Expression Levels Of LC3 Research Articles

Reduced expression of NUPR1 alleviates epilepsy progression via attenuating ER stress

Epilepsy is a neurological disorder characterized by recurring seizures. It is necessary to further understand the mechanisms of epilepsy in order to develop novel strategies for its prevention and treatment. Abnormal endoplasmic reticulum stress (ERS) activation is related to the pathogenesis of epilepsy. Nuclear protein 1, transcriptional regulator (NUPR1) is involved in ERS and it might play a role in epilepsy progression. In the present study, we generated an epileptic mouse model using pilocarpine induction. After 72 h of pilocarpine treatment, the expression of NUPR1 was increased in epileptic mice. Furthermore, NUPR1 knockdown reduced the number of spontaneous recurrent seizures and alleviated hippocampal damage in these mice. Interestingly, NUPR1 knockdown also reduced the protein expression levels of LC3, PINK1, and Parkin in the mitochondria, and decreased the PINK1 expression in hippocampus. Additionally, the expression of ERS-related proteins—cleaved caspase-12, ATF4, and CHOP—decreased in epileptic mice following NUPR1 knockdown. In vitro experiments showed that the absence of NUPR1 reduced the expression of ATF4, CHOP, and cleaved caspase-12 in hippocampal neurons and inhibited the neuron apoptosis. In all, our study suggested that NUPR1 maybe a potential molecular target for epilepsy therapy.

Prx1/PHB2 axis mediates mitophagy in oral leukoplakia cellular senescence

BackgroundOral leukoplakia (OLK) is the most common oral potentially malignant disorder (OPMD), which can be malignantly transformed into oral squamous cell carcinoma (OSCC). Peroxiredoxin1(Prx1) has been predicted to bind to Prohibitin2 (PHB2), which confers to affect OLK progression; however, the mechanism of Prx1/PHB2 mediated mitophagy involved in OLK remains unclear. MethodsThis study aimed to explore the mechanism of the Prx1/PHB2 axis on senescence in OLK through mediating mitophagy. The positive rate of Ki67 and the expression of p21, p16, PHB2, and LC3 in human normal, OLK, and OSCC tissues were detected by immunohistochemical staining. The mitophagy and mitochondrial function changes were then analyzed in Prx1 knockdown and Prx1C52S mutations in dysplastic oral keratinocyte (DOK) cells treated with H2O2. In situ Proximity Ligation Assay combined with co-immunoprecipitation was used to detect the interaction between Prx1 and PHB2. ResultsClinically, the positive rate of Ki67 progressively increased from normal to OLK, OLK with dysplasia, and OSCC. Higher p21, p16, PHB2, and LC3 expression levels were observed in OLK with dysplasia than in normal and OSCC tissues. In vitro, PHB2 and LC3II expression gradually increased with the degree of DOK cell senescence. Prx1/PHB2 regulated mitophagy and affected senescence in H2O2-induced DOK cells. Furthermore, Prx1C52S mutation specifically reduced interaction between Prx1 and PHB2. Prx1Cys52 is associated with mitochondrial reactive oxygen species (ROS) accumulated and cell cycle arrest. ConclusionPrx1Cys52 functions as a redox sensor that binds to PHB2 and regulates mitophagy in the senescence of OLK, suggesting its potential as a clinical target.

Muscle-derived Stem Cell Exosomes Enhance Autophagy through the Regulation of the mTOR Signaling Pathway to Attenuate Glucolipotoxicity-induced Pancreatic Β-cell Injury

