Bioavailability and Metabolism of Bioactive Peptide IRW with Angiotensin-Converting Enzyme 2 (ACE2) Upregulatory Activity in Spontaneously Hypertensive Rats.

Peptide IRW is the first food-derived angiotensin-converting enzyme 2 (ACE2) upregulator. This study aimed to investigate the pharmacokinetic characteristics of IRW and identify the metabolites contributing to its antihypertensive activity in spontaneously hypertensive rats (SHRs). Rats were administered 100 mg of IRW/kg of the body weight via an intragastric or intravenous route. The bioavailability (F %) was determined to be 11.7%, and the half-lives were 7.9 ± 0.5 and 28.5 ± 6.8 min for gavage and injection, respectively. Interestingly, significant blood pressure reduction was not observed until 1.5 h post oral administration, or 2 h post injection, indicating that the peptide's metabolites are likely responsible for the blood pressure-lowering activity. Time-course metabolomics revealed a significant increase in the level of kynurenine, a tryptophan metabolite, in blood after IRW administration. Kynurenine increased the level of ACE2 in cells. Oral administration of tryptophan (W), but not dipeptide IR, lowered the blood pressure and upregulated aortic ACE2 in SHRs. Our study supports the key role of tryptophan and its metabolite, kynurenine, in IRW's blood pressure-lowering effects.

The role of FPR2-mediated ferroptosis in formyl peptide-induced acute lung injury against endothelial barrier damage and protective effect of the mitochondria-derived peptide MOTS-c.

BACKGROUND: Acute lung injury (ALI) has garnered significant attention in the field of respiratory and critical care due to its high mortality and morbidity, and limited treatment options. The role of the endothelial barrier in the development of ALI is crucial. Several bacterial pathogenic factors, including the bacteria-derived formyl peptide (fMLP), have been implicated in damaging the endothelial barrier and initiating ALI. However, the mechanism by which fMLP causes ALI remains unclear. In this study, we aim to explore the mechanisms of ALI caused by fMLP and evaluate the protective effects of MOTS-c, a mitochondrial-derived peptide. METHODS: We established a rat model of ALI and a human pulmonary microvascular endothelial cell (HPMVEC) model of ALI by treatment with fMLP. In vivo experiments involved lung histopathology assays, assessments of inflammatory and oxidative stress factors, and measurements of ferroptosis-related proteins and barrier proteins to evaluate the severity of fMLP-induced ALI and the type of tissue damage in rats. In vitro experiments included evaluations of fMLP-induced damage on HPMVEC using cell activity assays, assessments of inflammatory and oxidative stress factors, measurements of ferroptosis-related proteins, endothelial barrier function assays, and examination of the key role of FPR2 in fMLP-induced ALI. We also assessed the protective effect of MOTS-c and investigated its mechanism on the fMLP-induced ALI in vivo and in vitro. RESULTS: Results from both in vitro and in vivo experiments demonstrate that fMLP promotes the expression of inflammatory and oxidative stress factors, activates ferroptosis and disrupts the vascular endothelial barrier, ultimately contributing to the development and progression of ALI. Mechanistically, ferroptosis mediated by FPR2 plays a key role in fMLP-induced injury, and the Nrf2 and MAPK pathways are involved in this process. Knockdown of FPR2 and inhibition of ferroptosis can attenuate ALI induced by fMLP. Moreover, MOTS-c could protect the vascular endothelial barrier function by inhibiting ferroptosis and suppressing the expression of inflammatory and oxidative stress factors through Nrf2 and MAPK pathways, thereby alleviating fMLP-induced ALI. CONCLUSION: Overall, fMLP disrupts the vascular endothelial barrier through FPR2-mediated ferroptosis, leading to the development and progression of ALI. MOTS-c demonstrates potential as a protective treatment against ALI by alleviating the damage induced by fMLP.

Unraveling the potential of segment scan mass spectral acquisition for chemical isotope labeling LC-MS-based metabolome analysis: Performance assessment across different types of biological samples.

