S-9-PAHSA ameliorates cognitive decline in a Type 2 Diabetes mouse model by inhibiting oxidative stress and apoptosis via CAⅢ modulation

Xin-Ru Wang^1,2,3Shan-Shan Huang¹Meng Wang¹Jin-Hong Lin⁴Jian-Tao Wang¹Jiao-Qi Ren¹Cheng-Feng He²Wen-Jiao Xue²Yin Wang¹Xue-Chun Wang¹Yan-Li Zhang¹Ji-Chang Xiao^4*Jing-Chun Guo^2*Hou-Guang Zhou^1*

• ¹Huashan Hospital, Fudan University, Shanghai, China
• ²Fudan University Institutes of Brain Science, Shanghai, China
• ³Qilu Hospital of Shandong University, Jinan, China
• ⁴Chinese Academy of Sciences Shanghai Institute of Organic Chemistry, Shanghai, China

Introduction Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by hyperglycemia. Diabetes-related cognitive disorder (DRCD) is one of the common complications of diabetes that attracts widespread attention (1). Evidence indicates that diabetes is an independent risk factor for cognitive impairment, with both prediabetes and diabetes accelerating cognitive deterioration (2). Increasing research suggests that multiple neuropathologic mechanisms may be involved in the development and progression of DRCD (3). Inhibition of neurogenesis, electrophysiological deficits, oxidative stress, and neuronal apoptosis induce structural changes and participate in dysfunction in the brains of individuals with diabetes (4), contributing to cognitive function decline. The subsequent deterioration of patients' self-care ability adversely affects their quality of life, imposing huge burdens on both families and society. However, no targeted clinical therapies for DRCD have been established yet (5). Palmitic acid esters of hydroxy-stearic acids (PAHSAs), a recently identified class of fatty acids, have demonstrated the capacity to enhance insulin sensitivity and possess anti-inflammatory properties (6). 9-PAHSA is the predominant isomer in serum in both insulin-resistant and insulin-sensitive humans. It is also the most abundant isomer in subcutaneous adipose tissue of humans and mice (7). Prior studies have shown that S-9-PAHSA (SP) can enhance glucose-stimulated insulin secretion (GSIS) and glucose uptake (6), suggesting a potential benefit in diabetic complications. The impact of SP on DRCD, however, remains to be elucidated. Carbonic anhydrases (CAs), a class of zinc-containing metalloproteases, are ubiquitously found in prokaryotes and eukaryotes, with diverse isoforms localized across cellular compartments such as the cell membrane, cytoplasm, and mitochondria (8, 9). Carbonic anhydrase Ⅲ (CAⅢ), as one of the isoenzymes, is widely expressed in the brain and is highly expressed in the cytoplasm in neurons. It exhibits low catalytic activity for CO2 hydration, suggesting additional roles in the brain beyond its catalytic function (10, 11). The role of CAⅢ in cognitive disorders and its underlying mechanisms warrant further investigation, as current CA inhibitors do not selectively target specific isoforms (12, 13). In the present study, in vivo and in vitro experiments were performed to explore the impacts of SP on DRCD and the potential involvement of CAⅢ. Exploring the pathogenesis and identifying novel targets for DRCD may pave the way for innovative clinical interventions. Materials and methods 2.1 Animal and experimental procedure Male C57BL/6 mice (6-8 weeks old) were procured from Shanghai Lingchang Biotechnology and housed under a controlled temperature (21-23°C) with a 12-hour light/dark cycle. All the animal protocols were performed according to the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (NIH) and were approved by the Animal Welfare and Ethics Group of the Department of Laboratory Animal Science of Fudan University (2020-Huashan hospital-JS190). Cohort 1: Mice (6-8 weeks old) were assigned to either a normal diet (ND, n=15) or high-fat diet (HFD, n=30) for five months. Glucose levels were measured, and mice with fasting blood glucose ≥8.0 mmol/l were classified as T2DM. (Roche, Switzerland). T2DM mice were randomly assigned to either the HFD (n=14) or SP (n=14) group, receiving either a vehicle (50% PEG-400 + 0.5% Tween-80 + 49.5% H2O) (14) or SP (30 mg/kg/d) in drinking water for one month, respectively. The groups were designated as ND, HFD, and SP. Cohort 2: Mice (6-8 weeks old) were fed either ND (n=15) or HFD (n=80) for five months. HFD mice randomly received bilateral hippocampal injections of AAV carrying either CAⅢ shRNA (HFD+AAV-CAⅢ sh, n=40) or control shRNA (HFD+AAV-Con, n=40) via stereotaxic surgery. CAⅢ knockdown was confirmed by real-time PCR and western blotting three weeks post-injection. Mice that received either AAV injection were subsequently randomized to receive either vehicle or SP (30 mg/kg/d) in drinking water for one month. Body weight, fasting blood glucose, and daily water/food intake were monitored (Figure S1). 2.2 Fasting blood glucose (FBG), intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) Mice were fasted for 12 hours to test fasting blood glucose (FBG). For the intraperitoneal glucose tolerance test (IPGTT), mice underwent a 14-hour fast followed by an intraperitoneal injection of 20% glucose (2 mg/kg body weight) in PBS. Blood glucose levels were measured at 0, 30, 60, 90, and 120 minutes post-injection, and the area under the curve (AUC) was calculated. For the intraperitoneal insulin tolerance test (IPITT), mice were fasted for 5 hours before receiving an intraperitoneal insulin injection (1 U/kg body weight, Novo Nordisk, Denmark). Blood glucose was measured at 0, 30, 60, 90, and 120 minutes after injection, and AUC was calculated. 