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Manganese (Mn) is an essential microelement for broiler breeding and its deficiency causes tibial dyschondroplasia (TD). Tibial growth plate (TGP) development and metaphyseal vascularization are crucial for tibia growth in fast-growing broiler chickens, but their roles in Mn deficiency-induced TD in chicks remain unclear. This study was designed to clarify this issue. A total of 36 one-day-old broilers were divided into the control group and Mn-deficiency (Mn-D) group, which were fed with a standard diet (60 mg Mn/kg) and Mn deficiency diet (22 mg Mn/kg) for 42 days, respectively. TGP and proximal tibial metaphysis were collected to perform the related assays. This study found that Mn deficiency decreased the tibia length and TGP thickness in the TD model. Also, Mn deficiency increased the irregular and white tibial dyschondroplasia lesions (TDL) region under the TGP, and reduced the expression levels of vascular endothelial growth factor (VEGF) and macrophage migration inhibitory factor (MIF). Combined with histological assessment, it was suggested that Manganese deficiency inhibited angiogenesis in the proximal tibial metaphysis. Meanwhile, Mn deficiency enhanced the expression levels of hypoxia-inducible factor-1 α (HIF-1α), autophagy-related protein 5 (ATG5), and microtubule-associated protein 1 light chain 3 β (LC3-II) in TGP, but decreased the expression level of SQSTM1 (P62), which suggested that autophagy was activated during this process. Collectively, these data indicate that HIF-1α up-regulation and concurrent autophagy activation exert a protective effect against Mn deficiency-induced angiogenesis inhibition, which may provide useful guidance to prevent TD in broilers.

Manganese (Mn) is an essential trace element for broiler chickens; its deficiency causes tibial dyschondroplasia (TD) characterized by lameness and growth retardation. Inorganic and organic manganese sources are used in global poultry production, but there is a lack of systematic investigations to compare the bioavailability among them. In this study, 120 1-day-old Arbor Acres (AA) broilers were randomly divided into four groups (n = 30), i.e., control group (Mn sulfate, 60 mg/kg), Mn-D group (Mn deficiency, 22 mg/kg), Mn-Gly group (Mn glycinate, 60 mg/kg), and Mn-Pro group (Mn proteinate, 60 mg/kg). During the 42-day experiment, growth performance, tibial bone parameters, pathological index changes, serum biochemical changes, and oxidative stress indicators were evaluated. These results not only suggested that Mn plays a crucial role in the normal development of tibia and the maintenance of redox homeostasis in broilers, but also proved that organic Mn supplementation, especially Mn proteinate, improved the tibia development and the absorption efficiency, as well as overall oxidative stress status of broilers, suggesting that it had greater bioavailability than inorganic Mn. Thus, application of organic Mn source may be an effective way to reduce economic losses and resolve animal welfare concerns due to TD in commercial poultry farming.

Manganese (Mn) is an essential trace element for bone growth, and its deficiency has been shown to increase the incidence of leg abnormalities in fast-growing broilers, such as tibial dyschondroplasia (TD). Proliferation and differentiation of growth plate chondrocyte are critical for tibia development, but their roles in Mn deficiency-induced TD remains to be elucidated. Thirty 1-day-old Arbor Acres chicks were randomly divided into two groups and fed with control diet (60 mg Mn/kg diet) and Mn-deficiency diet (22 mg Mn/kg diet) for 42 days, respectively. Mn deficiency-induced TD model was successfully established and samples from proximal tibia metaphysis and growth plate were collected for assays. Pathological observation showed that Mn deficiency induced morphological abnormality and irregular arrangement of chondrocytes in proliferative and hypertrophic zone of tibial growth plate. Also, Mn deficiency decreased mRNA and protein expression levels of type II collagen and type X collagen in tibial growth plate, indicating the impairment of proliferating and hypertrophic chondrocytes. Moreover, down-regulated gene expression levels of Sox9, Tgf-β, Ihh, Runx2, Mef2c and Bmp-2 were shown in tibial growth plate of Mn-deficiency group, demonstrating that Mn deficiency inhibited the transcription levels of key regulators to disrupt chondrocyte proliferation and differentiation. Collectively, these findings confirmed that Mn deficiency affected the proliferation and differentiation of chondrocytes in tibial growth plate via inhibiting related regulatory factors, leading to TD in broilers.

Manganese (Mn) is a crucial trace element for poultry nutrition, and its deficiency compromises tibial cartilage development, leading to perosis and a higher incidence of slipped tendon. Tibial dyschondroplasia (TD) is a metabolic cartilage disease characterized by disruption of endochondral bone formation, which is closely related to extracellular matrix (ECM) degradation, in which Mn deficiency plays an important role. Previous studies have confirmed the role of matrix metalloproteinases (MMPs) in the pathogenesis of TD, but whether dysregulated ECM degradation and MMP expression profiles in growth plate are involved in Mn deficiency-induced avian TD has not been fully elucidated yet. Thus, this study was conducted to clarify these issues. Firstly, we successfully established TD model induced by Mn deficiency in broiler chicks. Mn deficiency decreased the number of chondrocytes, contents of proteoglycan, and type II collagen in tibial growth plate, demonstrating the tibial growth plate damage with enhanced ECM degradation. Also, Mn deficiency inhibited the Nrf2 signaling pathway and enhanced the protein levels of NLRP3, active caspase-1, and active IL-1β in tibial growth plate, indicating the oxidative stress and inflammatory response in Mn deficiency-induced TD. Additionally, upregulated expression levels of MMPs (MMP1, 9, and 13) were observed in tibial growth plate of Mn deficiency group. In summary, these findings suggest that Mn deficiency-enhanced ECM degradation is involved in avian TD, which may be correlated with oxidative stress, inflammatory response, and upregulation of MMPs.

Glyphosate (Gly) is a globally spread herbicide that can cause toxic injuries to hepatocytes. Dietary trehalose (Tre) exerts cytoprotective effect in numerous liver diseases through anti-oxidant and anti-inflammatory properties. However, it is yet to be investigated whether Tre affords protection against Gly-induced hepatotoxicity. To evaluate the negative effect of Gly in liver and assess the possible protective role of Tre, sixty Hy-line Brown roosters were allocated into three groups: the first group presented the control with a normal diet, the second group fed normal feed containing 200mg/kg Gly, and the third group fed normal feed containing 200 mg/kg Gly and 5 g/kg Tre. Plasma and liver tissues were collected and analyzed after 120 days. Firstly, Gly-elevated serum levels of hepatic injury markers and liver histopathological damages were evidently alleviated by Tre administration. Also, Tre normalized Gly-altered serum and hepatic lipid profiles and Oil Red O-stained lipid levels, suggesting the improvement of hepatic steatosis. The severely accumulated malondialdehyde levels and impaired antioxidant status in Gly-exposed roosters were markedly improved by administration with Tre. Simultaneously, Gly-inhibited nuclear factor erythroid 2-related factor 2 (Nrf2) level and consequent reduced levels of Nrf2-downstream targets in liver were markedly normalized by Tre treatment. Additionally, Tre treatment evidently mitigated Gly-induced inflammasome response via inhibiting NLRP3 inflammasome activation. Overall, these observations provide novel insights that the protective action of Tre against Gly-induced hepatic steatosis is attributed to activation of Nrf2 pathway and inhibition of NLRP3 inflammasome activation.

