Jinshan Hospital of Fudan University

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is thatthere is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the completeprocess including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as forreviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagyrelated protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

AIMS: To investigate alterations in protein expression associated with deep brain stimulation (DBS) in an attempt to elucidate possible mechanisms of action . METHODS: Cerebrospinal fluid (CSF), obtained from six Parkinson's disease (PD) patients (pre- and post-DBS) and from six normal healthy controls, was studied for differentially expressed proteins. 2-D DIGE, in combination with MALDI-TOF and TOF-TOF Mass Spectrometry (MS) or ESI-MS, was used to identify the changed proteins (3 PD patients and 3 controls). Selected proteins were further studied using western blotting (6 PD patients and 6 controls). RESULTS: Twenty-one proteins were identified after MS and protein database interrogation. Apart from apolipoprotein A-I (apoA-I), the expression levels of complement C4 (C4), IgA, tetranectin, and extracellular superoxide dismutase (EC-SOD), detected by western blotting, correlated well with the 2-D DIGE results. In the follow-up period, the expression levels of C4, apoA-I and IgA were stable whereas EC-SOD and tetranectin were significantly elevated. In addition, when DBS was ceased in one patient due to a suicide attempt, the levels of EC-SOD and tetranectin significantly decreased. CONCLUSION: Our preliminary results suggest that variations in the expression levels of EC-SOD and tetranectin in CSF is related to DBS.

Pickering emulsion, a kind of emulsion stabilized only by solid particles locating at oil-water interface, has been discovered a century ago, while being extensively studied in recent decades. Substituting solid particles for traditional surfactants, Pickering emulsions are more stable against coalescence and can obtain many useful properties. Besides, they are more biocompatible when solid particles employed are relatively safe in vivo. Pickering emulsions can be applied in a wide range of fields, such as biomedicine, food, fine chemical synthesis, cosmetics and so on, by properly tuning types and properties of solid emulsifiers. In this article, we give an overview of Pickering emulsions, focusing on some kinds of solid particles commonly serving as emulsifiers, three main types of products from Pickering emulsions, morphology of solid particles and as-prepared materials, as well as applications in different fields.

the development of curative and regenerative therapies that go beyond the largely symptomatic treatment options available; and (6) deployment of public health preventive strategies for banning smoking and maintaining clean air. We are fully cognisant that the scientific evidence to support some of our proposals is lacking. However, it is the intention of the Commission to generate discourse and debate, catalyse momentum, and provide a much-needed new vision to set the course towards the elimination of COPD.

BACKGROUND: Depression is a heterogeneous disorder, with the exact neuronal mechanisms causing the disease yet to be discovered. Recent work suggests it is accompanied by neuro-inflammation, characterized, in particular, by microglial activation. However, microglial activation and its involvement in neuro-inflammation and stress-related depressive disorders are far from understood. METHODS: We utilized multiple detection methods to detect the neuro-inflammation in the hippocampus of rats after exposure to chronic mild stress (CMS). Male Sprague Dawley (SD) rats were subjected to chronic mild stressors for 12 weeks. Microglial activation and hippocampal neuro-inflammation were detected by using a combinatory approach of in vivo [18F] DPA-714 positron emission computed tomography (PET) imaging, ionized calcium-binding adapter molecule 1 and translocator protein (TSPO) immunohistochemistry, and detection of NOD-like receptor protein 3 (NLRP3) inflammasome and some inflammatory mediators. Then, the rats were treated with minocycline during the last 4 weeks to observe its effect on hippocampal neuro-inflammation and depressive-like behavior induced by chronic mild stress. RESULTS: The results show that 12 weeks of chronic mild stress induced remarkable depressive- and anxiety-like behavior, simultaneously causing hippocampal microglial activation detected by PET, immunofluorescence staining, and western blotting. Likewise, activation of NLRP3 inflammasome and upregulation of inflammatory mediators, such as interleukin-1β (IL-1β), IL-6, and IL-18, were also observed in the hippocampus after exposure to chronic stress. Interestingly, the anti-inflammatory mediators, such as IL-4 and IL-10, were also increased in the hippocampus following chronic mild stress, which may hint that chronic stress activates different types of microglia, which produce pro-inflammatory cytokines or anti-inflammatory cytokines. Furthermore, chronic minocycline treatment alleviated the depressive-like behavior induced by chronic stress and significantly inhibited microglial activation. Similarly, the activation of NLRP3 inflammasome and the increase of inflammatory mediators were not exhibited or significantly less marked in the minocycline treatment group. CONCLUSION: These results together indicate that microglial activation mediates the chronic mild stress-induced depressive- and anxiety-like behavior and hippocampal neuro-inflammation.

