Research Paper Volume 13, Issue 1 pp 813—830

Carnitine promotes recovery from oxidative stress and extends lifespan in C. elegans

L-carnitine promotes recovery from oxidative stress induced by paraquat and juglone. (A) L-carnitine facilitated the clearance of ROS. N2 wild-type C. elegans were synchronized at L1 larvae stage and raised on NG medium supplemented with or without 10 μM L-carnitine to L4/young adult stage. The ROS generator paraquat was added to the medium to the final concentration of 1mM. After 24 hours, animals were transferred to new paraquat-free plate with and without L-carnitine for recovery. After recovery for 12, 24, 48 hours, worms were stained with ROS dye dihydroethidium (DHE) and imaged with fluorescent microscope. Representative images at 48-hour recovery were shown. (B) Quantification of DHE signal from at least 3 independent experiments in (A) by ImageJ and relative expression levels were plotted. Statistical analysis was performed by two-tailed, unpaired student’s t-test (ns, not significant. *, PC) Thrashing assay for C. elegans. Worms were pick from agar plate to 1 mL M9 buffer in 24-well plate and examined under dissecting microscope. Worms were moving left and right rapidly and the movement from one side to the other side then back to the original position was counted as 1 thrash. (D) L-carnitine mitigated the toxicity of paraquat on mobility. Worms were treated with paraquat and L-carnitine as in (A) and recovered for 48 hours. Trashing speed (thrash/min) under different treatments were measured for at least 3-independent experiments with 10 animals/experiment. Statistical analysis was performed by two-tailed, paired student’s t-test (**, PE) L-carnitine rescued the H2O2 hypersensitivity of paraquat-treated worms. Worms were treated with paraquat and L-carnitine as in (A) and recovered for 48 hours. Worms were then incubated in 50mM H2O2 for 1 hour. Data were pooled from 2 independent experiments and survival rates under different treatment were compared. Statistical analysis was performed by two-tailed, paired student’s t-test (*, PF) L-carnitine mitigated the toxicity of paraquat on embryonic survival. Worms were treated with paraquat and L-carnitine as in (A) and recovered for 48 hours.10 worms were transferred to a new agar plate to allow egg laying for 2 hours and total number of eggs were counted. Hatching were examined after 24 hours. Experiments were conducted 2 times with 5 replicates each time. Data were normalized to control group for comparison. Statistical analysis was performed by two-tailed, unpaired student’s t-test (**, PG) L-carnitine mitigated paraquat-induced amyloid protein aggregation. Worms expressing human amyloid protein fragment Aβ(1-42) in body wall muscle (CL2006) were treated with paraquat and L-carnitine as in (A) and recovered for 48 hours. Worms were then homogenized in high-salt RAB buffer. Soluble and insoluble fraction were analyzed by western blot using anti-Aβ antibody. Total lysate was analyzed by western using anti-actin antibody. Shown was representative results of 2 independent experiments. (H) L-carnitine mitigated Aβ(1-42)-induced paralysis. Animals were raised at 25° C to young adult stage (day-0) and examined every day thereafter for paralysis. Data pooled from 2 independent experiments (n>120) were plotted. Log-rank test was performed (P

Figure 2. L-carnitine promotes recovery from oxidative stress induced by paraquat and juglone. (A) L-carnitine facilitated the clearance of ROS. N2 wild-type C. elegans were synchronized at L1 larvae stage and raised on NG medium supplemented with or without 10 μM L-carnitine to L4/young adult stage. The ROS generator paraquat was added to the medium to the final concentration of 1mM. After 24 hours, animals were transferred to new paraquat-free plate with and without L-carnitine for recovery. After recovery for 12, 24, 48 hours, worms were stained with ROS dye dihydroethidium (DHE) and imaged with fluorescent microscope. Representative images at 48-hour recovery were shown. (B) Quantification of DHE signal from at least 3 independent experiments in (A) by ImageJ and relative expression levels were plotted. Statistical analysis was performed by two-tailed, unpaired student’s t-test (ns, not significant. *, P<0.05. ***, P<0.001). Error bars indicate standard deviation of the mean. (C) Thrashing assay for C. elegans. Worms were pick from agar plate to 1 mL M9 buffer in 24-well plate and examined under dissecting microscope. Worms were moving left and right rapidly and the movement from one side to the other side then back to the original position was counted as 1 thrash. (D) L-carnitine mitigated the toxicity of paraquat on mobility. Worms were treated with paraquat and L-carnitine as in (A) and recovered for 48 hours. Trashing speed (thrash/min) under different treatments were measured for at least 3-independent experiments with 10 animals/experiment. Statistical analysis was performed by two-tailed, paired student’s t-test (**, P<0.01. ***, P<0.001). Error bars indicate standard deviation of the mean. (E) L-carnitine rescued the H2O2 hypersensitivity of paraquat-treated worms. Worms were treated with paraquat and L-carnitine as in (A) and recovered for 48 hours. Worms were then incubated in 50mM H2O2 for 1 hour. Data were pooled from 2 independent experiments and survival rates under different treatment were compared. Statistical analysis was performed by two-tailed, paired student’s t-test (*, P<0.05. **, P<0.01). Error bars indicate standard deviation of the mean. (F) L-carnitine mitigated the toxicity of paraquat on embryonic survival. Worms were treated with paraquat and L-carnitine as in (A) and recovered for 48 hours.10 worms were transferred to a new agar plate to allow egg laying for 2 hours and total number of eggs were counted. Hatching were examined after 24 hours. Experiments were conducted 2 times with 5 replicates each time. Data were normalized to control group for comparison. Statistical analysis was performed by two-tailed, unpaired student’s t-test (**, P<0.01. ***, P<0.001). Error bars indicate standard deviation of the mean. (G) L-carnitine mitigated paraquat-induced amyloid protein aggregation. Worms expressing human amyloid protein fragment Aβ(1-42) in body wall muscle (CL2006) were treated with paraquat and L-carnitine as in (A) and recovered for 48 hours. Worms were then homogenized in high-salt RAB buffer. Soluble and insoluble fraction were analyzed by western blot using anti-Aβ antibody. Total lysate was analyzed by western using anti-actin antibody. Shown was representative results of 2 independent experiments. (H) L-carnitine mitigated Aβ(1-42)-induced paralysis. Animals were raised at 25° C to young adult stage (day-0) and examined every day thereafter for paralysis. Data pooled from 2 independent experiments (n>120) were plotted. Log-rank test was performed (P<0.01). Error bar indicated the standard deviation of the mean.