A new dihydroflavone and a new polyacetylene glucoside from Bidens parviflora.

A new dihydroflavone, 2(S)-isookanin-4'-methoxy-8-O-ß-D-glucopyranoside (1), and a new polyacetylene glucoside, (10S)-tridecane-2E-ene-4,6,8-triyne-1-ol-10-O-ß-D-glucopyranoside (2), along with seven known compounds (3-9), were isolated from the herb of Bidens parviflora Willd. The structures of all the extracted compounds were elucidated by HR-ESI-MS, 1 D and 2 D NMR spectra, as well as circular dichroism (CD).

Elsholtzia ciliata (Thunb.) Hyland: A Review of Phytochemistry and Pharmacology.

In this paper, the confusion of the sources of medicinal materials was briefly expounded, and the differences among the varieties were pointed out. At the same time, the chemical components and pharmacological properties of Elsholtzia ciliata (Thunb.) Hyland (E. ciliata) were reviewed. The structures of 352 compounds that have been identified are listed. These mainly include flavonoids, terpenoids, phenylpropanoids, alkaloids, and other chemical components. They have antioxidant, anti-inflammatory, antimicrobial, insecticidal, antiviral, hypolipidemic, hypoglycemic, analgesic, antiarrhythmic, antitumor, antiacetylcholinesterase, and immunoregulator activities. At present, there are many researches using essential oil and alcohol extract, and the researches on antioxidant, anti-inflammatory, anti-microbial, and other pharmacological activities are relatively mature. This paper aims to summarize the existing research, update the research progress regarding the phytochemicals and pharmacology of E. ciliate, and to provide convenience for subsequent research.

The Genus Broussonetia: An Updated Review of Phytochemistry, Pharmacology and Applications.

The Broussonetia genus (Moraceae), recognized for its value in many Chinese traditional herbs, mainly includes Broussonetia papyrifera (L.) L'Hér. ex Vent. (BP), Broussonetia kazinoki Siebold (BK), and Broussonetia luzonica (Blanco) Bureau (BL). Hitherto, researchers have found 338 compounds isolated from BP, BK, and BL, which included flavonoids, polyphenols, phenylpropanoids, alkaloids, terpenoids, steroids, and others. Moreover, its active compounds and extracts have exhibited a variety of pharmacological effects such as antitumor, antioxidant, anti-inflammatory, antidiabetic, anti-obesity, antibacterial, and antiviral properties, and its use against skin wrinkles. In this review, the phytochemistry and pharmacology of Broussonetia are updated systematically, after its applications are first summarized. In addition, this review also discusses the limitations of investigations and the potential direction of Broussonetia. This review can help to further understand the phytochemistry, pharmacology, and other applications of Broussonetia, which paves the way for future research.

Cynanchum auriculatum Royle ex Wight., Cynanchum bungei Decne. and Cynanchum wilfordii (Maxim.) Hemsl.: Current Research and Prospects.

Cynanchum auriculatum Royle ex Wight. (CA), Cynanchum bungei Decne. (CB) and Cynanchum wilfordii (Maxim.) Hemsl. (CW) are three close species belonging to the Asclepiadaceous family, and their dry roots as the bioactive part have been revealed to exhibit anti-tumor, neuroprotection, organ protection, reducing liver lipid and blood lipid, immunomodulatory, anti-inflammatory, and other activities. Until 2021, phytochemistry investigations have uncovered 232 compounds isolated from three species, which could be classified into C21-steroids, acetophenones, terpenoids, and alkaloids. In this review, the morphology characteristics, species identification, and the relationship of botany, extraction, and the separation of chemical constituents, along with the molecular mechanism and pharmacokinetics of bioactive constituents of three species, are summarized for the first time, and their phytochemistry, pharmacology, and clinical safety are also updated. Moreover, the direction and limitation of current research on three species is also discussed.

Sulforaphane Prevents Neuronal Apoptosis and Memory Impairment in Diabetic Rats.

