Riptortus pedestris possesses a symbiotic bacterium, Burkholderia insecticola, in its midgut crypts. This symbiont is a typical rod-shaped bacterium under in vitro culture conditions, but changes to a spherical shape inside the gut symbiotic organ of the host insect, suggesting the induction of morphological alterations in B. insecticola by host factors. The present study revealed that a deletion mutant of a peptidoglycan amidase gene (amiC), showing a filamentous chain form in vitro, adapted a swollen L-form-like cell shape in midgut crypts. Spatiotemporal observations of the ΔamiC mutant in midgut crypts revealed the induction of swollen cells, particularly prior to the molting of insects. To elucidate the mechanisms underlying in vivo-specific morphological alterations, the symbiont was cultured under 13 different conditions and its cell shape was examined. Swollen cells, similar to symbiont cells in midgut crypts, were induced when the mutant was treated with fosfomycin, an inhibitor of peptidoglycan precursor biosynthesis. Collectively, these results strongly suggest that the Burkholderia symbiont in midgut crypts is under the control of the host insect via a cell wall-attacking agent.Bacterial cell shapes may be altered by the cell cycle, nutrient availability, environmental stress, and interactions with other organisms. The bean bug  Bacterial cell shape is intrinsically controlled by the cell wall, which is composed of the peptidoglycan sacculus, and by cytoskeleton protein complexes, such as the tubulin homolog FtsZ and actin-like MreB, which function during cell division and elongation , which harbors more than 100 million symbiont cells (R. pedestris are aposymbiotic (i.e. symbiont-free) and the insect acquires B. insecticola specifically from ambient soil during its development  deletion mutant of B. insecticola (ΔamiC) has a filamentous form composed of chains of unseparated cells because cell division is incomplete. Consequently, the mutant has less motility and infection ability  medium, it forms separate cells in minimal medium and its motility and infection ability are restored  and fed dry soybean seeds and a cotton pad containing distilled water with 0.05% ascorbic acid. The container was replaced twice a week. In infection experiments, newborn insects were placed in a Petri dish and fed as described above.The bean bug B. insecticola wild-type strain RPE225  or in MMGlc medium (minimum medium with glucose as the sole carbon source)  supplemented with rifampicin 10 μg mL–1 and kanamycin 30 μg mL–1. Bacto agar 15.0 g L–1 was added for solid media.The GFP-labeled n RPE225  and GFP-C mutant . These bB. insecticola wild type and ΔamiC mutant were pre-cultured in 3 mL MMGlc medium containing 30 μg mL–1 kanamycin at 27°C and 150 rpm in a rotary incubator; 200 μL of the overnight culture was inoculated into 3 mL MMGlc and incubated at 27°C and 150 rpm until the exponential growth phase. After the confirmation of bacterial motility by microscopic observations, bacterial density was adjusted to 107 cells mL–1 by measuring optical density, and the bacterial suspension was provided to insects as their drinking water. These insects were maintained until dissection and further analyses.Three days after hatching, water was removed from the plastic dish containing newly molted 2nd instar nymphs and they were kept overnight without water. This water deprivation stimulated the instant drinking of a bacterial suspension the next day, resulting in the efficient establishment of symbiotic infection. The B. insecticola symbiont cells colonizing M4 crypts, DNA extraction was performed from dissected M4 crypts infected with the wild type or ΔamiC mutant using the QIAmp DNA Mini kit (Qiagen). A 150-base pair fragment of the dnaA gene was amplified by real-time quantitative PCR using KAPA SYBR Fast qPCR polymerase (KAPA Biosystems) and the primer set BSdnaA-F (5′-AGC GCG AGA TCA GAC GGT CGT CGA T-3′) and BSdnaA-R (5′-TCC GGC AAG TCG CGC ACG CA-3′) . The number of B. insecticola symbiont cells was calculated based on a standard curve for the dnaA gene with 10, 102, 103, 104, 105, 106, and 107 copies per reaction of the target PCR fragment.To assess the number of G CA-3′) . The PCRB. insecticola wild type and ΔamiC mutant were pre-cultured in MMGlc medium, with 30 μg mL–1 kanamycin for the mutant, at 30°C and 150 rpm overnight. The overnighter was diluted with fresh MMGlc medium and incubated until the exponential phase. Bacterial cells were collected by centrifugation at 15,000 rpm at room temperature for 5 min. The bacterial pellet was washed with MMnoC medium (minimum medium without any carbon source). Bacterial density was adjusted to OD600=0.05 by MMnoC medium supplemented with a carbon source: glucose 0.5%, fructose 0.5%, mannitol 0.5%, yeast extract 0.3%, or maleic acid 0.2%. In the case of the culture with MMnoC medium without any carbon source, bacterial density was adjusted to OD600=0.5, and these bacteria were then incubated at 27°C with agitation at 150 rpm. Regarding stress exposure conditions, after washing with MMGlc medium, bacterial density was adjusted to OD=0.05 using MMGlc medium. Stress compounds were added as follows: lysozyme from chicken egg white  2.0 mg mL–1, polymyxin B (Sigma) 25 μg mL–1, H2O2 125 μM, or sodium dodecyl sulfate  125 μM. In the case of exposure to –1, bacterial density was adjustedfosfomycin  50 μg mL to OD600=0.05 in MMMSM  medium  modified slightly from NBMSM  medium  buffer under a binocular , and M4 crypts were collected. To observe amiC mutant cells, symbiotic insects at the early 3rd instar stage (a few hours after molting from the 2nd instar), 3rd instar middle stage (1~2 days after molting from the 2nd instar), and 3rd instar late stage (3~4 days after molting from the 2nd instar) were dissected and the posterior region of the midgut was collected. Tissue was stained by 3.75 μg mL–1 4,6′-diamidino-2-phenylindole (DAPI), washed with PBS, fixed by 1% paraformaldehyde, washed again with PBS, mounted in 20% glycerol, and then observed by confocal laser scanning microscopy using the Leica TCS SP8 instrument.In spatiotemporal observations of M4-colonizing ΔBacterial cells were randomly selected from microscopy images and cell shapes were evaluated using ImageJ software (ver.1.51) , and S3.B. insecticola wild-type strain was rod-shaped when cultured in vitro  and exposure to four cell membrane stresses , oxidative stress (hydrogen peroxide), and three osmotic stresses . Among the 13 different culture conditions examined, only fosfomycin exposure induced spherical cells in the B. insecticola wild type and swollen cells in the ΔamiC mutant , which is involved in the biosynthesis of the peptidoglycan precursor UDP-N-acetylglucosamine enolpyruvate .Many bacteria change their cell morphology based on environmental conditions, such as nutrient availability and bacterial stress  , membrane-integrated hopanoids, and several efflux pump mechanisms contribute to this cell envelope stability  . This plin vitro . L-form in vitro . Previouen cells . Fosfomyen cells . It is ten cells . We spectability . C-type amiC mutant in midgut crypts revealed that symbiont cells became swollen, particularly before molting  are secreted in M4 crypts  A Peptidoglycan Amidase Mutant of https://doi.org/10.1264/jsme2.ME20107Supplementary Material


We demonstrated that sirtuins are globally deregulated in BlCa, and specifically SIRT7 downregulation is implicated in EMT, fostering BlCa invasiveness through EZH2-CDH1 axis.Sirtuins are emerging players in cancer biology and other age-related disorders, and their putative role in bladder cancer (BlCa) remains elusive. Further understanding of disease biology may allow for generation of more effective pathway-based biomarkers and targeted therapies. Herein, we aimed to illuminate the role of sirtuins’ family in BlCa and evaluate their potential as disease biomarkers and therapeutic targets. SIRT1-7 transcripts and protein levels were evaluated in a series of primary BlCa and normal bladder mucosa tissues. SIRT7 knockdown was performed through lentiviral transduction in MGHU3, 5637 and J82 cells and its functional role was assessed. SIRT1, 2, 4 and 5 expression levels were significantly lower in BlCa, whereas SIRT6 and 7 were overexpressed, and these results were corroborated by TCGA cohort analysis. SIRT7 transcript levels were significantly decreased in muscle-invasive vs. papillary BlCa. In vitro studies showed that SIRT7 downregulation promoted cells migration and invasion. Accordingly, increased EMT markers expression and decreased E-Cadherin (CDH1) was observed in those BlCa cells. Moreover, increased EZH2 expression and H3K27 Bladder cancer (BlCa) is the 9th most common cancer type worldwide, with an estimated 400,000 new cases and 160,000 deaths per year, in both genders ,3,4.+-dependent deacetylases highly conserved among all living organisms. Seven different SIRTs (SIRT1–7) are described in mammals, also known as Class III histone deacetylases (HDACs), which differing among each other in substrate specificity and catalytic activity .RNA was extracted from tissues and from MGHU3, 5637 and J82 sh-scramble and sh-SIRT7 cells using TRIzolSIRT7 and NCAD transcript levels were quantified using TaqMan® Gene Expression Assay, as described above, in 396 well plates LightCycler480II . For ECAD and EZH2 genes, transcription levels were also evaluated in J82 sh-scramble and sh-SIRT7 cells in 396 well plates LightCycler480II (Roche) using Xpert Fast SYBER Mastermix Blue  with specific primers (S2). Transcript levels for studied genes were then evaluated using ΔΔCt method, with HPRT and BGUS housekeeping genes as reference genes.Concerning cell lines, 1000 ng of RNA were reverse transcribed using RevertAid RT kit , according to manufacturer’s instructions. For 100 ng of cDNA, n = 25, consisting of normal urothelial mucosa collected from the urether of nephrectomy specimens with renal cell tumors) were cut, deparaffinized and rehydrated. Antigen retrieval was accomplished by microwaving the specimens at 800 W for 20 min in 10 mM sodium citrate buffer, pH = 6. Endogenous peroxidase activity was blocked by incubating the s tions in 0.6% hydrogen peroxide solution for 20 minutes. Primary monoclonal antibody for SIRT7  was used in 1:500 dilution, and incubated at room-temperature (RT) for one hour. Then, 3,3′-diaminobenzidine (Sigma-Aldrich™) was used as chromogen for visualization and slides were mounted with Entellan® . Normal testicular tissue, showing intense SIRT7 immunoreactivity was used as positive control. SIRT7 immunoexpression was evaluated by a dedicated uropathologist and cases were classified using a semi-quantitative scale for both staining intensity  and percentage of positive cells , in each tumor. Staining intensity and percentage of positive cell results were then combined in a single score (Score S = staining intensity x percentage of positive cells) assigned to each tumor, and further stratified into low expression (S < 4 = IHC−) and high expression (S ≥ 4 = IHC+ ) groups, which correspond to cases with less than 33% stained cells or staining intensity lower than normal urothelium, and cases with at least 33% stained cells with an intensity equal to or higher than normal urothelium.Immunohistochemistry was performed using the Novolink™ Max Polymer Detection System  and controls . Biospecimen Core Resources (BCRs) provided the clinical data of each patient. This data is available for download through the GDC data portal  (The Cancer Genome Atlas (TCGA) dataset was interrogated for data on cer.gov) .2. Mycoplasma test was regularly performed for all cell lines using TaKaRa PCR Mycoplasma Detection Set .5637, J82, T24 and TCCSUP BlCa cell lines and normal bladder cell line SV-HUC1 were selected for this study. All cell lines were purchased from ATCC and grown using recommended medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin  at 37 °C and 5% COSIRT7 knockdown was performed through lentiviral transduction in J82 cell line using GIPZTM Lentiviral shRNA particles , and in MGHU3 and 5637 cell lines using SMARTvectorTM Inducible Lentiviral shRNA particles (target sequence: 5’-CCCTGCGTGCTGGTGAAGA-3’). All sh-SIRT7 vectors included the green fluorescent protein (GFP). Briefly, cells were seeded in 12 well/plate at density of 4 × 104 cells/well and incubated during 24 h in humidified chamber at 37 °C and 5% CO2. Then, culture medium was removed and 500 µL of completed medium with 8 µg/mL polybrene and lentiviral sh-SIRT7 particles with MOI 10 concentration were added. After 48 h, 1 µg/mL of puromycin dihydrochloride (Clontech Laboratories) was added to select stably transfected cells. For MGHU3 and 5637 cells, after puromycin selection, a treatment was performed with 100 ng/mL doxycycline in order to induce the Tet-On 3 G bipartite induction system. Additionally, J82, MGHU3 and 5637 control cells were generated using a non-target scramble shRNA under the same previously described conditions.For clone selection, 10, 20 and 50 cells/well were seeded in 96 well plate after stable selection, and the isolated clones were grown until confluence for protein extraction, and subsequent western blot analysis for SIRT7 expression. Moreover, sh-SIRT7 cells were observed under the fluorescence microscope for GFP expression.