BACKGROUND AND PURPOSE: When the coronavirus disease 2019 (COVID-19) outbreak became paramount, medical care for other devastating diseases was negatively impacted. In this study, we investigated the impact of the COVID-19 outbreak on stroke care across China. METHODS: Data from the Big Data Observatory Platform for Stroke of China consisting of 280 hospitals across China demonstrated a significant drop in the number of cases of thrombolysis and thrombectomy. We designed a survey to investigate the major changes during the COVID-19 outbreak and potential causes of these changes. The survey was distributed to the leaders of stroke centers in these 280 hospitals. RESULTS: <0.0001), respectively, in February 2020 as compared with February 2019. We retrieved 227 valid complete datasets from the 280 stroke centers. Nearly 50% of these hospitals were designated hospitals for COVID-19. The capacity for stroke care was reduced in the majority of the hospitals. Most of the stroke centers stopped or reduced their efforts in stroke education for the public. Hospital admissions related to stroke dropped ≈40%; thrombolysis and thrombectomy cases dropped ≈25%, which is similar to the results from the Big Data Observatory Platform for Stroke of China as compared with the same period in 2019. Many factors contributed to the reduced admissions and prehospital delays; lack of stroke knowledge and proper transportation were significant limiting factors. Patients not coming to the hospital for fear of virus infection was also a likely key factor. CONCLUSIONS: The COVID-19 outbreak impacted stroke care significantly in China, including prehospital and in-hospital care, resulting in a significant drop in admissions, thrombolysis, and thrombectomy. Although many factors contributed, patients not coming to the hospital was probably the major limiting factor. Recommendations based on the data are provided.

, we elucidated and confirmed the critical role of ferroptosis in NETs-induced ALI. These findings support a role for NETs-induced METTL3 modification and the subsequent induction of ferroptosis in the pathogenesis of sepsis-associated ALI.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death in many Asian and African countries. Lack of early diagnosis tools is one of the clinical obstacles for effective treatment of HCC. Thus, enhanced understanding of the molecular changes associated with HCC is urgently needed to develop novel strategies for the diagnosis and treatment of this dismal disease. While aberrant expression of long noncoding RNAs (lncRNAs) has been functionally associated with certain cancers, the expression profiles and biological relevance of lncRNAs in HCC remain unclear. Highly upregulated in liver cancer (HULC) lncRNA has been implicated in the regulation of hepatoma cell proliferation. In this study, we demonstrate that HULC expression is significantly higher in HCC tumors compared to normal liver tissues. Among the tumor tissues, higher HULC expression is positively associated with Edmondson histological grades or with hepatitis B (HBV) positive status. Moreover, HULC lncRNA is detected with higher frequency in the plasma of HCC patients compared to healthy controls. Higher HULC detection rates are observed in the plasma of patients with higher Edmondson grades or with HBV+ status. These findings indicate for the first time that the expression of HULC in plasma can be used as a noninvasive promising novel biomarker for the diagnosis and/or prognosis of HCC.

Growing evidence has indicated that iron deposition in the substantia nigra (SN) plays an important role in Parkinson’s disease (PD). However, the underlying mechanism is still elusive. Using primary dopaminergic neurons and SH-SY5Y cells cultured in vitro, we observed that iron loading increased α-synuclein and reactive oxygen species (ROS) levels in these cells but did not affect the intracellular α-synuclein mRNA levels. Furthermore, iron loading significantly downregulated Beclin-1 levels and decreased the ratio of microtubule-associated protein 1 light chain 3 isoforms (LC3 II/LC3 I). However, a significant change in the levels of autophagy-related gene 5 (Atg5) was not observed in either neurons or SH-SY5Y cells after iron treatment. After treatment with rapamycin, the iron loading-induced increase in the α-synuclein level was significantly reversed and ROS generation was alleviated in both cultured neurons and SH-SY5Y cells. These results indicate that the inhibition of autophagy is critical for the pathological alterations in α-synuclein induced by iron loading. Moreover, treatment with vitamin E did not affect the increase in the α-synuclein levels but significantly eliminated the iron-induced ROS production. Together, our study shows that autophagy dysfunction contributes to iron-induced α-synuclein pathology.

