Structural Basis of Smoothened Activation in Hedgehog Signaling.

The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.

Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling.

In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.

Multidomain integration in the structure of the HNF-4α nuclear receptor complex.

The hepatocyte nuclear factor 4α (HNF-4α; also known as NR2A1) is a member of the nuclear receptor (NR) family of transcription factors, which have conserved DNA-binding domains and ligand-binding domains. HNF-4α is the most abundant DNA-binding protein in the liver, where some 40% of the actively transcribed genes have a HNF-4α response element. These regulated genes are largely involved in the hepatic gluconeogenic program and lipid metabolism. In the pancreas HNF-4α is also a master regulator, controlling an estimated 11% of islet genes. HNF-4α protein mutations are linked to maturity-onset diabetes of the young, type 1 (MODY1) and hyperinsulinaemic hypoglycaemia. Previous structural analyses of NRs, although productive in elucidating the structure of individual domains, have lagged behind in revealing the connectivity patterns of NR domains. Here we describe the 2.9 Å crystal structure of the multidomain human HNF-4α homodimer bound to its DNA response element and coactivator-derived peptides. A convergence zone connects multiple receptor domains in an asymmetric fashion, joining distinct elements from each monomer. An arginine target of PRMT1 methylation protrudes directly into this convergence zone and sustains its integrity. A serine target of protein kinase C is also responsible for maintaining domain-domain interactions. These post-translational modifications lead to changes in DNA binding by communicating through the tightly connected surfaces of the quaternary fold. We find that some MODY1 mutations, positioned on the ligand-binding domain and hinge regions of the receptor, compromise DNA binding at a distance by communicating through the interjunctional surfaces of the complex. The overall domain representation of the HNF-4α homodimer is different from that of the PPAR-γ-RXR-α heterodimer, even when both NR complexes are assembled on the same DNA element. Our findings suggest that unique quaternary folds and interdomain connections in NRs could be exploited by small-molecule allosteric modulators that affect distal functions in these polypeptides.

Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity.

CD4(+) T helper lymphocytes that express interleukin-17 (T(H)17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in T(H)17 cell differentiation and function with susceptibility to Crohn's disease, rheumatoid arthritis and psoriasis. Thus, the pathway towards differentiation of T(H)17 cells and, perhaps, of related innate lymphoid cells with similar effector functions, is an attractive target for therapeutic applications. Mouse and human T(H)17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORγt, which is required for induction of IL-17 transcription and for the manifestation of T(H)17-dependent autoimmune disease in mice. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORγt transcriptional activity. Digoxin inhibited murine T(H)17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene specifically inhibited induction of IL-17 in human CD4(+) T cells. Using these small-molecule compounds, we demonstrate that RORγt is important for the maintenance of IL-17 expression in mouse and human effector T cells. These data indicate that derivatives of digoxin can be used as chemical templates for the development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease.

Dual Epidemics of Drug Use and Syphilis Among Chinese Female Sex Workers: Results of Eight Consecutive Cross-Sectional Surveys from 2006 to 2013 in Qingdao, China.

Eight consecutive annual cross-sectional surveys were conducted to examine the trend of the prevalence of HIV, syphilis, drug use and their correlates among female sex workers (FSWs) in Qingdao, China. Among sampled FSWs over the 8 years, a higher proportion of older, married or cohabited, higher education levels and more on-call FSWs were observed in recent years. The syphilis prevalence increased significantly from 1.0 % in 2006 to 13.5 % in 2013, with illicit drug use rate ranging from 21.8 % in 2007 to 55.5 % in 2010. Multivariate logistic regression analyses showed that drug use, syphilis and unprotected vaginal sex predicted each other. The dual epidemics of illicit drug use and syphilis among FSWs underscore the urgency to implement a tailored intervention to curb the dual epidemics while also preventing an HIV epidemic in the context of diversified commercial sex dynamic.

Structure of the intact PPAR-gamma-RXR- nuclear receptor complex on DNA.

Nuclear receptors are multi-domain transcription factors that bind to DNA elements from which they regulate gene expression. The peroxisome proliferator-activated receptors (PPARs) form heterodimers with the retinoid X receptor (RXR), and PPAR-gamma has been intensively studied as a drug target because of its link to insulin sensitization. Previous structural studies have focused on isolated DNA or ligand-binding segments, with no demonstration of how multiple domains cooperate to modulate receptor properties. Here we present structures of intact PPAR-gamma and RXR-alpha as a heterodimer bound to DNA, ligands and coactivator peptides. PPAR-gamma and RXR-alpha form a non-symmetric complex, allowing the ligand-binding domain (LBD) of PPAR-gamma to contact multiple domains in both proteins. Three interfaces link PPAR-gamma and RXR-alpha, including some that are DNA dependent. The PPAR-gamma LBD cooperates with both DNA-binding domains (DBDs) to enhance response-element binding. The A/B segments are highly dynamic, lacking folded substructures despite their gene-activation properties.

