LC3-II Protein Expression Research Articles

Exploring molecular mechanisms of fucoidan in improving human proximal renal tubular epithelial cells aging by targeting autophagy signaling pathways

Fucoidan(FPS) is an effective component of the Chinese patent medicine named Haikun Shenxi, which treats schronic renal failure in clinics, and has the potential anti-aging effects. However, it is still unclear whether FPS can improve renal aging, especially the molecular mechanism of its anti-aging. The human proximal renal tubular epithelial cells(HK-2) in vitro were divided into normal group(N), D-gal model group(D), low dose of FPS group(L-FPS), high dose of FPS group(H-FPS) and vitamin E group(VE), and treated by the different measures, respectively. More specifically, the HK-2 cells in each group were separately treated by 1 mL of 1% fetal bovine serum(FBS) or D-galactose(D-gal, 75 mmol·L~(-1)) or D-gal(75 mmol·L~(-1))+FPS(25 μg·mL~(-1)) or D-gal(75 mmol·L~(-1))+FPS(50 μg·mL~(-1)) or D-gal(75 mmol·L~(-1))+VE(50 μg·mL~(-1)). After the treatment for 24 h, firstly, the effects of D-gal on senescence-associated β-galactosidase(SA-β-gal) staining characteristics and klotho, P53 and P21 protein expression le-vels, as well as adenosine monophosphate activated protein kinase(AMPK)-uncoordinated 51-like kinase 1(ULK1) signaling pathway activation in the HK-2 cells were detected, respectively. Secondly, the effects of FPS and VE on SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels in the HK-2 cells exposed to D-gal were investigated, respectively. Finally, the effects of FPS and VE on microtubule-associated protein 1 light chain 3(LC3) protein expression level and AMPK-ULK1 signaling pathway activation in the HK-2 cells exposed to D-gal were examined severally. The results indicated that, for the HK-2 cells, the dose of 75 mmol·L~(-1) D-gal could induce the changes of SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels. That is causing cells aging. FPS and VE could both ameliorate the changes of SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels in the HK-2 cells exposed to D-gal. That is anti-cells aging, here, the functions of FPS and VE are similar. D-gal could not only induce cell aging but also increase LC3Ⅱ, phosphorylated-AMPK(p-AMPK) and phosphorylated-ULK1(p-ULK1) protein expressions, and activate autophagy-related AMPK-ULK1 signaling pathway. FPS and VE could both improve the changes of LC3Ⅱ, p-AMPK and p-ULK1 protein expression levels in the HK-2 cells exposed to D-gal. That is inhibiting autophagy-related AMPK-ULK1 signaling pathway activation. On the whole, for the human proximal renal tubular epithelial cells aging models induced by D-gal, FPS similar to VE, can ameliorate renal cells aging by possibly inhibiting autophagy-related AMPK-ULK1 signaling pathway activation. This finding provides the preliminary pharmacologic evidences for FPS protecting against renal aging.

Effect of moxibustion on expression of autophagy related molecules in synovial tissues of rheumatoid arthritis rats

To observe the effect of moxibustion on expression of autophagy related gene(Atg), serine/threonine protein kinase-uncoordinated 51 like kinase-1 (ULK1), Beclin1 and microtubule associated proteins light chain 3 (LC3) and ultrastructure of synovium in rheumatoid arthritis (RA) rats, so as to explore its mechanisms underlying improvement of RA. Forty SD rats were randomly divided into normal control, RA model, moxibustion, cigarette-roasting and medication groups (n=8 rats in each group). The RA model was established by keeping the rats in wind, cold and wet environment for 12 h, once a day for 20 days and subcutaneous injection of Freund's adjuvant complete into the sole of the left hind paw. Moxibustion was applied to the left "Zusanli" (ST36) for 20 min, once a day for 15 days. Rats of the cigarette-roasting group was treated by ignited cigarettes instead of moxa strips. Rats of the medication group was treated by gavage of Tripterygium wilfordii polyglycoside tablet suspension (0.8 mg/100 g) once a day for 15 days. The rats' paw volume of the left hindlimb was measured by using a water-based paw plethysmometer. The synovial tissue of the left plantar joint was harvested at the end of experiments for observing changes of the ultrastructure with transmission electron microscope, and the expression of ULK1, Atg3, Atg5, and Atg12 mRNAs was detected with quantitative real-time PCR and the expression of LC3-Ⅱ and Beclin-1 proteins were detected with Western blot. Following modeling, the paw volume of the left hindlimb was significantly increased (P<0.01), while the expression levels of Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins of the synovial tissue were notably down-regulated in the model group relevant to the normal control group (P<0.01). The increase of the paw volume in the moxibustion and medication groups and the down-regulation of synovial Atg3, Atg12 and ULK1 mRNAs in the 3 intervention groups, and Atg5 mRNA , and LC3-Ⅱ and Beclin-1 proteins in both moxibustion and medication groups were considerably suppressed (P<0.01, P<0.05). The therapeutic effect of moxibustion was apparently superior to that of cigarette-roasting in down-regulating the paw volume, and up-regulating the expression levels of Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins (P<0.05, P<0.01), and notably weaker than that of medication in up-regulating Atg3 and ULK1 mRNAs (P<0.01), but was comparable to that of medication in up-regulating the expression levels of Atg5 and Atg12 mRNAs, LC3-Ⅱand Beclin-1 proteins (P＞0.05). Results of the ultrastructural observation showed an obvious injury of synovial cells, such as unclear and incomplete nuclear membrane, chromatin condensation, swollen mitochondria with broken crests, cavitation-like degeneration of cytoplasma, and appearance of autophagosomes and lysosomes in the model group, which was relatively milder in the 3 intervention groups. Moxibustion can reduce the paw edema and inflammatory injury of the plantar synovial tissue in RA rats, which may be related to its effects in up-regulating Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins to enhance the cellular autophagy. The therapeutic effect of moxibustion is obviously superior to that of cigarette-roasting and medication in relieving swelling.

