High-throughput screening of novel TFEB agonists in protecting against acetaminophen-induced liver injury in mice

Macroautophagy (referred to as autophagy hereafter) is a major intracellular lysosomal degradation pathway that is responsible for the degradation of misfolded/damaged proteins and organelles. Previous studies showed that autophagy protects against acetaminophen (APAP)-induced injury (AILI) via selective removal of damaged mitochondria and APAP protein adducts. The lysosome is a critical organelle sitting at the end stage of autophagy for autophagic degradation via fusion with autophagosomes. In the present study, we showed that transcription factor EB (TFEB), a master transcription factor for lysosomal biogenesis, was impaired by APAP resulting in decreased lysosomal biogenesis in mouse livers. Genetic loss-of and gain-of function of hepatic TFEB exacerbated or protected against AILI, respectively. Mechanistically, overexpression of TFEB increased clearance of APAP protein adducts and mitochondria biogenesis as well as SQSTM1/p62-dependent non-canonical nuclear factor erythroid 2-related factor 2 (NRF2) activation to protect against AILI. We also performed an unbiased cell-based imaging high-throughput chemical screening on TFEB and identified a group of TFEB agonists. Among these agonists, salinomycin, an anticoccidial and antibacterial agent, activated TFEB and protected against AILI in mice. In conclusion, genetic and pharmacological activating TFEB may be a promising approach for protecting against AILI.

Cathepsin D overexpression in the nervous system rescues lethality and Aβ42 accumulation of cathepsin D systemic knockout in vivo

The lysosome is responsible for protein and organelle degradation and homeostasis and the cathepsins play a key role in maintaining protein quality control. Cathepsin D (CTSD), is one such lysosomal protease, which when deficient in humans lead to neurolipofuscinosis (NCL) and is important in removing toxic protein aggregates. Prior studies demonstrated that CTSD germ-line knockout-CtsdKO (CDKO) resulted in accumulation of protein aggregates, decreased proteasomal activities, and postnatal lethality on Day 26 ± 1. Overexpression of wildtype CTSD, but not cathepsin B, L or mutant CTSD, decreased α-synuclein toxicity in worms and mammalian cells. In this study we generated a mouse line expressing human CTSD with a floxed STOP cassette between the ubiquitous CAG promoter and the cDNA. After crossing with Nestin-cre, the STOP cassette is deleted in NESTIN + cells to allow CTSD overexpression-CTSDtg (CDtg). The CDtg mice exhibited normal behavior and similar sensitivity to sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced neurodegeneration. By breeding CDtg mice with CDKO mice, we found that over-expression of CTSD extended the lifespan of the CDKO mice, partially rescued proteasomal deficits and the accumulation of Aβ42 in the CDKO. This new transgenic mouse provides supports for the key role of CTSD in protecting against proteotoxicity and offers a new model to study the role of CTSD enhancement in vivo.

Role of lysosomal damage in senescence of SH-SY5Y cells induced by methylmercury and intervention of melatonin / 环境与职业医学

