Network pharmacology, molecular docking and experimental study on the mechanism of Curcumin's anti-ferroptosis in melanocytes.

Ferroptosis is a form of regulated nonapoptotic cell death associated with iron-dependent lipid peroxidation, closely associated with Vitiligo. Although the impact of Curcumin (Cur), a polyphenolic compound derived from the plant Curcuma longa Linn, on vitiligo has been established, the specific role and potential mechanistic pathways through which Cur modulates ferroptosis in vitiligo remain elusive. In this study, the critical targets and potential mechanisms of Cur in treating vitiligo were predicted by network pharmacology and molecule docking. Then, the effects of Cur on Erastin-induced ferroptosis were investigated in melanocytes induced by Erastin in vitro. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of Cur acting on Vitiligo found that these intersection genes are associated with the vitiligo oxidative stress pathway, including nuclear factor erythroid 2-related factor 2(Nrf2)/Heme Oxygenase 1(HO-1) signaling pathway. Further molecular docking shows that Cur has a good binding effect with Nrf2(the binding energy of Cur and Nrf2 protein is -6 kcal/mol). Through the CCK8 assay, showed that 10 µM Cur treatment 24 h after Erastin significantly improved cell viability In vitro. Then we found that Erastin induced cell death, ROS production, the mitochondrial membrane potential(MMP) decreased, Superoxide dismutase (SOD) and Glutathione (GSH) levels reduced, Malonaldehyde (MDA) and iron ion accumulation in melanocytes. In addition, the expression of glutathione peroxidase 4(GPX4) mRNA and protein was inhibited, while the expression of acyl-CoA synthetase long-chain family member 4(ACSL4), Transferrin Receptor Protein 1(TFR1) mRNA and protein was increased. However, the damage induced by Erastin was significantly relieved by Cur and Fer-1 treatment. Mechanistically, Cur treatment significantly promoted nuclear translocation of transcriptional factor Nrf2 and HO-1 expression. Interestingly, pretreatment with ML385, a selective Nrf2 inhibitor, counteracted anti-ferroptosis effects induced by Cur treatment. Taken together, these results demonstrate that Cur inhibits ferroptosis by regulating the Nrf2/HO-1 pathway to protect melanocytes.

The Role of Regulatory Cell Death in Vitiligo.

Vitiligo is one of the common chronic autoimmune skin diseases in clinic, which is characterized by localized or generalized depigmentation and seriously affects the physical and mental health of patients. At present, the pathogenesis of vitiligo is not clear; mainly, heredity, autoimmunity, oxidative stress, melanocyte (MC) self-destruction, and the destruction, death, or dysfunction of MCs caused by various reasons are always the core of vitiligo. Regulatory cell death (RCD) is an active and orderly death mode of cells regulated by genes, which widely exists in various life activities, plays a pivotal role in maintaining the homeostasis of the organism, and is closely related to the occurrence and development of many diseases. With the deepening of the research and understanding of RCD, people gradually found that there are many different forms of RCD in the lesions and perilesional skin of vitiligo patients, such as apoptosis, autophagy, pyroptosis, ferroptosis, and so on. Different cell death modes have different mechanisms in vitiligo, and different RCDs can interact and regulate each other. In this article, the mechanism related to RCD in the pathogenesis of vitiligo is reviewed, which provides new ideas for exploring the pathogenesis and targeted treatment of vitiligo.

Increased expression of heat shock protein 105 in rat uterus of early pregnancy and its significance in embryo implantation.

BACKGROUND: Heat shock proteins (Hsps) are a set of highly conserved proteins, Hsp105, has been suggested to play a role in reproduction. METHODS: Spatio-temporal expression of Hsp105 in rat uterus during peri-implantation period was examined by immunohistochemistry and Western blot, pseudopregnant uterus was used as control. Injection of antisense oligodeoxynucleotides to Hsp105 into pregnant rat uteri was carried out to look at effect of Hsp105 on embryo implantation. RESULTS: Expression of Hsp105 was mainly in the luminal epithelium on day 1 of pregnancy, and reached a peak level on day 5, whereas in stroma cells, adjacent to the implanting embryo, the strongest expression of Hsp105 was observed on day 6. The immunostaining profile in the uterus was consistent with that obtained by Western blot in the early pregnancy. In contrast, no obvious peak level of Hsp105 was observed in the uterus of pseudopregnant rat on day 5 or day 6. Furthermore, injection of antisense oligodeoxynucleotides to Hsp105 into the rat uterine horn on day 3 of pregnancy obviously suppressed the protein expression as expected and reduced number of the implanted embryos as compared with the control. CONCLUSION: Temporal and spatial changes in Hsp105 expression in pregnant rat uterus may play a physiological role in regulating embryo implantation.