Effect of Combined Sorafenib/Cisplatinum Treatment on the Autophagy and Proliferation of Hepatocellular Carcinoma hepG2 Cells in Vitro.

Objective:To explore the effect of combined Sorafenib/ cisplatinum treatment on the autophagy and proliferation of hepatocellular carcinoma (HepG2) cells in vitro. Methods:HepG2 cells were cultured and treated with different concentrations of Sorafenib, cisplatinum, or a combination of both over a 24-hour period. Cell proliferation was evaluated using a CCK8 assay, and the mRNA expression of the autophagy-related proteins AKT, mTOR, and LC3 were detected using quantitative PCR (qPCR). AKT, pAKT (Ser473), mTOR, pmTOR (Ser2448), LC3I, and LC3II protein expression levels were evaluated by western blot. Results:We found that the survival rate of HepG2 cells was 47.42% when treated with Sorafenib (10 μmol/L) monotherapy, and 46.04% when treated with cisplatinum (10 mg/L) monotherapy. When Sorafenib(10 μmol/L) was combined with cisplatinum (10 mg/L), the cellular proliferation and survival rate was only 16.71% ( P <0.05). qPCR and western blot revealed that a combination of Sorafenib (10 μmol/L) and cisplatinum (10 mg/L) reduced the transcription and protein expression of autophagy-related AKT and mTOR but increased that of LC3 (P <0.05). Conclusion: Combining Sorafenib and cisplatinum can effectively induce cell autophagy and reduce cellular proliferation via the PI3K/AKT/mTOR signal pathway.

Impairment of the autophagy-lysosomal pathway and activation of pyroptosis in macular corneal dystrophy

Macular corneal dystrophy (MCD) is ascribed to mutations in the carbohydrate sulfotransferase (CHST6) gene affecting keratan sulfate (KS) hydrophilicity and causing non-sulfated KS to precipitate in keratocytes and the corneal stroma. We investigated roles for inflammatory responses in MCD pathogenesis by examining the lysosomal-autophagy pathway and activation of pyroptosis in MCD keratocytes. Normal and lesioned keratocytes were obtained from MCD patients undergoing corneal transplantation. The keratocytes were subjected to gene sequencing, RT-PCR, western blotting, transmission electron microscopy, histological staining, induction and inhibition assays of autophagy and pyroptosis, CCK-8 and LysoTracker Green DND-26 labeling, and flow cytometry. A novel homozygous MCD mutation was identified in a family from Northeast China; the mutation was distinguished by cytoplasmic vacuolation, cell membrane disruption, electron dense deposits, and deposition of a band of Periodic acid-Schiff and Alcian blue-positive material in the keratocytes and stroma layer. KS protein levels were decreased, expression of p62 and LC3-II proteins was enhanced, cathepsin D expression was declined and the LysoTracker Green DND-26 signal was dramatically reduced in MCD keratocytes. Bafilomycin-A1 treatment significantly increased caspase-1 and Pro-IL-1β expression in normal and MCD keratocytes. Nod-like receptors pyrins-3 (NLRP3), caspase-1, Pro-IL-1β, and IL-1β levels were pronouncedly elevated in cells exposed to H2O2. Ac-YVAD-CMK treatment reversed this expression in normal and MCD keratocytes. Suppression of the autophagic degradation of non-sulfated KS by impaired autophagic flux in MCD keratocytes triggers pyroptosis. Amelioration of impaired autophagy and restraint of pyroptosis may, therefore, have therapeutic efficacy in the treatment of MCD.