Background: Diabetes mellitus (DM) refers to a series of metabolic disorders, including elevated blood glucose level diseases due to insufficient insulin secretion or insulin resistance. Objective: To investigate the effect and protective mechanism of muscle-derived stem cell exosomes (MDSC-Exo) on glucolipotoxicity-induced pancreatic β cell injury. Methods: Primary rat muscle-derived stem cells (MDSCs) were isolated and cultured. After the completion of the third-generation culture for MDSCs, MDSC-Exo was isolated. Then, the morphology and diameter of exosomes were observed by means of electron microscopy and nanoparticle tracking analysis (NTA) instrument. The expression of exosome-related proteins CD63, TSG101 and Calnexin was detected by western blot. After stimulation of rat insulinoma cell line INS- 1 with high glucose/palmitic acid (HG/PA) and/or MDSC-Exo, cell viability and apoptosis were measured through MTT and flow cytometry (FCT), respectively. Biochemical reagents were utilized for the examination of the levels of superoxide dismutase (SOD) and malondialdehyde (MDA); enzyme-linked immunosorbent assay (ELISA) for the levels of cellular insulin secretion, and the western blot for the expression level of LC3, p62, AKT, p-AKT, mTOR and p-mTOR. Results: MDSC-Exo was successfully isolated and identified, and it was found that MDSC-Exo could reduce HG/PA-induced apoptosis as well as MDA levels in INS-1 cells. Also, MDSC-Exo could significantly increase cell viability, insulin secretion ability within 24 hours and SOD level. Besides, MDSC-Exo was able to significantly increase the LC3-II/I ratio, decrease the expression level of p62, and promote autophagy in the cells. Aside from what has been mentioned, MDSC- Exo showed a significant reduction effect on p-Akt and p-mTOR level as well as p-Akt/Akt and p-mTOR/mTOR ratios. Conclusion: MDSC-Exo can alleviate oxidative stress and enhance autophagy by inhibiting Akt/ mTOR signaling pathway activation. Then, the inhibition of apoptosis and the promotion of insulin secretion can be achieved to alleviate glucolipotoxicity-induced pancreatic β cell injury.

Activation of the cGMP/PKG/ERK signaling pathway associated with PDE5Is inhibits fibroblast activation by downregulating autophagy in early progressive benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is one of the most prevalent diseases affecting aging males. However, approximately, 8% of the BPH patients under 50-year-old experience remarkably early progression, for reasons that remain elusive. Among the various factors implicated in promoting BPH advancement, the activation of fibroblasts and autophagy hold particular importance. Our research endeavors to explore the mechanisms behind the accelerated progression in these patients. Immunohistochemistry and immunofluorescence were performed to detect the expression levels of LC3, p62, PDE5, and α-SMA in diverse BPH tissues and prostate stromal cells. The autophagy activator rapamycin, the autophagy suppressor chloroquine, and siRNA transfection were used to identify the impact of autophagy on fibroblast activation. Prostatic stromal fibroblasts in early progressive BPH tissues displayed activation of autophagy with an upregulation of LC3 and a concurrent downregulation of p62. After starvation or rapamycin treatment to a heightened level of autophagy, fibroblasts exhibited activation. Conversely, chloroquine treatment and ATG-7-knockdown effectively suppressed the level of autophagy and fibroblast activation. High expression of PDE5 was found in early progressive BPH stromal cells. The administration of PDE5 inhibitors (PDE5Is) hindered fibroblast activation through suppressing autophagy by inhibiting the ERK signaling pathway. Our findings suggest that autophagy plays a pivotal role in promoting BPH progression through fibroblast activation, while PDE5Is effectively suppress autophagy and fibroblast activation via the ERK signaling pathway. Nevertheless, further investigations are warranted to comprehensively elucidate the role of autophagy in BPH progression.

