The study was performed on a Waters H-Class UPLC system. Chromatographic separation was achieved on a Waters ACQUITY UPLC®HSS T3 (2.1 mm × 100 mm, 1.8 μm), protected by VanGuandTM BEHC C18 at 40. The mobile phase was composed of ultra-pure water with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.4 mL/min. The gradient elution profile was as follows: 0–4 min, 5% B; 4∼9 min, 5%–10% B; 9∼24 min, 10%–20% B; 24∼26 min, 20%–25% B; 26∼28 min, 25%–40% B; 28∼34 min, 40%–90% B; 34∼37 min, 90% B; 37∼39 min, 90%–5% B. The injection volume was 2 μL. The temperature of the autosampler was kept at 4°C.

The UPLC-MS/MS analysis on HLTP extract was performed on Waters H-Class UPLC system coupled with an Agilent 6,560 quadrupole time-of-flight tandem mass spectrometer. MS analysis was performed at electrospray ionization in negative and positive ion mode and the acquisition parameters were as follows: drying gas, N₂; drying gas flow rate, 10 L/min; drying gas temperature, 225°C; nebulizer pressure, 25 psi; Electrospray ion source with capillary voltages of 3,500 V and 4,000 V in positive and negative ion modes, respectively; scan range m/z 20∼1,700; fragmentor voltage, 80 V; collision energy, 10, 20, 40 V; Data acquisition was performed using a Masshunter workstation.

This study first queried the SMILES numbers of the 32 identified phenolic components in H. citrina leaves through the DrugBank database (http://www.drugbank.ca), as shown in Table 1. Compounds not found in the database were drawn using ChemDraw and converted to SMILES format using the OpenBabel molecular format conversion tool. The SMILES numbers were uploaded to the Swiss Target Prediction database (http://www.swisstargetprediction.ch), with the attribute set to “Homosapiens,” to obtain target information. Using “Neuroprotection” as a keyword, we retrieved databases such as GenCards (https://www.genecards.org/) and OMIM database (https://omim.org/) to search target information. Removing the same targets and using Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) to obtain common targets. The obtained common targets were uploaded to the STRING database (https://www.string-db.org/) to retrieve the PPI network. Using Cytoscape 3.9.1 software, we drew the network diagram of “H. citrina leaves Phenolic Components - Common Targets - Neuroprotection” and selected core chemical components based on the Degree value, Betweenness Centrality, and Closeness Centrality of active ingredients.

The common targets were imported into the Metascape database (https://metascape.org/) for GO function and KEGG pathway enrichment analysis. GO analysis yielded three parts: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF), and generated bar charts. The top 20 signal pathways related to neuroprotection were visualized in a functional enrichment bubble chart based on the enriched KEGG pathways. Using the selected common targets and enriched KEGG pathways, an analytical network of “H. citrina leaves Phenolic Components - Common Targets - Pathways” was constructed using Cytoscape 3.9.1 software.

Autodock software was used to perform molecular docking between the targets associated with neuroprotection (including screened targets and the targets reported in the literature related to neuroprotection) and H. citrina leaves phenolic components. The binding activities between active components of H. citrina leaves and common targets were compared. The results with optimal conformations and energy were selected for further analysis, and the PyMOL software was used to interpret and visualize the macromolecule-ligand complexes.

Cort-induced PC12 cell injury model was used for in vitro screening of neuroprotective activity of the core components selected through network pharmacology and molecular docking (specific PC12 cell culture conditions and dosing methods are detailed in Section 2.5). For the screening of the core phenolic components of H. citrina leaves, the concentrations were set at 1, 10, 20, and 50 μmol/L based on literature (Zhou et al., 2017). According to the results, concentrations that had no impact on cell viability were selected for the activity screening experiment of phenolic components against Cort-induced PC12 cell damage.

