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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., (© 2023 Wiley Periodicals LLC.)

The imbalance between osteogenesis and osteoclastogenesis is a feature of bone metabolic disease. Cadmium (Cd) exposure causes human bone loss and osteoporosis (OP) through bioaccumulation of the food chain. However, the impact of Cd on bone tissues and the underlying molecular mechanisms are not well-characterized. In the current study, we found that the Cd concentration in bone tissues of OP patients was higher than normal subjects; meanwhile, the nuclear silent information regulator of transcription 1 (SIRT1) protein expression level was significantly decreased, which is a new star molecule to treat OP. It is further revealed that SIRT1 activation markedly reprograms bone metabolic and stress-response pathways that incline with osteoblast (OB) apoptosis. Suppressing reactive oxygen species (ROS) release with N -acetyl-l-cysteine (NAC) abolished Cd-induced reduction of SIRT1 protein, deacetylation of P53, OB apoptosis, and attenuated OP. Conversely, overexpression of SIRT1 suppressed Cd-induced ROS release. SIRT1 overexpression in vivo and in vitro dampened PGC-1α protein, acetylation of P53 at lysine 382, and caspase-dependent apoptosis. These results reveal that ROS/SIRT1 controls P53 acetylation and coordinates OB apoptosis involved in the onset of OP.

A pivotal clinical trial is often necessary to assess drug efficacy in the intended to use (IU) population. Ideally, patients should be enrolled based on a positive test result from a well-characterized companion diagnostic (CDx). However, the central challenge is that patients are instead recruited on the basis of a clinical trial assay (CTA) result. This challenge arises because, CTA is available at all local labs; the time delay to enable enrollment based on CDx could result in a significant proportion of patients being unable to participate, adversely affecting precision and/or bias. The difficulty is therefore that patients are recruited on the basis that their CTA result is positive (CTA+) but the goal is to assess the drug efficacy in patients positive by the companion diagnostic (CDx+). In this commentary, we will examine an apparent weakness of a variance formula that is proposed in the context of a sensitivity analysis. We will develop an alternative formula, and argue that this should be used instead.

Various factors cause animal bone malnutrition disease during intensive culture. Osteoclasts play an important role in regulating bone metabolism disease. Osteoprotegerin (OPG) modulates osteoclast function; however, the mechanism underlying this effect is unknown. Therefore, the present study aimed to explore whether OPG affects duck embryo osteoclast function via purinergic receptor P2X7. OPG significantly inhibited duck embryo osteoclast differentiation and bone resorption, and suppressed F-actin formation. In addition, OPG remarkably impaired duck embryo osteoclasts' adhesive structure. After OPG treatment, the expression of P2X7R significantly reduced, the ATP level and Ca 2+ -ATPase activity decreased rapidly, and concomitantly suppressed calcium and MAPK signaling. A438079 (a selective P2X7R inhibitor) significantly inhibited duck embryo osteoclast differentiation and bone resorption, and the phosphorylation of Ca 2+ regulated proteins (CAM, CAMKII, CAMKIV) and MAPKs (ERK, JNK, and P38) were markedly suppressed. Pretreatment of duck embryo osteoclasts with BzATP, a P2X7R agonist, activated Ca 2+ and MAPK signaling. BzATP alleviated OPG-induced duck embryo osteoclast differentiation and adhesive structure damage, and recovered the distribution of adhesion-related proteins in mature duck embryo osteoclasts. Thus, P2RX7-mediated Ca 2+ and MAPK signaling has a key function in OPG-induced duck embryo osteoclast differentiation and adhesive structure damage. P2X7R might be an ideal target to treat bone diseases through regulating bone cell activation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The present study was performed to investigate the effects of Cd exposure on lipid metabolism and mitochondrial dysfunction and to explore the role of mitophagy in Cd-induced dysregulation of lipid metabolism in chicken embryo liver tissues and hepatocytes. To this end, seven-day-old chicken embryos were exposed to different concentrations of Cd for 7 days, and primary chicken embryo hepatocytes were treated with Cd at four different concentrations for 6 h. Furthermore, the mitophagy inhibitor cyclosporine A (CsA) was used to investigate the role of mitophagy in Cd-induced disruption of lipid metabolism. Lipid accumulation, the expression levels of genes involved in lipid metabolism, mitochondrial dysfunction, and mitophagy were measured. The results demonstrated that Cd exposure increases hepatic triglyceride (TG) accumulation and the expression levels