Expression Of LC3-II Research Articles

Follistatin-like protein 1 attenuates doxorubicin-induced cardiomyopathy by inhibiting MsrB2-mediated mitophagy.

Doxorubicin (DOX) is a potent chemotherapeutic drug; however, its clinical use is limited due to its cardiotoxicity. Mitochondrial dysfunction plays a vital role in the pathogenesis of DOX-induced cardiomyopathy. Follistatin-like protein 1 (FSTL1) is a potent cardiokine that protects the heart from diverse cardiac diseases, such as myocardial infarction, cardiac ischemia/reperfusion injury, and heart failure. However, its role in DOX-induced cardiomyopathy is unclear. Therefore, the present study investigated whether administering recombinant FSTL1 could mitigate DOX-induced cardiomyopathy and clarified the underlying molecular mechanisms. FSTL1 treatment attenuated DOX-induced cardiac dysfunction, cardiac fibrosis, and cellular apoptosis by inhibiting excess mitochondrial matrix protein methionine sulfoxide reductase B2 (MsrB2)-mediated mitophagy. Furthermore, FSTL1 administration reduced the expression of apoptotic proteins, including MsrB2, Bax, caspase 3, mitochondrial Parkin, and LC3-II, increased myocardial ATP content, and decreased cardiac malondialdehyde levels, thus protecting mitochondrial function against DOX-induced cardiac injury. Furthermore, FSTL1 treatment protected the contractile properties of adult cardiomyocytes against DOX-induced injury in vitro. Furthermore, carbonyl cyanide m-chlorophenylhydrazone, a mitophagy inducer, impaired the protective effects of FSTL1 in DOX-treated H9c2 cardiomyocytes. In conclusion, these results show that FSTL1 is a novel therapeutic agent against DOX-induced cardiotoxicity that improves mitochondrial function and decreases mitophagy.

Baicalein Promotes Acute Myeloid Leukemia Cell Autophagy via miR-424 and the PTEN/PI3K/AKT/mTOR Pathway

Objective: To explore the autophagic effect of baicalein on acute myeloid leukemia (AML) cell lines, HL-60 and THP-1, and miR-424, which regulates the baicalein effect on HL-60 and THP-1 in which autophagy was observed. Methods: The cell counting kit-8 (CCK-8) assay was used to detect the optimal concentration of baicalein in the HL-60 and THP-1 cell lines. miR-424 was detected by qPCR. The influence of baicalein on the autophagy of the HL-60 and THP-1 cells was demonstrated by detecting the expression of Beclin-1, LC3- I, and LC3-II using western blot. The phosphatase and tensin homolog (PTEN)/PI3K/AKt/mTOR pathways were determined by western blot. Results: The optimum concentration of baicalein used and the time of treatment in the HL-60 and THP-1 cell lines were 40 μM and 48 hours, respectively. The expression of miR-424 in the baicalein-treated cells was lower than that in the blank group both in the HL-60 cells and THP-1 cells. The expression of PTEN was promoted by baicalein. However, baicalein inhibited PI3K expression, mTOR phosphorylation, and AKT phosphorylation in the two cell lines. LC3-Ⅰ/Ⅱ, which is the biomarker for autophagy, increased after the cells were treated with baicalein. The baseline expression also increased after the cells were treated with baicalein. Conclusion: Baicalein could promote the autophagy of the HL-60 and THP-1 cells via miR-424 and the PTEN/ PI3K/AKT/mTOR pathway.

Ginsenoside Rg_1 ameliorates OGD/R-induced PC12 cell injury by inhibiting autography via IRE1-JNK-CHOP pathway

This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12) cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP) signaling pathway. An OGD/R model was established in PC12 cells, and PC12 cells were randomly classified into control, model, OGD/R+GRg_1(0.1, 1, 10 μmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+3,5-dibromosalicylaldehyde(DBSA, IRE1 inhibitor) groups. Except the control group, the other groups were subjected to OGD/R treatment, i.e., oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h. Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Apoptosis was detected by Hoechst 33342 staining, and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC) assay. Western blot was employed to determine the expression of autophagy-related proteins(Beclin1, LC3-Ⅱ, and p62) and the pathway-related proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently increased the viability of PC12 cells and down-regulated the expression of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, compared with the model group. Furthermore, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 μmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity, up-regulated protein levels of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, decreased relative cell survival rate, and down-regulated protein level of p62. The 3-MA, 4-PBA, and DBSA groups exerted the opposite effects. Taken together, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.