BACKGROUND: Chemical isotope labeling (CIL) LC-MS is a powerful tool for metabolome analysis with high metabolomic coverage and quantification accuracy. In CIL LC-MS, the overall metabolite detection efficiency using Orbitrap MS can be further improved by employing a segment scan method where the full m/z range is divided into multiple segments for spectral acquisition with a significant increase in the in-spectrum dynamic range. Considering the metabolic complexity in different types of biological samples (e.g., feces, urine, serum/plasma, cell/tissue extracts, saliva, etc.), we report the development and evaluation of the segment scan method for metabolome analysis of different sample types. RESULTS: It was found that sample complexity significantly influenced the performance of the segment scan method. In metabolically complex samples such as feces and urine, the method yielded a substantial increase (up to 94 %) in detected peak pairs or metabolites, compared to conventional full scan. Conversely, less complex samples like saliva exhibited more modest gains (approximately 25 %). Based on the observations, a 120-m/z segment scan method was determined as a routine approach for CIL LC-Orbitrap-MS-based metabolomics with good compatibility with different types of biological samples. For this method, a further investigation on relative quantification accuracy was done. The peak area ratios of 12C-/13-labeled metabolites were slightly reduced with 72%-84 % of peak pairs falling within the ±25 % range of the anticipated peak ratio of 1.0 among different samples, as opposed to 81%-90 % in the full scan, which was attributed to the inclusion of more low-abundance peak pairs within the narrow MS segments. However, the overall peak ratio measurement precision was not significantly affected by the segment scan. SIGNIFICANCE AND NOVELTY: The segment scan method was found to be useful for CIL LC-Orbitrap-MS-based metabolome analysis of different types of samples with significant improvement in metabolite detectability (25-94 % increase), compared to the conventional full scan method.

Integration of Protein Nanocage with CpG Motifs: A Virus-Mimicked Core-Shell Nanostructure to Ignite Antitumor Immunity.

The tumor microenvironment typically possesses immunosuppressive properties that hinder the effectiveness of antitumor immune responses, even in the context of immunotherapies. However, it is observed that pathogenic microorganisms can trigger strong immune responses during infection, offering a potential means to counteract the immunosuppressive environment of tumors. In this study, a protein nanocage called CpG@HBc nanocages (NCs) is developed, which mimics the structure of the hepatitis B virus and combines with an immunostimulatory component known as cytosine phosphoguanosine oligonucleotide (CpG). By delivering these immunostimulatory agents, CpG@HBc NCs are able to effectively reverse the suppressive tumor microenvironment, resulting in the inhibition of poorly immunogenic tumors in mice. Through high-dimensional mass cytometry (CyTOF) analysis, remarkable alterations in immune responses is observed induced by CpG@HBc. Treatment with immunogenic CpG@HBc NCs, along with co-injection of an OX40 agonist, sensitized colorectal cancer tumors to T cell immune responses, resulting in significant impairment of tumor growth and robust immune activation. Furthermore, CpG@HBc NCs induced long-term antitumor immunological memory, protecting tumor-cured mice from tumor rechallenge. Overall, these findings highlight the potential of a virus-inspired protein nanocage to mimic anti-viral immunity and offer a unique therapeutic approach for cancer immunotherapy.

The mitochondrial-derived peptide MOTS-c suppresses ferroptosis and alleviates acute lung injury induced by myocardial ischemia reperfusion via PPARγ signaling pathway.

Acute lung injury (ALI) is a life-threatening complication of cardiac surgery that has a high rate of morbidity and mortality. Epithelial ferroptosis is believed to be involved in the pathogenesis of ALI. MOTS-c has been reported to play a role in regulating inflammation and sepsis-associated ALI. The purpose of this study is to observe the effect of MOTS-c on myocardial ischemia reperfusion (MIR)-induced ALI and ferroptosis. In humans, we used ELISA kits to investigate MOTS-c and malondialdehyde (MDA) levels in patients undergoing off-pump coronary artery bypass grafting (CABG). In vivo, we pretreated Sprague-Dawley rats with MOTS-c, Ferrostatin-1 and Fe-citrate(Ⅲ). We conducted Hematoxylin and Eosin (H&E) staining and detection of ferroptosis-related genes in MIR-induced ALI rats. In vitro, we evaluated the effect of MOTS-c on hypoxia regeneration (HR)-induced mouse lung epithelial-12 (MLE-12) ferroptosis and analyzed the expression of PPARγ through western blotting. We found that circulating MOTS-c levels were decreased in postoperative ALI patients after off-pump CABG, and that ferroptosis contributed to ALI induced by MIR in rats. MOTS-c suppressed ferroptosis and alleviated ALI induced by MIR, and the protective effect of MOTS-c- was dependent on PPARγ signaling pathway. Additionally, HR promoted ferroptosis in MLE-12 cells, and MOTS-c inhibited ferroptosis against HR through the PPARγ signaling pathway. These findings highlight the therapeutic potential of MOTS-c for improving postoperative ALI induced by cardiac surgery.

