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Keloid is commonly regarded as a benign skin tumour. Some keloids clinically exhibit hard tissue texture similar to that of cartilage or bone. We hypothesized that the keloid pathological niche environment is likely to induce keloid MSCs towards chondrogenic or osteogenic differentiation and leads to cartilage or bone-like tissue formation. The differences in tissue ossification, histology, mechanical properties, abnormal extracellular matrices and chondrogenic/osteogenic gene expression among sclerous keloids (SKs), regular keloids (RKs) and normal skins (NKs) were carefully examined. The sporadic ossified islets existed in SK group whereas no ossified/chondrified islet was found in other groups by micro-CT reconstruction. HE, Masson trichrome and safranin O staining revealed lacuna-like structures in SKs, which were featured as bone/cartilage histology. Immunohistochemical staining showed overproduction of osteoprotegerin, type I and III collagen in SK group but similar production level of aggrecan among three groups. The biomechanical analysis demonstrated the weakest compliance of SK tissues. In addition, SK fibroblasts exhibited a relatively slower proliferation rate but higher expression levels of osteogenic and chondrogenic genes among all three groups. These cell populations also showed the strongest potential for lineage transformation. In conclusion, we first reported the presence of ossified and chondrified matrices in some extremely hard keloids in the present study.

Efficient and sustainable technologies for cleaning of contaminated water and sediments are in urgent demand. In this study, a new type of sediment microbial fuel cell coupled floating bed (FB-SMFC) was developed to repair eutrophic water and sediment in a cleaner way. The effect of electrode spacing on the power generation capacity and the synchronous remediation of pollutants from eutrophic water and sediment were studied. When the electrode distance was 60 cm, the maximum power generation and pollutant removal effects were obtained. At the end of the experiment, the maximum output voltage was 0.4 V, and the chemical oxygen demand (CODCr, potassium dichromate method), total nitrogen (TN), and total phosphorus (TP) contents in the overlying water were 8 mg/L, 0.7 mg/L, and 0.39 mg/L. The corresponding removal rates were 88.2%, 78.8%, and 59.0%, respectively. The removal rates of organic matter and TN in the sediment were 12.8% and 86.4%, respectively, and the fixation rate of TP was 29.2%. Proteobacteria was the dominant phylum of bacteria in the sediment and anode. Many anaerobic bacteria were found in the overlying water, which facilitated denitrification. Overall, the results of this research revealed a highly efficient and reliable strategy for eutrophic water and sediment remediation, aquatic ecosystems restoration, and human health protection.

Clinically used inhibitors of mammalian target of rapamycin (mTOR) negatively impacts endothelial-dependent vasodilatation (EDD) through unidentified mechanisms. Here we show that either the endothelium-specific deletion of Mtor to inhibit both mTOR complexes, or depletion of Raptor or Rictor to disrupt mTORC1 or mTORC2, causes impaired EDD, accompanied by reduced NO in the serum of mice. Consistently, inhibition of mTOR decreases NO production by human and mouse EC. Specifically, inhibition of mTORC1 suppresses eNOS gene expression, due to impairment in p70S6K-mediated posttranscriptional regulation of the transcription factor KLF2 expression. In contrast to mTORC1 inhibition, a positive-feedback between MAPK (p38 and JNK) activation and Nox2 upregulation contributes to the excessive generation of reactive oxygen species (ROS), which causes eNOS uncoupling and decreased NO bioavailability in mTORC2-inhibited EC. Adeno-associated virus-mediated EC-specific overexpression of KLF2 or suppression of Nox2 restores EDD function in endothelial mTORC1- or mTORC2-inhibited mice.

