Identification of a Novel Peptide with Alcohol Dehydrogenase Activating Ability from Ethanol-Induced Lactococcus lactis: A Combined In Silico Prediction and In Vivo Validation.

Alcohol dehydrogenase (ADH) is a crucial rate-limiting enzyme in alcohol metabolism. Our previous research found that ethanol-induced intracellular extracts of Lactococcus lactis (L. lactis) could enhance alcohol metabolism in mice, but the responsible compounds remain unidentified. The study aimed to screen potential ADH-activating peptides from ethanol-induced L. lactis using virtual screening and molecular docking calculation. Among them, the pentapeptide FAPEG might bind to ADH through hydrophobic interaction and hydrogen bonds, then enhancing ADH activity. Spectroscopy analysis further investigated the peptide-enzyme interaction between FAPEG and ADH, including changes in the amino acid residue microenvironment and secondary structural alterations. Furthermore, FAPEG could protect against alcoholic liver injury (ALI) in mice by reducing blood alcohol concentration, enhancing the activity of antioxidant and alcohol metabolism enzymes, and attenuating alcohol-induced hepatotoxicity, which was related to the activation of the Nrf2/keap1/HO-1 signaling pathway. The study provided preliminary evidence that the generation of ADH-activating peptides in ethanol-induced L. lactis has the potential in preventing ALI in mice using in silico prediction and in vivo validation approaches.

Structural characterization of a glycoprotein from white jade snails (Achatina Fulica) and its wound healing activity.

Snail mucus is rich in proteins and polysaccharides, which has been proved to promote wound healing in mice in our previous research. The aim of this study was to investigate the effective component in snail mucus that can exert the wound healing potential and its structural characterization. Here, the glycoprotein from the snail mucus (SM1S) was obtained by DEAE-Sepharose Fast Flow and Sephacryl S-300 columns. The structural characteristics of SM1S were investigated via chromatographic techniques, periodic acid oxidation, FT-IR spectroscopy and NMR spectroscopy. Results showed that SM1S was a glycoprotein with a molecular weight of 3.8 kDa (83.23 %), consists of mannose, glucuronic acid, glucose, galactose, xylose, arabinose, fucose at a ratio of 13.180:4.875:1043.173:7.552:1:3.501:2.058. In addition, the periodic acid oxidation and NMR analysis showed that SM1S contained 1,6-glycosidic bonds, and might also contain 1 â†’ 4 and 1 â†’ 2 glycosidic or 1 â†’ 3 glycosidic bonds. Furthermore, the migration experiment of human skin fibroblasts in vitro suggested that SM1S had a good effect to accelerate the scratch healing of cells. This study suggested that SM1S may be a prospective candidate as a natural wound dressing for the development of snail mucus products.

AMPK-Dependent YAP Inhibition Mediates the Protective Effect of Metformin against Obesity-Associated Endothelial Dysfunction and Inflammation.

Hyperglycemia is a crucial risk factor for cardiovascular diseases. Chronic inflammation is a central characteristic of obesity, leading to many of its complications. Recent studies have shown that high glucose activates Yes-associated protein 1 (YAP) by suppressing AMPK activity in breast cancer cells. Metformin is a commonly prescribed anti-diabetic drug best known for its AMPK-activating effect. However, the role of YAP in the vasoprotective effect of metformin in diabetic endothelial cell dysfunction is still unknown. The present study aimed to investigate whether YAP activation plays a role in obesity-associated endothelial dysfunction and inflammation and examine whether the vasoprotective effect of metformin is related to YAP inhibition. Reanalysis of the clinical sequencing data revealed YAP signaling, and the YAP target genes CTGF and CYR61 were upregulated in aortic endothelial cells and retinal fibrovascular membranes from diabetic patients. YAP overexpression impaired endothelium-dependent relaxations (EDRs) in isolated mouse aortas and increased the expression of YAP target genes and inflammatory markers in human umbilical vein endothelial cells (HUVECs). High glucose-activated YAP in HUVECs and aortas was accompanied by increased production of oxygen-reactive species. AMPK inhibition was found to induce YAP activation, resulting in increased JNK activity. Metformin activated AMPK and promoted YAP phosphorylation, ultimately improving EDRs and suppressing the JNK activity. Targeting the AMPK-YAP-JNK axis could become a therapeutic strategy for alleviating vascular dysfunction in obesity and diabetes.

Nanoparticles prepared by polysaccharides extracted from Biyang floral mushroom loaded with resveratrol: Characterization, bioactivity and release behavior under in vitro digestion.

