mRNA Expression Of Beclin1 Research Articles

Tetrahydropalmatine promotes macrophage autophagy by inhibiting the AMPK/mTOR pathway to attenuate atherosclerosis.

Atherosclerosis (AS) is a chronic progressive arterial disease that is associated with macrophage autophagy and AMP-activated protein kinase (AMPK)/mechanistic target of the rapamycin (mTOR) pathway. Tetrahydropalmatine (THP) can activate AMPK-dependent autophagy. We aim to study the mechanism of macrophage autophagy mediated by THP in the treatment of AS via the AMPK/mTOR pathway. High-fat diet apolipoprotein E-deficient mice and ox-LDL-induced RAW264.7 cells were used to mimic the AS model, then THP was administered. Cell viability was detected by MTT. Pathological aorta lesions were detected using Hematoxylin and Eosin, Masson, and oil red staining. Lipid metabolism indices and inflammatory factors were measured using ELISA. A transmission electron microscope was used to observe autophagosomes. Autophagy and AMPK/mTOR pathway protein expression was detected by immunofluorescence and Western blot. The AMPK inhibitor 9-β-d-Arabinofuranosyl Adenine (Ara-A) was used to validate the effect of THP. The mRNA expression of Beclin-1 and MCP-1 was detected by q-PCR. THP administration regulated lipid metabolism by lowering total cholesterol, triacylglycerol, low-density lipoprotein, and high-density lipoprotein levels, and suppressed aortic damage. THP suppressed aortic damage and regulated lipid metabolism by altering serum lipid levels. THP reduced inflammation and macrophage CD68 expression. Twenty μg/mL THP reduced cell viability. THP decreased cholesterol uptake and increased efflux, promoting autophagy. THP increased autophagosome number, LC3B expression, and autophagy markers p-AMPK/AMPK and LC3-II/LC3-I. THP also decreased p-mTOR/mTOR and P62. THP increased Beclin-1 mRNA expression and decreased MCP-1 mRNA expression. Ara-A reversed THP's effects. THP promotes macrophage autophagy by inhibiting the AMPK/mTOR pathway to attenuate AS.

Short-peptide-based enteral nutrition affects rats MDP translocation and protects against gut-lung injury via the PepT1-NOD2-beclin-1 pathway in vivo.

Peptide transporter 1 (PepT1) transports bacterial oligopeptide products and induces inflammation of the bowel. Nutritional peptides compete for the binding of intestinal bacterial products to PepT1. We investigated the mechanism of short-peptide-based enteral nutrition (SPEN) on the damage to the gut caused by the bacterial oligopeptide product muramyl dipeptide (MDP), which is transported by PepT1. The gut-lung axis is a shared mucosal immune system, and immune responses and disorders can affect the gut-respiratory relationship. Sprague-Dawley rats were gavaged with solutions containing MDP, MDP + SPEN, MDP + intact-protein-based enteral nutrition (IPEN), glucose as a control, or glucose with GSK669 (a NOD2 antagonist). Inflammation, mitochondrial damage, autophagy, and apoptosis were explored to determine the role of the PepT1-nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-beclin-1 signaling pathway in the small intestinal mucosa. MDP and proinflammatory factors of lung tissue were explored to determine that MDP can migrate to lung tissue and cause inflammation. Induction of proinflammatory cell accumulation and intestinal damage in MDP gavage rats was associated with increased NOD2 and Beclin-1 mRNA expression. IL-6 and TNF-α expression and apoptosis were increased, and mitochondrial damage was severe, as indicated by increased mtDNA in the MDP group compared with controls. MDP levels and expression of proinflammatory factors in lung tissue increased in the MDP group compared with the control group. SPEN, but not IPEN, eliminated these impacts. Gavage of MDP to rats resulted in damage to the gut-lung axis. SPEN reverses the adverse effects of MDP. The PepT1-NOD2-beclin-1 pathway plays a role in small intestinal inflammation, mitochondrial damage, autophagy, and apoptosis.

Abstract Mo004: The Protective Effects of Empagliflozin in Carfilzomib-induced Cardiotoxicity in Mice: Unveiling the Crosstalk between Oxidative Stress, Inflammation, Endoplasmic Reticulum Stress, and Autophagy Axes.