Mercuric chloride (HgCl2) is a widely distributed environmental pollutant with multiorgan toxicity including immune organs such as spleen. Selenium (Se) is an essential trace element in animal nutrition and exerts biological activity to antagonize organ toxicity caused by heavy metals. The objective of this study was to explore the underlying mechanism of the protective effects of Se against spleen damage caused by HgCl2 in chicken. Ninety male Hyline brown chicken were randomly divided into 3 groups namely Cont, HgCl2, and HgCl2+Se group. Chicken were provided with the standard diet and nontreated water, standard diet and HgCl2-treated water (250 ppm), and sodium selenite-treated diet (10 ppm) plus HgCl2-treated water (250 ppm), respectively. After being fed for 7 wk, the spleen tissues were collected, and spleen index, the microstructure of the spleen, and the indicators of oxidative stress, inflammation, apoptosis as well as heat shock proteins (HSP) were detected. First, the results of spleen index and pathological examination confirmed that Se exerted an antagonistic effect on the spleen injury induced by HgCl2. Second, Se ameliorated HgCl2-induced oxidative stress by decreasing the level of malondialdehyde and increasing the levels of glutathione, glutathione peroxidase, and total antioxidant capacity. Third, Se attenuated HgCl2-induced inflammation by decreasing the protein expression of nuclear factor kappa-B, inducible nitric oxide synthase, and cyclooxygenase-2, and the gene expression of interleukin (IL)-1β, IL-6, IL-8, IL-12β, IL-18 as well as tumor necrosis factor-α. Fourth, Se inhibited HgCl2-induced apoptosis by downregulating the protein expression of BCL2 antagonist/killer 1 and upregulating the protein expression of B-cell lymphoma-2. Finally, Se reversed HgCl2-triggered activation of HSP 60, 70, and 90. In conclusion, Se antagonized HgCl2-induced spleen damage in chicken, partially through the regulation of oxidative stress, inflammatory, and apoptotic signaling.

Objectives of this study were to evaluate the alleviating effects of a commercial beta-1,3-glucan product (Aleta, containing 50% beta-1,3-glucan, Kemin Industries) on metabolic stress in transition Holstein cows as reflected by circulating metabolites and enzymes. Fifty-four multiparous Holstein cows were randomly allocated to three groups with 18 cows each. Cows in each group received a commercial basal diet or the basal diet supplemented with Aleta calculated to supply 5 or 10 g of Aleta per cow per day. Blood samples were collected at day −21, 1, and 21 relative to calving for determination of serum triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) , high density lipoprotein cholesterol (HDL C) , very low density lipoprotein (VLDL), glucose, insulin, β-hydroxybutyric acid (BHBA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamyl transpeptidase (GGT), and non-esterified fatty acid (NEFA). Supplementation with Aleta markedly elevated serum concentrations of TG, TC, HDL C, LDL-C and VLDL, implying its positive effect on lipid metabolism in transition dairy cows. Aleta treatment significantly decreased the serum concentrations of NEFA and BHBA, but markedly elevated the serum concentrations of glucose and insulin. Also, Aleta treatment significantly elevated the dry matter intake and milk production in postpartum cows, indicating the alleviating effect of Aleta on negative energy balance in transition cows. Moreover, Aleta treatment significantly reduced the serum activities of AST, ALT and GGT, indicating its hepatoprotective effect on transition cows. These results suggest that Aleta supplementation may help to improve fat metabolism disorder initiated by negative energy balance in transition dairy cows.

Mercuric chloride (HgCl

INTRODUCTION: Lead (Pb) is an environmental toxicant that poses severe health risks to humans and animals, especially renal disorders. Pb-induced nephrotoxicity has been attributed to oxidative stress, in which apoptosis and autophagy are core events. OBJECTIVES: Nuclear factor erythroid 2-related factor 2 (Nrf2) acts as a major contributor to counteract oxidative damage, while hyperactivation or depletion of Nrf2 pathway can cause the redox imbalance to induce tissue injury. This study was performed to clarify the function and mechanism of Nrf2 in Pb-triggered kidney injury. METHODS AND RESULTS: First, data showed that Pb exposure activates Nrf2 pathway in primary rat proximal tubular cells. Next, Pb-induced Nrf2 activation was effectively regulated by pharmacological modulation or siRNA-mediated knockdown in vitro and in vivo assays. Notably, Pb-triggered cytotoxicity, renal injury and concomitant apoptosis were improved by Nrf2 downregulation, confirming that Pb-induced persistent Nrf2 activation contributes to nephrotoxicity. Additionally, Pb-triggered autophagy blockage was relieved by Nrf2 downregulation. Mechanistically, we found that Pb-induced persistent Nrf2 activation is attributed to reduced Nrf2 ubiquitination and nuclear-cytoplasmic loss of Keap1 in a p62-dependent manner. CONCLUSIONS: In conclusion, these findings highlight the dark side of persistent Nrf2 activation and potential crosstalk among Pb-induced persistent Nrf2 activation, apoptosis and autophagy blockage in Pb-triggered nephrotoxicity.

Mercury (Hg) is a persistent environmental and industrial pollutant that accumulated in the body and induces oxidative stress and inflammation damage. Selenium (Se) has been reported to antagonize immune organs damage caused by heavy metals. Here, we aimed to investigate the prevent effect of Se on mercuric chloride (HgCl

Cadmium (Cd), as a kind of ubiquitous and highly toxic heavy metal pollutants, has been known to result in immunotoxicity in animals. As a multifunctional bioactivity disaccharide, trehalose (Tre) is characterized by antioxidative, antiapoptotic, and accelerating autophagy. In this study, Sprague-Dawley (SD) rats were fed with cadmium chloride (CdCl2) and/or Tre to explore the molecular mechanisms of Tre-protected against spleen injury caused by Cd exposure. Firstly, the results showed that Tre partially recovered splenic pathological changes induced by Cd exposure. Secondly, Tre dramatically declined the level of methane dicarboxylic aldehyde (MDA) and elevated the level of total antioxidant capacity (T-AOC) to weaken oxidative stress caused by Cd exposure in spleen tissue. Moreover, the results showed that Tre significantly suppressed Cd-induced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and up-regulated the protein expression of nuclear Nrf2. Thirdly, Tre remarkably reduced the protein expression of sequestosome 1 (p62/SQSTM1) and microtubule-associated protein light chain 3II (LC-3II) to restore autophagy inhibition induced by Cd exposure. Finally, the results of TUNEL and the expression of apoptosis marker proteins showed that Tre significantly inhibited Cd-induced apoptosis in spleen tissue to exert its protective effects. In summary, the results indicated that Tre modulated Nrf2 signaling pathway, which interacted with apoptosis and autophagy to against Cd-induced spleen injury, providing potential therapeutic strategies for the prevention and treatment of Cd-related immune system diseases.