Abstract The aim of this study was to investigate the effect of quercetin on hepatic fibrosis, a characteristic response to acute or chronic liver injury. Mice were randomized to bile duct ligation (BDL) or carbon tetrachloride (CCl 4 ) cirrhosis models. Quercetin (100 mg/kg or 200 mg/kg daily) was administered by gavage for 2 or 4 weeks. Liver tissue and blood samples were collected for histological and molecular analysis. The results of our experiments showed that quercetin reduced BDL or CCl 4 liver fibrosis, inhibited extracellular matrix formation, and regulated matrix metallopeptidase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1. Quercetin attenuated liver damage by suppressing the TGF-β1/Smads signaling pathway and activating the PI3K/Akt signaling pathway to inhibit autophagy in BDL- or CCl 4 - induced liver fibrosis. Quercetin prevented hepatic fibrosis by attenuating hepatic stellate cell activation and reducing autophagy through regulating crosstalk between the TGF-β1/Smads and PI3K/Akt pathways.

Background: To help inform decision making in the clinical setting, we carried out a systematic review and meta-analysis to estimate the association of thyroid disease risks with obesity. Methods—Pubmed, Embase, Web of Science, Cochrane database and Google Scholar electronic databases were searched from inception to October 31, 2018 without language restrictions to explore the relationship between thyroid disorders and obesity. The relative risk (RR) or odds risk (OR) for thyroid disorders were pooled using the SPSS and STATA software. Results—A total of 22 studies were included in the study. 1) Meta-analysis showed that obesity is significantly associated with increased risk of hypothyroidism (RR=1.86, 95%CI 1.63–2.11, P<0.001). Meta-analyses after stratification further showed that obese population had increased risks of overt hypothyroidism (RR=3.21, 95%CI 2.12–4.86, P<0.001) and subclinical hypothyroidism (RR=1.70, 95%CI 1.42–2.03, P<0.001). 2) Further meta-analysis also showed obesity was clearly associated with Hashimoto's thyroiditis (RR=1.91, 95%CI 1.10–3.32, P=0.022), but not Graves’ disease. 3) In the meta-analysis of antibodies, obesity was correlated with positive thyroid peroxidase antibody (TPOAb) (RR=1.93, 95%CI 1.31–2.85, P=0.001), but not positive thyroglobulin antibody (TGAb). Conclusions: Obesity was significantly related to hypothyroidism, HT and TPOAb, implying that prevention of obesity is crucial for thyroid disorders.

Microglia-induced neuroinflammation plays a vital role in the etiology and progression of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and multiple sclerosis. The neuroprotective role of androgens, including testosterone and its metabolite dihydrotestosterone (DHT), has been increasingly demonstrated in these diseases, but few studies investigated the effects of androgen on neuroinflammation. This study investigated the role of DHT in lipopolysaccharide (LPS)-induced neuroinflammation, neuronal damage and behavioral dysfunction, as well as underlying mechanisms. We showed that DHT inhibited LPS-induced release of proinflammatory factors, including TNF-α, IL-1β, IL-6; iNOS, COX-2, NO, and PGE2 in BV2 cells and primary microglia by suppressing the TLR4-mediated NF-κB and MAPK p38 signaling pathways, thus protecting SH-SY5Y neurons from inflammatory damage induced by activated microglia. In an LPS-induced neuroinflammation mouse model, endogenous DHT depletion by castration exacerbated inflammatory responses by upregulating the levels of TNF-α, IL-1β, IL-6, iNOS, and COX-2 in the serum and brain by increasing the LR4-mediated NF-κB and MAPK pathway activation, but these effects were restored by exogenous DHT supplementation. Moreover, DHT also regulated the mRNA levels of the anti-inflammatory cytokines IL-10 and IL-13 in the brain. In addition, DHT modulated the expression of Aβ, the apoptotic proteins caspase-3, Bcl-2, and Bax, and synaptophysin, as well as neuronal damage in LPS-treated mouse brains. Further behavioral tests revealed that DHT ameliorated LPS-induced spatial and learning impairment and motor incoordination, and partly improved the locomotor activity in LPS-injected mice. Therefore, this study suggests that DHT exerts anti-neuroinflammatory and neuroprotective effects; thus, androgen replacement therapy is a potential therapeutic strategy for improving cognitive and behavioral function in neuroinflammation-related diseases.

