P < 0.05), accompanied with the decreased intestinal permeability and the tighter gut barrier. Moreover, the efficiency of DMF was better than TC (P < 0.05). 16S rRNA gene sequence analysis revealed that the richness and diversity of bacteria was increased in DMF treatment. The most abundant OTUs belonged to Firmicutes and Bacteroidetes, and their changes in DMF were more moderate than the TC group, suggesting a more stable micro-ecology and the positive impact of DMF on the biodiversity of intestine. Specifically, the increased abundance of bacteria producing short-chain fatty acids (SCFAs), such as Gemella, Roseburia, Bacillus and Bacteroides in DMF group and prebiotics such as Lactobacillus in TC group, suggested a more healthier microbial composition and distribution. These findings supported that DMF had significant effects on animal's growth performance and intestinal barrier function by modulating the pathway of nutrient absorption and increasing the diversity and balance of gut microbes, which also illuminate that DMF is more efficient than traditional anti-mildew method.The effects of dimethyl fumarate (DMF) on mycotoxins and animal growth performance are well documented. However, its mechanism of anti-mildew effects is still unknown. The current study investigated how DMF detoxified the mycotoxin and improved the growth performance using BALB/c mice model, especially its effects on intestinal barrier function and gut micro-ecology. Our study also compared with the ultraviolet radiation (UR) treatment, a traditional anti-mildew control (TC). The results indicated that the DMF treatment had a lower contents of mycotoxin, better growth performance and improved mucosal morphology ( S) and intensifying the systemic inflammation [Attentions have focused on mycotoxins for their infection on monocotyledon-based crops such as maize, wheat and barley  which leammation . Actuallammation . Howeverammation .It is worth to pay attention that, enterocytes exposed to mycotoxins in gut lumen are particularly susceptible to the contamination of mycotoxins. Mechanistically, the mycotoxin-induced disruption of the gut barrier is an orchestrated process that occurs in association with increased phosphorylated MAPKs and subsequently decreased the expression of tight junction proteins such as Claudins , which lGenerally, several mycotoxin-detoxifying agents can be classified into physical removal methods, sequestering (absorbing) agents, and bio-transforming agents that are based on their mode of action. In our research, DMF is the effective additive belonging to biotransformation agents, it is not only able to bind mycotoxins and reduce their gastrointestinal absorption and bioavailability, but also restore a harmonious intestinal micro-environment due to its antioxidant and anti-inflammatory property . The traThe IECs can also maintain immune homeostasis via contacting with commensal bacteria. If the intestinal epithelial barrier is disrupted, the gut microbiota will pose a risk of infection and inflammatory responses . ConsideSeveral studies have been carried out to investigate the influence of traditional methods on physically removing, in which the innovative disinfectants, such as DMF, were not developed. Moreover, the alteration of gut barrier and microbiota was ignored during the detoxication process. Nowadays, with the development and application of high throughput 16S rRNA sequence analysis, more comprehensive variations of intestinal bacteria caused by the mycotoxin and its disinfectants, such as DMF, could be discovered. In the present study, we aimed at revealing the potential mechanism of DMF that weakened the damage caused by mycotoxins and improved the gut function via modulating intestinal microbiota and barrier. Furthermore, whether the DMF treatment or the TC had a better effect was also compared, in order to make a rational choice between traditional anti-mildew methods and novel anti-mildew additives when detoxicating mycotoxins.1 (AFB1), fumonisin B1 (FB1) and zearalenone (ZEA), were all decreased by treating with DMF. The most significantly decreased mycotoxin was AFB1, differences among each group were markedly observed (P < 0.05). As to FB1, DMF still showed the best detoxification effect (P < 0.05). However, a significant decrease of ZEA only existed in the traditional control (TC) group when compared to the negative control (NC) group (P < 0.05), while the DMF treatment had unmarked effect (P > 0.05)  Figure .P < 0.05) with a non-different initial body weight (IBW) (P > 0.05). Meanwhile, the increased level of the DMF group was larger than the TC group when compared with the NC one  were observed between the DMF and NC group, even between the DMF and TC treatment  . Although the increased level of the TC group was lower than the DMF one, its increasing was also significant (P < 0.05) on the basis of the NC group. In addition, DMF had non-marked influence on the crypt depth (C) and the ratio of the villi length and crypt depth (V/C) both in jejunum and ileum (P > 0.05)  Figure .P < 0.05), when compared with the NC group  .To evaluate the effect of the DMF treatment to the signal pathway of gut barrier, the expression of mucosal barrier protein, such as Occludin, Claudin-1 and zonula occludens protein-1 (ZO-1) were tested by western blot Figure . The expp Figure . While i) Figure . The abuTo further study the mechanism of dietary anti-mildew technologies on improved intestinal mucosal morphology and permeability, the intestinal bacterial richness and diversity in digesta was determined. In total, after size filtering, quality controlling and chimera removalling, 278,713 valid sequences were obtained, with an average of 96,567 sequences, 51,769 sequences and 130,377 sequences separately in per jejunal, ileal and colonic sample. The overall operational taxonomic unit (OTU) number classified at a distance level of 0.03 (97% similarity) was 1,570, among them, 481 was detected in jejunal sample and 402 and 687 were detected separately in ileal and colonic samples, thus, conlonic bacteria community had much more OUT numbers than jejunal and ileal ones. All samples reached a stable plateau based on rarefaction curve analysis, indicating the sampling was sufficient for the majority of the bacterial communities.The bacterial richness estimated by the Chao-1 estimate (Table P < 0.05). In addition, in the large gut, such as the colon, the Shannon index after DMF treatment was similar to both control groups.The Shannon index analysis Table  indicateJejunal bacterial community of three groups shared with 249 OTUs as shown in Venn diagrams, which accounted for 33.29% of the proportion Figure . FirmicuAt family level, Lactobacillaceae, Lachnospiraceae, Bacillaceae, Streptococcaceae, Ruminococcaceae and Family_XI constituted the main components of Firmicutes, while Bacteroidetes were mainly composed of Bacteroidales_S24-7_group (S24-7) and Proteobacteria were consisted of Desulfovibrionaceae. Lactobacillaceae (33.37%) and S24-7 (39.25%) were the most dominating families in the NC group with different changing tendencies between TC and DMF groups. Lactobacillaceae was significantly increased in TC group from 33.37% to 78.03%, while the proportion of S24-7 dropped dramatically from 39.25% to 0.14% and 26.69% after applying TC and DMF separately. Notably, the proportion of Desulfovibrionaceae and Lachnospiraceae were dramatically decreased in TC treatment, from 12.22% to 0.04%, and from 9.49% to 0.15%, separately. Meanwhile, the increased abundance of Bacillaceae was also significant in the DMF group, which was detected from 2,49% to 9.45%.Lactobacillus, Desulfovibrio and Bacillus were predominant genera of Lactobacillaceae, Desulfovibrionaceae and Bacillaceae, respectively. Meanwhile, Lachnoclostridium and Lachnospiraceae_NK4A136_group were both main genera of Lachnospiraceae. Specifically, Lactobacillus was dramatically increased in the TC group, which rose from 33.37% to 78.03%, while there was no obvious changing in the DMF treatment. On the contrary, Desulfovibrio, Lachnoclostridium and Lachnospiraceae_NK4A136_group were all significantly decreased in the TC group with the proportion from 12.18% to 0.04%, from 3.78% to 0.02%, and from 2.25% to 0.05%, respectively, and the degree of this decline was all definitely higher than the DMF treatment. As to Bacillus, its significant increase in the DMF group, was up to 9.36% on the basis of 2.45% in the control one. The proportional variation in these bacteria with anti-mildew treatments mainly led to the change of proportion at family level. In addition, about 42.35% jejunal bacteria community were unidentified at genus level.Down to genus levels, About 29.68% OUTs were shared among three treatments of bacterial community in ileum as shown in Venn diagram Figure . The DMFAs to family level, Firmicutes in ileum were mainly composed of Lactobacillaceae and Lachnospiraceae. The TC group had a significant larger proportion of Lactobacillaceae but the lowest proportion of Lachnospiraceae than other two groups. The abundance of Ruminococcaceae, Clostridiaceae_1 and Family_XI, which were all belonged to Firmicutes, was increased and only observed in DMF. The Desulfovibrionaceae belonged to Proteobacteria and the Coriobacteriaceae belonged to Actinobacteria were both dramatically decreased in the TC group compared to the NC one, which from 13.70% to 1.18%, and from 3.03% to 0.09%, respectively. In addition, Bacteroidetes mainly consisted of S24-7, were significantly decreased both in the TC and DMF treatments.Lactobacillus, Lachnoclostridium and Desulfovibrio were dominant genera of Lactobacillaceae, Lachnospiraceae and Desulfovibrionaceae, respectively. The proportion of their changes was consistent with the variation of their affiliated family. In addition, a significant increasing from 0.01% to 9.49% in the TC group was detected although there was no marked changes at family level. However, approximate 1.48% to 10.93% of ileal bacteria community was unidentified at genus level.Down to genus level, the The colonic bacterial community was further analyzed