Huang-Pu-Tong-Qiao Formula Ameliorates Tau Phosphorylation by Inhibiting the CaM-CaMKIV Pathway

Alzheimer's disease (AD) is a complex neurodegenerative disease. It is a chronic, lethal disease in which brain function is severely impaired and neuronal damage is irreversible. Huang-Pu-Tong-Qiao (HPTQ), a formula from traditional Chinese medicine, has been used in the clinical treatment of AD for many years, with remarkable effects. However, the neuroprotective mechanisms of HPTQ in AD have not yet been investigated. In the present study, we used AD models in vivo and in vitro, to investigate both the neuroprotective effect of HPTQ water extracts (HPTQ-W) and the potential mechanisms of this action. For the in vivo study, after HPTQ intervention, the Morris water maze test was used to examine learning and memory in rats. Transmission electron microscopy and immunofluorescence methods were then used to investigate neuronal damage. For the in vitro experiments, rat primary hippocampal neurons were cultured and cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Additionally, mRNA levels of CaM, CaMKK, CaMKIV, and tau were examined using qRT-PCR, and protein expression of CaM, CaMKK, p-CaMKIV, and p-tau were examined using western blot. In vivo, we revealed that HPTQ significantly improved learning and memory deficits and attenuated neuronal damage in the AD rat model. Furthermore, in vitro results showed that HPTQ significantly increased cell viability in the AD cell model. We also demonstrated that HPTQ significantly decreased the mRNA levels of CaM, CaMKK, CaMKIV, and tau and significantly decreased the protein expressions of CaM, CaMKK, p-CaMKIV, and p-tau. In conclusion, our results indicated that HPTQ improved cognition and ameliorated neuronal damage in AD models and implicated a reduction in tau phosphorylation caused by inhibition of the CaM-CaMKIV pathway as a possible mechanism.


Introduction
Alzheimer's disease (AD) is an age-related neurodegenerative disease [1] that is characterized by cognitive deficits, memory loss, language impairments, and mental and behavioral alterations [2,3]. Extracellular Aβ deposition forms senile plaques, and intracellular hyperphosphorylated tau produces neurofibrillary tangles (NFTs); these are the two hallmark histopathological lesions of AD. e World Alzheimer's Report of 2018 reported that 50 million people worldwide were living with dementia in 2018, and this number is expected to more than triple to 152 million by 2050. e cost of dementia is enormous, having reached $1 trillion by 2018, and is still increasing [4]. However, the mechanisms of AD are complex and may include the cholinergic hypothesis, the amyloid-beta (Aβ) hypothesis, the tau hypothesis, the neurotoxicity hypothesis, and the oxidative stress hypothesis, among others [5].
Tau is a microtubule-associated protein that plays an important role in the synthesis and stabilization of the neuronal cytoskeleton to maintain cell integrity [6]. Abnormal hyperphosphorylation of tau (p-tau) decreases the binding affinity of microtubules, resulting in NFTs. ese changes have been associated with multiple neurodegenerative disorders and are closely related to AD severity [7,8]. Tau can be phosphorylated in 85 residues [9], by kinases such as glycogen synthase kinase 3 (GSK-3), mitogen-activated protein kinase (MAPK), and Ca 2+ -calmodulin-dependent protein kinase II (CaMKII) [10]. ese kinases are considered to be targets for anti-p-tau therapies. Studies have shown that Tideglusib, a GSK-3 inhibitor, improves cognition in double transgenic mice expressing human tau and amyloid precursor protein (APP) by reducing tau phosphorylation, amyloid deposition, and neuronal loss [11].
However, the treatment of AD has historically been very difficult and remains challenging in the present. ere are currently four FDA-approved medications for improving AD symptoms, including cholinesterase inhibitors, such as donepezil, and the uncompetitive NMDA receptor modulator memantine. In addition, some novel approaches (including those involving BChE inhibition, antiamyloid agents, tau protein and related enzymes, and antioxidants) can alleviate patients' conditions, but do not modify AD progression. So far, amyloid-based therapies have fallen into the bottleneck in improving the course of AD, and tauoriented therapies are still in the early stages of development [12].
Huang-Pu-Tong-Qiao (HPTQ) is a hospital formula from the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine and is mainly composed of Shi Chang-pu, Yi-zhi, He Shou-wu, Chuang-xiong, Da-huang, and Ren-sheng.