Glyphosate (GLY), one of the most extensively used herbicides in the world, has been shown to inhibit testosterone synthesis in male animals. Mitochondria are crucial organelles for testosterone synthesis and its dysfunction has been demonstrated to induce the inhibition of testosterone biosynthesis. However, whether low-dose GLY exposure targets mitochondria to inhibit testosterone synthesis and its underlying mechanism remains unclear. Here, an in vitro model of 10 μM GLY-exposed mouse Leydig (TM3) cells was established to elucidate this issue. Data firstly showed that mitochondrial malfunction, mainly manifested by ultrastructure damage, disturbance of mitochondrial dynamics and mitochondrial reactive oxygen species (mtROS) overproduction, was responsible for GLY-decreased protein levels of steroidogenic enzymes, which leads to the inhibition of testosterone synthesis. Enhancement of autophagic flux and activation of mitophagy were shown in GLY-treated TM3 cells, and further studies have revealed that GLY-activated mitophagy is parkin-dependent. Notably, GLY-inhibited testosterone production was significantly improved by parkin knockdown. Finally, data showed that treatment with mitochondria-targeted antioxidant Mito-TEMPO (M-T) markedly reversed GLY-induced mitochondrial network fragmentation, activation of parkin-dependent mitophagy and consultant testosterone reduction. Overall, these findings demonstrate that GLY induces mtROS overproduction to activate parkin-dependent mitophagy, which contributes to the inhibition of testosterone synthesis. This study provides a potential mechanistic explanation for how GLY inhibits testosterone synthesis in mouse Leydig cells.

Mercuric chloride (HgCl

Cadmium (Cd) is a known nephrotoxic heavy metal and proximal tubules are the major target of Cd-induced acute kidney injury (AKI). We previously demonstrated that lysosomal dysfunction and dysregulated autophagy contribute to Cd-induced AKI. Recent studies have revealed that bromodomain-containing protein 4 (BRD4) is a transcriptional repressor of autophagy and lysosomal function. Hence, in vivo and in vitro studies were performed to clarify the role of BRD4 in Cd-induced AKI. Firstly, Cd has no effect on BRD4 expression levels, but increases H4K16 acetylation. Resultantly, Cd promotes the recruitment of BRD4 to lysosomal gene promoter regions to make it as a transcriptional regulator. Pharmacological and genetic inhibition of BRD4 alleviates Cd-inhibited lysosomal gene transcript levels and lysosomal function, leading to the alleviation of Cd-induced autophagy inhibition. Moreover, inhibition of BRD4 relieves Cd-induced oxidative stress and concurrent cytotoxicity, which is counteracted by the inhibition of autophagy via Atg5 knockdown, indicating that alleviation of oxidative stress by BRD4 inhibition is ascribed to its restoration of autophagic flux. Collectively, these results demonstrate that BRD4 acts as a transcriptional repressor to mediate lysosomal dysfunction, autophagy blockade and oxidative stress during Cd exposure, which may be a potential therapeutic target for Cd-induced AKI.

Autophagy dysregulation plays a pivotal role in cadmium (Cd)-induced nephrotoxicity. Quercetin (Qu), a flavonoid antioxidant with autophagy-enhancing effect, has protective effect on Cd-induced toxicity, but whether it can prevent Cd-induced nephrotoxicity via restoration of autophagy remains unknown. Here, primary rat proximal tubular (rPT) cells were exposed to Cd and/or Qu in vitro to clarify this issue. Data first showed that Cd-impaired autophagic flux was markedly alleviated by Qu, including decreased levels of autophagy marker proteins and recovery of autophagosome-lysosome fusion targeted for lysosomes. Meanwhile, Cd-induced lysosomal alkalization due to v-ATPases inhibition was prominently recovered by Qu. Accordingly, Qu enhanced Cd-diminished lysosomal degradation capacity and lysosome-related gene transcription levels. Notably, Qu improved Cd-inhibited TFEB nuclear translocation and its gene transcription level. Furthermore, data showed that the restoration of Cd-impaired autophagy-lysosome pathway and resultant alleviation of cytotoxicity by Qu are TFEB-dependent using TFEB gene silencing and overexpression technologies. In summary, these data provide novel evidences that the protective action of Qu against Cd-induced autophagy inhibition is attributed to its restoration of lysosomal dysfunction, which is dependent on TFEB.

Cadmium (Cd) poisoning is characterized by multiple organ dysfunction in organisms, and the kidney is the main target organ of Cd toxicity. Trehalose (Tr), a multifunctional bioactive disaccharide, possesses potential kidney protective properties. Nevertheless, the specific biological function of Tr in antagonizing kidney injury induced by Cd remains to be elucidated. Herein, an in vivo model of Tr antagonizing Cd nephrotoxicity was established and the indictors related to kidney function, oxidative stress, and apoptosis were detected to investigate the molecular mechanism underlying the Tr-protection against Cd-induced kidney injury of rats. Firstly, Tr significantly declined the levels of blood urea nitrogen (BUN) and serum creatinine, and partially restored renal pathological changes caused by Cd. Secondly, Cd exposure significantly increased the malondialdehyde (MDA) content, and decreased the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and glutathione (GSH) in serum. However, Tr significantly ameliorated these abnormal alterations. Moreover, Tr regulated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway to suppress the Cd-induced nuclear translocation of Nrf2 and the up-regulation of heme oxygenase-1 (HO-1) and NAD (P) H quinone reductase-1 (NQO1). Meanwhile, Tr significantly reversed the increased Sequestosome-1(SQSTM1/p62) and decreased Kelch-like ECH associated protein-1 (Keap1) protein levels induced by Cd. Thirdly, further mechanistic exploration suggested that Tr inhibited the mitochondrial apoptotic signaling pathway induced by Cd. Collectively, the results indicated that Tr exerts antioxidant and anti-apoptosis functions involving the Nrf2 and mitochondrial apoptotic signaling pathways to protect against Cd-induced kidney injury in rats.