®, Diagenode, Liège, Belgium). After centrifugation, the supernatant was collected, and total protein was quantified according the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.), according to the manufacture procedure. BlCa cell lines, sh-scramble/CTRL and sh-SIRT7 cells were grown until 80% confluence and homogenized in Kinexus lysis buffer supplemented with proteases inhibitors cocktail. Then, cells were sonicated for 5 cycles of 30 s ON and 30 s OFF  was used. Briefly, bladder cancer cell lines, sh-scramble/CTRL and sh-SIRT7 cells were washed in 1 X PBS with phosphate inhibitors and scrapped. Subsequently, cells were suspended in hypotonic buffer and incubated on ice during 15 min. Additionally, a detergent was added, and samples were centrifuged at 14,000 rpm during 30 s at 4 °C. Supernatant (cytoplasmic fraction) was collected and stored at −80 °C until use. Then, cell pellets were resuspended and incubated in a complete lysis buffer solution (lysis buffer with proteases inhibitor cocktail and 10 mM DTT), following centrifugation and supernatant (nuclear fraction) collection and storage at −80 °C. Nuclear and cytoplasmic proteins were then quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.), according to manufacture procedure. Aliquots of 30 µg total protein was separated in 10% polyacrylamide gel by SDS-PAGE and transferred onto an immunoblot PVDF membrane  in a 25 mM Tris-base/glycine buffer using a Trans-Blot Turbo Transfer system (Bio-Rad Laboratories). Membranes were blocked with 5% milk in TBS/0.1% Tween (TBS/T pH = 7.6) for 1 hour at RT. After incubation with primary antibodies for SIRT7  or EZH2  for 1 h 30 min at RT, the membranes were washed in TBS/T and incubated with s ondary antibody coupled with horseradish peroxidase for 1 h at RT. The bound was visualized by chemiluminescence  and quantification was performed using band densitometry analysis from the ImageJ software . β-Actin  for total protein and cytoplasmic protein analysis, and Laminin B1  for nuclear protein, were used as loading controls. For co-immunoprecipitation assays, 200µg of total protein from cell lysates were incubated with anti-acetylated-lysine antibody  and immunoprecipitated with Protein A/G magnetic beads  overnight at 4 °C. The final eluates were blotted with EZH2 primary antibody, as detailed above. Detailed information about western blot can be found at Wild-type MGHU3, 5637 and J82, sh-scramble/CTRL and/or sh-SIRT7 cells were seeded on cover slips at 20,000 cells/well, overnight. Briefly, cells were fixed in methanol during 10 min and then blocked with 5% bovine serum albumin (BSA) during 30 min. After overnight SIRT7 , ECAD  and/or NCAD  incubation at RT, cells were incubated with s ondary antibody anti-rabbit IgG-TRITC  during 1 h at RT. Finally, after 1× PBS wash, cells were stained with 4’,6-diamidino-2-phenylindole (DAPI)  r in mounting medium. Pictures were taken on a IX51 fluorescence microscope  equipped with an Olympus XM10digital camera using CellSens software.7) media at 1% final concentration, and after an 8 minutes’ incubation at RT, reaction was immediately stopped by adding 1.5 mL of 2.5 M glycine and incubating for 5 min. Cells were then washed twice with ice-cold 1× PBS, scraped, harvested and centrifuged at 4 °C. Chromatin immunoprecipitation (ChIP) analysis was performed in sh-scramble/CTRL and sh-SIRT7 cells. For the crosslink step, formaldehyde solution (Sigma) was added to adherent cells  for 15 cycles of 30 s ON and 30 s OFF. Soluble chromatin was then centrifuged and stored at −80 °C until further use. Cell pellets were homogenized with cell lysis buffer  and left on ice for 1 h30, with intermittent vortexing, and then centrifuged at 4 °C. At this point, pellets were re-suspended in nuclei lysis buffer  and incubated for 15 min on ice, followed by adding of 2× volumes of IP dilution buffer . Chromatin was solubilized and sheared to 200–400 bp fragments using an ultra-sonicator , positive control (RNA polymerase II) and negative control (mouse IgG), at assay dependent concentration. IPs were incubated overnight at 4 °C with rotation. After incubation, magnetic beads were precipitated using 1.5 mL tubes magnet rack and washed with four different salt concentration buffers. At this point, elution buffer  was added to all samples and input control, as well as 200 µg/mL of RNAse A, following an incubation of 30 min at 37 °C. After this, samples were incubated with proteinase K for 2 h at 62 °C, followed by an incubation of 10 min at 95 °C, for cross-link reversion. Before immunoprecipitation (IP), each 50 µL of chromatin was 1:10 diluted in dilution buffer , and 5 µL of this solution was reserved in a new tube for the input control. After this, 20 µL of protein A+G magnetic beads (Millipore) were added to each IP sample, as well as ChIP-grade antibodies for Histone H3 , tri-methylation of lysine 27 of histone H3 , according to manufacture procedures, and stored at −20 °C until further use. For qPCR, two pairs of primers for CDH1 promoter were designed, both for ~325 bp before TSS  and for ~600 bp before TSS . RT-qPCR was performed as mentioned above, and the relative amount of promoter DNA was normalized using Input Percent method.2 coverslips and allowed to grow overnight. Then, cells were fixed in 4% formaldehyde (Sigma) for 10 min and permeabilized in 0.5% Triton X-100 (Sigma), for 5 min, at RT and gently stirred. PLA assay was performed using the commercial kit Duolink In Situ , according to manufacturer’s instructions. The antibodies used were Histone H3 , tri-methylation of Lysine 27 of Histone H3 , SIRT7  and EZH2 . After the procedure, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI)  in mounting medium. Pictures were taken on an Olympus IX51 fluorescence microscope equipped with an Olympus XM10 digital camera using CellSens software. Sh-scramble/CTRL and sh-SIRT7 cells, were seeded in 1 cmTo assess the role of SIRT7 in cell growth, 3--2,5-diphenultetrazolium (MTT) assay (Sigma-Aldrich) was performed. Briefly, sh-scramble/CTRL and sh-SIRT7 cells were seeded at 3000 cell/well density, overnight, in 96 well plate. Then, 5 µg/mL MTT solution in completed MEM medium was incubated during 1 h at 37 °C for 0 h, 24 h, 48 h and 72 h. Then, formazan crystals formed were dissolved in dimethyl sulfoxide (DMSO) and spectrophotometric measurement was done at 540 nm, using 655 nm as a reference absorbance . The optical density (OD) obtained for 24 h, 48 h and 72 h was normalized for the 0 h time point. At least three independent experiments were performed.TM kit . This assay uses a dye that is integrated by cells undergoing at early stage of apoptosis due to phosphatidylserine transmembrane movements, which results in APOPercentage dye incorporation by cells. Briefly, sh-scramble/CTRL and sh-SIRT7 cells were seeded in 24 well plate at density of 25,000 cell/well and incubated during 72 h in a humidified chamber at 37 °C and 5% CO2. At this time point, cells were incubated with 300 µL/well of APOPercentage dye solution at ratio 1:20 respectively, during 20 min at 37 °C. Then, cells were washed with PBS1 X and detached from well plate with TrypleTM Express (GBICO) during 10 min at 37 °C. After that, APOPercentage Dye Release reagent was added and plate were vigorously agitated during 15 min, following colorimetric measurement at 550 nm with 620 nm reference filter (Fluostar Omega). The H2O2 was used as a positive control. The OD obtained for apoptosis assay was normalized for the OD obtained by viability assay at the same time point. At least three independent experiments were performed. Apoptosis was assessed using the APOPercentage5 cell/well and allowed reach confluence at 37 °C, 5% CO2. Then, a “wound” was made by manual scratching with a 200 µL pipette tip and cells were gently washed with 1× PBS. The “wounded” areas were photographed in specific wound sites (two sites for each wound) at 40× magnification using an Olympus IX51 inverted microscope equipped with an Olympus XM10 Digital Camera System every 24 h until wound closure. The relative migration distance (5 measures by wound) was calculated with the following formula: relative migration distance (%) = (A–B)/C × 100, where A is the width of cell wound at 0 h incubation, B is the width of cell wound after specific h of incubation, and C is the width mean of cell wound for 0 h of incubation. For relative migration distance, the results were analyzed using the beWound-Cell Migration Tool (Version 1.5) [Cells were seeded in 6 well plate at a density of 7.5 × 10ion 1.5) . At leas2. Then, the non-invading cells were removed by with swab and invaded cells were fixed with methanol and staining with DAPI. Invaded cells were counted on an Olympus IX51 fluorescence microscope equipped with an Olympus XM10 digital camera using CellSens software. The % invasion normalized for total of amount cell seeded in BD Matrigel Chamber.Invasion capacity of sh-scramble/CTRL and sh-SIRT7 cells was evaluated using a 24 well BD Biocoat Matrigel Invasion Chambers . After rehydration of BD Matrigel Chambers during 2 h with MEM medium at 37 °C, cells at a density of 25,000 cells/ insert were seeded and incubated during 24 h at 37 °C in 5% CO® SPSS® Statistic software version 23  and graphs were built using GraphPad Prim 7.0 . Significance level was set at p < 0.05, and Bonferroni’s correction was used when appropriate. All statistical analyses were performed using IBMFor both BlCa cohorts (IPO’s and TCGA), when applicable, Mann-Whitney U test (MW) was used to test for differences in sirtuins expression levels between NB and UCC tissue samples, pathological stages of cases divided in Ta-1 (NMIBC) and T2-4 (MIBC), and patients’ gender, and to assess differences in sh-scramble versus sh-SIRT7 conditions. Kruskall-Wallis test (KW) was performed to test for differences among UCC subtypes . Spearman’s rho was used to assess the correlation between SIRTs expression levels and age of the patients at diagnosis, and between SIRT7 and ECAD or NCAD expression levels. Associations between clinical grade or pathological stage and immunoexpression results were assessed by chi-square or Fisher’s exact test, and Somers’d directional measure was also computed. Disease-specific and disease-free survival curves (Kaplan-Meier with log rank test) were computed for standard variables (tumor stage and grade) and for categorized SIRTs transcript levels. Moreover, the same analyses were also performed separately for NMIBC and MIBC cases. A Cox-regression model comprising all significant variables (univariable and multivariable model) was computed to assess the relative contribution of each variable to the follow-up status.In conclusion, this study provides a global view on sirtuin family expression deregulation in BlCa. Specifically, SIRT7 overexpression seems to play an important role in the first steps of urothelial carcinogenesis, whereas subsequent downregulation is associated with acquisition of an invasive and aggressive phenotype, through stimulation of EMT phenotype involving the SIRT7-EZH2-CDH1 axis. Although further studies are required to clarify the mechanism underlying SIRT7 deregulation in BlCa, it might constitute an attractive target for innovative therapeutic strategies.


