Effects and mechanisms of salidroside on the behavior of SPS-induced PTSD rats.

Post-traumatic stress disorder (PTSD) is a complex mental disorder, closely associated with stress and traumatic events. Salidroside (Sal) has been reported to possess neuroprotective effects. However, the behavioral effects and mechanisms of Sal on PTSD remain unknown. In this study, we utilized a rat model of PTSD induced by single prolonged stress (SPS) and administered Sal intraperitoneally (25, 50, 75 mg/kg/d) for 14 days. We then examined the behavioral effects and underlying mechanisms of Sal on SPS-induced PTSD rats. Our findings demonstrated that Sal alleviated anxiety-like behavior and spatial learning and memory impairment in SPS-induced PTSD rats. Furthermore, Sal treatment preserved the histomorphology of the hippocampal region. It was observed that Sal protected against hippocampal neuronal apoptosis in PTSD rats by reducing the number of TUNEL-positive cells and modulating apoptosis-related proteins (Bcl-2 and Bax). Additionally, Sal inhibited the activation of the NF-κB/iNOS/COX-2 signaling pathway in the hippocampus of PTSD rats, thereby suppressing the release of inflammatory factors (TNF-α and IL-1ß) and the activation of microglia. Notably, Sal increased the expression of synapse-associated proteins PSD95 and Synapsin I in the hippocampus, while also enhancing dendritic density in the region. In conclusion, our results demonstrated that Sal could attenuate SPS-induced PTSD-like behaviors by inhibiting hippocampal neuronal apoptosis, enhancing hippocampal synaptic plasticity, and reducing neuroinflammatory responses. These findings may provide a foundation for the potential clinical application of Sal in the treatment of PTSD.

Emerging V1 neuronal ensembles with enhanced connectivity after associative learning.

Introduction: The visual stimulus-specific responses in the primary visual cortex (V1) undergo plastic changes after associative learning. During the learning process, neuronal ensembles, defined as groups of coactive neurons, are well known to be related to learning and memory. However, it remains unclear what effect learning has on ensembles, and which neuronal subgroups within those ensembles play a key role in associative learning. Methods: We used two-photon calcium imaging in mice to record the activity of V1 neurons before and after fear conditioning associated with a visual cue (blue light). We first defined neuronal ensembles by thresholding their functional connectivity in response to blue (conditioned) or green (control) light. We defined neurons that existed both before and after conditioning as stable neurons. Neurons which were recruited after conditioning were defined as new neurons. The graph theory-based analysis was performed to quantify the changes in connectivity within ensembles after conditioning. Results: A significant enhancement in the connectivity strength (the average correlation with other neurons) was observed in the blue ensembles after conditioning. We found that stable neurons within the blue ensembles showed a significantly smaller clustering coefficient (the value represented the degree of interconnectedness among a node's neighbors) after conditioning than they were before conditioning. Additionally, new neurons within the blue ensembles had a larger clustering coefficient, similar relative degree (the value represented the number of functional connections between neurons) and connectivity strength compared to stable neurons in the same ensembles. Discussion: Overall, our results demonstrated that the plastic changes caused by conditioning occurred in subgroups of neurons in the ensembles. Moreover, new neurons from conditioned ensembles may play a crucial role in memory formation, as they exhibited not only similar connection competence in relative degree and connectivity strength as stable neurons, but also showed a significantly larger clustering coefficient compared to the stable neurons within the same ensembles after conditioning.

The latest role of nerve-specific splicing factor PTBP1 in the transdifferentiation of glial cells into neurons.

Central nervous system injury diseases can cause the loss of many neurons, and it is difficult to regenerate. The field of regenerative medicine believes that supplementing the missing neurons may be an ideal method for nerve injury repair. Recent studies have found that down-regulation of polypyrimidine tract binding protein 1 (PTBP1) expression can make glial cells transdifferentiate into different types of neurons, which is expected to be an alternative therapy to restore neuronal function. This article summarized the research progress on the structure and biological function of the PTBP family, the mutual regulation of PTBP1 and PTBP2, their role in neurogenesis, and the latest research progress in targeting PTBP1 to mediate the transdifferentiation of glial cells into neurons, which may provide some new strategies and new ideas for the future treatment of central nervous system injury and neurodegenerative diseases. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing.