to reveal the role of DMF on the large gut microbiota. All sequences were classified and identified from phylum to genus Figure .16S rRNA profiles of each experimental group in the colon were very dissimilar even in phylum level distributions. The most abundant phyla associated with the colonic digesta were Firmicutes and Bacteroidetes. It has been detected that in the TC group, the Firmicutes was increased from 48.07% to 52.41%, while after treating with DMF, this phyla dropped to 28.57%. The changes in Bacteroidetes were not significant. However, we could still detect the decreased trend in the TC (from 50.27% to 45.84%) and the increased trend in the DMF group (from 50.27% to 69.80%).Within these two dominating phyla, there were some significant changes of each treatment at family level when compared to the NC group. The TC treatment decreased the Lachnospiraceae and Ruminococcaceae, both of which belonged to Firmicutes, and the decreased level was from 21.93% to 2.71% and from 9.06% to 1.82% separately. However, Lactobacillaceae was also a major family of Firmicutes, which significantly increased in the TC group, while the decline in DMF treatment was not obviously different from the NC group. S24−7, Bacteroidaceae and Prevotellaceae were all main families of Bacteroidetes, and were increased significantly after treating with DMF. However, the Rikenellaceae were decreased in both anti-mildew treatments.Lachnospiraceae_NK4A136_group, Incertae_Sedis and Roseburia were dominant genera of Lachnospiraceae, and their respective changes were consistent with the overall change of Lachnospiraceae, that were decreased in both anti-mildew treatments. Ruminiclostridium_9 and Ruminococcaceae_UCG-014 had the similar tendency of variation to the family of Ruminococcaceae. In addition, Lactobacillus, Alistipes, Prevotellaceae_UCG-001 and Bacteroides were major genus of Lactobacillaceae, Rikenellaceae, Prevotellaceae and Bacteroidaceae, respectively. What's more, the changes of these genus proportion, with exposing to the TC and DMF treatments, were almost as the same as the changing of family.In the analysis of the bacterial composition down to the genus level, The present work was undertaken to compare the different germicidal efficiency of DMF, a novel kind of anti-mildew technology in the BALB/c mice model. In contrast to applying UR which was a traditional and widely-used method on physically removal, adding germicidal additives such as DMF was a more efficient anti-mildew treatment. Our data demonstrated that DMF could detoxify mycotoxins. It is the first time to study the alteration of microbiota and find a promotion of harmonious gut micro-organism especially due to the DMF treatment. Moreover, considering the crosstalk between barrier and microbiota, it's rational for us to infer that DMF can improve the gut barrier and microbiota to gain a more efficient influence on mycotoxin than traditional anti-mildew method.1 (ppb), FB1(ppm) and ZEA (ppb), were all significantly deceased in the TC group. Our study also showed that the detoxification function of TC in ZEA was even much better than DMF, indicating that there were some individual functions between TC and ZEA. Consistent with our hypothesis, a previous study found that ZEA possessed the C10–C20 double bond which was essential to induce neutrophil suppression. Interestingly, the present TC treatment was prefer to damage or destroy the bond at least in part, thereby inhibiting the ZEA proliferation as well as alleviating toxic activity of ZEA [UR was a common pattern in physical removal methods against mycotoxins and was acted as the TC in our current study. It has been applied to the sterilization of agricultural products which was known to be effective for killing pathogenic moulds that contaminated the surface of grain . These ry of ZEA . On the y of ZEA  and amelP > 0.05) when compared with the control groups, which might result from the lower renewal and maturing rate of enterocyte. Accumulated studies reported that the mycotoxin could damage the intestinal structure and cause the toxic effect on gut morphology. Moreover, the mycotoxin also disrupted the tight junction, impaired the intestinal carrier functions, and leaded to an imbalance in the antioxidant system [The villus height and the crypt depth are important indicators to reflect the digestive and absorptive functions of the small intestine. Villus is a main micro-structure of nutrient absorption and transportation, and the area of villi will be increased when its height rising that consequently means an improved ability of nutrient absorption . The patt system . Parallet system . The inct system , 18 and t system . We belit system .Indeed, data from human and animal studies suggested that DMF had anti-oxidant properties. Moreover, the physiological maintenance of gut integrity and the function of intestinal epithelium were positively related to the anti-oxidant activity. Thus DMF might improve the gut barrier with the mechanism of its anti-oxidant property. Recently, it has been demonstrated that DMF reduced the production of reactive oxygen species (ROS) and also reduced the oxidative stress . With ROPrevious study observed that the epithelial disruption was less to be statistically significant in mice at the histological level after treating with DMF . The remThe relation between the gut microbiota and human health is being increasingly recognized. Intestinal microbiota imparts specific function in host nutrient absorption, drug metabolism, maintenance of structural integrity of the gut mucosal barrier, immunomodulation, and the protection against pathogens , 30. ChaGemella, Roseburia and Bacillus were stimulated to increase in jejunum and ileum after treated with DMF. Previous studies have shown that Gemella was obligatory fermentative, producing either a mixture of acetic and lactic acids or an equimolar molar mixture of acetic acid and CO2 depending on the abundance of oxygen [Roseburia prefer to express enzymes favoring the production of butyrate over propionate [Roseburia in DMF treatment is beneficial to gut health. Bacillus are capable of expressing spore-forming proteins, and recent studies indicated that bacteria genera which were capable of both butyrate production and spore formation might have characteristics expected in probiotic strains. Moreover, the ileal Bacillus increment raised by the DMF treatment could be attributed to the increment in ileal barrier function. After supplemented Bacillus subtilis PB6, an increased villus height and the abundance of zonula occludin-1 (ZO-1) and Claudin-1 in the ileum were found, indicating a better intestinal development [What is crucial, the anti-fungal treatments have been found to improve the intestinal bacterial communities, which might help to reveal the mechanism that improved the growth performance and intestinal barrier function of anti-mildew treatments. Results of our studies with bacteria producing short-chain fatty acids (SCFAs) in the small intestine indicated that bacteria belonging to the f oxygen . Generalopionate , which wopionate  but is aelopment , 38, whiDesulfovibrio was also obvious in jejunum and ileum. Desulfovibrio belongs to a kind of sulfate-reducing bacteria (SRB), which can induce production of lipopolysaccharides (LPS) and are associated with the microbiota inflammatory properties [Desulfovibrionaceae in our results meaned a restrained inflammation, which was paralleled to our intestinal morphology trial, the villi height in ileum was improved by DMF and TC group. Thus the anti-mildew treatment could be effective in reducing the inflammatory response by changing the intestinal structure as a result of controlling the Desulfovibrio in the upper gut.In the traditional anti-mildew treatment, a drop of operties . The decLactobacillus was also observed in the colon in TC group. Lactobacillus was capable to rapidly establish a complex bacterial community, which prevent host from infected of pathogenic bacteria [Bacteroides was also observed, and this change was limited in the colon, which indicated a specific function of anti-mildew treatments on the lower gut. Bacteroides were known to metabolize oligosaccharides of host origin derived from mucins [Bacteroides were also reported to produce both propionate and succinate as terminal products of its metabolism [Besides the small intestine, the significant increasing of the abundance of bacteria . This prbacteria , which mm mucins , which wtabolism . Furthertabolism .In addition, a combination of multiple dietary anti-mildew technologies was suggested to reduce toxins in the feed due to their respective effects on the growth performance, intestinal barrier functions and the gut ecology. Based on the theory that UR (TC) could remove the surface mold while DMF could alleviate harmful effects of mold in gut, it is a highlight that a combination of the traditional physical removal method and the innovative additives provide insight into new areas and may lead to new strategies to control mycotoxins in feed. Preferentially, we characterize that the crosstalk between gut microbiota and barrier may be the potential mechanism of DMF to enhance the gut health and food safety. The study we conducted is aimed at a stable gut micro-ecology and is expected to be beneficial to the health improvements in humans.Lactobacillus, Clostridium, Gemella and Blautia. They not only inhibit pathogenic bacteria but also derive some beneficial metabolites to maintain and restore the harmony of gut micro-ecology. Thus we rationally rank DMF as a better anti-mildew method.In summary, we found the DMF was effective in reducing the negative effect of mycotoxins on diets for BALB/c mice. Specifically, we tentatively put forward that fungicides particularly DMF can decrease the oxidative stress and inflammatory responses by modulating the intestinal ecology through regulating the absorption of nutrition, shitting the intestinal microbiota and enhancing the expression of tight junction proteins to improve the intestinal barrier function. What's more, we found a promotion of several prebiotics such as 36 BALB/c mice  were divided to three groups, male and female half, each group of three repeats, each repeat contained four mice, 28 d for a trial period. All the mice were housed in cages with a squirrel cage nipple drinkers and a self-feeding trough . Observations and manure treatments were routinely done. The three groups were individually treated by basal diet (the NC group) , ultraviolet radiation treatment of feed (the TC group)  and the feed of adding fumaric acid dimethyl ester (0.05%) (the DMF group). Dietary composition and nutrient contents of the basal diet was showed in Table 1, Zearalenone, Fumonisins B1 in the feed by different mould prevention treatments were quantitatively tested by ELISA kit , with the reference to the national standard. 