Preparation and Ultra-Performance Liquid Chromatography (UPLC) Analysis of HPTQ.
e HPTQ solution was prepared fresh every 3 days by boiling the following six components in water: Shi Chang-pu (20 g), Yi-zhi (20 g), He Shou-wu (20 g), Chuang-xiong (20 g), Da-huang (6 g), and Ren-sheng (10 g). Tenfold water was added and refluxed for 2 h for the first time, and for the second time, 8fold water was added and refluxed for 1.5 h. e solution was then collected, concentrated to 50 mg/mL, and stored at 4°C.
e Acquity BEH C18 (2.1 mm × 100 mm, 1.7 μm; Waters, Milford, MA, USA) analytical column was coupled with a column filter, and the temperature was set at 30°C. e gradient elution with two mobile phase systems consisting of 0.05% (v/v) phosphoric acid in distilled water (A) and acetonitrile (B) was performed as follows: 5%-25% B for 0-2 min, 25%-67% B for 2-8 min, 67%-82% B for 8-10 min, and 82%-5% B for 10-12 min, with a reequilibrium time of 2 min. e flow rate and injection volume were 0.2 mL/min and 1 μL, respectively. e (photodiode array, PDA) detector wavelength was monitored at 210 nm. After excluding animals with outlying results in the Morris water maze test, the rats were randomly divided into four groups: the normal control group (CTRL), the model group (MODEL), the HPTQ group (1.41 g/kg, corresponding to three times the clinical equivalent according to the volume surface area folding algorithm), and the nimodipine group (NMDP; 20 mg/kg, corresponding to three times the clinical equivalent according to the volume surface area folding algorithm). Except for the CTRL group, rats in the other groups were intraperitoneally injected with 100 mg/kg D-galactose once daily for 42 days. When they had undergone 21 days of D-galactose treatment, the rats were fastened to a brain stereotaxic instrument (Stoelting Co., Wood Dale, IL, USA) and injected using a microsyringe (Anting Scientific Instrument, Shanghai, China) with 5 μL (10 μg) Aβ [25][26][27][28][29][30][31][32][33][34][35] into the bilateral hippocampus. e injection point was located at 4.4 mm posterior to and 2.2 mm from the anterior fontanelle as the origin point, and the needle was placed 3.0 mm from the rat hippocampus [13]. e CTRL group was injected with an equal volume of saline. After treatment with D-galactose for 14 days, the HPTQ group and NMDP group rats were fed by gavage according to body mass.

Morris Water Maze Test.
After intracerebral injection of Aβ 25-35 for 7 days in rats, the classical Morris water maze method was used [14]. e experiment lasted for 6 days. For the first 5 days, the navigation experiment was performed. Rats were placed into the water from one of four quadrants, facing the wall of the pool, and the time taken to find the platform within 90 s was recorded. If the rats did not find the platform within 90 s, they were placed on the platform and left there for 20 s. On day 5, no guidance was given, and the time taken to find the platform was recorded as the escape latency. On day 6, the platform was removed and the same parameters were set as for the navigation experiments. e number of times that the rats crossed the area where the platform had been located was recorded.

Animal Sample Preparation.
After the Morris water maze test, rats were sacrificed by anesthesia with an intraperitoneal injection of 1% sodium pentobarbital (40 mg/ kg). Brain tissue was quickly removed and placed on saline ice. Some hippocampal tissue was cut to 1 mm 3 size and stored in glutaraldehyde, to be used in transmission electron microscopy (TEM) experiments, and the remaining hippocampal tissue was isolated from both sides of the brain and stored at −80°C for western blot and qRT-PCR experiments.

Determination of the Fluorescent Expression of p-Tau.
Brain tissue was fixed in 4% paraformaldehyde, then dehydrated, embedded, sectioned, and placed into pH 8.8 for repair and sealing. Tissue was then incubated overnight at 4°C in primary rabbit anti-rat p-tau antibody. e following day, the brain tissue was incubated in fluorescent secondary FITC-rabbit anti-goat IgG antibody at room temperature for 2 h, washed three times in phosphate-buffered solution (PBS), and incubated in DAPI for 10 min. After washing the slides, an antifluorescent quencher was used to coverslip the tissue, and images were collected using an inverted fluorescence microscope.

