Undesirable ER stress induced by bavachin contributed to follicular atresia in zebrafish ovary.

Fructus psoraleae (FP) is a commonly used herb with potential reproductive toxicity. Bavachin (BV), one of essential active ingredients of FP, was found to exhibit estrogenic activity, but its effect on female reproductive system remains unknown. In this study, the impact of BV on the female zebrafish reproductive system and underlying molecular mechanism were determined in vivo and ex vivo. The results showed that BV could accumulate in zebrafish ovary, leading to obvious follicular atresia and increase in gonadal index and vitellogenin content. Endoplasmic reticulum (ER) swelling and hypertrophy were observed in the BV-treated zebrafish ovary, accompanied by an increase in the expressions of ER stress and unfolded protein response (UPR) related genes, namely atf6, ire-1α and xbp1s. In the ex vivo study, BV was found to decrease the survival rate and maturation rate of oocytes, while increasing the expression of Ca2+. Additionally, BV led to an elevation in the level of estrogen receptor ESR1 and the expressions of genes involved in ER stress and UPR, including atf6, ire-1α, xbp1s, chop and perk. Moreover, molecular docking revealed that BV could directly bind to immunoglobulin heavy chain binding protein (BiP) and estrogen receptor 1 (ESR1). Besides, the alterations induced by BV could be partially reversed by fulvestrant (FULV) and 4-phenylbutyric acid (4-PBA), respectively. Thus, long-termed BV-containing medicine treatment could generate reproductive toxicity in female zebrafish by causing follicular atresia through BiP- and ESR-mediated ER stress and UPR, providing a potential target for the prevention of reproductive toxicity caused by BV.

Ginsenoside Rb1 Ameliorated Bavachin-Induced Renal Fibrosis via Suppressing Bip/eIF2α/CHOP Signaling-Mediated EMT.

The nephrotoxicity of Fructus Psoraleae, an effective traditional Chinese medicine for vitiligo treatment, has been reported. As one of the main toxic components in Fructus Psoraleae, bavachin (BV) was considered to be related to Fructus Psoraleae-caused adverse outcomes, but the direct evidence and molecular mechanism underlying BV-induced nephrotoxicity are not well elucidated. Therefore, this study was designed to confirm whether BV would cause toxic effects on the kidney and explore the possible mode of action. Our results demonstrated that days' treatment with 0.5 µM BV indeed caused obvious renal fibrosis in the zebrafish kidney. The obvious E- to N-cadherin switch and the expressions of proteins promoting epithelial-mesenchymal transition (EMT) were observed in BV-treated human renal tubular epithelial and zebrafish kidneys. In addition, elevated reactive oxygen species (ROS) levels and Bip/eIF2α/CHOP-mediated endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) were caused by BV, both of which could be reversed by ROS scavenger N-acetyl-L-cysteine (NAC). Also, blocking ER stress-caused cytoplasmic Ca2+ overload with 4-PBA notably alleviated BV-induced alterations in key molecular events related to EMT and renal fibrosis. Furthermore, of the natural compounds subjected to screening, ginsenoside Rb1 significantly downregulated BV-induced ER stress by inhibiting ROS generation and following the activation of Bip/eIF2α/CHOP signaling in HK2 cells. Subsequently, BV-triggered EMT and renal fibrosis were both ameliorated by ginsenoside Rb1. In summary, our findings suggested that BV-induced ROS promoted the appearance of EMT and renal fibrosis mainly via Bip/eIF2α/CHOP-mediated ER stress. This ER stress-related toxic pathway might be a potential intervention target for BV-caused renal fibrosis, and ginsenoside Rb1 would be a promising drug against BV- or Fructus Psoraleae-induced nephrotoxicity.

Low-dose radiation-induced demethylation of 3ß-HSD participated in the regulation of testosterone content.

The effects of low-dose radiation (LDR, ≤0.1 Gy) on living organisms have been the hot areas of radiation biology but do not reach a definitive conclusion yet. So far, few studies have adequately accounted for the male reproductive system responses to LDR, particularly the regulation of testosterone content. Hence, this study was designed to evaluate the effects of LDR on Leydig cells and testicular tissue, especially the ability to synthesize testosterone. We found that less than 0.2-Gy 60 Co gamma rays did not cause significant changes in the hemogram index and the body weight; also, pathological examination did not find obvious structural alterations in testis, epididymis, and other radiation-sensitive organs. Consistently, the results from in vitro showed that only more than 0.5-Gy gamma rays could induce remarkable DNA damage, cycle arrest, and apoptosis. Notably, LDR disturbed the contents of testosterone in mice serums and culture supernatants of TM3 cells and dose dependently increased the expression of 3ß-HSD. After cotreatment with trilostane (Tril), the inhibitor of 3ß-HSD, increased testosterone could be partially reversed. Besides, DNA damage repair-related enzymes, including DNMT1, DNMT3B, and Sirt1, were increased in irradiated TM3 cells, accompanying by evident demethylation in the gene body of 3ß-HSD. In conclusion, our results strongly suggest that LDR could induce obvious perturbation in the synthesis of testosterone without causing organic damage, during which DNA demethylation modification of 3ß-HSD might play a crucial role and would be a potential target to prevent LDR-induced male reproductive damage.

