*3.5. PAPZ Protected Neurons In Vitro*

The HT22 cell line is a widely used model to evaluate the pharmacological effects of potential antidepressant drugs [37,38]. To detect the effect of PAPZ on CORT-induced HT22 cells death (Figure 5), we selected 400 μM CORT, which decreased the viability of the HT22 cells by 30% (Supplementary Figure S1), as the optimal dose of the cell model to detect the effect of PAPZ on CORT-induced HT22 cells death. At these experimental conditions, the viability of the HT22 cells was significantly reduced in the CORT group when compared to the control group. Co-treatment with PAPZ increased the viability of HT22 cells in a dose-dependent manner when compared with CORT-only treated HT22 cells. The viability of HT22 cells in the PAPZ+CORT treated group was significantly higher than that in the CORT group, which indicated that PAPZ promoted the cell proliferation of HT22 cells (Figure 5a).

**Figure 5.** PAPZ attenuated CORT-induced apoptosis in HT22 cells. (**a**) Effects of different concentrations of PAPZ on CORT-induced HT22 cell viability determined by cell counting kit-8 (CCK-8 assay). (**b**) Fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI) staining followed by flow cytometry was performed to evaluate cell apoptosis of the HT22 cells. Living cells can be categorized as double negative (Q1). Early apoptotic cells can be classified as single positive or FITC-Annexin V positive (Q2). Late apoptotic cells are double positive (Q3). Dead cells can be categorized as single positive or PI positive (Q4). (**c**) The statistical analysis of total apoptosis in flow cytometry test. Data from the CORT group and PAPZ+CORT group were normalized to the control group and data are presented as mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 represent significant differences.

Flow cytometry analysis using conventional FITC-Annexin V and PI staining was performed to characterize three types of neuronal death. Total apoptosis in HT22 cells treated with CORT showed a significant increase when compared with the control group. PAPZ treatment decreased the percentage of both early and late apoptosis in HT22 cells when compared to the CORT group (Figure 5b). The statistical analysis of total apoptosis is provided in Figure 5c. The results suggested that HT22 cells were likely to undergo apoptotic rather than necrotic death when treated with CORT. Treatment with PAPZ could prevent against apoptosis induced by CORT. Our results suggested that PAPZ exerted a neuroprotective effect through inhibiting the apoptosis of HT22 cells induced by CORT.