Pseudolaric acid B suppresses NSCLC progression through the ROS/AMPK/mTOR/autophagy signalling pathway

Pseudolaric acid B (PAB), an acid isolated from the roots of Pseudolarix kaempferi gorden, has shown antitumour effects through multiple mechanisms of action. The objective of this study was to investigate the anticancer effect of PAB on non-small cell lung cancer (NSCLC) and its underlying mechanism. In our experiments, we observed that PAB decreased cell viability, inhibited colony formation, induced cell cycle arrest, impeded scratch healing, and increased apoptosis in H1975 and H1650 cells. Additionally, PAB treatment enhanced the fluorescence intensity of MDC staining in NSCLC cells, upregulated the protein expression of microtubule-associated protein light chain 3 II (LC3 II), and downregulated the expression of sequestosome 1 (SQSTM1/P62). Combined treatment with PAB and chloroquine (CQ) increased the protein expression levels of LC3 II and P62 while decreasing the apoptosis of H1975 and H1650 cells. Moreover, treatment with PAB led to significant mTOR inhibition and AMPK activation. PAB combined with compound C (CC) inhibited autophagy and apoptosis. Furthermore, PAB treatment increased intracellular reactive oxygen species (ROS) levels in NSCLC cells, which correlated with the modulation of the AMPK/mTOR signalling pathway and was associated with autophagy and apoptosis. Finally, we validated the antitumour growth activity and mechanism of PAB in vivo using athymic nude mice bearing H1975 tumour cells. In conclusion, our findings suggest that PAB can induce apoptosis and autophagic cell death in NSCLC through the ROS-triggered AMPK/mTOR signalling pathway, making it a promising candidate for future NSCLC treatment.

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.

Evaluation of angiogenesis and microvascular remodeling in the subventricular zone of the brain of mice with experimental Alzheimer’s disease

BACKGROUND: Under the influence of external factors (learning), processes of microvasculature remodeling occur in the neurogenic niche to meet the metabolic needs of activated cells. However, it remains unclear how these mechanisms of brain plasticity are disrupted during neurodegeneration.
 AIM: To study the expression features of markers of angiogenesis and microvascular remodeling in the subventricular zone of the brain during training of animals, including against the background of the development of Alzheimer’s-type neurodegeneration in them.
 MATERIAL AND METHODS: The studies were carried out on C57BL/6 mice at the age of 8 months. Modeling of Alzheimer’s disease was carried out by intrahippocampal injection of 2 μl of a 1 mM solution of β-amyloid Aβ25-35. To assess cognitive deficits, a conditioned passive avoidance test using an aversive stimulus was used. The expression of LC3B, ZO1, VEGFR2, VEGFR3, CD146, ICAM2, Dll4, Tie2 in the subventricular zone per 100 DAPI-positive cells was assessed. The test results were processed using one-way ANOVA and Fisher's test, the Mann–Whitney U test, the results were considered significant at p 0.05.
 RESULTS: On the 9th day after the administration of β-amyloid, before the application of the aversive stimulus, an increase in the expression level of LC3 (7.95±5.83%, p=0.045), CD146 (18.35±0.01%, p=0.045) was recorded, as well as VEGFR3 (17.13±5.05%, p=0.045), which continued to increase after the presentation of the stimulus (26.61±0.01%, p=0.045). By the beginning of registration of cognitive impairment (38th day of the experiment), the expression level of VEGFR2 (20.61±2.8%, p=0.045) and ICAM2 (126.61±41.28%, p=0.045) increased, the content of Dll4 (29.66±8.72%, p=0.045) and Tie2 (36.39±7.8%, p=0.045) decreased in animals with experimental Alzheimer’s disease.
 CONCLUSION: An aversive stimulus stimulates microvascular remodeling mechanisms in the subventricular zone of the animal’s brain, but when exposed to β-amyloid, these processes are significantly disrupted.

