Proteomic analysis reveals that cigarette smoke exposure diminishes ovarian reserve in mice by disrupting the CREB1-mediated ovarian granulosa cell proliferation-apoptosis balance.

Exposure to cigarette smoke (CS) adversely affects ovarian health and it is currently unknown how CS exposure causes ovarian injury. This study compared the differences in proteomics between CS exposure and healthy control groups using liquid chromatography-tandem mass spectrometry quantitative proteomics to further understand the molecular mechanism of ovarian cell injury in mice exposed to CS. Furthermore, western blotting and qPCR were carried out to validate the proteomic analysis outcomes. CREB1 was selected from the differentially expressed proteins, and then the down-regulation of CREB1 and phosphorylated CREB1(Ser133) expressions were confirmed in mice ovarian tissue and human ovarian granulosa cells (KGN cells) after CS exposure. In addition, the expressions of apoptosis-related proteins BCL-2 and BCL-XL were downregulated, and BAX expression was up-regulated. Moreover, the results of cellular immunofluorescence, flow cytometry, and transmission electron microscopy (TEM) showed that cigarette smoke extract (CSE) efficiently stimulated the production of reactive oxygen species, apoptosis, G1 phase arrest, mitochondrial membrane potential decreases, and ultrastructural changes in KGN cells. KG-501 (CREB inhibitor) aggravated CSE-induced mitochondrial dysfunction and apoptosis-proliferation imbalance in KGN cells mediated by down-regulated CREB1/BCL-2 axis. In addition, CREB1 over-expression partially restores mitochondrial dysfunction and apoptosis-proliferation imbalance of KGN cells induced by CSE. The results suggested that CSE diminished ovarian reserve in mice by disrupting the CREB1-mediated ovarian granulosa cell (GCs) proliferation-apoptosis balance and provided possible therapeutic targets for the clinical intervention of premature ovarian failure (POI) caused by CS exposure.

Pulmonary embolism induces pneumonia-like lung injury beyond pulmonary infarction.

Patients with pulmonary embolism (PE) commonly manifest concomitant "pneumonia," which is generally believed to be either a cause (infection) or a consequence (infarction) of PE. This study aimed to clarify the relationship between PE and "pneumonia-like" lesions beyond pulmonary infection and infarction. Chest computed tomography (CT) images of patients with PE and deep vein thrombosis (DVT) were retrospectively analyzed to compare the incidence of pneumonia lesions. The pathological damage and wet/dry ratio of lung tissues were observed in PE rats and PE plasma-injected rats. In total, 793 and 914 inpatients were enrolled in the PE and DVT groups, respectively. Pneumonia lesions were observed in 36.9% and 26.3% of patients in the PE and DVT groups, respectively (p < 0.0001). Among PE rats, 33.3% exhibited focal severe lung injury, which closely resembled the pathological damage of community-acquired pneumonia. The wet/dry ratio was significantly higher in the PE group than in the PE-control group (4.98 ± 0.08 vs. 4.39 ± 0.06, p < 0.0001). Among PE plasma-injected rats, individuals with focal proven lung injury were found at all experimental points, with an incidence of 27.6%. The lung wet/dry ratio was significantly higher in the PE plasma group than in the PE-control plasma group at 1 and 2 h postinjection (5.02 ± 0.12 vs. 4.61 ± 0.06 and 4.76 ± 0.16 vs. 4.34 ± 0.09, respectively; p < 0.05). In conclusion, the manifestation of pneumonia lesions in chest CT images was higher among PE patients than among DVT patients. Plasma of PE rats could induce focal pneumonia-like lung injury in healthy rats.

Down-regulated Smyd1 participated in the inhibition of myoblast differentiation induced by cigarette smoke extract.