Resveratrol (RES) is a common active factor in the functional food field, but poor water solubility and low bioavailability have limited its application. In the present study, the novel nanoparticles (RES-CBFMP NPs) using floral mushroom polysaccharide as the wall material have been developed for delivering RES, aiming to overcome its application shortcomings. After ratio optimization, RES-CBFMP NPs (RES-CBFMP,1:8 w/w), which combined through the hydrogen bonds between RES and CBFMP, showed the best overall performance, with the encapsulation efficiency (EE) of 49.74 ± 0.16%, loading efficiency (LE) of 5.53 ± 0.02%, particle size of 158.56 ± 1.97 nm and zeta-potential of -17.56 ± 0.24 mV. In addition, RES-CBFMP NPs exhibited good physicochemical stabilities, sustained gastrointestinal digestive release property, as well as improved in vitro antioxidant and anticancer activities. This study may contribute to the development of RES oral delivery systems and the application of hydrophobic active molecules in the functional food field.

Probiotic properties and proteomics analysis of ethanol-induced Lactococcus lactis subsp. lactis IL1403.

Our previous study indicated that ethanol-induced intracellular extracts (E-IEs) of Lactococcus lactis subsp. Lactis IL1403 (L. lactis IL1403) alleviated hangovers more effectively in mice than untreated intracellular extracts (U-IEs), but the material basis was unclear. Considering that stress-related proteins might play a significant role, the effects of ethanol induction on probiotic properties of L. lactis IL1403 and the associated stress response mechanism were initially explored in this study. E-IEs of L. lactis IL1403 showed better biological activities, significantly increased bacteria survival rates in oxidative stress environments, increased ADH activity, and enhanced proliferation in RAW264.7 and AML-12 cells. Proteomic analyses revealed that 414 proteins were significantly changed in response to ethanol induction. The expression of proteins involved in the universal stress response, DNA repair, oxidative stress response, and ethanol metabolism was rapidly upregulated under ethanol stress, and quantitative real-time PCR (qRT-PCR) results were consistent with proteomic data. KEGG pathway analysis indicated that citrate metabolism, starch and sucrose metabolism, and pyruvate metabolism were significantly enriched during ethanol stress to increase energy requirements and survival rates of stressed cells. Based on this observation, the active induction is an effective strategy for increasing the biological activity of L. lactis IL1403. Exploring the molecular mechanism and material basis of their functions in vivo can help us understand the adaptive regulatory mechanism of microorganisms.

Cognition of polysaccharides from confusion to clarity: when the next "omic" will come?

With the accelerated pace of modern life, people are facing more and more health pressure. The study of polysaccharides seemed a good choice as a potential treasure trove. Polysaccharides, one of the four basic substances (proteins, nucleic acids, lipids and carbohydrates) that constitute life activities, are obviously an underrated macromolecular substance with great potential. Compared with protein and nucleic acid, the research of polysaccharides is still in the primary stage. The relationship between structure and function of polysaccharides is not clear. In this review, we highlighted the main methods of extraction, purification and structure identification of polysaccharides; summarized their biological activities including immunoregulation, hypoglycemic, anti-tumor, anti-virus, anti-coagulation, and so on. Particularly, the relationship between their structures and activities was described. In addition, the applications of polysaccharides in health food, medicine and cosmetics were also reviewed. This review can help polysaccharide researchers quickly understand the whole process of polysaccharides research, and also provide a reference for the comprehensive utilization of polysaccharides. We need to standardize the research of polysaccharides to make the experimental data more universal, and take it as important references in the review process. Glycomic may appear as the next "omic" after genomic and proteomic in the future. This review provides support for the advancement of glycomics.

Co-assembling nanoparticles of Asiatic acid and Caffeic acid phenethyl ester: Characterization, stability and bioactivity in vitro.

Caffeic acid phenethyl ester (CAPE) is an efficient bioactive polyphenol ester derived from propolis. However, its poor water solubility, bioavailability, and stability significantly limit its application. Based on the assembly properties of some natural small molecules (NSMs), asiatic acid-caffeic acid phenethyl ester nanoparticles (ASA-CAPE NPs) were prepared to overcome the above defects. After proportion optimization, the encapsulation and loading efficiencies of ASA-CAPE NPs reached 47.72 ± 0.17 % and 11.62 ± 0.42 %, respectively. Characterization results showed that ASA-CAPE NPs, mainly assembled by hydrogen bonds and hydrophobic forces, possessed regular spherical morphology with a diameter size of less than 300 nm. Additionally, ASA-CAPE NPs presented improved water solubility, stability, and bioactivities than free CAPE. Besides, ASA-CAPE NPs also exhibited good sustained release of CAPE during the gastrointestinal digestion in vitro. Above all, ASA-CAPE NPs provide a new idea for efficiently utilizing hydrophobic active compounds in the functional food field.