Inflammation is actively involved in the clinical manifestation of peripheral artery disease (PAD). Sonodynamic therapy (SDT), a novel non-invasive, plaque-based, macrophage-targeted anti-inflammatory regimen for atherosclerosis has the potential to improve walking performance by reducing plaque inflammation.This phase-2, randomized, sham-controlled, double-blind clinical trial enrolled 32 participants with symptomatic femoropopliteal PAD. The primary outcome was the 30-day change in the target-to-background ratio (TBR) within the most diseased segment (MDS) of the femoropopliteal artery assessed through positron emission tomography/computed tomography (PET/CT). The secondary outcomes were changes in walking performance, limb perfusion, lesional morphology and quality of life measurements.The mean age was 64.7 years and 63% were male. Thirty-one completed follow-up. SDT significantly decreased the MDS TBR by 0.53 (95% CI, -0.70 to -0.36, P 0.001) compared with control. Furthermore, SDT increased peak walking time by 118.6 s (95% CI, 74.3 to 163.0, P 0.001), increased ankle-brachial index by 0.11 (95% CI, 0.07 to 0.14, P 0.001), decreased lesional diameter and area stenosis by 7.2% (95% CI, -8.6 to -4.5, P 0.001) and 9.6% (95% CI, -24.5 to -5.3, P = 0.005), respectively, and increased the walking speed score of the Walking Impairment Questionnaire by 16.1 (95% CI, 2.6 to 29.5, P = 0.021) and the physical functioning score of the 36-item Short-Form Health Survey by 10.0 (95% CI, 5.0 to 20.0, P = 0.003) compared with control. These improvements were maintained in the SDT group up to 6-month.SDT rapidly reduced plaque inflammation and improved walking performance among patients with symptomatic PAD.Clinical Trials NCT03457662.

Aims Mammalian target of rapamycin (mTOR) inhibitors used in drug-eluting stents (DES) to control restenosis have been found to delay endothelialization and increase incidence of late-stent thrombosis through mechanisms not completely understood. We revealed that mTOR inhibition (mTORi) upregulated the expression of cell growth suppressor IRF-1 in primary human arterial endothelial cells (HAEC). This study aimed to examine how mTOR-regulated IRF-1 expression contributes to the suppressive effect of mTORi on arterial endothelial proliferation. Methods and results Western blotting, quantitative PCR, and a dual-luciferase reporter assay indicated that mTOR inhibitors rapamycin and torin 1 upregulated IRF-1 expression and increased its transcriptional activity. IRF-1 in turn contributed to the suppressive effect of mTORi by mediating HAEC apoptosis and cell cycle arrest in part through upregulation of caspase 1 and downregulation of cyclin D3, as revealed by CCK-8 assay, Annexin V binding assay, measurement of activated caspase 3, BrdU incorporation assay, and matrigel tube formation assay. In a mouse model of femoral artery wire injury, administration of rapamycin inhibited EC recovery, an effect alleviated by EC deficiency of IRF-1. Chromatin immunoprecipitation assay with HAEC and rescue expression of wild type or dominant-negative IRF-1 in EC isolated from Irf1−/− mice confirmed transcriptional regulation of IRF-1 on the expression of CASP1 and CCND3. Furthermore, mTORi activated multiple PKC members, among which PKCζ was responsible for the growth-inhibitory effect on HAEC. Activated PKCζ increased IRF1 transcription through JAK/STAT-1 and NF-κB signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKCζ activation in mTOR-inhibited HAEC. Conclusions The study reveals an IRF-1-mediated mechanism that contributes to the suppressive effects of mTORi on HAEC proliferation. Further study may facilitate the development of effective strategies to reduce the side effects of DES used in coronary interventions.