Combined treatment of menadione and calcitriol increases the antiproliferative effect by promoting oxidative/nitrosative stress, mitochondrial dysfunction, and autophagy in breast cancer MCF-7 cells

The aim of this study was to determine new insights into the molecular mechanisms involved in the antiproliferative action of menadione + calcitriol (MEN+D) on MCF-7 cells. After 24 h, MEN+D inhibited the cell growth but was not observed with each single treatment. The combined drugs reduced the mitochondrial respiration at that time, as judged by an increase in the proton leak and a decrease in the ATP generation and coupling efficiency. At longer times, 48 or 96 h, either D or MEN reduced the proliferation, but the effect was higher when both drugs were used together. The combined treatment increased the superoxide anion ([Formula: see text]) and nitric oxide (NO•) contents as well as acidic vesicular organelles (AVOs) formation. The percentage of cells showing the lower mitochondrial membrane potential (ΔΨm) was highly increased by the combined therapy. LC3-II protein expression was enhanced by any treatment. In conclusion, the antiproliferative action of MEN+D involves oxidative/nitrosative stress, mitochondrial alteration, and autophagy. This combined therapy could be useful to treat breast cancer cells because it inhibits multiple oncogenic pathways more effectively than each single agent.

Differential capacity of CD90+ cells in autophagy activation following chemotherapy in hepatocellular carcinoma

Analysis of cancer biomarkers is an important tool in developing targeted-therapy and in modulating chemoresistance. Here, we analyze the relevance of CD90, a marker of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) and its correlation with autophagy. For in vivo study, 86 specimens were collected from 43 patients undergoing liver resections. In each patient, HCC nodule (HCC) and surrounding non-tumor (SNT) were collected. For in vitro study, HCC cells JHH6 subpopulations expressing CD90+ and CD90- were isolated using magnetic-sorter and confirmed by flow-cytometry. Upon doxorubicin treatment, autophagy turn-over was analyzed by RTqPCR for mRNA expression, Western blot for protein expression, and autophagosome staining for autophagy-flux. Cytotoxicity test was performed by MTT assay. Gene and protein analysis were performed in clinical samples together with immunohistostaining. CD90 mRNA expression was higher in HCC than in SNT for 8-fold (p < 0.001). LC3-II protein was up-regulated in the HCC in comparison with the SNT (p < 0.05). In vitro model showed that CD90+ and CD90- cells had diverse expressions of autophagy-related genes. Upon doxorubicin treatment, autophagy was activated in both cells by increasing LC3-II protein expression, autophagic vacuoles, and dysregulation of autophagy-related mRNAs. A differential autophagic capacity was noticed between two subpopulations and it was correlated with cellular toxicity assay. We demonstrated the relevance of differential autophagy capacity of CD90+ cells in HCC. Autophagy was involved in cancer-defense mechanism against doxorubicin. Cancer promoting function of autophagy in CD90+ cells was also related to cancer environment.

Berberine Attenuates Arterial Plaque Formation in Atherosclerotic Rats with Damp-Heat Syndrome via Regulating Autophagy.

PurposeBerberine (BBR) is an effective component of Huanglian and has shown to attenuate atherosclerosis (AS); however, the detailed mechanism of BBR-mediated protective actions against AS remains elusive. This study was undertaken to examine the effects of BBR on aortic atherosclerotic plaque stability and the expression of autophagy-related proteins in AS rats with damp-heat syndrome or yang deficiency.MethodsThirty SD rats were randomly divided into (1) control (CON); (2) damp-heat syndrome atherosclerosis (AS + DH); (3) yang deficiency syndrome atherosclerosis (AS + YX); (4) damp-heat syndrome atherosclerosis + BBR (AS + DH + BBR); (5) yang deficiency syndrome, atherosclerosis + BBR (AS + YX + BBR); and (6) damp-heat syndrome, atherosclerosis + BBR + 3-methyladenine (AS + DH + BBR + 3-MA) (n = 5/group) groups. Pathological morphology, macrophage plaque infiltration, inflammation, and LC3-II and P62 expression were assessed.ResultsCompared with the CON group, the AS + DH and AS + YX groups had an increased plaque area in the aortic tissue with substantial foam cell and macrophage infiltration, and increased levels of IL-1β and TNF-α (P < 0.01). After four weeks of BBR intervention, the plaque area in the AS + DH + BBR group was reduced with decreased foam cells and macrophage infiltration, and decreased levels of TNF-α and IL-1β, whereas LC3-II protein expression was increased and P62 protein expression was decreased in the AS + DH + BBR group when compared to AS + DH group. In addition, the AS + DH + BBR + 3-MA group exhibited a significantly enlarged plaque, substantial foam cell and macrophage infiltration, increased levels of IL-1β and TNF-α, and decreased LC3-II and P62 (P < 0.01) expression when compared to the AS + DH + BBR group.ConclusionOur results indicated that the BBR could inhibit arterial plaque formation and alleviate the inflammatory response in the aortic tissues in the AS rats with damp-heat syndrome possibly via promoting autophagy. The molecular mechanisms of BBR-mediated protective effects in this animal model still require further investigation.

