Components of Goutengsan in Rat Plasma by Microdialysis Sampling and Its Protection on Aβ1–42-Induced PC12 Cells Injury

Goutengsan, a Chinese herbal formula, potential protection on Alzheimer's disease (AD) has been less reported. In current study, we investigated the protection of Goutengsan on Aβ1–42-induced pheochromocytoma-derived cells (PC12). Furthermore, the components from Goutengsan in rat plasma were identified by microdialysis (MD) for in vivo sampling. Meanwhile, the protection of components identified was also verified. At last, we found that Goutengsan has a potential protective effect on Aβ1–42-induced PC12 cells via reducing cells damage and increasing cells vitality as well as six components (pachymic acid, liquiritin, rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine) which may be effective components. This study helps to understand the treatment of Goutengsan for AD and would facilitate the clinical and further studies for this formula.


Introduction
Alzheimer's disease (AD), also known as senile dementia, was a neurodegenerative disease and influenced a population of approximately 26 million worldwide with the number growing twice in 20 years [1]. The production and accumulation of amyloid (A ) were proved to be the central pathogenesis of AD, particularly soluble A oligomers which had been characterized by the progressive decline of cognitive function and behavioral derangement [2,3]. Nowadays, the mainstream medication for AD is typically acetylcholine esterase inhibitor (AchEI), but symptomatic improvement is limited [4].
Traditional Chinese medicine (TCM) has been used for preventing and treating cognitive decline and the anti-AD agents have been made to develop from TCM for a long time [5][6][7][8]. Goutengsan, a Chinese herbal formula, consists of 11 medicinal herbs including Gouteng, Chenpi, Maidong, Banxia, Fuling, Renshen, Fangfeng, Juhua, Shengjiang, Gancao, and Shigao (Table 1). It has remarkable functions on chronic headache and hypertension [9]. Studies have suggested that Goutengsan has a function on AD and its antidementia effect was due to antihypertensive, free radical scavenging, and antiexcitotoxic effects, which were attributed to phenolic compounds and indole alkaloids [9][10][11]. Our early exploratory research also indicated that Goutengsan had a protective effect on AlCl 3 -induced AD rats via improving brain index [10], and Goutengsan also had obvious protective 2 Evidence-Based Complementary and Alternative Medicine effect on glutamic-acid-induced and H 2 O 2 -induced PC12 cells injury [12]. Therefore, the protection of Goutengsan on AD treatment is worthy of further study. Microdialysis (MD), a well-established technique for the sampling of the extracellular space, has the merits of sampling for continuous and simultaneous monitoring of changes in the local biochemical environment [13,14]. This technique uses a catheter consisting of the perfusate, the surrounding medium, and substances, surrounded by a semipermeable membrane, which is perfused by fluid and inserted into a region of interest [15]. In recent years, MD has been proposed for the applications of pharmacological and pharmacokinetic studies. Thus, in this study, we investigated the protection of Goutengsan on A 1-42 -induced pheochromocytoma-derived cells (PC12). Furthermore, the components from Goutengsan in rat plasma were identified by MD for in vivo sampling. Meanwhile, the protection of components identified was also verified.  ). The remaining voucher specimens were deposited at the Jiangsu Provincial Academy of Chinese Medicine. Gouteng, Chenpi, Maidong, Banxia, and Fuling, 300 g for each; Renshen, Fangfeng, and Juhua, 200 g for each; Shengjiang and Gancao, 100 g for each; and 500 g Shigao were extracted together by refluxing with boiling water. They were boiled with 24 L water twice with 1.5 h for each time. Finally, all extract fractions were pooled and concentrated to 350 mL by rotary evaporator. The concentrated solution was stored at 4 ∘ C until use.

Western Blot Analysis.
After treating with drug, A 1-42induced PC12 cells were washed with PBS. Cell lysates were obtained using ice-cold lysis buffer for proteins extract and were centrifuged with 13,000 ×g for 10 min. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto a Polyvinylidene Fluoride (PVDF) membrane blocking with 5% skim milk. Sequentially, membranes were incubated with the primary antibodies Bax (1 : 200), Bcl-2 (1 : 200), caspase-3 (1 : 200), and caspase-9 (1 : 200) at 4 ∘ C. Additionally, membranes were rinsed with Tris Buffered Saline Tween (TBST) for three times (10 min/time) and then incubated with the horseradish peroxidase-bound secondary antibody (1 : 40) in a shaker for 1 h at room temperature. Chemiluminescence reagents were added for the visualization of the protein bands. The quantification of proteins was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Quantitative Real-Time PCR.
Total RNA of PC12 cells were extracted by TRIzol reagent. Then it was reverse transcribed with a SuperScript III First-Strand Synthesis System for quantitative real-time polymerase chain reaction (q-PCR) following manufacturer's indications. The sense primer for Bax was 5 -GAGCTGGTGGTTGACTTTCTC-3 and the antisense primer was 5 -TCCATCTCCGATTCA-GTCCCT-3 . The sense primer for caspase-3 was 5 -TAA-ATGAATGGGCTGAGCTG-3 and the antisense primer was 5 -ATGGAGAAATGGGCTGTAGG-3 . The sense primer for Bcl-2 was 5 -AATATCCAATCCTGTGCTGCTA-3 and the antisense primer was 5 -GTCCACGTTCTTCATTGT-TACTTC-3 . The sense primer for caspase-9 was 5 -AGG-GAAGAGGAGGAGATGAGA-3 and the antisense primer was 5 -GCTGGGTTTGTCGGTGTT-3 . q-PCR was performed with an ABI 7900 sequence detector (Life Technologies, Carlsbad, CA, USA) using the SYBR Green method and ( ) 6 random hexamer with primers purchased from Invitrogen. PCR thermocycling parameters were 95 ∘ C for 10 min, 40 cycles of 95 ∘ C for 15 s, and 60 ∘ C for 1 min. Each sample was run in triplicate and was normalized to 18S RNA. Fold changes were determined using the DDCt method. Primer sequences are available upon request.