Introduction: Multiple myeloma ranks as the second most prevalent hematologic cancer in the US. Survival rates have significantly improved owing to advances in chemotherapy treatment. Carfilzomib, an irreversible proteasome inhibitor, has been used globally for the treatment of multiple myeloma. Unfortunately, its clinical use has been linked to cardiovascular complications. Empagliflozin is a sodium glucose co-transporter-2 inhibitor with potential cardioprotective activities, for which it is approved for heart failure management. Aims: The current study determined the beneficial effects of Empagliflozin against Carfilzomib-induced cardiotoxicity in C57BL/6 mice and the possible underlying mechanisms. Methods: Thirty-two mice were randomly divided into four groups and treated for two days as follows: Control (vehicle), Empagliflozin (10 mg/kg, oral), Carfilzomib (8 mg/kg, i.p.), and Carfilzomib + Empagliflozin groups. Results: The impact of Carfilzomib and Empagliflozin treatment on markers of cardiotoxicity and heart tissue architecture was assessed. Empagliflozin corrected histological alterations, CK-MB activity, and troponin I level induced by Carfilzomib. Additionally, Empagliflozin mitigated Carfilzomib-induced oxidative deficits via its effect on catalase activity, reduced glutathione level, and superoxide dismutase activity, restoring the balance of redox circuit. Moreover, Empagliflozin averted Carfilzomib-induced inflammatory response as evidenced by reducing nuclear factor-κB (p65), tumor necrosis factor-α, and interleukin-1β levels. Mechanistically, a substantial amelioration in the activating transcription factor 6-mediated endoplasmic reticulum stress was demonstrated with Empagliflozin treatment owing to its effect on glucose regulated protein-78, activating transcription factor-6, and C/EBP homologous protein. Intriguingly, the induction of autophagy by Carfilzomib was notably amplified following Empagliflozin administration as evidenced by higher mRNA expression of LC3B and beclin-1, and lower expression of p62. Accordingly, the apoptotic marker cleaved caspase-3 expression was markedly reduced following Empagliflozin treatment. Importantly, Empagliflozin did not alter the cytotoxic effect of Carfilzomib on human U266B1 myeloma cell line. Conclusion: This research suggests Empagliflozin could offer cardioprotection against Carfilzomib-induced cardiotoxicity in multiple myeloma patients.

Effect of electroacupuncture on autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency

To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI), and explore the mechanism of EA in improving POI. Thirty-two female SD rats were randomly divided into a blank group (n=8) and a model making group (n=24). The rats in the model making group were injected intraperitoneally with cyclophosphamide for 15 days to establish the POI model (the dosage on the 1st day was 50 mg/kg, and 8 mg/kg from the 2nd day to 15th day). The successfully modeled rats were then randomly divided into a model group, an EA group, and an estradiol (E2) group, with 8 rats in each group. Rats in the EA group received EA at bilateral "Gongsun" (SP 4) with continuous wave, frequency of 2 Hz, and current intensity of 0.1 to 1 mA, 20 min per treatment, once daily for 14 days. Rats in the E2 group were administered with E2 (0.01 mg/mL) by gavage (10 mL/kg), once daily for 14 days. The changes in estrous cycle were observed by rapid Giemsa staining before and after modeling. After intervention, ovarian tissue morphology was observed by HE staining; serum levels of follicle-stimulating hormone (FSH), E2, anti-Mullerian hormone (AMH), and inhibin B (INHB) were detected by ELISA; immunofluorescence staining was used to observe the expression of p62 in ovarian granulosa cells; the ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy, and the number of autophagosomes and autolysosomes was compared; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of p62, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) in ovarian tissue. The results of vaginal smears in the blank group showed regular cyclical changes; the rats in the model group showed prolonged estrous cycle or cycle arrest, mostly in proestrus or metestrus, with overall ovarian atrophy, disordered structure, and decreased granulosa cells. Compared with the blank group, rats in the model group showed increased serum FSH level (P<0.01), decreased serum levels of E2, AMH, and INHB (P<0.01), decreased positive expression of p62 in ovarian granulosa cells (P<0.01), with obvious swelling of ovarian granulosa cells, mild to moderate swelling of mitochondria, slight expansion of rough endoplasmic reticulum, and hypertrophy of Golgi apparatus; the number of autophagosomes and autolysosomes in the ovaries was increased (P<0.01), the expression of p62 protein and mRNA was decreased (P<0.01), and the expression of Beclin-1 and LC3 protein and mRNA in ovarian tissue was increased (P<0.01). Compared with the model group, rats in the EA group and the E2 group showed decreased serum FSH levels (P<0.01), increased levels of E2, AMH, and INHB (P<0.01), increased positive expression of p62 in ovarian granulosa cells (P<0.01), alleviated degree of ovarian granulosa cell damage, with relatively intact organelle morphology, and decreased number of autophagosomes and autolysosomes in the ovaries (P<0.01); the rats also showed increased expression of p62 protein and mRNA (P<0.01), and decreased expression of Beclin-1 and LC3 protein and mRNA (P<0.01) in ovarian tissue. EA at "Gongsun" (SP 4) could improve ovarian reserve function in POI rats by reducing the number of autophagosomes and autolysosomes, up-regulating p62 expression, and down-regulating Beclin-1 and LC3 expression, thus inhibiting autophagy of ovarian granulosa cells, and regulating the serum levels of FSH, E2, AMH, and INHB.