Characterization and correlation of bacterial community and volatile flavor compounds in xiguajiang, a Chinese traditional fermented condiment.

Xiguajiang (XGJ) is one kind of Chinese traditionally fermented soybean food. The aim of this study was to identify core bacterial communities and volatile compounds and explore their relationships in XGJ samples obtained from different manufacturers. Results showed that Bacillus, Staphylococcus, Weissella, and Chromohalobacter were the predominant bacterial genus, although their relative abundance is quite diverse. Larger relative contents of esters and alcohols were detected in XGJ. Moreover, the results of E-nose analysis indicated that nitrogen oxides compounds, pyrazines, and ketones compounds also played a critical role in XGJ unique flavor. The correlation analysis suggested that 3-methyl-butanol, ethoxybenzene, ethyl acetate, acetaldehyde, and 2-(4-methyl-3-cyclohexen-1-yl)-2-propanyl acetate had a significant correlation with Enterobacter, Clostridium, Pseudomonas, Streptomyces, Weissella, Staphylococcus, and Bacillus. These results may provide vital information to understand the role of the microbiota in developing flavor in XGJ products, and improve the quality and safety of XGJ production in industries.

Extraction optimization, purification, and biological properties of polysaccharide from Chinese yam peel.

In this study, the Chinese yam peel polysaccharide (CYPP) was obtained under the extraction conditions optimized by the Response Surface Methodology (RSM). Further biological properties of CYPP-1 purified from CYPP were also determined. The results indicated that the optimum extraction conditions were an extraction temperature of 90.5°C, a liquid-solid ratio of 28.0 ml/g, and an extraction time of 2.94 h, along with a yield of 8.81 ± 1.48%. CYPP-1 was identified as a kind of heteropolysaccharide mostly composed of glucose and galactose (59.4:1.0). The molecular weights were two main parts of 50.5 kDa (54.77%) and 4.4 kDa (21.02%), and the triple-helix conformation was not formed in CYPP-1. Besides, CYPP-1 showed good biological properties including in vitro antioxidant activity and immunomodulatory function on RAW264.7 cells, as well as favorable hypoglycemic effect. Overall, the high-value utilization of CYPP-1 reveals a broad application prospect in the industrial production of functional foods and pharmaceuticals. PRACTICAL APPLICATIONS: Yam peel, which is discarded in large quantities during postharvest processing, results in the production of tremendous by-products and is a great waste of resources. In this study, the yield of water-soluble polysaccharide from yam peel reached 8.81 ± 1.48%. Besides, the purified CYPP-1 exhibited excellent antioxidant activity, favorable immunomodulatory function, and hypoglycemic effect. The high productivity and bioactive effects are both great merits for Chinese yam peel polysaccharide as a promising candidate for foods and medicines industrial production.

Lef1 is transcriptionally activated by Klf4 and suppresses hyperoxia-induced alveolar epithelial cell injury.

PURPOSE: Bronchopulmonary dysplasia (BPD) is a long-term respiratory condition. More than a quarter of extremely premature newborns are harmed by BPD. At present, there are no apparent effective drugs or treatments for the condition. In this study, we aimed to investigate the functional role and mechanism of lymphoid enhancer-binding factor 1 (Lef1) in BPD in vitro. MATERIALS AND METHODS: Blood samples from BPD patients and healthy volunteers were gathered, and an in vitro model of BPD was developed in alveolar epithelial cells (AECs) MLE-12 induced by hyperoxia. Then expression of krüppel-like factor 4 (KLF4/Klf4) and LEF1/Lef1 were evaluated. After Lef1 overexpressing plasmid and the vector were transfected into hyperoxia-induced MLE-12 cells, cell proliferation assays were carried out. Cell apoptosis was investigated by a flow cytometry assay, and apoptosis related proteins Bcl-2, cleaved-caspase 3 and 9 were analyzed by a western blot assay. The binding between Klf4 and Lef1 promoter predicted on the JASPAR website was verified using luciferase and ChIP assays. For further study of the mechanism of Klf4 and Lef1 in BPD, gain-of-function experiments were performed. RESULTS: The mRNA levels of KLF4/Klf4 and LEF1/Lef1 were diminished in clinical BPD serum samples and hyperoxia-induced MLE-12 cells. Overexpression of Lef1 stimulated AEC proliferation and suppressed AEC apoptosis induced by hyperoxia. Mechanically, Klf4 bound to Lef1's promoter region and aids transcription. Moreover, the results of gain-of-function experiments supported that Klf4 could impede AEC damage induced by hyperoxia via stimulating Lef1. CONCLUSION: Klf4 and Lef1 expression levels were declined in hyperoxia-induced AECs, and Lef1 could be transcriptionally activated by Klf4 and protect against hyperoxia-induced AEC injury in BPD. As a result, Lef1 might become a prospective therapeutic target for BPD.