Animal
Model. Female Sprague Dawley (SD) rats (180 ± 20 g) were purchased from SLAC experimental animals Co., Ltd. (Shanghai, China). The animal experiment protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Jiangsu Provincial Academy of Chinese Medicine. Before experimentation, the rats were allowed one-week acclimation period using the independent isolated feeding cages kept at a temperature of 25 ∘ C and a relative humidity at 45%. The rats were fed with normal diet and distilled water ad libitum. 20 rats with lateral cerebral ventricle injected with 5 L A 1-42 (20 M) were used for modeling.

Microdialysis Experiment.
After modeling for 24 h, the rats were anesthetized with chloral hydrate (300 mg/kg, i.p.) and rat body temperature was maintained at 37 ∘ C during the experiment. The blood microdialysis system is composed of the probes for blood CMA/12 microdialysis probe (membrane length, 2 mm; molecular weight cut-off 20 kDa), CMA/402 microinjection pump, and CMA/470 automatic collector (CMA, Stockholm, Sweden). The blood microdialysis probes were fixed in the jugular vein/right atrium and then Ringer's solution (122 mM sodium chloride, 3 mM potassium chloride, 0.4 mM monopotassium phosphate, 1.2 mM magnesium sulfate, 25 mM sodium bicarbonate, and 1.2 mM calcium chloride; pH = 7.0) was perfused at a flow rate of 1 L/min with the CMA microinjection pump for the postsurgical balancing for 1 h. Then they were orally gavaged with Goutengsan extraction (20 g crude drugs/kg).
Two hours later, the blood microdialysis samples (30 L) were collected for 15 min, 30 min, 45 min, and 60 min. The samples were added with four times volume of methanol and vortexed for 5 min. To remove the precipitation of proteins, all samples were centrifuged at 11,000 rpm for 10 min. Then the supernatants were evaporated until dryness at room temperature by nitrogen. The residue was dissolved in 100 L methanol followed by centrifugation at 11,000 rpm for 10 min. At last the samples were analyzed by using the validated UPLC/Q-TOF-MS system.

Ultra-Performance Liquid Chromatography (UPLC).
The UPLC/Q-TOF-MS analysis was performed on a Q-TOF Synapt system (Waters, Milford, MA, USA), equipped with electrospray ionization (ESI) source. Positive ion detection mode was conducted in this analysis. Nitrogen (N 2 ), the desolvation gas, was set at the temperature of 350 ∘ C with a flow rate of 700 L/h, and the source temperature was 120 ∘ C. The capillary and cone voltages were 2.5 kV and 40 V, respectively. The mass data were recorded within the range of 50-1200 Da with a scan time of 0.2 s. The transfer collision energy ( ) was 4 V.

Statistical Analysis.
All data were taken from three independent experiments and then expressed as means ± standard deviation (SD). One-way ANOVA was performed to compare the statistical analysis by GraphPad Prism 5.0 (San Diego, CA, USA). Tukey's test was then followed to determine the difference between groups. Statistical significance was indicated by the value which was less than 0.05.

Identification of Components in Rat Blood Samples.
After being sampled with MD, the main compounds in rat plasma were analyzed and identified by Ultra-Performance Liquid Chromatography (UPLC). UPLC chromatogram of Goutengsan was shown in Figure 4(a). The chromatogram showed that the sample could be eluted completely under the used UPLC conditions within 14 min and separated satisfactorily. Six components (1-6) (Figure 4(b)) in plasma were identified and tentatively characterized by UPLC/Q-TOF-MS. The results indicated that six components (1-6) could be absorbed into blood.
Liquiritin (1, 11.23 g/mL) whose molecular formula was determined to be C 21

Protection of Six Components on A 1-42 -Induced PC12
Cells. To verify the role of components (pachymic acid, liquiritin, rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine) identified in rat plasma on cell apoptosis, we used western blotting to evaluate the protein expression. As can be seen from  (Figure 5(a)), the protein expression of Bcl-2 was significantly increased comparing with model group. Thus, it appears that pachymic acid, liquiritin, rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine could regulate A 1-42induced PC12 cells apoptosis via enhancing the expression of Bcl-2.