BACKGROUND/AIMS: To explore the effects of sulforaphane (SFN) on neuronal apoptosis in hippocampus and memory impairment in diabetic rats. METHODS: Thirty male rats were randomly divided into normal control, diabetic model and SFN treatment groups (N = 10 in each group). Streptozotocin (STZ) was applied to establish diabetic model. Water Morris maze task was applied to test learning and memory. Tunel assaying was used to detect apoptosis in hippocampus. The expressions of Caspase-3 and myeloid cell leukemia 1(MCL-1) were detected by western blotting. Neurotrophic factor levels and AKT/GSK3ß pathway were also detected. RESULTS: Compared with normal control, learning and memory were apparently impaired, with up-regulation of Caspase-3 and down-regulation of MCL-1 in diabetic rats. Apoptotic neurons were also found in CA1 region after diabetic modeling. By contrast, SFN treatment prevented the memory impairment, decreased the apoptosis of hippocampal neurons. SFN also attenuated the abnormal expression of Caspase-3 and MCL-1 in diabetic model. Mechanically, SFN treatment reversed diabetic modeling-induced decrease of p-Akt, p-GSK3ß, NGF and BDNF expressions. CONCLUSION: SFN could prevent the memory impairment and apoptosis of hippocampal neurons in diabetic rat. The possible mechanism was related to the regulation of neurotropic factors and Akt/GSK3ß pathway.

Identification of key differentially methylated genes regulating muscle development in chickens: insights from Jingyuan breed.

Skeletal muscle development is a complex, regulated physiological process that involves myoblast proliferation and differentiation and the fusion of myotubes. In this study, phenotypic differences in the breast and leg muscles of 180-day-old Jingyuan chickens were investigated. Differentially methylated genes (DMG) that regulate muscle development were identified through differential expression analysis and weighted gene co-expression network analysis. Moreover, myoblasts were used as test material and treated with cycloleucine to investigate the effect of N6-methyladenosine (m6A) modification on their proliferation and differentiation. The results revealed that the myofiber diameter and cross-sectional area in the breast muscle of Jingyuan chickens were significantly smaller than those in the leg muscle, while myofiber density in the breast muscle was significantly higher. A total of 484 DMG were identified in both muscle types. Module gene association analysis with DMGs revealed multiple DMG associated with muscle development. In vitro cell model analysis revealed that cycloleucine treatment significantly downregulated the m6A modification level of myoblasts and inhibited their proliferation and differentiation. Additionally, stage-specific differences in LDHA, LDHB, and GAPDH expressions were observed during myoblast differentiation. Cycloleucine treatment significantly inhibited LDHA, LDHB, and GAPDH expression. These findings indicate that m6A methylation modifications play significant regulatory roles in muscle development, with LDHA, LDHB, and GAPDH being potential candidate genes for regulating muscle development. This study provides an essential theoretical basis for further study on the functional mechanisms of m6A modifications involved in muscle development.

A scientometrics analysis and visualization of refractory gastroesophageal reflux disease.

Background: Refractory gastroesophageal reflux disease (refractory GERD) is a heterogeneous disease characterized by unresponsiveness or poor efficacy to proton-pump inhibitors (PPIs). This chronic disorder substantially weakens patients' mental wellbeing and quality of life, increasing the financial burden on society. Multiple articles have been reported in this area. However, literature involving scientometric analysis of refractory GERD is absent. Therefore, it is necessary to understand the evolution of research themes and the main hotspots of refractory GERD through bibliometric methods. Methods: All documents related to refractory GERD based on the WOS Core Collection from January 2000 to November 2023 were selected for analysis. Citespace V 6.1 R6, VOSviewer V 1.6.20, and Scimago Graphica V 1.0.38 were used to perform bibliometric analysis. Results: We collected a total of 241 research articles from 36 countries and 322 institutions, contributed by over 1,000 authors. Over the last 20 years, the number of articles in this field has increased year by year, and since 2011, the number of publications has increased dramatically, with 85.89% of the papers. These countries are led by the United States and Japan. GUT had the highest number of citations and DIGESTION had the highest number of publications. Research on standardized diagnosis and management, mechanisms, novel monitoring methods, and innovative drugs and procedures for refractory GERD are the main topics and hotspots in this field. This study also found that neuroimmune interaction is closely related to refractory GERD, which may be a new direction for future mechanism research. Conclusion: Our study is the first bibliometric analysis of the global literature on refractory GERD. This research provides valuable insights for researchers, enabling them to quickly understand the research frontier and hot topics of this field.

Effects of depression on cognitive function in patients with diabetes in different age groups.

