Mitigating Effects of Liriope platyphylla on Nicotine-Induced Behavioral Sensitization and Quality Control of Compounds

In this study we investigated the mitigating effects of Liriope platyphylla Wang et Tang extract on behavioral sensitization and the quantification of its major compounds. The extract of L. platyphylla reduces the expression of tyrosine hydroxylase (TH) protein, which is increased by nicotine, back to normal levels, and increases the expression of dopamine transporter (DAT) protein, which is reduced by nicotine, back to normal levels in PC12 cells. In this study, rats received nicotine (0.4 mg/kg, subcutaneously) only for seven days and then received extract of L. platyphylla (200 or 400 mg/kg, oral) 1 h prior to nicotine administration for an additional seven days. The extract of L. platyphylla reduced locomotor activity compared to the nicotine control group in rats. The extract of L. platyphylla significantly attenuated the repeated nicotine-induced DAT protein expression in the nucleus accumbens (NAc), but there was no effect on increased TH protein expression in the dorsal striatum. These findings suggest that L. platyphylla extract has a mitigating effect on nicotine-induced behavioral sensitization by modulating DAT protein expression in the NAc. For quality control of L. plathyphylla, spicatoside A and D, which are saponin compounds, were quantified in the L. platyphylla extract. The amounts of spicatoside A and D in L. platyphylla extract obtained from ultra-high-performance liquid chromatography with tandem mass spectrometry were 0.148 and 0.272 mg/g, respectively. The identification of these compounds in L. platyphylla, which can be used for quality control, provides important information for the development of drugs to treat nicotine dependence.


Introduction
Cigarette smoking is one of the major global public health problems. Nicotine, a major active alkaloid in tobacco products, acts as a psychoactive compound in the brain and causes alterations in of spicatoside A and D, L. platyphylla roots were ground and extracted ultrasonically with methanol at 40 • C for 1 h. The extract was filtered through a 0.22 µm polytetrafluoethylene (PTFE) membrane syringe filter and lyophilized. Then, 400 mg of the extract was re-suspended in 10 mL H 2 O and extracted twice with 10 mL n-butanol (n-BuOH). The n-BuOH layer was concentrated under reduced pressure. For quantifying small amounts of compounds, L. platyphylla roots were homogenized using a ball mill (MM400, Retsch, Haan, Germany). Fine powder of L. platyphylla (100 mg) was suspended in 1 mL of methanol and ultrasonically extracted for 1 h at 40 • C. The extract was centrifuged at 15,000 rpm for 5 min at 4 • C, and the supernatant was filtered through a 0.22 µm PTFE membrane syringe filter.

Preparation of Sample and Standard Solutions
Spicatoside A and D isolated from the roots of L. platyphylla grown in Cheongyang, South Korea, were identified using medium-pressure liquid chromatography (MPLC) and NMR techniques (results published elsewhere). Two accurately weighed standard compounds were dissolved in methanol to prepare stock solutions containing 1.00 mg/mL. The stock solutions were diluted with methanol to obtain calibration solutions in the range of 0.019-5 µg/mL. Finally, diluted stock solutions were filtered through 0.22 µm PTFE membrane syringe filter and kept at 4 • C. Nicotine tartrate was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Nicotine was dissolved in phosphate-buffered saline (PBS, pH 7.4) for the in vitro study or 0.9% physiological saline for the in vivo study. The extract of L. platyphylla was dissolved in distilled water for the in vitro and in vivo studies. All solutions were freshly prepared just before each experiment.

Cell Culture
Since the PC12 cell line expresses TH and DAT proteins well [21,22], we used the PC12 cell line in this study. The PC12 cell line was purchased from American Type Culture Collection (University Boulevard Manassas, VA, USA). PC12 cells were cultured and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with heat-inactivated 5% horse serum, 10% fetal bovine serum, and 1% streptomycin/penicillin in a humidified atmosphere at 37 • C and 5% CO 2 , as previously described [21].