2.3 The synthesis of S-9-PAHSA (SP) SP was synthesized by the Institute of Organic Chemistry, Chinese Academy of Sciences based on a previous study (15). The molecular structures of 9-PAHSA and SP are illustrated in Figure 1A. 2.4 Morris water maze (MWM) The Morris water maze (MWM) was used to evaluate mouse spatial learning and memory. The latency on the training days (d 1 to d 5) was defined as the first time to reach the platform within 60 seconds. Mice unable to find the platform within 60 seconds were gently guided to the platform for 15 seconds. On day 6 (probe trial day), the platform was removed, with trajectories recorded by the video and analyzed by EthoVision software (version 8.5). Y maze was utilized to assess the short-term spatial working memory of mice. Mice were positioned at the end of the designated arm (start arm) and allowed to explore freely for 8 minutes. The course was recorded via video, with subsequent analysis performed by EthoVision software (version 14.0). The measurement of SP in tissues Concentrations of SP in tissues were determined using ultra-high performance liquid chromatography (UPLC-MS/MS). The procedure was conducted by Maixi Medical Technology Co. according to the instructions. Histology and immunohistochemistry Mice from four groups (n=4 per group) were perfused with PBS followed by 4% paraformaldehyde. The brains were postfixed in the same fixative overnight at 4°C. After progressively dehydrated, embedded in paraffin, and sliced into 4 μm-thick sections, brain morphology was observed by Hematoxylin and Eosin (HE) and Nissl staining as previously described (16). Sections were incubated with a primary antibody against neuronal nuclei (NeuN, 1:200 dilution, Beyotime, China) at 4 °C overnight, followed by PBS washing. Then, a Cy3-conjugated secondary antibody (1:500, Servicebio, China) was applied for 1 hour at room temperature. Fluorescence was detected under a fluorescence microscope (Nikon, Japan). Enzyme-Linked Immunosorbent Assay (ELISA) LDH (Beyotime, China), superoxide dismutase (SOD, Dojindo, Japan), catalase (CAT), glutathione peroxidase (GSH-Px), and hydrogen peroxide (H2O2) concentrations were measured by respective ELISA kits (Nanjing Jiancheng Biological Engineering Research Institute, China), following the provided protocols. Hippocampal CAⅢ gene knockdown in mice Recombinant AAV vectors encoding CAⅢ shRNA (5'-GGTTCACTGGAATCCAAAGTA-3') or non-specific control shRNA (5'- CGCTGAGTACTTCGAAATGTC-3') were synthesized by Shanghai Genechem Co., Ltd. Mice were anesthetized with 1.2% isoflurane and placed on a stereotaxic apparatus (RWD, China) with a heating pad to maintain body temperature at 37 °C. A cranial incision was created by an electric drill (RWD, China), followed by the precise injection of 2×10^9 v.g. AAV into each hippocampus (AP: −1.85 mm, ML: ±1.60 mm, DV: −1.90 mm) at 0.08 μl/min via a glass micropipette. The syringe (Gaoge, China) remained for 10 minutes before being slowly withdrawn. After bilateral injections, the incision was sutured, and the mice were kept warm until recovery. PC12 cells, obtained from Shanghai Fuheng Biotechnology, were authenticated before use through standard cell line identification methods. The CAⅢ knockdown (CAⅢ sh), CAⅢ overexpressing (CAⅢ OE), and negative control (NC) PC12 cells were transfected with lentiviral vectors. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml puromycin, and maintained in a humidified 5% CO2 incubator at 37°C. Previous studies indicated that PC12 cell growth was inhibited by elevated glucose and palmitic acid levels. A combination of 100 mM glucose and 200 μM palmitic acid (G100F200) was identified as the threshold for significant growth inhibition, establishing an in vitro diabetes model. The CAⅢ sh, CAⅢ OE, and NC cells were exposed to four conditions for 24 hours: normal culture medium (Con group), high-glucose/high-lipid medium (G100F200 group), solvent control (DMSO group), and 60 μM SP (SP group). 2.11 RNA sequencing (RNA-seq) Total RNA was extracted from PC12 cells using the RNA Extraction Kit (Takara, Japan) according to the manufacturer's protocol. RNA quantity and integrity were evaluated with a NanoDrop ND-2000/Qubit 2.0, and Agilent 4200 TapeStation (Agilent Technologies, USA). Only high-quality samples were used for transcriptome sequencing. Double-stranded cDNA libraries were constructed using the TruSeq® RNA Sample Preparation Kit (Illumina, USA), following the recommended protocol. In brief, mRNA containing polyadenylated tails was enriched using magnetic beads conjugated with poly-T oligonucleotides. The resulting libraries were quantified with a Qubit® 2.0 Fluorometer (Life Technologies, USA), and library quality—including fragment size and molar concentration—was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Sequencing