The histone methyltransferase Smyd1 is essential for muscle development; however, its role in smoking-induced skeletal muscle atrophy and dysfunction has not been investigated thus far. In this study, Smyd1 was overexpressed or knocked down in C2C12 myoblasts by an adenovirus vector and cultured in differentiation medium containing 5% cigarette smoke extract (CSE) for 4 days. CSE exposure resulted in inhibition of C2C12 cell differentiation and downregulation of Smyd1 expression, whereas Smyd1 overexpression reduced the degree of inhibition of myotube differentiation caused by CSE exposure. CSE exposure activated P2RX7-mediated apoptosis and pyroptosis, caused increased intracellular reactive oxygen species (ROS) levels, and impaired mitochondrial biogenesis and increased protein degradation by downregulating PGC1α, whereas Smyd1 overexpression partially restored the altered protein levels caused by CSE exposure. Smyd1 knockdown alone produced a phenotype similar to CSE exposure, and Smyd1 knockdown during CSE exposure aggravated the degree of inhibition of myotube differentiation and the degree of activation of P2RX7. CSE exposure suppressed H3K4me2 expression, and chromatin immunoprecipitation confirmed the transcriptional regulation of P2rx7 by H3K4me2 modification. Our findings suggest that CSE exposure mediates C2C12 cell apoptosis and pyroptosis through the Smyd1-H3K4me2-P2RX7 axis, and inhibits PGC1α expression to impair mitochondrial biosynthesis and increase protein degradation by inhibiting Smyd1 expression, ultimately leading to abnormal C2C12 myoblasts differentiation and impaired myotube formation.

Construction of Selenium Nanoparticle-Loaded Mesoporous Silica Nanoparticles with Potential Antioxidant and Antitumor Activities as a Selenium Supplement.

Excessive reactive oxygen species (ROS) can damage cells and affect normal cell functions, which are related to various diseases. Selenium nanoparticles are a potential selenium supplement for their good biocompatibility and antioxidant activity. However, their poor stability has become an obstacle for further applications. In this study, mesoporous silica nanoparticles (MSNs) were prepared as a carrier of selenium nanoparticles. Pluronic F68 (PF68) was used for the surface modification of the compounds to prevent the leakage of the selenium nanoparticles. The prepared MSN@Se@PF68 nanoparticles were characterized by transmission electron microscopy, energy-dispersive X-ray spectroscopy, dynamic light scattering, X-ray photoelectron spectroscopy, confocal micro-Raman spectroscopy, and Fourier transform infrared spectroscopy. The MSN@Se@PF68 nanoparticles showed excellent antioxidant activity in HeLa tumor cells and zebrafish larvae. The cytotoxicity of MSN@Se@PF68 nanoparticles was concentration- and time-dependent in HeLa tumor cells. The MSN@Se@PF68 nanoparticles showed a negligible cytotoxicity of ≤2 µg/mL at 48 h. At a concentration of 50 µg/mL, the cell viability of the HeLa tumor cells decreased to about 50%. The results indicated that the MSN@Se@PF68 nanoparticles could be a potential antitumor agent. The embryonic development of zebrafish cocultured with the MSN@Se@PF68 nanoparticles showed that there was no lethal or obvious teratogenic toxicity. The results implied that the MSN@Se@PF68 nanoparticles could be a safe selenium supplement and have the potential for antioxidant and antitumor activity.

Single-nucleus RNA Sequencing reveals the mechanism of cigarette smoke exposure on diminished ovarian reserve in mice.

The systematic toxicological mechanism of cigarette smoke (CS) on ovarian reserve has not been extensively investigated. Female 8-week-old C57BL/6 mice at peak fertility were exposed to CS or indoor air only for 30 days (100 mice per group) and the effects of CS on ovarian reserve were assessed using Single-Nucleus RNA Sequencing (snRNA-seq). In addition, further biochemical experiments, including immunohistochemical staining, ELISA, immunofluorescence staining, transmission electron microscopy, cell counting kit-8 assay, flow cytometry analysis, senescence-associated ß-galactosidase staining, and western blotting, were accomplished to confirm the snRNA-seq results. We identified nine main cell types in adult ovaries and the cell-type-specific differentially expressed genes (DEGs) induced by CS exposure. Western blot results verified that down-regulation of antioxidant genes (Gpx1 and Wnt10b) and the steroid biosynthesis gene (Fdx1) occurred in both ovarian tissue and human granulosa cell-like tumor cell line (KGN cells) after CS exposure. Five percent cigarette smoke extract (CSE) effectively stimulated the production of reactive oxygen species (ROS), DNA damage, cellular senescence and markedly inhibited KGN cell proliferation by inducing G1-phase cell cycle arrest. Moreover, down-regulation of Gja1, Lama1 and the Ferroptosis indicator (Gpx4) in granulosa cells plays a significant role in ultrastructural changes in the ovary induced by CS exposure. These observations suggest that CS exposure impaired ovarian follicle reserve might be caused by REDOX imbalance in granulosa cells. The current study systematically determined the damage caused by CS in mouse ovaries and provides a theoretical basis for early clinical prediction, diagnosis and intervention of CS exposure-associated primary ovarian insufficiency (POI), and is of great significance in improving female reproductive health.