Expression of ZKSCAN3 protein suppresses proliferation, migration, and invasion of pancreatic cancer through autophagy.

Elevated autophagy activity enhances the malignancy of pancreatic cancer (PaCa), and autophagy is recognized as a novel therapeutic target. Zinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3) is a transcription factor that suppresses autophagy, but its association with PaCa is unknown. We analyzed the function of ZKSCAN3 in PaCa and investigated whether autophagy regulation through ZKSCAN3 could become a new therapeutic target for PaCa. Using reverse transcription-quantitative polymerase chain reaction and western blotting, we observed that ZKSCAN3 expression was upregulated in several PaCa cell lines compared with normal pancreatic ductal epithelial cells. Additionally, comparing ZKSCAN3 expression with the prognosis of PaCa patients using web databases, we found that higher ZKSCAN3 expression in PaCa was associated with extended overall survival. Knocking down ZKSCAN3 promoted the proliferation of PaCa cells. Moreover, following ZKSCAN3 knockdown, PaCa cells exhibited significantly enhanced migratory and invasive properties. Conversely, overexpression of ZKSCAN3 significantly suppressed the proliferation, migration and invasion of PaCa cells. Additionally, the knockdown of ZKSCAN3 increased the expression of LC3-II, a marker of autophagy, whereas ZKSCAN3 overexpression decreased LC3-II expression. In a xenograft mouse model, tumors formed by MIA PaCa-2 cells in which ZKSCAN3 was knocked down significantly increased in size compared with the control group. In conclusion, ZKSCAN3 expression was upregulated in several pancreatic cancer cells. Additionally, it was revealed that ZKSCAN3 is negatively correlated with the malignancy of PaCa through autophagy. These results suggest that autophagy regulation via ZKSCAN3 may be a new therapeutic target for PaCa.

Luteolin, apigenin, and chrysin inhibit lipotoxicity-induced NLRP3 inflammasome activation and autophagy damage in macrophages by suppressing endoplasmic reticulum stress.

Lipotoxicity leads to numerous metabolic disorders such as nonalcoholic steatohepatitis. Luteolin, apigenin, and chrysin are three flavones with known antioxidant and anti-inflammatory properties, but whether they inhibit lipotoxicity-mediated NLRP3 inflammasome activation was unclear. To address this question, we used J774A.1 macrophages and Kupffer cells stimulated with 100 μM palmitate (PA) in the presence or absence of 20 μM of each flavone. PA increased p-PERK, p-IRE1α, p-JNK1/2, CHOP, and TXNIP as well as p62 and LC3-II expression and induced autophagic flux damage. Caspase-1 activation and IL-1β release were also noted after 24 h of exposure to PA. In the presence of the PERK inhibitor GSK2656157, PA-induced CHOP and TXNIP expression and caspase-1 activation were mitigated. Compared with PA treatment alone, Bcl-2 coupled to beclin-1 was elevated and autophagy was reversed by the JNK inhibitor SP600125. With luteolin, apigenin, and chrysin treatment, PA-induced ROS production, ER stress, TXNIP expression, autophagic flux damage, and apoptosis were ameliorated. Moreover, TXNIP binding to NLRP3 and IL-1β release in response to LPS/PA challenge were reduced. These results suggest that luteolin, apigenin, and chrysin protect hepatic macrophages against PA-induced NLRP3 inflammasome activation and autophagy damage by attenuating endoplasmic reticulum stress.