5g crushed samples  were homogenized and 1g of sodium chloride and 25 μL 70% aqueous methanol were added. And then all the samples were oscillated for 3 minutes and centrifuged at 8000 rpm / min for 15 minutes, 100μl Enzyme labeled conjugate was added into the holes of the Elisa plate, each with 50 μL standard sample and 50 μL sample along with 3 times percussion with pipettes, After being mixed, 100μl mixed sample was transferred to the corresponding test hole for 10 minutes reaction and the chromogenic agent was added for 100 μL / hole after 3 times washing, Finally, 50 μL sulfate was added for termination / hole, and the absorbance was measured at 450 nm using an ultraviolet spectrophotometer.The contents of aflatoxin BAll mice were sacrificed for later excision of intestinal segments. On d 28, for all mice's jejunum, ileum and colon's intermediate segments, two 3 cm long samples of each segment were quickly removed. After being thoroughly flushed with ice-cold saline to remove the intestinal fluids and chyme, one part was wrapped with aluminum foil and frozen in liquid nitrogen, and then was stored at -80C° for subsequent assay. The other part was preserved in 10% neutral buffered formalin solution for further test and histological analysis.All samples were fixed in 4% paraformaldehyde for 48h as previously , samples in jejunum, ileum and colon were fixed and embedded by paraffin. Sections of 2-3μm were cut and stained in hematoxylin and eosin. Each slide was divided into three single segments and the microstructures of jejunum and ileum was analyzed by using an microscope . The villus height and the crypt depth of 6 randomly chosen villi were measured, and the ratio of villus height and crypt depth of each sample was calculated.The protein levels of tight junction proteins, Occludin, Claudin-1 and ZO-1, were analyzed using immunoblot due to their crucial functions in intestinal mucosal barrier. The jejunal and ileal tissue samples were removed from -80C° and grinded in liquid nitrogen. RIPA / protease inhibitor / phosphatase inhibitor was added to the finely grinding samples to organize small study in an ice bath reaction for subsequent extraction of total protein. The total protein concentration of ileal and jejunal tissue samples was determined by the use of BCA Protein Assay Kit (Pierce Company). 10 μL was added to each sample of the prepared SDS-PAGE for protein electrophoresis. After that same amounts of protein extracts were transferred onto PVDF membranes  and blocked with 5 % skim milk powder solution at room temperature for 2 h. Then the membranes were incubated with diluted primary antibodies of β-actin  and Claudin-1 (1:200) overnight at 4°C. The primary antibodies against Occludin, Claudin-1 and ZO-1 were obtained from Santa Cruz , as well as β-actin from Sigma-Aldrich . After primary antibody incubation, membranes were carefully washed with 1 × TBST buffer  for three times, each time 5 min. Then horseradish peroxidase-labeled secondary antibody  (Beijing Zhongshan Golden Bridge Biotechnology) was added and incubated at room temperature for 1 h, the membranes were washed four times with 1 × TBST buffer, each time 5min, and then washed with PBS solution for 15 min, After incubating with HRP-conjugated secondary antibody, signals were visualized by the LI-COR Infrared Imaging System . Blots were then analyzed by “Quantity One” Software from BioRad Laboratories , with subsequent calculation of the ratio between the band intensities of β-actin and Claudin-1 in small intestinal segments like jejunum and ileum.°C for 5 min and then 95°C for 30 s and then 56°C for 30 s, 25 Cycles and then 72°C for 40 s and then 72°C for 10 min, finally was ending in 4°C. Products were examined on a 2%  agarose gel. The primers were 338F (5’ACTCCTACGGGAGGCAGCA-3’) and 806R (5’GGACTACHVGGGTWTCTAAT-3’). All procedures were operated according to the manufacture's protocol (Allwegene Company). Two hypervariable regions of 16S rRNA, the V3 and V4 region were used to identify the vast majority of bacteria based on 16S rRNA sequencing. Illumina HiSeq 2000 platform's V3, V4 region through positive and negative reading. Only sequences longer than 50 bp were used for phylotype analysis at the 0.03 OTU level.Four replicates from per treatment were blended in equimolar ratios based on concentration for PCR amplicons. DNA samples taken from the chyme of jejunum, ileum and colon of BALB/c mice were subjected to 16S rRNA gene sequence-based analysis to examine the characteristic bacterial communities along the mouse intestinal tract, including those presented in the jejunum, ileum and colon. Small fragment libraries whose concentration was more than 30 ng / μL were used for PCR amplification. The samples were detected before treatment after being thawed on ice, centrifuged and mixed thoroughly, Qubit test was used to test the sample concentration. The component and volume of the reagents in PCR amplification system concludes DNA sample X (30ng); Forward Primer (10uM), 2 μL; Reverse Primer (10uM) 2 μL; dNTPs (2.5mM) 4μ L; 10 * Pyrobest Buffer 5μL; Pyrobest DNA Polymerase 3 μL; ddH2O 36.7-X μL . The PCR procedure condition was setting in 95P value < 0.05 was considered to be statistically significant.All other data were analyzed using the analysis of variance (ANOVA) procedure of SAS system . The significant difference in the occurrence of growth and other targets among the three groups was tested by Duncan's multiple comparisons test. All indicators were analyzed in each repeat. Statistical results were represented by the mean and standard error,


The hippocampus plays important roles in memory processing. However, the hippocampus is not a homogeneous structure, which consists of several subfields. The hippocampal subfields are differently affected by many neurodegenerative diseases, especially mild cognitive impairment (MCI). Amnestic mild cognitive impairment (aMCI) and subcortical vascular mild cognitive impairment (svMCI) are the two subtypes of MCI. aMCI is characterized by episodic memory loss, and svMCI is characterized by extensive white matter hyperintensities and multiple lacunar infarctions on magnetic resonance imaging. The primary cognitive impairment in svMCI is executive function, attention, and semantic memory. Some variations or disconnections within specific large-scale brain networks have been observed in aMCI and svMCI patients. The aim of this study was to investigate abnormalities in structural covariance networks (SCNs) between hippocampal subfields and the whole cerebral cortex in aMCI and svMCI patients, and whether these abnormalities are different between the two groups. Automated segmentation of hippocampal subfields was performed with FreeSurfer 5.3, and we selected five hippocampal subfields as the seeds of SCN analysis: CA1, CA2/3, CA4/dentate gyrus (DG), subiculum, and presubiculum. SCNs were constructed based on these hippocampal subfield seeds for each group. Significant correlations between hippocampal subfields, fusiform gyrus (FFG), and entorhinal cortex (ERC) in gray matter volume were found in each group. We also compared the differences in the strength of structural covariance between any two groups. In the aMCI group, compared to the normal controls (NC) group, we observed an increased association between the left CA1/CA4/DG/subiculum and the left temporal pole. Additionally, the hippocampal subfields  significantly covaried with the orbitofrontal cortex in the svMCI group compared to the NC group. In the aMCI group compared to the svMCI group, we observed decreased association between hippocampal subfields and the right FFG, while we also observed an increased association between the bilateral subiculum/presubiculum and bilateral ERC. These findings provide new evidence that there is altered whole-brain structural covariance of the hippocampal subfields in svMCI and aMCI patients and provide insights to the pathological mechanisms of different MCI subtypes. The hippocampus is part of the limbic system. It plays important roles in memory processing, especially spatial memory . StudiesHowever, the hippocampus is not a homogeneous structure, which consists of several subfields, specifically the cornu ammonis (CA) areas 1–4, the dentate gyrus (DG), the subiculum, and the presubiculum . The hipMild cognitive impairment is a diagnosis given to older adults who have cognitive impairments but that does not interfere significantly with their daily activities . It is rStructural covariance networks (SCNs), based on voxel-based morphometry (VBM), generate a map of correlation between the gray matter (GM) volume of a region of interest and the other regions , 28. SCNPatients with svMCI and aMCI were recruited through the memory clinic of the neurology department of Xuanwu Hospital, Capital Medical University, Beijing, China. Two experienced neurologists diagnosed all patients using the Petersen criteria . HealthyStructural MR images were acquired using sagittal magnetization-prepared rapid gradient echo (MP-RAGE) three-dimensional T1-weighted imaging sequence on a 3.0 T Siemens scanner at Xuanwu Hospital, Capital Medical University. The image parameters included repetition time (TR) = 1,900 ms; echo time (TE) = 2.2 ms; inversion time = 900 ms; flip angle = 9°; field of view = 224 mm × 256 mm; matrix size = 448 × 512; 