Upregulation Of Autophagy Research Articles

Specific gene expression in type 1 diabetic patients with and without cardiac autonomic neuropathy

We hypothesized that some molecular pathways might interact to initiate the process of nervous tissue destruction, promoting cardiac autonomic neuropathy (CAN) in the course of diabetes type 1 (T1D). The study group consisted of 60 T1D patients (58.33% women/41.67% men), on standard therapy. The control group consisted of twenty healthy volunteers recruited in accordance with age, gender and body weight. The presence of CAN was documented by the Ewing test method (ProSciCard apparatus). A microarray data analysis was performed using Gene Spring version 13. The microarray results for selected genes were confirmed by real-time PCR (qRT-PCR), using specific TaqMan Gene Expression Assays. Plasma IL‐6 content was measured by an enzyme-linked immunosorbent assay (ELISA). The p < 0.05 value was considered as statistically significant. The microarray analysis, confirmed by qRTPCR, showed significant up-regulation of autophagy, quantity of mitochondria, quality regulatory genes (mTOR, GABARAPL2) apoptosis, ER-stress and inflammation (NFKB1, IL1b, IL1R1, SOD1), in T1D when compared to the control group. A significantly higher IL-6 protein level was observed in T1D patients, in comparison to the control group. We concluded that the observed changes in gene expression and activation of intracellular pathways give a coherent picture of the important role of oxidative stress in inflammation and the activation of apoptosis in the pathomechanism of DM. The significance of the inflammatory process, confirmed by the increased level of the inflammation biomarker IL-6 in the pathomechanisms of CAN was shown even in patients with properly treated T1D.

Necroptosis mediated by impaired autophagy flux contributes to adverse ventricular remodeling after myocardial infarction

Loss of functional cardiomyocytes by cell death after myocardial infarction is most critical for the subsequent left ventricular remodeling, cardiac dysfunction and heart failure. Numerous studies have implicated that dysregulation of autophagy might contribute to cardiomyocyte death. However, the underlying mechanisms by which autophagy dysregulation-mediated cell death remains to be elusive. Herein, we showed that,in response to myocardial ischemic damage in vivo and in vitro, autophagy activity was increased quickly but followed by the process of impaired autophagic degradation as evidenced by the sustained higher level of beclin1 until 12 weeks after myocardial infarction, while, increased accumulation of LC3 and p62. The results from both tandem mRFP-GFP-LC3 adenovirus and lysosomal inhibitor chloroquine supported defective autophagy induction by ischemia injury. Importantly, we found that the impaired autophagy flux, induced not only pharmacologically by CQ but also genetically by beclin1 knockdown, upregulated the expression of RIP3 and aggravated OGD-induced necroptotic cardiomyocyte death and cardiac dysfunction. While, upregulation of autophagy by cardiac-specific beclin1 overexpression partially ameliorated cardiac dysfunction after MI. Furthermore, constitutive activation of necroptosis by forced cardiac-specific overexpression of RIP3 aggravated necrotic cardiomyocyte death, post-MI cardiac remodeling and cardiac dysfunction, but all of which could be ameliorated by inhibition of necroptosis by RIP3 knockdown. In conclusion, these results suggested that autophagy dysfunction-mediated necroptosis mechanistically contributed to loss of cardiomyocytes, adverse ventricular remodeling and progressive heart failure after myocardial Infarction. Inhibition of necroptosis might be the potential target for preventing post-infarction cardiac remodeling and heart failure.

SUN-047 PROTECTION AFFORDED BY ANGIOTENSIN II RECEPTOR ACTIVATION AGAINST ACUTE KIDNEY INJURY IS ASSOCIATED WITH UPREGULATION OF TUBULAR AUTOPHAGY