Western Immunoblotting
Western immunoblotting was performed as previously described [23].
For western immunoblotting, PC12 cells were plated in 6-well plates at a density of 1 × 10 4 cells/well in the culture medium for 24 h. The treated doses of L. platyphylla extract and nicotine following in vitro study were determined from the results of a cell viability test. Cells were co-treated with L. platyphylla extracts (10 µg/mL) and a nicotine solution (1000 µM/mL) for an additional 24 h. After incubation, cells were collected in 0.1 mL of a mixture of radioimmunoprecipitation assay (RIPA) buffer and protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific), after which they were incubated at 4 • C for 30 min. After incubation, lysates were centrifuged at 13,000 rpm for 30 min at 4 • C. The pellet, which primarily contained nuclei and large debris, was discarded and the supernatant was obtained. The concentration of the solubilized proteins in the supernatant fraction was determined based on the bicinchoninic acid (BCA) assay using a BCA assay kit (Thermo Fisher Scientific). The proteins in the supernatant fraction were resolved using 10% bisacrylamide gel electrophoresis and then the separated proteins were transferred to a polyvinylidene fluoride membrane (PVDF; Bio-Rad, Hercules, CA, USA). The protein-transferred PVDF membrane was treated with blocking buffer containing 5% bovine serum albumin in a mixture of Tris-buffered saline and 0.1% Tween-20 (TBST), and washed three times for 10 min each with TBST. After washing, the membrane was probed with a rabbit primary antiserum for tyrosine hydroxylase (TH, 1:1000, Cell Signaling Technology, Danvers, MA, USA) and dopamine transporter (DAT, 1:1000; abcam, Cambridge, MA, USA) for 18 h at 4 • C on a shaker. Then, the membrane was re-washed three times, and incubated with goat anti-rabbit immunoglobulin G horse radish peroxidase-labeled secondary antiserum (Thermo Fisher Scientific) for 1 h at room temperature. Immunoreactive protein bands were detected by enhanced chemiluminescence reagents (Thermo Fisher Scientific) using Image Lab software (version 5.2.1, Bio-Rad). After stripping, the same membranes were re-probed with a mouse primary antiserum for β-actin (1:2000; #A5316, Sigma-Aldrich, St. Louis, MO, USA) for blot normalization. Immunoreactive protein bands on the membrane were semi-quantified using ImageJ software (version 1.52a, National Institutes of Health, Bethesda, MD, USA).

Animals
Adult male Sprague-Dawley rats weighing between 200 and 230 g (6 weeks old) were purchased from Orient Bio. Inc. (Seongnam, South Korea). Rats were separated into pairs and acclimated to their home cages for a minimum of 5 days. Food and water were provided ad libitum. Animals were housed in a temperature (21-23 • C) and humidity (45-55%) controlled vivarium under a 12/12 h light/dark cycle (light on at 8:00 a.m.). Experimental treatments were applied in a quiet room to minimize environmental stress. All animal procedures were approved by the Institutional Animal Care and Use Committee of Korea Institute of Toxicology (Approval No. 1811-0443) and conducted in accordance with the provisions of the Guide for the Care and Use of Laboratory Animals issued by the U.S. National Institute of Health.

Locomotor Activity
The behavioral assessment for locomotor activity test was performed as described previously [5]. Rats were acclimated to a square seamless open-field arena (43 × 43 × 30 cm, #ENV-515S, Med Associates, Georgia, VT, USA) in an illuminated and sound-attenuated cubicle (#SAC-283422-NIR, Med Associates) for at least 3 days (for 30 min/day) to avoid the influence of environmental factors prior to experiments. Locomotor activity was recorded using a computer-based monochrome/near infrared video camera (#VID-CAM-MONO-1, Med Associates). On the test day, to determine the mitigating effects of L. platyphylla on nicotine-induced locomotor activity, rats were pretreated with vehicle or L. platyphylla extract solution 1 h before measuring locomotor activity. After pretreatment, rats were placed in the open-field arena and basal activity was measured for 30 min. Rats were given the final administration of saline or nicotine after recording basal activity. Locomotor activity was recorded for an additional 1 h. Changes in locomotor activity were quantified using a computer-based video tracking system (Ethovision XT14, Noldus, Wageningen, The Netherlands) and values are expressed as total distance traveled (centimeter) for 1 h.

Brain Tissue Collection
After the measurement of locomotor activity for 1 h following the final saline or nicotine administration, rats were deeply anesthetized with 2-4% isoflurane, decapitated, and brains were rapidly removed. Tissue sections were serially cut using a stainless-steel coronal brain matrix (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, USA) at 4 • C. The dSTR and NAc were collected bilaterally for western immunoblotting analysis in vivo. Tissues were transferred to RIPA buffer with protease and phosphatase inhibitor cocktails, homogenized, and were then incubated at 4 • C for 30 min. The detailed western blotting procedures were the same as described above in the western immunoblotting section.

Statistics
Data are represented as mean ± standard error of the mean (SEM) of cell viability, immunoreactivity of protein expression in western blotting and travelled distance in the behavioral sensitization test. Tukey's post hoc test was used for all one-way analysis of variance (ANOVA) with repeated measures, and Bonferroni's post hoc test was used for all repeated measures two-way ANOVA. Statistical analysis was conducted using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). Statistical significance was accepted for p-values < 0.05. Each experiment of quantitative analyses was performed in triplicate. The data are reported as the mean ± standard deviation (SD) and were analyzed by SPSS (version 19.0, IBM Inc., Armonk, NY, USA).