Background Methylmercury (MeHg) is a neurotoxin, and melatonin (MT) has a protective effect on the nervous system, but whether it can antagonize MeHg-induced nerve cell damage and the associated mechanism remain unknown. Objective Human neuroblastoma cells (SH-SY5Y cells) were used as research objects. A MeHg-induced SH-SY5Y cell senescence model was established to observe autophagy related protein, lysosomal number, and function changes, as well as potential intervention role and associated mechanism of MT. Methods (1) After SH-SY5Y cells were treated with different doses of MeHg (0, 0.125, 0.25, 0.5, 1, 2, and 4 μmol·L−1) for 48 h, the cell viability was detected using a cell viability detection kit (CCK-8 method) and the viability rate was calculated. Senescent cells were detected by an acidic senescence-associated-β-galactosidase (SA-β-gal) staining. (2) A MeHg dose of 0.5 μmol·L−1 that significantly induced senescence of SH-SY5Y cells was screened, and a half and a quarter of the dose (0.25 and 0.125 μmol·L−1) were used for the middle and low dose groups, respectively. (3) In the MT intervention experiments, SH-SY5Y cells were divided into four groups, including control group (0.1% DMSO), MeHg group (0.5 μmol·L−1 MeHg), MT group (1 mmol·L−1 MT), and MT intervention group (1 mmol·L−1 MT+0.5 μmol·L−1 MeHg). In the MT intervention group, cells were exposed to 0.5 μmol·L−1 MeHg for 48 h after 24 h of 1 mmol·L−1 MT pretreatment. (4) SA-β-gal staining was conducted to observe cell senescence; Western blotting for the expression levels of senescence-associated protein p16, autophagy-associated protein p62, LC3Ⅱ, and lysosomal-associated proteins LAMP1, LAMP2, and TFEB; Lyso-Tracker Red for the quantity of lysosomes; LysoSensor Green DND-189 for lysosomal pH changes; electron microscope for the morphological changes of lysosomes. Results The results of CCK-8 indicated that the viability rate of cells decreased with the increase of MeHg exposure concentration. Compared with the control group, the SA-β-gal positive cell ratio in the 0.5 μmol·L−1 MeHg group increased by 48% (P<0.01), p16, p62, as well as LC3Ⅱ protein expressions were significantly increased (P<0.05), LAMP1 and LAMP2 protein levels, as well as the fluorescence intensities of lysosomal red and green fluorescent probes decreased with the increase of MeHg concentration (P<0.05), and the volume of lysosomes increased under the electron microscope. Compared with the MeHg group, the expression of p16 protein was decreased in the 1 mmol·L−1 MT + 0.5 μmol·L−1 MeHg group and the SA-β-gal positive cell ratio was significantly decreased by 19% (P<0.05), the protein levels of p62 and LC3Ⅱ were significantly decreased, the LAMP1 and LAMP2 protein levels and the fluorescence intensities of lysosomal red and green fluorescent probes were increased respectively, the nuclear entry of TFEB was significantly increased, and the differences were statistically significant (P<0.05). Conclusion MeHg may cause cellular senescence by reducing the number of lysosomes and impairing lysosomal activity in SH-SY5Y cells, and MT may ameliorate MeHg-induced lysosomal abnormalities in SH-SY5Y cells, thereby intervening cell senescence.

Nucleic acid drug delivery strategies without the lysosomal pathway / 中国药科大学学报

@#By regulating gene expression, nucleic acid drugs functioning in the cytoplasm or nucleus are of great significance in the treatment of acquired or inherited diseases and vaccine development.A variety of nucleic acid delivery vectors currently developed are suffering from low transfection efficiency due to endosome/lysosome entrapment.This paper introduces and summarizes the nucleic acid delivery strategies that bypass the endosomal/lysosomal pathway, including membrane translocation, membrane fusion, receptor/transporter-mediated non-endocytic uptake and caveolae-mediated endocytosis, and discusses the problems and challenges facing such strategies, aiming to facilitate the development of intracellular delivery of nucleic acid drugs bypassing lysosomal pathway.

BLOC1S1 promotes proliferation of goat spermatogonial stem cells / 生物工程学报

With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.

Biological effects and toxicity mechanisms of polystyrene nanoplastics: A review / 环境与职业医学

Polystyrene nanoplastics (PS-NPs) are widely used in industry, pharmaceutical and consumer packaging materials, and medical products. The biological health impacts of PS-NPs are receiving increasing attention. Therefore, it is necessary to conduct a literature review of in vitro and in vivo experimental studies from a biological mechanism perspective. Based on the latest research results at home and abroad, this review introduced the characteristics and cell internalization of PS-NPs in cytotoxicity experiments, and summarized the effects of PS-NPs on cytotoxic targets such as mitochondria, lysosomes, proteins, and DNA. In addition, the influencing factors of the health effects of PS-NPs were analyzed from the aspects of physical and chemical properties and cell types. Finally, by discussing the current research hotspots of cytotoxicity mechanism and biological effects, it was anticipated to provide a reference for the health risk management and biological safety assessment of PS-NPs.

Ginsenoside Rg_3 based liposomes target delivery of dihydroartemisinin and paclitaxel for treatment of triple-negative breast cancer / 中国中药杂志