Ellagic acid inhibits gastric cancer cells by modulating oxidative stress and inducing apoptosis

Objective: To evaluate the anticancer effect of ellagic acid on gastric cancer cells. Methods: MTT assay was used to evaluate the effect of ellagic acid at different concentrations (0.5-100 μg/mL) on gastric cancer AGS cells. RT-qPCR and Western blot analyses were applied to assess apoptosis (BCL-2, CASP-3, and BAX) and autophagy (LC3, ATG5, and BECN1) in AGS cells treated with ellagic acid. The expression of invasion-related markers including TP53, CDKN2A, and PTEN was determined. In addition, cell cycle markers including cyclin A, B, D, and E were measured by ELISA. Oxidative stress markers were evaluated using spectrophotometry. Results: Ellagic acid inhibited the proliferation of AGS cells in a concentration-and time-dependent manner. The expression of BCL-2 was significantly decreased (P<0.05) and CASP-3 and BAX were markedly increased (P<0.01) in AGS cells treated with ellagic acid. However, this compound induced no significant changes in the expression levels of LC3, ATG5, and BECN1 (P>0.05). Moreover, the oxidative stress markers including SOD, TAC, and MDA were increased by ellagic acid (P<0.01). Conclusions: Ellagic acid can inhibit cell proliferation, induce apoptosis, and modulate oxidative stress in AGS cells. However, further in vivo and molecular studies are needed to verify its anticancer efficacy.

Resveratrol and quercetin protect from Benzo(a)pyrene-induced autophagy in retinal pigment epithelial cells.

This study aims to investigate the role of Resveratrol (RES) and quercetin (QR) treatments against Benzo(a)pyrene (B(a)p)-induced autophagy in retinal pigment epithelial cells. The IC50 doses of B(a)p, RES and QR in retinal pigment epithelial cells were determined by MTT assay and the relevant agents were administered singly or in combinations to ARPE-19 cells for 24h. Occurrence of autophagy in the cells was verified by detection of autophagosomes using fluorescence microscope. Also, the mRNA expression levels of LC3 and Beclin 1 genes were analyzed by RT-PCR to collect further data on autophagy. Caspase-3 and IL-1β levels in lysed cells were analyzed by ELISA. Autophagosomes were detected in B(a)p-treated ARPE-19 cell lines, as well as a 1.787-fold increase in LC3 mRNA expression levels. No autophagosome occurred in RES and QR treatments, and a significant decrease in their percentage amounts were observed in B(a)p + RES and B(a)p + QR. The mRNA expression levels of LC3 and Beclin 1 also supported these findings. B(a)p had no effect on Caspase-3 levels in ARPE-19 cells, but combined with RES and QR, it increased Caspase-3 levels significantly.IL-1β levels were higher in B(a)p, B(a)p + QR, B(a)p + RES, RES and QR than control group. This rise in IL-1β levels was correlated with suppression of mRNA expression levels of Beclin 1. B(a)p exposure caused autophagy in ARPE-19 cells, but did not induce apoptosis. RES and QR treatments prevented B(a)p-induced autophagy. Therefore, RES and QR treatments showed protective effect against potential degenerative diseases caused by chronic exposure to B(a)p.

Identifying tumor cell-released extracellular vesicles as biomarkers for breast cancer diagnosis by a three-dimensional hydrogel-based electrochemical immunosensor

Tumor cell-released LC3+ extracellular vesicles (LC3+ EVs) participate in immunosuppression during autophagy and contribute to the occurrence and development of breast cancer. In view of the strong association between the LC3+ EVs and breast cancer, developing an effective strategy for the quantitative detection of LC3+ EVs levels with high sensitivity to identify LC3+ EVs as new biomarkers for accurate diagnosis of breast cancer is crucial, but yet not been reported. Herein, an ultrasensitive electrochemical immunosensor is presented for the quantitative determination of LC3+ EVs using a three-dimensional graphene oxide hydrogel-methylene blue composite as a redox probe, showing a low detection limit and a wide linear range. With this immunosensor, the expression levels of LC3+ EVs in various practical sample groups including different cancer cell lines, the peripheral blood of tumor-bearing mice before and after immunotherapy, and the peripheral blood from breast cancer patients with different subtypes and stages were clearly distinguished. This study demonstrated that LC3+ EVs were superior as biomarkers for the accurate diagnosis of breast cancer compared to traditional biomarkers, particularly for cancer subtype discrimination. This work would provide a new noninvasive detection tool for the early diagnosis and prognosis assessment of breast cancer in clinics.