Dairy cows undergo dramatic physiological changes during the transition from late pregnancy to early lactation, which make them vulnerable to metabolic stress and immune dysfunction. The objective of this study was to evaluate the effects of a commercial beta-1,3-glucan product (Aleta™, containing 50% beta-1,3-glucan) on productivity, immunity and antioxidative status in transition cows. Fifty-four multiparous Holstein cows received a control diet or a diet supplemented with 5 or 10 g of beta-1,3-glucan per cow per day from 21 days before expected calving to 21 days after parturition. Blood samples were collected at day −21, 1, and 21 relative to calving. Colostrum and milk were collected at day 1 and 21 after calving, respectively. Data showed that supplementation with beta-1,3-glucan had no effect on milk composition, but increased milk production. Beta-1,3-glucan treatment also improved the milk quality, as shown by reduced milk somatic cell count and increased immunoglobulin levels in colostrum. Notably, beta-1,3-glucan markedly reduced serum levels of pro-inflammatory cytokines and C-reactive protein, while elevated serum immunoglobulin levels, indicating its immunity enhancement in transition cows. Moreover, beta-1,3-glucan addition reduced the serum malondialdehyde level and enhanced the activities of serum superoxide dismutase and catalase, which enhanced the antioxidative capacity in transition cows. In summary, supplementation with beta-1,3-glucan improves productivity, immunity and antioxidative status in transition dairy cows.

Cadmium (Cd) is a persistent environmental contaminant and induces neurotoxicity in animals. Trehalose (Tre) exhibits powerful neuroprotective effects in certain brain injury models. Herein, we revealed the specific molecular mechanism underlying the protective effects of Tre against Cd-induced brain damage in rats. Firstly, the results showed that Tre significantly ameliorated brain pathological injury induced by Cd. Secondly, Cd-induced down-regulation of total anti-oxidation capacity (T-AOC) and up-regulation of methane dicarboxylic aldehyde (MDA) in brain tissues were significantly reversed by Tre treatment. Importantly, the augmentation of nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) caused by Cd was significantly inhibited by Tre treatment. Thirdly, the levels of autophagy marker proteins were measured and the results showed that Tre significantly reversed the up-regulation of light chain 3II (LC-3II) and sequestosome 1 (SQSTM-1/p62) caused by Cd exposure. Finally, the apoptosis rate and the levels of apoptosis marker proteins including B cell leukemia/lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) were also measured and the results showed that Cd-induced apoptosis was markedly inhibited by Tre treatment. Collectively, our data suggested that Tre exerted its neuroprotective effects by ameliorating oxidative stress, autophagy inhibition, and apoptosis induced by Cd in rat brains. In addition, the Nrf2 signaling pathway, which is continuously activated by Cd, may contribute to brain injury.

Mercuric chloride (HgCl

Autophagy and apoptosis are two different biological processes that determine cell fates. We previously reported that autophagy inhibition and apoptosis induction are involved in lead(II)-induced cytotoxicity in primary rat proximal tubular (rPT) cells, but the interplay between them remains to be elucidated. Firstly, data showed that lead(II)-induced elevation of LC3-II protein levels can be significantly modulated by 3-methyladenine or rapamycin; moreover, protein levels of Autophagy-related protein 5 (Atg5) and Beclin-1 were markedly up-regulated by lead(II) treatment, demonstrating that lead(II) could promote the autophagosomes formation in rPT cells. Next, we applied three pharmacological agents and genetic method targeting the early stage of autophagy to validate that enhancement of autophagosomes formation can inhibit lead(II)-induced apoptotic cell death in rPT cells. Simultaneously, lead(II) inhibited the autophagic degradation of rPT cells, while the addition of autophagic degradation inhibitor bafilomycin A1 aggravated lead(II)-induced apoptotic death in rPT cells. Collectively, this study provided us a good model to know about the dynamic process of lead(II)-induced autophagy in rPT cells, and the interplay between autophagy and apoptosis highlights a new sight into the mechanism of lead(II)-induced nephrotoxicity.

Glyphosate (GLY), a ubiquitous environmental pollutant, can result in gut microbiota dysbiosis intimately involving various diseases. The latest research has shown an association between gut microbiota alteration and defective spermatogenesis. Here, we aimed to investigate whether GLY-induced gut microbiota dysbiosis contributed to male reproductive toxicity. Data showed that GLY-exposed rats exhibited male reproductive dysfunction, evidenced by impaired testis architectural structure, reduced sperm motility, together with increased sperm malformation ratio. 16S rDNA sequencing analysis indicated that GLY exposure altered the composition of gut commensal microbiota, of which the relative abundance of Bacteroidetes and Firmicutes phyla was significantly changed. Unexpectedly, the increased abundance of Prevotella_1 and Bacteroides genera was negatively correlated with sperm quality. Mechanistically, the pathological changes in GLY-exposed testis were accompanied by the increased interleukin (IL)-17A production, probably due to gut microbes-derived Th17 cell migration. Furthermore, activation of IL-17A signaling triggered testicular oxidative damage. Taken together, these findings uncover an underlying mechanistic scenario that gut microbiota dysbiosis-driven local IL-17A production is one reason responsible for male reproductive toxicity induced by GLY, which provides new insights into the male reproductive toxicity of GLY in mammals.

Nuclear factor erythroid 2-related factor 2 (Nrf2) serves as the master regulator of antioxidant signaling and inhibition or hyperactivation of Nrf2 pathway will result in the redox imbalance to induce tissue injury. Herein, we established cadmium (Cd)-exposed rat kidney injury model by intraperitoneal injection with CdCl2 (1.5 mg/kg body weight) and cytotoxicity model of NRK-52E cells by CdCl2 (5 μM) exposure to reveal the role of Nrf2 hyperactivation in Cd-induced nephrotoxicity. Data from the in vitro and in vivo study showed that Cd caused Nrf2 nuclear retention due to nuclear-cytoplasmic depletion of Kelch-like ECH-associated protein 1 (Keap1) and Sequestosome-1(SQSTM1/p62) accumulation, leading to the persistent activation of Nrf2. Moreover, we established inhibited models of Cd-induced prolonged Nrf2 activation using siRNA-mediated gene silencing in vitro and pharmacological inhibition in vivo for subsequent assays. First, Cd-induced cytotoxicity, renal injury and concomitant oxidative stress were markedly alleviated by Nrf2 inhibition. Second, Cd-induced autophagy inhibition was notably alleviated by Nrf2 inhibition. Further, we revealed underlying molecular mechanisms of the crosstalk between persistent activation of Nrf2 and autophagy inhibition in Cd-induced nephrotoxicity. Data showed that Cd-induced lysosomal dysfunction evidenced by impaired lysosomal biogenesis and degradation capacity was markedly recovered by Nrf2 inhibition. Meanwhile, Cd-impaired autophagosome-lysosome fusion was obviously restored by Nrf2 inhibition. In conclusion, our findings revealed that persistent activation of Nrf2 promoted a vicious cycle of oxidative stress and autophagy inhibition in Cd-induced nephrotoxicity.

