Metabolite acetyl-L-carnitine participates in Bifidobacterium animalis F1-7 to ameliorate atherosclerotic inflammation by downregulating theTLR4/NF-κB pathway

This study aimed to explore the effect of Bifidobacterium animalis F1-7 on the improvement of atherosclerotic inflammation.Arteriosclerosis model ApoE^(-/-)mice were orally administered with B.animalis F1-7 for 12 weeks.The probiotic intervention reduced the plaque areas in aorta and the accumulation of macrophages,and downregulated the expression of toll-like receptor 4(TLR4)/nuclear factorκB(NF-κB)pathway to reduce the levels of inflammatory factors.The widely-targeted metabolomics analysis showed that acetyl-L-carnitine(ALC)in the intestine of atherosclerotic mice was significantly increased after B.animalis F1-7 intervention.Correlation analysis proved that ALC was associated with atherosclerotic inflammatory response.By using oxidized low density lipoprotein induced macrophage foam cells,we further verified that ALC could reduce lipid accumulation and inflammatory response in foam cells by downregulating the TLR4/NF-κB pathway.Finally,our results revealed that B.animalis F1-7 upregulated the metabolite ALC to downregulate the inflammatory responses,leading to the reduction of plaque accumulation of atherosclerosis.

Acetyl-L-carnitine:An Effective Antioxidant against Cryo-damage on Human Spermatozoa with Asthenospermia

A variety of natural and artificial cryoprotectant extenders have been explored to enhance sperm recovery following cryopreservation-thawing process. The current investigation is aimed at evaluating the effect of acetyl-L-carnitine on human spermatozoa and reactive species oxygen（ROS） level after freezing-thawing process. The spermatozoa were collected from 35 male patients diagnosed as having asthenospermia. The cryopreservation of human spermatozoa treated with acetyl-L-carnitine at different concentrations（group B： 2.5 mmol/L, group C： 7.5 mmol/L, group D： 15 mmol/L） was compared with control（group A： no acetyl-L-carnitine given）. For the frozen-thawed spermatozoa, the viability, motility and DNA integrity were measured by comet assay, acrosome integrity by FITC-PNA staining and ROS level was determined in each group. The results showed that there were no significant differences in motility and viability between group A and group B, while the motility and viability of spermatozoa in group C and group D were significantly increased as compared with those in group A. As compared with group A, the values for DNA integrity parameters including comet rate（CR）, tail DNA percentage（TD）, tail length（TL） and Oliver tail moment（OTM） were significantly reduced in group C and group D. Group C and group D also displayed a higher proportion of intact acrosome than group A. No significant difference in ROS level was found between group A and group B, while with the increase in acetyl-L-carnitine concentration, the ROS level in groups C and D was significantly reduced as compared with that in group A. In conclusion, acetyl-L-carnitine at a concentration of 7.5 mmol/L is an effective antioxidant against cryo-damage on post-thawed human spermatozoa.

Supplementation with L-Carnitine Rescues Sperm Epigenetic Changes in Asthenospermic Male Semen with Altered Acetyl-L-Carnitine Levels

Objective:To investigate the relationship between the concentration of L-carnitine in semen and sperm parameters and investigate the epigenetic profile in sperm cell after L-carnitine usage.Methods:From February 2017 to February 2018,46 semen samples from asthenospermic males and 41 semen samples from healthy donors were acquired.Motility parameters were assessed using computer-assisted sperm analysis(CASA,n=78)and the DNA fragmentation index(DFI)was evaluated through flow cytometry(n=86),%DFI=%cells outside main population.Other oxidative stress markers,such as reactive oxygen species(ROS)levels(n=86)and the mitochondria DNA copy numbers,were detected(n=78).The concentration of L-carnitine and acetyl-L-carnitine was detected(n=82),and methylation was analyzed(n=30).After that,we collected 13 fresh semen samples from asthenospermic males and 23 fresh semen samples from healthy donors.These samples were used in a freeze-thaw model that was used to determine whether adding L-carnitine could change sperm progressive motility(n=23),apoptosis index(n=9),and methylation analysis(n=7).In total,we have done 13 asthenospermia samples for Western blot,and except for the poor Western result,we analyzed 6 samples for H3K9ac detection,7 samples for H3K9m3 and H3K27m3 detection,and immunofluorescence(n=3).Finally,we had recruited 30 volunteers,and they were given oral administration of L-carnitine for 3 months and then collected semen samples at different time points for methylation analysis.Results:The concentration of acetyl-L-carnitine is negatively correlated with the%DFI value(r^2=0.1090;P=0.0026),and the concentration of acetyl-L-carnitine is positively correlated with sperm forward motility(r^2=0.0543;P=0.0458)and ROS(r^2=0.1854;P<0.0001),and the acetyl-L-carnitine level is negatively correlated with%DFI in asthenospermia(r^2=0.1701;P=0.0066),and the level of acetyl-L-carnitine in asthenospermic semen is significantly lower than the normal group(P=0.0419).In addition,this study indicates that adding L-carnitine significantly improved sperm motility(P=0.0325)and reduced sperm apoptosis(P=0.0032).Importantly,Western blotting(P=0.0429)and immunofluorescence staining results showed that the addition of L-carnitine reduced H3K9Me3 methylation level in sperm,respectively.Furthermore,semen samples from asthenospermic patients had reduced methylation levels in a specific region(16^thP=0.0003;17^thP=0.0016)of the brain-derived neurotrophic factor(BDNF)promoter.The 16^th methylation decreased with age(r^2=0.1564;P=0.0306),and the 17^th methylation was decreased after treatment with L-carnitine for 28 days(P=0.0341).Conclusion:L-carnitine can reduce the%DFI and also affect the methylation of the histone modification marker in sperm as a possible epigenetic regulator.