AIMS: To investigate the effects of depression on cognitive function in patients of different ages with diabetes mellitus (DM). METHODS: 6,549 patients with DM who were assessed using the Mini-Mental State Examination (MMSE) and Self-Rating Depression Scale (SDS) in 2016 were selected from the 2016 Kailuan Group staff physical examinations. Generalized linear regression models were used to analyze the effects of SDS index score on MMSE score in DM patients in different age groups. We also analyzed the effects of SDS index score on MMSE score in DM patients with different risk factors. RESULTS: Generalized linear regression analysis showed that higher SDS index score was associated with lower MMSE score (ß = -0.06; P < 0.01). In addition, there was an interaction effect between SDS index score and age groups on cognitive function. Meanwhile, SDS index score also has an interaction effect with level of education. CONCLUSIONS: The negative association between the degree of depression and cognitive function level increases with age in DM patients.

Dendrophthoe falcata (L.f.) Ettingsh. and Dendrophthoe pentandra (L.) Miq.: A review of traditional medical uses, phytochemistry, pharmacology, toxicity, and applications.

Dendrophthoe falcata (L.f.) Ettingsh. (DF) and Dendrophthoe pentandra (L.) Miq. (DP) have been traditionally used for the treatment of various ailments, such as cancer, ulcers, asthma, paralysis, skin diseases, tuberculosis, and menstrual troubles, in the ethnomedicinal systems of India and Indonesia. Currently, the chemical structures of 46 compounds have been elucidated from DF and DP, including flavonoids, triterpenes, tannins, steroids, open-chain aliphatics, benzyl derivates, and cyclic chain derivatives. In vitro assays have revealed their anti-tumor and anti-microbial activities. In vivo studies have unraveled their pharmacological properties against tumors, depression, fertility disorders, inflammatory responses, and so on. Additionally, their weak toxicity to rats and brine shrimp, as well as their promising applications for pharmaceutical preparations and combined medication, were also revealed. Herein, we not only recapitulated traditional medical uses, phytochemistry, pharmacology, toxicity, and applications of DF and DP but also discussed current research limitations and future perspectives, which are instructive for those interested in them and are committed to advancing parasitic plants to the Frontier of phytomedicine. We highlighted that DF and DP will become promising medical plants rather than being discarded as notorious pests, provided that more and deeper research is undertaken.

Research progress on chemical components and pharmacological action of Solanum lyratum Thunb.

OBJECTIVES: Solanum lyratum Thunb (SLT) is a perennial plant of the Solanaceae family, and is extensively used in the clinical practice of traditional Chinese medicine. Malaria, oedema, gonorrhoea, cancer, wind and fever, jaundiced hepatitis, cholecystitis and rheumatoid arthritis are among the diseases that it is used to treat. To offer a foundation for further development and usage of SLT, the pieces of literature about the chemical composition and pharmacological action of SLT were reviewed and analysed. KEY FINDINGS: The chemical constituents of SLT mainly included steroids, alkaloids, flavonoids, terpenoids, anthraquinones, phenylpropanoids and others. Pharmacological action mainly contains anti-tumour, antibacterial, anti-inflammatory, anti-oxidation and other pharmacological actions, among them, the anti-tumour effect is particularly outstanding. SUMMARY: At present, studies on the pharmacological effects of SLT mainly focus on alkaloids and steroidal saponins. In the follow-up studies, studies on the pharmacological activities of other chemical components in SLT, such as flavonoids and terpenoids, should be strengthened. It has the potential to pave the way for more research and development of novel SLT medicines.

New polyacetylenes from Bidens procera.

A phytochemical investigation of Bidens procera L.C.Xu ex X.W.Zheng afforded two novel polyacetylenes, tridecane-2E-monoene-4,6,8-triyntylen-1,13-diol-12-O-ß-glucoside (1) and tetradecane-2E,8E-diene-4,6-diyne-1,14-diol-13-O-ß-glucoside (2), together with ten known compounds (3 - 12). Their chemical structures were elucidated by NMR and MS spectrums as well as the comparison of the published data. Furthermore, the chemotaxonomy of the yielded compounds was also discussed.

Tapinanthus species: A review of botany and biology, secondary metabolites, ethnomedical uses, current pharmacology and toxicology.