Low-intensity pulsed ultrasound activates autophagy in periodontal ligament cells in the presence or absence of lipopolysaccharide

ObjectiveThis study aims to determine if low-intensity pulsed ultrasound (LIPUS) activates autophagy in human periodontal ligament cells (PDLCs) irrespective of lipopolysaccharide. DesignSix groups were designed: control, LIPUS, lipopolysaccharide, LIPUS + lipopolysaccharide, LIPUS+3-Methyladenine, LIPUS + lipopolysaccharide+3-Methyl- adenine. LIPUS pretreated PDLCs for 2 h and lipopolysaccharide treated for different times. Real-time PCR and Western-blot were performed to evaluate mRNA and protein expression levels of autophagic genes Beclin-1 and LC3 respectively. A transmission electronic microscope was used to observe the autophagosome. ELISA was used to test interleukin-6 expression. ResultsCompared with the non-treatment, LIPUS pretreatment increased mRNA expression levels of LC3 (P < 0.05) and Beclin-1 (P < 0.05) at 4 h and 8 h, and enhanced the protein expression levels of LC3-Ⅱ at 8 h (P<0.05) and Beclin-1 at 4 h, 8 h and 16 h(P<0.05). After LIPUS pretreatment and lipopolysaccharide treatment for 8 h, LC3-Ⅱ and Beclin-1 protein expression levels were elevated (P < 0.05) compared with the control. Following further treatment by 3-Methyladenine, Beclin-1 protein expression was decreased (P < 0.05) compared with the LIPUS plus lipopolysaccharide group, but LC3-Ⅱ protein expression was not. Autophagosomes were not found in the LIPUS+3-Methyladenine and LIPUS+lipopolysaccharide+3-Methyladenine groups. After LIPUS pretreatment and lipopolysaccharide treatment for 36 h, intreleukin-6 expression was decreased (P<0.05) compared with the lipopolysaccharide group. However, after addition of 3-Methyladenine, intreleukin-6 expression was elevated (P < 0.05) compared with the LIPUS +lipopolysaccharide group. ConclusionsLIPUS can promote autophagy in PDLCs irrespective of lipopolysaccharide. Autophagy might be involved in LIPUS anti-inflammatory mechanism in PDLCS.

Lentiviral-Mediated Beclin-1 Overexpression Inhibits Cell Proliferation and Induces Autophagy of Human Esophageal Carcinoma Eca109 Cell Xenograft in Nude Mice.

Esophageal carcinoma is one of the common malignant tumors in digestive tract. BECLIN-1 is a key gene that regulates autophagy, and its abnormal expression may be related with many human tumors. However, the mechanism of BECLIN-1 in esophageal carcinoma remains unknown. In this study, we explored the effect of BECLIN-1 overexpression on tumor growth in mice with esophageal carcinoma and its mechanism. Recombined lentiviral vector containing BECLIN-1 was used to transfect human esophageal carcinoma Eca109 cells and establish stable cell line. qRT-PCR was used to detect BECLIN-1 mRNA level in the transfected Eca109 cells, CCK-8 assay was used to detect cell proliferation. Beclin-1, P62 and LC3-II protein expression levels in Eca109 cells were detected using Western blot analysis. Subcutaneous xenograft nude mice model of human esophageal carcinoma was established, and the tumor growths in Beclin-1 group, control group and empty vector group were monitored. Beclin-1 protein expression in vivo was detected by immunohistochemistry. Beclin-1 mRNA and protein were overexpressed in Eca109 cells. Compared with empty vector group, the growth rate of cells transfected with BECLIN-1 decreased significantly. Compared with the control group and empty vector group, the expression level of P62 protein in beclin-1 group was significantly decreased, while the expression level of LC3-II protein was significantly increased. The tumor growth rate in nude mice of Beclin-1 group was significantly lower than that of the control group and empty vector group, and Beclin-1 protein was mainly expressed in Beclin-1 group in vivo. BECLIN-1 can induce autophagy in esophageal carcinoma Eca109 cells, and it can significantly inhibit the growth of esophageal carcinoma.

