Exploring the impact of osteoprotegerin on osteoclast and precursor fusion: Mechanisms and modulation by ATP.

Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG's influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG's inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG's multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG's inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism.

Inefficient Sox9 upregulation and absence of Rspo1 repression lead to sex reversal in the B6.XYTIR mouse gonad†.

Sry on the Y-chromosome upregulates Sox9, which in turn upregulates a set of genes such as Fgf9 to initiate testicular differentiation in the XY gonad. In the absence of Sry expression, genes such as Rspo1, Foxl2, and Runx1 support ovarian differentiation in the XX gonad. These two pathways antagonize each other to ensure the development of only one gonadal sex in normal development. In the B6.YTIR mouse, carrying the YTIR-chromosome on the B6 genetic background, Sry is expressed in a comparable manner with that in the B6.XY mouse, yet, only ovaries or ovotestes develop. We asked how testicular and ovarian differentiation pathways interact to determine the gonadal sex in the B6.YTIR mouse. Our results showed that (1) transcript levels of Sox9 were much lower than in B6.XY gonads while those of Rspo1 and Runx1 were as high as B6.XX gonads at 11.5 and 12.5 days postcoitum. (2) FOXL2-positive cells appeared in mosaic with SOX9-positive cells at 12.5 days postcoitum. (3) SOX9-positive cells formed testis cords in the central area while those disappeared to leave only FOXL2-positive cells in the poles or the entire area at 13.5 days postcoitum. (4) No difference was found at transcript levels of all genes between the left and right gonads up to 12.5 days postcoitum, although ovotestes developed much more frequently on the left than the right at 13.5 days postcoitum. These results suggest that inefficient Sox9 upregulation and the absence of Rspo1 repression prevent testicular differentiation in the B6.YTIR gonad.

Cadmium Induces Kidney Iron Deficiency and Chronic Kidney Injury by Interfering with the Iron Metabolism in Rats.

Cadmium (Cd) is a common environmental pollutant and occupational toxicant that seriously affects various mammalian organs, especially the kidney. Iron ion is an essential trace element in the body, and the disorder of iron metabolism is involved in the development of multiple pathological processes. An iron overload can induce a new type of cell death, defined as ferroptosis. However, whether iron metabolism is abnormal in Cd-induced nephrotoxicity and the role of ferroptosis in Cd-induced nephrotoxicity need to be further elucidated. Sprague Dawley male rats were randomly assigned into three groups: a control group, a 50 mg/L CdCl2-treated group, and a 75 mg/L CdCl2-treated group by drinking water for 1 month and 6 months, respectively. The results showed that Cd could induce renal histopathological abnormalities and dysfunction, disrupt the mitochondria's ultrastructure, and increase the ROS and MDA content. Next, Cd exposure caused GSH/GPX4 axis blockade, increased FTH1 and COX2 expression, decreased ACSL4 expression, and significantly decreased the iron content in proximal tubular cells or kidney tissues. Further study showed that the expression of iron absorption-related genes SLC11A2, CUBN, LRP2, SLC39A14, and SLC39A8 decreased in proximal tubular cells or kidneys after Cd exposure, while TFRC and iron export-related gene SLC40A1 did not change significantly. Moreover, Cd exposure increased SLC11A2 gene expression and decreased SLC40A1 gene expression in the duodenum. Finally, NAC or Fer-1 partially alleviated Cd-induced proximal tubular cell damage, while DFO and Erastin further aggravated Cd-induced cell damage. In conclusion, our results indicated that Cd could cause iron deficiency and chronic kidney injury by interfering with the iron metabolism rather than typical ferroptosis. Our findings suggest that an abnormal iron metabolism may contribute to Cd-induced nephrotoxicity, providing a novel approach to preventing kidney disease in clinical practice.

Cadmium promotes nonalcoholic fatty liver disease by inhibiting intercellular mitochondrial transfer.

