Ganoderma lucidum Prevents Cisplatin-Induced Nephrotoxicity through Inhibition of Epidermal Growth Factor Receptor Signaling and Autophagy-Mediated Apoptosis

Background. Cisplatin (cis-diaminedichloroplatinum, CDDP) is a broad-spectrum antineoplastic agent. However, CDDP has been blamed for its nephrotoxicity, which is the main dose-limiting adverse effect. Ganoderma lucidum (GL), a medicinal mushroom, has antioxidant and inflammatory activities. Therefore, this study is aimed at finding out the potential nephroprotection of GL against CDDP-induced nephrotoxicity in rats and the possible molecular mechanisms including the EGFR downstream signaling, apoptosis, and autophagy. Methods. Rats were given GL (500 mg/kg) for 10 days and a single injection of CDDP (12 mg/kg, i.p). Results. Nephrotoxicity was evidenced by a significant increase in renal indices and oxidative stress markers. Additionally, CDDP showed a plethora of inflammatory and apoptotic responses as evidenced by a profound increase of HMGB-1, NF-κB, and caspase-3 expressions, whereas administration of GL significantly improved all these indices as well as the histopathological insults. Renal expression of EGFR showed a similar trend after GL administration. Furthermore, activation of autophagy protein, LC3 II, was found to be involved in GL-mediated nephroprotection correlated with the downregulation of apoptotic signaling, caspase-3 and terminal deoxynucleotidyl transferase (TDT) renal expressions. Conclusion. These results suggest that GL might have improved CDDP-induced nephrotoxicity through antioxidant, anti-inflammatory, and autophagy-mediated apoptosis mechanisms and that inhibition of EGFR signaling might be involved in nephroprotection

IL-17/Notch1/STAT3 Pathway Contributes to 5-Fluorouracil-Induced Intestinal Mucositis in Rats: Amelioration by Thymol Treatment

5-Fluorouracil (5-FU) is an anticancer drug with intestinal mucositis (IM) as a deleterious side effect. Thymol is a monoterpene phenol which has been reported to possess an antioxidant and anti-inflammatory activity versus 5-FU-induced IM. The Notch pathway affects multiple cellular activities, such as cellular proliferation, in addition to inflammatory responses modulation. Accordingly, this work was carried out in order to elucidate the role of the Notch pathway in 5-FU-induced IM and to further elucidate the immunomodulatory protective mechanisms of thymol. Experimental rats were divided randomly into four groups: Control, 5-FU, 5-FU+thymol (60 mg/kg/day), and 5-FU+thymol (120 mg/kg/day). 5-FU was injected intraperitoneally at a dose of 150 mg/kg on days 6 and 7, while thymol was orally administered daily for 11 days. By the end of the study, intestinal tissues were collected for the determination of IL-17, CD4, CD8, Notch1, Hes-1, pSTAT3, and STAT-3 protein expressions. The effect of thymol on 5-FU cytotoxicity was also examined using WST1 assay. 5-FU induced a marked increase in IL-17 levels, along with a marked downregulation of CD4 and the upregulation of CD8, Notch1, Hes-1 protein expressions, and activation of STAT3 in the intestinal tissue when compared with the control group. Thymol ameliorated the changes that occurred in these parameters. Additionally, cytotoxicity testing revealed that thymol augmented the antiproliferative action of 5-FU against breast and colorectal human cancer cell lines. This study was the first to show that the IL-17/Notch1/STAT3 pathway is involved in the molecular mechanism of 5-FU-induced IM, as well as the immunomodulatory activity of thymol

Growth Hormone Ameliorates the Radiotherapy-Induced Ovarian Follicular Loss in Rats: Impact on Oxidative Stress, Apoptosis and IGF-1/IGF-1R Axis.