Cell Culture and Intervention.
e methods of culturing primary hippocampal neurons were performed according to Korkotian et al. [15,16]. Neonatal pups were sacrificed after immersion in 75% alcohol for 20 s. Hippocampi were dissected out and cut into 1 mm 3 pieces after separating the blood vessels and meninges. e tissue was digested with 0.125% trypsin for 20 min at 37°C, before DMEM/F12 medium with 10% fetal bovine serum (FBS) was added to terminate the digestion. e cells were separated and collected by centrifuging the cell suspension at 1500 rpm for 5 min. Next, the suspension was swirled gently and filtered through a 200-mesh sieve. Isolated primary hippocampal neurons were cultured in DMEM/F12 nutrient solution containing 20% FBS. Subsequently, the cells were seeded onto poly-D-lysinecoated plates at a density of 1 × 10 6 /mL and maintained at 37°C in an incubator with 5% CO 2 .
e medium was replaced with NM supplemented with 2% B27 and 0.5 mM/L L-glutamine, and half of the medium was changed every 3 days.
Based on previous experimental results from our research group, the neurons were assigned to the following treatment groups after being cultured for 7 days

Cell Viability.
After the cells were treated and cultured at 37°C in a 5% CO 2 incubator, the supernatant was removed and 20 μL of 5 mg/mL MTT was added to each well and incubated for 4 h. e supernatant was removed and 150 μL DMSO was added to each well. e optical density at 490 nm was measured using a microplate reader (318C+, Shanghai Peiou Co., Ltd., Shanghai, China).

Mechanism of HPTQ on AD In Vitro and In Vivo
e total RNA of hippocampal tissue or treated cells was extracted according to the instructions using a TRIzol kit (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from total cellular RNA using a RevertAid First Strand cDNA Synthesis Kit ( ermo Fisher Scientific, San Jose, CA, USA). qRT-PCR was performed using an ABI PRISM 7500 System (Applied Biosystems, Foster City, CA, USA; Life Technologies) and QuantiNova SYBR Green PCR Kit (Qiagen, Dusseldorf, Germany). e mRNA levels of CaM, CaMKK, CaMKIV, tau, and β-actin in hippocampal tissues and neurons were measured using qRT-PCR. e expression levels of each gene were calculated using the 2 −ΔΔCT method [17]. e mRNA expression levels were displayed as fold changes compared with the internal control, β-actin.
e primer sequences are shown in Table 1. e total cellular protein was extracted from hippocampal tissue or treated cells using a protein extraction kit, and the concentrations were measured using a Bicinchoninic Acid Kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were separated by 10%-12% SDS-PAGE (Bio-Rad) and transferred onto polyvinylidene fluoride (PDVF) membranes (Millipore, Billerica, MA, USA). After subsequently blocking in 5% nonfat dry milk for 2 h, the membranes were incubated overnight at 4°C with the relevant primary antibody, followed by incubation with secondary antibodies for 1.5 h at 37°C. e primary rabbit anti-rat monoclonal antibodies against CaM, CaMKK, p-CaMKIV, and p-tau were all obtained from Abcam (1 : 1000, Cambridge, UK). e secondary antibody, HRP-conjugated goat anti-rabbit IgG, was obtained from AmyJet Scientific Inc. (1 : 20,000, Abbkine, Wuhan, China). Rabbit anti-rat β-actin (1 : 2000, Abbkine, AmyJet Scientific Inc.) was used as an internal control. e protein bands were visualized using electrogenerated chemiluminescence (ECL; ermo Fisher Scientific) and analyzed on a gel imager (FluorChem M, ProteinSimple, USA).

Statistical Analysis.
e measured data are presented as mean ± standard deviation (SD). Multiple group means were compared by one-way analysis of variance and the LSD method. All analyses were performed using SPSS version 23.0 software, with a significance level set at P < 0.05.