ETHNOPHARMACOLOGICAL RELEVANCE: Tapinanthus species are hemiparasites that grow on diverse hosts in African regions. Tapinanthus species are locally known as "all purpose herbs" as they are traditionally used to treat various diseases such as diabetes, hypertension, cancer, inflammation, malaria, anemia, anxiety, itching, and so on. AIM OF THE STUDY: A comprehensive review on research outcomes and future perspectives of Tapinanthus species are presented to provide a reference for relevant researchers. MATERIALS AND METHODS: The references regarding Tapinanthus species were retrieved from Google Scholar, Web of Science, Sci-finder, PubMed, Elsevier, Wiley, China National Knowledge Infrastructure, Open Access Library, and SpringerLink between 1963 and 2022. Scientific plant names were provided by "The Plant List" (www.theplantlist.org) and "The world Flora Online" (www.worldfloraonline.org). RESULTS: Even though Tapinanthus species are regarded as notorious pests that can undermine various hosts, they are, as omnipotent herbs in folklore, meaningful for the development of potential phytomedicine sources. Phytochemistry screening has revealed the presence of glycosides, triterpenoids, flavonoids, alkaloids, tannins, steroids, anthraquinones. Among them, the chemical structures of 40 compounds have been elucidated by phytochemical methods without alkaloids and anthraquinones. These secondary metabolites might be responsible for ethnomedical uses and bioactivities of Tapinanthus species. Current research has provided scientific evidence for traditional uses of Tapinanthus species, especially unraveling hypoglycemic, hepatoprotective, antioxidant, antibacterial, anti-anxiety, anti-depression, anti-inflammatory, and other pharmacological properties. Given the fact that ethnomedical uses served as a valuable reference for pharmacology, however, some records to treat arthritis, fever, itching, dysentery, stomach pain, and anemia, have not been confirmed in current research. Furthermore, the toxic effects of Tapinanthus species were susceptible to the dosages, with relative safety across a wide range. CONCLUSIONS: To reasonably yield Tapinanthus species, artificial culture might be a promising method to develop in the future. The discrepancies between phytochemistry screening and structure elucidation, as well as between ethnomedical uses and current pharmacology, need to be further clarified. The identification of bioactive compounds in crude extracts and fractions, the illustration of the underlying mechanisms of pharmacology, along with the addition of cytotoxicity, genotoxicity, and clinical trials of toxic tests, should be carried out in depth. This review highlights that Tapinanthus species can be considered promising phytomedicine sources as long as we adhere to digging more deeply into their potential role.

Liraglutide improves cognitive impairment via the AMPK and PI3K/Akt signaling pathways in type 2 diabetic rats.

Liraglutide is a type of glucagon­like­peptide 1 receptor agonist, which has been reported as a novel type of antidiabetic agent with numerous benefits, including cardiovascular and neuroprotective effects. To the best of our knowledge, few studies to date have reported the potential mechanism underlying the neuroprotective effects of liraglutide on rats with type 2 diabetes mellitus (T2DM). The present study aimed to investigate the neuroprotective actions of liraglutide in diabetic rats and to determine the mechanisms underlying these effects. A total of 30 male T2DM Goto­Kakizaki (GK) rats (age, 32 weeks; weight, 300­350 g) and 10 male Wistar rats (age, 32 weeks; weight, 300­350 g) were used in the present study. Wistar rats received vehicle treatment, and GK rats randomly received treatment with vehicle, low dose of liraglutide (75 µg/kg) or high dose of liraglutide (200 µg/kg) for 28 days. Cognitive deficits were evaluated using the Morris water maze test. The expression levels of phosphoinositide 3­kinase (PI3K), protein kinase B (Akt), phosphorylated (p)­Akt, AMP­activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), Beclin­1, microtubule­associated protein light chain 3 (LC)­3 II, caspase­3, B­cell lymphoma 2 (Bcl­2)­associated X protein and Bcl­2 were assessed by western blot analysis. The results demonstrated that diabetic GK rats exhibited cognitive dysfunction, whereas treatment with liraglutide alleviated the learning and memory deficits, particularly in the high­dose liraglutide group. The expression levels of Beclin­1 and LC­3 II were decreased in GK rats; however, this decrease was alleviated in the presence of liraglutide. Liraglutide also reversed T2DM model­induced increases in mTOR, and decreases in p­AMPK, PI3K and p­Akt expression, and modulated the expression of apoptosis­associated proteins. Furthermore, the administration of liraglutide inhibited apoptosis and exerted a protective effect against cognitive deficits via the activation of autophagy. In conclusion, the protective effects of liraglutide may be associated with increased mTOR expression via activation of the AMPK and PI3K/Akt signaling pathways.