Construction and utilization of human genetic resources in large population cohorts.

In the era of big data and precision medicine, large population cohort studies are one of the preferred designs for studying the etiology of chronic diseases, and cohort genetic resources have become important strategic resources of China. Promoting the standardized construction and utilization of cohort genetic resources can effectively promote the original innovation of research and technological development in the field of biomedicine, and make full use of the rich genetic resources of China. To provide a reference for the construction and utilization of genetic resources in the cohort research in China, we took the Taizhou Longitudinal Study (TZL) as an example and introduced the principles, methods, standard system, and practical experience of the collection, preservation, and shared utilization of genetic resources in the process of the cohort construction.

Chlorogenic Acid Improves PTSD-like Symptoms and Associated Mechanisms.

Chlorogenic acid (CGA) is a kind of traditional Chinese medicine, abundant in honeysuckle and eucommia, and has a wide range of biological activities, and pharmacological effects. Previous studies have shown that CGA can regulate learning, memory, cognitive ability, coupled with improvement to anxiety, depression, and other post-traumatic stress disorder (PTSD)-like symptoms. This article explores the protective effects of CGA on neurons through its anti-apoptotic effect, inhibition of neuroinflammation and oxidative stress, which may be the mechanisms of its improvement of PTSD-like symptoms. It may provide a new therapeutic strategy for the treatment of PTSD and its comorbidities.

The Effects of P75NTR on Learning Memory Mediated by Hippocampal Apoptosis and Synaptic Plasticity.

Neurological diseases bring great mental and physical torture to the patients, and have long-term and sustained negative effects on families and society. The attention to neurological diseases is increasing, and the improvement of the material level is accompanied by an increase in the demand for mental level. The p75 neurotrophin receptor (p75NTR) is a low-affinity neurotrophin receptor and involved in diverse and pleiotropic effects in the developmental and adult central nervous system (CNS). Since neurological diseases are usually accompanied by the regression of memory, the pathogenesis of p75NTR also activates and inhibits other signaling pathways, which has a serious impact on the learning and memory of patients. The results of studies shown that p75NTR is associated with LTP/LTD-induced synaptic enhancement and inhibition, suggest that p75NTR may be involved in the progression of synaptic plasticity. And its proapoptotic effect is associated with activation of proBDNF and inhibition of proNGF, and TrkA/p75NTR imbalance leads to pro-survival or proapoptotic phenomena. It can be inferred that p75NTR mediates apoptosis in the hippocampus and amygdale, which may affect learning and memory behavior. This article mainly discusses the relationship between p75NTR and learning memory and associated mechanisms, which may provide some new ideas for the treatment of neurological diseases.

The Effect of Glycemia on Choroidal Thickness in Different Stages of Diabetic Retinopathy.

OBJECTIVE: The purpose of this study was to evaluate the influence of renal and glycemic parameters on choroidal thickness (CT) in patients with diabetes with and without diabetic retinopathy (DR). METHODS: This cross-sectional study included patients with and without diabetes. Patients underwent comprehensive ocular examination. CT was obtained using spectral-domain optical coherence tomography (SD-OCT) with enhanced depth imaging (EDI) mode. Clinical parameters were body mass index, mean arterial pressure, glycated hemoglobin, fasting plasma glucose, estimated glomerular filtration rate, and capillary plasma glucose (CPG) a few minutes before EDI-SD-OCT. RESULTS: The study included 275 participants: 42 with diabetes and no DR, 43 with mild nonproliferative diabetic retinopathy (NPDR), 46 with moderate NPDR, 39 with severe NPDR, 24 with proliferative diabetic retinopathy (PDR), 40 with previous panretinal photocoagulation (PRP) treatment for DR, and 41 without diabetes. The diabetic patients had thinner subfoveal CT than the nondiabetic participants (280.5 ± 83.4 vs. 327.1 ± 48.8 µm, p < 0.001). After multivariable adjustment, CT was significantly correlated with age, DR stage, and CPG. In patients with mild and moderate NPDR, a higher level of CPG was associated with thicker CT. This relationship was not found in patients with PDR. CONCLUSION: CPG had the strongest correlation with CT in patients with NPDR (mild, moderate, and severe), but not in PDR and PRP PDR patients. Our study suggests that the glucose level at the time of the test should be aggregated to other systemic and ocular parameters, such as age and axial length, when studying the choroid using SD-OCT.