Blocking retrograde axonal transport of autophagosomes contributes to sevoflurane-induced neuron apoptosis in APP/PS1 mice

Autophagy, a crucial pathway for the degradation of proteins in eukaryotic cells, is linked to the development of Alzheimer’s disease (AD), and the accumulated autophagosomes in the cells resulting in the death of cells. Sevoflurane can impair spatial learning and memory in mice with AD and lead to the apoptosis of nerve cells; however, the underlying mechanisms remain unknown. We aim to explore the effects and underlying mechanisms of sevoflurane in APPswe/PS1ΔE9 double-transgenic mice. 51 heterozygous APPswe/PS1ΔE9 double-transgenic mice were involved and divided into three groups, including control group, sham group and sevoflurane group. Morris water maze experiment was used to test the learning and memory abilities of mice, flow cytometry was conducted to detect apoptosis and mitochondrial membrane potential of brain cells in mice, transmission electron microscopy was used to observe the number of autophagosomes at the axon in mice, and western blot was carried out to detect the expression of Bax, Bcl-2, LC3II, P62, KIF3B and DIC proteins of brain cells in mice. In our study, we found that significantly longer escape latencies, fewer crossings of the platform and shorter time spent in the target quadrant of the morris water maze experiment in the sevoflurane group. Flow cytometry showed cellular apoptosis was increased and the membrane potential of the mitochondria was reduced of brain cells in the sevoflurane group. Transmission electron microscopy displayed that there was a remarkable upregulation of autophagosomes at the axon of brain cells in mice after treatment of sevoflurane. Western blot demonstrated that the expression of Bax, LC3II, P62 and KIF3B proteins were elevated, and the expression of Bcl-2 and DIC proteins were reduced in the sevoflurane group. Sevoflurane impaired acquisition learning and memory function, promoted the apoptosis of hippocampal neurons in APPswe/PS1ΔE9 double-transgenic mice, and the mechanism might be related to the activation of autophagy along with the disruption of autophagosomes retrograde transport in axons.

Regulation of taurine in OTA-induced apoptosis and autophagy

Ochratoxin A (OTA), one of the most deleterious mycotoxins, could cause a variety of toxicological effects especially nephrotoxicity in animals and humans. Taurine, a wide-distributed cytoprotective amino acid, plays an important role as a basic factor for maintaining cellular integrity homeostasis. However, the potential effect of taurine in OTA-induced nephrotoxicity remains unknown. In the present study, we demonstrated that OTA treatment at 4.0–8.0 μM increased apoptosis in PK-15 cells as shown by increased the ratio of apoptosis and protein expression of Bax and cleaved-caspase-3, decreased protein expression of Bcl-2. Meantime, OTA treatment triggered autophagy, as indicated by markedly increased the protein expression of LC3-II and fluorescence intensity of GFP-LC3 dots. Taurine supplementation decreased OTA-induced cytotoxicity and attenuated apoptosis as shown by the decreased Annexin V/PI staining and the decreased expression of apoptosis-related proteins including Bax and caspase-3. Meanwhile, taurine attenuated OTA-induced autophagy by decreased the protein expression of LC3-II and fluorescence intensity of GFP-LC3 dots to maintain cellular homeostasis. In conclusion, taurine treatment could alleviate OTA-induced apoptosis and inhibit the triggered autophagy in PK-15 cells. Our study provides supportive data for the potential roles of taurine in reducing OTA-induced renal toxicity.

Research on autophagy induced by two xanthone compounds in HepG2 cells

To investigate the inhibitory effects of two xanthone compounds, 1-hydroxy-2,3,4,8-4 methoxy xanthone(here in after referred to as Fr15) and 1-hydroxy-2,3,4,6-4 methoxy xanthone(here in after referred to as Fr17), on the proliferation of hepatocellular carcinoma cells HepG2, and to further investigate their mechanism in combination with transcriptomics. Cell counting was used to detect the effects of two kinds of xanthone compounds Fr15 and Fr17(0, 0.03, 0.15, 0.3 mmoL·L~(-1)) on the proliferation of HepG2 cells; the effects of the two compounds Fr15 and Fr17 on HepG2 cell cycle were detected by flow cytometry; the changes of autophagosomes count in cells were observed under fluorescence microscope; the expression of autophagy marker proteins autophagy marker proteins SQSTM 1(p62) and microtubule associated protein 1 light chain 3 Ⅰ/Ⅱ(LC3 Ⅰ/Ⅱ) in the cells was detected by Western blot; the differentially expressed genes between the control group and the experimental group were analyzed by RNA-seq transcriptome sequencing; qRT-PCR was used to verify the differentially expressed genes in sequencing. The results showed that compounds Fr15 and Fr17 inhibited the proliferation of HepG2 cells with the increase of drug concentration and time. Flow cytometry showed that compounds Fr15 and Fr17 had little effect on HepG2 cell cycle. Fluorescence microscopy results showed that the number of autophagosomes in cells increased with the increase of drug concentration. Western blot showed that the expression of p62 protein was decreased and the expression of LC3-Ⅱ protein was significantly increased after drug addition. The results of RNA sequencing showed that 26 102 and 52 351 differentially expressed genes were obtained in Fr15 and Fr17 respectively. Analysis of KEGG showed that drug treatment had a great effect on autophagy pathway. qRT-PCR verified that 6 up-regulated genes were related to autophagy, and their trend was consis-tent with sequencing results, where all 6 genes showed an up-regulated trend. Two xanthone compounds Fr15 and Fr17 may inhibit proliferation of HepG2 cells by inducing autophagy.