Autophagy reportedly plays a protective role in acute kidney injury (AKI), and there is in vitro evidence to indicate that the renin-angiotensin system is involved in regulation of autophagy. Here we examined whether activation of the angiotensin II (Ang II) receptor protects the kidney from ischemia-reperfusion (I/R) injury by upregulation of autophagy in vivo. In Study 1, male Sprague-Dawley rats were assigned into treatments with vehicle (Veh) or Ang II (200 ng/kg/min). Each drug was subcutaneously infused for 72 hrs. At the end of the infusion, kidneys were removed for the assessment of autophagosomes determined as LC3 dots by immunohistochemistry. In Study 2, rats received treatments with Veh or Ang II (n=10 for each) as Study 1 and then underwent 30-min renal artery occlusion/reperfusion or sham surgery. Blood and kidneys were collected for biochemical and histological analyses. Acute tubular necrosis (ATN) score was determined in PAS stained histology at 4, and 24 h after surgery. In Study 1, the number of autophagosomes in proximal tubular cells was larger in the Ang II group than in the Veh group (0.88 vs. 0.29 a.u.; P=0.002). In Study 2, autophagosomes were increased at 4 h after I/R in the Veh group. Blood urea nitrogen (BUN) level and ATN score were significantly lower in the Ang II group than in the Veh group at 24 h after I/R (99.2 vs. 123.3 mg/dl; P=0.004, 4.0 vs. 4.6; P=0.011, respectively). The number of autophagosomes was larger in the Ang II group than in the Veh group at baseline and 4 h after I/R (0.54 vs. 4.68; P ULK1-Ser555 phosphorylation was increased in the Ang II group at 4 h after I/R. RIP1 and RIP3 levels at baseline and after I/R were not affected by Ang II (Fig). Short term infusion of Ang II increases autophagosomes in the renal tubular cells and alleviates I/R kidney injury. The renoprotective effect of Ang II may be associated with upregulation of autophagy without suppression of necroptosis.

Pathogenesis of Photoaging in Human Dermal Fibroblasts

Pathogenesis of Photoaging in Human Dermal Fibroblasts

Remote Ischemic Preconditioning induces Cardioprotective Autophagy and Signals through the IL-6-Dependent JAK-STAT Pathway.

Autophagy is a cellular process by which mammalian cells degrade and assist in recycling damaged organelles and proteins. This study aimed to ascertain the role of autophagy in remote ischemic preconditioning (RIPC)-induced cardioprotection. Sprague Dawley rats were subjected to RIPC at the hindlimb followed by a 30-min transient blockade of the left coronary artery to simulate ischemia reperfusion (I/R) injury. Hindlimb muscle and the heart were excised 24 h post reperfusion. RIPC prior to I/R upregulated autophagy in the rat heart at 24 h post reperfusion. In vitro, autophagy inhibition or stimulation prior to RIPC, respectively, either ameliorated or stimulated the cardioprotective effect, measured as improved cell viability to mimic the preconditioning effect. Recombinant interleukin-6 (IL-6) treatment prior to I/R increased in vitro autophagy in a dose-dependent manner, activating the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway without affecting the other kinase pathways, such as p38 mitogen-activated protein kinases (MAPK), and glycogen synthase kinase 3 Beta (GSK-3β) pathways. Prior to I/R, in vitro inhibition of the JAK-STAT pathway reduced autophagy upregulation despite recombinant IL-6 pre-treatment. Autophagy is an essential component of RIPC-induced cardioprotection that may upregulate autophagy through an IL-6/JAK-STAT-dependent mechanism, thus identifying a potentially new therapeutic option for the treatment of ischemic heart disease.

Puerarin Attenuates Diabetic Nephropathy by Promoting Autophagy in Podocytes.

Puerarin, an active compound of radix puerariae, is a major compound used in Chinese herbal medicines to treat patients with diabetic nephropathy (DN). In the previous studies, we showed that puerarin exerts renoprotective effects in Streptozocin (STZ)-induced diabetic mice through activation of Sirt1 and anti-oxidative effects. Here, we further investigated the underlying mechanism mediating the renal protective effects of puerarin in DN. We studied the effects and mechanism of puerarin in STZ-induced diabetic mice and in cultured immortalized mouse podocytes treated with high glucose. We confirmed that puerarin ameliorated urinary albumin creatinine ratio and kidney injury in STZ-induced DN mice. We found that expression of heme oxygenase 1 (HMOX-1) and Sirt1 was suppressed in diabetic glomeruli but restored by puerarin treatment at both mRNA and protein levels. Additionally, we found that puerarin induced autophagy in the kidney of DN mice. In conditionally immortalized mouse podocytes, puerarin inhibited HG-induced apoptosis and restored the mRNA and protein levels of HMOX-1 and Sirt1. Interestingly, we showed that puerarin decreased liver kinase B1 (LKB1) acetylation, thereby promoting adenosine 5′-monophosphate-activated protein kinase-dependent autophagy. Knockdown of HMOX-1 and Sirt1 expression or treatment with the autophagy inhibitor 3-methyladenine abolished the protective effects of puerarin in HG-treated podocytes. Taken together, these results suggest that puerarin protects podocytes from diabetes-induced injury through HMOX1 and Sirt1-mediated upregulation of autophagy, a novel mechanism explaining its renal protective effects in DN.

Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

ABSTRACT Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis. CMA modulates proteomic organization through selective protein degradation, with targets including metabolic enzymes, cell growth regulators, and neurodegeneration-related proteins. CMA activity is low in ad libitum-fed rodents but is increased by prolonged fasting. AKT negatively regulates CMA at the lysosomal membrane by phosphorylating and inhibiting the CMA regulator GFAP. We have previously reported that long-lived Pou1f1/Pit1 mutant (Snell) mice and ghr (growth hormone receptor) knockout mice (ghr KO) have lower AKT activity when fed compared to littermate controls, suggesting the hypothesis that these mice have increased baseline CMA activity. Here, we report that liver lysosomes from fed Snell dwarf mice and ghr KO mice have decreased GFAP phosphorylation and increased CMA substrate uptake activity. Liver lysosomes isolated from fed Snell dwarf mice and ghr KO mice injected with the protease inhibitor leupeptin had increased accumulation of endogenous CMA substrates, compared to littermate controls, suggesting an increase in CMA in vivo. Mice with liver-specific ablation of GH (growth hormone) signaling did not have increased liver CMA, suggesting that a signaling effect resulting from a loss of growth hormone in another tissue causes enhanced CMA in Snell dwarf and ghr KO mice. Finally, we find Snell dwarf mice have decreased protein levels (in liver and kidney) of CIP2A, a well-characterized CMA target protein, without an associated change in Cip2a mRNA. Collectively, these data suggest that CMA is enhanced downstream of an endocrine change resulting from whole-body ablation of GH signaling. Abbreviations: CMA: chaperone-mediated autophagy; GH: growth hormone; ghr KO: growth hormone receptor knockout; LAMP2A: splice variant 1 of Lamp2 transcript; LC3-I: non-lipidated MAP1LC3; LC3-II: lipidated MAP1LC3; Li-ghr KO: liver-specific ghr knockout; MA: macroautophagy; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; PBS: phosphate-buffered saline.

TORC1 regulates vacuole membrane composition through ubiquitin- and ESCRT-dependent microautophagy.

Cellular adaptation in response to nutrient limitation requires the induction of autophagy and lysosome biogenesis for the efficient recycling of macromolecules. Here, we discovered that starvation and TORC1 inactivation not only lead to the up-regulation of autophagy and vacuole proteins involved in recycling but also result in the down-regulation of many vacuole membrane proteins to supply amino acids as part of a vacuole remodeling process. Down-regulation of vacuole membrane proteins is initiated by ubiquitination, which is accomplished by the coordination of multiple E3 ubiquitin ligases, including Rsp5, the Dsc complex, and a newly characterized E3 ligase, Pib1. The Dsc complex is negatively regulated by TORC1 through the Rim15-Ume6 signaling cascade. After ubiquitination, vacuole membrane proteins are sorted into the lumen for degradation by ESCRT-dependent microautophagy. Thus, our study uncovered a complex relationship between TORC1 inactivation and vacuole biogenesis.

Association of SMAD4 loss with response to neoadjuvant chemotherapy with the autophagy inhibitor hydroxychloroquine in patients with pancreatic adenocarcinoma.