Protective effect of the stressed supernatant from Lactococcus lactis subsp. lactis and its metabolic analysis.

There are numerous factors restricting wide application of lactic acid bacteria (LAB) in dairy industry, causing urgent demands for novel bioprotectants. Protective effects and metabolites of Lactococcus lactis subsp. lactis (L. lactis) from ultraviolet (UV)-induced supernatant were investigated and the protective mechanism was explored. The strain viability of the group treated with the supernatant of continuous UV irradiation (V1) and the group with intermittent UV irradiation (V2) was 8.45 and 14.13 times of the control group, respectively. Further exploration on the protective of L. lactis supernatant, under different dose of UV treatment, showed it was dose-dependent. The condition for the supernatant with best protective effect was vertical distance 50.00 cm, horizontal distance 25.00 cm, intermittent UV irradiation (30 s interval 30 s) for 4.5 min (V2), which was chose for untargeted metabolite analysis. And that in V1 was for comparative study. There were 181 up-regulated metabolites in V1 and 161 up-regulated metabolites in V2, respectively. Most of the up-regulated metabolites were related to secondary metabolite synthesis, environmental microbial metabolism, antibiotic synthesis and amino acid biosynthesis. Notably, production of dithiothreitol (DTT) in V2 was 65.2-fold higher than that in the control group. Trehalose in ABC transporter pathway was also up-regulated in the metabolites induced by UV. Results indicated that L. lactis could adapt to the UV stress by adjusting metabolic pathways and producing special metabolites to protect itself. This research offers the basis for robust strain development and contributes to initial study on potential bioprotectant.

Biochemical analysis reveals the systematic response of motion sickness mice to ginger (Zingiber officinale) extract's amelioration effect.

ETHNOPHARMACOLOGICAL RELEVANCE: As a common medicinal and edible plant, Zingiber officinale Roscoe (ginger) is often used for the prevention of motion sickness. However, the mechanism of its anti-motion sickness remains to be elucidated. AIM OF THE STUDY: To explore novel treatment for motion sickness with less side effects, anti-motion sickness effect of ginger (Zingiber officinale) extract (GE) and the possible molecular mechanisms were investigated. MATERIALS AND METHODS: The anti-motion sickness effect of ginger was evaluated through mice animal experimental models. Components of ginger that might contribute to the anti-motion sickness effect were analyzed by LC-MS/MS. Subsequently, biochemical analysis integrated with serum metabolomic profiling were performed to reveal the systematic response of motion sickness mice to ginger extract's amelioration effect. RESULTS: Exhaustive swimming time of mice in the GE group reached 8.9 min, which was 52.2% longer than that in the model group. Motion sickness index scores and time taken traversing balance beam of mice in the GE group were decreased by 53.2% and 38.5%, respectively. LC-MS/MS analysis suggested that various active ingredients in GE, such as gingerol, ginger oil and terpenoids, might contribute to its appealing anti-motion sickness activity. Biochemical analysis revealed that GE can relieve motion sickness through reducing histamine and acetylcholine release in vestibular system, regulating fatty acid oxidation, sugar metabolism and bile acid metabolism in mice. CONCLUSION: Gavage of mice with GE can effectively relieve the symptoms of autonomic nervous system dysfunction, improve the balance and coordination ability and ameliorate the ability to complete complex work after rotation stimulation. GE has attractive potential for development and utilization as novel anti-motion sickness food or drugs.

Protective effect of coix seed seedling extract on 60 Co-γ radiation-induced oxidative stress in mice.