IGF2BP3 enhances the mRNA stability of E2F3 by interacting with LINC00958 to promote endometrial carcinoma progression.

Long noncoding RNAs (lncRNAs) play important regulatory roles in a variety of pathological processes involving cancer. However, the exact molecular mechanisms of lncRNA regulation in endometrial carcinoma (EC) remain poorly defined. The aim of this study was to illustrate the mechanism of LINC00958 in regulating the function of IGF2BP3, an RNA binding protein involved in mRNA stability, and their clinical implications in EC. First, we investigated the clinical role of IGF2BP3 in EC and demonstrated its prognostic value. Loss-of-function and gain-of-function studies showed that IGF2BP3 promoted EC cell proliferation, migration and invasion. Then, we carried out RNA immunoprecipitation sequencing (RIP-seq) analysis, RNA pulldown and immunofluorescence-RNA fluorescence in situ hybridization to identify LINC00958 that interacted with IGF2BP3 in the cytoplasm of EC cells. Rescue experiments indicated that knockdown of LINC00958 partially offset the EC cell progression mediated by IGF2BP3. After that, RNA sequencing was used to screen out the downstream genes of IGF2BP3 and LINC00958. The results revealed that IGF2BP3 upregulated E2F3 expression by interacting with LINC00958. Furthermore, RNA stability assays demonstrated that silencing LINC00958 partially rescued the IGF2BP3-mediated promoting effect on the mRNA stability of E2F3. Collectively, this study suggests that LINC00958, as an oncogene, assists IGF2BP3 in stabilizing E2F3 mRNA and ultimately promotes EC progression, providing a promising therapeutic target for patients with EC.

Corrigendum: Downregulation of GLYAT Facilitates Tumor Growth and Metastasis and Poor Clinical Outcomes Through the PI3K/AKT/Snail Pathway in Human Breast Cancer.

[This corrects the article DOI: 10.3389/fonc.2021.641399.].

Antitumor effects of the small molecule DMAMCL in neuroblastoma via suppressing aerobic glycolysis and targeting PFKL.

BACKGROUND: Neuroblastoma (NB) is a common solid malignancy in children that is associated with a poor prognosis. Although the novel small molecular compound Dimethylaminomicheliolide (DMAMCL) has been shown to induce cell death in some tumors, little is known about its role in NB. METHODS: We examined the effect of DMAMCL on four NB cell lines (NPG, AS, KCNR, BE2). Cellular confluence, survival, apoptosis, and glycolysis were detected using Incucyte ZOOM, CCK-8 assays, Annexin V-PE/7-AAD flow cytometry, and Seahorse XFe96, respectively. Synergistic effects between agents were evaluated using CompuSyn and the effect of DMAMCL in vivo was evaluated using a xenograft mouse model. Phosphofructokinase-1, liver type (PFKL) expression was up- and down-regulated using overexpression plasmids or siRNA. RESULTS: When administered as a single agent, DMAMCL decreased cell proliferation in a time- and dose-dependent manner, increased the percentage of cells in SubG1 phase, and induced apoptosis in vitro, as well as inhibiting tumor growth and prolonging survival in tumor-bearing mice (NGP, BE2) in vivo. In addition, DMAMCL exerted synergistic effects when combined with etoposide or cisplatin in vitro and displayed increased antitumor effects when combined with etoposide in vivo compared to either agent alone. Mechanistically, DMAMCL suppressed aerobic glycolysis by decreasing glucose consumption, lactate excretion, and ATP production, as well as reducing the expression of PFKL, a key glycolysis enzyme, in vitro and in vivo. Furthermore, PFKL overexpression attenuated DMAMCL-induced cell death, whereas PFKL silencing promoted NB cell death. CONCLUSIONS: The results of this study suggest that DMAMCL exerts antitumor effects on NB both in vitro and in vivo by suppressing aerobic glycolysis and that PFKL could be a potential target of DMAMCL in NB.