Ginsenoside Rg_3, an active component of traditional Chinese medicine(TCM), was used as the substitute for cholesterol as the membrane material to prepare the ginsenoside Rg_3-based liposomes loaded with dihydroartemisinin and paclitaxel. The effect of the prepared drug-loading liposomes on triple-negative breast cancer in vitro was evaluated. Liposomes were prepared with the thin film hydration method, and the preparation process was optimized by single factor experiments. The physicochemical properties(e.g., particle size, Zeta potential, and stability) of the liposomes were characterized. The release behaviors of drugs in different media(pH 5.0 and pH 7.4) were evaluated. The antitumor activities of the liposomes were determined by CCK-8 on MDA-MB-231 and 4T1 cells. The cell scratch test was carried out to evaluate the effect of the liposomes on the migration of MDA-MB-231 and 4T1 cells. Further, the targeting ability of liposomes and the mechanism of lysosome escape were investigated. Finally, H9c2 cells were used to evaluate the potential cardiotoxicity of the preparation. The liposomes prepared were spheroid, with uniform particle size distribution, the ave-rage particle size of(107.81±0.01) nm, and the Zeta potential of(2.78±0.66) mV. The encapsulation efficiency of dihydroartemisinin and paclitaxel was 57.76%±1.38% and 99.66%±0.07%, respectively, and the total drug loading was 4.46%±0.71%. The accumulated release of dihydroartemisinin and paclitaxel from the liposomes at pH 5.0 was better than that at pH 7.4, and the liposomes could be stored at low temperature for seven days with good stability. Twenty-four hours after administration, the inhibition rates of the ginsenoside Rg_3-based liposomes loaded with dihydroartemisinin(70 μmol·L~(-1)) and paclitaxel on MDA-MB-231 and 4T1 cells were higher than those of the positive control(adriamycin) and free drugs(P<0.01). Compared with free drugs, liposomes inhibited the migration of MDA-MB-231 and 4T1 cells(P<0.05). Liposomes demonstrated active targeting and lysosome escape. In particular, liposomes showed lower toxicity to H9c2 cells than free drugs(P<0.05), which indicated that the preparation had the potential to reduce cardiotoxicity. The findings prove that ginsenoside Rg_3 characterized by the combination of drug and excipient is an ideal substitute for lipids in liposomes and promoted the development of innovative TCM drugs for treating cancer.

Fibroblast growth factor 21 (FGF21) attenuates tacrolimus-induced hepatic lipid accumulation through transcription factor EB (TFEB)-regulated lipophagy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Tacrolimus (TAC), also called FK506, is one of the classical immunosuppressants to prevent allograft rejection after liver transplantation. However, it has been proved to be associated with post-transplant hyperlipemia. The mechanism behind this is unknown, and it is urgent to explore preventive strategies for hyperlipemia after transplantation. Therefore, we established a hyperlipemia mouse model to investigate the mechanism, by injecting TAC intraperitoneally for eight weeks. After TAC treatment, the mice developed hyperlipemia (manifested as elevated triglyceride (TG) and low-density lipoprotein cholesterol (LDL-c), as well as decreased high-density lipoprotein cholesterol (HDL-c)). Accumulation of lipid droplets was observed in the liver. In addition to lipid accumulation, TAC induced inhibition of the autophagy-lysosome pathway (microtubule-associated protein 1 light chain 3β (LC3B) II/I and LC3B II/actin ratios, transcription factor EB (TFEB), protein 62 (P62), and lysosomal-associated membrane protein 1 (LAMP1)) and downregulation of fibroblast growth factor 21 (FGF21) in vivo. Overexpression of FGF21 may reverse TAC-induced TG accumulation. In this mouse model, the recombinant FGF21 protein ameliorated hepatic lipid accumulation and hyperlipemia through repair of the autophagy-lysosome pathway. We conclude that TAC downregulates FGF21 and thus exacerbates lipid accumulation by impairing the autophagy-lysosome pathway. Recombinant FGF21 protein treatment could therefore reverse TAC-caused lipid accumulation and hypertriglyceridemia by enhancing autophagy.

Role of autophagy in the maintenance of skeletal muscle mass / 中国骨伤

As an important exercise and energy metabolism organ of the human body, the normal maintenance of skeletal muscle mass is essential for the body to perform normal physiological functions. The autophagy-lysosome (AL) pathway is a physiological or pathological mechanism that is ubiquitous in normal and diseased cells. It plays a key role in the maintaining of protein balance, removing damaged organelles, and the stability of internal environment. The smooth progress of the autophagy process needs to go through multiple steps, which are completed under the coordinated action of multiple factors. Autophagy maintains the muscle homeostasis of a healthy body by removing cell components such as damaged myofibrils and isolated cytoplasmic proteins. Autophagy could also provide the initial energy required for cell proliferation, promote muscle regeneration and remodeling after injury. At the same time, autophagy disorder is also an important cause of age-related skeletal muscle atrophy. Autophagy could affect the response of skeletal muscle to exercise, and increasing the level of basic autophagy is beneficial to improve the adaptive response of skeletal muscle to exercise. This article summarizes the role and pathways of autophagy in the maintenance of skeletal muscle quality, in order to provide effective rehabilitation strategies for clinical prevention and treatment of muscle atrophy.