761 Background: SMAD4, a tumor suppressor gene, is inactivated or deleted in 60-90% of pancreatic adenocarcinomas (PDA). Loss of SMAD4 allows tumor progression by limiting cell cycle arrest and apoptosis and increasing metastases. SMAD4 deficient PDA cells are resistant to radiotherapy by upregulation of autophagy, a cell survival mechanism that allows intracellular recycling of macromolecules and organelles. Hydroxychloroquine (HCQ) is a known autophagy inhibitor, suggesting that HCQ treatment in SMAD4 deficient PDA may prevent therapeutic resistance induced by autophagy upregulation. Methods: We retrospectively analyzed the SMAD4 status of PDA patients enrolled in two prospective clinical trials evaluating preoperative HCQ. The first dose escalation trial demonstrated the safety of preoperative gemcitabine with HCQ ( NCT01128296 ). More recently, a randomized trial of gemcitabine/nab-paclitaxel +/- HCQ evaluated Evans Grade histopathologic response ( NCT01978184 ). Immunohistochemistry of resected specimens for SMAD4 was previously performed. Patients not treated at the max HCQ dose (n = 5), not resected (n = 2) or with SMAD4 staining unavailable were excluded (n = 10). The effect of SMAD4 loss on response to HCQ and chemotherapy was studied for association with clinical outcome. Fisher’s exact test and log-rank test were used to assess response and survival. Results: 52 patients receiving HCQ with neoadjuvant chemotherapy and 24 patients receiving neoadjuvant chemotherapy alone were studied. Of the HCQ group, 25 patients had SMAD4 loss (48%), compared with 15 control patients (63%, p = 0.32). 76% of HCQ treated patients with SMAD4 loss obtained a histopathologic response ≥2A, compared to only 37% with SMAD4 intact (p = 0.006). In the control group, loss of SMAD4 was associated with a nonsignificant detriment in 3 year OS (25% vs. 78%, p = 0.3) that was less apparent in patients treated with HCQ (46% vs. 47%, p = 0.18). Conclusions: The addition of HCQ to neoadjuvant chemotherapy in PDA may improve treatment response in patients with SMAD4 loss. Further study of the relationship between SMAD4, autophagy and treatment outcomes in PDA is warranted.

The Role of Autophagy in Cancer Radiotherapy.

Autophagy, a pathway for lysosomal-mediated cellular degradation, is a catabolic process that recycles intracellular components to maintain metabolism and survival. It is classified into three major types: macroautophagy, microautophagy, and the chaperone-mediated autophagy (CMA). Autophagy is a dynamic and multistep process that includes four stages: nucleation, elongation, autophagosome formation, and fusion. Interestingly, the influence of autophagy in cancer development is complex and paradoxical, suppressive, or promotive in different contexts. Autophagy in cancer has been demonstrated to serve as both a tumour suppressor and promoter. Radiotherapy is a powerful and common strategy for many different types of cancer and can induce autophagy, which has been shown to modulate sensitivity of cancer to radiotherapy. However, the role of autophagy in radiation treatment is controversial. Some reports showed that the upregulation of autophagy was cytoprotective for cancer cells. Others, in contrast, showed that the induction of autophagy was advantageous. Here, we reviewed recent studies and attempted to discuss the various aspects of autophagy in response to radiotherapy of cancer. Thus, we could decrease the viability of cancer cell and increase the sensibility of cancer cells to radiation, providing a new basis for the application of autophagy in clinical tumor radiotherapy.

Exenatide Improves the Survival of Random-Pattern Skin Flap &lt;i&gt;via&lt;/i&gt; TFE3 Mediated Autophagy Augment