Protective Effect of HPTQ on Neuronal Damage and
Memory Improvement in AD Model Rats

Learning and Memory in AD Model
Rats. e Morris water maze was performed to evaluate spatial reference memory. In the escape latency test, the time taken to reach the platform was significantly higher in the MODEL group rats than in the CTRL group rats (P < 0.01). However, compared with the MODEL group rats, the time taken to reach the platform was significantly lower in the HPTQ and NMDP group rats (P < 0.05 and P < 0.01). When comparing the number of times crossing the original platform, MODEL group rats had significantly lower frequencies than the CTRL group rats (P < 0.05 or P < 0.01). Furthermore, compared with the MODEL group rats, the frequencies were significantly higher in the HPTQ and NMDP group rats (Figure 3).

Effect of HPTQ on Hippocampal CA3 Region Ultrastructure in AD Rats.
e TEM results revealed that, in the CTRL group, tissue characteristics included intact nuclear membranes and condensed chromatin, with abundant organelles in the cytoplasm; in particular, a large number of ribosomes and rough endoplasmic reticulum were clearly visible. e cristae of mitochondria were intact, normal in structure, and without damage. In the MODEL group, the structure of neuronal nuclear membranes was blurred and irregular, and there were cytoplasmic edema and severe organelle damage. e mitochondria were swollen with some broken cristae, and vacuolar degeneration was apparent. In addition, most rough endoplasmic reticula were dilated, empty gun state, and even partly disappeared, with greatly reduced numbers of ribosomes. In the HPTQ and NMDP groups, nuclear membranes were relatively intact, and there were more ribosomes in the rough endoplasmic reticulum in the cytoplasm of neurons. A small number of mitochondria were slightly swollen and their cristae processes were destroyed ( Figure 4).

Effect of HPTQ on Fluorescent p-Tau Expression in AD
Rats. Immunofluorescence was used to detect the fluorescent protein expression of p-tau in the hippocampal CA3 region of AD rats. Compared with the CTRL group, the fluorescent expression of p-tau was significantly higher in the MODEL group, although the number of neurons was lower. However, compared with the MODEL group, the fluorescent expression of p-tau in the HPTQ and NMDP groups was significantly lower, while the number of neurons was higher ( Figure 5).

Protective Effect of HPTQ-Medicated Serum on Aβ 25-35 -Induced Neurotoxicity in Rat Hippocampal Neurons.
To further determine the protective effect of HPTQ-medicated serum in rat hippocampal neurons, cell viability and cytotoxicity were measured using MTT. After the establishment of a cell model induced by Aβ 25-35 , either 5%,  10%, or 15% HPTQ-medicated serum and nimodipine solution was continued to intervene for another 24 h. Compared with the CTRL group, the cell survival rate was significantly lower in the MODEL group (P < 0.01).
However, compared with the MODEL group, the cell survival rate was significantly higher in the 10% and 15% HPTQ-medicated serum groups and the NMDP group (P < 0.05 or P < 0.01; Figure 6).