Structural overview of the nuclear receptor superfamily: insights into physiology and therapeutics.

As ligand-regulated transcription factors, the nuclear hormone receptors are nearly ideal drug targets, with internal pockets that bind to hydrophobic, drug-like molecules and well-characterized ligand-induced conformational changes that recruit transcriptional coregulators to promoter elements. Yet, due to the multitude of genes under the control of a single receptor, the major challenge has been the identification of ligands with gene-selective actions, impacting disease outcomes through a narrow subset of target genes and not across their entire gene-regulatory repertoire. Here, we summarize the concepts and work to date underlying the development of steroidal and nonsteroidal receptor ligands, including the use of crystal structures, high-throughput screens, and rational design approaches for finding useful therapeutic molecules. Difficulties in finding selective receptor modulators require a more complete understanding of receptor interdomain communications, posttranslational modifications, and receptor-protein interactions that could be exploited for target gene selectivity.

Short sleep time may be the main reason for irregular breakfast to cause overweight-a cross-sectional study.

Introduction: In recent years, the relationship between circadian rhythm and overweight and obesity has attracted the attention of many scholars. Methods: To evaluate association between the duration of sleep and the regularity of breakfast and overweight. A total of 1,178 students from Qingdao University were selected by stratified cluster sampling. There were 601 males (24.69 ± 0.80 years old) and 569 females (24.54 ± 0.70 years old). We used body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) to define overweight levels. Chi-square test, Pearson correlation test, and logistic regression were applied to test association among overweight, sleep duration, sleep onset time, and breakfast regularity. Pittsburgh sleep quality index was used to assess the overall sleep quality of the study subjects. Mediation effect and Sobel test were used to analyze the effect of sleep duration on breakfast regularity and overweight. Results: Only 34.1% of the population ate breakfast every day, and eating breakfast 1-3 times per week was associated with a higher risk of overweight (BMI: OR = 2.183, 95%CI: 1.369,3,481; WC: OR = 2.101, 95%CI: 1.232,3,583; WHR: OR = 2.108, 95%CI: 1.331,3,337). The effects of all types of Usual Breakfast Consumption Frequency on overweight were fully mediated by sleep duration (p < 0.05). In particular, the subjects exercised outdoors more than five times per week slept longer (p < 0.05). Conclusion: Short sleep duration may be the main reason for irregular breakfast leading to overweight. Adequate outdoor exercise is essential for weight maintenance.

Tobacco control knowledge and beliefs among healthcare workers in respiratory departments in Fujian Province, China: A cross-sectional study.

INTRODUCTION: Smoking prevalence is high in China, and healthcare workers are important for tobacco control. This study aimed to determine the smoking status, cognition of tobacco hazards, and smoking cessation-related knowledge among respiratory healthcare workers, and to explore their ability to provide smoking cessation assistance. METHODS: A cross-sectional study was conducted in 2021 among 1028 respiratory healthcare workers from 89 hospitals in Fujian Province, China. A self-designed electronic questionnaire was used to collect data on smoking status, knowledge of smoking hazards, and smoking cessation knowledge. Descriptive statistics were calculated for all questions. Logistic regression analysis was used to explore the relationship between awareness of the tobacco control goals of Healthy China 2030 and demographic characteristics. RESULTS: Among the healthcare workers surveyed, 3.4% were smokers, all of whom were male. Most respondents (99.4%) were aware of smoking as a cause of lung cancer, but awareness of smoking as a cause of non-respiratory cancer was lower. The awareness rate of smoking cessation support was high (>90%), but only 40.0% of participants were aware of the Healthy China 2030 tobacco control targets. Male (HR=2.16; 95% CI: 1.69-2.80) and participation in the cessation clinic (HR=1.47; 95% CI: 1.10-1.96) were associated with higher awareness of the targets. CONCLUSIONS: Respiratory healthcare workers in Fujian Province demonstrated a high level of awareness regarding behavioral and pharmacotherapy support for smoking cessation. In order to enable healthcare workers to play a more active role in tobacco control, there is a need to increase public awareness of smoking cessation services in Fujian Province.

Extracellular carriers control lipid-dependent secretion, delivery, and activity of WNT morphogens.