Paraquat (PQ), a widely used herbicide, could cause neurodegenerative diseases, yet the mechanism remains incompletely understood. This study aimed to investigate the direct effect of PQ on NSC in vivo and its possible mechanism. Adult C57BL/6 mice were subcutaneously injected with 2 mg/kg PQ, 20 mg/kg PQ or vehicle control once a week for 2 weeks, and sacrificed 1 week after the last PQ injection. Furthermore, extra experiments with Tauroursodeoxycholic Acid (TUDCA) intervention were performed to observe the relationship between ER stress, neuroinflammation and the neural stem cell (NSC) impairment. The results showed that 20 mg/kg PQ caused the NSC number decrease in both subgranular zones (SGZ) and subventricular zone (SVZ). Further analysis indicated that the 20 mg/kg PQ suppressed the proliferation of NSC, without affecting the apoptosis. Moreover, 20 mg/kg PQ also induced ER stress in microglia and caused neuroinflammation in SGZ and SVZ. Interestingly, the ER stress inhibitor could simultaneously ameliorate the neuroinflammation and NSC reduction. These data suggested that increased ER stress in microglia might be a possible pathway for PQ-induced neuroinflammation and NSC impairment. That is a previously unknown mechanism for PQ neurotoxicity. Indeed, the PQ toxicity has been widely discussed but is not yet fully clarified. The underlying mechanism may be associated with mitochondrial dysfunction, lipid peroxidation, and oxidative stress etc.5.Paraquat (PQ), a widely used non-selective herbicides in the world, is known for its potent neurotoxicant properties8. In this context, experimental evidences showed that PQ exposure could cause permanent nerve damage in mice10. NSCs in the adult mammalian brain are regarded as a useful tool to regenerate neurons in pathological conditions such as neurodegenerative diseases or acute brain injury, indicating that the NSC impairments may be closely correlated with the nervous system diseases11. It was reported that exogenous hNSCs could improve the behavioral function in the model of Parkinson’s disease. Actually, as most primordial cell in the central nervous system, the NSC appeared to promote function preservation and/or restoration in neurodegenerative disease12. Besides, PQ-induced functional impairments in progenitor cell in vitro were also reported15. However, to date, there is no study evaluating the direct toxicity of PQ to NSC in vivo.In line with these data, epidemiological studies reported that PQ could result in cognitive and behavioral impairments and increase the risk of neurodegenerative diseases, such as autism spectrum disorders and Parkinson’s disease16. NSCs generate the central nervous system (CNS) during development and persist in adult brains at the SGZ and SVZ to make and replenish differentiated cells lost during stress and injury19. Most CNS injuries elicit proliferation and migration of the NSCs toward the injury site, indicating the activation of a regenerative response. However, regeneration from NSC is incomplete, which could be due to detrimental cues encountered during inflammation20. Different CNS diseases can cause activation of the innate and adaptive immune responses that could influence the NSCs. Furthermore, NSCs in the brain react differently to inflammatory cues than their counterparts in the spinal cord20. Thus, it is necessary to study the relation between PQ-induced inflammatory and NSC in the brain.In the adult rodent brain, NSCs persist in the SGZ and SVZ, which are specialized niches with a particular architecture, allowing NSCs to survive, proliferate and generate new neurons21. The canonical UPR signaling is initiated by activation of three ER membrane-bound transducers: Inositol Requiring Enzyme 1 (IRE1), Activating Transcription Factor 6 (ATF6), and Protein Kinase-like ER kinase (PERK)21. The relationship between ER stress response and inflammation has gained a massive focus in biomedical research21.ER stress is a buildup of unfolded or misfolded proteins, which often happened when secreted and membrane-embedded protein translation increased or protein folding decreased in the ER lumen. In response to ER stress, the ER has evolved a capacity termed Unfolded Protein Response (UPR), which could alleviate ER stress by degrading unfolded and misfolded proteins23.Regarding the PQ toxicity to NSC, numerous studies discussed its possible mechanism, but most experiments were based on in vitro designs. Thus, the influences of PQ on NSC in particular regions in vivo remain incompletely defined. To date, the relationships between PQ-induced ER stress and neuroinflammation, NSC impairments are unknown. In this study, we especially focused on SGZ and SVZ where NSCs persist and found that PQ-induced NSC impairment and neuroinflammation, could be restored by an ER stress inhibitor–Tauroursodeoxycholic Acid (TUDCA)24 in flow cytometry analysis, we quantified the percentage of NSC. Consistently, the results confirmed the reduction in NSC of SGZ and SVZ  Caspase-3, a molecule driving cellular apoptosis28, we next measured the activation of microglia in SGZ and SVZ by western blots, based on their surface expression of CD11b, and their cytoskeletal protein Iba1 . As shown in Fig. PQ-induce neuroinflammation in mice have been widely discussed previously. Specially, we wanted to know whether the inflammation happened in SGZ and SVZ after PQ exposure in our study. Therefore, we measured the level of inflammatory cytokines, including IL-6, IL-1β and TNF-α, in both zones by ELISA. As predicted, 20 mg/kg PQ significantly increased the level of IL-6, IL-1β, and TNF-α in SGZ and SVZ Fig. a, b, indba1 Fig. c, d. Theba1 Fig. g, h. Mor30. ER stress is mainly composed of three fundamental branches, including IRE1, PERK and XBP-1s. First, using western blots, we measured the activated IRE1α (p-IRE1α) in SGZ but not SVZ because of the low protein levels in SVZ. The results showed that in comparison with controls, the p-IRE1 was remarkably higher in mice treated with 20 mg/kg PQ  in SGZ  old wild type (WT) C57BL/6 (B6) mice were purchased from Shanghai SLAC Laboratory Animal Co. LTD, China. Both males and females were equally used. Mice were housed in a specific pathogen free environment at Fudan University with constant temperature (23 °C) and humidity (50%) and an artificial 12-hour (hr)-light/12-hr-dark cycle. 6 mice were housed per cage with free access to food and water. The animal experiment was approved by the Ethics Committee of Animal Use at Fudan University. All methods were carried out in accordance with the guideline National Standard GB/T 35892 for Laboratory Animal Procedures of China.PQ was purchased from Sigma, St. Louis, MO, USA. Mice were subcutaneously injected with phosphate buffered saline (PBS), 2 mg/kg PQ or 20 mg/kg PQ once a week for 2 weeks. Due to the quality of PQ in high metabolic rate, we performed preliminary experiment and found that neuroinflammation caused by PQ could persist for at least 1 wk. To further study the relationship between neuroinflammation and NSC impairments, the mice were sacrificed 1 wk after the last PQ injection.In the ER stress function test, mice with 20 mg/kg PQ treatments were subcutaneously injected with 500 mg/kg TUDCA  once every 3 days during PQ exposure. Then, the slices of SGZ and SVZ were prepared to measure the number of NSC.The SGZ and SVZ were isolated from the brain after intracardiac perfusion with 20 ml of ice-cold PBS, then digested in the trypsin and ethylenediaminetetraacetic acid (EDTA) at 37 °C for 30 min (min). The specific process of obtaining SVZ was as follows: Transect the brain about 1/2, keep the forebrain (near olfactory bulb end), and divide it into two halves along the midline. Put the lateral side of the hemi-brain down and fix it with tweezers. Use another tweezer to remove the remaining hippocampus and the inner wall of the SVZ. The outer wall of the SVZ is crescent-shaped, connected to the white matter on the upper and lower sides, with clear boundaries. The specific process of obtaining SVZ is as follows: Use forceps to separate the mouse brain, the hippocampus is located at the bottom of the cerebral cortex. Use tweezers to dislodge the cerebral cortex to expose the hippocampus. Separate hippocampal tissue from cerebral cortex and surrounding brain tissue.Simple wash buffer (SWB), comprised by PBS and 5% fetal bovine serum (FBS) was used to stop digestion. After filtered, single cell suspensions were prepared with SWB. Antibodies (clone) and fluorochromes included V500 anti-CD45.2 (104), FITC anti-I-A (G155-178), unconjugated anti-CD16/32  ; APC-Cy7 anti-CD45.2 (104), PE anti-SOX2 (14A6A34), FITC anti-Ki67 (16A8), PE anti-CD45 (30-F11), anti-Iba1(10904), anti-CD11b (66519-1-Ig), anti-Ki67 (D3B5), PE anti-XBP-1s (Q3-695) ; Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423), Cyc3-labeled goat anti mouse IgG (A0521) ; Alexa Fluor 488 anti-p-PERK, FITC anti-cleaved Caspase-3, FITC anti-Bcl-2 (BCL/10C4) ; and 4′, 6-diamidino-2-phenylindole . Cells were quantified by an automatic cell counter. Surface staining were incubated with fluorochrome-conjugated antibodies on ice in the dark for 30 min after Fc block which were performed on ice for 20 min. The intracellular and nuclear staining kits were obtained from BD Biosciences and performed following its protocol. The stained cells were analyzed with BD LSRFortessa  and data were analyzed with FlowJo9.3.2.After fixed with 4% paraformaldehyde and dehydration with 30% sucrose solution , the brains were sliced (8 μm) and washed with PBS for 10 min, then permeabilized with 0.1 Triton X-100 . After that, the brain slices were blocked by 5% goat serum at room temperature for 2 hr. . The slices were incubated with SOX2 antibody at 4 °C overnight and were stained with DAPI at RT for 5 min followed by confocal analyzation. All the antibodies were purchased from Santa Cruz, California, USA. The stained slices were analyzed with confocal microscope .The cytokines IL-1β, TNF-α and IL-6 in SGZ and SVZ were detected by commercial ELISA kits, following by the manufacturer’s instructions . The total protein concentration was quantified with BCA protein assay kit , and evaluated as pg cytokine/mg protein.To prepare homogenate protein, the SGZ were lysed with radio immunoprecipitation assay (RIPA) buffer containing 1% protease inhibitors . Next, performed electrophoresis using SDS-PAGE gels and transferred the protein to polyvinylidene difluoride membranes . Then, the membranes were blocked in TBST containing 5% skimmed milk powder  at RT for 2 hr and following incubation with primary antibodies to IRE1α or phosphorylated IRE1α (p-IRE1α) at 4 °C overnight and horseradish peroxidase (HRP)-conjugated at RT for 2 hr to detect target antibody. Then ECL Western Blotting Substrate  was used to visualize and the images were analyzed with ImageJ gel analysis software  to quantify the protein. All the antibodies were purchased from Abcam, Cambridge, UK and β-actin were used as internal loading control.p < 0.05 was considered as the level of significant difference.As no differences were observed between male and female, data were pooled and presented as mean ± standard deviation. Student’s t-tests or one-way analysis of variance (ANOVA) were performed and post-hoc LSD t-tests were used if necessary. Supplementary Figures.