Vildagliptin, a DPP4 inhibitor, alleviates diabetes-associated cognitive deficits by decreasing the levels of apoptosis-related proteins in the rat hippocampus.

Cognitive impairment is a prevalent but underestimated complication of diabetes, which can cause spatial memory and learning deficits. In the present study, a streptozotocin-induced type 2 diabetic rat model was employed to investigate the effects of vildagliptin, a new oral hypoglycemic agent that acts by inhibiting dipeptidyl peptidase-4, on diabetes-associated cognitive impairments, as well as the molecular mechanisms involved. The present findings demonstrated that vildagliptin treatment prevented memory impairment and decreased the apoptosis of hippocampal neurons. It also attenuated the abnormal expression of caspase-3, B cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein in the diabetic model. Vildagliptin treatment also reversed diabetes-induced decreases in phosphorylated (p)-protein kinase B (Akt) and p-glycogen synthase kinase 3ß (GSK3ß), brain-derived neurotrophic factor and nerve growth factor expression levels. The results indicated that the administration of vildagliptin exerts a protective effect against cognitive deficits by decreasing the expression of apoptosis-related proteins in the hippocampus and that this protective effect was mediated via the Akt/GSK3ß signaling pathway.

Association between 1p13 polymorphisms and peripheral arterial disease in a Chinese population with diabetes.

AIMS/INTRODUCTION: Variants on chromosome 1p13 have been associated with coronary artery disease and acute myocardial infarction risk in different ethnic groups. The present study aimed to investigate the association between 1p13 polymorphisms and the development of peripheral artery disease (PAD) in a Chinese population with type 2 diabetes mellitus. MATERIALS AND METHODS: 1p13 polymorphisms, rs599839, rs646776 and rs12740374, were assessed in a cohort of 882 type 2 diabetes mellitus patients including 440 type 2 diabetes mellitus patients with PAD (DM + PAD group) and 442 patients without PAD (DM group). Genotyping was carried out using TaqMan assay. RESULTS: Compared with the DM group, the frequencies of the minor G allele of both rs599839 and rs646776 and the minor T allele of rs12740374 decreased (P = 0.013, P = 0.019 and P = 0.005, respectively), and the frequencies of rs599839 AG + GG, rs646776 AG + GG and rs12740374 CT+TT genotypes were statistically significantly decreased as well (P = 0.017, P = 0.011 and P = 0.007, respectively) in the dominant model in the DM + PAD group than in the DM group. Multivariate unconditional logistic regression analyses adjusted for age, glycated hemoglobin, triglyceride, low-density lipoprotein cholesterol, smoking, hypertension, diabetes duration, coronary heart disease and cerebral infarction showed that the genotypic distribution of rs599839 AG + GG, rs646776 AG + GG and rs12740374 CT + TT remained statistically different between the DM and DM + PAD group (P = 0.014, P = 0.003 and P = 0.004, respectively). The frequencies of haplotype GGT were statistically significantly different between groups (P = 0.08). CONCLUSIONS: The present study strongly supports that genotypes of rs599839, rs646776 and rs12740374 on 1p13 are protective factors for diabetic PAD in a Chinese population. Haplotype GGT generated by rs599839, rs646776 and rs12740374 might also decrease the risk of the disease.

Transcriptional Profiling and Identification of Heat-Responsive Genes in Perennial Ryegrass by RNA-Sequencing.

Perennial ryegrass (Lolium perenne) is one of the most widely used forage and turf grasses in the world due to its desirable agronomic qualities. However, as a cool-season perennial grass species, high temperature is a major factor limiting its performance in warmer and transition regions. In this study, a de novo transcriptome was generated using a cDNA library constructed from perennial ryegrass leaves subjected to short-term heat stress treatment. Then the expression profiling and identification of perennial ryegrass heat response genes by digital gene expression analyses was performed. The goal of this work was to produce expression profiles of high temperature stress responsive genes in perennial ryegrass leaves and further identify the potentially important candidate genes with altered levels of transcript, such as those genes involved in transcriptional regulation, antioxidant responses, plant hormones and signal transduction, and cellular metabolism. The de novo assembly of perennial ryegrass transcriptome in this study obtained more total and annotated unigenes compared to previously published ones. Many DEGs identified were genes that are known to respond to heat stress in plants, including HSFs, HSPs, and antioxidant related genes. In the meanwhile, we also identified four gene candidates mainly involved in C4 carbon fixation, and one TOR gene. Their exact roles in plant heat stress response need to dissect further. This study would be important by providing the gene resources for improving heat stress tolerance in both perennial ryegrass and other cool-season perennial grass plants.