Mitochondrial transfer regulates intercellular communication, and mitochondria regulate cell metabolism and cell survival. However, the role and mechanism of mitochondrial transfer in Cd-induced nonalcoholic fatty liver disease (NAFLD) are unclear. The present study shows that mitochondria can be transferred between hepatocytes via microtubule-dependent tunneling nanotubes. After Cd treatment, mitochondria exhibit perinuclear aggregation in hepatocytes and blocked intercellular mitochondrial transfer. The different movement directions of mitochondria depend on their interaction with different motor proteins. The results show that Cd destroys the mitochondria-kinesin interaction, thus inhibiting mitochondrial transfer. Moreover, Cd increases the interaction of P62 with Dynactin1, promotes negative mitochondrial transport, and increases intracellular lipid accumulation. Mitochondria and hepatocyte co-culture significantly reduced Cd damage to hepatocytes and lipid accumulation. Thus, Cd blocks intercellular mitochondrial transfer by disrupting the microtubule system, inhibiting mitochondrial positive transport, and promoting their negative transport, thereby promoting the development of NAFLD.

Cadmium exposure exacerbates kidney damage by inhibiting autophagy in diabetic rats.

The incidence of diabetes mellitus (DM) is gradually increasing, making it a widespread global health concern. Cadmium (Cd) is a common toxic heavy metal in the environment, and cadmium exposure may be associated with diabetic nephropathy (DN). However, the mechanism of Cd-induced DN remains unclear. In this study, we aimed to determine the effect of cadmium on diabetic kidney injury and the underlying mechanism in diabetic rats and a renal tubular epithelial cell line (NRK-52E cells). Our results could provide novel insights on the nephrotoxic mechanism of cadmium. HE, PAS, and Masson staining were used to observe pathological renal injury. COL-I, COL-IV, CTSB, and CTSD protein levels were detected by immunohistochemistry and western blotting. Immunofluorescence was used to detect the fluorescence intensity of p62 and LC3 proteins in kidney tissue. TEM was used to observe the ultrastructure of mitochondria and number of autophagosomes. After cadmium exposure, DM rats showed a dramatic decrease in body weight compared to the unexposed DM group. Relative kidney weight showed a contrasting trend after cadmium exposure. Urinary microalbumin/creatinine significantly increased in normal and DM rats after cadmium exposure. However, the trend was clearer in the DM groups than in the control groups. Endogenous creatinine clearance exhibited a contrasting trend. After cadmium exposure in DM rats, MDA content significantly increased and GSH, CAT, SOD, and GSH-PX activation reduced compared to normal controls. Pathological damage was more pronounced, and the expression of autophagy related proteins and apoptosis and fibrosis proteins was significantly higher in vivo and vitro in the cadmium-exposed groups than in unexposed controls. Further, lysosomal protein levels were lower, and ROS content and autophagosome count significantly higher in the cadmium exposed groups compared to the unexposed controls. Therefore, Cadmium exposure aggravates diabetic kidney injury via autophagy inhibition.

Nuclear factor-kappaB mediates the survival of rat kidney cells after cadmium exposure via promoting autophagy and inhibiting apoptosis.

Cadmium (Cd) is a heavy metal pollutant in the environment, and the kidney is one of the target organs after Cd exposure. Previous studies have shown that apoptosis and autophagy disorders are the main mechanisms of Cd-induced nephrotoxicity in rats. As a transcription factor that balances cell survival and death, nuclear factor-kappaB (NF-κB) protein plays dual regulatory effects on apoptosis and autophagy in multiple renal diseases. However, the regulatory mechanisms of NF-κB in Cd-induced kidney injury remain unclear. Therefore, the normal rat kidney cell line (NRK-52E cells) was applied to investigate the above questions in this study. Here, we found that Cd promotes the nuclear translocation and activation of NF-κB in a concentration-dependent manner, and activated NF-κB mediates NRK-52E cells survival after Cd exposure. Next, our study elaborated the mechanisms of NF-κB in antagonizing Cd-induced renal cytotoxicity. Inhibition of NF-κB by inhibitor BAY 11-7082 (BAY) and NF-κB p65 siRNA (siNF-κB p65) exacerbate Cd-induced apoptosis and autophagy inhibition, and then aggravate Cd-induced NRK-52E cells injury. Activation of NF-κB by activator phorbol-12-myristate-13-acetate (PMA) alleviates Cd-induced apoptosis and autophagy inhibition, and then attenuates Cd-induced NRK-52E cells injury. In conclusion, Cd exposure promotes the activation of NF-κB, and activated NF-κB mediates the survival of NRK-52E cells after Cd exposure via promoting autophagy and inhibiting apoptosis.