Downregulation of GLYAT Facilitates Tumor Growth and Metastasis and Poor Clinical Outcomes Through the PI3K/AKT/Snail Pathway in Human Breast Cancer.

BACKGROUND: The Glycine N-acyltransferase (GLYAT) gene encodes a protein that catalyzes the transfer of acyl groups from acyl CoA to glycine, resulting in acyl glycine and coenzyme A. Aberrant GLYAT expression is associated with several malignant tumors, but its clinical importance in human breast cancer (BC), has yet to be fully addressed. This study aims to evaluate the clinical function of GLYAT in BC patients. METHODS: GLYAT expression was determined by immune blot and immunohistochemistry in three BC cell lines and primary cancer tissues. The MDA-MB 231 cell line was used for GLYAT gene knockdown experiments while the MCF7 cell line for overexpression experiments. Colony formation experiments, soft agar experiments, and transwell assays were utilized for further inspection of cell proliferation and migration capabilities. Immunofluorescence and western blot were used to detect markers of the epithelial-mesenchymal transition (EMT) and changes in the PI3K/AKT/Snail pathway. The role of GLYAT in tumor growth and metastasis was also assessed in nude mice in vivo. Also, a correlation analysis was performed between clinicopathological features and GLYAT expression in BC patients. RESULTS: GLYAT was decreased in human BC tissues and cell lines. Functional analysis showed that knockdown of GLYAT augmented BC cell proliferation in vitro and in vivo. However, this phenomenon was reversed when GLYAT was overexpressed in the transfected cells. Moreover, downregulation of GLYAT promoted the migratory properties of BC cells, likely through the activation of PI3K/AKT/Snail signaling, which subsequently induced the EMT. IHC analysis indicated that GLYAT was decreased in human BC tissues and lower GLYAT expression was correlated with histological grade, tumor TNM stage, Ki-67 status, and poorer survival in BC patients. Furthermore, lower GLYAT expression seemed as an independent risk factor related to poor prognosis in BC patients based on Cox regression analyses. CONCLUSION: Our findings demonstrate that downregulation of GLYAT expression in human breast cancer is correlated with EMT via the PI3K/AKT/Snail pathway and is also associated with histological grade, tumor TNM stage, Ki-67 status, and poor survival in breast cancer patients.

Maternal smoking during pregnancy aggravated muscle phenotype in FHL1-/y offspring mice similar to congenital clubfoot through P2RX7-mediated pyroptosis.

Congenital clubfoot (CCF) is a common birth defect. Maternal smoking during pregnancy increases the risk of CCF. In previous research, we found muscle phenotypes similar to CCF in four and a half LIM domain protein 1 (FHLI) offspring mice (FHL1-/y). However, the role of P2RX7-mediated pyroptosis in the effect of cigarette smoke (CS) on the skeletal muscle of FHL1-/y mice during pregnancy is unclear. In the present study, pregnant mice at 11 days of gestation were exposed to CS and male offspring of wild-type (WT) and FHL1-/y mice were divided into four groups (Control-WT, Control-KO, CS-WT, CS-KO). The histomorphology of lower limb muscles was examined using hematoxylin and eosin (H&E) staining. P2RX7, indicators of pyroptosis (NLRP3, ASC, cleaved-caspase 1, IL-1ß), and cytoskeletal proteins (MYBPC2, LDB3) were also detected using immunoblotting. CS exposure during pregnancy aggravated the muscle phenotype similar to CCF in FHL1-/y offspring mice. FHL1 gene knockout (KO) or CS exposure during pregnancy each activated the expression of P2RX7, cell pyroptosis-related proteins (NLRP3, ASC, cleaved-caspase 1, IL-1ß), a muscle injury marker (MYOD1), and cytoskeletal proteins (MYBPC2, LDB3); these two factors had an additive effect. The results showed maternal smoking during pregnancy aggravated muscle phenotype similar to CCF in FHL1-/y offspring mice through P2RX7-mediated pyroptosis.

JUNB-FBXO21-ERK axis promotes cartilage degeneration in osteoarthritis by inhibiting autophagy.