Discussion
In the present study, we investigated the neuroprotective effect of HPTQ against Aβ-induced toxicity in rats and rat hippocampal neurons. We also examined the CaM-CaMKIV pathway mediation of tau phosphorylation. e results revealed that HPTQ improved hippocampal neuronal damage and increased cell viability. In addition, HPTQ decreased tau phosphorylation via inhibition of the CaM-CaMKIV pathway. In addition, the use of KN-62, a CaMKIV inhibitor, further demonstrated that HPTQ decreases tau phosphorylation via inhibition of the CaM-CaMKIV pathway.
We also specifically examined the CaM-CaMKIV pathway-mediated reduction of tau phosphorylation. In AD, there is a wide imbalance in Ca 2+ signaling homeostasis [24]. Previous research has shown that Aβ enhances Ca 2+ entry and that its overload aggravates Ca 2+ imbalance [25]. CaM is a cytosolic protein that is expressed in all eukaryotic cells. It is the main receptor for Ca 2+ in cells and combines with Ca 2+ to form a Ca 2+ / CaM complex that participates in modulating cellular signaling pathways [26]. CaMK is one kind of signaling protein that is regulated by Ca 2+ /CaM. Multifunctional CaMKs are abundant in the brain and are activated by combining with Ca 2+ /CaM, such as members of the CaMK cascade (CaMKK and CaMKIV) that alter and regulate the functionality of numerous protein substrates by phosphorylating them. For example, CaMKIV can be activated by CaMKK phosphorylating the kinase in its activation loop on r196 [27]. CaMKIV has a two-way regulatory function and can either promote or inhibit cell proliferation by regulating transcription factors. e multifunctional CaMKIV kinase has multiple substrates for activation, including tau. Tau hyperphosphorylation can lead to the formation of NFTs in the AD brain, and pathological tau-induced neurotoxicity therefore plays a key role in driving neurodegenerative dysfunction [28]. Some studies have reported that overactivation of kinases and inactivation of phosphatases can lead to tau hyperphosphorylation [29], such as glycogen synthase kinase-3β (GSK-3β), Janus kinase (JNK), mitogen-activated protein kinase (MAPK), and Ca 2+ /calmodulin-dependent kinase [30,31]. In previous studies, TianDiJingWan treatment significantly reduced p-tau aggregation and improved learning and memory in an AD rat model [32], and the Aβ-N-terminus monoclonal antibody (mAb) A8 decreased levels of both Aβ and p-tau in the brains of APP/ PS1 mice and alleviated cognitive dysfunction [33]. erefore, the inhibition of tau hyperphosphorylation can meaningfully improve both neuronal damage and cognitive function. e HPTQ used in the current study was mainly composed of Radix Rhei Et Rhizome, Chuanxiong Rhizoma, Panax Ginseng C. A. Mey, Acori Tatarinowii Rhizoma, Fructus Alpinae Oxyphyllae, and Radix Polygoni Multiflori Praeparata. Modern pharmacological  Evidence-Based Complementary and Alternative Medicine studies have indicated that ginseng is an effective drug for protecting the nervous system and that it can improve and relieve stress and fatigue and prevent neurodegenerative diseases [34]. Ginsenoside Rg1, an active ingredient of ginseng, was reported to improve cognitive impairment and memory in SAMP8 mice and may regulate PKA/ CREB activity via the blood-brain barrier [35]; the effective ingredients of rhubarb are anthraquinone Evidence-Based Complementary and Alternative Medicine 9 derivatives, including emodin, aloe-emodin, chrysophanol, rhein, physcion, and danthron, and these can improve central nervous system diseases such as traumatic brain injury, brain tumors, and Alzheimer's disease [36]. Furthermore, β-asarone can improve cognitive function by suppressing neuronal apoptosis in an AD rat model induced by Aβ [37], and combined with tenuigenin, it can significantly improve the efficacy of memantine in treating moderate-to-severe AD [38]. Tetrahydroxystilbene glucoside (TSG) is the major active component of Polygonum multiflorum, which can improve cognitive deficits in AD rats [39]. Ferulic acid, an antioxidant, is the main component of Ligusticum chuanxiong, which can protect the brain from Aβ neurotoxicity and neuronal death caused by ROS [40]. Results from our study suggest that the Aβ toxin activates the CaM-CaMKIV pathway, leading to tau hyperphosphorylation, whereas HPTQ and the positive control NMDP attenuated this effect. Moreover, treatment with KN-62 only, as well as cotreatment with HPTQ and KN-62, significantly inhibited tau phosphorylation. However, there were no statistically significant differences between treatment with HPTQ only and cotreatment with HPTQ and KN-62.

Conclusions
For more than 10 years, HPTQ has been used as an inhospital preparation for the clinical treatment of dementia. However, the mechanisms of its effect are unclear. Our results show that: (1) HPTQ is a multiflavored Chinese medicine composition that contains stilbene glycoside, ferulic acid, ginsenoside Rg1, aloe-emodin, β-asarone, emodin, and chrysophanol as its various chemical components (2) HPTQ is protective against hippocampal neuronal damage and reduces hippocampal neuronal loss and acts as a neuroprotective agent in an AD rat model (3) HPTQ reduces tau phosphorylation via inhibition of the CaM-CaMKIV pathway Overall, our study revealed a potential mechanism of HPTQ in the treatment of AD, thus providing new ideas for the development of therapeutic AD drugs based on eliminating the potentially pathological product tau.

Data Availability
e data used to support the findings of this study are available upon request by contact with the corresponding author.

Conflicts of Interest
e authors declare no conflicts of interest.