Rhizoma Alismatis Decoction improved mitochondrial dysfunction to alleviate SASP by enhancing autophagy flux and apoptosis in hyperlipidemia acute pancreatitis

BackgroundAcute pancreatitis (AP) is an inflammatory disorder of the exocrine pancreas, especially hyperlipidemia acute pancreatitis (HLAP) is the third leading cause of acute pancreatitis which is more severe with a greater incidence of persistent multiorgan failure. HLAP inflicts injury upon the organelles within the acinar cell, particularly mitochondria, the endolysosomal–autophagy system, and is accompanied by senescence-associated secretory phenotype (SASP). RAD, only two consists of Rhizoma Alismatis and Atractylodes macrocephala Rhizoma, which is best known for its ability to anti-inflammatory and lipid-lowering. Nevertheless, the mechanism by which RAD alleviates HLAP remains obscure, necessitating further investigation. PurposeThe study aimed to assess the effects of the RAD on HLAP and to elucidate the underlying mechanism in vivo and in vitro, offering a potential medicine for clinical treatment for HLAP. Study design and methodsC57BL/6 mice with hyperlipidemia acute pancreatitis were induced by HFD and CER, then administrated with RAD. AR42J were stimulated by cerulein or conditioned medium and then cultured with RAD. Serums were analyzed to evaluate potential pancreas and liver damage. Furthermore, tissue samples were obtained for histological, and protein investigations by H&E, Oil red staining, and Western blot. In addition, western blot and immunofluorescent staining were utilized to estimate the effect of RAD on mitochondrial function, autophagy flux, and SASP. ResultsIn vivo, RAD considerably alleviated systemic inflammation while attenuating TC, TG, AMY, LPS, inflammatory cytokines, histopathology changes, oxidative damage, mitochondrial fission, and autophagy markers in HLAP mice. Impaired autophagy flux and mitochondrial dysfunction resulted in a significant enhancement of NLRP3 and IL-1β in the pancreas. RAD could reverse these changes. In vitro, RAD significantly restored mitochondrial membrane potential and oxidative phosphorylation levels. RAD decreased Beclin-1 and LC3-II expression and increased LAMP-1 and Parkin-Pink expression, which showed that RAD significantly ameliorated HLAP-induced damage to the mitochondria function by suppressing mitochondrial oxidative damage and enhancing autophagy flux and mitophagy to remove the damaged mitochondria. In addition, we found that RAD could up-regulate the expression of BAX, and Bad and down-regulate the expression of p16, and p21, indicating that RAD could promote damaged cell apoptosis and alleviate SASP. ConclusionsThis study revealed that RAD ameliorates mitochondrial function to alleviate SASP through enhancing autophagy flux, mitophagy, and apoptosis which provided a molecular basis for the advancement and development of protection strategies against HLAP.

Autophagy and exosomes; inter-connected maestros in Alzheimer’s disease

Autophagy is a crucial process involved in the degradation and recycling of cytoplasmic components which are transported to the lysosomal compartment by autophagosomes. Exosomes are an important means of communication and signaling in both normal and diseased states, and they have a significant role in the transmission and propagation of proteins, especially proteins implicated in neurodegenerative disorders. Autophagy may affect exosomal processing, but whether autophagy controls the release of aggregated β-amyloid and tau proteins in exosomes of Alzheimer disease (AD) is unclear. Therefore, our study aimed to investigate how modulating autophagy affects the exosomal release of these proteins in animal models of AD. Isolated exosomes from brain tissues of 48 male albino mice were divided into four groups (Negative control, LPS, rapamycin (RAPA), and chloroquine (CQ). LC3 I and LC3 II as well as Aβ and Tau proteins levels were determined. All mice undergone Neuro-behavioral tests (Morris Water maze test, Y-maze test, and Novel Object Recognition). Both LPS and CQ groups showed reduced expression levels of LC3 II and LC3 II/LC3 I ratio. In contrast, RAPA group showed a significant increase in both LC3-II expression and LC3-II/LC3-I ratio. The levels of both Aβ & Tau in exosomes of CQ & LPS groups were higher. While RAPA group showed a significant diminished levels of tau & Aβ proteins. In conclusion, our findings suggest that autophagy alterations in AD can influence the release of Aβ and tau proteins through exosomes, which may impact the spread of misfolded proteins in AD. These results highlight a potential innovative therapeutic approach for combating AD.

