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Benzene, a known occupational and environmental contaminant, has been recognized as the hematotoxin and human carcinogen. Lipids have a variety of important physiological functions and the abnormal lipid metabolism has been reported to be closely related to the occurrence and development of many diseases. In the present study, we aim to utilize LC–MS/MS lipidomic platform to identify novel biomarkers and provide scientific clues for mechanism study of benzene hematotoxicity. Results showed that a total of 294 differential metabolites were obtained from the comparison of benzene-treated group and control group. The glycerophospholipid pathway was altered involving the down-regulation of the levels of phosphatidylcholine and phosphatidylserine. In addition, phosphatidylethanolamine (PE) and 1-Acyl-sn-glycero-3-phosphocholine levels were increased in benzene-treated group. Based on the relationship between PE and autophagy, we then found that effective biomarker of autophagy, Beclin1 and LC3B, were increased remarkably. Furthermore, following benzene treatment, significant decreases in glucosylceramide (GlcCer) and phytosphingosine (PHS) levels in sphingolipid pathway were observed. Simultaneously, the levels of proliferation marker (PCNA and Ki67) and apoptosis regulator (Bax and Caspase-3) showed clear increases in benzene-exposed group. Based on our results, we speculate that disturbances in glycerophospholipid pathway play an important role in the process of benzene-induced hematopoietic toxicity by affecting autophagy, while sphingolipid pathway may also serve as a vital role in benzene-caused toxicity by regulating proliferation and apoptosis. Our study provides basic study information for the future biomarker and mechanism research underlying the development of benzene-induced blood toxicity.

Aristolochic acid I (AAI) is a natural bioactive substance found in plants from the Aristolochiaceae family and impairs spermatogenesis. However, whether AAI-induced spermatogenesis impairment starts at the early stages of spermatogenesis has not yet been determined. Spermatogonial stem cells (SSCs) are undifferentiated spermatogonia that balance self-renewing and differentiating divisions to maintain spermatogenesis throughout adult life and are the only adult stem cells capable of passing genes onto the next generation. The objective of this study was to investigate whether AAI impairs SSCs during the early stages of spermatogenesis. After AAI treatment, we observed looser, smaller and fewer colonies, decreased cell viability, a decreased relative cell proliferation index, and increased apoptosis in SSCs in a concentration- and/or time-dependent manner. Additionally, AAI promoted apoptosis in SSCs, which was accompanied by upregulation of caspase 3, P53 and BAX expression and downregulation of Bcl-2 expression, and suppressed autophagy, which was accompanied by upregulation of P62 expression and downregulation of ATG5 and LC3B expression, in a concentration-dependent manner. Then we found that AAI impaired spermatogenesis in rats, as identified by degeneration of the seminiferous epithelium, and increased apoptosis of testicular cells. Taken together, our findings demonstrate that AAI causes damage to SSCs and implicate apoptosis and autophagy in this process. The impairment of SSCs may contribute to AAI-induced testicular impairment. Our findings provide crucial information for the human application of botanical products containing trace amounts of AAI.

Cigarette smoke (CS) is one of the severe risk factors for the development of the pulmonary disease. However, the underlying mechanisms, especially the CS-induced the human bronchial epithelial cells (BEAS-2B) apoptosis related to endoplasmic reticulum stress (ERS) and autophagy, remains to be studied. This study aims to investigate the relationship between ERS and autophagy in apoptosis induced by CS condensate (CSC). BEAS-2B cells were stimulated with 0.02, 0.04 and 0.08 mg/ml CSC for 24 h to detect the ERS, autophagy and apoptosis. Then, ERS and autophagy of BEAS-2B cells were inhibited, respectively, by using 4-PBA and 3-MA, and followed by CSC treatment. The results showed that CSC decreased cell viability, increased cell apoptosis, elevated cleaved-caspase 3/pro-caspase 3 ratio and Bax expressions, but decreased Bcl-2 expressions. The GRP78 and CHOP expressions and LC3-II/LC3-I ratio were dose-dependently increased. The structure of the endoplasmic reticulum was abnormal and the number of autolysosomes was increased in BEAS-2B cells after CSC stimulation. The LC3-II/LC3-I ratio was decreased after ERS inhibition with 4-PBA, but GRP78 and CHOP expressions were enhanced after autophagy inhibition with 3-MA. CSC-induced apoptosis was further increased, Bax expressions and cleaved-caspase 3/pro-caspase 3 ratio were improved, but Bcl-2 expressions were decreased after 3-MA or 4-PBA treatment. In conclusion, the study indicates that ERS may repress apoptosis of BEAS-2B cells induced by CSC via activating autophagy, but autophagy relieves ERS in a negative feedback. This study provides better understanding and experimental support on the underlying mechanisms of pulmonary disease stimulated by CS.