Several polymorphisms of KCNQ1 gene are associated with plasma lipid levels in general Chinese populations.

BACKGROUND: Potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) is thought to be an important candidate gene of diabetes. Several single nucleotide polymorphisms (SNPs) in a 40-kb linkage disequilibrium (LD) block in its intron 15 have been identified to be associated with diabetes in East Asian populations in recent genome-wide association studies. The aim of this study was to investigate whether KCNQ1 polymorphisms influence the levels of the metabolic phenotypes in general Chinese populations. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the associations of two SNPs (rs2237892 and rs2237895) in the aforementioned 40-kb LD block, a missense variant rs12720449 (P448R) in exon 10, and a synonymous variant rs1057128 (S546S) in exon 13 with metabolic phenotypes in a Uyghur population (n = 478) and replicated these associations in a Han population (n = 2,485). We found that rs2237892-T allele was significantly associated with decreased triglyceride levels (p(combined) = 0.001). The minor G allele of the rs12720449, with sharp difference of the allelic frequency between European and East Asian populations (0.2% versus 14%, respectively), was associated with a lower triglyceride levels than G allele in Uyghur subjects (p = 0.004), in Han subjects (p = 0.052), and in subjects of meta-analysis (p(combined) = 0.001). Moreover, the minor A allele of the rs1057128 was also associated with decreased triglyceride levels in meta-analysis (p(combined) = 0.010). CONCLUSIONS: To the best of our knowledge, this is the first report associating a missense mutation of KCNQ1, rs12720449, with triglyceride levels. Rs2237892, representing the 40-kb LD block, is also associated with triglyceride levels in Han population. Further studies are required to replicate these findings in other East Asian populations.

[Association between mannose-binding-lectin gene and type 2 diabetic patients in Chinese population living in the northern areas of China].

OBJECTIVE: To investigate whether the mannose-binding-lectin 2 (MBL2) gene was associated with type 2 diabetes in the populations living the northern part of China. METHODS: The study involved 318 type 2 diabetic patients and 448 normoglycemic controls. The variances of rs1800450, rs1800451 and rs11003125 were determined by the Multiplex SNaPshot method. Fasting blood-glucose, triglyceride and total cholesterol were also measured. All of these results were analyzed by logistic regression method. Linkage disequilibrium and Haplotype measures were computed in all samples using Haploview. RESULTS: There seemed no mutation on rs1800451 while the rs1800450 and rs11003125 polymorphism was consistent with Hardy-Weinberg expectations in both the case and the control groups. Genotypes and allele frequencies of rs1800450 as well as rs11003125 were observed (P = 0.006, P = 0.003) and (P = 0.010, P = 0.004), respectively. Data from logistic regression analysis revealed that factors as overweight, abdominal obesity, hypercholesterolemia, GG genotype frequencies of Exon1 rs1800450 polymorphism as well as (GC + CC) genotype frequencies of rs11003125 polymorphism in MBL2 conferred increased risks for type 2 diabetes. Haplotype analyses of the two SNPs (rs1800450, rs11003125) revealed similar effects as compared with the single SNP associations. Only haplotype constructed from GC alleles conferred increased trends for type 2 diabetes (OR = 2.21, 95%CI: 1.47 - 3.33, P = 0.000). CONCLUSION: Our result suggested that the Exon1 rs1800450 polymorphism and promoter region rs11003125 polymorphism in MBL2 gene were both associated with type 2 diabetes in the Chinese population living in the northern areas of China. The G allele of rs1800450 and C allele of rs11003125 might be the risk factors of type 2 diabetes.

[Association of the C1155547T polymorphism in WNK4 gene with essential hypertension in Xinjiang Kazakhs].