PC12 cells were cultured with RPMI 1640 complete medium (containing 5% fetal bovine serum, 5% horse serum, and 1% penicillin-streptomycin) in a 37°C, 5% CO₂ cell culture incubator. When the cells density reached 80%∼90%, they were passaged with 0.25% trypsin, and logarithmic phase cells were selected for the experiment. PC12 cells were seeded in a 96-well plate at a density of 5×10⁴/mL, with 0.2 mL per well, and cultured for 24 h. The control group cells were cultured with complete medium. The experimental group cells were treated with Cort at final concentrations of 50, 100, 200, 400, and 800 μmol/L for 24 h. After removing the culture medium, 200 μL of MTT (0.5 mg/mL) were added to each well, and after 4 h of incubation, the supernatant was removed, and 150 μL of DMSO was added to each well. Shake in the dark for 15 min on a microplate shaker, and the absorbance A value of each well was measured at 470 nm by a microplate reader. The cell viability was calculated as follows: Cell viability = (A model/A control) × 100%.

PC12 cells were seeded in a 96-well plate at a density of 5×10⁴/mL, with 0.2 mL per well, and cultured for 24 h. Control and experimental groups were established. The control group cells were cultured with complete medium. The experimental groups cells were treated with HLTP of 50, 100, 200, 500, and 1,000 μg/mL, referring to the research of Tian et al. (2017), and the cell viability was detected by the above MTT method to screen HLTP concentration. PC12 cells were seeded at a density of 5 × 10⁴/mL in a 96-well plate with 0.2 mL per well and cultured for 24 h. Cells were divided into control group, model group, low-dose group, medium-dose group, and high-dose group of HLTP. The control group added 200 μL of complete medium, the model group and HLTP groups added 200 μL of complete medium with the optimal concentration of Cort. After 24 h, the entire culture medium was aspirated, and the control and model groups added 200 μL of fresh medium. The HLTP groups added 200 μL of medium containing low, medium, and high concentrations of HLTP. After 24 h of incubation, cell viability was determined using the MTT assay mentioned above. The LDH content was determined following the instructions of the LDH assay kit. The LDH level in the extracellular fluid reflects the degree of cell damage.

PC12 cells were seeded in a 6-well plate at a density of 4 × 10⁵/mL, with 2 mL per well, and cultured for 24 h. Cells were treated with the experimental grouping and cell treatment methods described in Section 2.5.2. The cell supernatant was discarded, trypsinized and resuspended with PBS. Then, cells were treated with cell disruptor, and the supernatant was taken after centrifugation. Commercial assay kits were used to determine cellular MDA content, total SOD levels, CAT activity, and ROS levels. ROS changes were also measured using a fluorescence spectrophotometer, and the enzyme levels in cells were expressed as a percentage of the control group.

PC12 cells were seeded at a density of 4 × 10⁵/mL cells/mL in a 6-well plate with 2 mL per well and cultured for 24 h. Cells were treated according to the experimental grouping and cell processing methods mentioned Section 2.5.2. The treated cells were aspirated of the culture medium, washed twice with PBS. Then, 0.5 mL of Hoechst 33258 staining solution was added, stained at 37°C in the dark for 20 min, washed twice with PBS. 0.5 mL of PI staining solution was added, stained at 37°C in the dark for 20 min, washed twice with PBS. The morphology of cells was observed under a fluorescence microscope. The treated cells were aspirated of the culture medium, washed twice with PBS, resuspended in 200 μL Binding Buffer, and then transferred 200 μL of cell suspension from each group to a flow tube. Subsequently, 5 μL of Annexin V-FITC solution and 5 μL of PI staining solution were added, mixed well, and kept in the dark at room temperature for 10 min before detection and analysis using a flow cytometer. The treated cells were aspirated of the culture medium, and each group was made into 0.5 mL of cell suspension, added 10 μmol of Fura-3/AM (5 μmol/L), and incubated at 37°C for 30 min. After washing twice with D-hanks buffer, the cells were further incubated for 30 min. A 1 mL cell suspension was prepared using D-hanks buffer, and the fluorescence intensity was measured by flow cytometry. Simultaneously, the cells were placed on the slide and observed under a fluorescence microscope. The treated cells were aspirated of the culture medium, washed twice with PBS, and the JC-1 staining working solution was diluted with culture medium at a ratio of 1:1. Then, 2 mL of staining solution was added to each well, incubated at 37°C for 20 min in the cell culture incubator. After aspirating the supernatant, cells were washed twice with JC-1 staining buffer, and the mitochondrial membrane potential (MMP) was measured using a flow cytometer.

