Probe 1 is feasible for evaluation of Cys, GSH and Hcy levels in PC12 cells and in vivo
In order to evaluate the chemical stability of Probe 1, UPLC-MS was utilized to analyze the changes in the retention time (RT) and mass spectrum before and after deposition at room temperature for 48 h (Fig. S1a and b). There were no changes in retention time observed pre and post -deposition, with an RT of 2.13 min, and the difference between the two peak areas was less than 5%. The mass spectrum of the compound after deposition was also not changed, with a [M + H]+ m/z of 399, indicating that Probe 1 has excellent stability in practical application.
After PC12 cells were treated with 1–10 µM of Probe 1 for 48 h the cell survival rate was greater than 90% (Fig. S1c), indicating that, at these concentrations, Probe 1 is not cytotoxic to PC12 cells.
The accuracy and validity of Probe 1 for use in measuring clinical serum samples was also verified. We firstly used the commercial ELISA method to measure the serum Cys, GSH and Hcy concentrations in the clinical patient samples, and compared the results when Probe 1 was used for measurement. There was a similar trend in the data obtained using both methods and the data were closely matched (Table S1), indicating Probe 1 is suitable for serum Cys, GSH and Hcy detection in clinical applications.
Probe 1 can additionally be used to detect thiols in PC12 cells. NEM was added to remove the endogenous thiol, and when exogenous Hcy, Cys or GSH was added, Probe 1 could induce the production of corresponding blue, green and red fluorescence. Furthermore, after storage at room temperature for 48 h, the exogenous thiols in PC12 cells could still be detected by Probe 1 with no attenuation in fluorescence intensity (Fig. S2a and b).
Moreover, in this study we elucidate that Probe 1 can be used to detect thiol groups in the hippocampus of mice. Firstly, NEM was used to suppress signal from endogenous biothiols, before Hcy, Cys or GSH were added to stimulate the bright blue, green and red fluorescence through activity of Probe 1, which confirmed the excellent response of Probe 1 to Cys, Hcy and GSH in hippocampal tissue (Fig. S2c). Similarly, the intensity of fluorescence in hippocampal tissue was not attenuated after being deposited for 48 h at room temperature (Fig. S2d). however, in-vivo, we observed that the intensity of fluorescence of produced by Probe 1 activity in the brain peaked at 30 min before being attenuated, while, the fluorescent produced by Probe 1 activity did continue for 120 min before fading completely (Fig. S3). This data ensures that adequate testing time will be available when using Probe 1 to detect thiol groups in brain tissue.
The results and discussion may be presented separately, or in one combined section, and may optionally be divided into headed subsections.
Aβ 1−42 induced changes in Hcy, Cys and GSH expression in PC12 cells
Probe 1 can detect endogenous Hcy, Cys, and GSH in PC12 cell (Fig. 1a). Aβ1−42 can exacerbate oxidative stress by decreasing endogenous antioxidant levels and resulting in induction of apoptosis in PC 12 cells [2, 18–20]. When PC12 cells were treated with Aβ1−42 for 24–48 h, the fluorescence intensity from the blue channel (Hcy) was enhanced with increasing treatment time, and correspondingly, fluorescence from the green and red channels (Cys and GSH) broadly decreased (Fig. 1a). Next, we quantified intracellular levels of the three biothiols using Probe 1 and observed that treatment of PC 12 cells with Aβ1−42 for 48 h resulted in significantly increased levels of Hcy (~ 2.44 ± 0.37 fold, P < 0.01), and decreased Cys and GSH levels (~ 32.70%±0.03 and 61.57%±0.05) compared to levels identified in nontreated control cells (Fig. 1b).
Biothiol level changes in the brains of AD mice
The three different fluorescent signals resulting from detection of Cys, GSH and Hcy were shown in the different layers of the hippocampal tissue slices from the C57BL/6 mice imaged by the Z-stack mode of a confocal microscope (Fig. 2a). In these C57BL/6 hippocampal sections, there was greener and red fluorescence representing endogenous Cys and GSH, while there was less blue fluorescence representing Hcy, indicating that Hcy was not abundant in the hippocampus of normal mice (Fig. 2b). This was converse to the data indicating the Cys, GSH and Hcy levels in the hippocampal slices of APP/PS1 mice at different ages. The green (Cys) and red fluorescent (GSH) signals were gradually attenuated with the age in APP/PS1 mice, and this attenuation was more obvious in the APP/PS112M+ group compared to in the hippocampus of the C57BL/612M+ group. Meanwhile, the blue fluorescence (representing Hcy) was enhanced with increasing age, indicating that the level of Hcy in the hippocampus rises with the progression of AD pathology in this mouse model (Fig. 2b). Meanwhile, we quantified Cys, GSH and Hcy concentrations in the hippocampus of APP/PS1 and C57BL/6 mice using Probe 1, and found that Cys and GSH levels were reduced by 39.54%±0.05 and 15.24%±0.02, but Hcy level were increased by 1.46 ± 0.31-fold, in the APP/PS112M+ group when compared to the C57BL/612M+ group (Fig. 2c). These results are consistent with the trend of fluorescence staining observations we described using Probe 1 for detection.