Huanglian Jiedu Decoction (HLJDD) has the effect of clearing heat and detoxifying, and has been considered as an effective prescription for cerebral ischemia (CI) for thousands of years in traditional Chinese medicine (TCM). It can improve the quality of life of patients with ischemic stroke, but its pharmacological mechanism remains unclear.The study aimed to explore the pharmacological action and potential mechanism of HLJDD against CI by systems pharmacology, proteomics and in vivo experiments.In this study, databases such as TCMIP V2.0 and Genecards were used to predict compounds, targets and CI related targets, and network topology criteria of protein-protein interaction (PPI) network was used to screen core targets. The Database for Annotation, Visualization and Integrated Discovery database (DAVID) was used to discover biological processes and pathways. In addition, molecular docking was performed between the screened core biological active compounds and targets to verify the binding activity. Finally, proteomics and Western blot were performed on cerebral cortex tissues of middle cerebral artery occlusion (MCAO) model rats with HLJDD intervention to further verify the predicted results.77 compounds and 308 targets of HLJDD were identified, 54 of which were predicted to be associated with cerebral ischemia. PPI network and enrichment results showed that 8 targets, including AKT1, PTGS2 and TLR4, were core targets of HLJDD in CI. And 19 signaling pathways, including Rap1 signaling pathway, cAMP signaling pathway and arachidonic acid metabolism, were identified as key pathways to the therapeutic activity of HLJDD in CI. Combined with proteomics studies, we identified that Rap1 signaling pathway and upstream and downstream targets were the key mechanisms. Molecular biology experiments showed that RAP1A and AKT expression levels were significantly up-regulated in middle cerebral artery occlusion (MCAO) rats treated with HLJDD (P 0.0001), GRIN1 expression level was significantly down-regulated (P 0.0001). However, ACTB expression level was slightly down-regulated (P 0.05), which may be related to the biological function.This study confirms the pharmacological effect of HLJDD on cerebral ischemia. These results indicate that HLJDD mediates various pathways such as inhibition of apoptosis, regulation of oxygen balance, inhibition of excitatory toxicity and maintenance of basic cell functions to improve CI by regulating Rap1 signaling pathway.

Our initial studies detected elevated levels of 3,4-dihydroxyphenyllactic acid (DHPLA) in urine samples of patients with severe heart disease when compared with healthy subjects. Given the reported anti-inflammatory properties of DHPLA and related dihydroxylated phenolic acids (DPAs), we embarked on an exploratory multi-centre investigation in patients with no urinary tract infections to establish the possible pathophysiological significance and therapeutic implications of these findings. Chinese and Caucasian patients being treated for severe heart disease or those conditions associated with inflammation (WBC ≥ 10 ×10

Acrolein, a known toxin in tobacco smoke, has been demonstrated to be associated with inflammatory cardiovascular diseases, such as atherosclerosis. However, the definite mechanism of acrolein-induced inflammation remains unclear. Here, we report that acrolein induces reactive oxygen species (ROS) production in EAhy926 cells. Additionally, acrolein induces EAhy926 cells' inflammatory response and pyroptosis by activating NOD-like receptor protein 3 (NLRP3) inflammasome. Also, acrolein-induced cytotoxicity could be attenuated by N-acetyl-L-cysteine (NAC). Furthermore, acrolein upregulates the level of autophagy which can be reversed by NAC. Notably, the present study also indicates that autophagy inhibited by inhibitor 3-methyladenine (3MA) and siAtg7 exacerbate acrolein-induced NLRP3 inflammasome activation and pyroptosis. In summary, acrolein induced cytotoxicity by ROS-mediated NLRP3 inflammasome activation, and ROS upregulates the level of autophagy to inhibit the NLRP3 inflammasome excessive activation, indicating the bidirectional role of ROS in acrolein-induced cellular inflammation. Our results may provide novel mechanistic insights into acrolein-induced cardiovascular toxicity.

Acrolein is a common environmental pollutant that has been linked to cardiovascular diseases, such as atherosclerosis (AS). Increasing evidence demonstrates that acrolein impairs the cardiovascular system by targeting vascular endothelial cells, but the underlying mechanisms haven't been completely elucidated. In human umbilical vein endothelial cells (HUVECs), we observed that acrolein treatment induced cell reactive oxygen species (ROS) generation, autophagy, pyroptosis and reduced cell migration. In addition, exposure to acrolein resulted in NLRP3 inflammasome activation as evidenced by cleavage of caspase-1 and downstream mature interleukin (IL)-1β and IL-18 secretion. Knockdown of NLRP3 by small interfering RNA remarkably suppressed acrolein-induced pyroptosis and increased cell migration. Moreover, the scavenging ROS relieved the autophagy, NLRP3 inflammasome activation and pyroptosis. Furthermore, the role of autophagy in the acrolein-medicated pyroptosis and cell migration was investigated. In our study, 3-methyladenine (3-MA), an autophagy inhibitor, aggravated NLRP3 inflammasome activation, pyroptosis and decreased cell migration, rapamycin (Rapa), an autophagy inducer, alleviated aforementioned phenomenon under acrolein stress. Besides, we found damaged mitochondrion accentuated NLRP3 inflammasome and pyroptosis in acrolein-treated cells. In conclusion, it is possible that acrolein induced cell pyroptosis and suppressed cell migration via ROS-dependent autophagy. What's more, NLRP3 inflammasome activation plays a key role in this process.