// Min-Ge Wang 1, 2, 3 , Wen-Hui Li 1, 2, 3 , Xin-Yu Wang 1, 2, 3 , Du-Bao Yang 1, 2, 3 , Zhen-Yong Wang 1, 2, 3 and Lin Wang 1, 2, 3 1 College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai’an City, Shandong Province, 271018, China 2 Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Tai’an City, Shandong Province, 271018, China 3 Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Shandong Agricultural University, Tai’an City, Shandong Province, 271018, China Correspondence to: Lin Wang, email: wanglin2013@sdau.edu.cn Keywords: lead, calcium, proximal tubular cells, CaMKII, endoplasmic reticulum stress Received: June 15, 2017 Accepted: July 25, 2017 Published: August 08, 2017 ABSTRACT Lead (Pb) is a known nephrotoxic element. Recently we have proved that subcellular Ca 2+ redistribution is involved in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells, but the underlying mechanism remains to be elucidated. Firstly, data showed that Pb triggers endoplasmic reticulum (ER) stress response in rPT cells, as evidenced by the elevations of ER stress markers. Moreover, pharmacological modulation of Ca 2+ mobilization in ER and cytoplasm with three chemicals (2-APB or TG or BAPTA-AM) can effectively increase or decrease the protein expression of ER stress markers in Pb-exposed rPT cells, demonstrating that Pb-induced ER stress is Ca 2+ -dependent. We found that Pb stimulates phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) to activate its activity. Meanwhile, inhibition of CaMKII with KN93 or KN62 attenuated Pb-activated caspase-12 and CCAAT/enhancer-binding protein homologous protein (CHOP) in rPT cells, demonstrating that CaMKII activation promoted ER stress in rPT cells. Likewise, Pb-induced apoptosis can be effectively inhibited by CaMKII inhibitor KN93 or KN62. Furthermore, co-treatment with KN93 or KN62 significantly reversed Pb-induced ER Ca 2+ release and concomitant intracellular Ca 2+ overload in rPT cells. In summary, these results expound the mechanisms involving in ER stress, Ca 2+ dyshomeostasis and activated CaMKII, which all contribute to Pb-induced apoptosis. CaMKII acts as a critical mediator of ER stress and associated apoptosis via regulating intracellular Ca 2+ mobilization from ER to cytoplasm.

Previous studies have shown that subcellular Ca2+ redistribution is involved in Cd-induced autophagy inhibition in primary rat proximal tubular (rPT) cells, but the mechanism remains unclear. In this study, the status of autophagic flux was monitored by the GFP and RFP tandemly tagged LC3 method. Pharmacological inhibition of cytosolic Ca2+ concentration ([Ca2+]c) with 2-APB or BAPTA-AM significantly alleviated Cd-elevated yellow puncta formation and restored Cd-inhibited red puncta formation, while thapsigargin (TG) had the opposite regulatory effect, demonstrating that Cd-induced [Ca2+]c elevation inhibited the autophagic flux in rPT cells. Resultantly, Cd-induced autophagosomes accumulation was obviously modulated by 2-APB, BAPTA-AM and TG, respectively. Meanwhile, blockage of autophagosome-lysosome fusion and decreased recruitment of Rab7 to autophagosomes by Cd exposure was noticeably restored by 2-APB or BAPTA-AM, but co-treatment with Cd and TG further impaired Cd-induced autophagy arrest. Moreover, Cd-induced oxidative stress intimately correlated with cytosolic Ca2+ mobilization, and N-acetylcysteine (NAC) markedly rescued Cd-blocked autophagosome-lysosome fusion and recruitment of Rab7 to autophagosomes in rPT cells, implying that Cd-induced autophagy inhibition was due to [Ca2+]c elevation-triggered oxidative stress. In summary, these results suggest that Cd-mediated autophagy inhibition in rPT cells is dependent on cytosolic Ca2+ overload. Elevation of [Ca2+]c inhibited the autophagosome-lysosome fusion to block the degradation of autophagosomes, which aggravated Cd-induced cytotoxicity in rPT cells.

Previous study has demonstrated that mitochondrial-dependent apoptotic pathway is involved in the nephroprotective effect of puerarin (PU) against lead-induced cytotoxicity in primary cultures of rat proximal tubular (rPT) cells. To further clarify how PU exerts its antiapoptotic effects, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and subsequent apoptotic events in the process of PU against Pb-induced cytotoxicity in rPT cells. The results showed that Pb-mediated mitochondrial permeability transition pore (MPTP) opening together with mitochondrial cytochrome c release, activations of caspase-9 and caspase-3, and subsequent poly-ADP-ribose polymerase (PARP) cleavage can be effectively blocked by the addition of PU. Simultaneously, upregulation and downregulation of Bcl-2 and Bax with increased Bcl-2/Bax ratio due to PU administration further alleviated Pb-induced mitochondrial apoptosis. Moreover, PU can reverse Pb-induced ATP depletion by restoring mitochondrial fragmentation to affect ATP production and by regulating expression levels of ANT-1 and ANT-2 to improve ATP transport. In summary, PU produced a significant protection against Pb-induced mitochondrial apoptosis in rPT cells by inhibiting MPTP opening to ameliorate the mitochondrial dysfunction.

Cadmium (Cd), as one of the most toxic heavy metals, has become a widespread environmental contaminant and threats the food quality and safety. The protective effect of selenium (Se) on Cd-induced tissue lesion and cytotoxicity in chicken has been extensively reported. The objective of this study was to investigate the antagonistic effect of Se on Cd-induced damage of chicken pectoral muscles via analyzing the trace elements and amino acids profiles. Firstly, 19 trace elements contents were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that under Cd exposure, the contents of Cd, lead (Pb), mercury (Hg), aluminum (Al), and lithium (Li) were significantly elevated, and the contents of Se, iron (Fe), and chromium (Cr) were significantly reduced. However, supplementing Se significantly reversed the effects induced by Cd. Secondly, the amino acids contents were detected by L-8900 automatic amino acid analyzer. The results showed that supplementing Se increased significantly Cd-induced decrease of valine (Val), leucine (Leu), arginine (Arg), and proline (Pro). Thirdly, the results of principal component analysis (PCA) showed that cobalt (Co), manganese (Mn), silicium (Si), and Pro may play special roles in response to the process of Se antagonizes Cd-induced damage of pectoral muscles in chickens. In summary, these results indicated that different trace elements and amino acids possessed and exhibited distinct responses to suffer from Se and/or Cd in chicken pectoral muscles. Notably, Se alleviated Cd-induced adverse effects by regulating trace elements and amino acids profiles in chicken pectoral muscles.