Site-Specific Modification of Virus-Like Particles for Exogenous Tumor Antigen Display and Minimizing Preexisting Immunity.

The widespread preexisting immunity against virus-like particles (VLPs) seriously limits the applications of VLPs as vaccine vectors. Enabling technology for exogenous antigen display should not only ensure the assembly ability of VLPs and site-specific modification, but also consider the effect of preexisting immunity on the behavior of VLPs in vivo. Here, combining genetic code expansion technique and synthetic biology strategy, a site-specific modification method for hepatitis B core (HBc) VLPs via incorporating azido-phenylalanine into the desired positions is described. Through modification position screening, it is found that HBc VLPs incorporated with azido-phenylalanine at the main immune region can effectively assemble and rapidly conjugate with the dibenzocycolctyne-modified tumor-associated antigens, mucin-1 (MUC1). The site-specific modification of HBc VLPs not only improves the immunogenicity of MUC1 antigens but also shields the immunogenicity of HBc VLPs themselves, thereby activating a strong and persistent anti-MUC1 immune response even in the presence of preexisting anti-HBc immunity, which results in the efficient tumor elimination in a lung metastatic mouse model. Together, these results demonstrate the site-specific modification strategy enabled HBc VLPs behave as a potent antitumor vaccine and this strategy to manipulate immunogenicity of VLPs may be suitable for other VLP-based vaccine vectors.

Sequential administration of virus-like particle-based nanomedicine to elicit enhanced tumor chemotherapy.

Protein cages have played a long-standing role in biomedicine applications, especially in tumor chemotherapy. Among protein cages, virus like particles (VLPs) have received attention for their potential applications in vaccine development and targeted drug delivery. However, most of the existing protein-based platform technologies are plagued with immunological problems that may limit their systemic delivery efficiency as drug carriers. Here, we show that using immune-orthogonal protein cages sequentially and modifying the dominant loop epitope can circumvent adaptive immune responses and enable effective drug delivery using repeated dosing. We genetically modified three different hepadnavirus core protein derived VLPs as delivery vectors for doxorubicin (DOX). These engineered VLPs have similar assembly characteristics, particle sizes, and immunological properties. Our results indicated that there was negligible antibody cross-reactivity in either direction between these three RGD-VLPs in mice that were previously immunized against HBc VLPs. Moreover, the sequential administration of multiple RGD-VLP-based nanomedicine (DOX@RGD-VLPs) could effectively reduce immune clearance and inhibited tumor growth. Hence, this study could provide an attractive protein cage-based platform for therapeutic drug delivery.

High-Sensitivity Cardiac Troponin T in Prediction and Diagnosis of Early Postoperative Hypoxemia after Off-Pump Coronary Artery Bypass Grafting.

To investigate the relationship of preoperative high-sensitivity cardiac troponin T (hs-cTnT) with early postoperative hypoxemia (EPH) following off-pump coronary artery bypass grafting (OPCAB). Records of patients undergoing OPCAB between 2018 and 2022 were reviewed. Baseline characteristics and postoperative arterial blood gas analysis were derived from the cardiovascular surgery electronic medical records. Preoperative hs-cTnT levels were measured routinely in all patients. Logistic regression analyses were performed to test the association of preoperative hs-cTnT with EPH. A total of 318 OPCAB patients were included, who had a preoperative hs-cTnT test available for review. Before surgery, 198 patients (62%) had a rise in hs-cTnT level (≥14 ng/L) and 127 patients (40%) had a more severe hs-cTnT level (≥25 ng/L). The preoperative hs-cTnT level was associated with EPH (odds ratio per ng/L, 1.86; 95% confidence interval 1.30−2.68; p < 0.001), prolonged intensive care unit stay (odds ratio, 1.58; 95% confidence interval 1.08−2.32; p = 0.019), and delayed extubating time (odds ratio, 1.63; 95% confidence interval 1.15−2.34; p = 0.007). On multivariable analysis, adjusted for BMI, hypertension, smoking status, serum creatinine, and cardiac function, preoperative hs-cTnT remained an independent factor associated with EPH. Elevation of hs-cTnT concentrations are significantly associated with EPH after OPCAB. Review of presurgical hs-cTnT concentration may help identify patients who would benefit from OPCAB to improve surgical risk assessment.

IDH1 Promotes Foam Cell Formation by Aggravating Macrophage Ferroptosis.