176 slices; and slice thickness = 1.0 mm.in vivo MRI scans in 10 subjects  were overlaid to the probabilistic atlas (in Heat color) of hippocampal subfields , as showP < 0.05 with the false discovery rate (FDR) correction and reserved positive covariance. Finally, the results were displayed on the MNI template in the BrainNet Viewer softwareFor each group, the strength of structural covariance between each subfield seed and all other regions across the whole brain were obtained by applying multiple regression models in SPM12 to perform a voxel-based statistical analysis on the smoothed and modulated GM image. We imported the extracted mean GM volume from each seed as a covariate. As the age and gender would influence the GM volumes, we removed the effects of gender and age on the structural covariance networks by entering them as confounding covariates. The resulting covariance patterns were employed with thresholds at Many studies have indicated that the different slopes for any pair of voxels may represent the difference in their structural association , 44. To G was used as a grouping variable, and two groups were put into the same model, where G = 1 for the one group, and G = 0 for another group. The gender and age may affect the association of two voxels, so they were considered as independent variables in a linear model, where X represented the averaged GM volume in each seed, and y represented the GM volumes of each voxel in whole brain. Then, the linear regression model between y and X was adjusted by adding a gender term Gender, an age term Age, a group term G, and an interaction term G × X. Specific t-value contrasts were established to map the significant different voxels in slopes between any two groups. The significant differences between groups were obtained based on the two-tailed Gaussian random field (GRF) correction, with a voxel level of P < 0.01 and a cluster level of P < 0.05.F = 12.059, P < 0.001), AVLT-delayed recall , AVLT-recognition recall , MMSE , and Montreal Cognitive Assessment  through one-way analysis of variance. The following post hoc test revealed that AVLT-immediate recall, AVLT-delayed recall, and MoCa in patients of aMCI and svMCI were significantly lower than scores in controls. In addition, the score of MMSE was significantly lower in the aMCI group than in the control group, but there was no significant difference in score of MMSE between the svMCI and NC groups.Table The SCN patterns of the left and right hippocampal subfields in the three groups are shown in Figures In the aMCI group, in addition to the ERC and FFG, the left CA1 covaried with the left temporal pole (TP), right angular gyrus, subcallosal cortex, and thalamus. For the left CA2/3 network, the structural maps involved the ERC and FFG, subcallosal cortex, thalamus, and angular gyrus. The left CA4/DG correlated regions were similar to the regions in the left CA2/3 network in the aMCI group, but it additionally included the right superior frontal gyrus. For the aMCI group, the left subiculum and left presubiculum covariance maps involved the left TP, subcallosal cortex, thalamus, superior and middle frontal gyrus, right middle occipital gyrus, and bilateral angular gyrus.In the aMCI group, the right CA1 covaried with the ERC and FFG, right TP, PCC, and angular gyrus. CA2/3 showed significant correlation with entorhinal areas, thalamus, bilateral TP, middle frontal gyrus, PCC, and angular gyrus. The CA4/DG correlated regions were similar to the regions covaried with the right CA2/3 subfield in the aMCI group. For the right presubiculum networks, the covariance maps of the right presubiculum involved entorhinal areas, thalamus, bilateral TP, PCC, and angular gyrus. The subiculum showed significant covariance with the ERC, FFG, and right angular gyrus.The left CA1 showed significant correlations with the ERC, FFG, superior occipital gyrus, orbitofrontal cortex (OFC), and right TP in the svMCI group. The covariance maps of the left CA2/3 involved the FFG, right TP, OFC, occipital pole, and entorhinal areas in the svMCI group. The left CA4/DG covariance maps were similar to the covariance maps of the left CA2/3 in the svMCI group. In the svMCI group, the maps of the left presubiculum and left subiculum were virtually identical, and the covaried regions included the FFG, ERC, fusiform, and right TP.The right CA1 covaried with the ERC, FFG, superior occipital gyrus, right TP, and OFC in the svMCI group. The right CA2/3 covaried with entorhinal areas, right TP, and superior occipital gyrus. The right CA4/DG covaried with entorhinal areas, fusiform, right TP, superior occipital gyrus, and subcallosal cortex. The regions covaried with the right presubiculum were similar to those regions connected with the right CA4/DG subfield in the svMCI group. In addition, the covariance maps of the right subiculum hippocampal subfields involved entorhinal areas, fusiform, right TP, and superior occipital gyrus.The left CA1 covaried with the bilateral ERC and FFG in the NC group. In addition to the ERC and FFG, the covariance regions with the left CA2/3 also included the left precuneus cortex. The left CA4/DG covariance maps were extremely similar to the maps of the left CA1 subfield in the NC group. For the left subiculum and left presubiculum networks, both covariance maps involved the left precentral gyrus, FFG, and ERC in NC subjects.The right CA1 covaried with the ERC and FFG in the NC group. In addition to the ERC and FFG, the covariance regions with the right CA2/3 also involved the right OFC. The right CA4/DG correlated regions included the ERC and FFG in the NC group. The right presubiculum showed significant correlations with the right OFC, FFG, and ERC in NC subjects. The right subiculum covaried with the ERC and FFG.There were some significant differences observed between the aMCI group and NC group when the strength of the structural correlations was considered Table . There wThe significant differences of the association slope between the svMCI group and the NC group are shown in Table As shown in Table In this study, we selected hippocampal subfields as seeds to build SCNs among three groups. Specifically, hippocampal subfields correlated with the TP, thalamus, subcallosal cortex, and posterior cingula cortex in the aMCI group, while hippocampal subfields significantly covaried with the OFC in the svMCI group. Finally, we compared the differences in strength of structural covariance between groups. The results demonstrated that there were abnormal structural associations between hippocampal subfields and the cerebral cortex in aMCI and svMCI patients, and these abnormalities were different between them.In our study, positive correlations between hippocampal subfields and FFG, and ERC in GM volume were found in each group. To some extent, these positive correlations suggested synchronous GM changes in these regions , 32. TheAll the hippocampal subfields showed significantly positive structural covariance with the thalamus in the aMCI group. A previous study reported atrophy of the thalamus in aMCI patients . The posWe also observed positive structural associations between the left hippocampal subfields and subcallosal cortex in the aMCI group. This suggested right subcallosal cortex atrophy in aMCI patients . In addiWe found significantly positive structural associations between the right hippocampal subfields and PCC in the aMCI group. Many histopathological , structuWe observed that the increased connections between the right FFG and left presubiculum were stronger in svMCI than in the aMCI and NC groups. Previous studies have shown FFG atrophy in svMCI patients , 54. TheIn the aMCI group, compared to the svMCI group, we observed an increased association between the bilateral presubiculum/subiculum and the ERC. The pathway from CA1 to the subiculum and projections to the ERC form the principal output from the hippocampus. The connections between CA1, subiculum, and ERC were associated with episodic memory processing . TherefoThe left CA1/CA4/DG/subiculum showed significantly increased structural association with the left TP in aMCI patients compared to NC. The stronger structural covariance potentially indicates synchronous GM changes in these regions affected by the disease . Thus, wWe also observed increased structural associations between the left CA1/subiculum and left POG in aMCI compared to NC. Left POG atrophy was reported in aMCI patients . AdditioIn addition, bilateral CA1 and left CA2/3 showed significantly positive associations with the OFC in the svMCI group compared to the NC group. In addition, we found increased covariance between the right CA1 and right PFC in svMCI compared to NC. For PET imaging, the patients with svMCI showed hypometabolism in the inferior and medial frontal cortices adjacent to the cingulate gyrus . ImportaThis study still has several limitations. First, we did not study the structural connectivity based on diffusion-weighted imaging (DWI) of the white matter pathway between hippocampal subfields and the whole brain cortex. There was a lack of systematic comparisons of structural covariance networks and DWI-based networks. Our findings of SCNs among the three groups should be cautiously extended to other structural networks. Second, in this study, due to the limited segmentation accuracies of hippocampal subfields in regular T1 images, we focused on the SCN analysis of each subfield instead of volumetric analysis of subfields. In the future, the volumetric analysis of hippocampal subfields would be helpful to understand the hippocampal abnormalities in the two MCI subtypes with the improvement of segmentation accuracies of hippocampal subfields in regular T1 images.This study was approved by the medical research ethics committee and the institutional review board of Xuanwu Hospital, Capital Medical University, Beijing, China.Conceived and designed the experiments: SL and XL. Analyzed the data: XW. Contributed reagents/materials/analysis tools: YY, YH, CY, and WZ. Wrote the paper: XW, SL, XL, QL, and YY.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.