Endothelial dysfunction is a primary feature of several cardiovascular diseases. Endothelial colony-forming cells (ECFCs) represent a highly proliferative subtype of endothelial progenitor cells (EPCs), which are involved in neovascularization and vascular repair. Statins are known to improve the outcome of cardiovascular diseases via pleiotropic effects. We hypothesized that treatment with the 3-hydroxy-3-methyl-glutaryl–coenzyme A (HMG-CoA) reductase inhibitor pravastatin increases ECFCs’ functional capacities and regulates the expression of proteins which modulate endothelial health in a favourable manner. Umbilical cord blood derived ECFCs were incubated with different concentrations of pravastatin with or without mevalonate, a key intermediate in cholesterol synthesis. Functional capacities such as migration, proliferation and tube formation were addressed in corresponding in vitro assays. mRNA and protein levels or phosphorylation of protein kinase B (AKT), endothelial nitric oxide synthase (eNOS), heme oxygenase-1 (HO-1), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and endoglin (Eng) were analyzed by real time PCR or immunoblot, respectively. Proliferation, migration and tube formation of ECFCs were enhanced after pravastatin treatment, and AKT- and eNOS-phosphorylation were augmented. Further, expression levels of HO-1, VEGF-A and PlGF were increased, whereas expression levels of sFlt-1 and Eng were decreased. Pravastatin induced effects were reversible by the addition of mevalonate. Pravastatin induces beneficial effects on ECFC function, angiogenic signaling and protein expression. These effects may contribute to understand the pleiotropic function of statins as well as to provide a promising option to improve ECFCs’ condition in cell therapy in order to ameliorate endothelial dysfunction. Cardiovascular diseases (CVD) are considered a major healthcare problem and represent the leading cause of mortality and morbidity worldwide ,2. They Endothelial progenitor cells (EPCs) have been studied extensively since they were first described and isolated by Asahara et al. in 1997 ,9. EPCs Endothelial colony-forming cells (ECFCs), a subgroup of EPCs, are known for their high proliferative and clonal capacity and play an important role in angiogenesis and vascular repair ,23. TheyStatins are a class of drugs that reduce morbidity and mortality in patients at high cardiovascular risk ,31. TheyProtein kinase B (AKT) is a serine/threonine protein kinase and acts as multifunctional intracellular regulator of cell growth, metabolism and survival, whereas endothelial nitric oxide synthase (eNOS) is known to be a downstream target in the AKT pathway and is mainly responsible for NO synthesis and, therefore, for vascular relaxation ,43. TherIn light of the potential of ECFCs in cell-based therapies, the present study systematically explores on functional and mechanistic levels whether pravastatin, a hydrophilic statin variant, modulates ECFCs’ functional capacity and activates vasoprotective mechanisms of ECFCs.Umbilical cord blood from six healthy, uncomplicated singleton pregnancies was collected immediately after delivery. All women included were normotensive, without proteinuria throughout gestation, and had healthy babies at term. This research was approved by the local Ethic Committee of Hannover Medical School  and written informed consent was provided by each participant.7 cells/well on collagen coated 6-well plates  and incubated at 37 °C, 5% CO2 supply. After 10 to 20 days, first appearance of ECFC colonies was noted as well circumscribed monolayers of rapidly proliferating, cobblestone-appearing cells and characterized by flow cytometry with appropriate antibodies , CD45 , and CD133 ) and the corresponding isotype controls  [ECFCs were isolated as previously described by Grundmann et al. . Umbilic Biotec) ,52. ECFC4 ECFCs from 3–4 biologically distinct lines were seeded into the upper side of the chamber in serum-free EBM-2 medium with 1% P/S but without other supplements, and 10% FBS was used as chemoattractant in the lower side of the chamber. Different concentrations (2–2000 µM) of pravastatin were added to both sides of the chamber in the presence or absence of 200 µM mevalonate . The cells were allowed to migrate for 4 h until the inserts were removed and the non-migrated cells were detached from the upper surface of the membrane with a cotton bud. The inserts were placed in fixing solution as described above, followed by two washing steps with PBS. Afterwards, the migrated cells on the lower side of the membrane were counterstained with 4′,6-diamidino-2-phenylindole , followed by mounting in antifade fluorescence mounting medium . Image acquisition was realized with a Leica DMI 6000 B microscope . Five pictures per membrane were randomly taken and DAPI-stained cells were counted with ImageJ 1.50b .To analyze ECFCs’ ability of directional cell migration in the presence or absence of pravastatin , we performed a modified Boyden chamber assay. Transwell inserts with an 8 µm microporous membrane  were placed in a 12-well-plate. Then, 5 × 104 cells) from 4 biologically distinct lines were seeded on gelatine (Sigma-Aldrich) coated wells of 6-well culture plates with EGM-2 containing 10% FBS and 1% P/S. After cells reached optical confluence, they were kept in culture for an additional 24 h. The cell monolayers were scratched with a sterile P1000 pipette tip to create a wound, media was aspirated and, after washing the cells with PBS, replaced with treatment media  containing different doses (2–2000 µM) of pravastatin with or without an additional 200 µM mevalonate. Each treatment was performed in duplicate wells. Phase contrast microscopic images were immediately obtained after scratching and then again after 18 h. Non-populated scratch areas were quantified by ImageJ 1.50b and subtracted to obtain the remigrated area.A scratch wound healing assay was used to assess ECFC’s ability to migrate in order to close a wound. ECFCs . The cells were incubated for 6 h with different concentrations (2–2000 µM) of pravastatin with or without 200 µM mevalonate in EBM-2 with 1% P/S without growth supplements. Phase contrast microscopic images were obtained at 20x magnification with a Leica DMI 6000 B microscope. The total tubule length and number of branch points in each visual field were measured using ImageJ 1.50b. Branch points were defined as nodes with connections to at least 3 tubes.2 supply. After reaching a stable cell index, different concentrations (2–2000 µM) of pravastatin with or without 200 µM mevalonate were added. Cell growth was continuously monitored for 72 h after pravastatin addition.Cell proliferation was measured with the xCelligence Real-Time Cell Analyzer . The xCelligence system allows us to monitor cell proliferation continuously in real time. The electrical impedance caused by the adherent cells is converted into cell indices by the xCelligence software . A total of 2000 to 3000 cells from 4–5 biologically distinct ECFC lines were seeded in quadruplicates in EGM-2 with 8% FBS and 1% P/S onto a gold-coated E-Plate View 96-well plate (Roche) and then placed into the Real-Time Cell Analyzer SP station, positioned in a 37 °C incubator with 5% CO5 cells were blocked with Intraglobin  for 1 min, followed by incubation with an Annexin V antibody  or isotype control at 4 °C for 30 min. UV radiation for 30 min was used as positive control for apoptosis for gating strategy. Propidiumiodide  was added one min prior to measurement on a BD FACS Calibur Flow Cytometer (BD Biosciences), and results were analyzed using FlowJo X Software v.10 .Flow cytometry analysis was performed to measure apoptosis. After 48 h of treatment with different concentrations of pravastatin (2–2000 µM) or mevalonate (200 µM) in EGM-2 with 8% FBS and 1% P/S, ECFCs from 4–5 biologically distinct lines were harvested with 0.05% trypsin/EDTA and washed with flow cytometry buffer . Then, 1 × 102O, the membrane was incubated overnight at 4 °C with appropriate antibodies , 1:3000 anti–beta-actin , 1:3000 anti-alpha-tubulin , 1:1000 HO-1 , 1:1000 peNOS S1177, 1:1000 eNOS , and 1:500 VEGF-A ). After washing three times with PBS-T (0.1% v/v), the secondary antibody was added  goat anti-mouse or 1:3000 goat anti-rabbit horseradish peroxidase; GE Healthcare) in 1% Roti-Block for 2 h at room temperature. Visualization of immunoblot bands was performed by using ECL chemiluminescence  and analyzed with ImageJ 1.50b.For analyses of proteins, ECFCs from 2–5 biologically distinct cell lines were grown to 80–90% confluence in 10 cm dishes  and treated with 200 µM pravastatin for 2 h  or 24 h . Mevalonate (200 µM) was applied simultaneously to block potential modifications. ECFCs were detached with trypsin-EDTA, washed with PBS and lysed with lysis buffer as previously described. . ProteinECFCs from 3–5 biologically distinct lines were treated with 20 or 200 µM pravastatin in the presence or absence of 200 µM mevalonate for 24 h. RNA was isolated by using the RNeasy Plus Mini Kit . For the cDNA synthesis, RNA was diluted with diethylpyrocarbonate (DEPC)-treated water and denatured at 68 °C for 10 min in a thermocycler . Then, High Capacity cDNA Reverse transcription (RT) master mix was added, containing RT buffer, RT random primer, deoxyribonucleoside triphosphate (dNTP) mix (100 mM), MultiScribeTMReverse transcriptase  and DEPC-treated water. In each case, 1.5 µL cDNA and 10.5 µL master mix  were pipetted into the appropriate strip tubes (0.1 mL). Real-time PCR was performed on a Rotor-Gene 6000 (Qiagen) for 