392 Targeting One-Carbon Metabolism in Brain Cancer

OBJECTIVES/GOALS: Glioblastoma (GBM) is the most malignant brain tumor in adults and remains incurable with an average survival of 15 months after diagnosis. There is great need for treatment options without side effects that are devastating to the quality of life for patients. GBM tumors can circumvent cellular damage by upregulating antioxidant production. METHODS/STUDY POPULATION: Highly aggressive tumors tend to exhibit increased oxidative metabolism, and thus rely on a mechanism to eliminate reactive oxygen species (ROS) in order for cells to evade autophagy and cell death. We propose that recurrent GBM cells achieve this is by promoting methionine metabolism, upregulating glutathione production and preventing ROS accumulation. We investigated the expression of AHCY and MAT2A, two key enzymes in the methionine pathway, at the gene and protein level in both GBM and non-GBM tissues. We probed for markers of cell death following pharmacological inhibition and siRNA knockdown, and performed metabolite-mediated rescue experiments. Finally, we evaluated changes in cellular respiration using the Seahorse XFe96 real-time mitochondrial stress test following inhibitor treatment. RESULTS/ANTICIPATED RESULTS: The selective AHCY inhibitor markedly reduced cell viability in different cancer cell types, but significantly reduced cell viability in recurrent GBM cells compared to newly diagnosed GBM (p=.009; MD: -0.828, 95% CI -1.350 to -0.306) and normal astrocytes (p=.073; MD: -0.609, 95% CI -1.305 to 0.085). AHCY and MAT2a protein expression appeared to be higher in GBM cells compared to normal astrocytes, medulloblastoma cells and other cancer cell lines. Genetic knockdown of AHCY and MAT2A demonstrated reduced cell viability, increased Caspase, SOD2, LC3-II and Transferrin receptor expression. Acute treatment with the AHCY inhibitor induced cell death, markedly reduced oxygen consumption rate and ablated spare respiratory capacity in recurrent GBM cells compared to newly diagnosed GBM cells. DISCUSSION/SIGNIFICANCE: Oxidative damage was induced following interference with key methionine pathway enzymes by pharmacological inhibition, while a similar concentration of drug largely preserved normal astrocyte viability. These results point to a novel targetable mechanism of disease progression and expand the realm of treatment options for recurrent GBM.

Antiproliferative activities through accelerating autophagic flux by basidalin and its analogs in human cancer cells

Basidalin, isolated from the basidiomycete Leucoagaricus naucina, has previously demonstrated antibacterial and antitumor properties against murine cancer cells in vivo, but its effects on human cancer cells remain unknown. In this study, we found that basidalin possesses antiproliferative activity against human cancer cell lines. To elucidate the antiproliferative mechanism of basidalin, we focused on autophagy. Treatment with basidalin led to an increase in LC3-II expression level, and accelerated autophagic flux through an mTOR-independent pathway. Moreover, according to the structure–activity relationship analysis—including newly synthesized basidalin analogs—the formyl group, not the amino group, contributes to the antiproliferative activities of basidalin against human cancer cells. Additionally, the antiproliferative activity of basidalin analogs was strongly correlated with autophagy-inducing activity, indicating that basidalin exhibits antiproliferative activity through autophagy induction. These data suggest that basidalin, characterized by its ability to upregulate autophagic flux, emerges as a novel anticancer drug.

Unraveling DDIT4 in the VDR-mTOR pathway: a novel target for drug discovery in diabetic kidney disease.

Diabetic kidney disease (DKD) necessitates innovative therapeutic strategies. This study delves into the role of DNA damage-inducing transcription factor 4 (DDIT4) within the VDR-mTOR pathway, aiming to identify a novel target for DKD drug discovery. Transcriptome data from the Gene Expression Omnibus Database were analyzed to assess the expression of mTOR and VDR expression in human renal tissues. Clinical samples from DKD patients and minimal change disease (MCD) controls were examined, and a DKD animal model using 20-week-old db/db mice was established. DDIT4 plasmid transfection was employed to modulate the VDR-mTOR pathway, with its components evaluated using immunohistochemistry, real-time quantitative PCR (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Changes in the expression of the VDR-mTOR pathway were observed in both DKD patients and the animal model. Overexpression of DDIT4 increased VDR expression and decreased levels of mTOR, p70s6k, and 4E-BP1. Furthermore, DDIT4 treatment regulated autophagy by upregulating LC3I expression and downregulating LC3II expression. Notably, DDIT4 alleviated oxidative stress by reducing the levels of lipid peroxidation product MDA, while simultaneously increasing the levels of superoxide dismutase (SOD) and glutathione (GSH), underscoring the role of DDIT4 in the pathological process of DKD and its potential as a therapeutic target. Unraveling DDIT4's involvement in the VDR-mTOR pathway provides insights for innovative DKD drug discovery, emphasizing its potential as a therapeutic target for future interventions.