Activation of autophagy after blast-induced traumatic brain injury in mice.

Injury mechanism and treatment of blast-induced traumatic brain injury (bTBI) has not made a breakthrough so far. Previous reports demonstrate autophagy is involved in regulating the pathophysiological process after TBI. Therefore, this study explored whether autophagy was activated after bTBI. A total of 108 mice were divided randomly into six groups: 6 h, 1 d, 3 d, 7 d, 14 d after bTBI groups and sham group. The protein levels of anti-microtubule associated protein 1 light chain 3B (LC3B, hereafter referred to as LC3), beclin1 and p62 were detected using western blot. Moreover, HO-1 and Nrf2 were localized using histologic staining. Immunofluorescence of LC3 and immunohistochemistry of beclin1 were performed. The autophagy-related ultrastructure was observed by TEM. LC3-II and beclin1 reached their peak on day 3 after bTBI, while p62 showed a continuous downward trend. Immunofluorescence and immunohistochemistry also confirmed that the expression levels of LC3 and beclin1 were the highest at 3 days after bTBI. Autophagic vesicles containing lysosomes or digestive residual structures were observed then. Autophagy was induced in the frontal lobe tissues of bTBI mice induced by moderate-intensity explosion, with a peak at 3d and a gradual decline thereafter.

Xanol Promotes Apoptosis and Autophagy and Inhibits Necroptosis and Metastasis via the Inhibition of AKT Signaling in Human Oral Squamous Cell Carcinoma.

Angelica keiskei Koidzumi (A. keiskei) is used as a traditional medicine, anti-aging agent, and health food, as well as to restore vitality. Xanthoangelol (xanol), a prenylated chalcone, is the predominant constituent of A. keiskei. Oral squamous cell carcinoma (OSCC), the most common malignancy, has a high proliferation rate and frequent metastasis. However, it is unknown whether xanol has anti-OSCC effects on apoptosis, autophagy, and necroptosis. In the present study, we purified xanol from A. keiskei and demonstrated that it suppressed cell proliferation and induced cytotoxicity in human OSCC. Xanol triggered apoptotic cell death by regulating apoptotic machinery molecules but inhibited necroptotic cell death by dephosphorylating the necroptotic machinery molecules RIP1, RIP3, and MLKL in human OSCC. We also found that xanol inhibited the PI3K/AKT/mTOR/p70S6K pathway and induced autophagosome formation by enhancing beclin-1 and LC3 expression levels and reducing p62 expression levels. Furthermore, we showed that xanol prevented the metastatic phenotypes of human OSCC by inhibiting migration and invasion via the reduction of MMP13 and VEGF. Finally, we demonstrated that xanol exerted anticancer effects on tumorigenicity associated with its transformed properties. Taken together, these findings demonstrate the anticancer effects and biological mechanism of action of xanol as an effective phytomedicine for human OSCC.

Bilirubin, an endogenous antioxidant that affects p53 protein and its downstream apoptosis/autophagy-related genes in LS180 and SW480 cell culture models of colorectal cancer

BackgroundColorectal cancer (CRC) is one of the commonest neoplasms worldwide, which its pathogenesis is strongly correlated with p53 mutations. Antioxidants are believed to decelerate the CRC progression, possibly through interfering with p53 and its downstream target genes and mechanisms. Regarding the potential antioxidant effects of bilirubin, as an incredible endogenous antioxidant, we sought to investigate how bilirubin affected the expression levels of p53 protein and its downstream target genes, including Mdm2, Bcl-2, BECN1 and LC3, in LS180 and SW480 cell culture models of CRC. Methods and resultsUsing the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide) assay, 50 and 100 μM concentrations of bilirubin were determined to be non-toxic for both LS180 and SW480 cell lines. Western blot analysis was employed to evaluate the protein expression levels of p53. The results revealed that p53 protein levels were higher in LS180 cells treated with bilirubin compared to the control group. Notwithstanding, in SW480 cells, no considerable changes were observed in p53 protein levels of treated cells compared to the control ones. The quantitative reverse transcriptase-polymerase chain reaction (q RT-PCR) method was used to measure the mRNA expression levels of the apoptosis/autophagy-related genes, Mdm2, Bcl-2, BECN1, and LC3 , as the p53's downstream target genes. Consequently, the expression of Bcl-2 and Mdm2 genes were affected by p53, while BECN1 and LC3 expression levels were decreased in both cell lines. ConclusionBilirubin is an endogenous antioxidant with significant anti-tumor effects in the studied CRC cell lines, probably through the regulation of p53 protein expression levels and subsequent control of apoptosis and autophagy, as two key processes involved in cell survival and progression of tumor cells.