In order to further monitor the changes in Cys, GSH and Hcy levels in the brain of AD model mice at different ages, we used the in-vivo imaging technique and found differences in the fluorescence intensities of the 3 biothiols when comparing the brains of normal C57BL/6 mice and APP/PS1 mice with AD symptoms. When the APP/PS1 mice were 6 months old, there was little to no difference in biothiol level expression compared to the C57BL/612M+ group. However, the brains of 9 month APP/PS1 mice showed significant enhancement of Hcy fluorescence, while Cys and GSH signal was attenuated. In the brains of 12 month old APP/PS1 mice, the fluorescent signals indicating Cys and GSH presence were decreased by 62.50%±0.07 and 44.83%±0.05, while Hcy signal was increased by 1.60 ± 0.19-fold, when compared with those of C57BL/612M + group (Fig. 2d).
Cys, GSH, and Hcy levels are abnormal in the serum of AD patients
The levels of Cys, GSH, and Hcy in the serum samples acquired from healthy individuals and AD patients were measured using Probe 1. Table 2 shows that the average level of Hcy in the serum of healthy individuals was just below 8 µM, whereas Cys and GSH concentrations were just over 39 µM and 700 µM, respectively. Meanwhile, in AD patients, average Hcy was over 22 µM, which is an increase of 2.78 ± 0.83 fold, and Cys and GSH were just below 24 µM and 662 µM, which is a decrease 36.2%±0.04 and 43.3%±0.06, compared to the control cohort, respectively (Table 2, S2 and S3).
Table 2
The Hcy, Cys and GSH levels in the serum from AD patients and healthy controls (µM, mean ± SD).
Group
|
n
|
Hcy
|
Cys
|
GSH
|
Control
|
31
|
7.99 ± 3.24
|
39.40 ± 5.51
|
749.53 ± 20.14
|
AD
|
31
|
22.17 ± 4.02**
|
24.94 ± 4.28**
|
662.23 ± 25.51**
|
Note: *P < 0.005; **P < 0.001, compared with that of the Control group. |
VB 6 reverses the changes in Hcy, Cys and GSH levels in Aβ1−42-treated PC12 cells and brains of AD model mice
VB6, is an important coenzyme that can promote cystathionine beta-synthase -mediated catalysis of serine and Hcy into Cys [8, 9]. PC12 cells were pretreated with Aβ1−42 before being supplemented with VB6 for 2 h. This supplementation with VB6 resulted in significantly increased levels of Cys and GSH, by 1.73 ± 0.25 and 1.25 ± 0.18 fold, and a significantly decreased Hcy level, by 30.44%±0.06 fold, when compared to those Aβ1−42 treated cells with no VB6 supplementation (Fig. 3a). Furthermore, when detected by Probe 1, we found that VB6 treatment led to significantly increased amounts of fluorescence signal indicating Cys and GSH, and decreased levels of Hcy fluorescence, compared to the Aβ1−42 treated cells with no VB6 (Fig. 3b).
12-month-old APP/PS1 mice were treated with VB6. After 7 days of VB6 (100 mg/kg) intervention, compared with the APP/PS112M+ group with no treatment, the brain levels of Cys and GSH had increased by 1.84 ± 0.27-fold and 1.21 ± 0.14 -fold, and the level of Hcy had decreased by 9.80%±0.02, respectively, in the APP/PS112M + VB6+ group (Fig. 3c). Similarly, the fluorescent signals indicating Cys and GSH were significantly increased, while Hcy signal was decreased in the APP/PS112M + VB6+ group compared to the APP/PS112M+ group (Fig. 3d).
Brain imaging showed that pre-treatment with VB6 (100 mg/kg, 7 d) effectively changed the Epi-fluorescence levels of Cys, GSH, and Hcy in the brain compared of the APP/PS112M+ mice. Cys and GSH levels were increased by 1.80 ± 0.20-fold and 1.25 ± 0.16-fold, respectively, while the level of Hcy was found to be decreased by 27.08%±0.03, in the APP/PS112M + VB6+ group compared to the APP/PS112M+ group (Fig. 3e).