Osteoarthritis (OA) is a heterogeneous disease that is extremely hard to cure owing to its complex regulation network of pathogenesis, especially cartilage degeneration. FBXO21 is a subunit of ubiquitin E3 ligases that degrades P-glycoprotein and EID1 by ubiquitination and activates the JNK and p38 pathways; however, its role in OA remains unknown. Here, the main objective of this study was to evaluate the potential effects and mechanism of FBXO21 in OA degeneration, we revealed that FBXO21 is upregulated in the cartilage of patients with OA, aging, and monosodium iodoacetate-induced OA rats, and chondrocytes treated with interleukin-1ß, tumor necrosis factor-α, and lipopolysaccharide. Moreover, the in vivo and in vitro knockdown of FBXO21 suppressed OA-related cartilage degeneration, as evidenced by activated autophagy, upregulated anabolism, alleviated apoptosis, and downregulated catabolism. In contrast, its overexpression promoted OA-related cartilage degeneration. In addition, using mass spectrometry and co-immunoprecipitation assay, we demonstrated that the downstream mechanism of FBXO21 inhibits autophagy by interacting with and phosphorylating ERK. Furthermore, FBXO21 alleviated anabolism and enhanced apoptosis and catabolism by inhibiting autophagy in rat chondrocytes. Interestingly, for its upstream mechanism, JUNB promoted FBXO21 expression by directly targeting the FBXO21 promoter, thus further accelerating cartilage degeneration in SW1353 cells and rat chondrocytes. Overall, our findings reveal that the JUNB-FBXO21-ERK axis regulates OA apoptosis and cartilage matrix metabolism by inhibiting autophagy. Therefore, FBXO21 is an attractive target for regulating OA pathogenesis, and its knockdown may provide a novel targeted therapy for OA.

d-Mannose suppresses osteoarthritis development in vivo and delays IL-1ß-induced degeneration in vitro by enhancing autophagy activated via the AMPK pathway.

Osteoarthritis (OA) is a heterogeneous disease that is consistently difficult to treat due to the complexity of the regulatory network involved in OA pathogenesis, especially in terms of cartilage degeneration. As a C-2 epimer of glucose, d-mannose can alleviate bone loss and repress immunopathology by upregulating regulatory T cells; however, the role of d-mannose in OA-related cartilage degeneration remains unknown. In this study, we investigated the chondroprotective effect of d-mannose in vitro and in vivo on OA. We found that incubating interleukin (IL)-1ß-treated rat chondrocytes with d-mannose restrained OA degeneration by elevating cell proliferation, strongly activating autophagy, reducing apoptosis, and downregulating catabolism. Additionally, oral gavage administration of d-mannose to monosodium iodoacetate (MIA)-treated rats revealed that a median (1.25 g/kg/day) rather than high or low dose of d-mannose suppressed OA progression and attenuated OA development based on lower macroscopic scores for cartilage, decreased histological scores for cartilage and synovium, strongly activated autophagy, and downregulated catabolism. In terms of a downstream mechanism, we showed that d-mannose might attenuate OA degeneration by activating autophagy in IL-1ß-treated rat chondrocytes by promoting the phosphorylation of 5' AMP-activated protein kinase (AMPK). Our in vitro findings revealed that d-mannose delayed IL-1ß-induced OA degeneration in rat chondrocytes by enhancing autophagy activation through the AMPK pathway. Furthermore, the in vivo results indicated that a median dose of d-mannose suppressed MIA-induced OA development. These results suggested that d-mannose exhibits chondroprotective effects and represents a potential disease-modifying drug and novel therapeutic agent for OA.

Cordyceps sinensis attenuates HBx­induced cell apoptosis in HK­2 cells through suppressing the PI3K/Akt pathway.