40 cycles. For each treatment, runs were performed in triplicates. The primer sequences used to determine mRNA levels of VEGF-A, PlGF, sFlt-1, Eng and HO-1 are described in t test, Mann–Whitney test, Kruskal–Wallis test, one sample t test or Wilcoxon’s signed-rank test were used as appropriate. Experimental data are presented as mean and standard error. All replicates (n) from each experiment were analyzed with Prism 9 . A significant deviation is indicated by values of p < 0.05, p < 0.01, p < 0.001. Statistical analysis was performed after testing for normality distribution by the Shapiro–Wilk or D’Agostino normality test. Student’s unpaired n = 20, p < 0.001), n = 15, p < 0.001 compared to control and p = 0.003 compared to 2000 µM: 2.24 ± 0.17), whereas mevalonate alone caused a slight decrease in directional migration , p = 0.30; 20 µM: 1.19 ± 0.06, p = 0.007; 200 µM: 1.43 ± 0.06, p < 0.001; 2000 µM: 1.35 ± 0.06, p < 0.001, n = 14), n = 16, p = 0.16) but lowered the beneficial effect of pravastatin (200 µM) significantly , The directional migration of single cells is promoted by chemotaxis and was tested using a modified boyden chamber assay. Therefore, we explored how directional cell migration is impacted by different concentrations of pravastatin and if the effect can be revised by the addition of mevalonate, the product of HMG-CoA reductase which is inhibited by pravastatin. The chemotaxis ability of ECFCs treated with pravastatin was significantly higher compared with the control and increased with higher doses  and higher numbers of branch points , whereas a high dose of pravastatin led to a significant impairment of tube formation , n = 12, p = 0.42), but reduced or even reversed the positive pravastatin effect of lower and intermediate doses  and ameliorated the negative effect of the high pravastatin dose , Reflecting their ability of de novo vessel formation in vivo, we performed an in vitro angiogenesis assay to determine pravastatin effects on ECFCs’ ability to form capillary-like structures in Matrigel. After treatment with low and intermediate doses of pravastatin, ECFCs showed significantly higher tube length than controls , whereas treatment with 200 µM pravastatin attenuated cell growth  and led to a stable plateau after 24 h. The presence of 2000 µM pravastatin inhibited proliferation completely , n = 15, p = 0.04 compared to 20 µM pravastatin: 1.28 ± 0.06) and moderately improved the impeded proliferation in the presence of 200 µM pravastatin , n = 5, p > 0.99), 20 µM  or 200 µM pravastatin  compared to the control (6.94% ± 0.83%). In accordance with the impaired proliferation at high pravastatin levels, we found a 3.8-fold increase in apoptosis while treated with 2000 µM pravastatin , p = 0.72).Regarding one of their main characteristics, we addressed ECFCs’ proliferation in the presence or absence of different doses of pravastatin. The cell index was continuously monitored with the xCelligence technology. Treatment with 2 µM or 20 µM pravastatin significantly enhanced ECFCs’ proliferation . As the AKT-cascade is considered one of the most important signaling pathways in endothelial cells, we addressed possible pravastatin effects on AKT and its downstream target eNOS in ECFCs. Pravastatin (200 µM) induced AKT . The presence of mevalonate reduced the effect significantly for 20 µM pravastatin , but not consistently for 200 µM pravastatin , We further tested whether pravastatin induces HO-1 expression level. There was an increase in HO-1 mRNA expression level in ECFCs after 24 h treatment with pravastatin  in ECFCs after 24 h incubation with 200 µM pravastatin, shown by immunoblot analysis. The addition of mevalonate abrogated the pravastatin effect , whereas mevalonate alone decreased the HO-1 expression level , In accordance with the increased HO-1 mRNA level observed in qRT-PCR after pravastatin treatment, there was also a higher HO-1 protein expression level , n = 5, p = 0.03), n = 4, p = 0.89; PlGF: 1.02 ± 0.20, n = 4, p = 0.93). Regarding the protein expression, we observed a 1.56-fold higher VEGF-A expression after 24 h treatment with pravastatin (200 µM) , n = 4, p = 0.003; Eng: 0.31 ± 0.11, n = 4, p = 0.008), n = 3, p = 0.007; Eng: 0.51 ± 0.13, n = 3, p = 0.06); however, sFlt-1 and Eng mRNA expression levels remained lower than the control.To explore the mechanisms underlying the pro-angiogenic effects of pravastatin on ECFCs, we determined VEGF-A and PlGF mRNA levels after 24 h of treatment with pravastatin by using qRT-PCR and immunoblot. We also assessed mRNA expression levels of the anti-angiogenic factor sFlt-1 and the angiogenesis-related protein Eng. ECFCs that were treated with pravastatin (20 µM) showed a 2.03-fold higher expression level of VEGF-A (2.03 ± 0.27, In the present study, we systematically explored pravastatin effects on human ECFC biology on functional and mechanistic levels. Our results reveal a significant promoting effect of low and moderate pravastatin concentrations on ECFCs’ functional capacities and further on AKT, eNOS and HO-1 signaling. However, pravastatin at high doses led to functional impairment and increased apoptosis and cell death. Specifically, we observed enhanced proliferative, migratory and angiogenic abilities of this EPC subtype and an augmented AKT and eNOS phosphorylation. Expression levels of HO-1 and the angiogenic molecules VEGF-A and PlGF were increased, whereas anti-angiogenic factor sFlt-1 and angiogenesis-related protein Eng expression levels were decreased. Pravastatin induced effects were diminished by adding the HMG-CoA reductase product mevalonate.Endothelial progenitor cells have been addressed in several studies for more than 20 years . Their nWe showed that pravastatin enhances ECFC migration towards a chemoattractive focus as well as in wound closure. This enhancement may also be beneficial regarding ECFCs’ homing to areas of endothelial damage in vivo. Statins’ favorable effects on cell migration have been shown in early-outgrowth EPCs, where they led to improved migration to VEGF producing foci . In addiAssmus et al. referred that the statin induced rise of EPC numbers after statin treatment in vitro might also improve their capacity in cell-based therapy ,65. In oThere is a big bandwidth in literature of statin concentrations used in experimental studies ,63,67. TNearly all our observed effects were completely or partly reversed by the addition of mevalonate. Mevalonate is the product of the HMG-CoA reductase catalyzed reaction, which is inhibited by the application of statins. Cells require mevalonate, for example, for cholesterol-biosynthesis, isoprenylation and ubiquinone, which are involved in biomembrane composition, signal transduction and mitochondrial respiratory chain mediated energy production . This maIn endothelial cells, the majority of growth factor-induced reactions are considered to depend on AKT activation . We therEndothelial NOS is a downstream target in the AKT pathway. eNOS activity underlies complex regulatory processes and is determined, among other things, by different phosphorylation patterns . We demoAs we have shown that the addition of mevalonate not only abrogates the functional effects of pravastatin on ECFCs, but also the pravastatin-induced phosphorylation of AKT and eNOS, it is likely that this pathway is mainly involved in pravastatin effect mediation. Inhibited statin-induced AKT-activation by the addition of mevalonate was also reported by Nakao et al., whereas Rossoni et al. described a mevalonate-inhibitable phosphorylation of eNOS in animal models ,80.Increased HO-1 gene and protein expression levels in ECFCs after pravastatin treatment have been detected in our study. HO-1 activation provides cell protection against oxidative stress and apoptosis and further maintains cell homeostasis in vitro and in vivo ,81,82,83In our study, we found that pravastatin induced mRNA expression levels of VEGF-A and PlGF in ECFCs. This finding is consistent with a study in early-outgrowth EPCs, where pravastatin increased VEGF mRNA expression . FurtherAdditionally, we demonstrated that pravastatin reduced the mRNA expression level of sFlt-1 and Eng, whose soluble form is considered to have anti-angiogenic properties. sFlt-1 blocks the binding of VEGF and PlGF to their receptors and has further been reported to inactivate eNOS . An exceA disbalance of angiogenic and anti-angiogenic factors is also involved in the pathophysiology of preeclampsia with higher sFlt-1 as well as sEng concentrations in the circulation of women that develop the disease ,96,97,98To our knowledge, this is the first study that systematically analyzed the effects of pravastatin on human ECFCs and elucidated involved signaling mechanisms. Our findings are of clinical relevance for various reasons. Firstly, our observations contribute to deepen the understanding of statins’ pleiotropic effects. As ECFCs have been shown to be impaired in several cardiovascular diseases, our findings might provide another approach to restore ECFCs’ function as a prognostic target in the future. Secondly, we further identified pravastatin treatment as a potential strategy to enhance ECFCs’ numbers and capacities prior to cell therapy and prepare the ground for further research regarding experimental animal models, e.g., of hindlimb ischemia or acute myocardial infarction, in order to ameliorate tissue damage via neovascularization and regeneration. Thirdly, these data add new supporting evidence to the current efforts in the development of preventative approaches with regards to preeclampsia and statin treatment.