Zearalenone damages the male reproductive system of rats by destroying testicular focal adhesion.

Zearalenone (ZEA), a common mycotoxin in animal feed, is harmful to public health and causes huge economic losses. The potential target proteins of ZEA and its derivatives were screened using the PharmMapper database and the related genes (proteins) of the testis were obtained from Genecards. We obtained 144 potential targets of ZEA and its derivatives related to the testis using Venn diagrams. The PPI analysis showed that ZEA had the most targets in testis, followed by ZAN, α-ZAL, ß-ZEL, α-ZEL, and ß-ZAL. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses evaluated the metabolic and cancer pathways. We further screened four hub genes: RAC3, CCND1, EP300, and CTNNB1. Eight key biological processes were obtained by GO analysis, and four important pathways were identified by KEGG analysis. Animal and cell experimental results confirmed that ZEA could inhibit the expression of four key KEGG pathway protein components and four hub proteins that interfere with cell adhesion by inhibiting the focal adhesion structure of the testis, Leydig cells, and Sertoli cells. Collectively, our findings reveal that the destruction of the focal adhesion structure in the testis is the mechanism through which ZEA damages the male reproductive system.

Polystyrene nanoplastics aggravate reproductive system damage in obese male mice by perturbation of the testis redox homeostasis.

The potential impact of the combination of a high-fat diet (HFD) and polystyrene nanoplastics (PS-NPs) on fertility cannot be ignored, especially when the fertility rate is declining. However, it has not attracted considerable attention. In this study, an obese mouse model was established using an HFD, and the reproductive function of male mice was evaluated after intragastric administration of 100 µL of a 10 mg/mL PS-NP suspension for 4 weeks. By determining the morphology and vitality of sperm and related indicators of testosterone production, it was found that PS-NPs aggravated the destruction of sperm mitochondrial structure, decrease sperm activity, and testosterone production in HFD-fed mice. To comprehensively analyze the injury mechanism, the integrity of the blood testicular barrier (BTB) and the function of Leydig and Sertoli cells were further analyzed. It was found that PS-NPs could destroy BTB, promote the degeneration of Leydig cells, reduce the number of Sertoli cells, and decrease lactate secretion in HFD-fed mice. PS-NPs further interfered with redox homeostasis in the testicular tissues of HFD-fed mice. This study found that PS-NPs could aggravate the damage to the reproductive system of obese male mice by further perturbing its redox homeostasis and revealed the potential health risk of PS-NPs exposure under an HFD.

Cadmium induces mitochondrial dysfunction via SIRT1 suppression-mediated oxidative stress in neuronal cells.

Cadmium is a widespread environmental contaminant and its neurotoxicity has raised serious concerns. Mitochondrial dysfunction is a key event in Cd-induced nervous system disease; however, the exact molecular mechanism involved has not been fully elucidated. Increasing evidences have shown that Sirtuin 1 (SIRT1) is the key target protein impaired in Cd-induced mitochondrial dysfunction. In this study, the role of SIRT1 in Cd-induced mitochondrial dysfunction and cell death and the underlying mechanisms were evaluated in vitro using PC12 cells and primary rat cerebral cortical neurons. The results showed that Cd exposure caused cell death by inhibiting SIRT1 expression, thus inducing oxidative stress and mitochondrial dysfunction in vitro. However, inhibition of oxidative stress by the antioxidant puerarin alleviated Cd-induced mitochondrial dysfunction. Furthermore, activation of SIRT1 using the agonist Srt1720 significantly abolished Cd-induced oxidative stress and mitochondrial dysfunction and ultimately alleviated Cd-induced neuronal cell death. Collectively, our data indicate that Cd induced mitochondrial dysfunction via SIRT1 suppression-mediated oxidative stress, leading to the death of PC12 cells and primary rat cerebral cortical neurons. These findings suggest a novel mechanism for Cd-induced neurotoxicity.

Polystyrene exacerbates cadmium-induced mitochondrial damage to lung by blocking autophagy in mice.