PC12 cells were seeded at a density of 4 × 10⁵ cells/mL in a 6-well plate with 2 mL per well and cultured for 24 h. Cells were treated according to the experimental grouping and cell processing methods mentioned Section 2.5.2. The culture medium was aspirated, washed twice with PBS. Cells were then lysed on ice with RIPA lysis buffer containing a protease inhibitor. After protein extraction, the protein samples were denatured by heating at 99°C for 10 min. The protein samples were subjected to 10% SDS-PAGE, transferred to a PVDF membrane, and blocked in 5% skimmed milk for 2 h. The membrane was then incubated with specific antibodies (Bax, Caspase-3, Bcl-2, Cyt-C, β-actin, IL-1β, TNF-α, IL-10, PI3K, AKT) overnight at 4°C, washed with PBS three times, each 10 min, incubated with HRP-conjugated goat anti-rabbit secondary antibody at room temperature for 2 h, washed with PBS 3 times, visualized by ImageJ software with grayscale analysis.

Based on 32 components of H. citrina leaves, 204 unique component targets were obtained from the Swiss Target Prediction database. Using databases such as GenCards and OMIM, 1,510 neuroprotective-related targets were excavated. After Venn analysis, 90 common targets were identified, representing potential targets for the neuroprotective effects of H. citrina leave phenolic compounds, as shown in Figure 1A. The PPI network relationship diagram was constructed, as shown in Figure 1B. The network diagram consists of 90 nodes and 836 edges, with an average Degree of 18.6. Node size and color depth are positively correlated with the Degree values of the selected neuroprotective target proteins. Larger and darker nodes such as TNF, AKT1, TP53, CASP3, EGFR, ESR1, MMP9, ACE, MAPK8, and APP may play a crucial role in neuroprotection. The “H. citrina leaves Phenolic Components-Common Targets-Neuroprotection” network diagram was constructed, as shown in Figure 1C. This network diagram comprises 122 nodes, where light blue nodes represent the 32 components, and white nodes represent the 90 neuroprotective-related targets. Based on the Degree, Betweenness Centrality, and Closeness Centrality network topology analysis of active components (Supplementary Table S2), four core chemical components were selected: M32 (Apigenin), M31 (Quercetin), M17 (Rutin), and M21 (Isoquercitrin), which may be the main active components responsible for the neuroprotective effects of H. citrina leaves phenolic components.

The GO enrichment analysis resulting in 288 entries related to BP, 75 to CC, and 115 to MF. The top ten enriched entries were visualized, as shown in Figure 1D. In BP analysis, it mainly involves cellular response to organonitrogen compound, positive regulation of phosphorylation, and regulation of inflammatory response. In CC analysis, it mainly involves membrane raft, perinuclear region of cytoplasm, vesicle lumen, etc. In MF analysis, it mainly involves nuclear receptor activity, oxidoreductase activity, kinase binding, etc. These may be related to the complex pathogenesis of neural damage, suggesting that H. citrina leaves phenolic compounds can exert neuroprotective effects through different levels and multiple pathways. The KEGG pathway enrichment resulting in 179 pathway entries. These pathways involve various diseases such as cancer, lipid metabolism, atherosclerosis, and diabetic cardiomyopathy. The top 20 pathways related to neuroprotection were selected, as shown in Figure 1E. The main pathways involve the MAPK pathway, PI3K/AKT pathway, apoptosis pathway, and IL-17 pathway. The “H. citrina leaves Phenolic Components-Common Targets-Pathways” was constructed, as shown in Figure 1F. Based on comprehensive PPI analysis and KEGG enrichment analysis, it is speculated that H. citrina leaves phenolic components may exert neuroprotective effects by regulating the PI3K/AKT pathway and its downstream pathways.