MKP1 may be involved in the occurrence of depression by regulating hippocampal autophagy in rats

BackgroundMitogen-activated protein kinase (MAPK) phosphatase-1 (MKP1) is upregulated in the hippocampus of patients with depression, while pharmacological inhibition of hippocampal MKP1 can mitigate depression-like behaviors in rodents. In addition, MAPK signaling regulates autophagy, and antidepressants were recently shown to target autophagic signaling pathways. We speculated that MKP1 contributes to depression by enhancing hippocampal autophagy through dephosphorylation of the MAPK isoform ERK1/2. MethodsWe established a rat depression model by exposure to chronic unpredictable mild stress (CUMS), and then examined depression-like behaviors in the sucrose preference test (SPT) and forced swimming test (FST) as well as expression changes in hippocampal MKP1, ERK1/2, phosphorylated ERK1/2, and autophagy-related proteins LC3II by Western blotting and immunostaining. These same measurements were repeated in rats exposed to CUMS following hippocampal infusion of a MKP1-targeted shRNA. Finally, the effects of MKP1 expression level on autophagy we examined in rat GMI-R1 microglia. ResultsCUMS-exposed rats demonstrated anhedonia in the SPT and helplessness in the FST, two core depression-like behaviors. Expression levels of MKP1 and LC3II were upregulated in the hippocampus of CUMS rats, suggesting enhanced autophagy, while pERK/ERK was downregulated. Knockdown of hippocampal MKP1 mitigated depression-like behaviors, downregulated hippocampal LC3II expression, and upregulated hippocampal pERK/ERK. Similarly, MKP1 knockdown in GMI-R1 cells upregulated pERK/ERK and reduced the number of LC3II autophagosomes, while MKP1 overexpression had the opposite effects. ConclusionEnhanced hippocampal autophagy via MKP1-mediated ERK dephosphorylation may contribute to the development of depression.

Inhibition of SIRT1 in the nucleus accumbens attenuates heroin addiction-related behavior by decreasing D1 neuronal autophagy.

This study aimed to investigate the effects of SIRT1 modulation on heroin addiction-like behavior and its possible biological mechanisms. Wild-type C57BL/6J and Sirt1loxp/loxp D1-Cre mice were used in this experiment, and Sirt1 loxp/loxp D1-Cre(-) mice were used as a control for conditional knockout mice. Mice were divided into saline control and heroin-dependent groups. Behavioral methods were used to record the withdrawal response, conditioned place preference (CPP) changes, and open field test results. Transmission electron microscopy (TEM) was used to observe the structure of autophagosomes in nucleus accumbens (NAc) neurons. The expression of SIRT1 and autophagy-related proteins and genes, such as LC3Ⅱ, ATG5 , and ATG7 , was detected in the NAc of each mouse group via western blot, real-time quantitative PCR (qPCR) analyzes, and immunofluorescence. The results of this experiment showed that compared with the saline group, mice in the wild-type heroin-dependent group showed marked withdrawal symptoms, with more autophagosomes observed in NAc via TEM. Compared with wild-type and Sirt1loxp/loxp D1-Cre(-) heroin-dependent groups, CPP formation was found to be reduced in the conditional knockout mouse group, with a significant decrease in spontaneous activity. Western blot, qPCR, and immunofluorescence results indicated that the expression of LC3Ⅱ, ATG-5, and ATG-7 was significantly reduced in the NAc of the Sirt1loxp/loxp D1-Cre(+) group. It was still, however, higher than that in the saline control group. These results suggest that inhibition of Sirt1 expression may prevent heroin-induced addiction-related behaviors via reducing D1 neuronal autophagy.