Propofol reduces lipopolysaccharide‑induced cardiomyocyte injury in sepsis by activating SIRT1‑mediated autophagy.

Myocardial injury is an indicator of poor prognosis in sepsis, whereas propofol has been reported to protect the myocardium. Therefore, the present study investigated the effect of propofol on myocardial injury in sepsis and its mechanism. An in vitro model of myocardial cell injury was established in myocardial H9C2 cells using lipopolysaccharide (LPS). The Cell Counting Kit 8 (CCK8) assay was used to investigate the effect of propofol pretreatment on the viability of normal and LPS-challenged H9C2 cells, whereas the lactate dehydrogenase (LDH) detection kit was used to measure the levels of LDH. The expression levels of LC3 were analyzed using an immunofluorescence assay. Western blotting was performed to analyze the expression levels of autophagy-related proteins. Following treatment with the autophagy inhibitor 3-methyladenine, CCK8 assay, TUNEL assay, western blotting, 2,7-dichlorohydrofluorescein diacetate assay and ELISA were performed to investigate whether propofol exerted its effects on cell viability, apoptosis, oxidative stress and inflammation via autophagy. Moreover, to further explore the regulatory mechanism of propofol in myocardial injury, sirtuin 1 (SIRT1) was knocked down via transfection with small interfering RNA, and SIRT1 protein was inhibited via the addition of the SIRT1 inhibitor EX527. The present study demonstrated that propofol activated autophagy in LPS-induced cardiomyocytes, and reversed the effects of LPS on viability, apoptosis, oxidative stress and the inflammatory response. Moreover, SIRT1 knockdown and inhibition decreased the activation of autophagy and the protective effect of propofol on LPS-induced cardiomyocytes. In conclusion, propofol reduced LPS-induced cardiomyocyte injury by activating SIRT1-mediated autophagy.

Autophagy Inhibits Inflammation via Down-Regulation of p38 MAPK/mTOR Signaling Cascade in Endothelial Cells.

Autophagy, an intracellular process of self-digestion, has been shown to modulate inflammatory responses. In the present study, we determined the effects of autophagy on inflammatory response induced by M5 cytokines. Human umbilical vein endothelial cells (HUVECs) were treated with M5 cytokines to induce inflammation. Expression levels of mRNA for inflammatory cytokines and BIRC2 were compared in HUVECs with vs without induction of autophagy with rapamycin (RAPA) by PCR, while cell apoptosis was assessed by flow cytometry and caspase-3 activity assay kit. Expression levels of LC3, p62, p-p38 MAPK (Thr180/Tyr182), p-mTOR (Ser2445) and p-ULK1 (Ser555) proteins were measured by Western blotting. The nitric oxide (NO) content, NO synthase (NOS) activity and cell angiogenesis were also evaluated. Induction of autophagy with RAPA decreased expression levels of IL6, IL8 and CCL20, in addition to reduction in inflammation-induced apoptosis in HUVECs. Moreover, RAPA increased LC3II, while decreasing p62 expression. Likewise, expression levels of p-p38 MAPK and p-mTOR proteins were markedly decreased by the treatment with RAPA. Finally, RAPA treatment increased the NO content and the NOS activity, and inhibited angiogenesis. Induced autophagy can improve the function of endothelial cells in psoriasis, suggesting approaches to induce autophagy can be used to ameliorate psoriasis.

