Fig 2: Endoplasmic reticulum stress was induced in hippocampal tissue of SAE mice. (A) Western blot and (B, C) western blot analysis for GRP78 (df = 35. *p = 0.0372, 6 h versus sham; **p = 0.0014, 1d versus sham; *p = 0.0267, 2d versus sham, n = 6) and T980p-PERK/PERK (df = 35. **p = 0.0019, 1d versus sham; *p = 0.0100, 2d versus sham; *p = 0.0370, 3d versus sham, n = 6) expression in hippocampus after CLP surgery. An internal control was GAPDH. One-way ANOVA with Dunnett's multiple comparisons test was used for statistical analysis. (D) Immunofluorescence staining of p-PERK in the hippocampus CA1 region neurons 1 day after CLP. Magnification, 40 × Scale bar, 40 µm and (E) statistical results of fluorescence intensity of p-PERK in hippocampus (df = 10. **p = 0.0010, n = 6). **p < 0.01 versus Sham. The student's t-test was used for statistical analysis.

Fig 3: Effects of L-proline co-administration on the expression of ER stress/UPR genes in AZE-treated BV2 microglia. Gene expression was measured using real-time qPCR and quantified using the ?Ct method after normalization to the S18 housekeeping gene. Bar graphs show the differential expression of the following upstream and downstream regulators of the ER stress/UPR response: ATF6, PERK (also known as EIF2AK3), IRE1a (also known as ERN1), ATF4, GADD34, XBP1 and DDIT3. Relative expression levels of (A) ATF6, (B) PERK, (C) IRE1a, (D) ATF4, (E) GADD34, (F) XBP1 and (G) DDIT3 transcripts were measured in AZE-treated cells in the absence (Aze) or presence of L-proline (Aze + L-Proline) at various times (1, 6 and 24 h, respectively). Results are presented as mean fold changes in controls ± SEM of three independent experiments, each run using two biological replicates per experiment (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 or **** p < 0.0001 vs. control, as determined by repeated measures ANOVA followed by Tukey post hoc test. Aze = L-azetidine-2-carboxylic acid; ATF6 = Activating transcription factor 6; PERK = protein kinase R-like endoplasmic reticulum kinase (also known as EIF2AK3 = eukaryotic translation initiation factor 2-alpha kinase 3); IRE1a = inositol-requiring transmembrane kinase/endoribonuclease 1a (also known as ERN1 = endoplasmic reticulum to nucleus signaling 1); ATF4 = activating transcription factor 4; GADD34 = growth arrest and DNA damage-inducible protein (also known as PPP1R15A = protein phosphatase 1 regulatory subunit 15A); XBP1 = X-box binding protein 1; DDIT3 = DNA damage inducible transcript 3 (also known as CHOP = C/EBP homologous protein).

Fig 4: Effects of AZE treatment on the expression of ER stress/UPR genes. Gene expression was measured using real-time qPCR and quantified using the ?Ct method after normalization to the S18 housekeeping gene. Bar graphs show the differential expressions of the following upstream and downstream regulators of the ER stress/UPR response: ATF6, PERK (also known as EIF2AK3), IRE1a (also known as ERN1), ATF4, GADD34 (also known as PPP1R15A), XBP1 and DDIT3 (also known as CHOP). A schematic diagram showing the main ER stress/UPR pathways and biological outcomes (apoptosis/autophagy or survival) are shown on the right. Relative expression levels of (A) ATF6, (B) PERK, (C) IRE1a, (D) ATF4, (E) GADD34, (F) XBP1 and (G) DDIT3 transcripts were measured in untreated (control) and AZE-treated BV2 microglial cells at various times (1, 6 and 24 h, respectively). Results are presented as mean fold changes in controls ± SEM of three independent experiments, each run using two biological replicates per experiment (n = 6). * p < 0.05, ** p < 0.01 or *** p < 0.001 vs. control, as determined by repeated measures ANOVAs followed by Tukey post hoc tests. Aze = L-azetidine-2-carboxylic acid; ATF6 = activating transcription factor 6; PERK = protein kinase R-like endoplasmic reticulum kinase (also known as EIF2AK3 = eukaryotic translation initiation factor 2-alpha kinase 3); IRE1a = inositol-requiring transmembrane kinase/endoribonuclease 1a (also known as ERN1 = endoplasmic reticulum to nucleus signaling 1); ATF4 = activating transcription factor 4; GADD34 = growth arrest and DNA damage-inducible protein (also known as PPP1R15A = protein phosphatase 1 regulatory subunit 15A); XBP1 = X-box binding protein 1; DDIT3 = DNA damage inducible transcript 3 (also known as CHOP = C/EBP homologous protein); RIDD = regulated IRE1a-dependent decay; JNK = c-Jun N-terminal kinase.

Fig 5: Effects of AZE treatment on PERK expression are prevented upon L-proline co-administration in BV2 microglia. (A) Representative photomicrographs showing PERK immunofluorescence (IF) and (B) related IF quantification of untreated cells (control) or following exposure to AZE (1000 µM), L-proline (50 µM) or their combination after 24 h. Images were taken on an Olympus BX51 Fluorescence Microscope using the 20× objective. Scale bar = 60 µm. (B) PERK immunofluorescence intensity was determined using NIH ImageJ version 1.52. At least five independent images (n = 5) were analyzed for each experimental condition. Results shown are the mean ± SEM. ** p < 0.01 vs. control or # p < 0.05 vs. AZE. Statistical significance was computed using a one-way ANOVA, followed by Tukey post hoc test. IF = immunofluorescence; Aze = L-azetidine-2-carboxylic acid; PERK = protein kinase R-like endoplasmic reticulum kinase.