of lipogenic genes while decreasing those of lipolytic genes. Furthermore, Cd exposure was observed to alter mitochondrial morphology in terms of reduced size, excessive mitochondrial damage, and the formation of mitophagosomes. The co-localization of lysosome-associated membrane glycoprotein 2 and LC3 puncta was significantly increased in primary chicken embryo hepatocytes after Cd exposure. Moreover, Cd exposure increased LC3, PINK1, and Parkin protein expression levels. CsA effectively alleviated Cd-induced mitochondrial dysfunction, blocked mitochondrial membrane potential collapse, and suppressed PINK1/Parkin-mediated mitophagy. Furthermore, CsA treatment reversed the Cd-induced TG accumulation in liver tissues but further increased it in hepatocytes. Taken together, our findings demonstrate (for the first time) the importance of mitochondrial dysfunction and mitophagy via the PINK1/Parkin pathway in Cd-induced disruption of lipid metabolism., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cadmium (Cd) is a widespread environmental contaminant that causes severe bone metabolism disease, such as osteoporosis, osteoarthritis, and osteomalacia. The present review aimed to explore the molecular mechanisms of Cd-induced bone injury starting from bone cell function and teeth development. Cd inhibits the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts, and directly causes BMSC apoptosis. In the case of osteoporosis, Cd mainly affects the activation of osteoclasts and promotes bone resorption. Cd-induces osteoblast injury and oxidative stress, which causes DNA damage, mitochondrial dysfunction, and endoplasmic reticulum stress, resulting in apoptosis. In addition, the development of osteoarthritis (OA) might be related to Cd-induced chondrocyte damage. The high expression of metallothionein (MT) might reduce Cd toxicity toward osteocytes. The toxicity of Cd toward teeth mainly focuses on enamel development and dental caries. Understanding the effect of Cd on bone cell function and teeth development could contribute to revealing the mechanisms of Cd-induced bone damage. This review explores Cd-induced bone disease from cellular and molecular levels, and provides new directions for removing this heavy metal from the environment., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cadmium (Cd), an environmental pollutant, induces osteoporosis by directly destroying bone tissue, but its direct damaging effect on bone cells is not fully illustrated. Here, we treated mouse bone marrow stem cells (BMSC) and bone marrow macrophages (BMM) with Cd, and gave BALB/c mice Cd in water. Long-term Cd exposure significantly inhibited BMSC osteogenesis and osteoclast differentiation in vitro, and induced osteoporosis in vivo. Cd exposure also reduced P2X7 expression dramatically. However, P2X7 deletion significantly inhibited osteoblast and osteoclast differentiation; P2X7 overexpression obviously reduced the suppression effect of Cd on osteoblast and osteoclast differentiation. The suppression of P2X7-PI3K-AKT signaling aggravated the effect of Cd. In mice, short-term Cd exposure did not result in osteoporosis, but bone formation was inhibited, RANKL expression was increased, and osteoclasts were significantly increased in vivo. In vitro, short-term Cd exposure not only increased osteoclast numbers, but also promoted osteoclast adhesion function at late-stage osteoclast differentiation. Cd exposure also reduced P2X7 expression in vivo and in vitro. Our results demonstrate that short-term Cd exposure does not affect osteoblast and osteoclast apoptosis in vivo and in vitro, but long-term Cd exposure significantly increases bone tissue apoptosis. Overall, our results describe a novel mechanism for Cd-induced osteoporosis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cadmium is a common environmental pollutant that accumulates in the bone and kidneys and causes severe health and social problems. However, the effects of Cd on the occurrence of osteoporosis and its mechanism of action in this process are unclear. To test whether Cd-induced osteoporosis is mediated via P2X7/PI3K/AKT signaling, duck bone marrow mesenchymal stem cells (BMSCs) and bone marrow macrophage cells (BMMs) were treated with Cd for 5 days, and duck embryos were treated with Cd . Micro-CT analysis indicated that Cd-induced osteoporosis occurs in vivo, and histopathology and immunohistochemical analyses also revealed that Cd induced bone damage and the downregulation of osteogenic and bone resorption-related proteins. Cd exposure significantly inhibited the differentiation of BMSCs and BMMs into osteoblasts and osteoclasts in vitro, and promoted osteoblast and osteoclast apoptosis. Cd exposure significantly downregulated the P2X7/PI3K/AKT signaling pathway in vivo and in vitro, and inhibition of this signaling pathway significantly aggravated osteoblast and osteoclast differentiation. Cd exposure