We investigated the association between the use of inhaled bronchodilators and the risk of AMI. A nested case-control study using the nationwide insurance claims database was conducted. Overall, 11,054 AMI cases and 47,815 matched (up to 1:5) controls were identified from 1,036,119 subjects without acute major cardiovascular events in the past year. Long-acting and short-acting β-agonists (LABAs and SABAs) were associated with increase in the risk of AMI, although an inhaled corticosteroid combined with a long-acting β-agonist was not. Long-acting muscarinic antagonists (LAMAs) in a dry powder inhaler (DPI) were significantly associated with reduced risk of AMI, while LAMAs in a soft mist inhaler (SMI) didn’t decrease the risk of it. In hypertensive or diabetic patients, LAMAs in a DPI were associated with reduced risk of AMI, but LABAs were associated with increased risk. Among the β-blocker users, the reduction of AMI risk by LAMAs was the most significant. In conclusions, inhaled β-agonists were associated with increase in the risk of AMI, while LABAs accompanied by ICSs were not associated with increase in the risk of AMI. LAMAs in a DPI use were associated with lower risk of AMI. Although inhaled therapy has advantages, such as rapid onset and fewer side effects compared with systemic administration, there have been concerns about the possibility of systemic adverse effects, including cardiovascular adverse events, because the drugs could be absorbed systemically after inhalation3. Of the possible cardiovascular adverse events, acute myocardial infarction (AMI) has been regarded as one of the most important issues concerning drug safety.The efficacy of inhaled bronchodilator therapy has been proven in patients with airway diseases such as chronic obstructive pulmonary disease (COPD) and asthma9 and anti-cholinergics14, and the development of AMI. In addition, there are also debates regarding the impact that the drug-delivery device has on the patient outcome15. Although several randomized controlled trials (RCTs) yielded important information concerning drug safety, there are a limited number of RCTs with which to verify the differences in the development of adverse events. These studies often lack external validation18 and statistical power.However, there are debates about the link between the use of inhaled bronchodilators, including inhaled β2–agonistsWe investigated whether the use of inhaled bronchodilators affects the risk of AMI by using the nationwide database in South Korea.In total, 1,036,119 individuals with prescriptions of inhaled respiratory drugs for 30 days or longer between January 1, 2009, and December 31, 2011, were identified from the database. Among them, 221,891 individuals had previous prescriptions for inhaled respiratory drugs for 30 days or longer during the year prior to the initiation of the current therapy of inhaled respiratory medication; 58,782 individuals were diagnosed as having an AMI during the 1-year period before the index date; and 129,520 individuals were <20 years old, >100 years old, or of unknown age; all of these groups were excluded. Finally, a cohort of 792,687 new users of inhaled respiratory drugs were identified. During the study period, 12,110 individuals in this cohort were diagnosed with AMI. After excluding 1,056 (8.7%) cases who did not have matched controls, 11,054 cases with AMI and 47,815 matched controls were included in the analysis , patients with hypertension , diabetes mellitus  and who used β-blocker  Fig. . On the 7. Inhaled SABA use was also associated with increased risk of AMI in our study. In contrast, inhaled LAMAs in a DPI was significantly associated with reduced risk of AMI, which is in agreement with the results of the UPLIFT trial11.This nested case-control study showed that the use of inhaled LABAs but not ICSs with LABAs was associated with an increased risk of AMI, which contradicts the results of a post-hoc analysis of the TORCH trial19, maybe because β2-agonists likely have a stronger effect on the cardiovascular system than cholinergic antagonism does. In fact, β-antagonists are considered important cardioprotective agents because of their anti-ischemic effects20. β2-adrenergic receptor stimulation not only increases the heart rate, which increases myocardial demand but also induces hypokalemia21 and causes direct myocardial injury or necrosis that could lead to ischemia23. β2-adrenoceptors are also present on numerous inflammatory cells, which could contribute to their potential for inducing adverse effects25. Our study also showed that LABAs combined with ICSs, potent anti-inflammatory drugs, did not increase the risk of AMI. In fact, there was a study reporting that low dose ICSs reduces AMI risk in patients with COPD26. The analysis of large RCTs showed there was a lower risk of cardiovascular adverse events in ICS-LABA combination group than there was in the LABA alone group27. This finding supports our results that the combined use of ICS/LABA could be a safer option than treatment with LABA alone.The difference in the risk of AMI between inhaled β2-agonists and anti-cholinergics, which both increase the heart rates that might lead to ischemic events2–agonists, their vagolytic nature leads to arrhythmia28, and pro-inflammatory effects30 has been suggested as the mechanisms of higher cardiovascular events in anti-cholinergics users. Contrary to these hypotheses, our analysis revealed that inhaled LAMAs in a DPI  were associated with reduced risk of AMI in the total cohort and the COPD subgroup. This corresponds with the results of the UPLIFT trial11. However, inhaled LAMAs in an SMI were not associated with reduced risk of AMI, and the aOR was over 1.0, which is similar to the findings reported in several studies noting that LAMAs had a higher risk in an SMI than they did compared with LAMAs in a DPI31. The usual dosage (18 μg in LAMA DPI and 5 μg in LAMA SMI) might have been responsible for the different results. However, we did not find any dose-response relationship, and several studies showed similar safety profiles between these types of devices32. These effect of inhaled LABAs and LAMAs in a DPI on the risk of AMI was not observed in the patients without COPD. The protective effects of inhaled LAMAs were most predominant in β-blocker users, but the effect of LABAs on AMI did not significantly differ with the use of β-blockers.Although, the provocative effects of anti-cholinergics on cardiovascular events might be weaker than those of β16. In addition, a higher dropout rate in the placebo group than that in the test group could lead to an underestimation of the incidence of adverse events in RCTs18. In our population-based, nested case-control study, the protective effects of LAMAs in a DPI on AMI were observed in the patients with hypertension or diabetes mellitus, whereas LABAs significantly increased the risk of AMI among these subgroups. This finding suggests that LAMAs rather than LABAs could be the preferred option in the selection of an inhaled bronchodilator for these AMI-susceptible patients. Second, RCTs were not designed to evaluate the significant differences in adverse events because these are not the primary outcomes; therefore, the statistical power could be insufficient and type I errors might occur. Our nested case-control study based on nationwide claim data included more than 600,000 individuals and detected more than 10,000 new AMI cases without specific exclusion criteria, which may reflect the reality of the clinical situation and could have sufficient statistical power. There could be concerns that many confounders might result in the misinterpretation of the results of a retrospective analysis. However, the distribution of confounding variables after our extensive matching in the analysis was well balanced in this study, as shown in Table Our study has advantages over previous studies including the large number of included RCTs. First, an RCT does not reflect the clinical situation. RCTs are designed to answer one question designated as the primary outcome, which usually has a narrow scope focused on the efficacy of the drug, and therefore, the RCT study design can be impractical. The strict inclusion and exclusion criteria in efficacy trials prevent the extrapolation of the study results to the general population, and the intensive follow-up schedule seldom occurs in usual clinical practiceOur study also has several limitations. First, we did not find statistically significant dose-response in the associations between either LABAs or LAMAs and the risk of AMI. Second, the LABAs were either salmeterol or formoterol, and all the LAMAs were tiotropium. The new LABAs including indacaterol, vilanterol and olodaterol, and new LAMAs including glycopyrronium, umeclidinium and acclidinium, were not used in South Korea until 2011. Third, our database did not include information about smoking, which is an important risk factors of both COPD and AMI. Fourth, we used dispensed prescriptions for inhaler use and did not directly confirm documented use. Fifth, although we observed the different effects on AMI between LAMA DPIs and LAMA SMI, the plausible underlying mechanisms could not be verified.In conclusions, our population-based, nested case-control study revealed that inhaled β2–agonists alone were associated with increased risk of AMI, while LABAs in combination with ICSs were not associated with an increase in the risk of AMI. Inhalation of LAMAs using a DPI were associated with reduced risk of AMI. Finally, the results of our population-based, nested case-control study could facilitate the selection of appropriate inhaled drugs.We used the Health Insurance Review and Assessment Service  database, which included 50.9 million South Koreans from the National Health Insurance (NHI) and National Medical Aid (NMA) databases. The HIRA database contains information on the demographics and all of the medical services rendered, along with the diagnostic codes  and all of the medications prescribed. Values in key fields such as drug name, quantity, date dispensed, and duration are missing or out of range in <0.5% of the records. This study was approved by the ethics review committee of the National Evidence-based Healthcare Collaborating Agency, Seoul, Republic of Korea. Informed consent was waived because of the retrospective fashion by the ethics review committee. We followed the STROBE guideline for observational studies.A nested case-control study was conducted based on the information from the HIRA database. The source population consisted of all of the individuals who were dispensed inhaled respiratory drugs for 30 days or longer between January 1, 2009 and December 31, 2011. The initiation date was defined as the date of the first use of the inhaled respiratory drugs in the hospital or at an outpatient visit. We excluded the following patients from this cohort: those who had prescriptions for inhaled respiratory drugs for 30 days or longer during the year prior to the initiation date; those who were diagnosed as having cardiovascular disease during the year prior to the initiation date; and those who were under 20 years of age or over 100 years of age. The detailed patient selection flow is presented in Fig. Within the eligible cohort, we identified case individuals based on an ICD-10 diagnosis of AMI (I21-I24) that occurred after the initiation date of the inhaled respiratory drugs. The date of the first assignment of the AMI ICD-10 codes was called the index date.We performed individual matching to select control patients for each case. The control patients were selected from the patients without ICD-10 codes for AMI. Each case was matched with up to five controls based on matching variables such as age (±5 years old), sex, initiation date of inhalers (±15 days), diagnosis of hypertension (ICD-10 code I10-I15), diabetes mellitus , COPD (ICD-10 code J41), ischemic heart disease , diagnosis of other heart disease one year before the index date, or a Charson comorbidity index (CCI) of one year before the index date. Other heart disease was defined as rheumatic disease (ICD-10 code I00-I09) and cardiomyopathies, arrhythmias, valvular diseases, pericardial diseases (ICD-10 code I30-I52). The CCI variable was categorized into the following three groups: 0–1, 2–3, and ≥4. The index date for the controls was defined as the index date of the matched case.Inhaled drugs included ICSs , a short-acting inhaled β2 agonist , a long-acting inhaled β2 agonist , a short-acting inhaled muscarinic antagonist , a long-acting inhaled muscarinic antagonist , a combination of a SABA and SAMA , or a combination of a LABA and an ICS . Inhaler users were defined when they used inhaled drugs for 30 days or longer during one year, and respiratory drugs requiring a nebulizer were excluded in this study.When we assessed the risk of AMI for each inhaler, the patient was defined as an inhaler user if the inhaler prescription was for 30 days or longer and was identified during the 90 days period before index date. If each inhaler prescription was for less than 30 days during the 90-day period before the index date, the patient was considered a non-user.We considered the covariates for the AMI risk adjustment as the following: other chronic respiratory disease, comorbidities, health care utilization, and concomitant medications. The other chronic respiratory disease were classified as tuberculosis-lung (ICD-10 code B90), bronchiectasis (ICD-10 code J47), asthma (ICD-10 code J45–46), and others. Comorbidities included chronic kidney disease or dialysis (ICD-10 code N17-N19) and dyslipidemia . We used health care utilization, such as number of hospitalizations , outpatient visits , and emergency room (ER) visits , to adjust patient severity. Concomitant medications included angiotensin converting enzyme inhibitors (ACEI)/angiotensin receptor blockers (ARB), beta-blockers, statins, aspirins, thiazides, and calcium-channel blockers (CCB).The baseline characteristics of the cases and controls were summarized by descriptive statistics, such as proportion, mean, standard deviation (SD), median, first quartile (Q1), and third quartile (Q3). We also summarized the continuous variables into the appropriated categorical variables based on their distributions. Statistical significances were derived from an independent t-test for continuous variables and a χ2-test for categorical variables.The association between the use of inhaled respiratory medication and AMI was investigated by conditional logistic regression analysis. We adjusted for the following covariates: age, other chronic respiratory disease, chronic kidney disease or dialysis, dyslipidemia, use of concomitant medications, number of hospitalization, outpatients visit, and ER visits. Unadjusted odds ratios (ORs) and adjusted odds ratios (aORs) are presented with a 95% confidence interval (CI).Subgroup analyses for LAMAs and LABAs were conducted according to beta-blocker use, IHD, DM, hypertension, and COPD.A p-value less than 0.05 was regarded as statistically significance, and all of the statistical analyses were performed using SAS V.9.2 .