Background: Random skin flaps are widely applied to rebuild and restore soft-tissue damage in reconstructive surgery. Nevertheless, severe ischemia and ischemia/reperfusion injury lead to flap necrosis, a major complication after operation occurring in reconstructive surgery. Exenatide exert therapeutic role in diabetic wounds, cardiac injury, and nonalcoholic fatty liver disease. However, whether Exenatide has a protective effect against flap necrosis remains unknown, and the underlying mechanism should be explored further. Methods: One hundred and forty-four mice were stochastically divided into the Control (n=30), Exe(n=30), Exe + 3 methyladenine (3MA) (n=24), Exe + chloroquine (CQ) (n=24), Exe + Scramble control (n=18), and Exe + TFE3 shRNA groups (n = 18). Flap tissues were obtained on postoperative day 7 to evaluate tissue vitality, angiogenesis, pyroptosis, oxidative stress, and autophagy using Immunohistochemistry, Immunofluorescence staining and western blotting. Findings: We found Exenatide increased the flap vitality, enhanced angiogenesis, reduced oxidative stress and alleviated pyroptosis and activate autophagy in random skin flap. Inhibition of autophagy by 3MA and CQ reversed the above effects of Exenatide in random skin flap, suggesting that autophagy exert a major role in pro-survival effect in skin flap. In mechanistic investigation, activation and nuclear translocation of TFE3 was found to mediate Exenatide – induced autophagy. Besides, nuclear translocation of TFE3 may result from activation of AMPK-SPK2-CARM1 and AMPK-mTOR signaling axis. Interpretation: Our findings revealed that Exenatide boosted the viability, enhanced angiogenesis, reduced oxidative stress, alleviated pyroptosis via upregulation of autophagy. These indicated that Exenatide is a potent autophagy enhancer and capable for promoting the viability of random skin flap, providing a promising novel therapeutic strategy. Funding Statement: Financial supports from Public Welfare Technology Research Project of Natural Science Foundation of China (No. 81601705 to Kailiang Zhou, No.81873942 to Weiyang Gao, No. 81801930 to Jian Ding, No. 81572227 and No. 81873992 to Huazi Xu); Zhejiang Provincial Medicine and Health Technology Project (No. 2017KY472 to Kailiang Zhou); Wenzhou Science and Technology Bureau Foundation (No.2016Y0350 to Jian Ding). Declaration of Interests: The author states that there is no conflict of interest. Ethics Approval Statement: Experimental steps involving animals strictly abided by the Guide for the Care and Use of Laboratory Animals of the China National Institutes of Health, which were approved of the Animal Care and Use Committee of Wenzhou Medical University (wydw2017–0022).

Endothelial protective effect of rapamycin against simulated ischemia injury through up-regulation of autophagy and inhibition of endoplasmic reticulum stress

IntroductionRapamycin has been shown to have cytoprotective properties in some experimental models of ischemia. However, the precise molecular mechanisms underlying the positive effect of rapamycin on endothelial cells in ischemic injury remain unknown. It is very important because endothelial cells are firstly exposed to ischemia and play an important role in ischemic organ damage. Autophagy and endoplasmic reticulum stress are suggested to be implicated in hypoxic/ischemic injury of endothelial cells. This study aims to explore whether the endothelial protective effect of rapamycin is associated with exacerbation of autophagy and attenuation of endoplasmic reticulum stress.Material and methodsThe protective effects of rapamycin against oxygen and glucose deprivation (OGD)-induced cell injury were explored in human vascular endothelial cells (HUVECs). Cell viability was measured by MTT assay. The protein levels of Beclin 1, p62, p-mTOR, p-S6K, p-4EBP, GRP78, p-PERK and p-IRE1 were analyzed using immunoblotting.ResultsRapamycin in the simulated ischemia model increased the cell viability, indicating its cytoprotective effect (p &lt; 0.05). Experiments with 3-methyladenine as an inhibitor of autophagy and thapsigargin as an inducer of endoplasmic reticulum stress support that rapamycin exerts endothelial protective effects against OGD-induced damage via autophagy – endoplasmic reticulum stress pathway.ConclusionsThis study demonstrated that rapamycin protects ischemic HUVECs via down-regulation of the mTOR pathway, enhancement of autophagy and inhibition of endoplasmic reticulum stress.

The Acetone Indigo Red Dehydrating Agent IF203 Induces HepG2 Cell Death Through Cell Cycle Arrest, Autophagy and Apoptosis.

BackgroundIsatin derivatives have extensive biological activities, such as antitumor. IF203, a novel isatin derivative, has not previously been reported to have antitumor activity.MethodsAcid phosphatase assays (APAs) and Ki-67 immunohistochemistry were used to detect the proliferation of HepG2 cells. Transmission electron microscope (TEM) was applied to detect ultrastructural changes. Flow cytometry (FCM) was used to detect cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) of HepG2 cells in vitro. TUNEL, MMP and ROS immunofluorescence assays were applied to assess apoptosis, MMP, and ROS of HepG2 cells in vivo. Western Blotting was applied to assess the levels of apoptosis- and autophagy-related proteins.ResultsIn this study, in vivo and in vitro experiments showed that IF203 possesses antitumor activity. The results of APAs and Ki-67 immunohistochemistry demonstrated that IF203 could inhibit the proliferation of HepG2 cells. Cell cycle assays, downregulation of Cyclin B1 and Cdc2, and upregulation of P53 suggested that IF203 could lead to G2/M cell cycle arrest. In addition, ultrastructural changes, apoptosis assays, TUNEL immunofluorescence results, upregulated expression of Bax, and downregulated expression of Bcl-2 suggest that IF203 can induce apoptosis in HepG2 cells. After IF203 treatment, intracellular ROS levels increased, MMP decreased, JC-1 green fluorescence was enhanced, and the levels of Caspase-9, Caspase-3 and Cytochrome C expression were upregulated, suggesting that IF203 could induce apoptosis of HepG2 cells through the mitochondrial apoptosis pathway. Moreover, characteristic apoptotic ultrastructural changes were accompanied by the appearance of many autophagy bubbles and upregulation of Atg5, Atg12, ULK1, Beclin-1 and LC3-II proteins, suggesting that IF203 could induce autophagy in HepG2 cells.ConclusionThis study showed that IF203 leads to the death of HepG2 cells through cell cycle arrest, apoptotic induction, and autophagy promotion.

