Figure 1.
Chemical structure of n-butylidenephthalide.
Figure 2.
Concentration of n-butylidenephthalide for experimenting was determined with a food clearance assay.
Between 20 and 30 newly hatched L1 synchronized animals of N2, BZ555 or OW13 were incubated in E. coli (OD A595 = 0.6) in a 96-well plate at 25°C containing different n-butylidenephthalide concentrations to a total volume of 60 µL. The OD of the plate was measured daily for 6 days. Note the lower OD (∼0.6) in a well due to the reduced path length of the 60 µL final suspension compared to the 1 cm path length in a spectrophotometer. (A) The OD of E. coli recorded daily for each concentration of n-butylidenephthalide. (B) Animals treated with no n-butylidenephthalide and the indicated n-butylidenephthalide concentrations. Animals treated with 10 mM n-butylidenephthalide were alive but concentrations over 20 mM caused death. Scale bar is 100 µm.
Figure 3.
n-Butylidenephthalide rescues dopaminergic neurons of C. elegans from degeneration resulting from 6-OHDA treatment.
(A) GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain BZ555. The left side shows the differential interference contrast (DIC) image. The right side shows the fluorescence images. Scale bar, 50 µm. (B) Graphical representation for fluorescence intensity of GFP expression pattern in dopaminergic neurons of a transgenic C. elegans strain BZ555 as quantified using AxioVision software. The data represent the mean ± SD (n = 10). A hash (#) indicates significant differences between 6-OHDA-treated and untreated animals (p<0.01); an asterisk (*) indicates significant differences between the 6-OHDA-treated control samples and the n-butylidenephthalide/6-OHDA-treated samples (*p<0.05, **p<0.01).
Figure 4.
n-Butylidenephthalide reduces α-synuclein accumulation in the OW13 strain of C. elegans.
(A) YFP expression pattern in muscles of transgenic C. elegans strain OW13. The left side shows the differential interference contrast (DIC) image. The right side shows fluorescence images. Scale bar, 50 µm. (B) Graphical representation for fluorescence intensity of YFP expression pattern in muscles of transgenic C. elegans strain OW13 as quantified using AxioVision software. The data represent the mean ± SD (n = 10). An asterisk (*) indicates significant differences between the control samples and the n-butylidenephthalide-treated samples (*p<0.05, **p<0.01).
Figure 5.
n-Butylidenephthalide elevates lipid content in the OW13 strain of C. elegans.
(A) Nile red staining pattern in transgenic C. elegans strain OW13. The left side shows the differential interference contrast (DIC) image. The right side shows fluorescence images. Scale bar, 50 µm. (B) Graphical representation for fluorescence intensity of the Nile red pattern in transgenic C. elegans strain OW13 as quantified using AxioVision software. The data represent the mean ± SD (n = 10). A hash (#) indicates significant differences between N2 and OW13 animals (p<0.01); an asterisk (*) indicates significant differences between the OW13 control samples and the n-butylidenephthalide-treated samples (*p<0.05, **p<0.01).
Figure 6.
n-Butylidenephthalide improves food-sensing behavior in 6-OHDA-treated N2 C. elegans.
The locomotory rate (frequency of bending) of 6-OHDA-untreated animals, 6-OHDA-treated animals, or n-butylidenephthalide/6-OHDA-treated animals with or without bacteria lawns was assayed. Shown are the slowing rates calculated as the percentage decrease of the locomotory rate in the bacteria lawn as compared with that in no bacteria lawn. The data represent the mean ± SD (n = 10). A hash (#) indicates significant differences between 6-OHDA-treated and untreated animals (p<0.001); an asterisk (*) indicates significant differences between the 6-OHDA-treated control samples and the n-butylidenephthalide/6-OHDA-treated samples (*p<0.05, **p<0.01).
Figure 7.
n-Butylidenephthalide raises DA content in 6-OHDA-treated N2 C. elegans.
Shown is quantitation of DA levels (ng/g wet weight of animals) by HPLC-chemiluminescence detection in 6-OHDA-untreated animals, 6-OHDA-treated animals, or n-butylidenephthalide/6-OHDA-treated animals. Three-day-old animals synchronized for age were used. The data represent the mean ± SD (n = 3). A hash (#) indicates significant differences between 6-OHDA-treated and untreated animals (p<0.001); an asterisk (*) indicates significant differences between the 6-OHDA-treated control samples and the n-butylidenephthalide/6-OHDA-treated samples (*p<0.05, **p<0.01).
Figure 8.
n-Butylidenephthalide prolongs longevity in 6-OHDA-treated N2 C. elegans.
Cumulative survival curves of wild-type N2 animals grown on OP50, 6-OHDA-treated animals grown on OP50, and n-butylidenephthalide/6-OHDA-treated animals fed on n-butylidenephthalide/OP50.
Figure 9.
n-Butylidenephthalide lessens egl-1 expression in apoptosis modulation in 6-OHDA-treated C. elegans.
Quantitative real-time RT-PCR experiments show the expression levels of egl-1, ced-3, ced-4 and ced-9 using RNAs isolated from N2, 6-OHDA-treated, or n-butylidenephthalide/6-OHDA-treated animals. The data represent the mean ± SD (n = 3). An asterisk (*) indicates significant differences between the 6-OHDA-treated control samples and the n-butylidenephthalide/6-OHDA-treated samples (**p<0.01).
Figure 10.
n-Butylidenephthalide augments proteasome activity by enhancing the expression of rpn-6 in the OW13 strain of C. elegans.
(A) n-Butylidenephthalide augments proteasome activity in the OW13 strain of C. elegans. Chymotrypsin-like activity of the proteasome was monitored by Z-Gly-Gly-Leu-AMC digestion in a day 3 adult animal extract containing equal amounts of total protein. The data represent the mean ± SD (n = 3). A hash (#) indicates significant differences between N2 and OW13 animals (p<0.05); an asterisk (*) indicates significant differences between the OW13 control samples and the n-butylidenephthalide-treated OW13 samples (*p<0.05, **p<0.01). (B) n-Butylidenephthalide enhances the expression of rpn-6 of proteasome in the OW13 strain of C. elegans. Quantitative real-time RT-PCR experiments show the expression level of individual subunit of the 26S proteasome, using RNAs isolated from OW13 control or n-butylidenephthalide-treated animals. The data represent the mean ± SD (n = 3). An asterisk (*) indicates significant differences between the OW13 control samples and the n-butylidenephthalide-treated samples (**p<0.01).