Effect of 43 degrees treatment on expression of heat shock proteins 105, 70 and 60 in cultured monkey Sertoli cells.

AIM: To examine the possible effect of heat treatment on expression of heat shock proteins (Hsps) 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. METHODS: Western blot analysis, real-time polymerase chain reaction (PCR), and confocal immunohistochemistry were used to analyze mRNA and protein levels of the Hsps in response to 43 degrees treatment of Sertoli cells isolated from pubertal monkey testes. RESULTS: Staining with Hoechst 33342 indicated Sertoli cells did not undergo apoptosis after heat treatment. Hsp105 was expressed in cytoplasm of untreated Sertoli cells. Both Hsp105 mRNA and protein levels were increased approximately 20-fold compared to those of the untreated controls at 12 h after heat treatment. Untreated Sertoli cells did not express Hsp70, but heat stress induced its expression in the cell cytoplasm. The time-course of changes in Hsp70 was similar to that of Hsp105. In contrast to Hsp105 and Hsp70, the change in Hsp60 expression was much less obvious. The protein level between 12 h and 48 h after heat treatment was only approximately 1.5-fold that of the untreated control. Extracellular regulated kinase (ERK) 1/2 inhibitor (U0126) or phosphoinositide kinase-3 (PI3K) inhibitor (LY294002) could partially block the response of Hsp105 and Hsp70 induced by heat treatment. CONCLUSION: These results indicate that the heat-induced expression of the three types of Hsp in monkey Sertoli cells might be regulated by ERK and/or PI3K signal pathways, but the profile of their expression is different, suggesting that they might have different regulatory functions in Sertoli cells.

Expression and regulation of stanniocalcin 1 and 2 in rat uterus during embryo implantation and decidualization.

Stanniocalcin-1 (STC-1) is a recently discovered polypeptide hormone, while stanniocalcin-2 (STC-2) is a subsequently identified homologue of stanniocalcin-1. Although previous studies have shown that both STC-1 and -2 are involved in various physiological processes, such as ion transport, reproduction and development, their expression in the uterus and roles in implantation and early pregnancy are unclear. Here we have investigated the expression and regulation of both STC-1 and STC-2 in rat uterus during early pregnancy under various physiological conditions. We show that only basal levels of STC-1 and STC-2 mRNA were detected in the uterus from day one (D1) to day five (D5) of pregnancy. STC-2 immunostaining was gradually increased in the glandular epithelium from day two (D2), with a peak occurring on D5. High levels of both STC-1 and STC-2 mRNA were observed in the stoma cells at the implantation site on day six (D6) of pregnancy, whereas their immunostaining signals were also significant in the luminal epithelium. Basal levels of both STC-1 and STC-2 mRNA and STC-1 immunostaining were detected in the uterus with delayed implantation. After the delayed implantation was terminated by estrogen treatment, both STC-1 and STC-2 mRNA signals were significantly induced in the stroma underlying the luminal epithelium at the implantation site, and STC-2 immunostaining was also observed in the luminal epithelium surrounding the implanting blastocyst. Embryo transfer experiments further confirmed that STC-1 and STC-2 expression at the implantation sites was induced by the implanting blastocyst. Both STC-1 mRNA and immunostaining were seen in the decidualized cells from day seven (D7) to day nine (D9) of pregnancy. STC-2 mRNA was also found in the whole decidua from D7 to D9 of pregnancy; STC-2 protein, however, was strictly localized to the primary deciduas on D7 and D8, with a weak expression in the whole deciduas on D9. Consistent with the normal pregnancy process, strong STC-1 and STC-2 mRNA signals were detected in the decidualized cells under artificial decidualization, whereas only basal levels of STC-1 mRNA and immunostaining were observed in the control horn. These data suggest, for the first time, that STC-1 together with STC-2 may play important roles in the processes of implantation and decidualization in the rat.

Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys.

AIM: To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. METHODS: Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. RESULTS: The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 (CK18), a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. CONCLUSION: The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.

Expression of orphan receptors TR2, TR3, TR4, and p53 in heat-treated testis of cynomolgus monkeys (Macaca fascicularis).

To investigate the possible role of testicular orphan receptors (TR) TR2, TR3, and TR4 in the process of germ cell apoptosis in the heat-treated testis of monkey, we have examined the spatiotemporal expression of the 3 TR mRNAs in relation to p53 mRNA levels in the monkey testis by in situ hybridization and reverse transcription polymerase chain reaction techniques. The results showed that TR2 mRNA was confined to spermatocytes; TR4 and TR3 mRNAs were expressed in both spermatocytes and spermatids. The heat treatment did not change TR2 mRNA level but significantly reduced TR4 mRNA expression in spermatocytes on days 3 and 8 after the heat treatment. TR3 mRNA expression was affected by the heat treatment in a time-dependent manner, with the lowest level on day 30 after the heat shock. Low to moderate signal for p53 mRNA was detected in spermatocytes before treatment, which increased dramatically on days 3, 8, and 30 after the heat shock. The coincident expression of the testicular TR3 and p53 mRNA, spatially and time dependently, implied that the decrease in TR3 expression in the heat-treated testis might be closely related to the p53 signal pathway, whereas the temporal decrease in TR4 production in the testis at the early stage indicated that this orphan receptor might be also involved in germ cell apoptosis. The data suggest that TR3, TR4, and p53 could be important regulators of germ cell apoptosis induced by the heat treatment, whereas TR2 might not be a key regulator in this process.

Expression of HSP105 and HSP60 during germ cell apoptosis in the heat-treated testes of adult cynomolgus monkeys (macaca fascicularis).

To confirm that transient increase in temperature of the testis (43C for 30 minutes once daily for 2 consecutive days) could induce apoptosis of germ cells in non-human primates and to investigate the possible roles of Hsp105 and Hsp60 in regulation of germ cell loss, we conducted the study on eight cynomolgus monkeys. The sperm concentration on day 28 after heat shock decreased to 8.4% of pretreatment levels and recovered to baseline on day 144. Using the TUNEL assay, increased numbers of apoptotic spermatocytes and round spermatids were detected on days 3, 8, and 30 post heat treatment. Hsp105 and Hsp60 mRNA and protein levels were analyzed using in situ hybridization, RT-PCR, immunohistochemical and Western blot methods. Hsp105 was confined to nuclei of spermatids before treatment, decreased dramatically with the loss of spermatids on days 3, 8, and 30, before returning to baseline levels on days 84 and 144. The expression of Hsp60 was high on days 3, 8, 30 and was only detected in Sertoli cells and spermatogonia. These results suggested that exposure of the testis to heat resulted in selective, but reversible damage to the seminiferous epithelium via increased germ cell apoptosis. Temporal changes in the expression pattern of Hsp105 and Hsp60 in relation to germ cell death suggests they may be involved in key processes in regulation of germ cell apoptosis.

Extracellular Ca2+-sensing receptor expression and hormonal regulation in rat uterus during the peri-implantation period.

The extracellular Ca2+-sensing receptor (CaR) is a member of the superfamily of G protein-coupled receptors (GPCRs). It is an important mediator of a wide range of Ca2+-dependent physiological responses in various tissues. In reproductive tissues it has been reported to play a significant role in promoting or maintaining placentation. Meanwhile, another Ca2+ regulated gene stanniocalcin-1 (STC-1) has been documented to be involved in decidualization and uterine remodelling. The phenomenon that CaR mediates STC-1's transcription responding to extracellular calcium in fish urges us to suppose that CaR, like STC-1, may also play a role in implantation and decidualization. To resolve this conjecture, we have examined the expression and hormonal regulation of the CaR gene in rat uterus during peri-implantation period. CaR mRNA was expressed at a moderate level in the luminal epithelium of the early stage of pregnancy (from day 1 to day 3). From day 2-3 it began to be expressed more strongly in the stromal cells immediately underneath the luminal epithelium, but decreased to a basal level on day 4. From day 6 to day 9 continuously, both CaR mRNA and protein were highly expressed in the primary decidua. Expression of CaR mRNA and protein in these cells was also observed when a delayed implantation was terminated by estrogen treatment to allow the embryo implantation. In contrast, only basal level expression of the molecules was detected in the cells of animals subjected to a normal-delayed implantation or the pseudopregnant condition. Embryo transplantation experiment confirmed that CaR expression at the implantation site was induced by the implanting blastocyst. Consistent with the normal pregnant process, CaR mRNA and protein in the cells were also induced by an artificial decidualization procedure. Further experiments demonstrated that treatment of the ovariectomized rat with estrogen or/and progesterone stimulated a high level expression of CaR mRNA in the uterine epithelial and glandular epithelium. In conclusion, CaR was specifically induced during the processes of implantation and subsequent decidualization and may play a role in these processes.