also upregulated the OPG/RANKL ratio in vivo and in vitro, further inhibiting osteoclast differentiation. These results demonstrate that Cd causes osteoporosis in duck by inhibiting P2X7/PI3K/AKT signaling and increasing the OPG/RANKL ratio. These results establish a previously unknown mechanism of Cd-induced osteoporosis., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cadmium (Cd), a heavy metal produced by various industries, contaminates the environment and seriously damages the skeletal system of humans and animals. Recent studies have reported that Cd can affect the viability of cells, including osteoblasts, both in vivo and in vitro. However, the mechanism of Cd-induced apoptosis remains unclear. In the present study, primary rat osteoblasts were used to investigate the Cd-induced apoptotic mechanism. We found that treatment with 2 and 5 μM Cd for 12 h decreased osteoblast viability and increased apoptosis. Furthermore, Cd increased the generation of reactive oxygen species (ROS), and, thus, DNA damage measured via p-H2AX. The level of the nuclear transcription factor p53 was significantly increased, which upregulated the expression of PUMA, Noxa, Bax, and mitochondrial cytochrome c, downregulated the expression of Bcl-2, and increased the level of cleaved caspase-3. However, pretreatment with the ROS scavenger N-acetyl-l-cysteine (NAC) or the p53 transcription specific inhibitor PFT-α suppressed Cd-induced apoptosis. Our results indicate that Cd can induce apoptosis in osteoblasts by increasing the generation of ROS and activating the mitochondrial p53 signaling pathway, and this mechanism requires the transcriptional activation of p53., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose : To investigate the protective effect of naringin (Nar) on H 2 O 2 -induced apoptosis of nucleus pulposus-derived mesenchymal stem cells (NPMSC) and the potential mechanism in this process. Methods : Rat NPMSC were cultured in MSC culture medium or culture medium with different concentrations of H 2 O 2 . Nar or the combination of Nar and LY294002 was added into the culture medium to investigate the effects of Nar. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. The apoptosis rate was determined using Annexin V/PI dual staining and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays. Additionally, the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. ATP level in NPMSC was analyzed via ATP detection kit. Mitochondrial ultrastructure change was observed through transmission electron microscope (TEM). Levels of apoptosis-associated molecules (cleaved caspase-3, Bax and Bcl-2) were evaluated via RT-PCR and western blot, respectively. Results : The cells isolated from NP met the criteria for MSC. H 2 O 2 significantly promoted NPMSC apoptosis in a dose and time-dependent manner. Nar showed no cytotoxicity effect on NPMSC up to a concentration of 100 μM for 24 h. Nar exhibited protective effects against H 2 O 2 -induced NPMSC apoptosis including apoptosis rate, expressions of proapoptosis and antiapoptosis related genes and protein. Nar could also alleviate H 2 O 2 -induced mitochondrial dysfunction of increased mitochondrial ROS production, reduced MMP, decreased intracellular ATP and mitochondrial ultrastructure change. However, these protected effects were inhibited after LY294002 treatment. Conclusions : Our results demonstrated that Nar efficiently attenuated H 2 O 2 -induced NPMSC apoptosis and mitochondrial dysfunction. The activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

Cadmium (Cd) is one of worldwide environmental pollutants that causes bone homeostasis, which depends on the resorption of bones by osteoclasts and formation of bones by the osteoblasts (OB). However, the Cd toxicity on OB and its mechanism are unclear. Autophagy is an evolutionarily conserved degradation process in which domestic intracellular components are selectively digested for the recycling of nutrients and energy. This process is indispensable for cell homeostasis maintenance and stress responses. Dysregulation at the level of autophagic activity consequently disturbs the balance between bone formation and bone resorption and mediates the onset and progression of multiple bone diseases, including osteoporosis. TAK1 has been recently emerged as an activator of AMPK and hence an autophagy inducer. AMPK is a key molecule that induces autophagy and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by mTORC1. In this study, we found that Cd treatment caused the formation of autophagosomes, LC3-II lipidation and p62 downregulation, and the increased autophagic flux, indicating that Cd treatment induced autophagy in OBs. Cd treatment induced TAK1 activation mediated AMPK phosphorylation, which promoted autophagy via phosphorylation of ULK1 at S317. Meanwhile, Cd treatment dramatically decreased mTORC1, S6K1, 4E-BP1, S6, ULK1 S555 and ULK1 S757 