OBJECTIVE: To investigate whether the WNK lysine deficient protein kinase 4 (WNK4) gene C1155547T polymorphism is associated with essential hypertension (EH) in Xinjiang Kazakhs and to assess the effect of the interaction between this polymorphism and environment factors on EH. METHODS: The study covered 556 hypertension patients and 341 normotensive controls. The C1155547T was determined by Taqman probe real-time PCR method. Some biochemical index such as glucose, triglyceride and total cholesterol were also measured. All of these results were analyzed with Logistic regression analysis. Additive model was applied to assess the effect of interaction between the WNK4 gene C1155547T polymorphism and environment factors on hypertension. RESULTS: The C1155547T polymorphism was consistent with Hardy-Weinberg equilibrium in both the case and control groups. There was significant difference in the genotype frequencies (P=0.003). The T allele frequency was significantly higher in the patient group (P=0.002). Logistic regression analysis revealed that the age, body mass index (BMI), total cholesterol as well as the CT+TT genotype frequency conferred increased risks for EH. Positive interaction between the C1155547T polymorphism and gender, BMI, glucose was observed. The ORs were 3.85 (95%CI:1.23-12.04), 5.91 (95%CI:1.99-17.57) and 8.77 (95%CI:1.04-73.93), respectively. CONCLUSION: The result suggested that the exon 7 C1155547T polymorphism in WNK4 gene might be associated with EH in Xinjiang Kazakhs, the T allele might be the risk factor of essential hypertension. There were interactive effects between the WNK4 gene C1155547T polymorphism and gender, BMI and glucose.

[Study on the association between genetic polymorphism on WNK4 genes and essential hypertension among Kazakhs ethnic population, in Xinjiang].

OBJECTIVE: To investigate whether G1155942T polymorphism in WNK4 gene is associated with essential hypertension in a population with Kazakhs ethnicity, in Xinjiang. METHODS: This study covered 563 hypertension patients and 346 normotensive controls. The variant of G1155942T was determined by the TaqMan probe real-time PCR method. Some biochemical indices such as glucose (GLU), triglyceride (TG) and total cholesterol (TC) were also measured. All of these results were under logistic regression analysis. Addictive model was applied to assess the interactive effects between WNK4 gene G1155942T mutation and environmental factors on hypertension. RESULTS: The G1155942T polymorphism was consistent with Hardy-Weinberg expectations in both case and control groups. Genotype and allele frequencies of G1155942T were observed (P = 0.004, P = 0.003). Data through logistic regression analysis showed that factors as age, BMI, total cholesterol as well as the GT + TT genotype frequencies of Exon 8 G1155942T polymorphism in WNK4 were responsible for the increased risks for hypertension. Positive interactions between G1155942T mutation and gender, BMI, GLU, the OR were 3.75 (95%CI: 1.19 - 11.80), 5.77 (95%CI: 1.93 - 17.21) and 8.67 (95%CI: 1.03 - 72.99), respectively. CONCLUSION: Our result suggested that the Exon 8 G1155942T polymorphism in WNK4 gene was associated with hypertension in the studied Kazakhs ethnic group in Xinjiang and the T allele might be the risk factor for essential hypertension. There were interactive effects between WNK4 gene G1155942T mutation, gender, BMI, and GLU.

[Association between methylthioadenosine phosphorylase gene single nucleotide polymorphisms and myocardial infarction in Chinese Han ethnicity].

OBJECTIVE: To investigate the association between methylthioadenosine phosphorylase (MTAP) gene single nucleotide polymorphisms (SNP) and myocardial infarction (MI) in the Chinese Han ethnicity. METHODS: 432 patients suffered from myocardial infarction and 430 controls were involved for case and control groups, respectively. Nine tag SNPs in MTAP gene were selected and genotyped. RESULTS: We found no significant association of selected tag SNPs with MI in all of the samples. However, in stratified analysis, significant association was observed at rs7027989 in male subjects. The risk of MI increased by 26% (P = 0.005) for male subjects of minor allele carriers in a dominant model. The increased risk of MI at rs7027989 remained significant after adjusting for confounding factors. CONCLUSION: MTAP gene might be involved in the etiology of MI in Chinese Han ethnicity.

Angiotensinogen polymorphisms and acquired atrial fibrillation in Chinese.