Rats were killed 1, 3, and 7 days after surgery. NAC treatment prevented the CCI-induced increase in lipid hydroperoxide levels only at day 1, although the amount was higher than that found in naive rats. NAC treatment also prevented the CCI-induced increase in ascorbic acid content, which occurred at days 1, 3, and 7. No significant change was found in TAC with NAC treatment. The changes observed here may be related to the antinociceptive effect of NAC because modulation of oxidative-stress parameters seemed to help normalize the spinal cord oxidative status altered by pain.N-acetylcysteine (NAC) inhibits nociceptive transmission. This effect has been associated partly with its antioxidant properties. However, the effect of NAC on the levels of lipid hydroperoxides (a pro-oxidant marker), content of ascorbic acid (a key antioxidant molecule of nervous tissue) and total antioxidant capacity (TAC) is unknown. Thus, our study assessed these parameters in the lumbosacral spinal cord of rats with chronic constriction injury (CCI) of the sciatic nerve, one of the most commonly employed animal models of neuropathic pain. Thirty-six male Wistar rats weighing 200–300 g were equally divided into the following groups: Naive ; Sham , and CCI (rats in which four ligatures were tied loosely around the right common sciatic nerve). All rats received intraperitoneal injections of NAC (150 mg·kg Neuropathic pain, which is caused by a lesion or disease of the somatosensory system, has high clinical incidence  and seriously affects the quality of life of patients , and theN-acetylcysteine (NAC) is a well-tolerated and safe medication that has been used for several decades as a mucolytic agent and for the treatment of numerous disorders such as acetaminophen intoxication ,5. It ha2O2) and nitric oxide play an important role in the pathogenesis of neuropathic pain  precursor ,5. GSH ihic pain ,9,10,13.The main action site for ROS in neuropathic pain is the spinal cord , which i−1·day−1), given for 1, 3, and 7, days, on the lipid hydroperoxide and ascorbic acid levels in the lumbosacral spinal cord of rats with CCI. Since oxidative stress has also been determined by the measurements of a decrease in total antioxidant capacity (TAC)  was chosen because it showed antinociceptive effect in rats with CCI  and was performed daily at 5:00 pm by the same researcher . Adult male Wistar rats weighing 200–300 g were divided into three experimental groups , and each one was further divided into two subgroups (n=6 each), which received NAC  at a dose of 150 mg·kgsearcher . Rats weCCI was performed based on the procedure described by Bennett and Xie , with slg for 20 min at 4°C. The supernatant was used for assays of lipid hydroperoxides and ascorbic-acid levels and TAC.Rats were killed by decapitation and their lumbosacral spinal cord was promptly dissected, homogenized in 1.15% KCl diluted 1:5 (w/v) containing 1 mmol/L phenylmethylsulfonyl fluoride, and centrifuged at 1000 2+ by LOOH in an acid medium containing xylenol orange dye, which forms a complex with Fe3+, as described by Jiang et al.  content was determined according to the method described by Roe and Kuether . The ass+), which in an acid medium is decolorized by antioxidants, according to their concentration and antioxidant capacity  followed by the Tukey After CCI, all rats exhibited a decrease in mechanical threshold, which was prevented by NAC treatment, as shown in our previous study .After CCI, lipid hydroperoxide levels increased in the spinal cord at day 1. While the increase was 823% in the spinal cord from saline-treated CCI rats, the increase was only 142% in the NAC-treated CCI rats, compared to naive rats , showingCCI induced a significant increase in the ascorbic acid content in the spinal cord of rats that received saline. The increase was 137 and 53% at days 1 and 3, respectively, compared to naive rats . No signTAC did not show significant changes in the spinal cord from saline- and NAC-treated CCI rats at days 1 and 3 . At day Neuropathic pain is a disease of global burden and its prevalence has been estimated to be in the range of 7–10% . It is s2O2 to H2O and O2 (Our results showed that NAC treatment prevented the early CCI-induced increase in lipid hydroperoxide levels of the lumbosacral spinal cord, without changing the levels of ascorbic acid and TAC in this tissue, which were increased and decreased, respectively, after CCI . The higO and O2 , increasO and O2 .NAC treatment, in turn, prevented an accentuated increase in lipid hydroperoxides in the spinal cord from CCI rats at day 1. Lipids are important targets of ROS, as described above. The reduction in lipid hydroperoxide formation suggests that ROS decreased after NAC treatment. NAC is a precursor of GSH, an important intracellular antioxidant ,5,12, anInterestingly, lipid hydroperoxides were still significantly increased in the spinal cord of NAC-treated CCI rats at days 3 and 7 compared to naive rats, and the levels were similar to those found in saline-treated CCI rats. Excessive ROS formation needs to be corrected only to prevent the accumulation of oxidative damage, and a slight pro-oxidative balance is necessary for optimal cell-signaling processes . Thus, iThe lipid hydroperoxide levels were also elevated in the spinal cord of saline- and NAC-treated sham rats for 1, 3, and 7 days. This increase may be related to surgery-induced ROS formation. These rats were submitted to procedures involving manipulation of deep tissues, such as muscles and adjacent connective tissue, which induce pain . Thus, tIn our study, while ascorbic-acid levels increased in the spinal cord of the saline-treated CCI rats for 1, 3 and 7 days, NAC treatment prevented an increase in this tissue at these time points. This difference may be related to the roles described for ascorbic acid in nervous tissue. In this tissue, ascorbic acid acts as an important antioxidant molecule, a neuromodulator of synaptic activity, and it functions in the metabolic switch of the neurons during brain activity and resting conditions, having its levels increased in response to brain activity . AccumulNAC causes the activity of the L-cystine/L-glutamate membrane exchanger, and L-cysteine is required for the synthesis of the GSH ,30. ThesSince NAC is a precursor of GSH ,5, it apHowever, TAC decreased in the spinal cord from saline-treated CCI rats at day 7. It has been demonstrated that TAC represents the enzymatic and non-enzymatic antioxidant compounds in the body such as superoxide dismutase, catalase, glutathione peroxidase and GSH ,33. SomeThe lack of significant changes in ascorbic acid levels and TAC in the spinal cord of the sham rats may be related to differences in the degree of central sensitization in sham and CCI rats, as suggested above. According to Goecks et al. , CCI injIn conclusion, our study provides evidence that NAC treatment prevented the early CCI-induced increase in lipid hydroperoxide levels of the lumbosacral spinal cord while preventing the rise of ascorbic acid content, and the decrease of TAC at later stages. Since NAC has an antinociceptive effect in rats with CCI as shown in our previous study , the cha


Here, we performed a meta-analysis to evaluate whether promoter hypermethylation of the MGMT gene contributed to gastric pathogenesis.The association of MGMT  databases. The Newcastle–Ottawa Scale was applied to assess methodological quality of the included studies. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to evaluate the association of MGMT promoter hypermethylation with gastric pathogenesis. Moreover, STATA 12.0 software was used to summarize the extracted data in this meta-analysis.MGMT promoter hypermethylation in the GC group (32.97%) was significantly higher than those in the control group (18.00%) . When stratified by cancer subtype, the results indicated that the frequency of MGMT promoter hypermethylation was significantly higher in gastric adenocarcinoma than in control group . In addition, MGMT promoter hypermethylation significantly promoted distant metastasis and lymph node (LN) metastasis of gastric tumor . A significant association between MGMT promoter hypermethylation and TNM-stage was also found in the present meta-analysis .Seventeen studies, comprising 1736 cases and 1291 controls, were included in this meta-analysis. The frequency of MGMT gene-promoter hypermethylation was significantly associated with an increased risk of GC, especially in Asians. Furthermore, MGMT gene-promoter hypermethylation might be correlated with the distant metastasis and LN metastasis of GC.The results of this meta-analysis suggested that  Although advances of early detection have led to a decline in incidence rates of GC, a lot of GC patients were still diagnosed at late stage, and patients usually died of metastasis. Many studies have confirmed that the development of GC was a multifactorial process that involved some environmental factors, multiple genetic, and epigenetic alterations such as Helicobacter pylori (H. pylori) infection, high salt intake, smoking, gene variation, and gene methylation.,5 In terms of risk evaluation, H. pylori infection was significantly associated with the risk of GC. However, H. pylori infection was insufficient for predicting the risk of GC. Thus, other biomarkers were still needed to be identified to improve our understanding of gastric carcinogenesis. Epigenetic changes such as promoter methylation and histone acetylation played an important role in cancer development. A number of tumor-related genes were often silenced by those deregulated modifier that produced epigenetic changes. Most deoxyribonucleic acid (DNA) methylation events usually existed in the CpG dinucleotides and especially in gene promoters. DNA methylation was a major cause of gene silencing and normally appeared in X-chromosome inactivation, imprinted genes, and tumor-suppressor genes. For instance, mutation of CDH1 gene was the main cause of hereditary diffuse GC, and DNA methylation often occurred to inactivate this gene. Therefore, DNA methylation might be considered as a good marker to assess the tumorigenesis, which was also beneficial for cancer diagnosis and treatment.Gastric cancer (GC) is one of the most common malignancies and remains the second most common cause of cancer-related death worldwide.6-methyguanine DNA methyltransferase (MGMT), a DNA repair protein encoded by MGMT gene that located at 10q26, removed cytotoxic and mutagenic adducts from the O6-guanine of DNA. Alkylation at the O6 position of guanine in DNA contributed a lot to the emergence of gene variation in cancers, because thymine tended to replace cytosine to pair with O6-methylguanine in the replication of DNA, which eventually resulted in the G > A mutation. MGMT protein could protect DNA from methylation damages, thus it had the ability of tumor inhibition. In contrast, it might produce chemoresistance to anticancer treatment, therefore leading to treatment failure. The loss of MGMT expression was mainly due to epigenetic event other than the deletions or rearrangements of the gene. The hypermethylation