Exposure to ionizing radiation (IR) can cause oxidative damage to human body, leading to various diseases and even death. In this study, the potential radioprotective effect of coix seed seedling extract (CSS-E) was studied through a model of 60 Co-γ radiation-induced oxidative stress in mice. Overall radioprotective effect of CSS-E against radiation-induced damage was evaluated by biochemical analysis and histopathological analysis. The results showed that CSS-E could significantly reduce the IR-induced damage to the hematopoietic system. CSS-E-M (200 mg/kg BW) pretreatment could increase the activities of superoxide dismutase in serum, liver, and spleen increased by 31.68%, 45.10%, and 56.67%, respectively, and the glutathione peroxidase levels in serum, liver, and spleen of mice were improved by 19.17%, 41.97%, and 130.56%, respectively. Meanwhile, the glutathione levels of serum, liver, and spleen in CSS-E-M group were increased by 17.10%, 35.06%, and 40.71%, respectively. The contents of MDA in different tissues and serum could be reduced by CSS-E-M treatment to the normal level. Moreover, CSS-E could markedly reduce the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in radiation mice, among which CSS-E-M group showed maximum restoration with decreased AST and ALT levels by 20.13% and 32.76% as compared against IR group. In conclusion, these results indicated that CSS-E could be used as a potential natural radioprotectant against IR-induced damage.

60Coγ induction improves the protective effect of Acetobacter pasteurianus against ionizing radiation in mice.

Exposure to ionizing radiation (IR) tends to cause serious health concerns. Thus, radioprotective agents are vital for the population exposed to radiation. As microorganisms have the advantages of fast reproduction and no geographical restrictions, direct microbe-based and environmental induction compounds are thriving radioprotectants resources. Oxidative system and oxidase in Acetobacter pasteurianus are unique and intriguing, the radioprotective effect of the cell-free extract from A. pasteurianus (APE) and 60Coγ-treated extract (IRE) were comparatively investigated in the present study. The survival rate of A. pasteurianus with IRE addition was 149.1% in H2O2 damage test, while that with APE was only 10.4%. The viability of 60Coγ-treated AML-12 cells was increased by 18.8% with IRE addition, yet APE showed no significant radioprotective effect. Moreover, in 60Coγ-treated mice, IRE could significantly protect the white blood cell, improve the liver index, and attenuate the injuries of immune organs in mice. Administration of IRE significantly raised the activities of superoxide dismutase (SOD) and reduced the products of lipid peroxidation. These results clarified that gavage with APE and IRE presented notable antioxidant and radioprotective efficacy. A. pasteurianus showed appealing potential to be novel radioprotective bioagents and 60Coγ treatment on microbe could be a new method for the development of better radioprotectant. KEY POINTS: • 60Coγ induction could improve the radioprotective effect of APE. • IRE protected white blood cell in mice under IR. • IRE products have broad application prospects in radioprotection based on microbes.

Radioprotective effect of radiation-induced Lactococcus lactis cell-free extract against 60Coγ injury in mice.

Ionizing radiation (IR) is widely used in the diagnosis and treatment of various cancers. However, IR can cause damage to human health by producing reactive oxygen species. Lactococcus lactis is a type of microorganism that is beneficial to human health and has a strong antioxidant capacity. In this study, the protective effect of normal and IR-induced L. lactis IL1403 cell-free extracts (CFE and IR-CFE, respectively) against oxidative damage in vitro and the radioprotective effect of IR-CFE in vivo was evaluated using 60Coγ-induced oxidative damage model in mice. Results showed that IR-CFE exhibited a stronger oxidative damage-protective effect than CFE for L. lactis IL1403 under H2O2 in vitro. Moreover, IR-CFE also showed strong radioprotective effect on hepatocyte cells (AML-12) under radiation condition, and the effect was better than that of CFE. Animal experiment indicated that IR-CFE could reduce the IR-induced damage to the hematopoietic system by increasing the number of white blood cells and red blood cells in peripheral blood of irradiated mice. It was also observed that IR-CFE could markedly alleviate the 60Coγ-induced oxidative stress via increasing the activities of superoxide dismutase and glutathione peroxidase, enhancing the levels of glutathione, and decreasing the contents of malondialdehyde in serum, liver, and spleen. In addition, IR-CFE also could reduce the activities of alanine transaminase and aspartate aminotransferase in serum, thereby reducing radiation damage to the liver. These results suggested that IR-CFE could be considered as potential candidates for natural radioprotective agents. This study provides a theoretical basis for improving the application of lactic acid bacteria.

Potential of natural products as radioprotectors and radiosensitizers: opportunities and challenges.

