glands may inhibit apoptosis by suppressing the expression of Casp-3. In addition, iCR increases the expression of Cu-Zn SOD in duct system of submandibular gland. The results suggest that both estradiol and iCR confer a protective effect on the lacrimal and submandibular glands of ovariectomized rats via different mechanisms.</p></div

H&E staining of lacrimal gland.

<p>A, SHAM; B, OVX; C, OVX + E; D, OVX + iCR. All scale bars represent 100 μm (magnification ×400). Compared with the SHAM group, the OVX group showed a sparse acinar cells, and the intercellular space was increased, suggested that cell atrophy was present, while estradiol valerate and iCR treatment can protected the glands from ovariectomy-induced cell atrophy to a certain extent.</p

H&E staining of trabecular bone from the left femoral distal ends at 12-week postoperation.

<p>Histological sections (stained with H&E) of trabecular bone from the left femoral distal ends of the four groups at 12 weeks after surgery. The OVX+NS group rats showed sparse and thinning trabeculae, with loss of connectivity and presence of abundant adiposity cells, while estradiol valerate and Remifemin treatment significantly protected trabecular bone from ovariectomy-induced osteopenia.</p

Effects of ovariectomy, estrogen and iCR therapy on body weight, lacrimal gland and submandibular gland weight.

<p>BW, body weight; WGR, weight gain rate; UW, uterus weight; LGW, lacrimal gland weight; LGW/BW final, lacrimal gland and final body weight ratio; SW, submandibular gland weight; SW/BW final, submandibular gland and final body weight ratio. Data are expressed as mean± SEM.</p><p>a means p<0.05 vs. SHAM;</p><p>b means p<0.01 vs. SHAM;</p><p>c means p<0.05 vs. OVX;</p><p>d means p<0.01 vs. OVX;</p><p>e means p<0.01 vs. OVX+E;</p><p>f means p<0.05 vs. OVX+ iCR;</p><p>g means p<0.01 vs. OVX+ iCR.</p><p>Effects of ovariectomy, estrogen and iCR therapy on body weight, lacrimal gland and submandibular gland weight.</p

H&E staining of submandibular gland.

<p>A, SHAM; B, OVX; C, OVX + E; D, OVX + iCR. All scale bars represent 100 μm (magnification ×400). Coincidence with the lacrimal gland, sparse acinar cells and increased intercellular space was commonly seen in the OVX group, while both of the treatment can improve the condition of ovariectomy-induced lesions.</p

The percentage of acinar and duct cells area in the lobule of lacrimal gland.

<p>a means p<0.01 vs. SHAM; b means p<0.01 vs. OVX.</p

The expression intensity of caspase-3 in the two kinds of gland.

<p>A: The ratio of casp-3 positive acinar cells in lacrimal gland. B: The ratio of casp-3 positive acinar cells in submandibular gland. C: The IOD of casp-3 positive staining in GCT of the submandibular gland. D: The IOD of casp-3 positive staining in the striated ducts of the submandibular gland. a means p<0.05 vs. SHAM; b means p<0.01 vs. SHAM; c means p<0.05 vs. OVX; d means p<0.01 vs. OVX; e means p<0.05 vs. OVX+ E; f means p<0.01 vs. OVX+ E; g means p<0.05 vs. OVX+ iCR; h means p<0.01 vs. OVX+ iCR.</p

The percentage of acinar and duct cells area in the lobule of submandibular gland.

<p>A, The percentage of intercellular space area in the lobule of submandibular gland. B, The percentage of acinar cells area in the lobule of submandibular gland. C, The percentage of GCT area in the lobule of submandibular gland. D, The percentage of striated ducts area in the lobule of submandibular gland. a means p<0.01 vs. SHAM; b means p<0.01 vs. OVX; c means p<0.05 vs. OVX; d means p<0.05 vs. OVX+E; e means p<0.05 vs. OVX+ iCR.</p

The striated duct of the submandibular gland.

<p>A, SHAM; B, OVX; C, OVX+ E; D, OVX+ iCR. Scale bars represent 1 μm. In the SHAM group, there was no obvious swelling or crest fracture in the mitochondria of the striated ducts. In contrast, many of the mitochondria were swollen, with double membrane structure damage, crest fracture in OVX group, but the condensed chromatin morphology was rarely observed. Both treatments improved this deleterious effect, resulting in mitochondria of normal appearance.</p

Comparisons of the morphometric indicators of the femoral distal end among the four groups.

<p>Comparison of bone morphometric indicators (relative bone volume (BV/TV), trabecular number (TbN), trabecular spacing (TbSp), and trabecular thickness (TbTh)) of the femoral distal ends (A-D) in SHAM, OVX+NS, OVX+E and OVX+iCR groups at 4-, 8- and 12-week postoperation. Data represents the means ± standard deviation (SD), n=6 rats in each group. The x-axis represents time after surgery (weeks). Compared with SHAM group, a means <i>p</i><0.05, b means <i>p</i><0.01; Compared with OVX+NS group, c means <i>p</i><0.05, d means <i>p</i><0.01; Comparison between OVX+iCR and OVX+E group, e means <i>p</i><0.05, f means <i>p</i><0.01.</p