The authors' previous studies demonstrated that the major renal damage from hepatitis B virus infection is HBx­induced apoptosis of renal tubular epithelial cells. Cordyceps sinensis is one of the most valuable of traditional Chinese medicines and is extensively used to treat chronic renal diseases. However, there is no research on the potential renal protective effect of C. sinensis on HBx­induced apoptosis of renal tubular cells. The protective effect and underlying mechanism of C. sinensis were examined using a renal tubular epithelial cell line stably overexpressing HBx. HK­2 cells were stably transfected with pCMV­HBx to establish HBx­overexpression in an in vitro cell model and HK­2 cells transfected with an empty vector were generated as a control. The effect of C. sinensis on cell proliferation and apoptosis, the phosphatidylinositol­3­kinase (PI3K)/protein kinase B (Akt) signaling pathway, and the enzyme activity of caspase­3 and caspase­9 was measured. The present study demonstrated that HBx transfection inhibited cell proliferation; increased apoptosis, caspase­3 and caspase­9 activity; and increased the activity of the PI3K/Akt pathway. Treatment with C. sinensis attenuated all of these HBx­induced responses. HBx triggered apoptosis and activated the PI3K/Akt signaling pathway in HK­2 cells. C. sinensis treatment significantly attenuated the effect of HBx, at least in part by suppressing the PI3K/Akt signaling pathway.

17-DMAG disrupted the autophagy flux leading to the apoptosis of acute lymphoblastic leukemia cells by inducing heat shock cognate protein 70.

AIMS: B-lineage acute lymphoblastic leukemia (B-ALL) is most common in children. We had reported heat shock protein 90 (Hsp90) over-expressed in high risk B-ALL children. 17-DMAG is a water soluble Hsp90 inhibitor, which was proved to be effective for advanced solid tumors and hematological malignancy. However, there is little research on its application in newly diagnosed B-ALL. And the detailed mechanism is seldom discussed. MAIN METHODS: Primary blast cells from 24 newly diagnosed B-ALL pediatric patients and two B-ALL cell lines were used in this study. Cell viability was measured by MTS assay. Apoptosis was evaluated by flow cytometry after annexin V-PI double staining. Protein expression was detected by immunoblotting analysis and immunofluorescence imaging. Cyto-ID autophagy detection assay was performed to show the autophagosomes and LysoTracker labeling to show the lysosomes. Gene knockdown was performed by RNA interference, and mRNA expression was measured by RT-qPCR. KEY FINDINGS: We showed 17-DMAG induced apoptosis in newly diagnosed B-ALL blasts and cell lines effectively. 17-DMAG induced heat shock cognate protein 70 (Hsc70) expression significantly. High expressed Hsc70 inhibited cathepsin D post-transcriptionally to impede the autophagic flux, which lead to the cell death. SIGNIFICANCE: Our work added new information towards understanding the molecular pharmacology of 17-DMAG, and suggested the newly diagnosed B-ALL pediatric patients might be benefited from 17-DMAG. Furthermore, we proved Hsc70 participated in the mechanism of cell death 17-DMAG leading in B-ALL.

Trichostatin A alleviated ovarian tissue damage caused by cigarette smoke exposure.

Cigarette smoke (CS) has a negative impact on women's health and fertility. Studies have shown that histone deacetylases 1 and 2 (HDAC1/2) were involved in oocyte development. However, the roles of HDAC1/2 in ovarian toxicity caused by CS exposure and the therapeutic potential of trichostatin A (TSA, a HDAC inhibitor) for ovarian tissue damage have not been investigated. In this study, Female C57BL/6 mice were exposed to CS from six cigarettes mixed with indoor air for 120 min (one cigarette for 20 min) using a whole-body mainstream smoke exposure system twice daily for 30 days. TSA (0.6 mg/kg body weight) was injected intraperitoneally into mice in the Control + TSA group and CS + TSA group every two days for 30 days. We found that exposure to CS resulted in ovarian tissue damage and HDAC1/2 over-expression. TSA alleviated the structural changes of ovarian tissue induced by smoking and prevented the activation of HDAC1/2. Exposure to CS caused autophagy inhibition and pyroptosis activation. TSA treatment restored the expression of autophagy-associated proteins and decreased the levels of pyroptosis-related proteins induced by CS exposure. The TSA effect may be mediated by inhibition of HDAC1/2 involved in autophagy and pyroptosis process.

Muscle death participates in myofibrillar abnormalities in FHL1 knockout mice.