Owing to the human activities such as smelting and mining, arsenic (As), lead (Pb) and cadmium (Cd) seriously polluted the soil of non-ferrous metal mining areas, thus efficient methods for the simultaneous immobilization of the three heavy metals are urgently needed. In the present study, Mg-Al modified biochars (MABs) were synthesized through a simple one-pot pyrolysis method to immobilize the three heavy metals. According to the BET (Brunauer-Emmett-Teller) test method, MABs had larger specific surface areas than biochar. Compared to the materials obtained at 300 °C and 700 °C, MAB with a pyrolysis temperature of 500 °C (MAB 500) had a significant immobilization effect on As, Pb and Cd in the Gansu mining area. Compared with BC, the removal efficiencies of As, Pb and Cd increased from -62%, 17% and 5% to 52%, 100% and 66%, respectively. And the toxicity characteristic leaching procedure (TCLP) test showed that the leaching concentrations of the three heavy metals in the treated soil were all lower than the standard value. X-ray photoelectron spectroscopy and kinetic experiments showed that there were various mechanisms in the immobilization process of the three heavy metals, and the large specific surface area and the multi-Mg/Al-OH of MABs play an important role in this process. More charges were provided by larger specific surface for ion exchange with heavy metals. In addition, larger specific surface area also provided more adsorption sites. More complex sites were provided by Mg/Al-OH to form Mg/Al-O-M then immobilize the heavy metals. In summary, the immobilization mechanism may involve electrostatic attraction, precipitation/co-precipitation, and surface complexation.

BackgroundThe ongoing pandemic of coronavirus disease 2019 (COVID-19) has caused serious concerns about its potential adverse effects on pregnancy. There are limited data on maternal and neonatal outcomes of pregnant women with COVID-19 pneumonia.MethodsWe conducted a case-control study to compare clinical characteristics and maternal and neonatal outcomes of pregnant women with and without COVID-19 pneumonia.ResultsDuring the period 24 January–29 February 2020, there were 16 pregnant women with confirmed COVID-19 pneumonia and 18 suspected cases who were admitted to labor in the third trimester. Two had vaginal delivery and the rest were cesarean delivery. Few patients presented respiratory symptoms (fever and cough) on admission, but most had typical chest computed tomographic images of COVID-19 pneumonia. Compared to the controls, patients with COVID-19 pneumonia had lower counts of white blood cells (WBCs), neutrophils, C-reactive protein (CRP), and alanine aminotransferase on admission. Increased levels of WBCs, neutrophils, eosinophils, and CRP were found in postpartum blood tests of pneumonia patients. Three (18.8%) of the mothers with confirmed COVID-19 pneumonia and 3 (16.7%) with suspected COVID-19 pneumonia had preterm delivery due to maternal complications, which were significantly higher than in the control group. None experienced respiratory failure during their hospital stay. COVID-19 infection was not found in the newborns, and none developed severe neonatal complications.ConclusionsSevere maternal and neonatal complications were not observed in pregnant women with COVID-19 pneumonia who had vaginal or cesarean delivery. Mild respiratory symptoms of pregnant women with COVID-19 pneumonia highlight the need of effective screening on admission.