Expression of autophagy-associated proteins in papillary thyroid carcinoma.

The present study was designed to assess the protein expression of the autophagy-associated genes, Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)-II, as well as the association with clinicopathological features in papillary thyroid carcinoma (PTC). A total of 50 subjects were recruited, including 50 human PTC samples and paired adjacent noncancerous tissue samples. The protein expression of Beclin-1 and LC3-II was analyzed using immunohistochemistry and western blotting. Beclin-1 and LC3-II expression in PTC tissues significantly reduced compared with normal tissues (P<0.05). Expression of Beclin-1 and LC3-II was associated with lymph node metastasis of PTC (P<0.05), but had no association with age, gender, tumor size, tumor number and Tumor-Node-Metastasis stage (P>0.05). Expression of Beclin-1 and LC3-II were positively correlated (r=0.327;P=0.020) in PTC. In conclusion, the activity of autophagy was declined in PTC; this decrease in autophagic capacity may be associated with tumorigenesis and the development of PTC.

Long noncoding RNA are aberrantly expressed in human papillary thyroid carcinoma.

Long noncoding RNAs (lncRNAs) have emerged as key regulatory molecules at almost every level of gene expression regulation. The altered expression of lncRNAs is a characteristic of numerous types of cancer, and lncRNAs have been demonstrated to promote the development, invasion and metastasis of tumors through various mechanisms. However, the role of lncRNAs in papillary thyroid carcinoma (PTC) remain unclear. In the present study, differentially expressed lncRNAs and mRNAs were detected by human lncRNA microarray in three pairs of PTC and adjacent noncancerous samples. The microarray results revealed that 675 lncRNAs and 751 mRNAs were abnormally expressed in the three PTC samples compared with adjacent noncancerous samples (fold change ≥2.0; P<0.05). To validate the microarray results, 8 differentially expressed lncRNAs were randomly selected for quantitative polymerase chain reaction (qPCR). The results of qPCR were consistent with the microarray data; the 8 lncRNAs had an aberrant expression in the PTC samples compared with the adjacent noncancerous samples. Gene ontology and pathway analysis indicated that there were 7 downregulated pathways and 29 upregulated pathways in PTC. LncRNA classification and subgroup analysis revealed 7 pairs of enhancer-like lncRNA-mRNA, 9 pairs of antisense lncRNA-mRNA and 45 pairs of lncRNA-mRNA were differentially expressed between PTC and their paired noncancerous samples. In conclusion, the present study identified a series of novel PTC-associated lncRNAs. Further study with these lncRNAs is instrumental for the identification of novel target molecules that could lead to improved diagnosis and treatment for PTC.

9p21 polymorphisms increase the risk of peripheral artery disease in the Han Chinese population.

OBJECTIVE: A case-control study to investigate the association of the 9p21 single nucleotide polymorphisms (SNPs) rs10757274 and rs10757278 (known to be associated with coronary artery disease [CAD] risk) with peripheral arterial disease (PAD), in a Han Chinese population. METHODS: The rs10757274 and rs10757278 genotypes of patients with PAD, and age- and sex-matched control subjects, were determined. Multivariate unconditional logistic regression analyses were performed, with adjustments for age, sex, hypertension, dyslipidaemia, diabetes and smoking status. RESULTS: The study included 420 patients with PAD and 418 control subjects. Variant forms of both SNPs were associated with increased risk of PAD in the total study population, when excluding patients with CAD or stroke (additive genetic model). The GG haplotype increased the risk of PAD, but this association did not remain significant after further sensitivity analysis. Both SNPs were associated with PAD risk in patients aged <65 years, but not in those aged ≥ 65 years (additive model). CONCLUSIONS: 9p21 is associated with PAD. When stratified according to age, 9p21 increases PAD risk in individuals aged <65 years, but not in those aged ≥ 65 years.