clusters were generated on a cBot system after diluting the libraries to 10 pM, and paired-end sequencing was performed on the Illumina HiSeq X Ten platform (Illumina, USA). All steps related to library preparation and sequencing were conducted at Shanghai Biotechnology Corporation. Raw sequencing reads were initially subjected to quality control, during which rRNA sequences, adapter contaminants, short fragments, and low-quality reads were removed. Clean reads were then aligned to the human reference genome (GRCh38) using HISAT2 (version 2.0.4) (17), allowing up to two mismatches. Transcript assembly and quantification were performed with StringTie (version 1.3.0) (18, 19) using the corresponding gene annotation to calculate FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values for annotated genes. Differential gene expression was assessed using edgeR (20), followed by functional annotation using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses. The significance threshold for multiple comparisons was controlled using the false discovery rate (FDR) method. Fold change values were calculated based on FPKM levels in individual samples. Genes were considered differentially expressed if they met both criteria: FDR≤0.05 and fold change≥2. Data processing was conducted using R software (version 4.3.1). 2.12 Cell viability assay Cell viability was measured by CCK-8 assay kit (Dojindo, Japan). The CAⅢ sh, CAⅢ OE, and NC PC12 cells were plated at 4000 cells/well in a 96-well plate and cultured for 24 hours. They were then treated with normal culture medium (Con group), high-glucose and high-fat culture medium (100 mM glucose +200 μM palmitic acid, G100F200 group), solvent control (DMSO group), and 60 μM SP (SP group) for 24 hours. Following treatment, 10 μl of CCK8 reagent was added to each well and incubated for 2 hours in the dark. The absorption was determined at 450 nm using a microplate reader (Biotek, USA). 2.13 Flow cytometry Apoptosis was analyzed by flow cytometry using dual staining with Annexin V-FITC (to detect early apoptotic cells) and propidium iodide (PI) (to identify late apoptotic and necrotic cells) (Thermo Scientific, USA). The quadrants were interpreted as follows: Q1 (upper left): PI⁺/Annexin V⁻ (necrotic cells), Q2 (upper right): PI⁺/Annexin V⁺ (late apoptotic cells), Q3 (lower right): PI⁻/Annexin V⁺ (early apoptotic cells), and Q4 (lower left): PI⁻/Annexin V⁻ (viable cells). 2.14 Measurement of mitochondrial membrane potential and mitochondrial ROS Mitochondrial membrane potential was assessed by TMRE (tetramethylrhodamine ethyl ester) staining in PC12 cells seeded at 5000 cells/well on 12-well plates with coverslips and incubated for 24 hours. Following treatment with normal culture medium, G100F200, DMSO, or SP, cells were incubated with TMRE working solution (Beyotime, China) for 10 minutes. Cells were then stained with 10 μg/ml Hoechst 33258 (Yeasen, China) for 10 minutes. Fluorescent images were captured using a fluorescence microscope (Nikon, Japan). Mitochondrial ROS levels were measured using MitoSOX Red (Thermo Scientific, USA). After treatment with the respective media, cells were incubated with 5 μM MitoSOX for 10 minutes, followed by 0.1 μM Mito-Tracker Green (Beyotime, China) for 30 minutes and 10 μg/ml Hoechst 33258 for 10 minutes. After washing with HBSS, fluorescence was analyzed under a fluorescence microscope (Nikon, Japan). 2.15 RNA extraction and real-time PCR Total RNA from the brain and the cells were extracted using an RNA Extraction Kit (Takara, Japan) followed by the manufacturer's instructions. The cDNA was synthesized from 1000 ng RNA using the 1st Strand cDNA Synthesis SuperMix (Yeasen, China). mRNA levels were quantified via SYBR Green (Yeasen, China) real-time PCR and normalized to β-actin using the 2−∆∆Ct method. Primer sequences: mouse CAⅢ (F 5'- GAATCTCAGCACTCCTACTTTCA-3'; R 5'-GTCCGCATACTCCTCCATAC-3'), mouse β-actin (F 5'-CCTCTATGCCAACACAGT-3'; R 5'-AGCCACCAATCCACACAG-3'), rat CAⅢ (F 5'-TGCCGGGACTATTGGACCTA-3'; R 5'-ATTCTCTGCACTGGCGAACA-3'), rat β-actin (F 5'-CCTCTATGCCAACACAGT-3'; R 5'-AGCCACCAATCCACACAG-3'). 2.16 Western blotting Proteins from the brains and the cells were extracted using radioimmunoprecipitation assay (RIPA) buffer as previously described (1). Protein concentrations were determined by BCA method (Thermo Scientific, USA). Proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA), and probed with anti-CAⅢ (1:500; Santa Cruz, USA), anti-p-AMPK (1:1000; Cell Signaling, USA), anti-AMPK (1:1000; Cell Signaling, USA), anti-Sirt1 (1:1000; Abcam, UK), anti-PGC-1α (1:1000; Cell Signaling, USA), anti-Bcl-2 (1:1000; Cell Signaling, USA), anti-Bax (1:1000; Cell Signaling, USA), cleaved-Caspase 3 (1:1000; Cell Signaling, USA), Bcl-xl (1:1000; Cell Signaling, USA), and anti-β-actin (1:2000; Cell Signaling, USA) antibodies. ECL (Bio-Rad, USA) was used for imaging, and the protein bands were analyzed using Image J software. 