A distinctive feature of ferroptosis is intracellular iron accumulation and the impairment of antioxidant capacity, resulting in a lethal accumulation of lipid peroxides leading to cell death. This study was conducted to determine whether inhibiting isocitrate dehydrogenase 1 (IDH1) may help to prevent foam cell formation by reducing oxidized low-density lipoprotein (ox-LDL)-induced ferroptosis in macrophages and activating nuclear factor erythroid 2-related factor 2 (NRF2). Gene expression profiling (GSE70126 and GSE70619) revealed 21 significantly different genes, and subsequent bioinformatics research revealed that ferroptosis and IDH1 play essential roles in foam cell production. We also confirmed that ox-LDL elevates macrophage ferroptosis and IDH1 protein levels considerably as compared with controls. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, reduced ox-LDL-induced elevated Fe2+ levels, lipid peroxidation (LPO) buildup, lactate dehydrogenase (LDH) buildup, glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4), ferritin heavy polypeptide 1 (FTH1), and solute carrier family 7 member 11 (SLC7A11) protein downregulation. More crucially, inhibiting IDH1 reduced Fe2+ overload, lipid peroxidation, LDH, and glutathione depletion, and elevated GPX4, FTH1, and SLC7A11 protein expression, resulting in a reduction in ox-LDL-induced macrophage ferroptosis. IDH1 inhibition suppressed ox-LDL-induced macrophage damage and apoptosis while raising NRF2 protein levels. We have demonstrated that inhibiting IDH1 reduces ox-LDL-induced ferroptosis and foam cell formation in macrophages, implying that IDH1 may be an important molecule regulating foam cell formation and may be a promising molecular target for the treatment of atherosclerosis.

Instrument-type effects on chemical isotope labeling LC-MS metabolome analysis: Quadrupole time-of-flight MS vs. Orbitrap MS.

Chemical isotope labeling (CIL) LC-MS is a powerful tool for metabolome analysis with markedly improved metabolomic coverage and quantification accuracy over the conventional LC-MS technique. In addition, with differential isotope labeling, each labeled metabolite is detected as a peak pair in the mass spectra, offering the possibility of differentiating true metabolite peaks from the singlet noise or background peaks. In this study, we examined the effects of instrument type on the detectability of true metabolites with a focus on the comparison of quadrupole time-of-flight (QTOF) and Orbitrap mass spectrometers. Using the same ultra-high-performance liquid chromatography setup and optimized running conditions for QTOF and Orbitrap, we compared the total number of peak pairs detected and identified from the two instruments using human urine and serum as the test samples. Many common peak pairs were detected from the two instruments; however, there were a significant number of unique peak pairs detected in each type of instrument. By combining the datasets obtained using QTOF and Orbitrap, the total number of peak pairs detected could be significantly increased. We also examined the effect of mass resolving power on peak pair detection in Orbitrap (60,000 vs. 120,000 resolution). The observed differences in peak pair detectability were much less than those of QTOF vs. Orbitrap. However, the type of peak pairs detected using different resolutions could be somewhat different, offering the possibility of increasing the overall number of peak pairs by combining the two datasets obtained at two different resolutions. The results from this study clearly indicate that instrument type can have a profound effect on metabolite detection in CIL LC-MS. Therefore, comparison of metabolome data generated using different instruments needs to be carefully done. Moreover, future research (e.g., hardware modifications) is warranted to minimize the differences in order to generate more reproducible metabolome data from different types of instruments.

Segment Scan Mass Spectral Acquisition for Increasing the Metabolite Detectability in Chemical Isotope Labeling Liquid Chromatography-Mass Spectrometry Metabolome Analysis.

We report a segmented spectrum scan method using Orbitrap MS in chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) for improving the metabolite detection efficiency. In this method, the full m/z range is divided into multiple segments with the scanning of each segment to produce multiple narrow-range spectra during the LC data acquisition. These segmented spectra are separately processed to extract the peak pair information with each peak pair arising from a differentially labeled metabolite in the analysis of a mixture of 13C and 12C reagent-labeled samples. The sublists of peak pairs are merged to form the final peak pair list from the LC-MS run. Various experimental conditions, including automatic gain control (AGC) values, mass resolutions, segment m/z widths, number of segments, and total data acquisition time in the LC run, were examined to arrive at an optimal setting in the segment scan for increasing the number of detectable metabolites while maintaining the same analysis time as in the full scan. The optimal method used a segment width of 120 m/z with 60k resolution for a 16 min CIL LC-MS run. Using dansyl-labeled human urine samples as an example, we demonstrated that this method could detect 5867 peak pairs or metabolites (not features), compared to 3765 peak pairs detectable in a full scan, representing a 56% gain. Out of 5867 peak pairs, 5575 (95.0%) could be identified or mass-matched. The relative quantification accuracy was slightly reduced (81% peak pairs were within ±25% of the expected peak ratio of 1.0 in full, compared to 87% in the full scan) due to the inclusion of more low-abundance peak pairs in the segment scan. The peak ratio measurement precision was not significantly affected by the segment scan. We also showed the increase of the peak pair number detectable from 3843 in the full scan to 7273 (89% gain) using the Orbitrap operated at 120k resolution with a 60 m/z segment width when multiple repeat sample injections were used. Thus, segment scan Orbitrap MS is an enabling method for detecting coeluting metabolites in CIL LC-MS for increasing the metabolomic coverage.