Emodin-Induced Oxidative Inhibition of Mitochondrial Function Assists BiP/IRE1α/CHOP Signaling-Mediated ER-Related Apoptosis.

Cassiae Semen is a widely used herbal medicine and a popular edible variety in many dietary or health beverage. Emerging evidence disclosed that improper administration of Cassiae Semen could induce obvious liver injury, which is possibly attributed to emodin, one of the bioactive anthraquinone compounds in Cassiae Semen, which caused hepatotoxicity, but the underlying mechanisms are not completely understood. Hence, the present study firstly explored the possible role of oxidative stress-mediated mitochondrial dysfunction and ER stress in emodin-cause apoptosis of L02 cells, aiming to elaborate possible toxic mechanisms involved in emodin-induced hepatotoxicity. Our results showed that emodin-induced ROS activated ER stress and the UPR via the BiP/IRE1α/CHOP signaling pathway, followed by ER Ca2+ release and cytoplasmic Ca2+ overloading. At the same time, emodin-caused redox imbalance increased mtROS while decreased MMP and mitochondrial function, resulting in the leaks of mitochondrial-related proapoptotic factors. Interestingly, blocking Ca2+ release from ER by 2-APB could inhibit emodin-induced apoptosis of L02, but the restored mitochondrial function did not reduce the apoptosis rates of emodin-treated cells. Besides, tunicamycin (TM) and doxorubicin (DOX) were used to activate ER stress and mitochondrial injury at a dosage where obvious apoptosis was not observed, respectively. We found that cotreatment with TM and DOX significantly induced apoptosis of L02 cells. Thus, all the results indicated that emodin-induced excessive ROS generation and redox imbalance promoted apoptosis, which was mainly associated with BiP/IRE1α/CHOP signaling-mediated ER stress and would be enhanced by oxidative stress-mediated mitochondrial dysfunction. Altogether, this finding has implicated that redox imbalance-mediated ER stress could be an alternative target for the treatment of Cassiae Semen or other medicine-food homologous varieties containing emodin-induced liver injury.

Mitochondrial Iron Overload-Mediated Inhibition of Nrf2-HO-1/GPX4 Assisted ALI-Induced Nephrotoxicity.

Aristolactam I (ALI) is an active component derived from some Traditional Chinese medicines (TCMs), and also the important metabolite of aristolochic acid. Long-term administration of medicine-containing ALI was reported to be related to aristolochic acid nephropathy (AAN), which was attributed to ALI-induced nephrotoxicity. However, the toxic mechanism of action involved is still unclear. Recently, pathogenic ferroptosis mediated lipid peroxidation was demonstrated to cause kidney injury. Therefore, this study explored the role of ferroptosis induced by mitochondrial iron overload in ALI-induced nephrotoxicity, aiming to identify the possible toxic mechanism of ALI-induced chronic nephropathy. Our results showed that ALI inhibited HK-2 cell activity in a dose-dependent manner and significantly suppressed glutathione (GSH) levels, accompanying by significant increases in intracellular 4-hydroxynonenal (4-HNE) and intracellular iron ions. Moreover, the ALI-mediated cytotoxicity could be reversed by deferoxamine mesylate (DFO). Compared with other inhibitors, Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, obviously alleviated ALI-induced cytotoxicity. Furthermore, we have shown that ALI could remarkably increase the levels of superoxide anion and ferrous ions in mitochondria, and induce mitochondrial damage and condensed mitochondrial membrane density, the morphological characteristics of ferroptosis, all of which could be reversed by DFO. Interestingly, ALI dose-dependently inhibited these protein contents of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutathione peroxidase 4 (GPX4), which could be partly rescued by Tin-protoporphyrin IX (SnPP) and mitoTEMPO co-treatment. In conclusion, our results demonstrated that mitochondrial iron overload-mediated antioxidant system inhibition would assist ALI-induced ferroptosis in renal tubular epithelial cells, and Nrf2-HO-1/GPX4 antioxidative system could be an important intervention target to prevent medicine containing ALI-induced nephropathy.