BACKGROUND: Mutations in the four and-a-half LIM domain protein 1 (FHL1) gene or FHL1 protein deletion have been identified as the cause of rare hereditary myopathies or cardiomyopathies. In our previous study, autophagy activation was associated with myofibrillar abnormalities in FHL1 knockout (KO) mice. P2RX7 induces cell death, such as autophagy, pyroptosis or apoptosis via cell-specific downstream signaling; however, the roles of P2RX7 in pyroptosis or apoptosis in myofibrillar abnormalities in FHL1 KO mice have not been well elucidated. METHODS: In this study, skeletal muscle and heart of 2.5 months old WT and FHL1 KO male mice histomorphology were examined by hematoxylin and eosin staining. The indicators for pyroptosis (NLRP3; ASC; cleaved-caspase1; IL-1ß), apoptosis (Apaf-1; Bcl-2; caspase9; cleaved-caspase3), and P2RX7 were detected in the triceps (Tri), tibialis anterior muscles (TA), and heart by western blot and/or immunohistochemistry in WT and FHL1 KO male mice. RESULTS: Indicators for pyroptosis (ASC; cleaved-caspase1; IL-1ß) and apoptosis (Apaf-1 and cleaved-caspase3), as well as P2RX7 were upregulated in Tri, tibialis TA, and heart in FHL1 KO mice, indicating pyroptosis and apoptosis play important roles in myofibrillar abnormalities in FHL1 KO mice. CONCLUSIONS: P2RX7 may participate in myofibrillar abnormalities by activating pyroptosis and apoptosis in FHL1 KO mice. These findings have basic implications for the understanding of myopathies induced by FHL1 deficiency and provide new avenues for the treatment of these hereditary myopathies by modulating P2RX7.

Trichostatin A inhibits skeletal muscle atrophy induced by cigarette smoke exposure in mice.

AIMS: It is well known that cigarette smoke (CS) is the main risk factor for chronic obstructive pulmonary disease (COPD) accompanied by skeletal muscle atrophy. Histone deacetylases (HDACs) that remove acetyl groups from target proteins are necessary for the muscle atrophy associated with skeletal muscle disuse. However, the role of HDACs and trichostatin A (TSA), a HDAC inhibitor, in skeletal muscle atrophy caused by CS exposure remains poorly understood. MAIN METHODS: Female mice were exposed to CS twice daily for 40 days and TSA injected intraperitoneally into CS-exposed mice on alternate days. Skeletal muscles were weighed and gastrocnemius (Gas) muscle histomorphology examined by hematoxylin and eosin staining. Histone deacetylases 1 and 2 (HDAC1/2), and markers of ubiquitin degradation, muscle differentiation, apoptosis, pyroptosis, and the cytoskeletal proteins were assessed by western blot and immunohistochemistry in Gas. KEYFINDINGS: CS exposure decreased body and skeletal muscle weights and triggered an increase in the percentage of fiber with centralized nuclei in Gas. HDAC1/2 proteins were upregulated in the Gas of mice exposed to CS, while TSA effectively inhibited HDAC1/2 protein levels and attenuated the loss of body weight and skeletal muscle wet weight induced by CS exposure. Markers for ubiquitin degradation, muscle differentiation, cytoskeletal proteins, apoptosis and pyroptosis were all upregulated following CS exposure and effectively restored by TSA. SIGNIFICANCE: TSA may inhibit skeletal muscle atrophy and histomorphological alterations induced by CS exposure by downregulating markers of ubiquitin degradation, muscle fiber differentiation, cytoskeletal proteins, apoptosis and pyroptosis via HDAC1/2 inhibition.

Trichostatin A inhibits uterine histomorphology alterations induced by cigarette smoke exposure in mice.