Interleukin-22 Alleviates Caerulein-Induced Acute Pancreatitis by Activating AKT/mTOR Pathway

BackgroundAcute pancreatitis (AP) is one of the most common acute abdominal disorders; due to the lack of specific treatment, the treatment of acute pancreatitis, especially serious acute pancreatitis (SAP), is difficult and challenging. We will observe the changes of Interleukin -22 levels in acute pancreatitis animal models, and explore the mechanism of Interleukin -22 in acute pancreatitis.ObjectiveThis study aims to assess the potential protective effect of Interleukin -22 on caerulein-induced acute pancreatitis and to explore its mechanism.MethodsBlood levels of amylase and lipase and Interleukin -22 were assessed in mice with acute pancreatitis. In animal model and cell model of caerulein-induced acute pancreatitis, the mRNA levels of P62 and Beclin-1 were determined using PCR, and the protein expression of P62, LC3-II, mTOR, AKT, p-mTOR, and p-AKT were evaluated through Western blot analysis.ResultsInterleukin -22 administration reduced blood amylase and lipase levels and mitigated tissue damage in acute pancreatitis mice model. Interleukin -22 inhibited the relative mRNA levels of P62 and Beclin-1, and the Interleukin -22 group showed a decreased protein expression of LC3-II and P62 and the phosphorylation of the AKT/mTOR pathway. Furthermore, we obtained similar results in the cell model of acute pancreatitis.ConclusionThis study suggests that Interleukin -22 administration could alleviate pancreatic damage in caerulein-induced acute pancreatitis. This effect may result from the activation of the AKT/mTOR pathway, leading to the inhibition of autophagy. Consequently, Interleukin -22 shows potential as a treatment.

N-acetylcysteine and Hydroxychloroquine Ameliorate ADMA-Induced Fetal Growth Restriction in Mice via Regulating Oxidative Stress and Autophagy.

Fetal growth restriction (FGR) seriously threatens perinatal health. The main cause of FGR is placental malperfusion, but the specific mechanism is still unclear, and there is no effective treatment for FGR. We constructed a FGR mouse model by adding exogenous asymmetric dimethylarginine (ADMA) through in vivo experiments and found that ADMA could cause placental dysplasia and induce the occurrence of FGR. Compared with the control group, reactive oxygen species (ROS) production in the placenta was increased in mice with FGR, and the expression of autophagy-related proteins p-AKT/AKT, p-mTOR/mTOR, and P62 was significantly decreased, while the expression of Beclin-1 and LC3-II was significantly increased in the FGR group. Furthermore, ADMA had a favorable effect in promoting the formation of autophagosomes. Hydroxychloroquine (HCQ) and N-acetylcysteine (NAC) improved ADMA-induced disorders of placental development and alleviated ADMA-induced FGR. This study found that ADMA could cause excessive autophagy of trophoblasts by increasing the level of oxidative stress, ultimately leading to the occurrence of FGR, and HCQ and NAC had therapeutic effects on ADMA-induced FGR.

Mechanism of Shenling Baizhu Powder on treatment of alcoholic liver disease by regulating TLR4/NLRP3 pathway

This study aims to investigate the regulatory effects of Shenling Baizhu Powder(SBP) on cellular autophagy in alcoholic liver disease(ALD) and its intervention effect through the TLR4/NLRP3 pathway. A rat model of chronic ALD was established by gavage of spirits. An ALD cell model was established by stimulating BRL3A cells with alcohol. High-performance liquid chromatography(HPLC) was utilized for the compositional analysis of SBP. Liver tissue from ALD rats underwent hematoxylin-eosin(HE) and oil red O staining for pathological evaluation. Enzyme-linked immunosorbent assay(ELISA) was applied to quantify lipopolysaccharides(LPS), tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1β), and interleukin-18(IL-18) levels. Quantitative reverse transcription polymerase chain reaction(qRT-PCR) was conducted to evaluate the mRNA expression of myeloid differentiation factor 88(MyD88) and Toll-like receptor 4(TLR4). The effect of different drugs on BRL3A cell proliferation activity was assessed through CCK-8 analysis. Western blot analysis was performed to examine the protein expression of NOD-like receptor pyrin domain-containing 3(NLRP3), nuclear factor-kappa B P65(NF-κB P65), phosphorylated nuclear factor-kappa B P65(p-P65), caspase-1, P62, Beclin1, and microtubule-associated protein 1 light chain 3(LC3Ⅱ). The results showed that SBP effectively ameliorated hepatic lipid accumulation, reduced liver function, mitigated hepatic tissue inflammation, and reduced levels of LPS, TNF-α, IL-1β, and IL-18. Moreover, SBP exhibited the capacity to modulate hepatic autophagy induced by prolonged alcohol intake through the TLR4/NLRP3 signaling pathway. This modulation resulted in decreased expression of LC3Ⅱ and Beclin1, an elevation in P62 expression, and the promotion of autolysosome formation. These research findings imply that SBP can substantially enhance liver function and mitigate lipid irregularities in the context of chronic ALD. It achieves this by regulating excessive autophagic responses caused by prolonged spirit consumption, primarily through the inhibition of the TLR4/NLRP3 pathway.