Lead (Pb) is a known nephrotoxicant that causes damage to proximal tubular cells. PERK pathway plays an important role in the pathogenesis of renal diseases, but its role in Pb-induced nephrotoxicity remains largely unknown. In this study, data showed that Pb could induce ER stress as shown by increased phosphorylation of PERK with subsequent activation of the eIF2α-ATF4-CHOP axis in primary rat proximal tubular (rPT) cells, indicating the activation of PERK-eIF2α-ATF4-CHOP pathway due to excessive ER stress. Pb-activated PERK pathway can be effectively inhibited by 4-phenylbutyric acid and PERK gene silencing, respectively; whereas continuously up-regulated by tunicamycin (TM) treatment. Moreover, Pb-induced apoptosis and inhibition of autophagic flux in rPT cells were significantly augmented and aggravated by co-treatment with TM, respectively. Pharmacological or genetic inhibition of the PERK pathway results in alleviation of apoptosis and restoration of autophagy inhibition in Pb-exposed rPT cells. Mechanistically, activation of PERK-eIF2α-ATF4-CHOP axis triggered by excessive ER stress in rPT cells leads to Pb-induced apoptosis and blockage of autophagic flux, resulting in nephrotoxicity.

Previous studies have shown that cytosolic Ca(2+) ([Ca(2+)]c) overload was involved in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells, but the source of elevated Ca(2+) and the effect of potential subcellular Ca(2+) redistribution on apoptosis are still unknown. In this study, variations of [Ca(2+)]c in two culture media (Ca(2+)-containing and Ca(2+)- free) were analyzed, indicating that Pb-induced elevation of [Ca(2+)]c was primarily generated intracellularly. Fluo-4-AM, dihydro-Rhod-2-AM and Mag-Fluo-4-AM was loaded to Pb-exposed rPT cells to monitor the imaging of Ca(2+) concentrations in the cytoplasm ([Ca(2+)]c), mitochondria ([Ca(2+)]mit) and endoplasmic reticulum (ER) ([Ca(2+)]ER), respectively, under the confocal microscope. Data indicate that elevations of [Ca(2+)]c and [Ca(2+)]mit with depletion of [Ca(2+)]ER were revealed in Pb-treated rPT cells, but this subcellular Ca(2+) redistribution could be significantly suppressed by 2-APB, a specific inhibitor of inositol 1,4,5-trisphosphate receptor (IP3R) that functions to release Ca(2+) from ER stores. Simultaneously, Pb-mediated mitochondrial Ca(2+) overload can be partially suppressed by the cytosolic Ca(2+) chelator BAPTA-AM, suggesting that Ca(2+) uptake into mitochondria occurs via diverse pathways and ER Ca(2+) storage was the chief source. Furthermore, Pb-induced apoptosis was markedly inhibited by 2-APB and BAPTA-AM, respectively. Additionally, elevated IP3 levels with up-regulated IP3R-1 and IP3R-2 (mRNA and protein) levels were revealed in Pb-exposed rPT cells. In summary, IP3R-mediated ER Ca(2+) release promoted the elevations of [Ca(2+)]c and [Ca(2+)]mit in Pb-exposed rPT cells, which played a chief role in apoptosis induced by impaired calcium homeostasis.

Manganese (Mn) deficiency can result in perosis in chicks, but the mechanism of Mn deficiency on tibia development remains poorly understood. Ninety one-day-old Arbor Acres male broiler chickens administered with control diet (60 mg Mn/kg) and Mn-deficient diets (40 mg Mn/kg, 8.7 mg Mn/kg) to investigate the effects of Mn deficiency on morphology of tibia and related signal transduction pathways in broiler chickens. At the age of 42 days, the bone trabecula, damaged osteoblasts and OPG/RANKL mRNA expression levels were investigated by histological assessment, electron microscopic examination and real-time quantitative PCR analysis, respectively. Results of histological observations showed that decreased trabecular thickness, trabecular number and trabecular bone area (%) together with increased trabecular bone separation were involved in perosis induced by Mn deficiency. The most striking ultrastructural modifications involved disruption of nuclear membrane and mitochondria outer membrane, loss of mitochondrion cristae and alteration in endoplasmic reticulum in osteoblasts of the Mn-deficient groups. Likewise, Mn deficiency results in a significant (P < 0.05) decrease in the relative mRNA expression levels of OPG and RANKL with a significantly higher RANKL/OPG ratio (P < 0.05). In conclusion, Mn deficiency can affect the development of tibia in broiler chickens, leading to metaphyseal osteoporosis which may be due to decreased OPG/RANKL mRNA expression.

Autophagy has an important renoprotective function and we recently found that autophagy inhibition is involved in cadmium (Cd)-induced nephrotoxicity. Here, we aimed to investigate the protective effect of trehalose (Tre), a novel autophagy activator, against Cd-induced cytotoxicity in primary rat proximal tubular (rPT) cells. First, data showed that Tre treatment significantly decreased Cd-induced apoptotic cell death of rPT cells via inhibiting caspase-dependent apoptotic pathway, evidenced by morphological analysis, flow cytometric and immunoblot assays. Also, administration with Tre protected rPT cells against Cd-induced lipid peroxidation. Inhibition of autophagic flux in Cd-exposed rPT cells was markedly restored by Tre administration, demonstrated by immunoblot analysis of autophagy marker proteins and GFP and RFP tandemly tagged LC3 method. Resultantly, Cd-induced autophagosome accumulation was obviously alleviated by Tre treatment. Meanwhile, blockage of autophagosome–lysosome fusion by Cd exposure was noticeably restored by Tre, which promoted the autophagic degradation in Cd-exposed rPT cells. Moreover, Tre treatment markedly recovered Cd-induced lysosomal alkalinization and impairment of lysosomal degradation capacity in rPT cells, demonstrating that Tre has the ability to restore Cd-impaired lysosomal function. Collectively, these findings demonstrate that Tre treatment alleviates Cd-induced cytotoxicity in rPT cells by inhibiting apoptosis and restoring autophagic flux.