phosphorylation, suggesting that mTORC1 activity was inhibited and inactive mTORC1 prevents ULK1 activation by phosphorylating ULK1 at SerS555 and Ser757. Our data strongly suggest that TAK1 mediates AMPK activation, which activates ULK1 by phosphorylating ULK1 S317 and suppressing mTORC1-mediated ULK1 S555 and ULK1 S757 phosphorylation. Our study has revealed a signaling mechanism for TAK1 in Cd-induced autophagy in OBs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: When conservative treatments do not work, TKA may be the best option for patients with knee osteoarthritis, although a relatively large proportion of individuals do not have clinically important improvement after TKA. Evidence also suggests that women are less likely to benefit from TKA than men, but the reasons are unclear. Widespread pain disproportionately affects women and has been associated with worse outcomes after joint arthroplasty, yet it is unknown if the effect of widespread pain on TKA outcomes differs by patient gender., Questions/purposes: (1) Does the association between widespread pain and no clinically important improvement in osteoarthritis-related pain and disability 2 years after TKA differ between men and women? (2) Does the use of pain medications 2 years after TKA differ between those with widespread pain and those without widespread pain before surgery?, Methods: Osteoarthritis Initiative (https://nda.nih.gov/oai/) study participants were followed annually from March 2005 until October 2015. Participants who underwent TKA up to the 7-year follow-up visit with pain/disability assessment at the protocol-planned visit before TKA and at the second planned annual visit after surgery were included in the analysis. Among 4796 study participants, 391 had a confirmed TKA, including 315 with pain/disability assessment at the protocol-planned visit before TKA. Overall, 95% of participants (298) had the required follow-up assessment; 5% (17) did not have follow-up data. Widespread pain was defined based on the modified American College of Rheumatology criteria. Symptoms were assessed using the WOMAC pain (range 0 to 20; higher score, more pain) and disability (range 0 to 68; higher score, more disability) scores, and the Knee Injury and Osteoarthritis Outcome Score for pain (range 0 to 100; higher score, less pain). Improvements in pain and disability were classified based on improvement from established clinically important differences (decrease in WOMAC pain ≥ 1.5; decrease in WOMAC disability ≥ 6.0; increase in Knee Injury and Osteoarthritis Outcome Score for pain ≥ 9). At baseline, more women presented with widespread pain than men (45% [84 of 184] versus 32% [36 of 114]). Probability and the relative risk (RR) of no clinically important improvement were estimated using a logistic regression analysis in which participants with widespread pain and those without were compared. The analyses were done for men and women separately, then adjusted for depression and baseline outcome scores., Results: Among women, preoperative widespread pain was associated with an increased risk of no clinically important improvement 2 years after TKA, based on WOMAC pain scores (13.5% versus 4.6%; RR 2.93 [95% CI 1.18 to 7.30]; p = 0.02) and the Knee Injury and Osteoarthritis Outcome Score for pain (16.5% versus 4.9%; RR 3.39 [95% CI 1.34 to 8.59]; p = 0.02). Given the lower and upper limits of the confidence intervals, our data are compatible with a broad range of disparate associations between widespread pain and lack of clinically important improvement in WOMAC pain scores (RR 0.77 [95% CI 0.22 to 2.70]; p = 0.68) and the Knee Injury and Osteoarthritis Outcome Score for pain (RR 1.37 [95% CI 0.47 to 4.00]; p = 0.57) among men, as well as clinically important improvement in WOMAC disability scores among men (RR 0.72 [95% CI 0.20 to 2.55]; p = 0.61) and women (RR 1.98 [95% CI 0.92 to 4.26]; p = 0.08). Participants presenting with widespread pain before TKA were more likely than those without widespread pain to use medication for symptoms of knee osteoarthritis most days for at least 1 month 2 years after TKA (51% [61 of 120] versus 32% [57 of 178]; mean difference, 18.8 [95% CI 7.3 to 30.1]; p < 0.01)., Conclusions: Widespread pain before TKA was associated with an increased risk of no clinically important improvement in knee pain 2 years postoperatively among women. Because of the small number of men with widespread pain in the sample, the results for men were inconclusive. In clinical practice, screening TKA candidates for widespread pain may be useful, and expectations of surgical outcomes may need to be tempered if patients have a concurrent diagnosis of widespread pain. Future studies should include more men with widespread pain and investigate if treatment of widespread pain before or concurrent with TKA surgery may improve surgical outcomes., Level of Evidence: Level III, therapeutic study.