Effect of acetyl L-carnitine on human retinal pigment epithelium-19 cells in hypoxic conditions

AIM:To investigate the effect of acetyl-L-carnitine(ALCAR)on cell viability,morphological integrity,and vascular endothelial growth factor(VEGF)expression in human retinal pigment epithelium(ARPE-19)cells using a hypoxic model.METHODS:In the first set of experiments,the optimal CoCl_(2) dose was determined by exposing ARPE-19 cell cultures to different concentrations.To evaluate the effect of ALCAR on cell viability,five groups of ARPE-19 cell culture were established that included a control group,a sham group(200μM CoCl_(2)),and groups that received 1,10 and 100 mM doses of ALCAR combined with 200μM CoCl_(2),respectively.The cell viability was measured by MTT assay.The morphological characteristics of cells were observed by an inverted phase contrast microscope.The levels of VEGF and HIF-1α secretion by ARPE-19 cells were detected by enzyme linked immunosorbent assay(ELISA)assay.RESULTS:ARPE-19 cells were exposed to different doses of CoCl_(2) in order to create a hypoxia model.Nevertheless,when exposed to a concentration of 200μM CoCl_(2),a notable decrease in viability to 83% was noted.ALCAR was found to increase the cell viability at 1 mM and 10 mM concentrations,while the highest concentration(100 mM)did not have an added effect.The cell viability was found to be significantly higher in the groups treated with a concentration of 1 mM and 10 mM ALCAR compared to the Sham group(P=0.041,P=0.019,respectively).The cell viability and morphology remained unaffected by the greatest dose of ALCAR(100 mM).The administration of 10 mM ALCAR demonstrated a statistically significant reduction in the levels of VEGF and HIF-1α compared with the Sham group(P=0.013,P=0.033,respectively).CONCLUSION:The findings from the current study indicate that ALCAR could represent a viable therapeutic option with the potential to open up novel treatment pathways for retinal diseases,particular relevance for age-related macular degeneration(AMD).However,to fully elucidate ALCAR’s application potential in retinal diseases,additional investigation is necessary to clearly define the exact mechanisms involved.

Effect of Specific Nutrients on Ovulation, Oocytes Development, Gene Expression and Coupling Success in Mice

Introduction: The roles of genetic, epigenetic, metabolic and other environmental factors such as nutrition and stress, are becoming evident for a successful and healthy pregnancy. This raises the possibility and question, if and how, we improve the probability of pregnancy and of a healthy fetus? The present study examined the role of metabolic, antioxidant and minerals and the results suggest that these factors may positively influence the oocyte quality and the pregnancy rate. Methods: CD1 female mice aged 15 and 5 weeks were divided into four groups of ten each and treated by intragastric gavage daily for 3 weeks. G1: Vehicle;G2: Carnitines (L-carnitine 0.4 mg and acetyl-L-carnitine 0.12 mg/mouse);G3: Microelements (Zinc 4 ng, Copper 0.8 ng, Iron 7 ng/mouse);G4: G3+G2. At the end of the treatment period superovulation was induced and oocytes were collected to assess their quantity and quality. Further, in vitro fertilization (IVF) experiments were performed to assess the preimplantation embryo development. The birth success rate was also analyzed in old and young female. The mice were in vivo fertilized. qRT-PCR were performed to analyze a possible modulation in key genes of the reproductive process. Results: The number of oocytes was significantly higher in groups 2 and 4 compared to the control group. The oocyte number in group 3 was not affected. The level of degraded oocytes was 29.1% and 19.3% (group 2 and 4) versus 34.3% (control). Concomitantly, the numbers of embryos arriving to successful birth were also increased in G4, both in the old and young group of mice. Preliminary analysis of genes affected evidenced that AMH was up regulated in the ovary and KITL in the uterus in group 2. Conclusion: Results showed that L-carnitine, acetyl-L-carnitine and micronutrients were able to improve both oocytes quality and success rate of pregnancy. Further studies are planned to further examine ways to improve pregnancy and fetal health.

The Possible Mechanisms Involved in the Protection Strategies against Radiation-Induced Cellular Damage by Carnitines

There is constant low level background radiation from the cosmos but in certain situation the body may be subjected to increased acute or chronic exposure from other sources. This occurs in situations such as radiation accidents, medical use and could possibly occur in military/terrorist incident. Dependent on the type, strength of the actual source, degree of exposure and type of radiation different strategies may be employed to reduce damage to the body tissues. A number of pharmacological agents such as peroxisome proliferator-activated receptor (PPAR) gamma agonists, diltiazem, amifostine and palifermin as well as antioxidants and metabolic compounds have been shown to be effective in preventing and also in reducing the long-term damage of the exposure of the living cells to radiation. The major drawback of synthetic (pharmacological) compounds has been that they are highly toxic at the optimum protective dose. Studies have shown that various endogenously found compounds such as L-carnitine, and its derivative acetyl-L-carnitine, are able to protect tissues and organs against various forms of toxic insult including radiation damage. The radiation-induced chronic injury may also be counteracted by other metabolic compounds with amine groups and antioxidant properties similar to the carnitines such as cysteine, 3,3’-diindolylmethane (DIM) and N-acetylcysteine. This review discuses the radioprotective compounds as well as the potential mechanism of cellular protection against radiation by carnitines and other compounds.