Natural products can be used as natural radiosensitizers and radioprotectors, showing promising effects in cancer treatments in combination with radiotherapy, while reducing ionizing radiation (IR) damage to normal cells/tissues. The different effects of natural products on irradiated normal and tumor cells/tissues have attracted more and more researchers' interest. Nonetheless, the clinical applications of natural products in radiotherapy are few, which may be related to their low bioavailability in the human body. Here, we displayed the radiation protection and radiation sensitization of major natural products, highlighted the related molecular mechanisms of these bioactive substances combined with radiotherapy to treat cancer, and critically reviewed their deficiency and improved measures. Lastly, several clinical trials were presented to verify the clinical application of natural products as radiosensitizers and radioprotectors. Further clinical evaluation is still needed. This review provides a reference for the utilization of natural products as radiosensitizers and radioprotectors.

Anti-fatigue activity of hemp leaves water extract and the related biochemical changes in mice.

To explore novel sources of anti-fatigue drugs and food, the anti-fatigue activity of hemp leaves water extract (HLWE) was investigated through exhaustive swimming tests of mice. The median exhaustion swimming time of mice gavaged with HLWE reached 55.4 min, which was 156.8% and 87.8% longer than that of the control group and Rhodiola group, respectively. Then, several biochemical parameters related to fatigue were determined to explore the possible anti-fatigue reasons. The blood lactic acid concentration of mice in HLWE group was 0.76 mmol/L, which was 24.8% lower than that in the control group. Compared with the control group, the glutathione peroxidases activity of mice in HLWE group increased by 296.2%. Based on the results, HLWE exhibited outstanding anti-fatigue activity through reducing the accumulation of lactic acid and improving the activities of defense antioxidant enzymes. It shows appealing potential for development and utilization as novel anti-fatigue food or drugs.

Wound-healing activity of glycoproteins from white jade snail (Achatina fulica) on experimentally burned mice.

Burns are a global public health problem and the treatment of burn wounds is a major medical and economic issue. White jade snails (Achatina fulica) are now widely distributed in Asia, and they have been used to treat burns in folk medicine of China. In this study, the glycoproteins from white jade snails were investigated and their effect on burn healing was evaluated by a mouse burn model. The results showed that the snail mucus was mainly composed of proteins and polysaccharides, and it had good adhesion. The main component of snail mucus was glycoprotein from the results of DEAE Sepharose FF ion exchange chromatography. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging effect of 1 mg/mL snail mucus reached 13.77%. The wound healing rate of the snail mucus group was higher than that of the control group (p < 0.0001). Histopathological results showed that mice in the snail mucus group had a faster healing than that of the control group. The biochemical analysis was in agreement with the histopathological findings. These results suggested that glycoproteins from snail mucus showed effective wound healing activities in the skin of experimentally burned mice.

Correlation analysis between CARMEN variants and alcohol-induced osteonecrosis of the femoral head in the Chinese population.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a complicated disease associated with trauma, hormone abuse and excessive alcohol consumption. Polymorphisms of long non-coding RNAs have been also linked with the development of ONFH. Our research aimed to explore the relationship between CARMEN (Cardiac Mesoderm Enhancer-Associated Non-Coding RNA) variants and ONFH risk. METHODS: Our study used Agena MassARRAY Assay to genotype 6 selected single nucleotide polymorphisms (SNPs) in 731 participants (308 alcohol-induced ONFH patients and 423 controls). We used odds ratios (ORs) and 95% confidence intervals (CIs) to calculate the effect of gene polymorphisms on the occurrence of alcohol-induced ONFH by logistic regression analysis and haplotype analysis. RESULTS: Our overall analysis illustrated that rs13177623 and rs12654195 had an association with a reduced risk of ONFH after adjustment for age and gender. We also found that rs13177623, rs12654195 and rs11168100 were associated with a decreased susceptibility to alcohol-induced ONFH in people ≤45 years. In addition, the necrotic sites stratification analysis showed that rs12654195 was only found to be related to alcohol-induced ONFH risk in the recessive model. In patients with different clinical stages, rs353300 was observed to be associated with a higher incidence of ONFH. Individuals with different genotypes of rs13177623, rs12654195 and rs11168100 had significantly different clinical parameters (cholinesterase, globulin, percentage of neutrophils and the absolute value of lymphocytes). CONCLUSIONS: Our data provided new light on the association between CARMEN polymorphisms and alcohol-induced ONFH risk in the Chinese Han population.