Effect of Hei Xiaoyaosan regulating p38MAPK/Beclin-1/Bcl-2 pathway on autophagy level in Alzheimer's disease model rats

To explore the effect of Hei Xiaoyaosan on autophagy levels in Alzheimer's disease(AD). A total of 100 4-month-old Wistar male rats were randomly selected as a blank group, and 10 rats were taken as a sham operation group and injected with 1 μL of normal saline on both sides of the hippocampus. The other rats were injected with Aβ_(1-42) solution in the hippocampus to replicate the AD model. Fifty successfully modeled rats were selected and randomly divided into the model group, Aricatio group(0.5 mg·kg~(-1)), and high, medium, and low dose groups of Hei Xiaoyaosan(15.30, 7.65, and 3.82 g·kg~(-1)), with 10 rats in each group. The rats were administered by continuous gavage for 42 days. Morris water maze was used to detect the learning and memory ability of rats, and Hoechst staining was used to observe the pathological changes of nerve cells in the hippocampal CA1 region. The mRNA expression of p38MAPK, Beclin-1, and Bcl-2 was detected by RT-qPCR.Western blot was used to detect the expressions of p38MAPK, Beclin-1, Bcl-2, APP, and related proteins. The level of Aβ_(1-42) in the hippocampus was detected by ELISA, and the expression level of LC3Ⅱ in the hippocampus was detected by immunohistochemistry. The experimental results showed that compared with the blank group, the learning and memory ability of rats in the model group decreased(P&lt;0.01). The nuclei in the CA1 region of the hippocampus showed blue bright spots and were closely arranged. The mRNA expression of p38MAPK was up-regulated, and the mRNA expressions of Beclin-1 and Bcl-2 were down-regulated(P&lt;0.01). The expressions of p38MAPK, p-p38MAPK, and APP were increased, while those of Beclin-1, Bcl-2, and p-Bcl-2 were decreased(P&lt;0.01). The expression of Aβ_(1-42) was increased(P&lt;0.01). The relative expression of LC3Ⅱ decreased(P&lt;0.01). Compared with the model group, the learning and memory ability of rats in each administration group was improved(P&lt;0.05 or P&lt;0.01). The nuclei in the CA1 region of the hippocampus gradually became clear, showing light blue. The mRNA expression of p38MAPK was down-regulated(P&lt;0.01), and that of Beclin-1 and Bcl-2 was increased(P&lt;0.05 or P&lt;0.01). The expressions of p38MAPK, p-p38MAPK, and APP were down-regulated, while those of Beclin-1, Bcl-2, and p-Bcl-2 were up-regulated(P&lt;0.05 or P&lt;0.01). The expression of Aβ_(1-42) was decreased(P&lt;0.01). The relative expression of LC3Ⅱ was increased(P&lt;0.01). It can be concluded that Hei Xiaoyaosan can improve the cognitive ability of AD model rats, and its potential mechanism may be related to regulating the p38MAPK/Beclin-1/Bcl-2 signaling pathway, increasing the level of autophagy, and reducing the accumulation of Aβ_(1-42).

Empagliflozin Alleviates Carfilzomib-Induced Cardiotoxicity in Mice by Modulating Oxidative Stress, Inflammatory Response, Endoplasmic Reticulum Stress, and Autophagy.

Carfilzomib is an irreversible proteasome inhibitor used for multiple myeloma patients. However, carfilzomib treatment is associated with cardiovascular complications. Empagliflozin, an Sodium Glucose Co-transporter 2 inhibitor (SGLT-2) inhibitor, is an oral antidiabetic drug with proven antioxidant and anti-inflammatory properties. The aim of the present study was to determine the cardioprotective effects of empagliflozin against carfilzomib-induced cardiotoxicity. C57BL/6 mice were randomly divided into four groups: control, empagliflozin, carfilzomib, and carfilzomib + empagliflozin. Empagliflozin prevented carfilzomib-induced cardiotoxicity by ameliorating histological alterations, CK-MB, and troponin-I. Moreover, it inhibited carfilzomib-induced oxidative damage and inflammation via its action on catalase activity, reduced glutathione levels and superoxide dismutase activity, and reduced nuclear factor-κB (p65) and cytokine levels. Mechanistically, empagliflozin abrogated endoplasmic reticulum stress induced by carfilzomib, as evidenced by the effect on the Glucose Regulated Protein-78 (GRP-78)/Activating Transcription Factor 6 (ATF6)/C/EBP homologous protein (CHOP) axis. Intriguingly, carfilzomib significantly induced autophagy, an effect that was further enhanced by empagliflozin, evidenced by increased LC3B and beclin-1 mRNA expression and reduced p62 expression. The effect of empagliflozin on apoptosis was confirmed by reduced expression of active caspase-3. Importantly, empagliflozin did not alter the cytotoxic effect of carfilzomib on human U266B1 multiple myeloma cells. our findings suggest that empagliflozin may provide a new therapeutic strategy to mitigate carfilzomib-induced cardiotoxicity in multiple myeloma patients.