of the CpG islands on MGMT gene promoter significantly silenced the MGMT gene. In recent studies, significant associations between MGMT promoter hypermethylation and glioblastoma, breast cancer, and colorectal cancer were found. Many studies evaluating the correlation of MGMT promoter hypermethylation with GC were also performed. However, limitations of sample type, sample size, and race lowered the reliability of the results. Hence, in order to clarify the association between GC pathogenesis and MGMT promoter hypermethylation, we performed this meta-analysis.O22.1PubMed, Web of Science, Embase, and CNKI databases were searched up to September 2016 using the search terms “Stomach Neoplasms[Mesh],” “gastric cancer,” “gastric tumor,” “gastric carcinoma,” “MGMT protein, human” [supplementary concept], “MGMT,” and “‘Methylation’[Mesh].” The references of included articles and relevant review literatures were searched and scanned to acquire additional eligible studies. In this retrospective meta-analysis, institutional review board approval was not required.2.2Published studies included must meet the following criteria: studies that evaluated the association of MGMT promoter methylation with GC risk or clinical characteristics, studies that provided sufficient data on the promoter methylation of MGMT in control group and GC group, and all cancer patients were diagnosed according to the diagnostic criteria of GC. If studies did not meet these inclusion criteria, they were excluded.2.3Two investigators independently extracted the relevant data from included studies according to the inclusion criteria. Any discrepancy was discussed and settled by consulting with the team. The following information were collected in this meta-analysis: name of the first author, publication year of article, country of subjects, ethnicity of subjects, the number of controls and cases, GC type, clinical information, detection method of methylation, and sample type.2.4http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp) table. The NOS criteria included 3 parts: subjects selection, 0 to 4 scores; comparability of subjects, 0 to 2 scores; and exposure of subjects, 0 to 3 scores. The scores range from 0 to 9, and a score of ≥7 indicated a high-quality study.Two investigators independently assessed methodological quality of included articles using the Newcastle–Ottawa Scale (NOS)  and 95% confidence intervals (95% CIs) were calculated using STATA 12.0 software  software. The degree of heterogeneity present in different studies was performed based on Cochran Q statistic and I2 tests. When a P value of <.05 or I2 > 50% was presented, significant heterogeneity was considered existing among studies, and the random effects model was used to calculate the ORs and 95% CIs; otherwise, the fixed-effects model was applied.,16 Funnel plots were also drawn to investigate the potential publication bias of included studies. If funnel plots were distributed approximately symmetrically, results indicated an absence of obvious publication bias. Begg and Egger tests were also carried out to observe the publication bias. Furthermore, subgroup analysis based on sample subtype and race was performed to lower the between-study heterogeneity. Sensitivity analysis was conducted by excluding individual study to assess stability of results, and outlying studies were removed.In order to evaluate the strength of the association between 33.1The original searching yielded a total of 32 publications using the search terms. Thirty-one publications were obtained after 1 duplicated article was removed. Through reading title and abstract, 24 publications were remained. Finally, 16 articles (17 studies) with 1736 cases and 1291 controls were included in this meta-analysis by scanning the full texts. In these publications, 14 studies had sufficient data of both controls and cases group, and 3 studies only had sufficient data of cases. Thus, the remaining 3 studies were used to observe the association between MGMT promoter hypermethylation and clinical characteristics of GC. Moreover, 8 studies considered adjacent tissue as control group, while 4 studies put normal tissue as control group. All studies on hypermethylation of MGMT promoter were genotyped in GC tissue except 2, which used blood sample. On the other hand, 2 studies were in Asians and 15 studies were in Caucasians . In the subgroup analysis based on sample subtype, the heterogeneity significantly lowered in blood sample and normal tissue . However, significant associations were still found in the subgroup analysis . In the subgroup analysis based on GC subtype, the MGMT promoter hypermethylation significantly increased the risk of gastric adenocarcinoma . The results of subgroup analysis based on race, displaying on the forest plot, indicated that the race was one of the sources of heterogeneity. In addition, the frequency of MGMT promoter hypermethylation in M1-stage of GC was significantly higher than those in M0-stage . In a separate analysis, there was a significant association of MGMT promoter hypermethylation with lymph node (LN) metastasis of gastric tumor , especially in Asians . The probability of occurrence of diffuse GC was 1.81 times than that of intestinal GC . In the analysis for TNM-stage, the MGMT promoter hypermethylation accelerated the progress of GC . Moreover, no significant associations of MGMT promoter hypermethylation with tumor differentiation, T-stage, and gender were detected . The results indicated that sample type and race were not the main source of heterogeneity, especially in Asians. However, the 2 factors, cancer type and ethnicity, still lowered the heterogeneity among studies according to the subgroup analysis.Although a significant association of MGMT promoter hypermethylation with GC risk was found, heterogeneity among studies was found. Considering the existence of heterogeneity, in order to lower the heterogeneity present in different studies, subgroup analysis based on sample subtype and race was performed in this meta-analysis. Furthermore, meta-regression analysis was also carried out to find the source of heterogeneity   Fig. . In the 05) Fig. .4–19 It has been observed that almost half of the tumor suppressor genes were inactivated by gene-promoter hypermethylation in cancers. Therefore, many studies have focused on the discovery of gene-promoter hypermethylation and have found many epigenetic alterations such as p16INK4a, CDKN2B/p151NK2b, and p14ARF. Increasing evidence suggested that different tumors often had different epigenetic alterations such as clustering of gene hypermethylation on the CpG island methylator phenotype. Previous study has found that concordant hypermethylation of multiple locus occurred in 31% GC; however, hypermethylation of independent locus could not satisfactorily predict the risk and prognosis of GC. In fact, no ideal tumor markers for the detection of GC were found in present studies. However, these biomarkers were very important to screen, diagnose, and determine the stage and metastasis of GC. If methylation microarrays could be conveniently used in GC, it might be beneficial for the diagnosis and therapy of GC patients. But before this, a lot of gene methylation alterations should be found and verified in different populations to determine specificity strongly.Traditional genetic studies on GC have found many gene variations such as single-nucleotide polymorphism, copy number variation, and indel.hMLH1 gene was often found in GCs and had a significantly association with the loss of hMLH1 expression. Of note, CDH1 promoter hypermethylation had a high frequency in the diffuse histological type. It has been reported that this hypermethylation alteration contributed a lot to LN metastasis of GC.CDH4 gene hypermethylation has also been found in a higher frequency in GC tissue than adjacent mucosa, and it might have a significant association with progression of GC. Furthermore, APC, H-Cadherin, DAPK, RASSF1A, XIAP, VLDLR, RUNX3, RARβ, CASP8, and MGMT had abnormal epigenetic variations in the development of GC. Although previous studies have found that the hypermethylation of MGMT gene accounted for 31% in GC patients, the association between MGMT promoter hypermethylation and risk of GC was inconsistent. Thus, this meta-analysis was performed by summarizing the existing data to observe the correlation.Currently, many genes hypermethylated in human cell lines and primary tumors have been found. For instance, promoter hypermethylation of ,37,40 while 11 studies observed a significant association of MGMT promoter hypermethylation with risk of GC.–36,38,39 In the calculation of total ORs, the 3 studies contributed to 21.61% of weight; thus, it was necessary to conduct a meta-analysis to determine the association between MGMT promoter hypermethylation and GC risk. From the overall analysis, the MGMT promoter hypermethylation might significantly increase the risk of GC. According to the subgroup of gastric adenocarcinoma, a significant result was also detected, and the carriers of MGMT promoter hypermethylation have 3.47 times higher risk of GC than those of hypomethylation. Furthermore, the frequency of MGMT promoter hypermethylation between males and females did not indicate significant difference . This result showed that the occurrence of MGMT promoter hypermethylation was not related to the gender. Stomach carcinoma might be classified into the diffuse cancer and intestinal cancer. These 2 types of GCs had different pathways and mechanisms. The development of intestinal cancers could be divided into 4 steps: atrophic gastritis, intestinal metaplasia, dysplasia, and GC, while no clear steps of development existed in the diffuse cancer. On the basis of this meta-analysis, different frequencies of MGMT promoter methylation were also found between diffuse and intestinal types. The frequency of MGMT promoter hypermethylation in diffuse type was significantly higher than that in intestinal type. In the included studies on Lauren classification, only 1 study had a same significant result. The significant association of MGMT promoter hypermethylation with distant metastasis and LN metastasis was also observed in the forest plot. However, the 2 significant associations were only detected in Asians, and no between-study heterogeneity was observed. Therefore, the MGMT promoter hypermethylation might encourage the metastasis of gastric tumor in Asians and decrease the survival rate of GC. The result in Caucasians might not be accurate due to the small number of included studies; thus, more large-scale and well designed studies were still needed to be conducted. In the meantime, the GC patients’ number of MGMT promoter hypermethylation in stages III to IV was higher than the number of MGMT promoter hypermethylation in stages I and II. Finally, these results demonstrated that the MGMT promoter hypermethylation had a crucial role in the development of GC. Although heterogeneity was detected in the overall analysis, it was significantly decreased after subgroup analysis and random effects model were conducted. According to the