3.1 The neuroprotection effect of SSA and AF
The effect of SSA and AF alone were investigated in PC12 cells. In Fig. 1A/B, 0.312–5 µmol/L of SSA and 3.12–100 µmol/L of AF shown no obvious effects. Therefore, SSA and AF concentration of 0.156, 0.312, 0.625, 1.25, 2.5, 5 µmol/L and 1.56, 3.12, 6.25, 12.5, 25, 50 µmol/L were used to evaluate the neuroprotective effects. As shown in Figure C/D, SSA and AF treatment dose-dependently prevented the decrease in corticosterone-induced cell viability with maximum protective effects at 2.5 µmol/L and 25 µmol/L respectively.
3.2 The synergistic neuroprotection effect of SSA and AF
To examine whether the combination of SSA and AF would have a synergistic effect. To this end, corticosterone-induced PC12 cells were treated with indicated concentrations of SSA and AF, alone and in combination, and the cell viability was analyzed using MTT assay. As shown in Fig. 2A/B/C, CI values were determined using the CompuSyn software. CI < 1.0 for virtually all conditions (except one condition for SSA and AF combination) indicated synergism between SSA and AF, and the optimal synergistic dose was 2.5 µmol/L of SSA and 25 µmol/L of AF. The interaction relationship between SSA and AF was further studied using Combenefit programs by using two “classical” mathematical models (Loewe and HSA). The output is composed of scores that capture information about the synergy distribution and the synergy levels were depicted as surface plots. As shown in Fig. 2D/E/F/G/H/I (D/E/F - Loewe model, G/H/I - HAS model), the synergistic dose range was 1.25–2.5 µmol/L of SSA and 12.5–25 µmol/L of AF. Those three mathematical models agree in the overall evaluation of SSA and AF combination activity against corticosterone-induced neuro-injury in PC12 cells. These results revealed that the combination of SSA and AF has a synergistic effect. Thus, the concentrations of 2.5 µmol/L (SSA) and 25 µmol/L (AF) were chosen in subsequent experiments.
For further evaluation of the neuroprotection effect of SSA and AF, alone and in combination, the LDH assay, Hoechst 33342 staining assay, and Annexin V-FITC/PI flow cytometry assay was implemented. As depicted in Fig. 3A, the cells incubated with corticosterone (400 µmol/L) exhibited an increased level of LDH release compared with the control group (P < 0.01), SSA (2.5 µmol/L), AF (25 µmol/L) pretreatment resulted in a reduction of the LDH release. Moreover, when SSA was combined with AF, significantly decreases in LDH release (p < 0.01). And, the effect of the SSA and AF combination was significantly better than SSA or AF alone (p < 0.01, p < 0.01). Figure 3B/C/D showed that the population of apoptotic cells after corticosterone treatment was significantly increased relative to the control group. However, these changes were markedly reversed by SSA, AF, and SSA and AF combination (p < 0.01, p < 0.01, p < 0.01). Especially that SSA and AF combination treatment dramatically reduced the population of apoptotic cells as compared to the SSA and AF alone (p < 0.01). These results further revealed that the combination of SSA and AF has synergistic effects.
3.3 SSA and AF protected corticosterone-induced neuro-injury by regulating metabolic disorders
The metabolite profile of PC12 cells was obtained by LC-MS in positive and negative ion modes; the total peak intensity chromatograms of the cell sample was shown in Figure S1. The PCA score plots observed that the metabolic distribution of the model group was different from the control group. Besides, the QC group gathered together showed that the instrument was stable (Fig. 4A). The PLS-DA model was validated by the permutation test (Fig. 4B). The R2X (0.633), R2Y (0.997), and Q2 (0.993) showed that the OPLS-DA model established had good quality and predictive performance. The OPLS-DA score plots observed that significant separations between the model and the control group (Fig. 4C). The S-plots of OPLS-DA revealed a variety of metabolites (Fig. 4D). Future, the OPLS-DA score plots observed that all groups were separated. Among them, the drug treatment groups were closer to the control group than the model group, suggesting that the metabolic disturbances induced by corticosterone were reversed after drug treatment. Moreover, the combination group has a better separation effect than an alone agent (Fig. 4E).
According to VIP values (> 1.0) and T-test (P < 0.05), 30 differential metabolites were screened out (Table S1). Among them, 14 increased metabolites and 16 decreased metabolites in the model group. The SSA, AF, and the combination of SSA and AF could regulate 12, 10, and 16 differential metabolites respectively, and the combination has a stronger regulation effect on metabolites than an alone agent (Fig. 4F, Fig. 2S). The hierarchical clustering analysis heatmap observed that the combination group separated from the model group and gathered with the control group (Fig. 4G). As a result, these metabolites could be significantly regulated, and the metabolic disorders were ameliorated in corticosterone-induced PC12 cell injury after drug treatment. These differential metabolites were imported into MetaboAnalyst to explore the potential neuroprotection mechanisms. The metabolic pathways were selected according to the pathway impact Values > 0. The results showed that the combination regulated more metabolic pathways (7) than an alone agent (3 & 5) (Fig. 5A/B/C/D). The TCA cycle, purine metabolism, and glutamate metabolism were selected as the crucial metabolic pathways according to the number of metabolites contained in metabolic pathways and the relationship between metabolic pathways [34] (Fig. 5E).