Cadmium (Cd) is an environmental heavy metal, and its accumulation is harmful to animal and human health. The cytotoxicity of Cd includes oxidative stress, apoptosis, and mitochondrial histopathological changes. Furthermore, polystyrene (PS) is a kind of microplastic piece derived from biotic and abiotic weathering courses, and has toxicity in various aspects. However, the potential mechanism of action of Cd co-treated with PS is still poorly unclear. The objective of this study was to investigate the effects of PS on Cd-induced histopathological injury of mitochondria in the lung of mice. In this study, the results have showed that Cd could induce the activity of oxidative enzymes of the lung cells in mice, increasing the content of partial microelement and the phosphorylation of inflammatory factor NF-κB p65. Cd further destroys the integrity of mitochondria by increasing the expression of apoptotic protein and blocking the autophagy. In addition, PS solely group aggravated the lung damage in mice, especially mitochondrial toxicity, and played a synergistic effect with Cd in lung injury. However, how PS can augment mitochondrial damage and synergism with Cd in lung of mice requiring further exploration. Therefore, PS was able to exacerbate Cd-induced mitochondrial damage to the lung in mice by blocking autophagy, and was associated with the apoptosis.

Cadmium accelerates autophagy of osteocytes by inhibiting the PI3K/AKT/mTOR signaling pathway.

Cadmium (Cd) can damage bone cells and cause osteoporosis. Osteocytes are the most numerous bone cells and also important target cells for Cd-induced osteotoxic damage. Autophagy plays important role in the progression of osteoporosis. However, osteocyte autophagy in Cd-induced bone injury is not well characterized. Thus, we established a Cd-induced bone injury model in BALB/c mice and a cellular damage model in MLO-Y4 cells. Aqueous Cd exposure for 16 months showed an increase in plasma alkaline phosphatase (ALP) activity and increase in urine calcium (Ca) and phosphorus (P) concentrations in vivo. Moreover, expression level of autophagy-related microtubule-associated protein 1A/1B-light chain 3 II (LC3II) and autophagy-related 5 (ATG5) proteins were induced, and the expression of sequestosome-1 (p62) was reduced, along with Cd-induced trabecular bone damage. In addition, Cd inhibited the phosphorylation of mammalian target of rapamycin (mTOR), protein kinase B (AKT), and phosphatidylinositol 3-kinase (PI3K). In vitro, 80 µM Cd concentrations exposure upregulated LC3II protein expression, and downregulated of p62 protein expression. Similarly, we found that treatment with 80 µM Cd resulted in a reduction in the phosphorylation levels of mTOR, AKT, and PI3K. Further experiments revealed that addition of rapamycin, an autophagy inducer, enhanced autophagy and alleviated the Cd-induced damage to MLO-Y4 cells. The findings of our study reveal for the first time that Cd causes damage to both bone and osteocytes, as well as induces autophagy in osteocytes and inhibits PI3K/AKT/mTOR signaling, which could be a protective mechanism against Cd-induced bone injury.

The Mechanism of Osteoprotegerin-Induced Osteoclast Pyroptosis In Vitro.

Osteoprotegerin (OPG) is a new member of the tumor necrosis factor (TNF) receptor superfamily, which can inhibit the differentiation and activity of osteoclasts by binding to nuclear factor kappa B receptor activator (RANK) competitively with nuclear factor kappa B receptor activator ligand (RANKL). The previous experiments found that OPG can induce apoptosis of mature osteoclasts in vitro, which can inhibit the activity of mature osteoclasts, thereby exerting its role in protecting bone tissue. In addition, pyroptosis is a new type of cell death that is different from apoptosis. It is unclear whether OPG can induce mature osteoclast pyroptosis and thereby play its role in protecting bone tissue. In this study, the results showed that compared with the control group, the survival rate of osteoclasts in the OPG group was significantly reduced, and the contents of IL-1ß, IL-18, and LDH in the supernatant both increased. Many osteoclast plasma membranes were observed to rupture in bright fields, and OPG induced loss of their morphology. Flow cytometry was used to analyze the pyroptosis rate; OPG significantly increased the osteoclast pyroptosis rate. To further reveal the mechanism of OPG-induced osteoclast pyroptosis, we examined the expression level of pyroptosis-related genes and proteins, and the results found that OPG increased the expression of NLRP3, ASC, caspase-1, and GSDMD-N compared with the control group. In summary, OPG can induce osteoclast pyroptosis, and its mechanism is related to the expression levels of ASC, NLRP3, caspase 1 and GSDMD, which were included in the classical pathway of pyroptosis.