Lidocaine is one of the most common local anesthetics (LA) used in clinical practice and it is neurotoxic. Recent studies suggested that LA, including lidocaine, could exert protective effect over neurotoxicity by promoting autophagy. However, the underlying mechanism was not sufficiently elucidated. This study aimed to explore the mechanism behind. Human neuroblastoma cell line SH-SY5Y was used throughout the whole study. The effect of lidocaine on viability, toxicity of SH-SY5Y cells were analyzed by MTT and lactate dehydrogenase (LDH) assays, respectively. The relative expression of miR-145 was assessed by quantitative reverse transcription-polymerase chain reaction. The impact which lidocaine brought on PI3K/AKT/mTOR pathway and autophagy-related proteins were examined by the western blot assay. LC3B was assessed by immunofluorescence staining. The interaction between miR-145 and AKT3 was conducted by the dual-luciferase reporting assay. Lidocaine inhibited viability of SH-SY5Y cells in a time and dose dependent manner and enhanced the release of LDH in SH-SY5Y cells. Furthermore, the expression of miR-145 and autophagy were enhanced by lidocaine. Transfection with miR-145 inhibitor inhibited the release of LDH and autophagy. miR-145 targeted AKT3 to inhibit PI3K/AKT/mTOR pathway. Finally, lidocaine inactivated PI3K/AKT/mTOR pathways via upregulation of miR-145, and it subsequently promoted autophagy of SH-SY5Y cells. However, silence of miR-145 could reverse the promotion of the autophagy of SH-SY5Y cells. Our results showed that lidocaine promoted autophagy of nerve cells via regulating miR-145 expression and further inactivation of PI3K/AKT/mTOR signaling pathway.

Background: Mutations in LRRK2 are associated with familial and sporadic Parkinson’s disease (PD). Subjects with PD caused by LRRK2 mutations show pleiotropic pathology that can involve inclusions containing α-synuclein, tau or neither protein. The mechanisms by which mutations in LRRK2 lead to this pleiotropic pathology remain unknown. Objectives: To investigate mechanisms by which LRRK2 might cause PD. Methods: We used systems biology to investigate the transcriptomes from human brains, human blood cells and Caenorhabditis elegans expressing wild-type LRRK2. The role of autophagy was tested in lines of C. elegans expressing LRRK2, V337M tau or both proteins. Neuronal function was measured by quantifying thrashing. Results: Genes regulating autophagy were coordinately regulated with LRRK2. C. elegans expressing V337M tau showed reduced thrashing, as has been noted previously. Coexpressing mutant LRRK2 (R1441C or G2019S) with V337M tau increased the motor deficits. Treating the lines of C. elegans with an mTOR inhibitor that enhances autophagic flux, ridaforolimus, increased the thrashing behavior to the same level as nontransgenic nematodes. Conclusion: These data support a role for LRRK2 in autophagy, raise the possibility that deficits in autophagy contribute to the pathophysiology of LRRK2, and point to a potential therapeutic approach addressing the pathophysiology of LRRK2 in PD.

The goal of this investigation was to ascertain whether bone cells undergo autophagy and to determine if this process is regulated by environmental factors. We showed that osteocytes in both murine and human cortical bone display a punctuate distribution of microtubule-associated protein light chain 3, indicative of autophagy. In addition, we noted a basal level of autophagy in preosteocyte-like murine long bone-derived osteocytic (MLO)-A5 cells. Autophagy was upregulated following nutrient deprivation and hypoxic culture, stress conditions that osteocytes encounter in vivo. Furthermore, in response to calcium stress, the transcription factor hypoxia inducible factor 1 regulated MLO-A5 autophagy. Finally, we showed that the more differentiated MLO-Y4 osteocyte-like cells exhibited a significant basal autophagic flux. Based on these findings, we suggest that raising the level of autophagic flux is a mechanism by which differentiated bone cells survive in a stressful environment.

The overall goal of the investigation was to examine autophagy in the growth plate and to ascertain how this process was regulated. Herein, we show that in the postmitotic maturing zone of the growth plate, chondrocytes express an autophagic phenotype. This robust and particulate immunohistochemical response provides direct evidence that autophagy is a new and transient stage in the chondrocyte maturation pathway. We found that induction of autophagy was regulated by mTOR, a sensor of cellular metabolism. When mTOR was inhibited, changes in LC3 fluorescence indicated that this kinase regulated development of the autophagic state. To determine if AMP kinase was required for chondrocyte autophagy, we suppressed its expression in N1511 cells using siRNA technology. When these cells were serum starved, a condition that triggers autophagy, there was no change in LC3 distribution. This result confirmed that AMP kinase was required for the induction of the autophagic response. Based on the 2 studies described above, and our previous observation that HIF-1 is required for the induction of autophagy, we put forward the hypothesis that autophagy is regulated by the activities of AMP kinase and mTOR in a HIF-1-dependent manner. Once autophagy is activated, the postmitotic chondrocytes would be expected to remain viable in their unique microenvironment and complete their life cycle.