BECN1 mRNA expression in breast cancer tissue; significant correlation to tumor grade

Breast cancer (BC) is a heterogenous disease with multiple pathways implicated in its development, progression, and drug resistance. Autophagy, a cellular process responsible for self-digestion of damaged organelles, had been recognized as eminent player in cancer progression and chemotherapeutic resistance. The haploinsufficiency of Beclin 1 (BECN1), autophagy protein, is believed to contribute to cancer pathogenesis and progression. In our study, we investigated the expression of BECN1 in a BC female Egyptian patient cohort, as well as its prognostic role through evaluating its association with disease free survival (DFS) after 2 years follow up and association of tumor clinicopathological features. Twenty frozen female BC tissue samples and 17 adjacent normal tissue were included and examined for the expression levels of BECN1. Although the tumor tissues showed lower expression 0.73 (0–8.95) than their corresponding normal tissues 1.02 (0.04–19.59), it was not statistically significant, p: 0.463. BECN1 expression was not associated with stage, nodal metastasis or tumor size, p:0.435, 0.541, 0.296, respectively. However, statistically significant negative correlation was found between grade and BECN1 mRNA expression in the studied cases, p:0.028. BECN1 expression had no statistically significant association with DFS, P = 0.944. However, we observed that triple negative (TNBC) cases had significantly lower DFS rate than luminal BC patients, p: 0.022, with mean DFS 19.0 months, while luminal BC patients had mean DFS of 23.41 months. Our study highlights the potential role of BECN1 in BC pathogenesis, showing that BECN1 expression correlates with poorer differentiation of BC, indicating its probable link with disease aggressiveness. DFS two years follow up showed that TNBC subtype remains associated with less favorable prognosis.

The potential anticancer effect of bee venom in combination with sorafenib against HepG2 cell lines via induction of apoptosis and autophagy candidate genes

Abstract Background Hepatocellular carcinoma (HCC) is a severe threat and a main reason for cancer-related deaths around the world. Drug resistance to sorafenib (Sorf), the effective HCC first-line therapy, is very common. A number of natural compounds, notably bee venom (BV), have been claimed to show a great impact against cancer when administered on its own or in conjunction with chemotherapy. Thus, this study aimed to investigate the anticancer effect of BV alone and/or combined with Sorf on HepG2 liver cancer cell lines. Methods Both mRNA and protein expressions of Bax, Bcl-2 and Beclin-1 were investigated by quantitative real-time PCR (qPCR) and western blot respectively, to examine the apoptotic and autophagic regulatory effects of BV and Sorf single treatments plus BV/Sorf combination on HepG2 cell lines. Results Our findings showed that BV and Sorf had considerable dose-dependent anti-proliferative effects on HepG2 cells whether administered alone or in combination, with the greatest impact for the combined therapies. Single BV and Sorf treatments showed IC50 of 93.21 and 7.28 μg/ml respectively, while combined treatment showed IC50 of 6.73 μg/ml BV + 6.73 μg/ml Sorf. Moreover, both the pro-apoptotic gene Bax and the autophagy-related gene Beclin-1 showed significant up-regulation in their mRNA expression, while the anti-apoptotic Bcl-2 mRNA gene expression showed significant down-regulation after BV/Sorf treatment as compared to either BV or Sorf single treatment. These qPCR results were further confirmed by western blot. Conclusions These findings indicate that BV synergistically potentiates the anticancer effect of Sorf on HepG2 cells through induction of apoptotic and autophagic machineries.

Comparative Analysis of Autophagy and Apoptosis in Disc Degeneration: Understanding the Dynamics of Temporary-Compression-Induced Early Autophagy and Sustained-Compression-Triggered Apoptosis.