1α,25(OH)2D3 Promotes the Autophagy of Porcine Ovarian Granulosa Cells as a Protective Mechanism against ROS through the BNIP3/PINK1 Pathway

Vitamin D (VD) is one of the important nutrients required by livestock; however, VD deficiency is reported to be widespread. Earlier studies have suggested a potential role for VD in reproduction. Studies on the correlation between VD and sow reproduction are limited. The aim of the current study was aimed to determine the role of 1,25-dihydroxy vitamin D3 (1α,25(OH)2D3) on porcine ovarian granulosa cells (PGCs) in vitro to provide a theoretical basis for improving the reproductive efficiency of sows. We used chloroquine (autophagy inhibitor) and reactive oxygen species (ROS) scavenger N-acetylcysteine in conjunction with 1α,25(OH)2D3 to explore the effect on PGCs. The results showed that 10 nM of 1α,25(OH)2D3 increased PGC viability and ROS content. In addition, 1α,25(OH)2D3 induces PGC autophagy according to the gene transcription and protein expression levels of LC3, ATG7, BECN1, and SQSTM1 and promotes the generation of autophagosomes. 1α,25(OH)2D3-induced autophagy affects the synthesis of E2 and P4 in PGCs. We investigated the relationship between ROS and autophagy, and the results showed that 1α,25(OH)2D3-induced ROS promoted PGC autophagy. The ROS-BNIP3-PINK1 pathway was involved in PGC autophagy induced by 1α,25(OH)2D3. In conclusion, this study suggests that 1α,25(OH)2D3 promotes PGC autophagy as a protective mechanism against ROS via the BNIP3/PINK1 pathway.

Type III secretory protein SINC of Chlamydia psittaci promotes host cell autophagy by activating the MAPK/ERK signaling pathway

To investigate the effects of SINC, a secreted protein of Chlamydia psittaci, on autophagy of host cells and the role of MAPK/ERK signaling pathway in mediating SINC-induced autophagy. RAW 264.7 cells treated with recombinant SINC were examined for changes in expression levels of LC3-II, Beclin-1, phosphorylated and total ERK1/2 using Western blotting. The expression level of LC3 in the treated cells was detected using immunofluorescence analysis, and the formation of autophagosomes and autolysosomes was observed with transmission electron microscopy (TEM). The effect of pretreatment with U0126 (a specific ERK inhibitor) on the expression levels of LC3-II and Beclin-1 in RAW 264.7 cells exposed to different concentrations of SINC was examined using Western blotting, and LC3 puncta in the cells was detected with immunofluorescence analysis. The expression levels of LC3-II and Beclin-1 were the highest in RAW 264.7 cells treated with 2 μg/mL SINC for 12h. Immunofluorescence analysis showed exposure to SINC significantly increased the number of cells containing LC3 puncta, where the presence of autophagosomes and autolysosomes was detected. Exposure to 2 μg/mL SINC for 15 min resulted in the most significant increase of the ratios of p-ERK1/2/ERK1/2 in RAW 264.7 cells. Pretreatment of the cells with U0126 prior to SINC exposure significantly decreased the ratio of p-ERK1/2/ERK1/2, lowered the expression levels of LC3-II and Beclin-1, and decreased LC3 aggregation in the cells. SINC exposure can induce autophagy in RAW 264.7 cells by activating the MAPK/ERK signaling pathway.

The potential importance of autophagy genes expression profile dysregulation and ATG polymorphisms in COVID-19 pathogenesis.