Acrolein, a highly reactive α,β-unsaturated aldehyde, is a toxic component of cigarette smoke. As a lipid peroxidation biomarker, acrolein plays an important role in a wide variety of disease states, such as neurodegenerative, Alzheimer's disease, diabetes and atherosclerosis. Endothelial cell injury is one of the initiating factors of atherosclerosis, but the underlying molecular mechanisms remain unclear. Our study primarily focused on acrolein-induced autophagy–dependent apoptosis and the possible molecular mechanism. The results showed that treatment with acrolein increased the number of intracellular GFP-LC3 II punctuates and the expression of autophagosome biomarker LC3-II, with the low dose (25 μM) or at the early stage of treatment (3 h). Following treatment of EAhy926 cells with acrolein for 6 h, lysosomal permeabilization changed, and cathepsin B (CB) was released. Additionally, acrolein induced the collapse of mitochondrial transmembrane potential, and cytochrome c was released. Furthermore, caspase-3 and caspase-9 activation showed that acrolein induced EAhy926 cell apoptosis. Autophagy inhibitor 3MA and CB inhibitor CA-074 Me (CA) attenuated acrolein-induced apoptosis. Collectively, our results suggested that acrolein-induced apoptosis is autophagy-dependent, occurring via injury to lysosomes and mitochondria. This study provides new mechanistic insight toward understanding the pathogenesis of acrolein-related disorders.

The increasing wall stress as is elicited by arterial hypertension promotes their reorganization in the vessel wall which may lead to arterial stiffening and contractile dysfunction. The nuclear factor of activated T cells 5 (NFAT5) pathway plays a role in regulating growth and differentiation in various cell types. We investigated whether the NFAT5 pathway was involved in the regulation of biomechanical stretch-induced human arterial smooth muscle cell (HUASMC) proliferation, inflammation, and migration. Herein, we showed that stretch promoted the expression of NFAT5 in human arterial smooth muscle cells and regulated through activation of c-Jun N-terminal kinase under these conditions. This may contribute to an improved activity of HUASMCs and thus promote reorganization in vascular remodeling processes such as hypertension-induced arterial stiffening and contractile dysfunction.

The cardiotoxicity of doxorubicin (DOX) limits its clinical use in the treatment of a variety of solid tumors and malignant hematologic disease. However, the mechanism by which it causes cardiotoxicity is not fully understood. Apoptosis has been regarded as one of mechanisms underlying the cardiotoxic effects of DOX. In our study, we found that treatment of human umbilical vein endothelial cells (HUVECs) with DOX induced autophagy and apoptosis in a dose- and time-dependent manner. Treatment with DOX induced autophagy at earlier time (3 h), then lysosomal membrane permeabilization (LMP) altered after treatment for 12 h which followed by the release of cathepsin D (CTSD). Lysosome-associated membrane proteins-1 and -2 (LAMP1 and LAMP2) were decreased in DOX-treated cells. Additionally, DOX induced the collapse of mitochondrial transmembrane potential, reduction of translocase of the outer mitochondrial membrane-20 (TOM-20), and release of cytochrome c. Furthermore, autophagy inhibitor 3-MA relieved DOX-induced apoptosis as assessed by the expression of cleaved caspase-3, cleaved caspase-9 and TUNEL assay. CTSD inhibitor, pepstatin A, upregulated TOM-20 and suppressed the mitochondria release of cytochrome c as well as apoptosis under DOX stress. Pyrroloquinoline quinine (PQQ), a new B vitamin, ameliorated aforementioned phenomenon. In conclusion, our results suggested that DOX-induced apoptosis was autophagy-dependent via lysosomal-mitochondrial axis. PQQ had an ability to protect cell from autophagy-dependent apoptosis induced by DOX via lysosomal-mitochondrial axis to some extent. This study provided new mechanistic insight toward understanding the pathogenesis of DOX-induced cardiotoxicity and the protection effect of PQQ.

Stress urinary incontinence (SUI) is a common hygienic problem affecting the quality of women’s life worldwide. In this research, we revealed the involvement and regulation of extracellular matrix (ECM) remodeling, oxidative damage, and TGF-β1 signaling in the pathological mechanisms of mechanical trauma-induced SUI. We found that excessive mechanical strain significantly increased apoptosis rate, decreased cell viability and ECM production, and broke the balance of MMPs/TIMPs compared with the nonstrain control (NC) group. The expression levels of TGFβ1, p-Smad3, Nrf2, GPx1, and CAT were downregulated, the production of ROS, 8-OHdG, 4-HNE, and MDA was increased, and the nuclear translocation of Smad2/3 was suppressed after 5333 μstrain’s treatment. Both mTGF-β1 pretreatment and Nrf2 overexpression could reverse mechanical injury-induced TGFβ1/Smad3 signaling inhibition and ECM remodeling, whereas mTGF-β1 had no effect on Nrf2 expression. Nrf2 overexpression significantly alleviated mechanical injury-induced ROS accumulation and oxidative damage; in contrast, Nrf2 silencing exhibited opposite effects. Besides, vaginal distention- (VD-) induced in vivo SUI model was used to confirm the in vitro results; Nrf2 knockout aggravates mechanical trauma-induced LPP reduction, ECM remodeling, oxidative damage, and TGF-β1/Smad3 suppression in mice. Therefore, we deduce that mechanical injury-induced ECM remodeling might be associated with Nrf2/ARE signaling suppression mediating TGF-β1/Smad3 signaling inhibition. This might reflect a new molecular target for SUI researches.