Radiotherapy is one of the standard cytotoxic therapies for cancer. However, it has a profound impact on ovarian function leading to premature ovarian failure and infertility. Since none of the currently available methods for fertility preservation guarantees future fertility, the need for an effective radioprotective agent is highly intensified. The present study investigated the mechanisms of the potential radioprotective effect of growth hormone (GH) on γ irradiation-induced ovarian failure and the impact of the insulin like growth factor 1 (IGF-1) in the underlying protection. Immature female Sprague-Dawley rats were either exposed to single whole body irradiation (3.2 Gy) and/or treated with GH (1 mg/kg s.c). Experimental γ-irradiation produced an array of ovarian dysfunction that was evident by assessment of hormonal changes, follicular development, proliferation marker (PCNA), oxidative stress as well as apoptotic markers. In addition, IGF-1/IGF-1R axis expression was assessed using real-time PCR and immunolocalization techniques. Furthermore, after full maturity, fertility assessment was performed. GH significantly enhanced follicular development and restored anti-Mullerian hormone serum level as compared with the irradiated group. In addition, GH significantly ameliorated the deleterious effects of irradiation on oxidative status, PCNA and apoptosis. Interestingly, GH was shown to enhance the ovarian IGF-1 at transcription and translation levels, a property that contributes significantly to its radioprotective effect. Finally, GH regained the fertility that was lost following irradiation. In conclusion, GH showed a radioprotective effect and rescued the ovarian reserve through increasing local IGF-1 level and counteracting the oxidative stress-mediated apoptosis

Radioprotective Effects of Carvacrol and/or Thymol against Gamma Irradiation-Induced Acute Nephropathy: In Silico and In Vivo Evidence of the Involvement of Insulin-like Growth Factor-1 (IGF-1) and Calcitonin Gene-Related Peptide

Radiotherapy (RT) is an effective curative cancer treatment. However, RT can seriously damage kidney tissues resulting in radiotherapy nephropathy (RN) where oxidative stress, inflammation, and apoptosis are among the common pathomechanisms. Carvacrol and thymol are known for their antioxidative, anti-inflammatory, and radioprotective activities. Therefore, this study investigated the nephroprotective potentials of carvacrol and/or thymol against gamma (γ) irradiation-induced nephrotoxicity in rats along with the nephroprotection mechanisms, particularly the involvement of insulin-like growth factor-1 (IGF-1) and calcitonin gene-related peptide (CGRP). Methods: Male rats were injected with carvacrol and/or thymol (80 and 50 mg/kg BW in the vehicle, respectively) for five days and exposed to a single dose of irradiation (6 Gy). Then, nephrotoxicity indices, oxidative stress, inflammatory, apoptotic biomarkers, and the histopathological examination were assessed. Also, IGF-1 and CGRP renal expressions were measured. Results: Carvacrol and/or thymol protected kidneys against γ-irradiation-induced acute RN which might be attributed to their antioxidative, anti-inflammatory, and antiapoptotic activities. Moreover, both reserved the γ -irradiation-induced downregulation of CGRP- TNF-α loop in acute RN that might be involved in the pathomechanisms of acute RN. Additionally, in Silico molecular docking simulation of carvacrol and thymol demonstrated promising fitting and binding with CGRP, IGF-1, TNF-α and NF-κB through the formation of hydrogen, hydrophobic and alkyl bonds with binding sites of target proteins which supports the reno-protective properties of carvacrol and thymol. Collectively, our findings open a new avenue for using carvacrol and/or thymol to improve the therapeutic index of γ-irradiation

Schematic diagram of the experimental design.

<p>Schematic diagram of the experimental design.</p

Immunohistochemical localization and Real-time quantitative Rt-PCR of insulin like growth factor-1 receptor (IGF-1R).