Genetic predisposition may be underlying the prevalence of acquired atrial fibrillation (AF). We investigated the association between polymorphism in angiotensinogen (AGT) and angiotensin-converting enzyme gene and risk of acquired AF in a pair-matched case-control study conducted in Chinese Hans. We selected 9 single nucleotide polymorphisms (SNPs) in the AGT gene and 3 SNPs in the angiotensin-converting enzyme gene using a tagging-SNP strategy. We observed significant association between tagging-SNP rs699 (M235T), located in exon 2 of the AGT gene, and AF. The AA genotype of rs699 increased the risk of AF by 70% (95% confidence interval, 1.01-2.85; P = .044) under a recessive model (AA vs AG + GG). The significance remained after controlling for covariates age, smoking, body mass index, hypertension, diabetes, and left atrial dimension, with an increased risk of AF by 90% (95% confidence interval, 1.04-3.46; P = .036). We provide evidence that polymorphism in AGT gene may confer predisposition to acquired atrial fibrillation in Chinese Hans.

[The influencing factors of the postoperative survival in patients with glioma].

OBJECTIVE: To analyze the risk factors that associated with survival of postoperative patients with glioma. METHODS: From 2000 to 2006, 522 patients were enrolled in our hospital and were analyzed related risk factors by using Kaplan-Meier's Product-Limit Survival Estimates method, log-rank test and Cox's proportional-hazards model. Analysis of data were performed in SAS 9.1. Results In univariate analysis, age, sex, extent of resection in surgery and pathological grades appeared to be associated with survival rate of the patients (alpha = 0.05). Cox regression analysis showed these four factors were also significant (HR value 0.811, 1.553, 1.634 and 1.429, respectively). Multivariate Cox regression model also showed that age, pathological grades and extent of resection in surgery were main factors affecting the survival of the patients while HR value increased with the ascending class (2.349, 3.826, and 5.062, respectively)with only subtotal excision enter the model (HR = 1.459). Other factors had no statistical importance on survival rate. CONCLUSION: Age, extent of resection in surgery, pathological grades, chemotherapy after surgery, and radiotherapy after surgery might associate with the prognosis of the patients with glioma.

Genetic polymorphism of KCNH2 confers predisposition of acquired atrial fibrillation in Chinese.

INTRODUCTION: Nonfamiliar atrial fibrillation (AF) is usually associated with acquired structural heart disease, including valvular heart disease, coronary artery disease, and hypertension. Suggestive evidence indicates that these forms of acquired AF are more likely to occur in individuals with a genetic predisposition. We investigated the effect of the potassium channel voltage-gated subfamily member 2 (KCNH2) gene on the prevalence of acquired AF in a Chinese population. METHODS: In a pair-matched, hospital-based case control study (297 vs 297) conducted in Chinese Hans, we investigated 4 tagging single nucleotide polymorphisms (tSNPs), rs1805120, rs1036145, rs3807375, and rs2968857 in the KCNH2 gene, and determined their association with AF acquired from structural heart diseases. RESULTS: We did not observe the association of rs1036145, rs3807375, and rs2968857 with AF. However, we determined that the tSNP, rs1805120, in exon 6 confers the risk of AF in Chinese Hans. Both genotype and allele frequencies of rs1805120 were distributed differently in cases and controls (P = 0.0289 and P = 0.0172, respectively). The most significant association was observed under a recessive model for the minor GG genotype with a 1.45-fold risk of developing AF (95% confidence interval 1.09-1.93, P = 0.012). The significance remained after controlling for the covariates of age, smoking, BMI, hypertension, and diabetes. CONCLUSION: We report a new genetic variation (rs1805120) in the KCNH2 gene that predisposes Chinese Han individuals to the risk of acquired AF. Further genetic and functional studies are required to identify the etiological variants in linkage disequilibrium with this polymorphism.

MTAP and CDKN2B genes are associated with myocardial infarction in Chinese Hans.

OBJECTIVES: To investigate the association between cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, cyclin-dependent kinase inhibitor 2B (CDKN2B) gene, and methylthioadenosine phosphorylase (MTAP) gene and myocardial infarction (MI) in Chinese Hans. DESIGN AND METHODS: A total of 432 patients with MI and 430 controls were included in the study. Nine polymorphisms in the MTAP gene, two polymorphisms in the CDKN2A gene, and two polymorphisms in the CDKN2B gene were selected using a tagging single nucleotide polymorphism (tSNP) strategy. RESULTS: We observed that rs7027989 in the MTAP gene, and rs3217992 and rs1063192 in the CDKN2B gene were significantly associated with MI in male subjects. For rs7027989 and rs3217992, male subjects with the AA or AG genotypes had 1.26-fold and 1.24-fold increased risk of MI, respectively, compared with those with the GG genotype. For rs1063192, the G allele was associated with a reduced risk of MI with a per-allele OR of 0.71 in male subjects. The risk of rs7027989 and rs1063192 remained significant after adjusting for covariates. CONCLUSIONS: This study demonstrates for the first time that polymorphisms in CDKN2B and MTAP gene may influence the risk of MI in Chinese.