2.17 Statistical analysis Data were described as mean ± standard error of mean (SEM). Statistical comparisons were made using Student's t-test for two-group analysis and one-way ANOVA for multiple groups. Analysis and graphing were conducted with GraphPad Prism 9.0, EthoVision, Image J, and R software. P<0.05 was considered statistically significant. Results 3.1 SP attenuated hippocampal neuronal damage in diabetic mice The structure of 9-PAHSA and S-9-PAHSA (SP) are depicted in Figure 1A, and the experimental timeline is shown in Figure 1B. The hippocampus is an essential region for memory and the loss of hippocampal neurons is the main pathological feature of cognitive impairment. HE staining revealed that the ND group displayed clearly, normally arranged neurons with evenly distributed basophilic granules (Nissl bodies) (Figure 1C-D). Conversely, HFD-fed mice exhibited disorganized neurons in the CA1 and CA3 regions, with irregular morphology, reduced basophilic granules, highly eosinophilic cytoplasm and dense black basophilic nuclei, indicative of neuronal damage. SP treatment ameliorated this injury in diabetic mice, as evidenced by improved cellular organization (Figure 1C-G). Nissl staining confirmed that the significant Nissl body reduction in the HFD group was significantly reduced, suggesting neurodegeneration, which was attenuated by SP (Figure 1D-G). The results implied that SP might rescue the HFD-induced hippocampal neuronal damage. 3.2 SP ameliorated working memory in T2DM mice through Bcl-2/Bax and AMPK/Sirt1/PGC1α pathway Y maze test was used to assess working memory, and representative trajectories are shown in Figure 2A. HFD-fed mice showed reduced total distance, movement velocity, and alternation percentage compared to ND-fed mice, while SP treatment reversed these deficits (Figure 2B-D, all P<0.05). This indicated that SP enhanced working memory in diabetic mice. Next, we found that SP increased Bcl-2 (Figure 2E-F, P<0.01) and Bcl-xl (Figure 2I-J, P>0.05) expression, decreased Bax expression (Figure 2G, P>0.05) and cleaved-Caspase 3 (cl-casp3) (Figure 2K-L, P>0.05), and remarkably elevated the Bcl-2/Bax ratio (Figure 2H, P<0.05) in the hippocampus, attenuating neuronal apoptosis in the hippocampus. In addition, AMPK phosphorylation was significantly diminished in diabetic mice (Figure 2M-P, P<0.01). SP intervention rescued the inhibition of AMPK phosphorylation in the HFD group (Figure 2M-N, P<0.05), significantly increased downstream Sirt1 expression (Figure 2O, P<0.05), and slightly elevated PGC1α expression (Figure 2P, P>0.05). 3.3 Effects of SP on serum and hippocampal oxidative stress Mice on a high-fat diet showed significantly increased body weights (44.51±5.06 g vs. 30.97±1.19 g) and fasting blood glucose levels (9.82±1.39 mmol/l vs. 5.95±0.98 mmol/l) compared to those on a normal diet (Figure 3A-B, all P<0.0001). Of the 30 HFD-fed mice, 28 had fasting glucose levels exceeding 8.0 mmol/l, resulting in a 93.3% success rate for diabetes modeling. These findings confirm that chronic HFD feeding substantially elevated both body weight and blood glucose. In HFD-fed mice treated with SP, serum and brain SP levels were increased by 34.8% and 21.7%, respectively, while levels in the small intestine and colon were reduced by 36.7% and 83.6%, respectively (Figure 3C-F, P>0.05). The results suggested that SP is absorbed through the intestine and can reach the brain. Lactate dehydrogenase (LDH), a key marker of tissue damage, showed significantly increased activity in the serum of HFD mice compared to ND controls, which was attenuated by SP treatment (Figure 3G). Additionally, hippocampal reactive oxygen species (ROS) levels were lower in SP-treated mice compared to the HFD group (Figure 3K). In both serum and hippocampus, antioxidant enzyme activities of SOD and CAT were reduced in the HFD group but were elevated by SP treatment (Figure 3H-I, L-M). SP also tended to increase GSH-Px activity (Figure 3J, N), although it had no effect on H2O2 (Figure 3O). These results suggest that SP may alleviate oxidative stress and exert protective effects. 3.4 CAⅢ knockdown had no significant effects on body weight and fasting blood glucose in diabetic mice We observed a reduction in CAⅢ expression in the hippocampus of HFD-fed mice, whereas treatment with SP effectively mitigated the HFD-induced downregulation of CAⅢ (Figure S2A-B, P<0.01), suggesting that CAⅢ may be involved in the protective effects of SP. To further investigate this possibility, we performed bilateral hippocampal knockdown of CAⅢ in T2DM mice following the protocol (Figure 4A). HFD mice were bilaterally injected with AAV-Con sh or AAV-CAⅢ shRNA. After three weeks, mRNA and protein expression of CAⅢ in the hippocampus, cortex, and remaining brain regions (excluding the hippocampus and cortex) were significantly reduced in the AAV-CAⅢ sh group compared to the AAV-Con sh group (Figure 4B-G, S3A-C). These findings confirm the effective knockdown of CAⅢ in these brain areas following AAV-CAⅢ shRNA injection. AAV injection had no observable effect on weekly body weight or fasting blood glucose levels in any group (Figure S3D-E). Weekly body weight and fasting blood glucose of HFD-fed mice were significantly higher compared to ND-fed mice, with no significant variations observed within the HFD group (Figure 4H-I). Daily water intake and food intake showed no