Alzheimer’s disease (AD) is a common neurodegenerative disease in the elderly. In recent years, the number of AD patients worldwide has been on the rise (Fjell et al., 2014). The World Alzheimer’s Report of 2021 reported that over 55 million people live with dementia worldwide, and the number of AD patients is predicted to reach around 78 million by 2030. The cognitive impairment, memory loss, and behavioral disorders caused by AD inevitably reduce the quality of life in patients and increase the financial burden on families and the national healthcare system (Lane et al., 2018). Senile plaques formed by the deposition of amyloid-beta (Aβ) and neurofibrillary tangles formed by aggregation of hyperphosphorylated Tau are two pathological markers of AD. The presence of both these manifestations indicates the activation of multiple pathogenic mechanisms and neuronal damage in the brain (Sharma et al., 2019; Busche and Hyman, 2020). Most single-target drugs developed over the years to reduce Aβ or hyperphosphorylated Tau have failed in recent clinical trials. The reason can be attributed to the fact that AD is a multifactorial disease and therefore drugs targeting a single AD target cannot achieve substantial therapeutic effects (Huang and Mucke, 2012; Knopman et al., 2021). A growing number of studies have shown that a multi-target combination is an effective treatment for AD (Cummings et al., 2019; Veitch et al., 2019; Kabir et al., 2020). Therefore, determining the integrated protein changes in the hippocampal region before and after drug administration in AD patients is important to elucidate the potential molecular mechanisms of drug therapy. The hippocampus is an important area of the brain responsible for memory function and cognitive ability. The systematic study of protein expression in the hippocampus is key to discovering the actual pathogenic mechanisms of AD and developing effective therapeutic drugs (Poll et al., 2020). The hippocampal proteins show different expression levels at different stages of AD, and these changes undoubtedly include neurodegenerative processes triggered by pathophysiological abnormalities and adaptive responses. Although many researchers have performed extensive experimental research on hippocampal disease-related genes and proteins, studies on the differentially expressed proteins in the hippocampal tissue after drug administration are still lacking.

Huang-Pu-Tong-Qiao formula (HPTQ) has been widely used for decades in treating AD at the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine. HPTQ is composed of 6 herbs: Rheum, Acorus tatarinowiischott, Alpiniaeoxyphyllae fructus, Polygoni Multiflori Radix Preparata, Ligusticum wallichii, and Panax ginseng. Previous studies have found that HPTQ could reduce neuronal loss and improve AD symptoms. For example, it could reduce the over phosphorylated tau in AD model rats and primary hippocampal neurons cultured in vitro by inhibiting the calcium/calmodulin-dependent protein kinase IV (CAM-CAMKIV) pathway (Ye et al., 2020). The administration also reduced oxidative stress and inhibited the mitochondrial apoptosis pathway in AD model rats (Cai et al., 2018). In another study, it modulated the epidermal growth factor receptor (EGFR)- phospholipase Cγ (PLCγ) signaling pathway and inhibited calcium overload in neurons (Wang et al., 2020). Further, inhibition of the abnormal activation of the c-Jun N-terminal kinases (JNK) signaling pathway and reducing the inflammatory response in animal models were also documented (Jiang et al., 2019). These results confirm that HPTQ has a variety of pharmacological effects. However, the total protein expression changes in the hippocampus before and after HPTQ administration have not been documented to this date.

Proteomics is a method for systematically studying the total protein expression changes and an important tool for elucidating disease mechanisms and discovering potential therapeutic targets (Bai et al., 2020). With rapid biotechnological development, isobaric tags for relative and absolute quantification (ITRAQ), which is a quantitative proteomics method can be used to perform quantitative studies of protein expression (Li et al., 2017). Currently, proteomics is a major tool being used to study the therapeutic mechanism of Chinese Traditional Medicine and to identify disease-related proteins (Xu et al., 2019, 2020).