Discussion
Goutengsan, a widely used Chinese herbal formula, was first recorded in "Effective Prescriptions for Universal Relief" (Chinese name: Benshi Fang) for chronic headache and hypertension. At present, our researches of Goutengsan were mainly focused on the effect of A in brain for AD treatment. As a Chinese herbal formula, Goutengsan consisted of so more medicinal herbs that it is difficult to study its complicated composition. Different analysis techniques should be explored for rapid analysis of Chinese herbal formula.
Microdialysis (MD) is a powerful sampling technique that enables monitoring of dynamic processes in vitro and in vivo [16]. MD can be used for researching the diseases, pathology, and therapeutic methods in the fields of neurotransmitters coupled with all kinds of analysis technique for quantitative analysis [17]. The applications of MD and its hyphenated techniques play an important role in the analysis of endogenous substances, pharmacokinetic study of Traditional Chinese Medicine (TCM), and drug interactions [18]. MD combined with chromatographic or electrophoretic methods had an extensive application in recent years. Therefore, in order to analyze the components of Goutengsan in rat plasma, MD coupling with UPLC/Q-TOF-MS was used for sampling. We inferred that Goutengsan at least pachymic acid, liquiritin, rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine could be absorbed into blood. Amyloid-beta (A ) peptides, especially A 1-42 , cause neurotoxicity and cell death in the brain of Alzheimer's disease (AD) at higher concentrations [19]. Some soluble A oligomers, highly prone to aggregation and self-assemble to form a heterogeneous mixture of oligomers and protofibrils, are considered as the major neurotoxic species in AD [20][21][22]. A induced apoptosis occurs in the AD brain and plays a role in neuronal dysfunction and neurodegeneration, which contributes to the progression of AD [23]. In this study, A 1-42 exposed PC12 cells were used to investigate the protection mechanism of Goutengsan in vitro and rats lateral cerebral ventricle injected with A 1-42 was used in vivo.
Executioner caspases (caspase-3, caspase-6, and caspase-7) and upstream effector caspases (caspase-2, caspase-8,   110  120  130  140  150  160  180  190  170  200  210  220  240  230  260  250  280  270  300  290  320  330  310  340  360  350  380  390  370  110  120  130  140  150  160  170  180  190  200  210  220  230  240  250  260  270  280  290  300  310  320  330  340  350  360  370  380   caspase-9, and caspase-10) are well known as mediators in AD brains [23]. Caspase-3 and caspase-9 may also be potential therapeutic targets for AD [17]. It is well known that caspase-9 can activate caspase-3. Activated caspase-3 cleaves poly ADP-ribose polymerase (PARP) that helps cells to maintain their viability. Caspase-3 is also demonstrated to cleave amyloid precursor protein (APP) and tau [24]. Caspase-9 is reported to mediate a fall in synaptic plasticity and memory deficits [25]. A change of Bax/Bcl-2 ratio and subsequent activation of caspase-9 play a central role in the apoptosome-dependent intrinsic pathway of apoptosis [26]. Western blotting and q-PCR were used to evaluate the cell apoptosis in this experiment. Our results showed that Goutengsan could upregulate the expression of Bcl-2 and downregulate the expression of Bax, caspase-9, and caspase-3. In addition, Goutengsan could also regulate A 1-42 -induced PC12 cells apoptosis via reducing Bax, caspase-9, and caspase-3 levels, while increasing Bcl-2 level. As a Chinese herbal formula, Goutengsan consisted of 11 medicinal herbs. In the present study, 6 components from 3 medicinal herbs of Goutengsan were identified in rat plasma by MD sampling. This does not mean that other components may not play a role in the protective effect on A 1-42 -induced cell death. We just found these components because of the limitation of analytical approach for finding low content components existing in MD samples. And some components may change into other components or improve the absorption of other components. Four components including rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine were main alkaloids in Gouteng, while pachymic acid and liquiritin were the components from Fuling and Gancao, respectively. Early exploratory research indicated that alkaloids of Goutengsan had a protective effect on AD [9,10]. So we inferred that six components (pachymic acid, liquiritin, rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine) identified in rat plasma were effective components in Goutengsan. At last, western blotting was used for evaluating the protection of six components identified in rat plasma on A 1-42 induced PC12 cells; the result suggested that six components could protect A 1-42induced PC12 cells from apoptosis via upregulating the expression of Bcl-2 and downregulating the expression of caspase-3.

Conclusion
In conclusion, Goutengsan has a protective effect on A 1-42induced PC12 cells as well as 6 components (pachymic acid, liquiritin, rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine) which could be absorbed into blood via reducing cell damage and increasing the vitality of the cells. Thus, the results turned out that pachymic acid, liquiritin, rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine were effective components for the treatment of AD. This study provides pharmacological application of Goutengsan for the treatment of AD.