difference across all groups (Figure 4J-K, P>0.05). In IPGTT, blood glucose peaked 30 minutes post-injection, with HFD-fed mice showing higher levels than ND-fed mice at all time points, but no AUC differences among HFD-fed groups (Figure 4L-M). IPITT revealed no significant differences in insulin sensitivity among HFD-fed groups (Figure 4N-O). These results indicated that SP had no significant effect on glucose tolerance and insulin sensitivity. CAⅢ knockdown abolished the improvement of SP on diabetic mice cognition Y-maze test showed that compared to the ND group, the total distance (P>0.05), movement velocity (P>0.05), and alternation percentage (P<0.05) decreased in the HFD group and HFD+AAV-Con+vehicle group (Figure 5A-D). SP administration improved cognitive performance (Figure 5D, P<0.01), except for no change in alternation percentage between HFD+AAV-CAⅢ+vehicle and HFD+AAV-CAⅢ+SP groups. It suggested that SP ameliorates spatial memory in diabetic mice, with CAⅢ knockdown in the hippocampus negating this benefit. The trajectories of MWM test are shown in Figure 5E. HFD-fed mice demonstrated increased latency compared to ND controls across training days (Figure 5F). On the probe trial day, the escape latency elevated in the HFD and the HFD+AAV-Con+vehicle group, which was remarkably reduced by SP treatment, but this effect was abolished with CAⅢ knockdown (Figure 5G, P<0.05). HFD group and the HFD+AAV-Con+vehicle group also showed significant fewer platform crossings and decreased time in the platform and the target quadrant versus the ND group, improvements that were observed with SP administration (Figure 5H-J, P<0.05). No obvious difference in total distance and velocity were noted among groups (Figure 5K-L, P>0.05). These findings further confirmed that CAⅢ may mediate the protective effect of SP on cognitive function of diabetic mice. SP reduced ROS and ameliorated neuronal damage in mouse hippocampus via CAⅢ-mediated Bcl-2/Bax and AMPK/Sirt1/PGC1α Hippocampal ROS levels were significantly elevated in the HFD and HFD+AAV-Con+vehicle groups but were notably reduced with SP treatment. This SP-induced reduction in ROS was attenuated in CAⅢ knockdown mice (Figure 6A). SP also enhanced SOD activity and CAT, effects not seen in CAⅢ knockdown mice (Figure 6B-C). A similar trend was also found in GSH-Px (Figure 6D, P>0.05), while SP had no significant effect on H2O2 (Figure 6E, P>0.05). These findings indicate that SP's neuroprotection may involve CAⅢ-mediated modulation of oxidative stress. NeuN staining revealed disorganized neurons in the CA1 and CA3 regions of the HFD+AAV-Con+vehicle group, which were more orderly in the HFD+AAV-Con+SP group. This beneficial effect of SP was not observed in CAⅢ knockdown mice (Figure 6F). Additionally, the proportion of NeuN-positive cells was higher in both the CA1 (Figure 6G, P<0.05) and CA3 regions (Figure 6H, P>0.05) in the HFD+AAV-Con+SP group compared to the HFD+AAV-Con+vehicle group, with CAⅢ knockdown reducing this protective effect (Figure 6G-I, P>0.05). Furthermore, the SP-induced increases in Bcl-2/Bax ratio and Bcl-xl were diminished in CAⅢ knockdown mice (Figure 6J-O), while the cleaved-Capase 3 decreased by SP was increased after CAⅢ knockdown (Figure P-Q, P>0.05). Likewise, the inhibitory impact of SP on the AMPK pathway in diabetic mice was also decreased (Figure 6R-U, P>0.05). These results suggested that CAⅢ played a crucial role in mediating the neuroprotective and anti-apoptotic effects of SP. 3.7 CAⅢ mediated SP protection against cellular damage and mitochondrial dysfunction in PC12 cells Consistent with the in vivo findings, our in vitro experiments revealed that a diabetes-mimicking environment led to a reduction in CAⅢ expression, which was alleviated by SP treatment (Figure S2C-D, P<0.05). Lentiviral transfection successfully generated CAⅢ knockdown (CAⅢ sh), overexpressing (CAⅢ OE), and their negative control (NC) PC12 cells, confirmed at both the mRNA and protein levels (Figure S4A-F, P<0.05). Cell viability was reduced in the G100F200 and DMSO groups but increased in NC and CAⅢ OE cells (Figure 7A, C, D, P<0.05). SP did not significantly affect cell viability in CAⅢ sh cells under diabetic conditions (Figure 7B, P>0.05). Elevated LDH release was observed under high glucose and lipid conditions in NC cells (Figure 7E-H, P<0.001). SP intervention significantly lowered LDH release in NC cells exposed to diabetic conditions (Figure 7E, P<0.05), but this inhibitory effect was negated by CAⅢ knockdown (Figure 7F, P>0.05). Additionally, there was a tendency for SP to increase LDH in CAⅢ OE cells (Figure 7H, P>0.05). Flow cytometric analysis further demonstrated that SP significantly attenuated apoptosis induced by high-fat and high-glucose conditions (Figure 7I-J, P<0.01). In NC cells, antioxidant enzymes including SOD, CAT, and GSH-Px were significantly reduced in G100F200 and DMSO group compared to controls (Figure 7K-U, P<0.05). SP treatment notably reversed this decline (Figure 7K, O, S, P<0.05). However, in CAⅢ knockdown cells, SP showed no significant effect (Figure 7L, P, T, P>0.05). In contrast, in CAⅢ OE cells, SP continued to restore antioxidant