Si-Wei-Qing-Gan-Tang Improves Non-Alcoholic Steatohepatitis by Modulating the Nuclear Factor-κB Signal Pathway and Autophagy in Methionine and Choline Deficient Diet-Fed Rats.

Si-Wei-Qing-Gan-Tang (SWQGT) is a Chinese medicine formula that is widely used as a folk remedy of herbal tea for the treatment of chronic hepatitis, like non-alcoholic steatohepatitis (NASH), around Ganzhou City (Jiangxi province, China). However, the underlying mechanisms of this formula against NASH are still unknown. This study aimed to explore the effect and mechanisms of SWQGT against NASH. A network pharmacology approach was used to predict the potential mechanisms of SWQGT against NASH. Then a rat model of NASH established by feeding the methionine and choline deficient (MCD) diet was used to verify the effect and mechanisms of SWQGT on NASH in vivo. SWQGT (1 g/kg/d and 3 g/kg/d) were given by intragastric administration. Body weight, liver weight, serum biochemical indicators, liver triglyceride and total cholesterol were all measured. Tumor necrosis factor-α (TNF-α), Interleukin (IL)-1β, IL-6 levels in the livers were evaluated using ELISA. Hematoxylin and eosin (HE) and Oil Red O staining were used to determine histology, while western blot was used to assess the relative expression levels of the nuclear factor-κB (NF-κB) pathway- and autophagy-related proteins. Functional and pathway enrichment analyses revealed that SWQGT obviously influenced inflammation-related signal pathways in NASH. Furthermore, in vivo experiment showed that SWQGT caused a reduction in liver weight and liver index of MCD diet-fed rats. The formula also helped to reduce hepatomegaly and improve pathological liver changes and hepatic steatosis. SWQGT likewise reduced liver TNF-α, IL-1β, and IL-6 levels and down-regulated p-NF-κB p65, p-p38 MAPK, p-MEK1/2, p-ERK1/2, p-mTOR, and p62, while up-regulating p-ULK1 and LC3II protein expression levels. SWQGT could improve NASH in MCD diet-fed rats, and this effect may be associated with its down-regulation of NF-κB and activation of autophagy.

Endoplasmic Reticulum (ER) stress‐generated microparticles self‐perpetuate ER stress and mediate endothelial cell dysfunction independently of cell survival

Microparticles (MPs) are nanosized membrane‐shed vesicles released from activated cells. MPs can induce endothelial dysfunction, the first step in atherosclerosis, through endoplasmic reticulum (ER) stress. However, it is not known whether ER stress can lead to MPs release and what biological messages these MPs would carry. Therefore, we aimed to assess the impact of ER stress on MPs shedding from endothelial cells, and to investigate their effects on endothelial function.Human umbilical vein endothelial cells (HUVECs) were treated for 24h with ER stress inducer, thapsigargin, in the presence or absence of 4‐Phenylbutyric acid (PBA), a chemical chaperone to inhibit ER stress. Then, MPs were isolated and used to treat naïve HUVECs (20 g/mL) for 24‐48h before assessing ER‐stress response, autophagy and apoptosis.MPs generated under ER stress conditions did not differ quantitatively from controls; however, they carried deleterious messages for endothelial function. The treatment of naïve HUVECs with ER stress‐generated MPs (48h) increased mRNA and protein expression of key ER stress markers [activating transcription factor (ATF)‐4, and Binding immunoglobulin protein (BiP)], indicating a vicious circle between MPs production and ER stress activation. ER stress‐generated MPs impaired the angiogenic capacity of HUVECs to form tube‐like structures, indicating an impaired endothelial function. However, ER stress‐generated MPs did not affect autophagy (no changes in protein expression of LC3‐II) or apoptosis in HUVECs.Our data show that ER stress activation caused the shedding of MPs, which were capable of activating ER stress in HUVECs in a vicious circle and impaired angiogenic capacity of these cells. These MPs impaired endothelial function in a mechanism independent from cell death and autophagy.Support or Funding InformationThis work was supported by awards from Qatar University (QUUG‐CPH‐CPH‐15/16‐6; QUST‐2‐CPH‐2017‐19; QUST‐1‐CPH‐2018‐1) to Dr Abdelali Agouni. The findings achieved herein are solely the responsibility of the authors.

Bishonokiol A Induces Multiple Cell Death in Human Breast Cancer MCF-7 Cells.