Cisplatin (CP) is one of the most effective antitumor drugs in the clinic, but has serious adverse reactions, and its hepatotoxicity has not been fully investigated. Licorice (GC), a traditional herbal medicine, has been commonly used as a detoxifier for poisons and drugs, and may be an effective drug for CP-induced hepatotoxicity. However, its mechanism and the effector molecules remain ambiguous. Therefore, in this study, a network pharmacology and proteomics-based approach was established, and a panoramic view of the detoxification of GC on CP-induced hepatotoxicity was provided. The experimental results indicated that GC can recover functional indices and pathological liver injury, inhibit hepatocyte apoptosis, upregulate B-cell lymphoma/leukemia 2 (Bcl-2) and superoxide dismutase (SOD) levels, and downregulate cellular tumor antigen p53 (p53), caspase-3, malondialdehyde high mobility group protein B1 (HMGB1), tumor necrosis factor alpha (TNF-α), and interleukin 1β (IL-1β) levels. Proteomics indicated that GC regulates phosphatidylcholine translocator ABCB1 (ABCB1B), canalicular multispecific organic anion transporter 1 (ABCC2), cytochrome P450 4A2 (CYP4A2), cytochrome P450 1A1 (CYP1A1), cytochrome P450 1A2 (CYP1A2), estrogen receptor (ESR1), and DNA topoisomerase 2-alpha (TOP2A), inhibits oxidative stress, apoptosis, and inflammatory responses, and accelerates drug metabolism. In this study, we provide the investigation of the efficacy of GC against CP-induced hepatotoxicity, and offer a promising alternative for the clinic. Glycyrrhiza uralensis Fisch. ex DC.) is widely used for its antibacterial, anti-inflammatory, antioxidant, and anticancer properties. GC and its active components, including glycyrrhizin, glycyrrhetinic acid, and glycyrrhetinic, have remarkable detoxification effects on drugs and poisons by relieving the toxic symptoms and reducing the death rate (4 or acetaminophen (Cisplatin (CP) has been widely used in ovarian cancer, prostate cancer, testicular cancer, and others, due to its potent penetration and wide anticancer spectrum . Howeverath rate . Moreoveminophen . Some anminophen . Althougminophen , functioDue to synergistic effects by complex components of GC, it remains a challenge to determine the molecular targets and the mechanism involving detoxification of GC on CP-hepatotoxicity. To solve this problem, a molecular network with GC-effective compounds, targets, and CP toxic targets can be established by network pharmacology. Moreover, changes in protein expression in toxic organs can be analyzed by proteome to explain the biological process involved , which iGlycyrrhiza uralensis Fisch. ex DC. were obtained from Jinta, Jiuquan , and authenticated by Prof. Ling Jin (Gansu University of Chinese Medicine). Voucher specimens were deposited in the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences (NYBG Herbarium Code: IMD). 100 g wild GC powder was extracted with 1,000 ml of 70% ethanol that was refluxed for 3 h twice, filtered, combined the filtrate, concentrated to water evaporation for reserve (CP of over 98.5% purity (by HPLC) was obtained from Beijing Solarbio Technology Co., Ltd. . Wild roots of  reserve , and theThe chemical components of GC were obtained from the TCMSP database , in whicTissue localization was analyzed by the BioGPS gene annotation database , and tisThe 3D structures of active constituents of GC and critical common targets were collected. Then, the hydrogenation and atomic type definitions were completed by AutoDock tools 4.2, and the grid was used to set grid parameter of receptors as follows: Spacing (angstrom) to 1, exhaustiveness to 10,000, z-dimension value of number and the center value were set to contain the entire receptors with docking area, and the rest was set to default. The AutoDock Vina program was run to dock active constituents with critical common targets.n = 10 rats per group): 1) control, 2) CP, 3) CP + GC at 255 mg/kg daily , 4) CP + GC at 450 mg/kg daily (ig), 5) CP + GC at 900 mg/kg daily (ig), and 6) GC , and were continually fed for 8 days. On day 5, after 30 min of administration, except for the control and GC groups, rats were given an intraperitoneal injection of CP (8 mg/kg) to induce liver injury  animal laboratory of Gansu University of Chinese Medicine . All animal studies were approved by the Ethic Committee of Gansu University of Chinese Medicine  with approval number 2019-049, and followed related regulations. Rats were randomly divided into six groups . Its morphology was examined by pathologists and imaged by light microscopy . The degree of liver damage was evaluated by a semiquantitative Knodell liver score, independently scoring by four indicators of inflammation, including necrosis, fibrosis, inflammatory volume, and structural change as follows: 1) the degree of necrosis around the manifold: 0–10 points; 2) the degree of lobular degeneration and focal necrosis: 0–4 points; 3) the inflammatory volume in the portal area: 0–4 points; and 4) the degree of liver fibrosis: 0–4 points. 10 visual fields were randomly selected from each section .5-μm thick deparaffinized tissue sections were hydrated and incubated overnight with rabbit anti-HMGB1, anti-p53, and anti-caspase-3 antibodies at 4°C, washed, and incubated with the secondary antibody. After washing, chromogen liquid DAB was applied, and hematoxylin staining was conducted. And its histopathological changes were observed using a light microscope  and analyzed by Image Pro Plus .in situ apoptosis detection kit   .t-text and p < 0.05 was considered significant.A total of 0.1 g liver tissue was added to 0.9 ml physiological saline, a homogenate was prepared, the supernatant was collected, and its protein concentration was determined using a rat total protein assay kit . Subsequently, rat SOD activity assay kit , rat MDA content assay kits , rat Bcl-2 ELISA kit , rat TNF-α ELISA kit , and rat IL-1β ELISA kit  were used. All kits were performed according to the manufacturer’s guidelines. Each kit consisted a 96-well plate into which a specific antibody against a target protein, and wells were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for colorimetric quantification . The OD Liver tissues were homogenized in PBS with a tissue homogenizer , and each group contained liver tissues from three rats. A total of 0.1 g pooled tissues was lyzed with 400 μL urea lysis buffer , and 4 μL protease inhibitor  was added . Protein4HCO3, and digested overnight at 98.6°F by trypsin (an enzyme to protein mass ratio of 1:25). Then, peptides were extracted and dried in a SpeedVac (Eppendorf).A total of 100 μg liver proteins were digested by the FASP procedure. Protein samples were added to 5 mM final concentration by 1 M dithiothreitol and were incubated for 30 min at 132.8°F, then protein samples were added to 20 mM final concentration by iodoacetamide and were incubated in the dark at 77°F for 30 min, and to a 5 mM final concentration by adding DTT in the dark at 15 min. Protein samples were loaded into 10 kD Microcon filtration devices (Millipore) and centrifuged, then, were loaded into 10 kD Microcon filtration devices (Millipore) and centrifuged, washed twice, respectively, with Urea lysis buffer , and 50 mM NHLiquid chromatography (LC) (Bruker nanoElute) was coupled online to a hybrid TIMS quadrupole time-of-flight mass spectrometer (Bruker timsTOF Pro) via a CaptiveSpray nanoelectrospray ion source. LC was performed at 140°F and with a constant flow of 600 nL/min using a reversed-phase column  with a pulled emitter tip, packed with 1.9 μm C18-coated porous silica beads  . Mobile Raw MS files were managed by MaxQuant software (version 1.6.6.0), and its Andromeda search engine was used to identified MS/MS-based peptide, which uses a target-decoy approach to identify proteins at an FDR <1%. The rat ref-sequence protein database  was used as a forward database and the reverse database of decoy search was autogenerated by MaxQuant. “Trypsin” was set for enzyme specificity, and at least seven amino acids were required to identify peptides. Default settings were used for variable and fixed modifications (variable modification: acetylation (protein-N terminus) and oxidation methionine (M)), fixed modification: carbamidomethylation (C). A MaxQuant label-free quantification (LFQ) algorithm was used to quantitate the MS signals, and the intensities of proteins were represented in intensity based absolute protein quantification (iBAQ) . The iBAThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner The iBAQ value was used as the protein expression, and an average value of three samples in each group was taken. The variation trend of the expression of the same protein in each group was analyzed. The cellular component, molecular function, and biological processes were analyzed by DAVID Bioinformatics Resources 6.8 software.t-test, and p < 0.05 was considered significant.Proteins from liver samples were extracted by precooling RIPA lysis buffer , loaded on 10% SDS-PAGE gels, and proteins were transferred to PVDF membranes . Activated membranes were transferred in protein blots, blocked in 5% nonfat milk within 1.5 h, and then incubated overnight at 4°C with one of the following primary antibodies: anti-GAPDH , anti-cytochrome P450 4A1/2 , anti-CYP1A1 (1:100), anti-CYP1A2 (1:500), anti-Abcb1b , anti-ABCC2 (1:500), anti-ESR1 , and anti-TOP2A . Then, membranes were washed with Tris-buffered saline containing Tween 20 (TBST) and incubated at 24°C for 1 h with a second antibody, and visualized by an enhanced chemiluminescence kit . Results were analyzed by The relationships between GC and the toxicity of CP were investigated by network analysis as shown in Next, bioinformatics analysis of common targets was conducted . The detThe candidate critical components of GC were docked with the critical common targets to verify the accuracy of the prediction mentioned above. Glycyrrhizic acid and glycyrrhetinic acid were successfully docked with CYP1A1, CYP1A2, ESR1, and TOP2A , which sp < 0.01; The liver index and serum levels of ALT and AST were used to assess the detoxification of GC on CP-induced hepatotoxicity. After CP administration, the liver index and the serum levels of ALT and AST were significantly increased when compared with the control (p < 0.05) when compared with CP. Taken together, these results showed that GC alleviated liver injury to reduce CP-induced hepatotoxicity in rats. Moreover, hepatic pathological changes mentioned above were not found in GC administered alone group, which means liver injury was not induced by GC in 900 mg/kg.Histological staining of liver  showed tp < 0.01). The positive expression after GC pretreatment (900 mg/kg) was significantly decreased when compared to CP (p < 0.05). The ELISA results showed that levels of Bcl-2 in the liver were significantly decreased by CP (p < 0.01), and were effectively reversed after GC administration (p < 0.05).The predicted detoxification of GC on CP is most likely associated with apoptosis. Therefore, the protein expression of caspase-3, p53, and Bcl-2 was determined by immunohistochemistry