enzyme levels (Figure 7N, R, V, P<0.05). Therefore, CAⅢ might mediate the protective effects on neuronal viability and cellular damage mitigation by SP in vitro. Our in vivo experiments indicated that CAⅢ influences hippocampal ROS production. Given mitochondria's central role in ROS generation, we detected ROS levels in mitochondria. Mitochondrial ROS were markedly elevated in NC cells under high-glucose and high-fat stimulation but were attenuated by SP (Figure 8A-B, P<0.05). However, SP had no significant effect on mitochondrial ROS in CAⅢ knockdown cells (Figure 8C-D, P> 0.05). Subsequently, we assessed the mitochondrial membrane potential by TMRE staining, which revealed high intensity in controls and diminished intensity under diabetic conditions. SP treatment restored the loss of mitochondrial membrane potential in the diabetic environment (Figure S5A-B, P<0.0001). This beneficial effect of SP was absent in CAⅢ knockdown cells (Figure S5C-D, P>0.05). These results suggest that CAⅢ is essential for the impact of SP on mitochondrial function and ROS reduction in diabetic conditions. 3.8 CAⅢ Modulated SP-Induced Gene Expression and Pathway Activation in PC12 Cells Volcano plots and heatmaps were used to visualize differentially expressed genes (DEGs) between DMSO-and SP-treated groups. Thirteen genes were significantly altered in NC cells following SP treatment, including five upregulated and eight downregulated transcripts (Figure S6A–D, FDR-adjusted P<0.05). The most prominently upregulated genes included Pfkfb3, Mrfap1, RT1-M6-1, 1810024B03Rik, and Fbxo41. Among these, Pfkfb3 has been implicated in multiple biological processes, particularly in the regulation of the AMPK signaling pathway. In CAⅢ sh cells, five genes were differentially expressed in the SP group, with 3 upregulated (Srm, Cebpb, Oca2) and 2 downregulated (LOC361108, Mgp) (Figure S6A–D, FDR-adjusted P<0.05), involving in cellular process and various pathways. The UpSet plot further illustrates the intersections of DEGs in DMSO-or SP-treated NC cells and CAⅢ sh cells (Figure S6E). GO analysis revealed that in NC cells, SP and DMSO treatments induced differential gene expression across various biological processes, cellular components, and molecular functions (Figure 9A). In contrast, in CAⅢ sh cells, gene expression shifted, with a decrease in genes related to signaling and metabolic processes, an increase in organelle-associated genes, and a reduction in catalytic activity genes (Figure 9B). These changes suggest that SP modulates neuronal processes via CAⅢ. GO enrichment analysis showed that SP in NC cells significantly impacted terms related to multicellular organism development, anatomical structure development, response to stimulus, and intracellular membrane-bounded organelles (Figure 9C). In CAⅢ sh cells, SP's effects on protein binding and membrane-bounded organelles were greatly diminished (Figure 9D), indicating that CAⅢ mediates SP's regulation of cellular processes in PC12 cells. Pathway analysis revealed that SP influenced several pathways in NC cells, including immune response, endocrine signaling, carbohydrate metabolism, transcription, signal transduction, and cell growth and death (Figure 9E). However, following CAⅢ knockdown, the impact of SP on immune and signal transduction pathways was reduced, while effects on endocrine signaling, carbohydrate metabolism, and cell growth were less pronounced (Figure 9F). KEGG analysis further identified that SP affected pathways involved in diabetic complications, such as AGE-RAGE signaling, endocrine resistance, type 1 diabetes, and HIF-1 signaling (Figure 9G-H). CAⅢ knockdown reduced the number of pathways influenced by SP, reinforcing the role of CAⅢ in mediating SP's effects on oxidative stress, cell proliferation, and apoptosis in PC12 cells. Discussions PAHSAs are a new type of hydroxyl fatty acid synthesized endogenously in humans and mammals, with tissue-and isomer-specific distribution (7). According to the position of the branch chain of PAHSA connected to the carbon atom, PASHAs encompass multiple isomers. 9-PAHSA can improve diabetic cardiomyopathy through enhanced autophagy and attenuated cardiac hypertrophy in db/db mice (22). Exogenous supplementation of 9-PAHSA also shows promise in alleviating diabetes-related cognitive deficits (23). We've developed a novel synthesis route for S-9-PAHSA (SP), which enhances GSIS and glucose uptake, unlike its R-isomer (6). DRCD is a chronic complication characterized by deficits in memory, information processing speed, executive function, and attention (24). Its etiology is multifactorial, involving oxidative stress, neuronal injury, inflammation, insulin resistance, synaptic dysfunction, gut microbiota, autophagy, and more (3). The management of DRCD often mirrors that of cognitive impairment or dementia, with the efficacy of hypoglycemic agents remaining debatable (25). Carbonic anhydrase (CA) modulates memory in mice, with activators enhancing and inhibitors impairing memory (26). In the present study, SP ameliorates cognitive deficits in diabetic mice, which aligns with our previous work on 9-PAHSA's effects on working memory (23). This suggests SP is a potential DRCD therapeutic, with CAⅢ emerging as a crucial mediator of SP's cognitive effects. The impact of PAHSA on glycemic control in mice is currently controversial. Studies have reported the beneficial effects of PAHSA in improving glucose tolerance and insulin sensitivity, with reduced levels in insulin-resistant and obese humans (7, 27, 28). Positive correlations between PAHSA levels and insulin sensitivity have been documented in humans and mice (7, 29, 30). PAHSA has been found to prevent impaired GSIS, boost pancreatic β-cell proliferation and viability, and ameliorate glucose homeostasis (31). Prolonged intervention with 5-PAHSA promotes increased GSIS in mice and human islets (32). Oral 5-PAHSA or 9-PAHSA administration improved glucose tolerance and increased insulin and glucagon-like peptide 1 (GLP-1) secretion in mice on a normal diet or a high-fat diet (7, 14, 33, 34). The capacity of 5-PAHSA and 9-PAHSA to inhibit lipolysis by modulating circulating free fatty acids and to regulate hepatic glucose production has also been demonstrated in mice (30). Growing evidence shows that oxidative stress is a pivotal factor in DRCD pathogenesis (35-37), with heightened ROS levels correlating with neuronal damage and cognitive deficits in T2DM (38, 39). Oxidative stress escalation results from both augmented free radical production and compromised antioxidant defenses, such as diminished SOD and CAT activities in the diabetic brain (40). Our prior work showed that 5-PAHSA could reduce ROS in PC12 cells, exerting an antioxidant effect (21). SP similarly lowered ROS in PC12 cells under a diabetic environment in our previous research (data not shown). Echoing these results, hippocampal ROS was found to be decreased in this study. Additionally, SP inhibited oxidative stress by reducing serum LDH and enhancing serum and hippocampal SOD activity and CAT. There was also an elevated trend of GSH-Px after SP treatment. Our in vitro experiments further demonstrated that SP effectively mitigated the decrease in SOD, CAT, and GSH-Px levels induced by high glucose and high fat conditions. 9-PAHSA has been noted to reverse mitochondrial dysfunction and enhance the survival of steatotic primary hepatocytes (41). Research indicates a link between CA and oxidative stress, with CA inhibitors mitigating high-altitude oxidative stress (42) and preventing mitochondrial dysfunction along with caspase activation and cell death (43). Our results proposed that CAⅢ mediated the neuroprotective effects of SP against oxidative stress both in vivo and in vitro, underscoring CAⅢ's role in SP's antioxidant and mitochondrial-protective actions. AMPK, recognized as an "energy sensor", is implicated in cognitive decline in obese and diabetic patients (44, 45). Our study revealed that SP rescued the decrease in AMPK phosphorylation and the downregulation of Sirt1 and PGC1α via CAⅢ. These findings indicate that SP may preserve mitochondrial homeostasis and ameliorate DRCD via the CAⅢ-dependent AMPK/Sirt1/PGC1α pathway. RNA-seq analysis further supported this mechanism by revealing upregulation of gene involved in AMPK signaling pathway. It has been reported that increased Bax expression and cysteine asparaginase 3 activity were observed in diabetic rats, associated with reduced neuronal density and poor performance in the Morris water maze (46-49). Thus, apoptosis likely drives neuronal loss and the associated cognitive deficits observed in diabetic conditions. While CAⅢ has been shown to exert anti-apoptotic effects in cardiac and skeletal muscle cells (50, 51), its neuronal role remains elusive. Our findings demonstrate that SP, via CAⅢ, can elevate the Bcl-2/Bax ratio and Bcl-xl, and reduce terminal apoptotic marker cleaved caspase 3 (cl-casp 3) both in vivo and in vitro, indicating that SP's anti-apoptotic effects were CAⅢ-dependent. Flow cytometry further validated these findings in vitro. In this study, hippocampal CAⅢ knockdown did not affect body weight and fasting glucose. Similarly, SP treatment did not influence body weight, fasting glucose, food intake, and water intake of the mice, aligning with earlier findings where 5-PAHSA and 9-PAHSA administration in normal mice did not alter intake or body weight (14). Furthermore, PAHSA treatment in HFD-fed mice showed no impact on body weight or fat mass (30). Our study has several limitations. First, the sample size for tissue SP content was small, necessitating larger cohorts for validation. SP concentration across all the groups needs to be determined, as well as the enzymatic activity of CAⅢ. Second, further studies are required to assess SP's blood-brain barrier penetration both in vivo and in vitro. Additionally, the brain receptors for SP and the specific CAⅢ targets in SP's mechanism of action remain to be identified, with the precise role of CAⅢ in the pathway yet to be determined. Conclusion In this study, we demonstrated that CAⅢ plays a critical role in mediating the cognitive improvements induced by SP in type 2 diabetes mice. SP alleviated hippocampal damage and improved spatial memory through CAⅢ-dependent activating the AMPK/Sirt1/PGC1α pathway, enhancing