<p><b>(A)</b>: section of ovary obtained from the control rats shows modest degree of IGF-1R expression (brown color). <b>(B)</b>: section of ovary obtained from rats subjected to γ- irradiation (3.2 Gy) shows extensive IGF-1R expression (brown color) in granulosa cells of almost all follicles. <b>(C)</b>: section of an ovary obtained from rats treated with GH and exposed to γ –irradiation (3.2 Gy) shows high IGF-1R expression (brown color). <b>(D)</b>: section of ovary obtained from rats treated with GH alone (1mg/kg; once daily for one week) shows modest IGF-1R expression similar to control ovaries (brown color).Scale bar, 20 μm. <b>(E)</b>: Semi-quantitative expression of ovarian IGF-1R staining represents the mean optical density of immunopositive cells of 6 high power fields (20×) of at least three independent experiments. <b>(F)</b>: Real-time quantitative RT-PCR of ovarian IGF-1 and IGF-1R genes expressed as fold changes relative to control group. Each bar represents the Mean ± SEM for a group of 3 rats. a or b: Statistically significant from control or radiation group, respectively at P<0.05 using one-way ANOVA followed by Tukey–Kramer as a post-hoc test.</p

Follicular proliferation.

<p>Immunohistochemical localization of PCNA in ovarian follicles was studied 4 days after irradiation. <b>(A)</b>: Expression of PCNA in the control ovaries shows a large antral follicle (AnF) with positively stained (dark brown) granulosa cells (GC) and oocyte (O) and a cluster of primordial follicles with positively stained oocytes and stain-free pregranulosa cells around (oval). (<b>B)</b>: Expression of PCNA in ovaries of rats subjected to γ-irradiation (3.2 Gy) shows minimal expression. <b>(C) & (D)</b>: Expression of PCNA in ovaries of rats treated with GH 1 mg/kg for one week [either exposed to γ-radiation <b>(C)</b> or not <b>(D)]</b> shows a high PCNA expression of oocyte (O) and Granulosa cells (GC) (brown color). Scale bar, 20 μm. <b>(E)</b>: Quantitative Immunohistochemical staining of PCNA expressed as a percentage of immunopositive cells against the total number of granulosa cells across six high power fields (40×) from each rat section. Bars represent the Mean ± SEM of at least three independent experiments. a or b: Statistically significant from control or radiation group, respectively at P<0.05 using one-way ANOVA followed by Tukey–Kramer as a post-hoc test.</p

Effect of Growth hormone (1mg/kg s.c.; once daily for 1 wk) on different classes of ovarian follicles and the mean diameter of antral follicles in rats subjected to single dose whole-body irradiation (3.2 Gy).

<p>-Data expressed as Mean ± SEM of at least three rats.</p><p>-a, b: Significantly different from control, radiation group, respectively at p <0.05 using one-way ANOVA followed by Tukey–Kramer as a post-hoc test.</p><p>Effect of Growth hormone (1mg/kg s.c.; once daily for 1 wk) on different classes of ovarian follicles and the mean diameter of antral follicles in rats subjected to single dose whole-body irradiation (3.2 Gy).</p

Photomicrographs of uterine sections stained by hematoxylin and eosin.

<p><b>(A):</b> uterus from control rats shows normal histopathological structure of the mucosal lining epithelium (m) and the underlying lamina propria (p) with normal glandular structure (G). <b>(B):</b> uterine sections from rats subjected to γ- irradiation shows marked degeneration and stratification of the mucosal lining epithelium with multiple vacuoles (v) and thickening in the lamina propria <b>(C) & (D):</b> uterine sections taken from rats following GH treatment [either γ- irradiated <b>(C)</b> or not <b>(D)</b>] shows normal histological structure. Scale bar, 20 μm.</p

Effect of Growth hormone (1mg/kg s.c.; once daily for 1 wk) on oxidative stress markers in rats subjected to single dose whole-body irradiation (3.2 Gy).

<p>-Data are mean ± SEM (n = 6).</p><p>- a or b: Significantly different from control or radiation group, respectively at P <0.05 using one-way ANOVA followed by Tukey–Kramer as a post-hoc test</p><p>IR: irradiation; IR/GH: irradiation/Growth hormone; GH: Growth hormone; GSH, reduced glutathione; MDA, malondialdehyde; GPx, glutathione peroxidase; GSHRx, glutathione reductase.</p><p>Effect of Growth hormone (1mg/kg s.c.; once daily for 1 wk) on oxidative stress markers in rats subjected to single dose whole-body irradiation (3.2 Gy).</p