Objective:A dimeric neolignan, bishonokiol A (BHNKA) isolated from Magnolia grandiflora, significantly inhibits the proliferation of human breast cancer cells. However, the exact mechanism of BHNKA induced breast cancer cell death is unknown. In this study, we investigated the pharmacological mechanism underlying BHNKA induced MCF-7 cell death. Methods:Cell viability measurement was performed by the MTT assay. Flow cytometry with PI staining, DAPI staining, and electron microscopy were used to analyze cellular death modes. In addition, western blotting, siRNA transfection, ATP assay, and fluorescence microscopy were used to determine the mechanism of BHNKA induced MCF-7 cell death. Results:BHNKA induced cell death by apoptosis, necroptosis and autophagy at the same concentration and time in MCF-7 cells, and electron microscopy confirmed these results. The mechanism of BHNKA triggered apoptosis and autophagy in MCF-7 cells was primarily due to an increase in the Bax/Bcl-2 ratio and simultaneous up-regulation of LC3-II protein expression, respectively. BHNKA induced necroptosis by activation of the RIP1-RIP3-MLKL necroptosis cascade, up-regulation of cyclophilin D (CypD) protein expression to stimulate ROS generation. We further demonstrated that siRNA-mediated down-regulation of CypD protected against BHNKA induced cell death. Conclusions:These results suggest that BHNKA may be a potential lead compound for development as an anti-breast cancer agent for induction of multiple cell death pathways.

LncRNA FEZF1-AS1 promoted chemoresistance, autophagy and epithelial-mesenchymal transition (EMT) through regulation of miR-25-3p/ITGB8 axis in prostate cancer.

Accumulating evidence determined that lncRNA plays important roles in the development and occurrence of cancers. Prostate cancer is the second most common type of cancer and one of the top five cancers for the cause of male death in the world. Therefore, this study was to explore the regulatory mechanism of lncRNA in chemoresistance of PC. qRT-PCR was used to detect the mRNA expression of FEZF1-AS1, miR-25-3p and ITGB8. Western blot was applied to measure the protein expression of ITGB8 E-cadherin, N-cadherin, Vimentin, LC3I, LC3II, ATG5 and Beclin-1. In addition, CCK-8 assay was used to assess cell proliferation of transfected cells. Luciferase reporter assay and RIP assay were used to determine the relationship among FEZF1-AS1, miR-25-3p and ITGB8. In this study, the expression of FEZF1-AS1 and ITGB8 was upregulated, whereas the expression of miR-25-3p was downregulated in PC tumor tissues and PC/PTX cells. Luciferase reporter assay and RIP assay determined that miR-25-3p was a target of FEZF1-AS1 and ITGB8 was a target mRNA of miR-25-3p. Interestingly, knockdown of FEZF1-AS1 could inhibit cell viability and EMT and promoted cell autophagy in PC/PTX cells, but inhibition of miR-25-3p or promotion of ITGB8 could reverse the effects of si-FEZF1-AS1 on PC/PTX cells. In this study, we found that lncRNA FEZF1-AS1 promoted chemoresistance, autophagy and epithelial-mesenchymal transition (EMT) through regulation of miR-25-3p/ITGB8 axis in PC, providing a new regulatory mechanism of PC and a novel therapeutic target.

Role of autophagy in Puromycin Aminonucleoside-induced podocyte apoptosis

Objective: The aim of our study is to investigate the relationship between podocyte autophagy and apoptosis induced by Puromycin Aminonucleoside (PAN) and to clarify its mechanism.Methods: Podocytes were cultured in vitro. The apoptosis rates of each group were detected using flow cytometry. The expression of LC3-II protein and changes in distribution were detected through laser scanning confocal microscope, and the western blot protocol was employed for detection of protein expression of LC3-II. The autophagosomes were detected by transmission electron microscopy.Results: In this study, We found that autophagosome increased followed by apoptosis after podocyte injury. Furthermore, we conformed that the activation of autophagy could inhibit the apoptosis to alleviate the injury of podocyte at an early stage.Conclusions: Autophagy occurred earlier before apoptosis and autophagy mediated podocyte apoptosis induced by PAN. These findings indicate that autophagy may become a novel therapeutic target for the treatment of podocyte injury and proteinuria in the future.

Endoplasmic reticulum stress regulates autophagy and tumor necrosis factor-α secretion of RAW264.7 cells induced by silica