AIMS: Cigarette smoking results in well-known negative reproductive consequences. However, the role of histone deacetylase 1 and 2 (HDAC1/2) in the structural changes of uterine tissues induced by cigarette smoke (CS) exposure and the therapeutic potential of trichostatin A (TSA), a HDAC inhibitor, have not been investigated. MAIN METHODS: Female mice were exposed to CS twice daily for 30 days and TSA was injected intraperitoneally into CS-exposed mice on alternate days in the TSA-treated group. Uteri in the estrus phase were weighed and uterine histomorphology and HDAC1 cell distribution were examined by HE and immunohistochemistry. Markers associated with macro-autophagy (Beclin-1), autophagic flux (increased LC3-II and a lack of p62 accumulation), autophagy inhibiting factor (mTOR, phosphorylated mTOR and its upstream IRS, phosphorylated IRS), HDAC1/2, FOXO1 and FOXO3 were assessed by Western blot. KEY FINDINGS: CS exposure decreased body weight and triggered uterine histomorphologic alterations, including a thinner myometrium and a reduced number of glandular and interstitial cells. HDAC1/2 were activated in uterine tissues after CS exposure and TSA effectively inhibited HDAC1/2 activation and attenuated the loss of body weight and uterine wet weight induced by CS exposure. TSA effectively restored the thickness of the myometrium and number of glandular and interstitial cells. TSA also restored the expression of markers of macro-autophagy (LC3-II and Beclin-1) and reduced phosphorylated mTOR, phosphorylated IRS, FOXO1 and FOXO3 activation. SIGNIFICANCE: TSA inhibited uterine histomorphologic alterations induced by CS exposure. The TSA effect might be associated with resumption of macro-autophagy via HDAC1/2 inhibition.

HDAC-selective Inhibitor Cay10603 Has Single Anti-tumour Effect in Burkitt's Lymphoma Cells by Impeding the Cell Cycle.

Histone deacetylases (HDACs) inhibitors are novel in cancer therapy nowadays. HDAC6-selective inhibitors exert advantageous effects due to higher selectivity and less toxicity. We explored the anti-tumor effect and the molecular mechanism of cay10603, a potent HDAC6 inhibitor in Burkitt's lymphoma cells. Our study revealed cay10603 inhibited the proliferation of Burkitt's lymphoma cell lines, and induced caspase-dependent apoptosis. Cay10603 inhibited the expression of CDKs and cyclins to impede cell cycle progression in both Burkitt's lymphoma cell lines. Cay10603 also showed the additive effect with vp16 notably. Our data presented the promising anti-tumor effect of cay10603 in the Burkitt's lymphoma therapy.

Aberrant Protein Turn-Over Associated With Myofibrillar Disorganization in FHL1 Knockout Mice.

Mutations in the FHL1 gene, and FHL1 protein deletion, are associated with rare hereditary myopathies and cardiomyopathies. FHL1-null mice develop age-dependent myopathy and increased autophagic activity. However, the molecular pathway involved in contractile function and increased autophagic activity in the FHL1-null mouse has not yet been fully elucidated. In this study, FHL1 protein was knocked out in mice using Transcription Activator-like Effector Nucleases (TALENs) and the IRS1-FOXO1/mTOR signaling pathway was investigated in skeletal muscles and heart. TALEN constructs caused targeted mutations in 30% of newborn mice; these mutations caused a deletion of 1-13 base pairs which blocked synthesis of the full-length FHL1 protein. Furthermore, 2.5-month old FHL1-null male mice were not prone to global muscular fatigue when compared with WT littermates, but histological analysis and ultrastructural analysis by transmission electron microscopy confirmed the presence of myofibrillar disorganization and the accumulation of autophagosome or autolysosome-like structures in FHL1-null mice. Moreover, autophagy and mitophagy were both activated in FHL1 KO mice and the degradation of autophagic lysosomes was impeded. Enhanced autophagic activity in FHL1 KO mice was induced by FOXO1 up-regulation and protein synthesis was increased via mTOR. The cytoskeletal proteins, MYBPC2 and LDB3, were involved in the formation of pathological changes in FHL1 KO mice. Markers of early differentiation (MEF2C and MYOD1) and terminal differentiation (total MYH) were both up-regulated in tibialis anterior (TA) muscles in FHL1 KO mice. The number of type I and type II fibers increased in FHL1-null TA muscles, but the number of type| | b, and type | | d fibers were both reduced in FHL1-null TA muscles. The results obtained from the heart were consistent with those from the skeletal muscle and indicated autophagic activation by FOXO1 and an increase in protein synthesis via mTOR also occurred in the heart tissue of FHL1 knockout mice. In conclusion, aberrant protein turn-over associated with myofibrillar disorganization in FHL1 knockout mice. the up-regulation of FOXO1 was associated with enhanced autophagic activity and pathological changes in the muscle fibers of FHL1 KO mice. These results indicated that autophagy activated by FOXO1 is a promising therapeutic target for hereditary myopathies and cardiomyopathies induced by FHL1.