The purpose of this study was to investigate the initiation of autophagy activation and apoptosis in nucleus pulposus cells under temporary compression (TC) and sustained compression (SC) to identify ideal research approaches in intervertebral disc degeneration. Various techniques were used: radiography (X-ray), magnetic resonance imaging (MRI), transmission electron microscope (TEM), H&E staining, Masson's trichrome staining, immunohistochemistry (IHC) (LC3, beclin-1, and cleaved caspase-3), and real-time polymerase chain reaction (RT-qPCR) for autophagy-related (beclin-1, LC3, and P62) and apoptosis-related (caspase-3 and PARP) gene expression analysis. X-ray and MRI revealed varying degrees of disc degeneration, ranging from moderate to severe in both groups. The severity was directly linked to compression duration, with SC resulting in notably severe central NP cell degeneration. Surprisingly, TC also caused similar, though less severe, degeneration. Elevated expression of LC3 and beclin-1 was identified after 6 weeks, but it notably declined after 12 weeks. Central NP cells in both groups exhibited increased expression of cleaved caspase-3 that was positively correlated with the duration of SC. TC showed fewer apoptotic markers compared to SC. LC3, beclin-1, and P62 mRNA expression peaked after 6 weeks and declined after 12 weeks in both groups. Cleaved caspase-3 and PARP expression peaked in SC, positively correlating with longer compression duration, while TC showed lower levels of apoptosis gene expression. Furthermore, TEM results revealed different events of the autophagic degradation process after 2 weeks of compression. TCmay be ideal for studying early triggered autophagy-mediated degeneration, while SC may be ideal for studying late or slower-triggered apoptosis-mediated degeneration.

Plasminogen deficiency exacerbates skeletal muscle loss during mechanical unloading in developing mice.

Mechanical-unloading-induced skeletal muscle atrophy results in physical frailty and disability. Elucidating its mechanism is required to establish effective countermeasures for this muscle adaptation. First, we analyzed the proteome profile in the gastrocnemius (Gast) and soleus muscles of space-flown mice raised under microgravity or artificial 1-g for 30 days, and found that the expression levels of fibrinolysis-related proteins were significantly elevated in the mechanical-unloaded muscles. Next, we investigated the roles of the fibrinolytic system in skeletal muscle atrophy induced by mechanical unloading on the ground. Eight-week-old male mice with plasminogen gene deficiency (Plg-/-) and their wild-type littermates were divided into control and hindlimb-suspended groups and were raised for 21 days. Plasminogen deficiency significantly enhanced the decrease in muscle mass at the lower limbs of mice following hindlimb unloading, and the Gast muscle atrophy was more prominent in Plg-/- mice. In addition, plasminogen deficiency significantly increased the expression of autophagy-related markers, beclin1 mRNA and LC3B protein, in the mechanical-unloaded Gast muscles, but did not affect the increase in the gene expression of ubiquitin ligases, atrogin-1 and MuRF1. Neither plasminogen deficiency nor hindlimb unloading affected the Akt/mechanistic target of rapamycin pathway in the Gast muscles. These results suggested that plasminogen deficiency might accelerate protein breakdown via the autophagy-lysosome, but not the ubiquitin-proteasome, system in the mechanical-unloaded Gast muscles. In conclusion, we first showed that plasminogen deficiency exacerbated the Gast muscle atrophy in hindlimb-unloaded mice. Plasminogen and the fibrinolysis system might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice.NEW & NOTEWORTHY The expression levels of fibrinolysis-related proteins, including plasminogen, were significantly elevated in the gastrocnemius (Gast) and soleus muscles of mice following 30-day microgravity exposure. Plasminogen deficiency exacerbated atrophy of the Gast, but not the soleus, muscles in mice following 21-day hindlimb suspension. It was also suggested that protein breakdown via the autophagy-lysosome system was accelerated in the Gast muscles. Plasminogen might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice.

Pro-oxidation status of rotator cuff tissue and expression of Beclin-1 and mTOR in patients with rotator cuff injury

To explore pro-oxidative state of rotator cuff tissue and expression levels of Beclin-1 and mam-malian target of rapamycin(mTOR) in patients with acute and chronic rotator cuff injury, and then analyzed relationship between rotator cuff injury and oxidative stress and autophagy. Forty patients with rotator cuff injury were seleceted from July 2019 to December 2020, and divided into male chronic injury group, male acute injury group, female chronic injury group, and female acute injury group, 10 patients in each group. All patients were performed rotator cuff repair under arthroscopy. The sample of tendon at the rotator cuff injury site of the patient was taken during operation, and total reactive oxygen species (ROS) and superoxide dismutase(SOD) were detected by detection kit;expression of Beclin-1 and mTOR mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR), and Western-blot was applied to detect protein expression of Beclin-1 and p-mTOR/mTOR. There were no significant difference in expression of ROS, SOD, Beclin-1mRNA and mTOR mRNA between male and female chronic injury groups, and between male and female acute injury groups (P>0.05); ROS, SOD and Beclin-1mRNA in male chronic injury group were higher than those in male chronic injury group, while mTOR mRNAand protein decreased (P<0.05);ROS, SOD and Beclin-1 mRNA in female chronic injury group were up-regulated compared with female acute injury group, while mTOR mRNA was down-regulated (P<0.05). Chronic rotator cuff injury is more likely to stimulate the pro-oxidation state of rotator cuff tissue than acute rotator cuff injury, which could up-regulating expression of autophagy factor Beclin-1 and down-regulating expression of mTOR. Therefore, patients with chronic rotator cuff injury may have higher levels of oxidative stress and autophagy.