results of meta-regression, ethnicity and cancer subtypes were not the main causes of heterogeneity, and other factors might result in the heterogeneity. Furthermore, no significant publication bias was shown on the basis of Begg and Egger tests. Therefore, although some limitations existed in the meta-analysis, these results might provide a direction that MGMT promoter hypermethylation might contribute a lot to the pathogenesis of GC.From the forest plots, 3 studies did not show a significant correlation, Therefore, to a large extent, the negative result was same with most studies.In addition to these positive results, negative results were also shown in this meta-analysis. No significant associations of MGMT promoter hypermethylation with gastric tumor differentiation were found. In previous studies, the association was contradictory in included studies that were conducted in different sample sizes and races. For tumor differentiation, 1 study indicated that MGMT promoter hypermethylation encouraged the tumor differentiation.I2 = 84.1%, P = .000; for Caucasians, I2 = 66.8%, P = .083; for Asians, I2 = 0.0%, P = .376).In this meta-analysis, we applied the NOS table to evaluate the methodology quality of literatures. The high scores of studies ensured the efficiency and accuracy of statistical analysis. Finally, subgroup based on sample size, sample type, and race was conducted to reduce the heterogeneity, and the heterogeneity lowered obviously. The heterogeneity decreased a lot after subgroup analysis on the basis of race performed, especially in gastric adenocarcinoma (for total, Some limitations still existed in this meta-analysis. First, selection bias was inevitable due to restriction to articles in English or Chinese language. Second, although subgroup analysis and meta-regression were conducted, heterogeneity present in different study still existed. This result might be caused by individual difference; GC subtypes; or other environmental factors such as diet, smoking, and work environment. Third, the small sample size of studies involving clinical information restricted the statistical power. Fourth, the studied population in these articles only included Caucasians and Asians, and the Asians accounted for a lot. Hence, these results might not represent the overall tendency in the total crowd. Finally, the accuracy of results might be influenced by the small number of subjects and different sample type. Although the number of subjects was moderate, but it was still small after the subgroup analysis based on race and sample type was performed.MGMT gene-promoter hypermethylation was significantly associated with the risk of GC, especially in Asians and gastric adenocarcinoma. Furthermore, MGMT gene-promoter hypermethylation might promote the distant metastasis and LN metastasis of gastric tumor.In conclusion, the results of this study indicated that


P < 0.001). Meanwhile we also analyzed the following cancers independently: hepatocellular carcinoma  and urothelial carcinoma . Compared to the high GAS5 expression group, additionally, patients with low GAS5 expression in tumor tissues were more prone to lymph node metastasis  and high tumor stage . In conclusion, this meta-analysis showed that GAS5 might be served as a novel biomarker for predicting prognosis in various types of cancers.The growth arrest-specific transcript 5 (GAS5) is a long noncoding RNA with low expression in multiple cancers. This meta-analysis aims to explore the association between GAS5 expression levels and cancer patients' prognosis. We collected all the relevant literatures about GAS5 expression levels associated with overall survival (OS), lymph node metastasis (LNM) and high tumor stage (II/III/IV) (HTS) from the PubMed and Web of Science. The hazard ratio (HR) and the corresponding 95% confidence interval (CI) were calculated to evaluate the link strength between GAS5 and cancer prognosis. A total of 934 patients from 14 studies were included to the present meta-analysis, according to the inclusion and exclusion criteria. The results demonstrated that low expression of GAS5 could predict poor OS in cancer patients (HR = 1.955, 95% CI: 1.551–2.465,  Cancer has become a global health problem. In recent years, the incidence of cancer has been increased year by year. According to WHO estimates, 14.1 million new cancer patients and 8.2 million deaths from cancer occurred worldwide in 2012 and more than 20 million new cases of cancer will be expected as early as 2025  are noncoding RNAs with a length of more than 200 nucleotides that regulate gene expression  is a rising star among tumor-suppressive lncRNAs among all the kinds of lncRNAs , including articles referenced in the publications. We used “GAS5 or growth arrest specific 5” AND “cancer or tumor or carcinomas or neoplasm” as the keywords, in order to identify potentially relevant studies. Citation lists of retrieved articles were searched manually to ensure sensitivity of the search strategy.All the eligible study data elements were independently assessed and extracted by two investigators. For inclusion in this meta-analysis, the studies met the following criteria: the association between GAS5 and cancer prognosis (OS) was investigated; patients were grouped according to the expression levels of GAS5;related clinic pathologic parameters were described, such as LNM, TNM and sufficient original data for calculating a hazard ratio (HR) with its 95% confidence interval (CI). Exclusion criteria are as the following: Duplicate publications; irrelevant to cancer, GAS5, or cancer prognosis; animal studies, letters, editorials, expert opinions, abstracts, case reports and reviews; studies without usable data.According to the inclusion and exclusion criteria, two investigators extracted and reviewed the data independently , and disagreements were discussed with two investigators  in conference. The following data were extracted: first author, publication date, country of origin, tumor type, total number of patients, number of high GAS5 expression group and low GAS5 expression group, number of patients with LNM, number of patients with HTS, detection method of GAS5 expression levels, follow-up month and cut-off values, multivariate analysis, hazard ratios (HRs), and corresponding 95% CI for OS.2 statistics were used . The potential publication bias was assessed using the Eegg'stest and P < 0.05 was considered representative of statistically significant publication bias. Sensitivity analysis was performed by sequential omission of each individual study in order to validate the stability of outcomes in the present meta-analysis.Meta-analysis was performed using Stata12.0 software. Pooled hazard ratios (HRs) were extracted from the included studies; the log HR and standard error (SE) were used for aggregation of the survival results  performed the quality assessment of primary studies independently. We evaluated all eligible studies' quality by using the Newcastle-Ottawa Scale (NOS) for assessing the quality of studies in meta-analyses  = 0.728). We found an inverse relationship that low expression of GAS5 might be associated with poor overall survival outcome   = 0.902), UC  = 0.268) and HR for the subgroup of other cancers was 2.641  = 0.730). We didn't perform subgroup analyses for CRC, BRC, EOC, GC, CEC, and HNSCC, because there is only one paper investigating these associations between GAS5 and OS  of 730 patients according to GAS5 expression levels. In order to study the relationship between GAS5 expression level and prognosis, the fixed-effect model was used to calculate the pooled HR with corresponding 95% CI because heterogeneity analysis showed that low between-study heterogeneity among those nine studies for GAS5 expression was found , the random-effects model was adopted to calculate the odds ratio . It demonstrated that patients with low GAS5 expression in tumor tissues were more prone to lymph node metastasis . OR for the subgroup of other cancers was 0.115 . But the expression of GAS5 in NSCLC tumor tissues might not be a direct evidence of LNM . We didn't perform subgroup analyses for UC, BRC, HCC, EOC, GC, CEC, and HNSCC, there is only one paper investigating these associations between GAS5 and LNM . The odds ratio, expressed as high GAS5 expression group vs. low GAS5 expression group, was 0.185 . The result showed that patients with low GAS5 expression in cancerous tissues were more prone to high tumor stage   = 0.902), UC  = 0.268) which meant that the above conclusions applied equally in HCC and UC. Meanwhile, we investigated the association between the GAS5 expression levels and LNM and HTS, and we found that low GAS5 expression in cancerous tissues were more prone to LNM and HTS , and other types of cancer, while the heterogeneity still existed in NSCLC which might be caused by the different cut-off value methods which were adopted to define the high GAS5 expression group or low GAS5 expression group. In conclusion, all these results provided strong evidence for GAS5 as a potential biomarker for the prognosis of various cancers.As shown in Figure  Figures . HoweverNowadays, many lncRNAs have been found to be abnormally expressed in cancer. Therefore, many meta-analysis articles, like our study, have been used to reveal the correlation of lncRNAs and cancer prognosis. Several lines of studies, meanwhile, have revealed that a lot of lncRNAs play a important role in cancer prognosis, such as TUG1, SPRY4, MALAT1 (Wang et al., There are several limitations in our study that should be acknowledged. Firstly, the present study used the summary data rather than a specific patient data. Secondly, the methods for distinguishing the cut-off value of GAS5 in high and low expression groups were inconsonant which inevitably could cause heterogeneity. Thirdly, most of the HR values were not directly reported in these included studies. We extracted and calculated them according to the survival curves, so inevitably there might be errors. Fourthly, different treatment methods for different types of cancer patients after surgery might have great influence on the survival time, which leaded to the heterogeneity of the researches. Fifthly, we only included English related literatures that could not be so comprehensive. Sixthly, most of the studies were from China, so the conclusion might not necessarily apply in other areas. Seventhly, we only included related studies reporting OS, LNM and HTS, and the articles on other prognostic indicators were thus excluded. In the light of the above deficiencies, a more comprehensive study covering larger samples, more regions, and more indicators will be needed to confirm our results.In conclusion, our meta-analysis found that lncRNA GAS5 could sever as a molecular biomarker to predict the prognosis of various cancers and the low GAS5 expression could indicate the poor prognosis.QG and HX performed Data extraction, HZ and JL did the data analysis. YL and WH designed the project and QG wrote the paper. YL supervised the project. WH provided financial support for the project.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.