Research advances in new technologies in targeted protein degradation / 药学学报

In recent years, the targeted protein degradation technology has developed quickly, with proteolysis-targeting chimera (PROTAC) as the best-known strategy through exploring the ubiquitin-proteasome system. A number of new targeted protein degradation strategies have been emerging to expand the scope of protein degradation technology, including lysosome-targeting chimeras (LYTACs), autophagy-targeting chimeras (AUTACs), autophagosome-tethering compounds (ATTECs) and chimeras based on chaperone-mediated autophagy (CMA). The emerging methodologies have explored another important protein degradation system in eukaryotes-lysosomal systems, such as the endosome-lysosome pathway and the autophagy-lysosome pathway. This review summaries the mechanisms and features of different strategies for targeted protein degradation, with a special emphasis on the new targeted protein degradation technologies, such as their current status, advantages and limitations.

Effects of oxidative stress, PSMB5, TFEB, and lysosomes on sodium arsenite-induced liver injury in rats / 环境与职业医学

Background Liver damage presented in endemic arsenic poisoning is usually serious. Studies have shown that oxidative stress, proteasome beta 5 subunit (PSMB5), regulatory transcription factor EB (TFEB), and lysosomes are associated with liver injury, but their specific links to arsenic-induced liver injury remain unclear. Objective Using a sodium arsenite (NaAsO2)-induced rat liver injury model established earlier by the research group, the expressions of PSMB5, TFEB, and lysosomal associated membrane protein 1 (LAMP1) in liver tissues were detected. Methods Twenty-four SPF Wistar rats were randomly divided into control group, and low, medium, and high dose groups, with 6 rats in each group, half male and half female. The exposure concentrations were 0, 25, 50, and 100 mg·L−1 NaAsO2 solutions for 24 weeks. At the end of the experiment, liver was dissected after rats were anesthetized. The levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bile acid (TBA), and catalase (CAT) in liver tissues were detected by chemical colorimetry, and the levels of lipid peroxide (LPO), 4-hydroxynonenal (4-HNE), LAMP1, and cathepsin D (CTSD) in liver tissues were detected by enzyme-linked immunosorbent assay (ELISA); the transcriptional expression levels of PSMB5 and TFEB in liver tissues were detected by real-time fluorescence quantitative PCR (RT-qPCR), and the protein expressions of PSMB5, TFEB, and phosphorylated TFEB (p-TFEB) in liver tissues were detected by immunohistochemistry. Results The results of chemical colorimetry and ELISA showed that compared with the control group, the liver homogenate levels of ALP, TBA, and LAMP1 of each arsenic-exposed group, the ALT and LPO in the medium and high concentration groups, the 4-HNE and CTSD in the high concentration group were increased, while the CAT activity of each arsenic-exposed group was decreased (P<0.05). The results of real-time fluorescence quantitative PCR showed that the transcription levels of PSMB5 and TFEB in the liver tissues of each arsenic-exposed group were decreased compared with those of the control group (P<0.05). The results of immunohistochemistry showed that compared with the control group, the expression of PSMB5 of each arsenic-exposed group were decreased, the expression of TFEB in the medium and high concentration groups was decreased, while the expression of p-TFEB of each arsenic-exposed group was increased (P<0.05). The expression of TFEB protein gradually decreased in the nucleus, while the expression of p-TFEB protein gradually increased in the cytoplasm, but no expression of p-TFEB was found in the nucleus. The results of Pearson correlation analysis showed that PSMB5 in liver tissues was positively correlated with CAT (r=0.818, P＜0.05), and negatively correlated with 4-HNE and p-TFEB (r=−0.582, r=−0.899; P＜0.05); TFEB was negatively correlated with CTSD and LAMP1 (r=−0.457, r=−0.564; P＜0.05); CTSD was positively correlated with ALT and ALP (r=0.529, r=0.485; P＜0.05). Conclusion Long-term exposure to NaAsO2 can induce oxidative stress, inhibit the expression of PSMB5 and TFEB, promote the accumulation of p-TFEB in the cytoplasm, decrease the nuclear entry of active TFEB, damage the lysosome, and cause liver damage.