The Sonic hedgehog pathway inhibitor GDC0449 induces autophagic death in human Medulloblastoma Daoy cells

ABSTRACT Medulloblastoma (MB) is a frequently occurring malignant brain tumor in children, and many of these tumors are identified by the abnormal activation of the Sonic Hedgehog (SHH) pathway. Although the Shh inhibitor GDC0449 initially shows some effectiveness in certain tumors, they eventually recur due to drug resistance mechanisms, highlighting the need for new treatment options. In this study, we explore whether GDC0449 induces autophagy in the human MB cell lines. To investigate the ultrastructural pathology changes of GDC0449-treated Daoy and D283 cells, we employed Transmission Electron Microscopy (TEM) technology to identify the expression of autophagic vacuoles. Our results indicate that GDC0449 only increases autophagy in Daoy cells by increasing the LC3-II/LC3-I ratio and autophagosome formation.We also analyzed Beclin1, LC3, Bax, and Cleaved-caspase3 protein and mRNA expression levels of autophagic and apoptotic markers using fluorescence confocal microscopy, RT-PCR, and Western blot. We found that cell autophagy and apoptosis increased in a dose-dependent manner with GDC0449 treatment. Additionally, we observed increased mammalian target of rapamycin (mTOR) phosphorylation and decreased protein kinase B (AKT/PKB), Ribosomal Protein S6, eIF4E-binding protein (4EBP1) phosphorylation in GDC0449-treated Daoy cells. It was observed that inhibiting autophagy using Beclin1 siRNA significantly blocked the apoptosis-inducing effects of GDC0449, suggesting that GDC0449 mediates its apoptotic effects by inducing autophagy.Our data suggests that GDC0449 inhibits the growth of human MB Daoy cells by autophagy-mediated apoptosis. The mechanism of GDC0449-induced autophagy in Daoy cells may be related to the inhibition of the PI3K/AKT/mTOR signaling pathway.

Therapeutic Potential of Hydrogen-Rich Water on Muscle Atrophy Caused by Immobilization in a Mouse Model.

Skeletal muscle atrophy is associated with poor quality of life and disability. Thus, finding a new strategy for the prevention and treatment of skeletal muscle atrophy is very crucial. This study aimed to investigate the therapeutic potential of hydrogen-rich water (HRW) on muscle atrophy in a unilateral hind limb immobilization model. Thirty-six male Balb/C mice were divided into control (without immobilization), atrophy, and atrophy + hydrogen-rich water (HRW). Unilateral hind limb immobilization was induced using a splint for 7 days (atrophy) and removed for 10 days (recovery). At the end of each phase, gastrocnemius and soleus muscle weight, limb grip strength, skeletal muscle histopathology, muscle fiber size, cross-section area (CSA), serum troponin I and skeletal muscle IL-6, TNF-α and Malondialdehyde (MDA), and mRNA expression of NF-κB, BAX and Beclin-1 were evaluated. Muscle weight and limb grip strength in the H2-treated group were significantly improved during the atrophy phase, and this improvement continued during the recovery period. Treatment by HRW increased CSA and muscle fiber size and reduced muscle fibrosis, serum troponin I, IL-6, TNF-α and MDA which was more prominent in the atrophy phase. These data suggest that HRW could improve muscle atrophy in an immobilized condition and could be considered a new strategy during rehabilitation.

Infection of epithelioma papulosum cyprini (EPC) cells with spring viremia of carp virus (SVCV) induces autophagy and apoptosis through endoplasmic reticulum stress

Spring viremia of carp virus (SVCV) is a lethal freshwater pathogen of cyprinid fish that has caused significant economic losses to aquaculture. To reduce the economic losses caused by SVCV, its pathogenic mechanism needs to be studied more thoroughly. Here, we report for the first time that SVCV infection of Epithelioma papulosum cyprini (EPC) cells can induce cellular autophagy and apoptosis through endoplasmic reticulum stress. The presence of autophagic vesicles in infected EPC cells was shown by transmission electron microscopy. Quantitative fluorescence PCR and Western blot results showed that p62 mRNA expression was decreased, and the expression of Beclin1 and LC3 mRNA was increased. The p62 protein was decreased, and the Beclin1 protein and LC3 were increased in the endoplasmic reticulum stress activation state. To further clarify the mode of death of SVCV-infected EPC cells, we examined caspase3, caspase9, BCL-2, and Bax mRNA, which showed that they were all increased. Apoptosis of SVCV-infected cells increased upon activation of endoplasmic reticulum stress. Our results suggest that endoplasmic reticulum stress can regulate SVCV infection-induced autophagy and apoptosis. The results of this study provide theoretical data for the pathogenesis of SVCV and lay the foundation for future drug development and vaccine construction.