WNT morphogens trigger signaling pathways fundamental for embryogenesis, regeneration, and cancer. WNTs are modified with palmitoleate, which is critical for binding Frizzled (FZD) receptors and activating signaling. However, it is unknown how WNTs are released and spread from cells, given their strong lipid-dependent membrane attachment. We demonstrate that secreted FZD-related proteins and WNT inhibitory factor 1 are WNT carriers, potently releasing lipidated WNTs and forming active soluble complexes. WNT release occurs by direct handoff from the membrane protein WNTLESS to the carriers. In turn, carriers donate WNTs to glypicans and FZDs involved in WNT reception and to the NOTUM hydrolase, which antagonizes WNTs by lipid moiety removal. WNT transfer from carriers to FZDs is greatly facilitated by glypicans that serve as essential co-receptors in Wnt signaling. Thus, an extracellular network of carriers dynamically controls secretion, posttranslational regulation, and delivery of WNT morphogens, with important practical implications for regenerative medicine.

Nonalcoholic steatohepatitis critically rewires the ischemia/reperfusion-induced dysregulation of cardiolipins and sphingolipids in mice.

Background: Lipid dysregulation plays a fundamental role in nonalcoholic steatohepatitis (NASH), which is an emerging critical risk factor that aggravates hepatic ischemia/reperfusion (I/R) injury. However, the specific lipids that mediate the aggressive I/R injury in NASH livers have not yet been identified. Methods: The mouse model of hepatic I/R injury on NASH was established on C56B/6J mice by first feeding the mice with a Western-style diet to induce NASH, then the NASH mice were subjected to surgical procedures to induce hepatic I/R injury. Untargeted lipidomics were performed to determine hepatic lipids in NASH livers with I/R injury through ultra-high performance liquid chromatography coupled with mass spectrometry. The pathology associated with the dysregulated lipids was examined. Results: Lipidomics analyses identified cardiolipins (CL) and sphingolipids (SL), including ceramides (CER), glycosphingolipids, sphingosines, and sphingomyelins, as the most relevant lipid classes that characterized the lipid dysregulation in NASH livers with I/R injury. CER were increased in normal livers with I/R injury, and the I/R-induced increase of CER was further augmented in NASH livers. Metabolic pathway analysis revealed that the enzymes involved in the synthesis and degradation of CER were highly upregulated in NASH livers with I/R injury, including serine palmitoyltransferase 3 (Sptlc3), ceramide synthase 2 (Cers2), neutral sphingomyelinase 2 (Smpd3), and glucosylceramidase beta 2 (Gba2) that produced CER, and alkaline ceramidase 2 (Acer2), alkaline ceramidase 3 (Acer3), sphingosine kinase 1 (Sphk1), sphingosine-1-phosphate lyase (Sgpl1), and sphingosine-1-phosphate phosphatase 1 (Sgpp1) that catalyzed the degradation of CER. CL were not affected by I/R challenge in normal livers, but CL was dramatically reduced in NASH livers with I/R injury. Consistently, metabolic pathway analyses revealed that the enzymes catalyzing the generation of CL were downregulated in NASH-I/R injury, including cardiolipin synthase (Crls1) and tafazzin (Taz). Notably, the I/R-induced oxidative stress and cell death were found to be aggravated in NASH livers, which were possibly mediated by the reduction of CL and accumulation of CER. Conclusions: The I/R-induced dysregulation of CL and SL were critically rewired by NASH, which might potentially mediate the aggressive I/R injury in NASH livers.

The relationship between sleep quality and daytime dysfunction among college students in China during COVID-19: a cross-sectional study.

Objective: College Students' sleep quality and daytime dysfunction have become worse since the COVID-19 outbreak, the purpose of this study was to explore the relationship between sleep quality and daytime dysfunction among college students during the COVID-19 (Corona Virus Disease 2019) period. Methods: This research adopts the form of cluster random sampling of online questionnaires. From April 5 to 16 in 2022, questionnaires are distributed to college students in various universities in Fujian Province, China and the general information questionnaire and PSQI scale are used for investigation. SPSS26.0 was used to conduct an independent sample t-test and variance analysis on the data, multi-factorial analysis was performed using logistic regression analysis. The main outcome variables are the score of subjective sleep quality and daytime dysfunction. Results: During the COVID-19 period, the average PSQI score of the tested college students was 6.17 ± 3.263, and the sleep disorder rate was 29.6%, the daytime dysfunction rate was 85%. Being female, study liberal art/science/ engineering, irritable (due to limited outdoor), prolong electronic entertainment time were associated with low sleep quality (p < 0.001), and the occurrence of daytime dysfunction was higher than other groups (p < 0.001). Logistics regression analysis showed that sleep quality and daytime dysfunction were associated with gender, profession, irritable (due to limited outdoor), and prolonged electronic entertainment time (p < 0.001). Conclusion: During the COVID-19 epidemic, the sleep quality of college students was affected, and different degrees of daytime dysfunction have appeared, both are in worse condition than before the COVID-19 outbreak. Sleep quality may was inversely associated with daytime dysfunction.

