Fig 2: GSTM2 alleviates oxidative stress and mitochondrial defects in DACD models. (A) Representative images of the morphology of astrocytes mitochondria by TEM in hippocampus. Scale bar, 1.0 μm. (B) Mitochondrial aspect ratio, n = 3. (C) Mitochondrial cristae density, n = 3. (D-F) MDA, GSH and SOD contents in hippocampus. n = 5. (G) Western blotting of GSTM2 protein expression in primary cultured astrocytes, and quantification data. n = 3. (H) Representative images of ROS content by flow cytometry analysis, and quantification data. n = 5. (I–K) MDA, GSH and SOD contents in primary cultured astrocytes. n = 5. (L) Representative images of the morphology of primary astrocytes mitochondria by TEM. Scale bar, 500 nm. (M) Mitochondrial aspect ratio, n = 3. (N) Mitochondrial cristae density, n = 3. (O) Western blotting of Mfn1, p-Drp1 and Drp1 protein levels in primary cultured astrocytes, and quantification data. n = 3. (P, Q) Representative images of JC-1 staining by flow cytometry analysis used to assess mitochondrial membrane potential. (R–U) OCR, ATP production, Basal respiration and Maximal respiration in primary cultured astrocytes. n = 5. rAAV-NC (rAAV-GFAP-EGFP-WPRE-hGH pA); rAAV-GSTM2 (rAAV-GFAP-GSTM2-EGFP-WPRE-hGH pA). Vector (pEGFP-C1 plasmid empty vector); OE-GSTM2 (pEGFP-C1-GSTM2 plasmid vector). HG (High glucose, 50 mmol/L treated 48 h). Data are shown as mean ± SEM or SD. Statistical significance was defined as ∗P < 0.05, ∗∗P < 0.01 and ∗∗P < 0.001.

Fig 3: Icariin improves cognition through the GSTM2/STAT3 axis in DACD mice models.(A) Experimental paradigms. (B) Schematic representation of the virtual screening strategy utilized to discover potential small-molecule binding to GSTM2 protein. (C) Binding affinities of Tubuloside A, Icariin and Militarine by docking scores. (D) Visualization of the predicted binding pose of Tubuloside A docked to the GSTM2 protein. (E) Visualization of the predicted binding pose of Icariin to the GSTM2 protein. (F) Visualization of the predicted binding pose of Militarine to the GSTM2 protein. (G) Western blotting of p-STAT3(Ser727), STAT3 and GSTM2 protein levels in hippocampus, and quantification data. n = 3. (H) The represents of the exploration trajectory of db/m and db/db mice in the NOR test. (I) The preference index in the NOR test. n = 10. (J) The represents of the swimming trajectories in the MWM test. (K-M) The escape latency, across platform times and target quadrant retention time. n = 10. (N) Representative images of the morphology of astrocytes mitochondria by TEM in hippocampus. Scale bar, 500 nm. (O) Mitochondrial aspect ratio, n = 3. (P) Mitochondrial cristae density, n = 3. (Q-S) MDA, GSH and SOD contents in hippocampus. n = 5. Vehicle (gavage, 20% PEG400 solution); Icariin (gavage, 100 mg/kg/day). Data are shown as mean ± SEM. Statistical significance was defined as ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Fig 4: GSTM2 expression in astrocytes is significantly downregulated in the hippocampus of DACD mice models. (A) Experimental paradigms. (B) The represents of the exploration trajectory of db/m and db/db mice in the NOR test. (C) The preference index in the NOR test. n = 10. (D) The represents of the swimming trajectories in the MWM test. (E-G) The escape latency, across platform times and target quadrant retention time. n = 10. (H) Cluster analysis of proteomic sequencing. (I) Volcano plot volcanic map of proteomic sequencing. Red dots are raised, and green dots are decreased. Gstm2 was downregulated in hippocampus of db/db mice compared to db/m mice. (J) KEGG pathway enrichment analysis showed Glutathione metabolism was the key metabolic pathways. (K) Representative images of the cellular localization and expression of GSTM2 by immunofluorescence in astrocytes (GFAP), microglia (Iba-1), neurons (NeuN), oligodendrocytes (MBP) and nucleus (DAPI) of the CA1 region in hippocampus. Scale bar, 50 μm. And quantification data. n = 3. (L) Western blotting of GSTM2 protein expression in hippocampus, and quantification data. n = 3. (M) Representative images of the localization and expression of GSTM2 by immunofluorescence in HG primary cultured astrocytes (50 mmol/L concentration glucose treated for 48 h) compared to control. Scale bar, 50 μm. And quantification data. n = 3. (N) Western blotting of GSTM2 protein expression in primary cultured astrocytes, and quantification data. n = 3. HG (High glucose, 50 mmol/L treated 48 h). Data are shown as mean ± SEM. Statistical significance was defined as ∗∗P < 0.01 and ∗∗P < 0.001.

Fig 5: GSTM2 suppresses the phosphorylation of STAT3. (A) Experimental paradigms. (B) Top 15 list of GSTM2-binding proteins identified through GSTM2 IP-MS analysis in primary cultured astrocytes. STAT3 was identified with high level of interaction intensity. (C) Detection of the binding affinity between GSTM2 and STAT3 by SPR method. (D-E) Co-IP analysis for validation of the endogenous interaction between GSTM2 and STAT3 in primary cultured astrocytes. (F) Representative images of the cellular localization and expression of GSTM2 and STAT3 by immunofluorescence in primary cultured astrocytes. Scale bar, 50 μm. GSTM2 (red), STAT3 (green) and DAPI (blue). (G) Western blotting of p-STAT3(Ser727), STAT3 and GSTM2 protein levels in primary cultured astrocytes, and quantification data. n = 3. si-NC (scrambled negative control siRNA); si-GSTM2 (si-GSTM2 primer). Vector (pEGFP-C1 plasmid empty vector); OE-GSTM2 (pEGFP-C1-GSTM2 plasmid vector). HG (High glucose, 50 mmol/L treated 48 h). Data are shown as mean ± SEM. Statistical significance was defined as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.