Bloodletting Acupuncture at Jing-Well Points on Hand Induced Autophagy to Alleviate Brain Injury in Acute Altitude Hypoxic Rats by Activating PINK1/Parkin Pathway.

To explore the protective effect of bloodletting acupuncture at twelve Jing-well points on hand (BAJP) on acute hypobaric hypoxia (AHH)-induced brain injury in rats and its possible mechanisms. Seventy-five Sprague Dawley rats were divided into 5 groups by a random number table (n=15), including control, model, BAJP, BAJP+3-methyladenine (3-MA), and bloodletting acupuncture at non-acupoint (BANA, tail tip blooding) groups. After 7-day pre-treatment, AHH models were established using hypobaric oxygen chambers. The levels of S100B, glial fibrillary acidic protein (GFAP), superoxide dismutase (SOD), and malondialdehyde (MDA) in serum were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method were used to assess hippocampal histopathology and apoptosis. Transmission electron microscopy assay was used to observe mitochondrial damage and autophagosomes in hippocampal tissues. Flow cytometry was used to detect mitochondrial membrane potential (MMP). The mitochondrial respiratory chain complexes I, III and IV activities and ATPase in hippocampal tissue were evaluated, respectively. Western blot analysis was used to detect the protein expressions of Beclin1, autophagy protein 5 (ATG5), microtubule-associated protein 1 light chain 3 beta (LC3B), phosphatase and tensin homolog induced kinase 1 (PINK1), and Parkin in hippocampal tissues. The mRNA expressions of Beclin1, ATG5 and LC3-II were analyzed by quantitative real-time polymerase chain reaction. BAJP treatment reduced hippocampal tissue injury and inhibited hippocampal cell apoptosis in AHH rats. BAJP reduced oxidative stress by decreasing S100B, GFAP and MDA levels and increasing SOD level in the serum of AHH rats (P<0.05 or P<0.01). Then, BAJP increased MMP, the mitochondrial respiratory chain complexes I, III and IV activities, and the mitochondrial ATPase activity in AHH rats (all P<0.01). BAJP improved mitochondrial swelling and increased the autophagosome number in hippocampal tissue of AHH rats. Moreover, BAJP treatment increased the protein and mRNA expressions of Beclin1 and ATG5 and LC3-II/LC3-I ratio in AHH rats (all P<0.01) and activated the PINK1/Parkin pathway (P<0.01). Finally, 3-MA attenuated the therapeutic effect of BAJP on AHH rats (P<0.05 or P<0.01). BAJP was an effective treatment for AHH-induced brain injury, and the mechanism might be through reducing hippocampal tissue injury via increasing the PINK1/Parkin pathway and enhancement of mitochondrial autophagy.

Retraction Note to: Changes in Cathepsin D and Beclin-1 mRNA and protein expression by the excitotoxin quinolinic acid in human astrocytes and neurons.

Retraction Note to: Changes in Cathepsin D and Beclin-1 mRNA and protein expression by the excitotoxin quinolinic acid in human astrocytes and neurons.

Echinacoside ameliorates 5-fluorouracil-induced endothelial injury and senescence through SIRT1 activation

Echinacoside (ECH) is a natural bioactive component with antioxidant, anti-inflammatory, anti-apoptosis, and anti-tumor properties. In the current study, we explore the ECH-mediated protective effect and underlying mechanism of 5-fluorouracil (5-FU)-induced endothelial injury and senescence in the Human umbilical vein endothelial cells (HUVECs). In HUVECs, Cell viability, Apoptosis and Senescence assays evaluated 5-fluorouracil-induced endothelial injury and senescence. Protein expressions were assessed using RT-qPCR and Western blotting. Our results showed that 5-FU-induced endothelial injury and endothelial cell senescence could be improved when treated with ECH in HUVECs. ECH treatment potentially attenuated oxidative stress and ROS production in HUVECs. In addition, the effect of ECH on autophagy markedly reduced the percentage of HUVECs with LC3-II dots and suppressed the Beclin-1 and ATG7 mRNA expression but enhanced the p62 mRNA expression. Besides, ECH treatment significantly increased migrated cells and suppressed the adhesion of THP-1 monocytes in HUVECs. Furthermore, ECH treatment activated the SIRT1 pathway, and its related proteins (SIRT1, p-AMPK and eNOS) expression increased. Nicotinamide (NAM), an inhibitor of SIRT1, significantly attenuated the ECH-induced decrease in the apoptotic rate, increased SA-β-gal-positive cells and significantly reversed the ECH-induced reduction of endothelial senescence. Our results demonstrated that ECH employed endothelial injury and senescence in HUVECs via activation of the SIRT1 pathway.