In this study, we established an animal model of AD with an intraperitoneal injection of D-galactose and an intracranial hippocampal injection of Aβ25-35. Isobaric tag for relative and absolute quantitation (ITRAQ) method based on ITRAQ Labeling, HPLC Fractionation, and LC-MS/MS analysis was used to identify the full range of proteins in rat hippocampal tissue samples, and the differentially expressed proteins were obtained by bioinformatics analysis (Rhein et al., 2009; Yang et al., 2012). The proteomics results were then further validated by western blotting (WB) experiments. This study aimed to identify the potential therapeutic target proteins and related mechanistic pathways of HPTQ and to provide a basis for the therapeutic potential of HPTQ.

The SPSS version 23.0 was selected as the data analysis software for this experiment. Data results were expressed as mean ± standard deviation (SD). The Independent samples t-test was used to compare the means between two groups while one-way analysis of variance (ANOVA) and Least Significance Difference (LSD) were used to compare the means of multiple groups. The results with a p-value < 0.05 were considered to be statistically and significantly different while one-way analysis of variance (ANOVA) and Least Significance Difference (LSD) were used to compare the means of multiple groups. The results with a p-value < 0.05 were considered to be statistically and significantly different.

In this study, we applied the ITRAQ-based proteomics approach and performed a comparative proteomics analysis of the hippocampus in AD model rats after HPTQ treatment. The AD rat model was established using intraperitoneal injections of D-galactose combined with an intraventricular injection of Aβ25-35. The Morris water maze results pointed at the ability of HPTQ to improve learning memory in AD model rats. Subsequently, the ITRAQ results showed 57 differentially expressed proteins in hippocampal tissues of the HPTQ and MODEL groups, of which 35 proteins were upregulated (61%) and 22 proteins were downregulated (39%). This result indicated that the protein expression level in the hippocampal tissue of AD rats was enhanced after HPTQ treatment. To elucidate the potential molecular mechanisms of HPTQ for the treatment of AD, we analyzed the effect and mechanism of proteins by GO analysis and KEGG analysis and assessed the degree of association between proteins through the construction of PPI networks and KEGG enrichment analysis. AD-related biological functions of differentially expressed proteins obtained by bioinformatics analysis included DNA transcription and mRNA translation, coagulation, fibrin clot formation, regulation of autophagy, inhibition of cholinesterase, improvement of neuronal survival, anti-inflammation, and alleviation of oxidative stress. In addition, the PPI network showed significant associations between most of the differentially expressed proteins. Some differentially expressed proteins with potential neuroprotective effects have been selected for further analysis.

Ribosomal protein is an important constituent of the ribosome unit. The main function of the ribosomes is to participate in the assembly and translation of protein ribosomes. In neurodegenerative diseases, ribosomal dysfunction leads to impaired protein biosynthesis (Ding et al., 2005; Ding et al., 2006). For instance, pathological Tau protein formation in AD-induced ribosomal dysfunction led to reduced protein synthesis (Meier et al., 2016). It was found in another study that the levels of 60S ribosomal protein subunits were significantly decreased in Tau-induced frontotemporal dementia mice compared to the normal group (Evans et al., 2021). Moreover, after the Aβ1-42 intervention, the phosphorylation of Tau protein led to reduced protein transcription and translation levels by inducing ribosomal dysfunction in SHSY5Y cells (Maina et al., 2018). In this study, the KEGG enrichment analysis indicated that the ribosomal pathway was significantly enriched and the PPI network showed that several genes of the 60S ribosomal protein subunits occupied the center of the network and interacted with other differentially expressed proteins. The expression of L7, L14, L18, L18Aa, L19, and L13 of 60S ribosomal protein subunits was upregulated in the HPTQ group. Combined with the results of our previous study in which we found that HPTQ could reduce the level of pathologically phosphorylated Tau protein in AD rat models (Ye et al., 2020), we suggest that HPTQ could play a neuroprotective role by enhancing protein biosynthesis through restoring ribosomal dysfunction in AD. The complement and coagulation cascade pathways involved in Fga and Fgb proteins were found to be significantly enriched in the KEGG enrichment analysis. Fibrinogen alpha chain (Fga) and Fibrinogen beta chain (Fgb) are proteins that are closely associated with vascular diseases, and play an essential role in thrombosis, wound healing, and other biological functions (Simurda et al., 2020). The protein expression levels of Fga and Fgb were previously reported to be positively correlated with the degree of Aβ deposition in AD humans and mice brains (Bian et al., 2021). Interestingly, our proteomics results showed that the protein expression levels of Fga and Fgb were decreased after HPTQ treatment. Therefore, Fga and Fgb might be potential therapeutic targets for HPTQ in reducing Aβ deposition.