or ELISA. The positive expression of p53 located in the nucleus and that of caspase-3 in the cytoplasm was rare in liver tissue of control rats , and thestration . Moreovestration  revealedp < 0.01), and the high expression in the cytoplasm was significantly reduced after GC-treatment (900 mg/kg). Moreover, in CP-induced injured livers, the levels of TNF-α, IL-Iβ, and MDA were increased and SOD levels were reduced when compared with the control (p < 0.01), whereas they were effectively reversed in a dose-dependent manner after GC administration . Among them, molecular function and biological processes of 111 upregulated proteins were related to acute-phase responses, cellular responses to organic cyclic compounds, liver regeneration, vasodilation, and negative regulation of cell death. In addition, 47 downregulated proteins were related to oxidation-reduction processes, cholesterol biosynthetic processes, acetyl-CoA metabolic processes, NADP metabolic processes, and lipid metabolic processes (p < 0.05). Among them, 18 upregulated proteins were related to phosphatidylcholine metabolic processes, neuromuscular processes, regulation of protein localization, calmodulin binding, and calcium ion binding  and ABCC2 levels were significantly decreased (p < 0.05), whereas they were effectively reversed by GC pretreatment in a dose-dependent manner. Among them, the expression levels of Cyp4a2, ABCB1B, and ABCC2 were consistent with the data obtained by proteomics. Moreover, there was no significant difference in the expression levels of candidate critical proteins in GC administered alone group when compared with control.The expression levels of candidate critical proteins were determined by Western blot analysis. A network of critical proteins, known targets and cellular components, molecular functions, and biological processes was constructed to investigate the molecular mechanism of detoxification of GC against CP-induced hepatotoxicity . VerifieCurrently, CP is one of the most effective antitumor drugs, but has serious adverse reactions, including nephrotoxicity, ototoxicity, hepatotoxicity, and genetic toxicity . GC, a tIn this study, we confirmed that GC can recover functional indices and liver pathological injury in the injured liver to exert favorable effects against CP-induced hepatotoxicity. However, as a Traditional Chinese Medicine (TCM), it is a challenge for GC to determine the effector molecules involved, reveal the underlying molecular mechanism of action, and locate its detoxification to CP-induced hepatotoxicity in the clinic. In our study, a systematic approach based on network pharmacology and proteomics was established to provide a panoramic and accuracy view of the comprehensive efficacy of GC by predicting the detoxification, investigating the detoxification of GC in rats, and by analyzing the changes in protein expression in the liver.Apoptosis, oxidative stress, and inflammatory responses were evident after treatment with CP , which w4-induced liver injury could be improved by decreasing the expression of CYP4A2  expression, and accelerating excretion . Cytochrf CYP4A2 . ABCB1 cf CYP4A2 . P-gp haf CYP4A2 , and plaf CYP4A2 . ABCC2 ef CYP4A2 , and druf CYP4A2 . And thef CYP4A2 . Moreovef CYP4A2 , and in f CYP4A2 . As a kef CYP4A2 . ESR1 cof CYP4A2 . TOP2A wf CYP4A2 . Overexpf CYP4A2 , and is f CYP4A2 . The expf CYP4A2 .FIGURE 9In conclusion, GC plays a detoxifying role through multitargets and multifunction, and can be used as an adjuvant drug candidate for CP to effectively reduce hepatotoxicity by inhibiting oxidative stress, apoptosis, inflammatory responses, and accelerating metabolism.http://www.ebi.ac.uk/pride with the accession number: PXD021688.The original contributions presented in the study are publicly available. These data can be found here: The animal study was reviewed and approved by the Animal Care Welfare Committee of Gansu University of Chinese Medicine  and followed related regulations.QM conceived the study, conducted the proteomics, and wrote the manuscript. YD conceived the study. PL, JM, and XY performed the animal study. ZY, YZ, and XJY conducted ELISA and Western blot experiments. All authors reviewed and approved the final manuscript.This work was supported by National Natural Science Foundation of China (No. 81960723), Lanzhou science and the technology planning project (No. 2018‐4‐63), Open Foundation project of Key Laboratory of Pharmacology and Toxicology of Chinese Medicine in Gansu Province (No. ZDSYS‐KJ‐2018-09), and Foundation of Chengdu Medical college (No. CYZZD 20‐02).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SLC44A4) rs577272, notch receptor 4 (NOTCH4) rs3134931 SNPs and serum lipid levels in the Han and Maonan ethnic groups.The current research was to assess the relationship of the solute carrier family 44 member 4  was obtained by using polymerase chain reaction and restriction fragment length polymorphism technique, combined with gel electrophoresis, and confirmed by direct sequencing.The genetic makeup of the SLC44A4 rs577272 and NOTCH4 rs3134931 SNPs were different between Han and Maonan populations (P < 0.05); respectively. The SLC44A4 rs577272 SNP was associated with total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) levels in Maonan group. The NOTCH4 rs3134931 SNP was associated with triglyceride (TG) in Han; and TG and low-density lipoprotein cholesterol (LDL-C) levels in Maonan groups (P < 0.025–0.001). Stratified analysis according to gender showed that the SLC44A4 rs577272 SNP was associated with TC and HDL-C in Han and Maonan females; TC in Maonan males, meanwhile, the NOTCH4 rs3134931 SNP was associated with TG and HDL-C in Han males; TG in Han females; TG and LDL-C in Maonan males; and TG, HDL-C and LDL-C in Maonan females. Linkage disequilibrium analysis showed that the most common haplotype was rs577272G-rs3134931A (> 50%) in both Han and Maonan groups. The haplotype of rs577272G-rs3134931A was associated with TG and HDL-C in Han; and TC, TG and HDL-C in Maonan ethnic groups.The genotype frequencies of SLC44A4 rs577272, NOTCH4 rs3134931 SNPs and serum lipid parameters may vary depending on the gender and/or ethnicity/race in some populations. Haplotypes could explain more changes in serum lipid parameters than any single SNP alone particularly for TC, TG and HDL-C.These results suggest that the relationship among  Dyslipidemia is heritable risk factor of coronary heart disease (CHD), which has been a prominent reason of disability, mortality, morbidity, functional deterioration and expensive healthcare, and accounts for approximately 30% of all the deaths worldwide –4. PreviSLC44A4; also knows as: CTL4; NG22; TPPT; DFNA72; hTPPT1; C6orf29, GeneID:80736, HGNC ID: 13941, locus type: gene with protein product, located in chromosome 6p21.33) was associated with serum TC and C-reactive protein (CRP) levels, which are all risk factors for CHD [NOTCH4; also knows as: INT3, Gene ID: 4855, HGNC ID: 7884, locus type: gene with protein product, located in chromosome 6p21.32) may result in regulating serum myeloperoxidase (MPO) levels in Europeans [NOTCH signaling is impacted by lipid-mediated inflammatory status, and its down-regulation seems to correlate with an inflammatory state in the endothelium, and all NOTCH receptors (NOTCH1-4) are expressed in the vascular system [NOTCH4 expression is significantly reduced in patients with HLP, NOTCH4 is a pathogenic factor involved in the process that lipids lead to vascular endothelial inflammation [SLC44A4 rs577272, NOTCH4 rs3134931 SNPs and serum lipid levels in Han and Maonan ethnic groups is not clear and not reported in literature.Previous genome-wide association studies (GWASes) have demonstrated that the rs577272 SNP near the Solute carrier family 44 member 4 gene , the total population of the Maonan ethnic group was 107,166 (37th). Most of the Maonan people are located in Huanjiang Maonan Autonomous County, Guangxi Zhuang Autonomous Region. Although the population of Maonan is small, there are various differences in lifestyle and dietary habits between Maonan and local Han populations, the marriage custom in Maonan is relatively conservative. Maonan still maintain the custom of intra-ethnic marriages, thus, intermarriage with other ethnic groups is very rare [10, the tA total of 1254  unrelated participants of Han nationality and 1213 unrelated subjects  of Maonan nationality were arbitrarily chosen based on our previously stratified randomized samples. All of the subjects were farm workers. They were staying in Huanjiang Maonan Autonomous County, Guangxi Zhuang Autonomous Region of China. They were in the age range of 16–88 years. There was not any difference in age distribution (57.58 ± 12.94 vs. 57.20 ± 15.08) and gender ratio between Han and Maonan groups, respectively. The selection criteria for Maonan individuals have been described in detail in our previous epidemiological studies . All sub2), blood pressure, and waist circumference were measured [Universally standardized methods and protocols were used to conduct the epidemiological survey . By usinmeasured .A fasting venous blood sample (5 mL) was collected from each participant. A part of the sample (2 mL) was collected into glass tubes to measure serum lipid levels. The remaining 3 mL of the sample was collected in the tubes containing anticoagulants  and was utilized to extract deoxyribonucleic acid (DNA). Measurements of serum TG, TC, LDL-C, and HDL-C levels in the samples were performed by enzymatic methods with commercially available kits . Serum ApoA1 and ApoB levels were detected by the immunoturbidimetric immunoassay using a commercial kit  . FastingSLC44A4 rs577272 and NOTCH4 rs3134931 SNP genotypes. The primer sequences of the SLC44A4 rs577272 and NOTCH4 rs3134931 SNPs as follows: forward 5′-ACTGTAGGTGCTCACTGGAT-3′ and reversed 5′-GATTCGTATTGCCATCGCCC-3′; forward 5′-AGAAGAGGAAAGGTGGAGGC-3′ and reversed 5′-AAGCTGGGTGTCAATGGAGA-3′ ; respectively. The PCR reaction mixture  contained 2.0 μL of genomic DNA, 1.0 μL of each primer (10 μmol/L), 12.5 μL of 2 × Taq PCR Master Mix , and 8.5 μL of DNase/RNase-free ddH2O. The cycle details for the reaction are as follows: 95 °C for 5 min, 95 °C for 30 s for denaturing, 59 °C for 30 s for annealing, and elongation for 35 s at 72 °C for 35 cycles. The final extension of 72 °C for 7 min was used to finish amplification. Electrophoresis was done by using 2.0% agarose gels to run PCR products and bands were visualized by using ultraviolet light , and the PCR products located in 522- and 490-bp bands represent the target genes. The restriction enzyme reaction system includes 5.0 μL amplified DNA, 8.8 μL nuclease-free water, 1.0 μL of 10 × buffer solution and 0.2 μL RsaI restriction enzyme in a total volume of 15 µL digested at 37 °C overnight. Restriction enzyme was used to digest the amplified DNA. Next, the genotypes were recognized by running an electrophoresis with 2.0% agarose gel and were visualized under ultraviolet light . An experienced reader who was unaware of the epidemiological data and lipid levels scored genotypes. Different bands of