Polystyrene Microplastics Induce Oxidative Stress in Mouse Hepatocytes in Relation to Their Size.

Microplastics have become a new type of environmental pollutant that can accumulate in various tissues and organs of the body and cause chronic damage. In this study, two different size polystyrene microplastics (PS-MPs, 5 µm and 0.5 µm) exposure models were established in mice to investigate the effects of PS-MPs with different particle sizes on oxidative stress in the liver. The results showed that PS-MPs exposure caused a decrease in body weight and liver-to-body weight. The hematoxylin and eosin staining and transmission electron microscopy results showed that exposure to PS-MPs led to the disorganized cellular structure of liver tissue, nuclear crinkling, and mitochondrial vacuolation. The extent of damage in the 5 µm PS-MP exposure group was more extensive when compared with the other group. The evaluation of oxidative-stress-related indicators showed that PS-MPs exposure exacerbated oxidative stress in hepatocytes, especially in the 5 µm PS-MPs group. The expression of oxidative-stress-related proteins sirtuin 3(SIRT3) and superoxide dismutase (SOD2) was significantly reduced, and the reduction was more pronounced in the 5 µm PS-MPs group. In conclusion, PS-MPs exposure led to oxidative stress in mouse hepatocytes and caused more severe damage in the 5 µm PS-MPs group when compared with the 0.5 µm PS-MPs group.

Puerarin Prevents Cadmium-Induced Neuronal Injury by Alleviating Autophagic Dysfunction in Rat Cerebral Cortical Neurons.

Autophagic dysfunction is one of the main mechanisms of cadmium (Cd)-induced neurotoxicity. Puerarin (Pue) is a natural antioxidant extracted from the medicinal and edible homologous plant Pueraria lobata. Studies have shown that Pue has neuroprotective effects in a variety of brain injuries, including Cd-induced neuronal injury. However, the role of Pue in the regulation of autophagy to alleviate Cd-induced injury in rat cerebral cortical neurons remains unclear. This study aimed to elucidate the protective mechanism of Pue in alleviating Cd-induced injury in rat cerebral cortical neurons by targeting autophagy. Our results showed that Pue alleviated Cd-induced injury in rat cerebral cortical neurons in vitro and in vivo. Pue activates autophagy and alleviates Cd-induced autophagic blockade in rat cerebral cortical neurons. Further studies have shown that Pue alleviates the Cd-induced inhibition of autophagosome-lysosome fusion, as well as the inhibition of lysosomal degradation. The specific mechanism is related to Pue alleviating the inhibition of Cd on the expression levels of the key proteins Rab7, VPS41, and SNAP29, which regulate autophagosome-lysosome fusion, as well as the lysosome-related proteins LAMP2, CTSB, and CTSD. In summary, these results indicate that Pue alleviates Cd-induced autophagic dysfunction in rat cerebral cortical neurons by alleviating autophagosome-lysosome fusion dysfunction and lysosomal degradation dysfunction, thereby alleviating Cd-induced neuronal injury.

Fusarium Mycotoxins Zearalenone and Deoxynivalenol Reduce Hepatocyte Innate Immune Response after the Listeria monocytogenes Infection by Inhibiting the TLR2/NFκB Signaling Pathway.

Zearalenone (ZEA) and deoxynivalenol (DON) are two common mycotoxins produced by the genus Fusarium and have potential immunotoxic effects that may lead to a weak immune response against bacterial infections. Listeria monocytogenes (L. monocytogenes), a food-borne pathogenic microorganism ubiquitous in the environment, actively multiplies in the liver, where hepatocytes are capable of resistance through mediated innate immune responses. At present, it is not clear if ZEA and DON affect hepatocyte immune responses to L. monocytogenes infection or the mechanisms involved. Therefore, in this study, in vivo and in vitro models were used to investigate the effects of ZEA and DON on the innate immune responses of hepatocytes and related molecules after L. monocytogenes infection. In vivo studies revealed that ZEA and DON inhibited the toll-like receptors 2 (TLR2)/nuclear factor kappa-B (NFκB) pathway in the liver tissue of L. monocytogenes-infected mice, downregulating the expression levels of Nitric oxide (NO), in the liver and repressing the immune response. In addition, ZEA and DON inhibited Lipoteichoic acid (LTA)-induced expression of TLR2 and myeloid differentiation factor 88 (MyD88) in Buffalo Rat Liver (BRL 3A) cells in vitro, downregulating the TLR2/NFκB signaling pathway and resulting in the decreased expression levels of NO, causing immunosuppressive effects. In summary, ZEA and DON can negatively regulate NO levels through TLR2/NFκB, inhibiting the innate immune responses of the liver, and aggravate L. monocytogenes infections in mouse livers.