Autophagy is a clearance mechanism that degrades abnormal or redundant intracellular components to promote cell survival. The ability of autophagy activation in AD to eliminate abnormal Aβ deposition and phosphorylated Tau tangles makes autophagy a vital mechanism for neuronal cell protection (Nilsson et al., 2013; Liu et al., 2015). Among the differentially expressed proteins obtained by proteomics analysis, Histone cluster 1 H1 family member (Hist1h1d) and Cathepsin B (Ctsb) were closely associated with autophagy. Hist1h1d is a histone family member responsible for gene transcription and protein folding. The up-regulated expression of Hist1h1d in neurons promoted Aβ clearance through autophagy (Bastrup et al., 2021). As an important cysteine protease in lysosomes, Ctsb was shown to be involved in several pathways connected with neuroinflammation and injury, including the autophagy pathway, lysosomal pathway, apoptotic pathway, and nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway associated with the NLRP3 inflammasome. A previous study has shown that overexpression of Ctsb led to lysosomal damage, causing autophagy dysfunction and the activation of the NLRP3 signaling pathway, ultimately leading to neuronal inflammation (Wang et al., 2017). In addition, the antigen processing and presentation pathways involving Ctsb were significantly enriched in the KEGG enrichment analysis, where the MHC II pathway could eventually generate pro-inflammatory CD4+ T cells (Monsonego et al., 2013). A study showed that over-activated CD4+ T cells in AD lead to excessive immune responses and promote the formation of β-secretase, which cleaves APP and forms pathological Aβ (Dai et al., 2021), while Ctsb has been shown to positively affect the activity of the MHC II pathway (Ma et al., 2022). Therefore, the reduction of the Ctsb expression level can reduce the overexpression of CD4+ T cells in AD and alleviate the inflammatory response of neurons. Among the obtained proteomics data, we found that the Hist1h1d levels were significantly increased and the Ctsb levels were significantly decreased after HPTQ treatment. Taken together, the above results suggest that HPTQ can regulate the expression of Hist1h1d and Ctsb, both of which might be potential therapeutic targets for HPTQ to exert its neuroprotective effects.