Bushen Wenyang Huayu Decoction inhibits autophagy by regulating the SIRT1-FoXO-1 pathway in endometriosis rats.

Bushen Wenyang Huayu Decoction (BWHD) is a traditional Chinese medicine for tonifying kidney and warming Yang, thereby resolving blood stasis and relieving pain. BWHD can significantly improve the clinical symptoms of patients with endometriosis (EMs), but its mechanism is still unclear. We evaluated the expression and role of the SIRT1-FoxO-1 pathway and autophagy levels in EMs rats. The therapeutic effects and potential therapeutic mechanisms of BWHD were also investigated. Twenty rats were randomized into the sham group and eighty rats were used for model establishment by autologous transplantation. After successful modeling, they were randomized into the model, BWHD, EX527+BWHD and EX527 groups, with 20 rats in each group. All rats were intragastrically administered with for 3 weeks. Localization of Sirtuin 1 (SIRT1), Forkhead boxO-1 (FoXO-1), Beclin-1, autophagy-related 5 (Atg5) and autophagy-related 7 (Atg7) was determined by immunohistochemical staining. The expression of the above proteins was determined by Western blot and their messenger RNA (mRNA) levels were detected by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The protein and mRNA expressions of FoXO-1, Beclin-1, Atg5 and Atg7 in the model group were markedly increased, while that of SIRT1 was markedly decreased relative to the sham group (p<0.05 and p<0.01, respectively). Results showed that the protein and mRNA expressions of FoXO-1, Beclin-1, Atg5 and Atg7 in eutopic and ectopic endometrium of BWHD group were lower, while SIRT1 expression was higher than in the model group (p<0.05 and p<0.01, respectively). Furthermore, protein and mRNA expression levels of FoXO-1, Beclin-1, Atg5 and Atg7 in eutopic and ectopic endometrium of EX527 group were higher, while SIRT1 level was significantly lower than in the model group (p<0.05 and p<0.01, respectively). The EX527-induced changes in protein and mRNA expressions were reversed in the EX527+BWHD group (p<0.05 and p<0.01, respectively). BWHD inhibits autophagy by up-regulating SIRT1 and down-regulating FoXO-1 expression in EMs via the SIRT1-FoXO-1 signaling pathway. Therefore, it is a potential treatment for EMs.

Acteoside promotes autophagy and apoptosis of hepatoma cells by regulating JNK signaling pathway

This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P&lt;0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P&lt;0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P&lt;0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P&lt;0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P&lt;0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P&lt;0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P&lt;0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.

Acetylated Oligopeptide and N-acetyl cysteine Protected Against Oxidative Stress, Inflammation, Testicular-Blood Barrier Damage, and Testicular Cell Death in Iron-Overload Rat Model.

Multiple organs, including the testes, are damaged by iron overload. It has been shown that N-acetyl cysteine (NAC) influences oxidative stress in iron overload. The present study aimed to evaluate the roles of acetylated peptide (AOP) and NAC in the inhibition of iron-overload induced-testicular damage. At the beginning of the experiment, NAC (150 mg /kg) was given for a week to all 40 rats. Then, four groups were formed by dividing the animals (10 rats/group). Group I included healthy control rats. Group II (iron overload) was given intraperitoneal iron dextran (60 mg/kg/day) 5 days a week for 4 weeks. Group III (NAC) was given NAC orally at a dose of 150 mg/kg/day for 4 weeks in addition to iron dextran. Group IV (AOP) was given AOP orally at a dose of 150 mg/kg/day for 4 weeks besides iron dextran. When the experiment time was over, testosterone serum level, testicular B cell lymphoma-2 (BCL-2) and protein kinase B (PKB) protein levels, nuclear factor kappa-B (NF-κB), and Beclin1 mRNA expression levels, and malondialdehyde (MDA), and reduced glutathione (GSH) were determined by ELISA, quantitative reverse transcription-PCR, and chemical methods. Finally, histopathological examinations and immunohistochemical detection of claudin-1 and CD68 were performed. The iron overload group exhibited decreased testosterone, BCL-2, PKB, claudin-1, and GSH and increased MDA, NF-κB, Beclin1, and CD68, while both NAC and AOP treatments protected against the biochemical and histopathological disturbances occurring in the iron overload model. We concluded that NAC and AOP can protect against testes damage by iron overload via their antioxidant, anti-inflammatory, antiapoptotic, and ant-autophagic properties. The NAC and AOP may be used as preventative measures against iron overload-induced testicular damage.