Rutin Inhibits Ox-LDL-Mediated Macrophage Inflammation and Foam Cell Formation by Inducing Autophagy and Modulating PI3K/ATK Signaling.

Atherosclerosis (AS) is one of the leading causes of death among the elderly, and is primarily caused by foam cell generation and macrophage inflammation. Rutin is an anti-inflammatory, anti-oxidant, anti-allergic, and antiviral flavonoid molecule, known to have anti-atherosclerotic and autophagy-inducing properties, but its biological mechanism remains poorly understood. In this study, we uncovered that rutin could suppress the generation of inflammatory factors and reactive oxygen species (ROS) in ox-LDL-induced M2 macrophages and enhance their polarization. Moreover, rutin could decrease foam cell production, as shown by oil red O staining. In addition, rutin could increase the number of autophagosomes and the LC3II/I ratio, while lowering p62 expression. Furthermore, rutin could significantly inhibit the PI3K/ATK signaling pathway. In summary, rutin inhibits ox-LDL-mediated macrophage inflammation and foam cell formation by inducing autophagy and modulating PI3K/ATK signaling, showing potential in treating atherosclerosis.

A Meta-Analysis of the Effectiveness of Telemedicine in Glycemic Management among Patients with Type 2 Diabetes in Primary Care.

Introduction: Telemedicine interventions are gradually being used in primary health care to help patients with type 2 diabetes receive ongoing medical guidance. The purpose of this study was to analyze the effectiveness of using telemedicine in primary health care for the management of patients with type 2 diabetes. Methods: A systematic search was conducted from database inception to August 2021 in nine databases, including PubMed, Web of Science, Cochrane Library, EMBASE, EBSCO, CNKI, Wanfang Data, VIP, and CBM. Data extraction and quality assessment were performed for studies that met the inclusion criteria. The meta-analysis was performed using Review Manager 5.4 (Cochrane) and Stata v.16.0SE (College Station, TX, USA). Results: A total of 32 articles were included in this study. Analysis showed a reduction in glycated hemoglobin, fasting glucose, and postprandial glucose after the telemedicine intervention. Systolic blood pressure and self-efficacy improved significantly, but there was no significant improvement in weight, lipid metabolism, or diabetes awareness. Subgroup analysis based on the duration of intervention showed significant improvement in glycated hemoglobin at 6 months of intervention. Conclusions: Telemedicine interventions may help patients with type 2 diabetes to effectively control blood glucose and improve self-management in primary health care. There is only moderate benefit, and the benefit may not be sustained beyond 6 months. However, the evidence for the improvement in lipid metabolism is insufficient and further studies are needed.

Sorption behavior of Pb(II) onto polyvinyl chloride microplastics affects the formation and ecological functions of microbial biofilms.

Microplastics (MPs) are regarded as transport media for heavy metals in aquatic systems, whereas the effects of the heavy metal-enriched MPs on microbial biofilms are still unclear. In this study, Pb(II) sorption onto polyvinyl chloride (PVC) MPs and its effects on the formation and ecological functions of microbial biofilms were investigated. The results showed that the interaction between Pb(II) and PVC MPs was dominated by physisorption. The maximum sorption amount reached 1.25 mg/g. Afterward, microbial biofilms were exposed to the Pb(II)-enriched PVC particles. It is suggested that Pb(II)-enriched PVC exposure reduced productivities of polysaccharides and proteins in extracellular polymeric substances, which restricted the formation of microbial biofilms. Meanwhile, microbial community structure was reassembled accompanying the decline of capacities for nitrate and phosphate removal. Therefore, this study examines the ecological risk associated with the heavy metal-enriched MPs that can adversely affect microbial biofilms.