To validate the results of network pharmacology, we conducted molecular docking verification between the 32 phenolic components in H. citrina leaves with the proteins. These proteins including top ten target proteins (TNF, AKT1, TP53, CASP3, EGFR, ESR1, MMP9, ACE, MAPK8, and APP) and proteins that ranked lower or did not appear in the screening results but have been extensively studied and proven to be related to neuroprotection or serve as targets for some neuroprotective drugs (GSK3B, PIK3R1, MTOR, MAOB, TYR, APOA4, SLC6A4, PARP1, HSP90AA1, CREB1). The results are shown in Figure 2A. Generally, when the binding energy is <−5.0 kcal/mol, it indicates a strong binding activity between the compound and the target. Overall, most components exhibit good binding affinity with proteins. The phenolic acid components in H. citrina leaves generally have lower binding energies compared to flavonoid components, indicating better binding. Among all neuroprotective-related target proteins, the phenolic components that bind well to AKT1, PIK3R1, TP53, and PARP1 are the most abundant. Further visualization analysis was performed on the four components with the strongest binding to AKT1, PIK3R1, TP53, and PARP1, as well as the four core chemical components selected through comprehensive screening of network topology analysis. The results are shown in Figures 2B–I. Apigenin has good binding sites with TNF, Quercetin with TP53, Rutin with MAOB, Isoquercitrin with AKT1, 4-O-Caffeoylquinic acid with AKT1, 4-p-Coumaroylquinic acid with PARP1, 5-O-Caffeoylshikimic acid with PIK3R1, and 5-O-Feruloylquinic acid with AKT1. For instance, 4-O-Caffeoylquinic acid binds to a deep groove on the surface of the receptor protein AKT1, and they have good shape complementarity. The binding is mainly stabilized through hydrogen bonding, and the residues involved in hydrogen bonding with 4-O-Caffeoylquinic acid include LYS389, GLU49, GLN47, LYS30, ASP44, and GLU40.

The research results of the main phenolic components in H. citrina leaves on PC12 cells are shown in Figure 3. After the treatment of PC12 cells with 50 μmol/L Quercetin, Rutin, and 4-O-Caffeoylquinic acid, 20∼50 μmol/L Isoquercitrin and 4-p-Coumaroylquinic acid, and 10∼50 μmol/L Apigenin, 5-O-Caffeoylshikimic acid, and 5-O-Feruloylquinic acid for 24 h, the cell viability significantly decreased, all below 90%. However, after treating cells with remaining concentrations of individual phenolic components for 24 h, the cell viability was all above 95%, indicating no effect on the cells. Therefore, in the active screening experiment, the concentrations selected for Quercetin, Rutin, Isoquercitrin, 4-O-Caffeoylquinic acid, and 4-p-Coumaroylquinic acid were 10 μmol/L, while those for Apigenin, 5-O-Caffeoylshikimic acid, and 5-O-Feruloylquinic acid were 1 μmol/L. The research results on the protective effect of the main phenolic components in H. citrina leaves against Cort-induced damage to PC12 cells are shown in Figure 3I. Compared to the Control group, the cell viability significantly decreased after Cort (200 μmol/L, the concentration screening process is shown in Section 3.3) treatment of PC12 cells (P < 0.01). Subsequently, treating cells with the eight main individual components in H. citrina leave, as well as their equimolar mixture and the original proportion mixture, for 24 h resulted in a significant increase in cell viability for all components except Apigenin (P < 0.05 or P < 0.01), demonstrating a pronounced protective effect. Additionally, the study found that the original proportion mixture of the eight main individual components in H. citrina leave has a stronger improvement effect on the cell viability of PC12 cells. Therefore, subsequent in vitro activity evaluations chose HLTP as a therapeutic drug.

Firstly, the impact of Cort and HLTP at different concentrations on cell viability was analyzed, as shown in Figures 4A, B. After stimulating PC12 cells with 200 μmol/L Cort for 24 h, the cell viability was close to 50% (P < 0.01). Therefore, 200 μmol/L Cort stimulation for 24 h was chosen as the experimental condition for the neuronal cell damage model. HLTP (50, 100, and 200 μg/mL) had no significant effect on PC12 cell viability. When the HLTP concentration reached 1,000 μg/mL, the cell viability significantly decreased (P < 0.01). Therefore, the subsequent concentration of HLTP was controlled within 200 μg/mL. The impact of HLTP on the cell damage model was analyzed, as shown in Figure 4C. Compared to the Control group, the cell viability of the Model group significantly decreased (P < 0.01), with a reduction of 39.77%. After administering HLTP (50, 100, and 200 μg/mL), the cell viability significantly increased (P < 0.05 or 0.01) 9.50%, 10.42%, and 21.25%, respectively. It is suggested that HLTP have a good protective effect against Cort-induced neuronal damage. The LDH detection results are shown in Figure 4D. The addition of HLTP (50, 100, and 200 μg/mL) significantly reduced cell LDH release rates (P < 0.01). Compared to the Model group, the reductions were 18.86%, 29.96%, and 33.03%, respectively, indicating a decrease in the degree of cell damage. Cell morphology changes are shown in Figure 4E. The Control group exhibited good cell growth with spindle-shaped cells. In the Model group, the number of cells significantly decreased, accompanied by pronounced morphological changes such as cell shrinkage, rounding, and detachment. The cell status in different concentrations of HLTP groups was better than that in the model group. The phenomena of cell shrinkage, rounding, and detachment were reduced, indicating that HLTP can alleviate PC12 cell damage induced by Cort.