Durability of neutralization against Omicron subvariants after vaccination and breakthrough infection.

Booster immunizations and breakthrough infections can elicit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant neutralizing activity. However, the durability of the neutralization response is unknown. We characterize the sensitivity of BA.1, BA.2, BA.2.75, BA.4/BA.5, BF.7, BQ.1.1, and XBB against neutralizing antibodies from vaccination, hybrid immunity, and breakthrough infections 4-6 months after vaccination and infection. We show that a two-dose CoronaVac or a third-dose ZF2001 booster elicits limited neutralization against Omicron subvariants 6 months after vaccination. Hybrid immunity as well as Delta, BA.1, and BA.2 breakthrough infections induce long-term persistence of the antibody response, and over 70% of sera neutralize BA.1, BA.2, BA.4/BA.5, and BF.7. However, BQ.1.1 and XBB, followed by BA.2.75, are more resistant to neutralization, with neutralizing titer reductions of ∼9- to 41-fold, ∼16- to 63-fold, and ∼4- to 25-fold, respectively. These data highlight additional vaccination in CoronaVac- or ZF2001-vaccinated individuals and provide insight into the durability of neutralization against Omicron subvariants.

Modulation of the proteostasis network promotes tumor resistance to oncogenic KRAS inhibitors.

Despite substantial advances in targeting mutant KRAS, tumor resistance to KRAS inhibitors (KRASi) remains a major barrier to progress. Here, we report proteostasis reprogramming as a key convergence point of multiple KRASi-resistance mechanisms. Inactivation of oncogenic KRAS down-regulated both the heat shock response and the inositol-requiring enzyme 1α (IRE1α) branch of the unfolded protein response, causing severe proteostasis disturbances. However, IRE1α was selectively reactivated in an ER stress-independent manner in acquired KRASi-resistant tumors, restoring proteostasis. Oncogenic KRAS promoted IRE1α protein stability through extracellular signal-regulated kinase (ERK)-dependent phosphorylation of IRE1α, leading to IRE1α disassociation from 3-hydroxy-3-methylglutaryl reductase degradation (HRD1) E3-ligase. In KRASi-resistant tumors, both reactivated ERK and hyperactivated AKT restored IRE1α phosphorylation and stability. Suppression of IRE1α overcame resistance to KRASi. This study reveals a druggable mechanism that leads to proteostasis reprogramming and facilitates KRASi resistance.

Structural basis for catalyzed assembly of the Sonic hedgehog-Patched1 signaling complex.

The dually lipidated Sonic hedgehog (SHH) morphogen signals through the tumor suppressor membrane protein Patched1 (PTCH1) to activate the Hedgehog pathway, which is fundamental in development and cancer. SHH engagement with PTCH1 requires the GAS1 coreceptor, but the mechanism is unknown. We demonstrate a unique role for GAS1, catalyzing SHH-PTCH1 complex assembly in vertebrate cells by direct SHH transfer from the extracellular SCUBE2 carrier to PTCH1. Structure of the GAS1-SHH-PTCH1 transition state identifies how GAS1 recognizes the SHH palmitate and cholesterol modifications in modular fashion and how it facilitates lipid-dependent SHH handoff to PTCH1. Structure-guided experiments elucidate SHH movement from SCUBE2 to PTCH1, explain disease mutations, and demonstrate that SHH-induced PTCH1 dimerization causes its internalization from the cell surface. These results define how the signaling-competent SHH-PTCH1 complex assembles, the key step triggering the Hedgehog pathway, and provide a paradigm for understanding morphogen reception and its regulation.

Beliefs and perceptions of electronic cigarettes among medical staff in respiratory departments of Fujian Province, China, in 2021.