Autophagy is one of the important mechanisms in cell maintenance, which is considered associated with different pathological conditions such as viral infections. In this current study, the expression level and polymorphisms in some of the most important genes in the autophagy flux in COVID-19 patients were evaluated. This cross-sectional study was conducted among 50 confirmed COVID-19 patients and 20 healthy controls. The COVID-19 patients were divided into a severe group and a mild group according to their clinical features. The expression levels of ATG5, ATG16L1, LC3, and BECN1 were evaluated by the 2-∆∆CT method and beta-actin as the internal control. The polymorphisms of the ATG5 (rs506027, rs510432) and ATG16L1 (rs2241880 or T300A) were evaluated by the Sanger sequencing following the conventional PCR. The mean age of the included patients was 58.3 ± 17.9 and 22 (44%) were female. The expression levels of the LC3 were downregulated, while BECN1 and ATG16L1 genes represent an upregulation in COVID-19 patients. The polymorphism analysis revealed the ATG16L1 rs2241880 and AGT5 rs506027 polymorphism frequencies are statistically significantly different between COVID-19 and Healthy controls. The autophagy alteration represents an association with COVID-19 pathogenesis and severity. The current study is consistent with the alteration of autophagy elements in COVID-19 patients by mRNA expression-level evaluation. Furthermore, ATG16L1 rs2241880 and AGT5 rs506027 polymorphisms seem to be important in COVID-19 and are highly suggested for further investigations.

Betulinaldehyde exhibits effective anti-tumor effects in A549 cells by regulating intracellular autophagy

It is of great significance to find new effective drugs for an adjuvant therapy targeting lung cancer to improve the survival rate and prognosis of patients with the disease. Previous studies have confirmed that certain Chinese herbal extracts have clear anti-tumor effects, and in our preliminary study, betulinaldehyde was screened for its potential anti-tumor effects. The current study thus aimed to confirm the anti-tumor effect of betulinaldehyde, using in vitro experiments to explore its underlying molecular mechanism. It was found that betulinaldehyde treatment significantly inhibited the viability, proliferation, and migration of A549 cells in a dose-dependent manner. In addition, betulinaldehyde inhibited the activation of Akt, MAPK, and STAT3 signaling pathways in A549 cells in a time-dependent manner. More importantly, betulinaldehyde also decreased the expression level of SQSTM1 protein, increased the expression level of LC3 II, and increased the autophagy flux in A549 cells. The pretreatment of A549 cells with the autophagy inhibitor, 3-methyladenine, could partially negate the anti-tumor effects of betulinaldehyde. These findings suggest that betulinaldehyde could significantly inhibit the oncological activity of A549 cells by regulating the intracellular autophagy level, making it a potentially effective option for the adjuvant therapy used to treat lung cancer in the future.

Hexokinase inhibitor 2-deoxyglucose coordinates citrullination of vimentin and apoptosis of fibroblast-like synoviocytes by inhibiting HK2 /mTORC1-induced autophagy.

High hexokinase 2 (HK2) expression is associated with aberrant activation of fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA). However, the mechanism by which this occurs has not been fully elucidated. To investigate the role of HK2 and its underlying mechanism, adjuvant arthritis (AA) rats were treated with the HK2 inhibitor, 2-deoxyglucose (2-DG). In conjunction with HK2 knockdown experiments in FLSs, we evaluated the effect of HK2 on the citrullination of vimentin (cVIM), autophagy and apoptosis-associated protein expression, including that of cVIM, LC3, p62, Beclin1, Bax, Bcl2, and caspase 3. We further investigated the interaction of HK2 with downstream mTORC1 signaling effectors. Correlation analysis revealed that 2-DG treatment and HK2 knockdown upregulated the expression levels of caspase3, Bax, and p62 and downregulated the expression levels of LC3, Bcl2, and Beclin1, as well as decreasing vimentin citrullination. Furthermore, interactions between HK2 and mTOR decreased, coinciding with mTORC1 pathway activation. These findings suggest that the regulation of apoptosis and cVIM by HK2/mTORC1-dependent autophagy involves the inhibition of aberrant FLSs activation in the rat model of arthritis.