The objective of this study was to investigate correlations between oxidative stress, metabolism of mineral elements, and lameness in dairy cows. Forty multiparous Chinese Holstein dairy cows were selected and divided into two groups (healthy vs lame, n = 20) by gait score. The experiment lasted for 60 days and samples of hair, blood, and hoof were collected at days 0, 30, and 60 of experiment period, individually. Compared with healthy cows, elevation of MDA, CTX-II, COMP levels, and GSSG/GSH ratio together with depletion of SOD and MT levels in the serum were revealed in lame cows. Simultaneously, significant decreased contents of Zn, Cu, and Mn in the serum, hair, and hoof samples were shown in lame cows, but there was no obvious difference in contents of P, Mg, and Ca (except hoof Ca) in the serum, hair, and hoof between healthy and lame cows. In addition, histological examination and the hardness test demonstrated a poor hoof quality in lame cows. In summary, oxidative stress is implicated in the pathogenesis of lameness caused by imbalance of nutrients (especially selective minerals promoting healthy hoof growth) in dairy cows.

The aim of this study was to investigate the effects of manganese (Mn) deficiency on chondrocyte development in tibia growth plate. Ninety 1-day-old Arbor Acres chicks were randomly divided into three groups and fed on control diet (60 mg Mn/kg diet) and manganese deficient diets (40 mg Mn/kg diet, manganese deficiency group I; 8.7 mg Mn/kg diet, manganese deficiency group II), respectively. The width of the proliferative zone of growth plate was measured by the microscope graticule. Chondrocyte apoptosis was estimated by TUNEL staining. Gene expression of p21 and Bcl-2, and expression of related proteins were analyzed by quantitative real time reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Compared with the control group, manganese deficiency significantly decreased the proliferative zone width and Bcl-2 mRNA expression level, while significantly increased the apoptotic rates and the expression level of p21 gene in chondrocytes. The results indicate that manganese deficiency had a negative effect on chondrocyte development, which was mediated by the inhibition of chondrocyte proliferation and promotion of chondrocyte apoptosis.

Puerarin, a potent free radicals scavenger, has been demonstrated to have protective efficacy in oxidative damage induced by nephrotoxins. In the present study, the attenuating effect of puerarin (PU) on lead (Pb)-induced apoptosis and oxidative stress was investigated in cultured primary rat proximal tubular (rPT) cells. Results showed that exposure to 0.5 µM Pb induced a decrease in cell viability accompanied with obvious cellular morphological alterations and caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, depletion of mitochondrial membrane potential (Δ Ψ) and intracellular glutathione (GSH); elevation of caspase-3 activity, intracellular reactive oxygen species, and malondialdehyde levels; and inhibition of GSH peroxidase (GSH-Px) activity were revealed in the cells exposed to Pb alone. However, simultaneous supplementation with PU (50 and 100 µM) protected rPT cells from Pb-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function, and elevating the intracellular antioxidants (nonenzymatic and enzymic) levels. In conclusion, these findings suggested that PU, as a widely distributed dietary antioxidant, contributes potentially to inhibition of Pb-induced cytotoxicity in rPT cells.

Conduct experimental study on high temperature performance about asphalt concrete with 6mm basalt fiber and ordinary asphalt concrete, 6mm fibers whose dosage is 0.12%0.15% and 0.17%, test method is high temperature rut test, test temperature is 60°C. The results show that basalt fiber can improve the high temperature properties of asphalt concrete substrate, in which basalt fiber asphalt concrete maximum dynamic stability value is almost 1.27 times that of ordinary asphalt concrete, permanent deformation is 34.02% lower than that of the ordinary asphalt concrete, improved the shear performance of asphalt concrete;for high temperature performance, the optimum amount of value about 6mm basalt fiber is 0.15% .

Previous studies have shown that subcellular Ca

Ca2+ signaling plays a vital role in regulating apoptosis and autophagy. We previously proved that cytosolic Ca2+ overload is involved in cadmium (Cd)-induced apoptosis in rat proximal tubular (rPT) cells, but the source of elevated cytosolic Ca2+ concentration ([Ca2+]c) and the effect of potential subcellular Ca2+ redistribution on apoptosis and autophagy remain to be elucidated. Firstly, data showed that Cd-induced elevation of [Ca2+]c was primarily generated intracellularly. Moreover, elevations of [Ca2+]c and mitochondrial Ca2+ concentration ([Ca2+]mit) with depletion of endoplasmic reticulum (ER) Ca2+ levels ([Ca2+]ER) were revealed in Cd-treated rPT cells, but this subcellular Ca2+ redistribution was significantly suppressed by 2-Aminoethoxydiphenyl borate (2-APB). Elevated inositol 1,4,5-trisphosphate (IP3) levels with up-regulated IP3 receptor (IP3R) protein levels were shown in Cd-exposed cells, confirming that IP3R-mediated ER Ca2+ release results in the elevation of [Ca2+]c. Up-regulated sequestosome 1 (p62) protein levels and autophagic flux assay demonstrated that Cd impaired autophagic degradation, while N-acetylcysteine (NAC) markedly attenuated Cd-induced p62 and microtubule-associated protein 1 light chain 3-II (LC3-II) accumulation, implying that the inhibition of autophagic flux was due to oxidative stress. Furthermore, pharmacological modulation of [Ca2+]c with 1,2-Bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and 2-APB alleviated Cd-mediated apoptosis, inhibition of autophagic degradation and subsequent cytotoxicity, while thapsigargin (TG) had the opposite regulatory effect on them. In summary, cytosolic calcium overload originated from IP3R-mediated ER Ca2+ release has a negative impact on Cd nephrotoxicity through its promotion of apoptosis and inhibition of autophagic flux.

Puerarin (PU) is a potent free radical scavenger with a protective effect in nephrotoxin-mediated oxidative damage. Here, we show a novel molecular mechanism by which PU exerts its anti-apoptotic effects in cadmium (Cd)-exposed primary rat proximal tubular (rPT) cells. Morphological assessment and flow cytometric analysis revealed that PU significantly decreased Cd-induced apoptotic cell death of rPT cells. Administration of PU protected cells against Cd-induced depletion of mitochondrial membrane potential (ΔΨm) and lipid peroxidation. Cd-mediated mitochondrial permeability transition pore (MPTP) opening, disruption of mitochondrial ultrastructure, mitochondrial cytochrome c (cyt-c) release, caspase-3 activation and subsequently poly ADP-ribose polymerase (PARP) cleavage could be effectively blocked by the addition of PU. Moreover, up-regulation of Bcl-2 and down-regulation of Bax and hence increased Bcl-2/Bax ratio were observed with the PU administration. In addition, PU reversed Cd-induced ATP depletion by restoring ΔΨm to affect ATP production and by regulating expression levels of ANT-1 and ANT-2 to improve ATP transport. In summary, PU inhibited Cd-induced apoptosis in rPT cells by ameliorating the mitochondrial dysfunction.