Optimization of Nanostructured Copper Sulfide to Achieve Enhanced Enzyme-Mimic Activities for Improving Anti-Infection Performance.

Advanced antibacterial methods are urgently needed to deal with possible infectious diseases. As promising alternatives to antibiotics, enzyme-mimic nanocatalysts face bottlenecks of low activities and indistinct catalytic mechanisms, which seriously restrict their development for anti-infection treatment. Herein, metastable copper sulfide (Cu2-xS) nanozymes with diversiform sizes and compositions were selected to adjust the electronic structure for enhancing enzyme-mimic activities. The as-synthesized large and thin nanoplates (L/TN nanoplates), with the stoichiometric ratio of Cu1.25S, were proven to possess the optimal peroxidase (POD)-mimic activity. Using quantum mechanics, it was theoretically revealed that the sulfur vacancies could alter the electronic structure of copper active sites and thus reduce the reaction energy barrier of H2O2 to·OH to promote the POD-mimic performance. Moreover, through enhanced enzyme-mimic activities, L/TN nanoplates achieved efficient depletion of glutathione and ascorbic acid for improving antibacterial performances. Further, synergizing with the NIR irradiation, the satisfactory destruction capability for bacteria and biofilm was achieved for L/TN nanoplates under an inflammatory level of hydrogen peroxide (50 µM). Altogether, this work provides a deeper understanding of geometrical and electronic properties-dependent antibacterial performance, and paves the way toward precise compositions and structures engineering of nanozymes.

Dual-Antigen-Loaded Hepatitis B Virus Core Antigen Virus-like Particles Stimulate Efficient Immunotherapy Against Melanoma.

Tumor cells are rich in antigens, which provide a reliable antigen library for the design of personalized vaccines. However, an effective tumor vaccine vector that can efficiently deliver antigens to lymphoid organs to stimulate strong CD8+ cytotoxic T-lymphocyte immune response is still lacking. Here we designed a dual-antigen delivery system based on hepatitis B virus core antigen virus-like particles (HBc VLPs). We first confirmed that different antigen-loaded HBc VLP monomers could be assembled into nanoparticles (hybrid VLPs). Hybrid VLPs could slightly enhance bone marrow-derived dendritic cell maturation in vitro. Strikingly, hybrid VLPs could generate antigen-specific antitumor immunity and innate immunity in vivo which could significantly inhibit tumor growth or metastatic formation in a subcutaneous tumor or lung metastatic tumor model, respectively. Moreover, dual-epitope vaccination generated enhanced T-cell responses that potently inhibited tumor growth and metastatic formation. Together, this study provides a new powerful concept for cancer immunotherapy and suggests a novel design for VLP-based personalized nanomedicine.

Estimation of PM2.5 mortality burden in China with new exposure estimation and local concentration-response function.

The estimation of PM2.5-related mortality is becoming increasingly important. The accuracy of results is largely dependent on the selection of methods for PM2.5 exposure assessment and Concentration-Response (C-R) function. In this study, PM2.5 observed data from the China National Environmental Monitoring Center, satellite-derived estimation, widely collected geographic and socioeconomic information variables were applied to develop a national satellite-based Land Use Regression model and evaluate PM2.5 exposure concentrations within 2013-2015 with the resolution of 1 km × 1 km. Population weighted concentration declined from 72.52 µg/m3 in 2013 to 57.18 µg/m3 in 2015. C-R function is another important section of health effect assessment, but most previous studies used the Integrated Exposure Regression (IER) function which may currently underestimate the excess relative risk of exceeding the exposure range in China. A new Shape Constrained Health Impact Function (SCHIF) method, which was developed from a national cohort of 189,793 Chinese men, was adopted to estimate the PM2.5-related premature deaths in China. Results showed that 2.19 million (2013), 1.94 million (2014), 1.65 million (2015) premature deaths were attributed to PM2.5 long-term exposure, different from previous understanding around 1.1-1.7 million. The top three provinces of the highest premature deaths were Henan, Shandong, Sichuan, while the least ones were Tibet, Hainan, Qinghai. The proportions of premature deaths caused by specific diseases were 53.2% for stroke, 20.5% for ischemic heart disease, 16.8% for chronic obstructive pulmonary disease and 9.5% for lung cancer. IER function was also used to calculate PM2.5-related premature deaths with the same exposed level used in SCHIF method, and the comparison of results indicated that IER had made a much lower estimation with less annual amounts around 0.15-0.5 million premature deaths within 2013-2015.