Autophagy in kidney disease: Advances and therapeutic potential.

Autophagy is a highly conserved intracellular catabolic process for the degradation of cytoplasmic components that has recently gained increasing attention for its importance in kidney diseases. It is indispensable for the maintenance of kidney homeostasis both in physiological and pathological conditions. Investigations utilizing various kidney cell-specific conditional autophagy-related gene knockouts have facilitated the advancement in understanding of the role of autophagy in the kidney. Recent findings are raising the possibility that defective autophagy exerts a critical role in different pathological conditions of the kidney. An emerging body of evidence reveals that autophagy exhibits cytoprotective functions in both glomerular and tubular compartments of the kidney, suggesting the upregulation of autophagy as an attractive therapeutic strategy. However, there is also accumulating evidence that autophagy could be deleterious, which presents a formidable challenge in developing therapeutic strategies targeting autophagy. Here, we review the recent advances in research on the role of autophagy during different pathological conditions, including acute kidney injury (AKI), focusing on sepsis, ischemia-reperfusion injury, cisplatin, and heavy metal-induced AKI. We also discuss the role of autophagy in chronic kidney disease (CKD) focusing on the pathogenesis of tubulointerstitial fibrosis, podocytopathies including focal segmental glomerulosclerosis, diabetic nephropathy, IgA nephropathy, membranous nephropathy, HIV-associated nephropathy, and polycystic kidney disease.

Therapeutic effect of regulating autophagy in spinal cord injury: a network meta-analysis of direct and indirect comparisons.

Objective:An increasing number of studies indicate that autophagy plays an important role in the pathogenesis of spinal cord injury, and that regulating autophagy can enhance recovery from spinal cord injury. However, the effect of regulating autophagy and whether autophagy is detrimental or beneficial after spinal cord injury remain unclear. Therefore, in this study we evaluated the effects of autophagy regulation on spinal cord injury in rats by direct and indirect comparison, in an effort to provide a basis for further research.Data source:Relevant literature published from inception to February 1, 2018 were included by searching Wanfang, CNKI, Web of Science, MEDLINE (OvidSP), PubMed and Google Scholar in English and Chinese. The keywords included “autophagy”, “spinal cord injury”, and “rat”.Data selection:The literature included in vivo experimental studies on autophagy regulation in the treatment of spinal cord injury (including intervention pre- and post-spinal cord injury). Meta-analyses were conducted at different time points to compare the therapeutic effects of promoting or inhibiting autophagy, and subgroup analyses were also conducted.Outcome measure:Basso, Beattie, and Bresnahan scores.Results:Of the 622 studies, 33 studies of median quality were included in the analyses. Basso, Beattie, and Bresnahan scores were higher at 1 day (MD = 1.80, 95% CI: 0.81–2.79, P = 0.0004), 3 days (MD = 0.92, 95% CI: 0.72–1.13, P < 0.00001), 1 week (MD = 2.39, 95% CI: 1.85–2.92, P < 0.00001), 2 weeks (MD = 3.26, 95% CI: 2.40–4.13, P < 0.00001), 3 weeks (MD = 3.13, 95% CI: 2.51–3.75, P < 0.00001) and 4 weeks (MD = 3.18, 95% CI: 2.43–3.92, P < 0.00001) after spinal cord injury with upregulation of autophagy compared with the control group (drug solvent control, such as saline group). Basso, Beattie, and Bresnahan scores were higher at 1 day (MD = 6.48, 95% CI: 5.83–7.13, P < 0.00001), 2 weeks (MD = 2.43, 95% CI: 0.79–4.07, P = 0.004), 3 weeks (MD = 2.96, 95% CI: 0.09–5.84, P = 0.04) and 4 weeks (MD = 4.41, 95% CI: 1.08–7.75, P = 0.01) after spinal cord injury with downregulation of autophagy compared with the control group. Indirect comparison of upregulation and downregulation of autophagy showed no differences in Basso, Beattie, and Bresnahan scores at 1 day (MD = −4.68, 95% CI: –5.840 to −3.496, P = 0.94644), 3 days (MD = −0.28, 95% CI: −2.231–1.671, P = 0.99448), 1 week (MD = 1.83, 95% CI: 0.0076–3.584, P = 0.94588), 2 weeks (MD = 0.81, 95% CI: −0.850–2.470, P = 0.93055), 3 weeks (MD = 0.17, 95% CI: −2.771–3.111, P = 0.99546) or 4 weeks (MD = –1.23, 95% CI: −4.647–2.187, P = 0.98264) compared with the control group.Conclusion:Regulation of autophagy improves neurological function, whether it is upregulated or downregulated. There was no difference between upregulation and downregulation of autophagy in the treatment of spinal cord injury. The variability in results among the studies may be associated with differences in research methods, the lack of clearly defined autophagy characteristics after spinal cord injury, and the limited autophagy monitoring techniques. Thus, methods should be standardized, and the dynamic regulation of autophagy should be examined in future studies.