The effects of HLTP on the levels of intracellular oxidases and antioxidants in PC12 cells after Cort-induced damage were analyzed. The results are shown in Figures 4F–J. After Cort induction, the activities of SOD and CAT in PC12 cells were significantly reduced (P < 0.01), decreasing by 61.51% and 60.23%, respectively, compared to the Control group. This indicates that Cort induction can decrease the activity of intracellular antioxidants. However, after treatment with different concentrations of HLTP (50, 100, and 200 μg/mL), both SOD and CAT activities significantly increased (P < 0.01). Specifically, SOD activities increased by 14.77%, 31.36%, and 39.65%, and CAT activities increased by 23.45%, 25.27%, and 35.45%, respectively. Compared to the Control group, after Cort induction, the levels of MDA in PC12 cells significantly increased (P < 0.01). After treatment with different concentrations of HLTP (50, 100, and 200 μg/mL), the MDA levels significantly decreased (P < 0.05 or 0.01), showing reductions of 16.34%, 27.09%, and 54.66% compared to the Model group. The ROS levels followed a similar trend as MDA and after treatment with different concentrations of HLTP (50, 100, and 200 μg/mL), the ROS levels significantly decreased (P < 0.01), showing reductions of 80.78%, 116.9%, and 140.2% compared to the Model group, and exhibited a concentration-dependent trend. The same phenomenon can be observed in the detection of ROS by fluorescence spectrophotometry. It is suggested that HLTP can increase intracellular antioxidant enzyme activity, reduce intracellular reactive oxygen species, alleviate intracellular lipid peroxidation, effectively improve the oxidative stress state in PC12 cells, and achieve a protective effect on nerve cells.

The impact of HLTP on the degree of cell apoptosis was analyzed, and the results are shown in Figure 5. Hoechst 33324 is a nucleic acid dye that can enter the cell membrane of normal cells to stain them light blue. Apoptotic cells, with increased membrane permeability, exhibit bright blue fluorescence in their cell nuclei when stained with Hoechst 33324. PI dye cannot penetrate the intact cell membranes of normal cells and apoptotic cells, so live cells resist PI staining, while dead cells are stained red. The Hoechst 33324/PI staining results are shown in Figure 5A. Compared with the Control group, the number of cells emitting bright blue and red fluorescence in the Model group significantly increased (P < 0.01), indicating that Cort can cause damage to PC12 cells and induce cell apoptosis, even cell necrosis. In the HLTP group (50, 100, and 200 μg/mL), the number of cells emitting bright blue and red fluorescence significantly decreased (P < 0.01), indicating that HLTP can inhibit Cort-induced apoptosis of PC12 cells. After staining PC12 cells with Annexin V-FITC/PI double staining, flow cytometry was used to sort cells in different stages, and the results are shown in Figure 5B. Compared with the Control group, the Q2-2 quadrant (late-stage apoptosis) and Q2-4 quadrant (early-stage apoptosis) cells in the Model group significantly increased, and the cell apoptosis rate significantly increased (P < 0.01), indicating that Cort induces apoptosis of PC12 cells. Compared with the Model group, in the HLTP group (50, 100, and 200 μg/mL), the cells in Q2-2 and Q2-4 quadrants decreased, and the cell apoptosis rate decreased. Each concentration of HLTP showed significant differences (P < 0.01), indicating that HLTP can exert neuroprotective effects by inhibiting apoptosis of PC12 cells.