Lysosome TMEM175 Potassium Channel Protein and Parkinson’s Disease / 中国生物化学与分子生物学报

Parkinson’s disease (PD)is a complex neurodegenerative disorder by motor impairments and non-motor symptoms. While dopamine-based therapies are effective in fighting the symptoms in the early stages of the disease‚ a lack of neuroprotective drugs means that the disease continues to progress. New disease modifying therapies and novel therapeutic strategies are in high demand for PD patients. Genetic studies indicated that both rare and common genetic variants could induce the development PD. As a risk candidate gene for Parkinson’s disease‚ TMEM175 encodes a lysosomal potassium channel protein with new structures‚ and the protein plays an important role in maintaining lysosomal membrane potential and pH stability. With the in-depth understanding for its structure and function‚ TMEM175 deficiency results in decreased lysosomal catalytic activity and the pathological aggregation of α-synuclein. In view of the importance of lysosome potassium channel TMEM175‚ it could be an interesting target for the development of drugs to treat Parkinson’s disease and other neurodegenerative diseases. Herein we review the structure and function TMEM175‚ and focuses on its involvement in the occurrence and development of PD by affecting the function of lysosome as a homeostatic regulator. Future drug screenings based on lysosome TMEM175 may be carried out to maintain the active state or enhance the expression of TMEM175 to improve the condition of PD patients. Further investigations are needed to study how to maintain the balance between the open and closed state of TMEM175 channels to regulate the ion homeostasis of lysosomes. Studies of this ion channel protein will bring new strategies and ideas for the treatment of PD‚ and provide support for establishing the molecular status of TMEM175 in the diagnosis and treatment of PD.

Effect of erythrocytes, plasma proteins, and lysosomes on systemic exposure to tetrandrine and fangchinoline / 中国临床药理学与治疗学

AlM: The Chinese medicinal herb Hanfangji is dried roots of Stephania tetrandra S. Moore (Family, Menispermaceae). Tetrandrine and fangchinoline are two major constituents of Hanfangji and these bisbenzyltetrahydroisoquinoline alkaloids possess anti - cancer and other pharmacological activities. To facilitate further pharmacodynamic investigation of these compounds, a pharmacokinetic investigation was performed in rats and in vitro. METHODS: Pharmacokinetics of tetrandrine and fangchinoline were characterized in rats p.o. or i.v. dosing an aqueous extract of Hanfangji or the individual compound. Unbound levels of systemic exposure to these two alkaloids were assessed using in vitro studies of plasma protein binding, blood-plasma partition, and lysosomal trapping. All the study samples were analyzed by liquid chromatography/mass spectrometry.RESULTS: We found two pharmacokinetic features of tetrandrine and fangchinoline. First, the two compounds had blood levels of systemic exposure substantially higher than the respective plasma levels of systemic exposure. Second, the two compounds exhibited significantly higher systemic exposure levels after p.o. dosing an aqueous extract of Hanfangji than the respective exposure levels after p.o. dosing the individual compound, at the same compound dose levels and under the same conditions for analytical measurement and the same conditions for animal study. Unbound fractions of tetrandrine and fangchinoline in rat plasma were 2%-5% and the concentrations of the alkaloids in rat erythrocytes were 5-times higher than those in rat plasma. Lysosomal inhibitor could block their trapping in lysosomes and significantly reduce their concentrations in HEK-293 cells. CONCLUSlON: The following pharmacokinetic aspects should be noted in pharmacodynamic investigation of tetrandrine and fangchinoline: extensive binding with plasma proteins, extensive binding with erythrocytes, and trapping by lysosomes of tissue cells substantially reduce the levels of unbound tetrandrine and fangchinoline in the systemic circulation.

Nuciferine protects against high-fat diet-induced hepatic steatosis and insulin resistance via activating TFEB-mediated autophagy-lysosomal pathway

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and insulin resistance and there are currently no approved drugs for its treatment. Hyperactivation of mTOR complex 1 (mTORC1) and subsequent impairment of the transcription factor EB (TFEB)-mediated autophagy-lysosomal pathway (ALP) are implicated in the development of NAFLD. Accordingly, agents that augment hepatic TFEB transcriptional activity may have therapeutic potential against NAFLD. The objective of this study was to investigate the effects of nuciferine, a major active component from lotus leaf, on NAFLD and its underlying mechanism of action. Here we show that nuciferine activated ALP and alleviated steatosis, insulin resistance in the livers of NAFLD mice and palmitic acid-challenged hepatocytes in a TFEB-dependent manner. Mechanistic investigation revealed that nuciferine interacts with the Ragulator subunit hepatitis B X-interacting protein and impairs the interaction of the Ragulator complex with Rag GTPases, thereby suppressing lysosomal localization and activity of mTORC1, which activates TFEB-mediated ALP and further ameliorates hepatic steatosis and insulin resistance. Our present results indicate that nuciferine may be a potential agent for treating NAFLD and that regulation of the mTORC1-TFEB-ALP axis could represent a novel pharmacological strategy to combat NAFLD.