3.4 SSA and AF protected corticosterone-induced neuro-injury by regulating TCA cycle disorders
To clarify the regulation mechanisms of SSA and AF, alone and in combination, on the TCA cycle, the mitochondrial function was detected. Exposure to corticosterone (400 µmol/L) significantly reduced the level of mitochondrial membrane potential. In contrast, pretreatment with SSA, AF, and SSA and AF combination significantly reversed the phenomenon induced by corticosterone, meanwhile, the effect of the SSA and AF combination was significantly better than SSA or AF alone (Fig. 6A/B/C).
3.5 SSA protected corticosterone-induced neuro-injury by regulating purine metabolism disorders
To evaluate the regulation effects of SSA and AF, alone and in combination, on purine metabolism, the level of xanthine, and the activity of XOD in cells were detected. Exposure to corticosterone (400 µmol/L) significantly elevated the level of xanthine and increased the activity of XOD (P < 0.01, P < 0.01). In contrast, pretreatment with SSA and SSA and AF combination significantly reversed the phenomenon induced by corticosterone, meanwhile, the effect of the SSA and AF combination was significantly better than SSA (Fig. 6D/E). To verify the effect of SSA on purine metabolism, the XOD inhibitor febuxostat (FBX) was applied. The effect of FBX alone was assessed in PC12 cells. As shown in Fig. 3SA, 1–100 nmol/L FBX revealed no obvious effects. Therefore, FBX concentrations of 10, 25, 50, and 100 nmol/L were used to evaluate the effect on corticosterone-induced PC12 cells. As shown in Fig. 3SB, the cell viability was increased in a dose-dependent manner, when treated with 25, 50, and 100 nmol/L FBX (P < 0.05, P < 0.01, P < 0.01). Moreover, SSA had the same effect as FBX to reduce xanthine levels (P < 0.01, P < 0.01) and inhibit XOD activity (P < 0.01, P < 0.01) (Fig. 6D/E). In addition, FBX (100 nmol/L) enhanced the neuroprotective effect of AF (25 µmol/L) and SSA and AF combination (2.5 and 25 µmol/L) (P < 0.05, P < 0.05) (Fig. 6F). The above results indicated that SSA regulated purine metabolism disorders by inhibiting XOD activity.
3.6 AF protected corticosterone-induced neuro-injury by regulating glutamate metabolism disorders
To evaluate the regulation effects of SSA and AF, alone and in combination, on glutamate metabolism, the level of glutamate in cells culture supernatant, and the activity of GLS in cells were detected. Exposure to corticosterone (400 µmol/L) significantly elevated the level of glutamate and increased the activity of glutamate (P < 0.05, P < 0.01). In contrast, pretreatment with AF and SSA and AF combination significantly reversed the phenomenon induced by corticosterone, meanwhile, the effect of the SSA and AF combination was significantly better than AF (Fig. 6G/H). To verify the effect of AF on glutamate metabolism, the GLS inhibitor BPTES was applied. The effect of BPTES alone was assessed in PC12 cells. As shown in Fig. 3SC, 0.25–25 µmol/L BPTES revealed no obvious effects. Therefore, BPTES concentrations of 0.25, 0.5, 1, and 2.5 µmol/L were used to evaluate the effect on corticosterone-induced PC12 cells. As shown in Fig. 3SD, the cell viability was increased in a dose-dependent manner, when treated with 1, and 2.5 µmol/L BPTES (P < 0.05, P < 0.01). Moreover, AF had the same effect as BPTES to reduce glutamate levels (P < 0.01, P < 0.01) and inhibit GLS activity (P < 0.01, P < 0.01) (Fig. 6G/H). In addition, BPTES (25 µmol/L) enhanced the neuroprotective effect of SSA (2.5 µmol/L) and SSA and AF combination (2.5 and 25 µmol/L) (P < 0.05, P < 0.05) (Fig. 6I). The above results indicated that AF regulated glutamate metabolism disorders by inhibiting GLS activity.
3.7 SSA and AF protected corticosterone-induced neuro-injury by inhibiting oxidative stress and inhibiting inflammation
To evaluate the anti-oxidation effect of SSA and AF, alone and in combination, the levels of ROS were detected. Exposure of PC12 cells to corticosterone significantly elevated the level of ROS (P < 0.01). In contrast, treatment with SSA and SSA and AF combination significantly reversed the phenomenon induced by corticosterone (P < 0.01, P < 0.01), meanwhile, the effect of the SSA and AF combination was significantly better than SSA (P < 0.01) (Fig. 7A/B/C/D). To evaluate the anti-inflammatory effect of SSA, AF, and SSA and AF combination, the level of IL-1β, IL-6, and TNF-α were detected. Exposure of PC12 cells to corticosterone significantly elevated the level of IL-1β (P < 0.01), IL-6 (P < 0.01), and TNF-α (P < 0.01). In contrast, pretreatment with SSA, AF, and SSA and AF combination significantly reversed the phenomenon induced by corticosterone (P < 0.01, P < 0.01, P < 0.01), meanwhile, the effect of the SSA and AF combination was significantly better than SSA or AF alone (P < 0.01, P < 0.01) (Fig. 7D/E/F). To clarify the mechanism of SSA, AF, and SSA and AF combination on anti-inflammatory, the expression of NLRP3 was assessed. Figure 7G/H/ showed that corticosterone treatment significantly upregulated the expression of NLRP3. However, pre-treatment with SSA, AF, and SSA and AF combination blocked these effects, meanwhile, the effect of the SSA and AF combination was significantly better than SSA or AF alone (P < 0.01, P < 0.01).