Taurine Alleviates Cadmium-Induced Hepatotoxicity by Regulating Autophagy Flux.

Our previous studies have confirmed that cadmium (Cd) exposure causes hepatotoxicity; it also induces autophagy and blocks the autophagy flux. Therefore, we hypothesized that Cd hepatotoxicity could be alleviated through nutritional intervention. Taurine (Tau) has various biological functions such as acting as an antioxidant, acting as an anti-inflammatory, and stabilizing cell membranes. In order to explore the protective effect and internal mechanism of Tau on Cd-induced hepatotoxicity, normal rat liver cell line BRL3A cells were treated with Cd alone or in combination with Tau to detect cell injury and autophagy-related indexes in this study. We found that Tau can alleviate Cd-induced cell-proliferation decline and morphological changes in the cell. In addition, Tau activates autophagy and alleviates the blockage of Cd-induced autophagy flux. In this process, lysosome acidification and degradation were enhanced, and autophagosomes were further fused with lysosomes. Then, we found that Tau alleviated autophagic flux block by promoting the transfer of membrane fusion proteins STX17 and SNAP29 to autophagosomes and the translocation of VAMP8 to lysosomes, which in turn attenuated the hepatocyte injury induced by Cd exposure. This will further reveal the hepatotoxicity mechanism of Cd and provide the theoretical basis for the prevention and treatment of Cd poisoning.

Ca2+ transfer via the ER-mitochondria tethering complex in neuronal cells contribute to cadmium-induced autophagy.

Mitochondrial-associated endoplasmic reticulum (ER) membranes (MAMs) play a key role in several physiological functions, including calcium ion (Ca2+) transfer and autophagy; however, the molecular mechanism controlling this interaction in cadmium (Cd)-induced neurotoxicity is unknown. This study shows that Cd induces alterations in MAMs and mitochondrial Ca2+ levels in PC12 cells and primary neurons. Ablation or silencing of mitofusin 2 (Mfn2) in PC12 cells or primary neurons blocks the colocalization of ER and mitochondria while reducing the efficiency of mitochondrial Ca2+ uptake. Moreover, Mfn2 defects reduce interactions or colocalization between GRP75 and VDAC1. Interestingly, the enhancement of autophagic protein levels, colocalization of LC3 and Lamp2, and GFP-LC3 puncta induced by Cd decreased in Mfn2-/- or Grp75-/- PC12 cells and Mfn2- or Grp75-silenced primary neurons. Notably, the specific Ca2+ uniporter inhibitor RuR blocked both mitochondrial Ca2+ uptake and autophagy induced by Cd. Finally, this study proves that the mechanism by which IP3R-Grp75-VDAC1 tethers in MAMs is associated with the regulation of autophagy by Mfn2 and involves their role in mediating mitochondrial Ca2+ uptake from ER stores. These results give new evidence into the organelle metabolic process by demonstrating that Ca2+ transport between ER-mitochondria is important in autophagosome formation in Cd-induced neurodegeneration.

Beclin 1 positively regulates osteoprotegerin-induced inhibition of osteoclastogenesis by increasing autophagy in vitro.