Role of oxidative stress-induced AMPK/ULK1 pathway activation and lysosomal dysfunction in arsenic-induced liver injury in rats / 环境与职业医学

Background A prominent feature of endemic arsenic poisoning is severe liver damage. Studies have found that liver injury is closely related to oxidative stress, lysosomes, and autophagy. Objective Through establishing a liver injury model of rats by sodium arsenite (NaAsO2)administration in drinking water, this experiment is designed to explore the roles of oxidative stress, lysosomes, and AMP activated protein kinase (AMPK)/Unc-51 like kinase 1 (ULK1) pathway in this model. Methods Twenty-four Wistar rats were randomly divided into four groups with six rats in each group (half male and half female), including control group and 25, 50, 100 mg·L−1 NaAsO2 groups. A rat liver injury model was established by drinking water containing NaAsO2 freely for 24 weeks. Then liver of rats was dissected after sacrificed, and the levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bile acid (TBA), catalase (CAT), lipid peroxidation (LPO), and total antioxidant capacity (T-AOC) in liver tissues were detected by assay kits. The levels of lysosomal-associated membrane protein 2 (LAMP2), cathepsin B (CTSB), and acid phosphatase (ACP2) were determined by enzyme linked immunosorbent assay. The mRNA transcriptional expressions of AMPK, ULK1, microtubule-associated protein light chain 3 (LC3), and sequestosome 1 (p62) were detected by real-time fluorescence quantitative PCR (RT-qPCR). The protein expressions of p-AMPK, p-ULK1, LC3, and p62 were detected by immunohistochemistry. Results Following the NaAsO2 administration, significant differences were found in the levels of ALT, ALP, and TBA among the designed groups (F=12.09, 72.11, and 23.58, P<0.05). Compared with the control group, the levels of ALT in the 50mg·L−1 and 100 mg·L−1 NaAsO2 groups were increased (P<0.05); the levels of ALP and TBA in the 25, 50, and 100 mg·L−1 NaAsO2 groups were increased (P<0.05); the level of LPO in the 100 mg·L−1 group was increased (P<0.05); the levels of CAT and T-AOC in the 25, 50, and 100 mg·L−1 NaAsO2 groups were decreased (P<0.05). According to the results of enzyme linked immunosorbent assay, the levels of ACP2 in the 25, 50, and 100 mg·L−1 NaAsO2 groups, the level of CTSB in the 100 mg·L−1 NaAsO2 group, and the levels of LAMP2 in the 50 and 100 mg·L−1 NaAsO2 groups were decreased compared with the control group (P<0.05). Based on the results of RT-qPCR and immunohistochemistry, the mRNA transcriptional and protein expressions of AMPK, ULK1, and LC3 in some arsenic groups were elevated to varying degrees compared with the control group, and the increment in the 100 mg·L−1 NaAsO2 group was significant for all the indicators (P<0.05); the mRNA transcriptional expressions of p62 in the three arsenic groups and the protein expressions of p62 in the 50 and 100mg·L−1 NaAsO2 groups also increased compared with the control group (P<0.05). Besides, the results of Pearson correlation analysis showed that there was a positive correlation of T-AOC with LAMP2, CTSB, and ACP2 (r=0.651, 0.673, 0.626; P<0.05), a negative correlation of LPO with CTSB and ACP2 (r=−0468, −0.482; P<0.05), a negative correlation of p62 with LAMP2, CTSB, and ACP2 (r=−0.57, −0.626, −0.591; P<0.05), and a positive correlation of p62 with ALT, ALP, and TBA (r=0.709, 0.897, and 0.857, P<0.05). Conclusion Long-term arsenic exposure may induce oxidative stress, damage lysosomes, and activate the AMPK/ULK1 pathway, which can lead to the blockage of autophagy process, and eventually result in liver damage.