Signal transduction of stem cell factor in promoting early follicle development.

Stem cell factor (SCF), another alternative name is kit ligand, is essential for the development of early follicles. However, the underlying molecular mechanism remains to be defined. By using cultured ovaries that are rich in primordial follicles, the action of SCF (kit ligand) on early follicular development and the activated signal transduction pathways were investigated. SCF (kit ligand) promoted early follicle development. PKC and MEK but not PKA were involved in the signal transduction of SCF (kit ligand) as indicated by results using their specific pharmacological inhibitors. SCF (kit ligand) also enhanced the phosphorylation of two MEK substrates, Erk1 and 2 (Erk1/2) in thecal-interstitial cells where PKC might play an important role indicated by results using its inhibitors. SCF (kit ligand) elevated the expression of steroidogenic factor 1 (SF-1) in thecal-interstitial cells probably through a pathway that consists of Erk1/2. These results suggest that SCF (kit ligand) promotes follicular growth by stimulating the function of thecal-interstitial cells through the Erk1/2 pathway.

Localization of CD9 in pig oocytes and its effects on sperm-egg interaction.

CD9 is a cell surface protein that participates in many cellular processes, such as cell adhesion. Fertilization involves sperm and oocyte interactions including sperm binding to oocytes and sperm-oocyte fusion. Thus CD9 may play an essential role during fertilization in mammals. The present study was conducted to examine whether CD9 is present in porcine gametes and whether it participates in the regulation of sperm-oocyte interactions. The presence of CD9 in ovarian tissues, oocytes and spermatozoa was examined by immunohistochemistry, immunofluorescence and immunoblotting. Sperm binding and penetration of oocytes treated with CD9 antibody were examined by in vitro fertilization. The results showed that CD9 was present on the plasma membrane of oocytes at different developmental stages. A 24 kDa protein was found in oocytes during in vitro maturation by immunoblotting and its quantity was significantly (P < 0.001) increased as oocytes underwent maturation and reached the highest level after the oocytes had been cultured for 44 h. No positive CD9 staining was found in the spermatozoa. Both sperm binding to ooplasma and sperm penetration into oocytes were significantly (P < 0.01) reduced in anti-CD9 antibody-treated oocytes (1.2 +/- 0.2 per oocyte and 16.6% respectively) as compared with oocytes in the controls (2.5 +/- 0.4 per oocyte and 70.3% respectively). These results indicated that CD9 is expressed in pig oocytes during early growth and meiotic maturation and that it participates in sperm-oocyte interactions during fertilization.

Molsidomine and N-omega-nitro-L-arginine methyl ester inhibit implantation and apoptosis in mouse endometrium.

AIM: To investigate the possible effect of nitric oxide on receptivity and apoptosis of mouse endometrium and the possible pathway. METHODS: Female pregnant mice were treated with either molsidomine, a generator of nitric oxide (NO), or N-omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. The pregnancy rates of each group were calculated; 3'-end-labeling was used to detect DNA fragmention of apoptotic cells; immunohistochemistry, in situ hybridization, and Western blot were applied respectively to estimate expression levels of Fas/FasL proteins and mRNA. RESULTS: The pregnancy rate in the drug treated group was reduced in a dose-dependent manner; apoptosis, Fas protein and mRNA levels in the endometrium of drug treated mice were correlatively decreased during the peri-implantation period. CONCLUSION: The decreased pregnant rate in mice by abnormal levels of nitric oxide may be brought about by inhibiting the normally occurrence of apoptosis in the receptive endometrium.