The effects of HLTP on intracellular Ca²⁺ concentration and MMP in injured PC12 cells were analyzed, and the results are shown in Figure 6. When the MMP is high, JC-1 dye aggregates in the matrix of mitochondria, forming polymers that emit red fluorescence. When the MMP is low, JC-1 cannot aggregate in the matrix of mitochondria, and it exists as a monomer, emitting green fluorescence. As shown in Figure 6A, compared with the Control group, the number of cells in the P1 quadrant with lower membrane potential in the Model group significantly increased (P < 0.01), increasing by 48.10% compared to the Control group. This indicates that Cort can decrease the MMP in PC12 cells. Compared with the Model group, in the HLTP group (50, 100, and 200 μg/mL), the number of cells in the P1 quadrant decreased, and each concentration of HLTP showed significant differences (P < 0.01or 0.05). This suggests that HLTP can effectively restore the depolarization of MMP induced by Cort in PC12 cells, thereby exerting a neuroprotective effect. The Ca²⁺ fluorescence probe Fluo-4 AM itself has almost no fluorescence. Once it enters the cell and undergoes intracellular esterase hydrolysis, the generated Fluo-4 binds to Ca²⁺ and emits green fluorescence, as shown in Figures 6B, C. After Cort induction, the number of cells with green fluorescence increases, and flow cytometry analysis also indicates a significant increase in Ca²⁺ concentration (P < 0.01) of 138.0% compared to the Control group. However, after treatment with different concentrations of HLTP (50, 100, and 200 μg/mL), the number of cells with green fluorescence significantly decreases. Flow cytometry analysis also shows a significant reduction in intracellular Ca²⁺ concentration (P < 0.01), decreasing by 31.14%, 47.74%, and 51.34%, respectively, compared to the Model group. This indicates that Cort induction of apoptosis in PC12 cells leads to Ca²⁺ overload, and HLTP can effectively alleviate the Ca²⁺ overload effect, exerting a neuroprotective effect.

This study examined the expression of apoptosis-related proteins in PC12 cells after treatment with Cort and HLTP. The results, as shown in Figure 7A, indicate that Cort treatment significantly downregulates the expression of the anti-apoptotic protein Bcl-2 and upregulates the expression of pro-apoptotic proteins Bax, Cyt-C, and Caspase-3 (P < 0.01). However, treatment with HLTP at concentrations of 50, 100, and 200 μg/mL effectively reverses this effect (P < 0.01 or 0.05). The relative expression levels of Bcl-2/Bax proteins increased by 15.27%, 27.90%, and 28.54%, respectively, compared to the Model group. The relative expression levels of Caspase-3 proteins decreased by 28.12%, 36.47%, and 40.32%, respectively, and Cyt-C protein expression decreased by 40.28%, 52.86%, and 65.26%, suggesting that HLTP against Cort-induced apoptosis in PC12 cells by modulating the expression of apoptosis-related proteins.

This study examined the expression of inflammatory factors in PC12 cells after treatment with Cort and HLTP. As shown in Figure 7B, Cort treatment significantly increased the protein expression levels of the pro-inflammatory factors TNF-α and IL-1β (P < 0.01), while decreasing the anti-inflammatory factor IL-10 (P < 0.01). Treatment with different concentrations of HLTP (50, 100, and 200 μg/mL) effectively reversed the abnormal changes in inflammatory factors (P < 0.01). Compared to the Model group, the relative protein expression levels of TNF-α decreased by 38.17%, 70.76%, and 63.87%, IL-1β decreased by 24.12%, 42.89%, and 55.38%, and IL-10 increased by 44.61%, 61.32%, and 39.98%. These results suggest that HLTP protect against Cort-induced apoptosis in PC12 cells by modulating the expression of inflammatory factors, thereby alleviating the inflammatory response.

This study examined the expression of PI3K and AKT proteins in PC12 cells after treatment with Cort and HLTP. As shown in Figure 7C, after 24 h of Cort treatment, the protein expression levels of PI3K and AKT in the cells significantly decreased (P < 0.01), with reductions of 49.22% and 50.29% compared to the Control group. However, after treating the cells with different concentrations of HLTP (50, 100, and 200 μg/mL) for 24 h, the protein expression levels of PI3K and AKT in the cells significantly increased (P< 0.01). Compared to the Model group, the protein expression levels of PI3K and AKT increased by 15.24%, 30.44%, 41.03%, and 21.78%, 43.63%, 12.86%, respectively. This indicates that HLTP may regulate the PI3K/AKT signaling pathway to inhibit apoptosis in PC12 cells, exerting a neuroprotective effect.