SIRT1 Protects Against Apoptosis by Promoting Autophagy in the Oxygen Glucose Deprivation/Reperfusion-Induced Injury

Silent information regulator 1 (SIRT1) contributes to cellular regulation. Previous studies have reported SIRT1 to be abnormally expressed in the ischemic penumbra of cerebral ischemia/reperfusion (I/R) injury rat model. We investigated the effect of SIRT1 on oxygen and glucose deprivation/reperfusion (OGD/R) cell injury. Over-expressed or silenced SIRT1 pheochromocytoma 12 (PC12) cells were exposed to an in-vitro OGD/R injury. Western blot, TUNEL staining and immunofluorescence analyses were performed to assess apoptosis and autophagy. We found autophagy and apoptosis to be up-regulated and down-regulated, respectively, following the over-expression of SIRT1 in the OGD/R-induced PC12 cells. We also found the silencing of SIRT1 to culminate in the down-regulation and up-regulation of autophagy and apoptosis, respectively. On the basis of our results, we surmise that SIRT1 can promote autophagy and inhibit apoptosis in-vitro, and thus exhibit potential neuroprotection against OGD/R-induced injury. This could facilitate in the development of therapeutic approaches for cerebral I/R injury.

Autophagy up-regulation by ulipristal acetate as a novel target mechanism in the treatment of uterine leiomyoma: an in vitro study

To assess the effect of ulipristal acetate (UPA) on the autophagic process of uterine leiomyoma cells. Invitro study in primary cultures of leiomyoma and myometrial cells isolated from biopsy specimen, and gene expression evaluation in biopsy material. Cellular pathology laboratory. Premenopausal women (without hormonal treatment) undergoing myomectomy or hysterectomy for symptomatic leiomyomas. Surgical specimens collected from uterine leiomyomas and matched normal myometria. After treatment of myometrial and leiomyoma cells with UPA, autophagy was evaluated by Western blot analysis of the typical biochemical markers, LC3-II, LC3-II:LC3-I ratio, and p62/SQSTM1. The expression level of Atg7 and Atg4D proteins was also assessed by Western blot. The increase of LC3-II protein, LC3-II:LC3-I ratio, and p62/SQSTM1 protein indicates that UPA treatment up-regulates the autophagic response in leiomyoma cells, whereas these markers were almost unchanged in myometrial cells. Consistently, an increased level of Atg7 and Atg4D proteins was observed only in UPA-treated leiomyoma cells. The autophagic machinery is put into motion selectively in these cells, despite that the basal messenger RNA levels of LC3, SQSTM1, and ATG7 in leiomyoma biopsy specimen were not significantly different from those found in normal myometrial biopsy material. Invitro UPA treatment stimulates the autophagic response selectively in leiomyoma cells, which adds a novel indication for the clinical use of this selective P receptor (PR) modulator. Autophagy up-regulation may potentially contribute to the leiomyoma shrinkage occurring in UPA-treated patients and warrants further study.