Proteolysis Pathways via FoxO1 Reduce the Diameter of Myotube under Hypoxia Exposure / 中国生物化学与分子生物学报

The high altitude/hypoxic environment induced skeletal muscle atrophy is considered to be the interaction of multi-system and multi-organ, but the direct mechanism of hypoxia on muscle cells in this process is not clear. This study intended to investigate the effects of hypoxia exposure on proteins in ubiquitin and autophagy pathways, and explored the possible mechanism of hypoxia induced change of myotube diameter. The expression of myosin, hypoxia inducible factor-1 α (HIF-1α), forkhead box protein O1 (FoxO1), and ubiquitin protease pathway (MuRF1 and Atrogin1) and autophagy lysosomal pathway (p62, Beclin1, LC3) related proteins were detected by Western blot; The integrated optical density (IOD) of Myosin and LC3 was detected by IF. The results showed that the diameters of myotube at 6 h and 12 h were significantly reduced, and the expression of myosin was significantly reduced at 6 h after hypoxia exposure (P<0. 05); the protein levels of HIF-1α and FoxO1 were significantly increased at 6 h (P<0. 05); The expression of MuRF1 in each time points of hypoxia was significantly higher than 0 h (P<0. 05), but no difference of Atrogin1 expression was detected; Compared with 0 h, the expression of p62 was reduced significantly in response to hypoxia. The protein expression of Beclin1 and the IOD of LC3 was increased significantly at 6 h, and the LC3Ⅱ/Ⅰ ratio was significantly higher at 6 h, but significantly lower at 12 h and 24 h (P<0. 05).The results above indicated that the reduction of the myotube diameter of L6 skeletal muscle cells was induced by hypoxia exposure (1% O

Changes of expressions of tubulin and endosome-Iysosome system in neurons in hippocampus tissue of mice after status epilepticus and their significances / 吉林大学学报(医学版)

Objective: To investigate the changes of expressions of tubulin and endosome-lysosome in the neurons in hippocampus tissue of the mice after status epilepticus (SE), and to elucidate the change rule of microtubule and endosome-lysosome system in the process of delayed neuronal death. Methods: A total of 40 male ICR mice were divided into control group ( n= 7, given normal saline) and experiment group (w=33» give pilocarpine); the mice in experiment group met the SE standand were divided intoSE 1 d» SE 2 d, SE 3 d and SE 7 d groups according to the time after SE ( n=5). Nissl and Fluoro-Jade B (F-JB) staining methods were used to detect the damage of neurons in hippocampus tissue of the mice in various groups. The expression intensities of (3-tubulin? endosom protein Rab5 and lysosome constitutive protein LAMP1 and the percentages of (3-tubulin∗ Rab5 and LAMP1 positive areas in neurons in hippocampus tissue of the mice in various groups were detected by immunofluorescence method. The relationships between the expression of

Screening potential Chinese materia medica and their monomers for treatment diabetic nephropathy based on caspase-1-mediated pyroptosis / 南方医科大学学报

OBJECTIVE@#To screen potential traditional Chinese medicine and their active monomer ingredients for treatment of diabetic nephropathy (DN) through the mechanism of caspase-1-mediated pyroptosis.@*METHODS@#Using the Chinese Medicine System Pharmacology Analysis Platform (TCMSP), we screened traditional Chinese drugs and their active monomer components targeting caspase-1, and searched for the potential gene targets of the monomer components using GeneCards database. Cytoscape was used to construct the monomer compound-gene target network. Gene ontology (GO) functional enrichment analysis and Kyoto Gene and Gene Encyclopedia (KEGG) pathway enrichment analysis were used to predict the molecular mechanism of the screened traditional Chinese medicine and monomers. In SD rat models of diabetic mellitus (DM), we tested the therapeutic effect of ginsenoside Rh2 (daily dose of 20 mg/kg for 12 weeks) by examining renal pathology with HE staining and detecting the expressions of pyroptosis marker proteins caspase-1, GSDMD, IL-1β and IL-18 in the renal tissues using Western blotting, the serum levels of IL-1β and IL-18 and activities of cathepsin B and cathepsin L.@*RESULTS@#Ginsenoside Rh2 could effectively dock with caspase-1 molecule. Fourteen targets were identified in ginsenoside Rh2 target network. GO function enrichment analysis revealed 27 GO terms associated with molecular function (4 terms), cell component (10 terms) and biological process (13 terms). KEGG pathyway enrichment analysis identified 4 signaling pathways involving lysosomes, glycosaminoglycan degradation, galactose metabolism, and sphingolipid metabolism pathways. In the animal experiment, treatment with ginsenoside Rh2 significantly alleviated renal pathologies and down-regulated the expressions of pyroptosis marker proteins (cleaved caspase-1, GSDMD-N, IL-1β and IL-18) ( < 0.05 or 0.01), lowered serum levels of IL-1β and IL-18 ( < 0.01), and enhanced the activities of cathepsin B and cathepsin L in the serum of the diabetic rats.@*CONCLUSIONS@#Ginsenoside Rh2 may inhibit caspase-1-mediated pyroptosis through the lysosome pathway to improve kidney damages in rat models of DN.