Ziyin Bushen Fang improves Diabetic Osteoporosis by Inhibiting Autophagy and Oxidative Stress In vitro and In vivo.

Diabetic osteoporosis (DOP) belongs to the group of diabetes-induced secondary osteoporosis and is the main cause of bone fragility and fractures in many patients with diabetes. The aim of this study was to determine whether Ziyin Bushen Fang (ZYBSF) can improve DOP by inhibiting autophagy and oxidative stress. Type 1 diabetes mellitus (T1DM) was induced in rats using a high-fat high-sugar diet combined with streptozotocin. Micro-CT scanning was used to quantitatively observe changes in the bone microstructure in each group. Changes in the serum metabolites of DOP rats were analyzed using UHPLC-QTOF-MS. The DOP mouse embryonic osteoblast precursor cell model (MC3T3-E1) was induced using high glucose levels. After ZYBSF treatment, bone microstructure significantly improved. The bone mineral density, trabecular number, and trabecular thickness in the ZYBSF-M and ZYBSF-H groups significantly increased. After ZYBSF treatment, the femur structure of the rats was relatively intact, collagen fibers were significantly increased, and osteoporosis was significantly improved. A total of 1239 metabolites were upregulated and 1527 were downregulated in the serum of T1DM and ZYBSF-treated rats. A total of 20 metabolic pathways were identified. In cellular experiments, ZYBSF reduced ROS levels and inhibited the protein expression of LC3II / I, Beclin-1, and p-ERK. ZYBSF may improve DOP by inhibiting the ROS/ERK-induced autophagy signaling pathway.

Knockdown of ANGPTL4 inhibits adipogenesis of preadipocyte via autophagy.

It has been demonstrated that angiopoietin-like protein 4 (ANGPTL4) plays an important regulatory role in lipid metabolism and backfat deposition appears to vary in different pig breeds. However, the correlation between ANGPTL4 and backfat deposition have not been well characterized and the role of ANGPTL4 in regulating adipogenesis remains unclear. Therefore, this study aimed to investigate correlation between ANGPTL4 and backfat deposition and to explore the effects of ANGPTL4 on preadipocyte differentiation and the underlying mechanism. Our results showed that the backfat thickness and the ANGPTL4 gene expression of Laiwu pigs were significantly higher than those in DLY pigs and the ANGPTL4 gene expression was positively correlated with backfat thickness both in DLY pigs and Laiwu pigs. Moreover, an increase in ANGPTL4 expression and activation of autophagy were observed during the differentiation of stromal vascular fraction cells. In addition, knockdown of ANGPTL4 inhibited the differentiation of 3T3-L1 cells with decreased expression of LC3-II and ATG5 and increased expression of SQSTM1, suggesting the involvement of autophagy in ANGPTL4-mediated adipogenesis. In conclusion, these results suggested that ANGPTL4 is positively correlated with backfat deposition in pigs and knockdown of ANGPTL4 inhibits adipogenesis of preadipocyte via autophagy, providing new insights into the regulation of fat deposition and to improve the carcass quality and meat quality of porcine.

Betulinic acid induces apoptosis of HeLa cells via ROS-dependent ER stress and autophagy in vitro and in vivo.