Punicalagin Reversed the Hepatic Injury of Tetrachloromethane by Antioxidation and Enhancement of Autophagy.

Hepatic injury is significant in the pathogenesis and development of many types of liver diseases. Punicalagin (PU) is a bioactive antioxidant polyphenol found in pomegranates. To explore its protective effect against carbon tetrachloride (CCl4)-induced liver injury and the mechanism, Institute of Cancer Research (ICR) mice and L02 cells were used to observe the changes of serum biochemical indicators, histopathological liver structure, cell viability, antioxidative indices, and autophagy-related proteins were assessed. In ICR mice, PU ameliorated the CCl4-induced increase of the serum aspartate aminotransferase, alanine aminotransferase, the activity of liver lactate dehydrogenase, and the damage of histopathological structure, and exhibited a hepatoprotective effect against CCl4. PU attenuated oxidative stress by decreasing the liver malondialdehyde level and increasing the activities of liver superoxide dismutase, glutathione peroxidase, and the expression of the liver nuclear factor E2-related factor (Nrf2) protein. Furthermore, according to the vivo and vitro experiments, PU might activate autophagy through the mediation of the Akt/FOXO3a and P62/Nrf2 signaling pathway. Taken together, these results suggest that PU may protect against CCl4-induced liver injury through the upregulation of antioxidative activities and autophagy.

FGF21 augments autophagy in random-pattern skin flaps via AMPK signaling pathways and improves tissue survival

Random-pattern skin flap is commonly used for surgical tissue reconstruction due to its ease and lack of axial vascular limitation. However, ischemic necrosis is a common complication, especially in distal parts of skin flaps. Previous studies have shown that FGF21 can promote angiogenesis and protect against ischemic cardiovascular disease, but little is known about the effect of FGF21 on flap survival. In this study, using a rat model of random skin flaps, we found that the expression of FGF21 is significantly increased after establishment skin flaps, suggesting that FGF21 may exert a pivotal effect on flap survival. We conducted experiments to elucidate the role of FGF21 in this model. Our results showed that FGF21 directly increased the survival area of skin flaps, blood flow intensity, and mean blood vessel density through enhancing angiogenesis, inhibiting apoptosis, and reducing oxidative stress. Our studies also revealed that FGF21 administration leads to an upregulation of autophagy, and the beneficial effects of FGF21 were reversed by 3-methyladenine (3MA), which is a well-known inhibitor of autophagy, suggesting that autophagy plays a central role in FGF21’s therapeutic benefit on skin flap survival. In our mechanistic investigation, we found that FGF21-induced autophagy enhancement is mediated by the dephosphorylation and nuclear translocation of TFEB; this effect was due to activation of AMPK-FoxO3a-SPK2-CARM1 and AMPK-mTOR signaling pathways. Together, our data provides novel evidence that FGF21 is a potent modulator of autophagy capable of significantly increasing random skin flap viability, and thus may serve as a promising therapy for clinical use.

AMPK-ERK/CARM1 Signaling Pathways Affect Autophagy of Hepatic Cells in Samples of Liver Cancer Patients.

Liver cancer is one of the most common malignant tumors, with the death rate ranking fourth among all types of cancer. Over the past few decades, several studies have reported that liver tumorigenesis is associated with dysfunction in autophagy. However, the detailed mechanism remains unclear. In this paper, we used tissue micro-array (TMA) of liver cancer to detect proteins associated with the regulation of autophagic signaling in non-cancerous and cancerous regions by immunohistochemical staining. Those proteins contained 4-HNE, p-AMPK, Erk1/2, p-Erk1/2, CARM1, TFEB, LAMP1, and p62. According to the degrees of tumor differentiation in patients (well differentiated group vs. moderately and poorly differentiated group), we analyzed each protein's expression in the ratio of the “cancerous region/non-cancerous region” in two groups. Current data showed that there were AMPK-ERK/CARM1 autophagic signaling pathways during the formation of liver cancer. The above-mentioned changes in signals indicated an upregulation of autophagy in cancerous regions, which means overactivated autophagy plays an important role in liver cancer.