INTRODUCTION: Due to the popularity of e-cigarettes, more and more patients ask about e-cigarettes, and it is particularly important to understand doctors' beliefs and perceptions on e-cigarettes. The aim was to evaluate the belief and perception of electronic cigarettes among medical staff in the respiratory department of medical institutions located in Fujian Province. METHODS: The electronic questionnaires were conveyed to the medical staff of the respiratory department in Fujian Province during March to April 2021. Descriptive statistics were calculated for all questions, and the relationship between relevant factors and the perception of e-cigarette-related statements was analyzed by logistic regression analysis. RESULTS: Among 1028 medical staff in the respiratory departments of Fujian Province, 90.5% of medical staff agreed that electronic cigarettes are harmful to the human body; 61.4% of medical staff agreed that e-cigarettes cannot be regarded as a type of smoking cessation treatment; 71.7% of medical staff agreed that e-cigarettes could be a 'gateway' to other tobacco use; and 69.2% of medical staff agreed that electronic cigarettes are in 'Three No' states. The multivariate logistic regression analysis showed that the respondents' perception of 'e-cigarettes cannot be regarded as a type of smoking cessation treatment' were related to gender, professional title and whether they participated in the cessation clinic. CONCLUSIONS: The medical staff of the respiratory department in Fujian Province put more emphasis on the adverse effects of e-cigarettes on health, but lack the cognition of the effect of e-cigarette smoking cessation. In order to better carry out smoking cessation work, it is necessary to strengthen the training of respiratory medical staff at all levels of medical institutions on e-cigarette knowledge.

Ceramides and sphingosine-1-phosphate mediate the distinct effects of M1/M2-macrophage infusion on liver recovery after hepatectomy.

Post-hepatectomy liver dysfunction is a life-threatening morbidity that lacks efficient therapy. Bioactive lipids involved in macrophage polarization crucially regulate tissue injury and regeneration. Herein, we investigate the key bioactive lipids that mediate the cytotherapeutic potential of polarized-macrophage for post-hepatectomy liver dysfunction. Untargeted lipidomics identified elevation of ceramide (CER) metabolites as signature lipid species relevant to M1/M2 polarization in mouse bone-marrow-derived-macrophages (BMDMs). M1 BMDMs expressed a CER-generation-metabolic pattern, leading to elevation of CER; M2 BMDMs expressed a CER-breakdown-metabolic pattern, resulting in upregulation of sphingosine-1-phosphate (S1P). After infusing M1- or M2-polarized BMDMs into the mouse liver after hepatectomy, we found that M1-BMDM infusion increased M1 polarization and CER accumulation, resulting in exaggeration of hepatocyte apoptosis and liver dysfunction. Conversely, M2-BMDM infusion enhanced M2 polarization and S1P generation, leading to alleviation of liver dysfunction with improved hepatocyte proliferation. Treatment of exogenous CER and S1P or inhibition CER and S1P synthesis by siRNA targeting relevant enzymes further revealed that CER induced apoptosis while S1P promoted proliferation in post-hepatectomy primary hepatocytes. In conclusion, CER and S1P are uncovered as critical lipid mediators for M1- and M2-polarized BMDMs to promote injury and regeneration in the liver after hepatectomy, respectively. Notably, the upregulation of hepatic S1P induced by M2-BMDM infusion may have therapeutic potential for post-hepatectomy liver dysfunction.

Generation of sphingosine-1-phosphate by sphingosine kinase 1 protects nonalcoholic fatty liver from ischemia/reperfusion injury through alleviating reactive oxygen species production in hepatocytes.

BACKGROUND: Nonalcoholic fatty liver (NAFL) is emerging as a leading risk factor of hepatic ischemia/reperfusion (I/R) injury lacking of effective therapy. Lipid dyshomeostasis has been implicated in the hepatopathy of NAFL. Herein, we investigate the bioactive lipids that critically regulate I/R injury in NAFL. METHODS: Lipidomics were performed to identify dysregulated lipids in mouse and human NAFL with I/R injury. The alteration of corresponding lipid-metabolizing genes was examined. The effects of the dysregulated lipid metabolism on I/R injury in NAFL were evaluated in mice and primary hepatocytes. RESULTS: Sphingolipid metabolic pathways responsible for the generation of sphingosine-1-phosphate (S1P) were uncovered to be substantially activated by I/R in mouse NAFL. Sphingosine kinase 1 (Sphk1) was found to be essential for hepatic S1P generation in response to I/R in hepatocytes of NAFL mice. Sphk1 knockdown inhibited the hepatic S1P rise while accumulating ceramides in hepatocytes of NAFL mice, leading to aggressive hepatic I/R injury with upregulation of oxidative stress and increase of reactive oxygen species (ROS). In contrast, administration of exogenous S1P protected hepatocytes of NAFL mice from hepatic I/R injury. Clinical study revealed a significant activation of S1P generation by I/R in liver specimens of NAFL patients. In vitro studies on the L02 human hepatocytes consolidated that inhibiting the generation of S1P by knocking down SPHK1 exaggerated I/R-induced damage and oxidative stress in human hepatocytes of NAFL. CONCLUSIONS: Generation of S1P by SPHK1 is important for protecting NAFL from I/R injury, which may serve as therapeutic targets for hepatic I/R injury in NAFL.