A novel biopolymer, deoxyribonucleic acid-hexadecyltrimethylammonium chloride (DNA-HCTAC), is used as the core layer material in optical waveguide, and the cleanroom technology is successfully applied to fabricate the single-mode channel waveguides with low propagation loss. The prepared DNA-HCTAC material shows high optical quality at the optical telecommunication wavelengths, such as high transparency, relatively high refractive index and low birefringence. In the fabrication approach, polymethyl methacrylate (PMMA) is used as a barrier layer to protect the DNA-HCTAC material from the corrosive of photoresist developer, and the etching conditions are optimized to form the smooth wall and sharp cross-section of the waveguide. Lastly, the optical characteristics of DNA-HCTAC channel waveguides are measured. The results show that the DNA-HCTAC waveguide operates with single-mode propagation and has a low optical loss.

Babesiosis (piroplasmosis) is a zoonotic disease caused by an intraerythrocytic protozoan transmitted by Ixodes ticks. The aim of this study was to detect Babesia spp. infection using molecular methods in 377 blood samples from anemic patients. Sequence analysis showed that the 18S rRNA gene was 439 bases long by polymerase chain reaction (PCR) amplification and that the PCR products from the samples had an identical sequence (named Taian China, HM355854). BLAST search showed that the sequence was identical to the 18S rRNA sequences of Babesia divergens. The 18S rRNA sequence for Toxoplasma gondii was included as the outlier for phylogenetic analysis by using the program MEGA4.0 software. The results showed that the 18S rRNA gene sequences obtained from the present study was most closely related to B. divergens Switzerland (DQ312439) with 98.4% similarity (differing only by seven bases). The phylogenetic analysis also revealed that this sequence closely resembled B. divergens strains from other countries and belonged to the same clade. This is the first report of a human being infected by B. divergens in China.

Sixteen shorthorn cows from Xiazhuang farm were admitted to the Veterinary Teaching Hospital at the College of Animal Science and Veterinary Medicine, Shandong Agricultural University for evaluation of poor appetite, listlessness, fever, tachycardia, tachypnea, lethargy, positive jugular venous pulse and anemia. Blood smear examination and polymerase chain reaction analysis in these cows revealed an infection with Mycoplasma wenyonii. The subjects were divided into two groups: control group (three cows) treated with intramuscular injection with imidocarb dipropionate (3 mg/kg/day for 2 days) and the experimental group (13 cows), treated with injection-acupuncture (Imidocarb Dipropionate, 1 mg/kg, once every 3 days for 6 days) at BL17, BL18, BL20, BL25, ST36, SP06 and CV04. At day 15, negative results were found using blood smear examination in all control and experimental groups.

Previous studies have already demonstrated that mitochondria play a key role in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells. To further clarify the underlying mechanism of Pb-induced mitochondrial apoptosis, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and its regulatory components in Pb-induced apoptosis in rPT cells. Mitochondrial permeability transition pore (MPTP) opening together with disruption of mitochondrial ultrastructure, translocation of cytochrome c from mitochondria to cytoplasm and subsequent caspase-3 activation were observed in this study, suggesting that MPT is involved in Pb-induced apoptosis in rPT cells. Simultaneously, Pb-induced caspase-3 activation and apoptosis can be significantly inhibited by three MPTP inhibitors (CsA, DIDS, BA), which target different regulatory components of MPTP (Cyp-D, VDAC, ANT), respectively, demonstrating that Cyp-D, VDAC and ANT participate in MPTP regulation during lead exposure. Moreover, decreased ATP levels and increased ADP/ATP ratio induced by lead treatment can be significantly reversed by BA, indicating that Pb-mediated ANT dysfunction resulted in ATP depletion. In addition, up-regulation of VDAC-1, ANT-1 together with down-regulation of Cyp-D, VDAC-2 and ANT-2 at both the levels of transcription and translation were revealed in rPT cells under lead exposure conditions. In conclusion, Pb-mediated mitochondrial apoptosis in rPT cells is dependent on MPTP opening. Different expression levels in each isoform of three regulatory components contribute to alteration in their functions, which may promote the MPTP opening.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates a cluster of oxidative stress-inducible genes in cells. Here, we aimed to investigate whether trehalose (Tre) protects primary rat proximal tubular (rPT) cells against cadmium (Cd)-induced oxidative stress via Nrf2 antioxidant pathway. Data showed that Tre treatment inhibited Nrf2 nuclear translocation and restored the decline in Kelch-like ECH-associated protein 1 (Keap1) protein level in Cd-exposed rPT cells. Moreover, Cd-activated Nrf2 target genes, including phase II detoxifying enzymes, that is, NAD(P)H quinone oxidoreductase 1 and heme oxygenase-1, direct antioxidant proteins, that is, glutathione peroxidase, superoxide dismutase, catalase, and glutathione biosynthesis-related proteins, that is, glutamatecysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione reductase, were all downregulated by co-treatment with Tre. Collectively, these findings demonstrate that Tre treatment alleviates Cd-induced oxidative stress in rPT cells by inhibiting the Nrf2-Keap1 signaling pathway.

Lead (Pb) is a known nephrotoxicant that causes damage to proximal tubular cells. Autophagy has an important protective role in various renal injuries, but the role of autophagy in Pb-elicited nephrotoxicity remains largely unknown. In this study, Pb promoted the accumulation of autophagosomes in primary rat proximal tubular (rPT) cells, and subsequent findings revealed that this autophagosome accumulation was caused by the inhibition of autophagic flux. Moreover, Pb exposure did not affect the autophagosome–lysosome fusion in rPT cells. Next, we found that Pb caused lysosomal alkalinization, may be through suppression of two V-ATPase subunits. Simultaneously, Pb inhibited lysosomal degradation capacity by affecting the maturation of cathepsin B (CTSB) and cathepsin D (CTSD). Furthermore, translocation of CTSB and CTSD from lysosome to cytoplasm was observed in this study, suggesting that lysosomal membrane permeabilization (LMP) occurred in Pb-exposed rPT cells. Meanwhile, Pb-induced caspase-3 activation and apoptosis were significantly but not completely inhibited by CTSB inhibitor (CA 074) and CTSD inhibitor (pepstatin A), respectively, demonstrating that LMP-induced lysosomal enzyme release was involved in Pb-induced apoptosis in rPT cells. In conclusion, Pb-mediated autophagy blockade in rPT cells is attributed to the impairment of lysosomal function. Both inhibition of autophagic flux and LMP-mediated apoptosis contribute to Pb-induced nephrotoxicity in rPT cells.