The liver is essential for metabolic homeostasis. The onset of liver cancer is often accompanied by dysregulated liver function, leading to metabolic rearrangements. Overwhelming evidence has illustrated that dysregulated cellular metabolism can, in turn, promote anabolic growth and tumor propagation in a hostile microenvironment. In addition to supporting continuous tumor growth and survival, disrupted metabolic process also creates obstacles for the anticancer immune response and restrains durable clinical remission following immunotherapy. In this review, we elucidate the metabolic communication between liver cancer cells and their surrounding immune cells and discuss how metabolic reprogramming of liver cancer impacts the immune microenvironment and the efficacy of anticancer immunotherapy. We also describe the crucial role of the gut-liver axis in remodeling the metabolic crosstalk of immune surveillance and escape, highlighting novel therapeutic opportunities.

BACKGROUND: Radiation-induced lung fibrosis (RILF) is a severe and life-threatening complication of thoracic radiotherapy. Cell death is the key issue in RILF. Ferroptosis is a form programmed cell death implicated in the pathologies of inflammation. This study aimed to investigate the role of ferroptosis in RILF, and the effectiveness and the potential underlying mechanism of ferroptosis inhibitor on RILF. METHODS: Immunofluorescence, western blot and RT-PCR assays were performed to examine the ferroptosis maker glutathione peroxidase 4 (GPX4) in a mice RILF model. The lung tissue sections were stained with hematoxylin and eosin (H&E), Masson trichrome staining and Sirius-Red staining to evaluate the histopathological changes in RILF mice. Reactive oxygen species (ROS) and hydroxyproline (HYP) in lungs were measured by the relevant kits. The serum levels of inflammatory cytokines (TNF-α, IL-6, IL-10, and TGF-β1) were measured with Elisa. The protein and mRNA levels of GPX4, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), hemeoxygenase-1 (HO1) and quinone oxidoreductase 1 (NQO1) in lungs were examined by western blot and RT-PCR. RESULTS: GPX4 levels of the irradiated lungs were significantly down-regulated than the groups with no irradiation, and the ferroptosis inhibitor, liproxstatin-1, increased GPX4 levels significantly in RILF mice. Treatment with liproxstatin-1 lowered the Szapiel and Ashcroft scores significantly, down-regulated the levels of ROS and HYP in lungs and reduced the serum inflammatory cytokines levels in RILF mice. The protein and the mRNA levels of Nrf2, HO1 and NQO1 were up-regulated by liproxsratin-1 in RILF. CONCLUSIONS: Our data suggested that ferroptosis played a critical role in RILF, ferroptosis inhibitor liproxstatin-1 alleviated RILF via down-regulation of TGF-β1 by the activation of Nrf2 pathway. The effectiveness of ferroptosis inhibition on RILF provides a novel therapeutic target for RILF.

OBJECTIVE: Hepatocellular carcinima is one of the most common tumors in clinic and also one of the leading causes of death from cancer worldwide. Quercetin shows significant effects on blocking the development of various cancers. METHODS: We used the human hepatocellular carcinoma LM3 and nude mice tumor model to assess the effects of quercetin in hepatocellular carcinoma and clarify its mechanism of action. We collected LM3 cell line treated with different doses of quercetin at different time periods and determined the vital indexes. The liver tissues of mice were collected and used for western boltting (WB), Hematoxylin and Eosin (H&E) and TUNEL staining. RESULTS: Results indicated that quercetin suppressed the Hepatocellular carcinoma (HCC) growth both in vivo and in vitro. Quercetin could disturb LM3 cells proliferation and cell cycle distribution, thus inducing apoptosis. At the same time, quercetin inhibited LM3 cells migration and invasion and promoted HCC autophagy. These effects at least partly depended on the down-regulation of the activation of JAK2 and STAT3 by quercetin. CONCLUSION: Quercetin inhibited hepatocellular carcinoma progression by modulating cell apoptosis, migration, invasion, and autophagy; and its effects were at least partly related with the JAK2/STAT3 signaling pathway.

microRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by base pairing with their target messenger RNAs. Dysregulation of miRNAs is involved in the pathological initiation and progression of many human diseases. miR-221 and miR-222 (miR-221/222) are two highly homologous miRNAs, and they are significantly overexpressed in several types of human diseases. Silencing miR-221/222 could represent a promising approach for therapeutic studies. In the present review, we will describe the potential value of miR-221/222 as diagnostic, prognostic, and therapeutic biomarkers in various diseases including cancer and inflammatory diseases.