Osteoclastogenesis is induced by receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), and can be suppressed by osteoprotegerin (OPG). Beclin1 has a dual role in osteoclastogenesis. However, the role of Beclin1-mediated autophagy during OPG-induced inhibition of osteoclastogenesis remains unclear. Here, we found that Beclin1 and matrix metalloproteinase 9 (MMP-9) expression were increased during osteoclastogenesis. OPG (20, 40, and 80 ng/mL) decreased Src and MMP-9 expression, but augmented Beclin1 expression and fluorescence intensity. Similarly, treatment with the autophagy activator rapamycin increased Beclin1 expression during OPG-induced inhibition of osteoclastogenesis. Further, Beclin1 knockdown restored osteoclast numbers by reducing autophagy during OPG-induced inhibition of osteoclastogenesis. These results indicate that Beclin1 has a positive role during OPG-induced inhibition of osteoclastogenesis by regulating autophagy, which might provide a potential basis for osteoclastogenesis.

Role of endoplasmic reticulum stress in cadmium-induced hepatocyte apoptosis and the protective effect of quercetin.

Cadmium (Cd) is one of the most toxic environmental pollutants. Quercetin (Que) is a kind of natural flavonoid with neuroprotective, antioxidant, and free-radical scavenging pharmacological activities. However, whether Que has the protective effect of on Cd-induced rat hepatocyte injury is unclear. This study aimed to determine the protective effect of Que on Cd-induced hepatotoxicity in vivo and in vitro. For in vivo, 36 4-week-old male SD rats were randomly divided into six groups and were treated with CdCl2 (2 mg/kg b.w.) and/or Que (50 or 100 mg/kg b.w.). Four weeks later, the rats were sacrificed and livers were collected. The levels of alanine aminotransferase, aspartate aminotransferase, glutathione, malondialdehyde, catalase, and superoxide dismutase were measured. Liver histopathological sections were made, and TUNEL method was performed to detect cell apoptosis. The mRNA and protein expression levels of endoplasmic reticulum stress (ERS) signaling pathway-related factors and apoptosis-related factors were detected. For in vitro, BRL-3A rat cells were treated with CdCl2 (12.5 µM) and/or Que (5 µM Que). The mRNA and protein expression levels of ERS signaling pathway-related factors and apoptosis-related factors were detected. Results showed that Cd led to liver injury, disorder of hepatocyte morphology and structure, decreased BRL-3A cells viabilities, increased oxidative damage. The mRNA and protein expression levels of ERS related factors GRP78, PERK, eIF2α, ATF4, CHOP, IRE1α, XBP1, and ATF6 increased. The mRNA and protein levels of apoptosis related factors Caspase12, Caspase3, and Bax increased, whereas Bcl2 decreased. It indicated that cadmium could activate PERK-eIF2α-ATF4-CHOP, IRE1α-XBP1, and ATF6-CHOP ERS-related signal pathways and lead to apoptosis. Moreover, Que can improve the vitality of hepatocytes, and effectively reduce hepatocytes damage, and reduce oxidative damage by Cd. As a result, the mRNA and protein expression levels of ERS related factors were reduced and hepatocyte apoptosis related factors decreased. Therefore, Que can be used as an effective component in daily diet to prevent Cd toxicity.

Puerarin alleviates cadmium-induced mitochondrial mass decrease by inhibiting PINK1-Parkin and Nix-mediated mitophagy in rat cortical neurons.

Cadmium (Cd) has well-known central nervous system toxicity, and mitochondria are direct targets of Cd-induced neuronal toxicity. However, how Cd induces mitochondrial mass decrease in terms of its neurotoxic effects remains unknown. Puerarin, an isoflavone extracted from kudzu root, can cross the blood-brain barrier and exert protective effects in nervous system disease. The purpose of the study was to determine the mechanism of Cd-induced mitochondrial mass decrease and the protective role of puerarin in rat cortical neurons. The results indicated that Cd induced mitochondrial mass decrease by activating mitophagy mediated by the PTEN-induced putative kinase protein 1 (PINK1)-E3 ubiquitin ligase (Parkin) and Nip3-like protein X (Nix) pathways in rat cortical neurons. Puerarin improved the Cd-induced decrease in mitochondrial membrane potential (MMP) in vitro, and blocked PINK1-Parkin and Nix-mediated mitophagy, inhibiting Cd-induced mitochondrial mass decrease in rat cortical neurons in vitro and in vivo. In summary, our data clearly indicated that puerarin protects rat cortical neurons against Cd-induced neurotoxicity by ameliorating mitochondrial damage, inhibiting mitophagy-mediated mitochondrial mass decrease. Puerarin appears to have great potential as a neuroprotective agent.