To validate the results of ITRAQ quantitative proteomics, four proteins showing high variation in expression, ACHE, CAMKV, Pxmp4, and Rims4, were screened and validated by western blotting analysis. ACHE hydrolyzes acetylcholine, and a normal ACHE expression is necessary for the brain to maintain healthy neuron signal transmission. The cholinergic system of the AD brain is defective, and over-activation of cholinesterase leads to decreased synaptic acetylcholine levels and ultimately to various neurodegenerative symptoms (Schliebs and Arendt, 2011; Ferreira-Vieira et al., 2016; Ahmed et al., 2017). Currently, cholinesterase inhibitors are an important class of drugs for the treatment of AD and have shown efficacy in clinical trials for the treatment of mild and moderate AD. In this study, the expression level of ACHE was decreased in the HPTQ group, indicating that the administration of HPTQ in AD has the effect of inhibiting ACHE and restoring the normal function of the cholinergic system. CAM kinase-like vesicle-associated protein (CAMKV) is a synaptic protein that belongs to the calmodulin-dependent protein kinase family and does not possess kinase activity. CAMKV plays an essential role in learning and memory by regulating dendritic spine generation, synaptic transmission, and neural plasticity (Liang et al., 2016). Combined with the recovery of spatial memory ability shown in the water maze experiment and increased levels of CAMKV expression in the HPTQ group in the proteomics data, we surmised that CAMKV is a potential therapeutic target of HPTQ to improve spatial memory deficits in AD rats. Peroxisome membrane protein 4 (Pxmp4) is an essential component of the peroxisome and is responsible for many redox reactions (Reguenga et al., 1999). KEGG pathway analysis showed that Pxmp4 is involved in the metabolism of reactive oxygen species (ROS) through the peroxisome pathway. A recent study has reported that excessive elevation of ROS could lead to oxidative stress and neuron loss in vitro (Angelova and Abramov, 2018). Pxmp4 has an important role in reducing the excess ROS in AD and maintaining redox homeostasis in neuron cells (Singh et al., 2019). The upregulation of peroxisome level in AD mice was shown to improve spatial memory, reduce Aβ deposition, and restore the impaired synaptic function (Inestrosa et al., 2013). In our results, the high expression of Pxmp4 after HPTQ treatment indicates that HPTQ could attenuate oxidative stress and promote the maintenance of redox homeostasis in neuronal cells of AD rats, which is consistent with our previous findings (Cai et al., 2018). The protein regulating synaptic membrane exocytosis protein 4 (Rims4) is critical in regulating neurosynaptic activity (Mittelstaedt et al., 2010). Previous studies have confirmed the important role of Rims4 in neuronal arborization and dendritic spine development as the knockdown of Rims4 protein reduced the complexity of neuronal arborization and caused abnormal axonal and dendritic growth (Alvarez-Baron et al., 2013). Up to now, the relationship between Rims4 and AD has not been studied, but the important role of Rims4 in neuronal axons and dendrites combined with the decrease of Rims4 expression level after HPTQ treatment suggest that Rims4 is a potential therapeutic target of HPTQ in AD treatment. Finally, our results showed S100-A4(S100a4), a protein that belongs to the multifunctional S100 protein family, is another potential therapeutic target (Donato et al., 2013). S100A4 has multiple functions in the nervous system. For example, it promoted neuronal survival in brain injury by regulating neuronal plasticity through the NRG/ErbB4 and ErbB2/Akt pathways (Pankratova et al., 2018). In another study, the interaction of S100a4 with cell surface heparan sulfate proteoglycans could increase the Ca2+ concentration in primary neuronal cells and modulate neuronal plasticity (Kiryushko et al., 2006). In addition, S100A4 could also exert antioxidant effects through the IL-10 receptor/Janus kinase/Signal Transducers and Activation Transcription system (IL-10R/JAK/STAT3) pathway to treat neuronal loss induced by brain injury (Dmytriyeva et al., 2012). Combining the neuroprotective role of S100a4 in neurodegeneration with our proteomics results, we believed that S100a4 could be an important therapeutic target of HPTQ in the treatment of AD.

Previous clinical results and studies have demonstrated that HPTQ can alleviate neuronal damage and improve AD symptoms by modulating multiple cellular mechanisms. However, the molecular targets of HPTQ have not yet been comprehensively studied. Hence, this study analyzed the data obtained from ITRAQ-based proteomic experiments conducted on the HPTQ and the Model groups to identify potential HPTQ therapeutic targets in AD rats. The proteomics analysis suggested that the effect of HPTQ in AD was achieved by improving the deposition of Aβ, regulating autophagy, inhibiting ACHE, regulating neuronal synaptic function and plasticity, reducing ROS damage, and alleviating oxidative stress. Furthermore, our findings suggested that HPTQ could promote protein biosynthesis in neurons and upregulate protein expression in the hippocampus of AD model rats. In conclusion, we analyzed several potential therapeutic targets and therapeutic mechanisms of HPTQ in AD treatment. In light of our findings, we believe that this study could provide a theoretical basis for the use of HPTQ in the treatment of AD.