enzyme-digested products represent different genotypes of SLC44A4 rs577272 polymorphism ; NOTCH4 rs3134931 polymorphism . Six samples detected by PCR–RFLP were also established by direct sequencing with an ABI Prism 3100 .The phenol–chloroform method was used to isolate genomic DNA from the peripheral blood leucocytes of the blood samples , 29. TheSerum ApoB (0.80–1.05 g/L), TG (0.56–1.70 mmol/L), LDL-C (2.70–3.10 mmol/L), TC (3.10–5.17 mmol/L), HDL-C (1.16–1.42 mmol/L), ApoA1 (1.20–1.60 g/L) levels and the ApoA1/ApoB ratio (1.00–2.50) were defined as normal values at our Clinical Science Experiment Center. The participants with TC > 5.17 mmol/L and/or TG > 1.70 mmol/L were defined as HLP . The diat test. Covariance analysis (ANCOVA) was used to test the relationship between blood lipid parameters and genotypes, and P < 0.025  was considered significantly statistical significance. The correlation between haplotypes/genotypes and the occurrence of HLP was detected by unconditional logistic regression analysis. Age, gender, BMI, alcohol consumption, cigarette smoking, and blood pressure were adapted for the statistical analysis. In order to estimate the link between the genotypes and some environmental elements with blood lipid levels in males and females of Han and Maonan populations, multivariable linear regression analysis with stepwise modeling was used. P value of < 0.05 was considered as statistically significant. Interactive heat map with several parameters related to blood lipid levels was drawn by R software (version 3.3.0) [All data were evaluated by using SPSS (Version 22.0). The values of quantitative variables were presented as mean ± SD. Only serum TG levels were reported as medians and interquartile ranges. Direct counting was used to determine allele frequency. The standard goodness-of-fit test was utilized to verify the Hardy–Weinberg equilibrium (HWE). Chi-square test was used to assess the differences in the genotype distribution of selected 2 SNPs, the proportion of smokers and alcohol consumption between the two populations. The difference in general characteristics between Han and Maonan was analyzed by the independent-samples n 3.3.0) .P < 0.05). The levels of serum TG, TC, LDL-C and ApoB, systolic and diastolic blood pressure, pulse pressure, the proportion of smokers and alcohol consumption were lesser in the Han than in the Maonan nationalities (P < 0.05–0.001). There was no obvious difference in age distribution, gender, height, BMI, weight, waist circumference and glucose between Han and Maonan nationalities. Subgroup analysis also found that the levels of ApoB, TC, weight, glucose, BMI, waist circumference, TG, systolic blood pressure, LDL-C, the proportion of smokers, diastolic blood pressure, alcohol consumption and pulse pressure were higher in HLP than in normal subjects in both Han and Maonan groups; the levels of ApoA1, HDL-C and the ApoA1/ApoB ratio were less in HLP than in normal subjects in both Han and Maonan groups; there was no any obvious difference in following factors such as gender, height, and age distribution in HLP than in normal subjects in both Han and Maonan groups.As mentioned in Table Results from PCR and electrophoresis showed that each sample had the presence of 522-bp Fig. a1 nucleoSLC44A4 rs577272 and NOTCH4 rs3134931 SNPs in both Han and Maonan populations conformed to HWE (P > 0.05). As shown in Table SLC44A4 rs577272 and NOTCH4 rs3134931 SNPs were different between Han and Maonan populations (P < 0.05); respectively. As shown in Table P = 0.0011); dominant model: GA/GG versus AA ; overdominant model: AA/GG versus GA  and log-additive model: G versus A  in Maonan ethnic group. The genotypes of the rs3134931 SNP were associated with the risk of HLP in different genetic models: co-dominant model: AG versus GG ; dominant model: AG/AA versus GG ; recessive model: GG/AG versus AA  and log-additive model: A versus G  in Han ethnic group and co-dominant model: AG versus GG ; dominant model: AG/AA versus GG ; recessive model: GG/AG versus AA  and log-additive model: A versus G  in Maonan ethnic group. As shown in Table SLC44A4 rs577272 SNP was associated with TC and HDL-C in Maonan group, the NOTCH4 rs3134931 SNP was associated with TG in Han; TG and LDL-C in Maonan group (P < 0.025–0.001). Stratified analysis according to gender showed that the SLC44A4 rs577272 SNP was associated with TC in Maonan males; TC and HDL-C in Han and Maonan females; TC in Maonan males, meanwhile, the NOTCH4 rs3134931 SNP was associated with TG and HDL-C in Han males; TG in Han females; TG and LDL-C in Maonan males; TG, HDL-C and LDL-C in Maonan females (P < 0.025–0.001).The genotypic scattering of the P < 0.05–0.001, respectively). In addition, multivariate logistic analysis showed that the rs577272G-rs3134931A haplotype was positively correlated with the incidence of HLP in Han and Maonan according to stratified risk factors ; respectively.Table P < 0.05–0.001 for all).As shown in Tables SLC44A4 rs577272 and NOTCH4 rs3134931 SNPs were connected with serum lipid levels. Several environmental factors such as weight, gender, height, age, waist circumference, alcohol consumption, cigarette smoking, BMI and blood pressure levels were also correlated with serum lipid parameters in both ethnic groups.As shown in Fig. SLC44A4 rs577272 and NOTCH4 rs3134931 SNPs were significantly different between Han and Maonan populations. (2) The SLC44A4 rs577272 SNP was associated with TC and HDL-C in Maonan ethnic group, the NOTCH4 rs3134931 SNP was associated with TG in Han, TG and LDL-C in Maonan ethnic groups. (3) Stratified analysis according to gender showed that the SLC44A4 rs577272 SNP was associated with TC and HDL-C in Han and Maonan females; TC in Maonan males, meanwhile, the NOTCH4 rs3134931 SNP was associated with TG and HDL-C in Han males; TG in Han females; TG and LDL-C in Maonan males; and TG, HDL-C and LDL-C in Maonan females.The main findings of the current research included the following aspects: (1) It revealed that the genotype frequencies of SLC44A4 rs577272 and NOTCH4 rs3134931 SNPs was diverse amongst various origins. As per the HapMap data, the occurrence of rs577272G allele was 42.7% in Chinese, 48.3% in American, 33.5% in Italian, 35.0% in Kenyan and 34.3% in Japanese and 45.6% in European population. At the same time, the occurrence of rs3134931A allele was 47.6% in Chinese, 43.6% in Japanese, 55.3% in Yoruba, 67.6% in Italian, 53.5% in Kenyan, 57% in Mexican, and 69.5% in European population. However, the genotypic and allelic frequencies of the SLC44A4 rs577272 and NOTCH4 rs3134931 SNPs have not been reported previously in Maonan group. In this study, we firstly reported that the frequencies of rs577272G allele and AG, GG genotypes were 50%, 50% and 25%; rs3134931A allele and AG, AA genotypes were 46%, 49% and 22%; respectively. It means the frequencies of the rare homozygous genotype and minor allele of two selected SNPs were different between European and Asian. The above results indicated that the frequencies of minor allele or rare homozygous genotype of selected 2 SNPs would be shared a racial/ethnic-specificity. We speculated that the differences in blood lipid levels between the two ethnic groups might partly be attributed to the differences in the genotype frequencies of the two SNPs.A lot of studies have showed that HLP as a severe risk factor for CHD, may be due to the combined effects of various elements, just as the age, gender, lifestyle, genetic background, environmental factors and their interactions , 36. HLPSLC44A4 rs577272 SNP is associated with serum TC and CRP levels. In addition, the NOTCH4 rs3134931 SNP was highly associated with circulating serum or plasma MPO levels, which were responsible for the incidence as well as development of the CHD and ischemic stroke [SLC44A4 rs577272, NOTCH4 rs3134931 SNPs and blood lipid parameters was not previously documented in the Maonan population, the results of the current research clearly indicated that the levels of TC were higher and those of HDL-C were lower in the rs577272G allele carriers than in the rs577272G allele non-carriers in Maonan ethnic group. Meanwhile, the rs3134931A allele carriers had higher TG levels in Han nationality and higher TG and LDL-C levels in Maonan ethnic group than the rs3134931A allele non-carriers.Previous studies suggested that plasma concentrations of TG, TC, LDL-C, HDL-C were the most important risk factors for CHD and targets for therapeutic intervention . At the c stroke , 42. At c stroke –46. Furtc stroke –49. MPO-c stroke –52. AlthImportant inter-genetic LD associations were also found in the current study. A strong linkage imbalance was detected between the two loci in both Han and Maonan ethnic groups. The haplotype of rs577272G-rs3134931A was the commonest one and accounted for more than 50% of the samples. The haplotype of the rs577272G-rs3134931A was related to an increased morbidity of HLP in the both Han and Maonan groups. At the same time, the haplotype of rs577272G-rs3134931A was associated with TG and HDL-C levels in Han; TC, TG and HDL-C levels in Maonan ethnic groups. We also noticed that haplotypes could explain more changes in serum lipid parameters than any single SNP alone particularly for TC, TG and HDL-C.Previous studies indicated that several environmental factors were significantly associated with blood lipid spectrums, including hypertension, obesity, daily exercise, diet and lifestyle –56. In tRice acts as a staple food of Maonan people. In addition, corn, potato, wheat, sorghum and so on are also be components of their diet. Maonan people especially like to eat some food that rich of oil, spicy, acid and salt. This type of diet rich in long-term high saturated fat might contribute to obesity, hypertension, high blood glucose levels, atherosclerosis and HLP . PreviouSLC44A4 rs577272, NOTCH4 rs3134931 SNPs and serum lipid levels, other genomic as well as environmental factors are necessary to be considered. The future studies need to be done to study the effects of either gene–gene or gene-environment or environment-environment on serum lipid levels. In order to further demonstrate our findings, some efficient studies on the natural functions of the SLC44A4 rs577272 and NOTCH4 rs3134931 mutations are essential.This study may have several limitations. To begin with, in the statistical analysis, we were not in a position to mitigate the effects of diet and some environmental factors. Secondly, other serum lipid parameters such as HDL2, small dense LDL, large buoyant LDL etc. had not been measured in our study. Thirdly, regardless of the fact that we observe a significantly correlation between the SLC44A4 rs577272, NOTCH4 rs3134931 SNPs and serum lipid levels were not similar between Han and Maonan populations as well as among men and women in both ethnic groups. There might be a race- and/or gender-specific relationship of the SLC44A4 rs577272, NOTCH4 rs3134931 SNPs and serum lipid levels. Haplotypes could explain more changes in serum lipid parameters than any single SNP alone particularly for TC, TG and HDL-C.The associations of the