Immunohistochemical study of amelogenin binding proteins in an amelogenin point mutation mouse / Estudio inmunohistoquímico de proteínas de unión de la amelogenina en un ratón con mutación puntual de amelogenina

Amelogenin is one of the enamel matrices secreted by ameloblasts. A mutation of the amelogenin gene can cause hereditary dental enamel defects known as amelogenesis imperfecta (AI). Since lysosome-associated membrane protein-1 (LAMP-1), -3 (LAMP-3), and 78kDa glucose-related protein (Grp78) were identified as binding proteins of amelogenin, several studies have suggested the involvement of these binding proteins with the cell kinetics of ameloblasts in normal or abnormal conditions. The purpose of this study is to investigate the distribution of these amelogenin binding proteins in the ameloblast cell differentiation of mice with a point mutation of the amelogenin gene (Amelx*). The incisors of Amelx* mice had a white opaque color and the tooth surface was observed to be rough under a scanning electron microscope. Among the sequential ameloblast cell differentiation in the Amelx* mice, the shape of ameloblasts at the transition stage was irregular in comparison to those in wild-type (WT) mice. Immunostaining of Grp78 revealed that the whole cytoplasm of the transition stage ameloblasts was immunopositive for Grp78 antibody, while only the distal part of cell was positive in the WT mice. Furthermore, in the Amelx* mice, the cytoplasm of the transition stage ameloblasts was immunopositive for LAMP-1 and LAMP-3. These results suggest that Amelx* may cause the abnormal distribution of amelogenin binding proteins in the cytoplasm of ameloblasts.

La amelogenina es una de las matrices de esmalte secretadas por los ameloblastos. Una mutación del gen de amelogenina puede causar defectos hereditarios del esmalte dental conocidos como amelogénesis imperfecta (AI). Dado que la proteína de membrana asociada a lisosoma-1 (LAMP-1), -3 (LAMP-3) y la proteína relacionada con la glucosa de 78 kDa (Grp78) se identificaron como proteína de unión a amelogenina, varios estudios han sugerido la participación de estas proteínas con la cinética celular de los ameloblastos en condiciones normales o anormales. El objetivo del estudio fue investigar la distribución de LAMP-1, LAM-3 y Grp78 durante la diferenciación celular de ameloblastos de ratones con una mutación puntual del gen de amelogenina (Amelx*). Los incisivos de los ratones Amelx* presentaron un color blanco opaco y se observó en microscopio electrónico de barrido que la superficie del diente era áspera. La diferenciación celular secuencial y la forma de los ameloblastos en la etapa de transición en los ratones Amelx* fue irregular en comparación con los ratones silvestres (RS). La inmunotinción de Grp78 reveló que todo el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para el anticuerpo Grp78, mientras que solo la parte distal de la célula fue positiva en los ratones RS. Además, en ratones Amelx*, el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para LAMP-1 y LAMP-3. Estos resultados sugieren que Amelx* puede causar distribución anormal de proteínas de unión a amelogenina en el citoplasma de los ameloblastos.

Cell Death and Its Different Modes: History of Understanding and Current Trends

Discussions about what is life continue to struggle; there are pros and cons for whether a virus is alive. However, an opposite thing –cell death –appears to be tantamount important and equally not-easygoing to define. Nevertheless, our current knowledgeabout eukaryotic cell death has made a long way and resulted in a fruitful outcome: starting from three types of cell death (type I, II and III which are mainly applicable to eukaryotic cells of organisms from the biological kingdom animalia) in 1970s, Nomenclature Committee on Cell Death has named already twelve cell death forms in 2018, including the above mentioned apoptosis, autophagy and necrosis among them. How the scientific attitude towards cellular demise evolved and various aspects of different cell death modes are reviewed in this article.