Curcumin is extracted from the rhizomes of the traditional Chinese herbCurcuma longaand has been proposed to function as a photosensitizer. The potential use of curcumin as a sonosensitizer for sonodynamic therapy (SDT) requires further exploration. This study investigated the sonodynamic effect of curcumin on macrophages, the pivotal inflammatory cells in atherosclerotic plaque. THP-1-derived macrophages were incubated with curcumin at a concentration of 40.7 μmol/L for 2 h and then exposed to pulse ultrasound irradiation (2 W/cm2with 0.86 MHz) for 5–15 min. Six hours later, cell viability was decreased in cells that had been treated with ultrasound for 10 and 15 min. After ultrasound irradiation for 15 min, the ratio of apoptotic and necrotic cells in SDT group was higher than that in ultrasound group, and the ratio of apoptotic cells was higher than that of necrotic cells. Both loss of mitochondrial membrane potential and morphological changes of cytoskeleton were apparent 2 h after treatment with curcumin SDT. These findings support that curcumin had sonodynamic effect on THP-1-derived macrophages and that curcumin SDT could be a promising treatment for atherosclerosis.

Objective To investigate the relationship between the genetic polymorphisms of apolipoprotein M (ApoM) and the susceptibility to rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS) among Chinese Han population in Lanzhou. Methods Primers for the two single nucleotide polymorphism (SNP) sites (rs805296 and rs805297) in ApoM gene were designed and their genotyping methods of polymerase chain reaction-high resolution melting (PCR-HRM) assay were established. Case-control studies were performed among the 599 cases of RA, 194 cases of SLE, 179 cases of AS and 273 matched healthy controls to analyze the correlations between the two SNPs and the susceptibility to rheumatic diseases. Results The genotype frequencies of rs805296 were AA 87.0%, AG 12.7%, GG 0.3% in RA cases, AA 84.5%, AG 15.0%, GG 0.5% in SLE cases, AA 91.6%, AG 7.3%, GG 1.1% in AS cases, AA 85.0%, AG 15.0%, GG 0% in healthy controls. The ones of rs805297 were GG 38.2%, GT 51.8%, TT 10.0% in RA cases, GG 44.3%, GT 45.4%, TT 10.3% in SLE cases, GG 37.4%, GT 47.5%, TT 15.1% in AS cases, GG 40.7%, GT 46.1%, TT 13.2% in healthy controls. Statistical analyses showed that only the genotype distribution of rs805296 was significantly different between the AS cases and the healthy controls. Under the dominant model, the G allele carriers of rs805296 (AG heterozygote and GG homozygote) were found to significantly decrease the risk for AS development. Conclusion The established PCR-HRM genotyping assays in the present study can successfully achieve the molecular diagnosis of the two SNPs sites (rs805296 and rs805297) from clinical samples, and the study found a significant association between the SNP of rs805296 and the susceptibility to AS among Chinese Han population in Lanzhou.

Skin aging principles play important roles in skin disease diagnosis, the evaluation of skin cosmetic effect, forensic identification and age identification in sports competition, etc. This paper proposes a new method to evaluate the skin aging objectively and quantitatively by skin texture area. Firstly, the enlarged skin image was acquired. Then, the skin texture image was segmented by using the iterative threshold method, and the skin ridge image was extracted according to the watershed algorithm. Finally, the skin ridge areas of the skin texture were extracted. The experiment data showed that the average areas of skin ridges, of both men and women, had a good correlation with age (the correlation coefficient r of male was 0.938, and the correlation coefficient r of female was 0.922), and skin texture area and age regression curve showed that the skin texture area increased with age. Therefore, it is effective to evaluate skin aging objectively by the new method presented in this paper.