Betulinic acid (BA), a naturally occurring lupane-type triterpenoid, possesses a wide range of potential activities against different types of cancer. However, the molecular mechanisms involved in anti-cervical cancer about BA were rarely investigated. Herein, the role of BA in cervical cancer suppression by ROS-mediated endoplasmic reticulum stress (ERS) and autophagy was deeply discussed. The findings revealed that BA activated Keap1/Nrf2 pathway and triggered mitochondria-dependent apoptosis due to ROS production. Furthermore, BA increased the intracellular Ca2+ levels, inhibited the expression of Beclin1 and promoted the expression of GRP78, LC3-II, and p62 associated with ERS and autophagy. Besides, BA initiated the formation of autophagosomes and inhibited autophagic flux by the co-administration of BA with 3-methyladenine (3-MA) and chloroquine (CQ), respectively. The in vivo experiment manifested that hydroxychloroquine (HCQ) enhanced the apoptosis induced by BA. For the first time, we demonstrated that BA could initiate early autophagy, inhibit autophagy flux, and induce protective autophagy in HeLa cells. Thus, BA could be a potential chemotherapy drug for cervical cancer, and inhibition of autophagy could enhance the anti-tumor effect of BA. However, the interactions of signaling factors between ERS-mediated and autophagy-mediated apoptosis deserve further attention.

Cross-talk between Vimentin and autophagy regulates blood-testis barrier disruption induced by cadmium

The blood-testis barrier (BTB) plays a vital role in mammalian spermatogenesis by separating the seminiferous epithelium into an adluminal and a basal compartment. Cadmium (Cd) is a toxic heavy metal that is widely present in the environment. We observed that Cd can induce BTB disruption, leading to apoptosis of testicular cells. However, the molecular mechanisms contributing to BTB injury induced by Cd have not yet been fully clarified. Vimentin (Vim) is an important desmosome-like junction protein that mediates robust adhesion in the BTB. In this study, we investigated how Vim responds to Cd. We found that Cd treatment led to a significant decrease in Vim expression, accompanied by a marked increase in LC3-II expression and a higer number of autophagosomes. Interestingly, we also observed that Cd-induced autophagy was associated with decreased Vim activity and enhanced apoptosis of testicular cells. To further investigate the role of autophagy in Vim regulation under Cd exposure, we treated cells with an autophagy inhibitor called 3-MA. We found that 3-MA treatment enhanced Vim expression and improved the disruption of the BTB under Cd exposure. Additionally, the inhibition of Vim confirmed the role of autophagy in modulating Vim expression. These results reveal a previously unknown regulatory mechanism of Cd involving the interplay between a heavy metal and a protein.

Empagliflozin Ameliorates the Impaired Osteogenic Differentiation Ability of Adipose-Derived Stem Cells in Diabetic Osteoporosis by Activating Autophagy.

Adipose-derived stem cells (ASCs) from diabetic osteoporosis (DOP) mice showed impaired osteogenic differentiation capacity. Recent studies have shown that in addition to antidiabetic drugs, sodium-glucose co-transporter inhibitor-2 (SGLT-2), empagliflozin, can play multipotent roles through various mechanisms of action. In this study, we aimed to investigate the effects and underlying mechanisms of empagliflozin on osteogenic differentiation of ASCs in DOP mice. Our results showed that osteogenic differentiation potential and autophagy activity weakened in DOP-ASCs when compared to controls. However, empagliflozin enhanced autophagy flux by promoting the formation of autophagosomes and acidification of autophagic lysosomes, resulting in an increase in LC3-II expression and a decrease in SQSTM1 expression. Furthermore, empagliflozin contributed to the reversal of osteogenesis inhibition in DOP-ASCs induced by a diabetic microenvironment. When 3-methyladenine was used to block autophagy activity, empagliflozin could not exert its protective effect on DOP-ASCs. Nonetheless, this study demonstrated that the advent of cellular autophagy attributed to the administration of empagliflozin could ameliorate the impaired osteogenic differentiation potential of ASCs in DOP mice. This finding might be conducive to the application of ASCs transplantation for promoting bone fracture healing and bone regeneration in patients with DOP.