Objective: To investigate the role of endoplasmic reticulum stress (ERS) in the autophagy of RAW264.7 cells induced by SiO(2) and its effect on the secretion of tumor necrosis factor-α. Methods: RAW264.7 cells stimulated by 200 μg/ml SiO(2) were used as an vitro cell model, and different treatment times of SiO(2) were used as variables. They were divided into 0 h treatment group (blank control group) , 6 h, 12 h, 24 h, and 48 h treatment group. The formation of autophagospores was detected by acridine orange and mondane-sulfonate (MDC) staining. Application of real-time quantitative PCR (Real-time PCR) to detect autophagy related molecular Beclin1 mRNA expression and protein immunoblot (Western Blotting) detecting autophagy related proteins LC3Ⅰ, LC3Ⅱ and expression of Beclin1. Real-time PCR and Western blotting were used to detect the expression of ERS specific marker BiP. Secretion of RAW 264.7 cell transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) was detected by enzyme-linked immunosorbent assay (ELISA) . ERS inhibitors 4-PBA intervention experiment, including blank control group, SiO(2), 1 μmol/L 4-PBA+SiO(2), 10 μmol/L 4-PBA+SiO(2), 20 μmol/L 4-PBA+SiO(2) treatment group, Western blotting testing LC3Ⅰ, LC3Ⅱ and expression of Beclin1 changes. Results: Compared with the control group, SiO(2)-induced fluorescence intensity in RAW264.7 cells was significantly increased, with statistically significant differences (P<0.05) . Compared with control group, with SiO(2) processing time prolonged, LC3Ⅰ, LC3Ⅱ Beclin1 mRNA and protein expression and protein expression increased, 6 h, 24 h, the height of the differences were statistically significant (P<0.05) ; Compared with the control group, the mRNA and protein expression level of BiP reached the peak for 6 h, and the expression level in 6 h, 12 h and 24 h groups increased significantly, and the difference was statistically significant (P<0.05) . Compared with the SiO(2) stimulation group, the LC3Ⅱand Beclin 1 protein levels of RAW264.7 cells were gradually down-regulated by increasing the dose of 4-PBA. With the increase of 4-PBA concentration, the down-regulated levels were more significant, and the difference was statistically significant (P<0.05) . Compared with the SiO(2) stimulation group, the TNF-α secretion level of RAW264.7 cells significantly decreased of 1, 10, 20 μmol/L 4-PBA+SiO(2) treatment group, and the difference was statistically significant (P<0.05) . Conclusion: ERS induced by SiO(2) is involved in the secretion of autophagy and TNF-α in RAW264.7 cells.

Baicalin reverses radioresistance in nasopharyngeal carcinoma by downregulating autophagy

BackgroundRadiation resistance is the main cause of recurrence after radiotherapy, and increased autophagy after radiotherapy is related to radiotherapy resistance. This study aims to investigate the reversal effect of baicalin on radioresistance and its related mechanism.MethodsCCK-8 and flow cytometry were used to detect the effect of proliferation and apoptosis by baicalin. Clone formation test was used to verify the effect of baicalin radiosensitization. Western blot analysis and electron microscopy were employed to observe the effect of baicalin on autophagy.ResultsCompared with the radiation therapy (RT) group, the RT combined baicalin (RT + BA) group showed a significantly low 2 Gy survival fraction of radiation therapy (P < 0.05). LC3-II protein expression in the RT group was significantly higher than which in the RT + BA group (P < 0.05). Electron microscopy showed that more autophagic vacuoles were observed in the RT group than those in the RT + BA group.ConclusionsOverall, baicalin can reverse the radioresistance of human nasopharyngeal carcinoma CNE-2R cells by downregulating RT-enhanced autophagy.

Anthocyanins isolated from Oryza Sativa L. protect dermal fibroblasts from hydrogen peroxide-induced cell death

Background: Oxidative stress, cellular toxicity, and inflammation lead to skin damage, which results in premature skin aging. Recently, anthocyanins (ANT) have received much attention as dietary anti-oxidants involved in the prevention of oxidative damage. Materials and Methods: This study investigated the effects of ANT extracted from black rice (Oryza sativa L.) on the survival of rat dermal fibroblasts (RDFs) after oxidative stress-induced cellular damage by hydrogen peroxide (H2O2) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We further investigated the apoptosis-inducing effects of ANT using 4′,6-diamidino-2-phenylindole and Annexin V staining. The effect of ANT extract on autophagy was confirmed by reverse transcription polymerase chain reaction of the autophagy-related microtubule-associated protein 1B light chain 3 (LC3-II) and ffluorescence microscopy of the LC3-II protein. Results: The high-performance liquid chromatography results indicated the presence of cyanidin-3-O-glucoside in both extracts. The study demonstrated that the addition of crude or purified ANT extract before H2O2 treatment increased RDF cell viability. Pretreatment with ANT decreased the number of cells exhibiting dense chromatin fragments and DNA condensation, which are characteristics of apoptotic cell death. ANT decreased the number of late apoptotic/necrotic (Annexin + and propidium iodide (PI) +) cells and early apoptotic (Annexin V + and PI-) cells. Furthermore, ANT inhibited the H2O2-mediated induction of LC3-II gene expression in RDFs. Conclusion: The contribution of autophagy induction to the protective effects of ANT was verified by the observed decrease in the mRNA and protein expression of LC3-II. These results suggest the therapeutic potential of polyphenolic compounds extracted from O. sativa L. in oxidative damage-induced skin aging.