OPRM1 gene is associated with BMI in Uyghur population.

To test the hypothesis that micro-opioid receptor (OPRM1) gene might be involved in the prevalence of obesity, a population-based association study was carried out in Uyghur population. Overall 10 tagging single-nucleotide polymorphisms (tSNPs) in OPRM1 gene were genotyped. We showed that genotypes of rs1799971 in exon 1, and rs514980 and rs7773995 in intron 1 were significantly associated with the BMI. The BMI significantly decreased by the copy of minor allele carriers of rs1799971 which is a nonsynonymous functional polymorphism, whereas the BMI significantly increased by the copy of minor allele carriers of rs514980 and rs7773995. Subsequently, subjects were subsequently divided into case (BMI >or= 28) and control group (BMI < 24). Significant associations were again observed at rs1799971, rs514980, and rs7773995, regardless of controlling for covariates age and gender or not. The stronger evidence for association was found under the additive model for each of the three SNPs. The per-allele odds ratio of the minor allele for obesity was 0.75 (95% confidence interval 0.58-0.96, P = 0.023) for rs1799971, 1.68 (95% confidence interval 1.14-2.49, P = 0.009) for rs514980, and 1.80 (95% confidence interval 1.14-2.85, P = 0.012) for rs7773995, respectively. Our observations give the evidence that OPRM1 gene is involved in the prevalence of obesity in Uyghurs.

[Associations of the genetic polymorphisms in CYP11B2 gene with nonfamilial structural atrial fibrillation].

OBJECTIVE: To investigate whether polymorphisms in CYP11B2 gene are associated with nonfamilial structural atrial fibrillation (AF) in Chinese Han population. METHODS: A fine-designed pair-matched hospital based case-control study was performed in 297 cases and 297 controls. We investigated two tagging single nucleotide polymorphisms (tSNPs)-rs4545, rs3802228 in CYP11B2 gene by using GenomeLab SNPstream technique. RESULTS: Two tSNPs were consistent with Hardy-Weinberg expectations in case and control groups. Compared with controls, the left atrial diameter of cases was significantly higher (P < 0.0001). No significant difference in genotype or allele frequencies of tSNPs in CYP11B2 gene was observed. However, at the site of rs3802228 in 3'UTR of the case group, the left atrial diameter in AF patients with GG genotype was significantly higher than others. After adjusted for covariates age, smoking, Body mass index and hypertension, we did not observe the association of rs4545, rs3802228 with AF. CONCLUSION: Our result suggested that polymorphisms of rs4545 in CYP11B2 gene might not be associated with atrial fibrillation but polymorphism of 3'UTR rs3802228 locus in CYP11B2 gene might be associated with atrial structural remodeling.

[Study on the association of predisposing genes with essential hypertension among Kazakhs ethnic group in Xinjiang].

OBJECTIVE: To investigate the association between M235T and G-6A polymorphism of AGT gene, insertion/deletion (I/D) polymorphism of ACE gene, Gly460Trp polymorphism of ADD1 gene, C825T polymorphism of GNB3 gene and essential hypertension in Xinjiang Kazakhs group as well as to identify the interactions of gene-gene and gene-environment. METHODS: A case-control study (n = 441) was performed in 241 cases and 202 controls. Polymerase chain reaction and restrict fragment length polymorphism (PCR-PFLP) technique were used to detect the genotypes polymorphism. RESULTS: Comparing the frequencies of alleles and genotypes, there were no statistical significances except frequency of allele of M235T (P = 0.0483) identified. In logistic regression analysis, there were significant differences in all of the loci. The 4 loci model (AGE/CHO/G-6A/ACE) appeared the best model in MDR analysis. CONCLUSION: Our research data showed that the polymorphisms of all the four genes might be associated with hypertension in the Kazakhs group of Xinjiang while there might be interactions existed in AGT, ACE, AGE and CHO.