Human SCGB1A1 protein has been shown to be significantly reduced in BAL, sputum, and serum from humans with asthma as compared with healthy individuals. However, the mechanism of this reduction and its functional impact have not been entirely elucidated. By mining online datasets, we found that the mRNA of SCGB1A1 was significantly repressed in brushed human airway epithelial cells from individuals with asthma, and this repression appeared to be associated with reduced expression of FOXA2. Consistently, both Scgb1A1 and FoxA2 were downregulated in an ovalbumin-induced mouse model of asthma. Furthermore, compared with wild-type mice, Scgb1a1 knockout mice had increased airway hyperreactivity and inflammation when they were exposed to ovalbumin, confirming the antiinflammatory role of Scgb1a1 in protection against asthma phenotypes. To search for potential asthma-related stimuli of SCGB1A1 repression, we tested T-helper cell type 2 cytokines. Both IL-4 and IL-13 repressed epithelial expression of SCGB1A1 and FOXA2. Importantly, infection of epithelial cells with human rhinovirus similarly reduced expression of these two genes, which suggests that FOXA2 may be the common regulator of SCGB1A1. To establish the causal role of reduced FOXA2 in SCGB1A1 repression, we demonstrated that FOXA2 was required for SCGB1A1 expression at baseline. FOXA2 overexpression was sufficient to drive promoter activity and expression of SCGB1A1 and was also able to restore the repressed SCGB1A1 expression in IL-13-treated or rhinovirus-infected cells. Taken together, these findings suggest that low levels of epithelial SCGB1A1 in asthma are caused by reduced FOXA2 expression.

Ca 2+ is an important ion in various intracellular metabolic pathways. Endoplasmic reticulum (ER) is a major intracellular calcium store and ER calcium homeostasis plays a key part in the regulation of apoptosis. We have previously shown that Cadmium (Cd) induces apoptosis in osteoblasts (OBs), accompany by increased cytoplasmic calcium. As the role of calcium in OBs apoptosis induced by Cd has not been clarified we investigated the effects of Cd exposure in rat OBs on intracellular Ca 2+ , CaMKII phosphorylation, and the pathways implicated in inducing apoptosis. The results showed that cadmium(Cd) induced elevation of intracellular Ca 2+ ([Ca 2+ ] i ) in OBs by the release of Ca 2+ from ER and the inflow of Ca 2+ from the extracellular matrix. Cd induced [Ca 2+ ] i elevation and phosphorylation of CaMKII which might be involved in activation of MAPKs and participated in Cd-induced mitochondrial apoptosis through the alteration of the ratio of Bax/Bcl-2 expression. Meanwhile, CaMKII phosphorylation activated unfolded protein response (UPR) during cadmium treatment and could enable the ER apoptosis pathway through the activation of caspase-12. These results indicated that CaMKII plays an important role in Cd induced ER apoptosis and MAPK activation. Our data provide new insights into the mechanisms underlying apoptosis in OBs following Cd exposure. This provides a theoretical basis for future investigations into the clinical therapeutic application of CaMKⅡ inhibitors in osteoporosis induced by Cd exposure., (Copyright © 2018 Elsevier B.V. All rights reserved.)