Abstract: Porous microspheres have drawn great attention in the last two decades for their potential applications in many fields, such as carriers for drugs, absorption and desorption of substances, pulmonary drug delivery, and tissue regeneration. The application of porous microspheres has become a feasible way to address existing problems. In this essay, we give a brief introduction of the porous microsphere, its characteristics, preparation methods, applications, and a brief summary of existing problems and research tendencies. Keywords: pore, porosity, porogen, suspension polymerization, seed swelling, pulmonary drug delivery, tissue regeneration

BACKGROUND: Genistein is a natural isoflavone with many health benefits, including antitumour effects. Increased hypoxia-inducible factor 1 α (HIF-1α) levels and glycolysis in tumour cells are associated with an increased risk of mortality, cancer progression, and resistance to therapy. However, the effect of genistein on HIF-1α and glycolysis in hepatocellular carcinoma (HCC) is still unclear. METHODS: Cell viability, apoptosis rate, lactate production, and glucose uptake were measured in HCC cell lines with genistein incubation. Lentivirus-expressed glucose transporter 1 (GLUT1) or/and hexokinase 2 (HK2) and siRNA of HIF-1α were used to test the direct target of genistein. Subcutaneous xenograft mouse models were used to measure in vivo efficacy of genistein and its combination with sorafenib. RESULTS: Genistein inhibited aerobic glycolysis and induced mitochondrial apoptosis in HCC cells. Neither inhibitors nor overexpression of HK2 or GLUTs enhance or alleviate this effect. Although stabiliser of HIF-1α reversed the effect of genistein, genistein no longer has effects on HIF-1α siRNA knockdown HCC cells. In addition, genistein enhanced the antitumour effect of sorafenib in sorafenib-resistant HCC cells and HCC-bearing mice. CONCLUSIONS: Genistein sensitised aerobic glycolytic HCC cells to apoptosis by directly downregulating HIF-1α, therefore inactivating GLUT1 and HK2 to suppress aerobic glycolysis. The inhibitory effect of genistein on tumour cell growth and glycolysis may help identify effective treatments for HCC patients at advanced stages.

Effective control of hepatic stellate cell (HSC) activation and proliferation is critical to the treatment of liver fibrosis. Long non-coding RNAs have been shown to play a pivotal role in the regulation of cellular processes. It has been reported that growth arrest-specific transcript 5 (GAS5) acts as a crucial mediator in the control of cell proliferation and growth. However, little is known about the role and underlying mechanism of GAS5 in liver fibrosis. In this study, our results indicated that GAS5 expression was reduced in mouse, rat, and human fibrotic liver samples and in activated HSCs. Overexpression of GAS5 suppressed the activation of primary HSCs in vitro and alleviated the accumulation of collagen in fibrotic liver tissues in vivo. We identified GAS5 as a target of microRNA-222 (miR-222) and showed that miR-222 could inhibit the expression of GAS5. Interestingly, GAS5 could also repress miR-222 expression. A pulldown assay further validated that GAS5 could directly bind to miR-222. As a competing endogenous RNAs, GAS5 had no effect on primary miR-222 expression. In addition, GAS5 was mainly localized in the cytoplasm. Quantitative RT-PCR further demonstrated that the copy numbers of GAS5 per cell are higher than those of miR-222. GAS5 increased the level of p27 protein by functioning as a competing endogenous RNA for miR-222, thereby inhibiting the activation and proliferation of HSCs. Taken together, a new regulatory circuitry in liver fibrosis has been identified in which RNAs cross-talk by competing for shared microRNAs. Our findings may provide a new therapeutic strategy for liver fibrosis.

The molecular mechanism of intervertebral disc degeneration (IVDD) remains unclear. This study aimed to investigate the role of circular RNAs (circRNAs) in the pathogenesis of IVDD. We sued nucleus pulposus (NP) tissues of patients, tert-butyl hydroperoxide (TBHP) stimulated NP cells (NPCs), and IVDD rat model to explore the interaction between circERCC2 and miR-182-5p/SIRT1 axis. The results showed that downregulation of circERCC2 increased the level of miR-182-5p and decreased the level of SIRT1 in degenerative NP tissues in vivo as well as in TBHP-stimulated NPCs in vitro. Treatment of SIRT1-si activated apoptosis and inhibited mitophagy. Moreover, miR-182-5p-si could regulate the mitophagy and the apoptosis of NPCs by targeting SIRT1. The effects of circERCC2 on NPCs and IVDD rat model were mediated by miR-182-5p/SIRT1 axis. In conclusion, this study provides the first evidence that circERCC2 could ameliorate IVDD through miR-182-5p/SIRT1 axis by activating mitophagy and inhibiting apoptosis, and suggests that circERCC2 is a potentially effective therapeutic target for IVDD.