cellular viability and reducing oxidative stress. These findings suggest that CAⅢ will be a promising therapeutic target for diabetes-related cognitive disorders. Figure legends Figure 1. SP ameliorates neuronal damage in the hippocampus of T2DM mice. (A). Chemical structures of 9-PAHSA and S-9-PAHSA; (B). Schematic illustration of the experimental protocol; (C). Hippocampal HE staining in mice across groups, including whole hippocampus (× 20) and CA1, CA3, and DG regions (× 200), n=4; (D). Hippocampal Nissl staining in mice across groups, similarly magnified, n=4; (E-G). Mean percentage of Nissl-positive cells in the CA1, CA3, and DG regions, normalized to the ND group; Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 2. SP ameliorates cognitive disorders of T2DM mice by inhibiting apoptosis through the AMPK/Sirt1/PGC1α pathway. (A). Representative Y-maze track images from mice, n=8; The total distance moved (B), mean velocity (C), and alternation (D) of the mice in three groups in the Y maze test. (E-L). Western blot analysis of Bcl-2 and Bax, Bcl-xl, and cleaved-Caspase 3 (cl-casp3) expression in mouse hippocampus, n=3; (M-P). Western blot analysis of p-AMPK, AMPK, Sirt1, and PGC1α expression, n=3. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 3. SP improves oxidative stress in T2DM mice. Body weight (A) and fasting glucose (B) in mice on normal (n=15) or HFD (n=30) for 5 months; Tissue SP levels in brain (C), serum (D), colon (E), and intestine (F), detected by UPLC-MS/MS after the administration of vehicle or SP (30 mg/kg/d), n=3; Serum LDH activity (G), SOD activity (H), CAT (I), and GSH-Px activity (J) in ND, HFD, and SP groups, n=5; Hippocampal ROS (K), SOD activity (L), CAT (M), GSH-Px (N) and H2O2 (O) levels, n=3~4; Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 4. The knockdown of CAⅢ in the hippocampus and the effects of SP during the administration. (A). Overview of the experimental design; (B-G) protein expression and statistical analysis of CAⅢ in the hippocampus (B, E), cortex (C, F) and the remaining brain (excluding the hippocampus and cortex) (D, G) of mice following injection with control or CAⅢ shRNA, n=5; Body weight (H), fasting glucose (I), water intake (J), and food intake (K) in mice during SP treatment, n=15; (L, M). IPGTT results and AUC, n=9; (N, O). IPITT results and AUC, n=9. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 5. CAⅢ-mediated the improvement of SP in the cognitive impairment of T2DM mice. (A). Y-maze test track images from mice, n=10~11; The total distance moved (B), velocity (C), and alternation (D) for different groups in the Y maze test; (E). Representative tracks of each group on the probe trial day in the Morris water maze test; (F) latency to platform over 5 training days; Escape latency (G), platform crossings (H), time at target platform (I), time in target quadrant (J), total distance (K) and movement velocity (L) of each group on the probe trial day, n=8~10; Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 6. SP ameliorated neuronal injury and apoptosis via CAⅢ-mediated Bcl-2/Bax and AMPK/Sirt1/PGC1α pathway. Hippocampal ROS (A), SOD activity (B), CAT (C), and GSH-Px (D) and H2O2 (E) in mice, n=4~5. (F). NeuN immunostaining in hippocampal sections across groups; NeuN-positive neuron percentages in CA1 (G), CA3 (H), and DG (I) regions, normalized to the HFD+AAV-Con+vehicle group, n=4; (J-Q). Western blot analysis of Bcl-2, Bax, Bcl-xl, and cleaved-Caspase 3 (cl-casp3) in mouse hippocampus, n=3; (R-U). Western blot analysis of p-AMPK, AMPK, Sirt1 and PGC1α, n=3. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 7. The effects and mechanism of SP on cell viability, LDH release, and mitochondrial production in PC12 cells. NC, CAⅢ sh, and CAⅢ OE cells were exposed to standard medium (Con), high-glucose and high-fat medium (100 mM glucose + 200 μM palmitic acid, G100F200), G100F200 with DMSO, or G100F200 with 60 μM SP for 24 hours. (A-D). Cell viability of CAⅢ sh cells, CAⅢ OE cells, and NC cells, n=6; (E-H). LDH release of CAⅢ sh cells, CAⅢ OE cells and NC cells, n=5~6; (I). Flow cytometric analysis of apoptosis using Annexin V-FITC/PI staining. (J). Quantification of apoptosis rate based on flow cytometry results. SOD activity (K-N), CAT (O-R), and GSH-Px (S-V) of CAⅢ sh cells, CAⅢ OE cells, and NC cells, n=6. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 8. The effects and mechanism of SP on mitochondrial production in PC12 cells. MitoSOX Red immunofluorescence for mitochondrial ROS and quantification in NC (A, B) and CAⅢ sh (C, D) cells, n=3. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 9. Differentially expressed genes of biological processes and pathways after SP treatment in NC and CAⅢ sh PC12 cells. Graphical representation of GO classification for biological processes, cellular components, and molecular functions (A, B) and GO enrichment analysis (C, D) for differential gene expression between DMSO and 60 μM SP groups in NC and CAⅢ sh cells, n=3; KEGG pathway classification (E, F) and KEGG enrichment scatterplots (G, H) for the same comparison, n=3. Data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.