Jiali Cheng,1,* Xin Sun,1,2,* Shuyuan Guo,1,* Wei Cao,1 Haibo Chen,1 Yinghua Jin,1 Bo Li,1 Qiannan Li,1 Huan Wang,1 Zhu Wang,3 Qi Zhou,3 Peng Wang,3 Zhiguo Zhang,3 Wenwu Cao,3,4 Ye Tian1,21Division of Cardiology, the First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, Harbin, People’s Republic of China; 2Division of Pathophysiology, the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education, Harbin, People’s Republic of China; 3Laboratory of Photo- and Sono-theranostic Technologies and Condensed Matter Science and Technology Institute, Harbin Institute of Technology, Harbin, People’s Republic of China; 4Materials Research Institute, The Pennsylvania State University, University Park, PA, USA*These authors contributed equally to this workBackground: Inflammatory cells exhibit an elevated level of protoporphyrin IX (PpIX) after the administration of 5-aminolevulinic acid (ALA). Here, we investigate the sonodynamic effects of ALA-derived PpIX (ALA-PpIX) on macrophages, which are the pivotal inflammatory cells in atherosclerosis.Methods and results: Cultured THP-1 macrophages were incubated with ALA. Fluorescence microscope and fluorescence spectrometer detection showed that intracellular PpIX increased with the concentration of ALA in the incubation solution in a time dependent manner; the highest level of intracellular PpIX was observed after 3-hour incubation. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays demonstrated that lower concentrations (less than 2 mM) of ALA have no influence on cell viability (more than 90% of cells survived), but sonodynamic therapy (SDT) with a low concentration of ALA significantly decreased the survival rate of cells, and the effect was increased with both ALA concentration and ultrasound exposure time. Cell apoptosis and necrosis induced by ALA-mediated SDT (ALA-SDT) were measured using Hoechst 33258 and propidium iodide assay. ALA-SDT induced both cell apoptosis and necrosis, and the maximum apoptosis/necrosis ratio was observed at 6 hours after SDT with 1 mM of ALA and 5 minutes of ultrasound exposure. Flow cytometry analysis showed that ALA-SDT significantly increased late stage apoptotic cells (about 10-fold control). Furthermore, ALA-SDT induced reactive oxygen species generation in THP-1 macrophages immediately after the treatment and a conspicuous loss of mitochondrial membrane potential (MMP) at 6 hours compared with that of the control, ALA alone, and ultrasound alone groups.Conclusion: ALA-SDT exhibited synergistic apoptotic effects on THP-1 macrophages, involving excessive intracellular reactive oxygen species generation and MMP loss. Therefore, ALA-SDT is a potential treatment for atherosclerosis.Keywords: 5-aminolevulinic acid, protoporphyrin IX, sonodynamic therapy, macrophage, atherosclerosis

OBJECTIVE: To investigate relationships between serum levels of retinol binding protein 4 (RBP4) and clinical and metabolic variables in Chinese patients with type 2 diabetes mellitus. METHODS: A total of 513 patients (286 males/227 females) provided clinical and lifestyle data and blood and urine samples for analysis. Patients were stratified into four quartile groups according to serum RBP4 concentrations. RESULTS: RBP4 concentration was independently associated with gender, systolic blood pressure, serum triglyceride and creatinine levels, and estimated glomerular filtration rate (eGFR). Hypertension, dyslipidaemia, microalbuminuria and impaired eGFR (< 60 ml/min per 1.73 m2) were significantly more prevalent in patients with the highest RBP4 levels than in those with the lowest levels. Increased serum RBP4 was associated with increased risk of hypertension, dyslipidaemia, microalbuminuria and impaired eGFR after adjusting for gender, age, body mass index and duration of diabetes. CONCLUSION: Serum RBP4 may be a useful marker of overall metabolic control in Chinese patients with type 2 diabetes mellitus.