Analyses of Standard Compounds in L. platyphylla Extract by a UPLC-MS System
The extract of L. platyphylla was analyzed using optimized UPLC-QTOF/MS. Spicatosides A and D were separated by the UPLC system.
Fractionation was performed because we found low intensities of overall compounds in the extract. After fractionation, two standards were confirmed in the UPLC chromatogram of the n-butanol fraction. A typical base peak intensity (BPI) chromatogram and the mass spectra of the identified standard compounds are shown in Figure 1.
The intensities of spicatoside A and D were low in the n-butanol fraction for quantification using UPLC-QTOF/MS. Therefore, QTRAP ® MS/MS was used for quantifying small amounts of compounds. With the optimized instrument, the retention time of spicatoside A and D were 9.53 and 3.97 min, respectively. The precursor ions of spicatoside A and D for quantification using MRM mode were selected at m/z 869.385 and 1049.423, respectively (Table 3). After formation of optimum energies for fragmentation, the product ions with the best sensitivity were selected and quantified. The product ion of spicatoside A was m/z 737.4 and that of spicatoside D was m/z 917.2. Linear calibration curves were constructed using five different concentrations of two compounds. The values of the calibration plots are shown in Table 4. Spicatoside A and D showed excellent correlation coefficients. D were separated by the UPLC system.
Fractionation was performed because we found low intensities of overall compounds in the extract. After fractionation, two standards were confirmed in the UPLC chromatogram of the nbutanol fraction. A typical base peak intensity (BPI) chromatogram and the mass spectra of the identified standard compounds are shown in Figure 1.

Effects of L. platyphylla Extract and Nicotine on PC12 Cell Viability
We performed a cell viability test to determine whether L. platyphylla extract and nicotine induce cytotoxicity in PC12 cells. The results showed that treatment with 100 and 1000 µg/mL of L. platyphylla extract, but not 1 and 10 µg/mL, significantly reduced cell viability in PC12 cells compared with the vehicle control group (p < 0.05; Figure 2A). However, various concentrations of nicotine treatment (1, 10, 100 and 1000 µM/mL) did not cause cytotoxicity in PC12 cells ( Figure 2B). Based on these results,

Co-Treatment of L. platyphylla Extract with Nicotine Significantly Mitigated Nicotine-Induced Alterations in TH and DAT Protein Expression in PC12 Cells
Since treatment with psychostimulants such as nicotine alters the expression level of TH and DAT in cell culture [24,25], we performed western immunoblotting to examine whether L. platyphylla extract attenuates the nicotine-induced alterations in the expression of TH and DAT protein in PC12 cells. The co-treatment of L. platyphylla extract with nicotine significantly mitigated the nicotineinduced increase in TH protein expression in PC12 cells compared to the vehicle control group (p < 0.05, Figure 3A,B). Similarly, the co-treatment of L. platyphylla extract with nicotine significantly rescued the nicotine-induced decrease in DAT protein expression in PC12 cells compared to the vehicle control group (p < 0.05; Figure 3A,C). However, treatment with L. platyphylla extract alone did not alter the levels of TH and DAT protein expression in PC12 cells (Figures 3A-C).

Pretreatment with L. platyphylla Extract Significantly Atteunuated Repeated Nicotine-Induced Increase in Locomotor Activity in Rats
Since repeated nicotine exposure induces behavioral sensitization [2,5], we measured locomotor activity to examine whether L. platyphylla extract attenuated the nicotine-induced behavioral sensitization in rats. The timeline for measuring locomotor activity following repeated saline or nicotine administration after the pretreatment with L. platyphylla extract is shown in Figure 4A. The

Co-Treatment of L. platyphylla Extract with Nicotine Significantly Mitigated Nicotine-Induced Alterations in TH and DAT Protein Expression in PC12 Cells
Since treatment with psychostimulants such as nicotine alters the expression level of TH and DAT in cell culture [24,25], we performed western immunoblotting to examine whether L. platyphylla extract attenuates the nicotine-induced alterations in the expression of TH and DAT protein in PC12 cells. The co-treatment of L. platyphylla extract with nicotine significantly mitigated the nicotine-induced increase in TH protein expression in PC12 cells compared to the vehicle control group (p < 0.05, Figure 3A,B). Similarly, the co-treatment of L. platyphylla extract with nicotine significantly rescued the nicotine-induced decrease in DAT protein expression in PC12 cells compared to the vehicle control group (p < 0.05; Figure 3A,C). However, treatment with L. platyphylla extract alone did not alter the levels of TH and DAT protein expression in PC12 cells ( Figure 3A-C).

Co-Treatment of L. platyphylla Extract with Nicotine Significantly Mitigated Nicotine-Induced Alterations in TH and DAT Protein Expression in PC12 Cells
Since treatment with psychostimulants such as nicotine alters the expression level of TH and DAT in cell culture [24,25], we performed western immunoblotting to examine whether L. platyphylla extract attenuates the nicotine-induced alterations in the expression of TH and DAT protein in PC12 cells. The co-treatment of L. platyphylla extract with nicotine significantly mitigated the nicotineinduced increase in TH protein expression in PC12 cells compared to the vehicle control group (p < 0.05, Figure 3A,B). Similarly, the co-treatment of L. platyphylla extract with nicotine significantly rescued the nicotine-induced decrease in DAT protein expression in PC12 cells compared to the vehicle control group (p < 0.05; Figure 3A,C). However, treatment with L. platyphylla extract alone did not alter the levels of TH and DAT protein expression in PC12 cells (Figures 3A-C).

Pretreatment with L. platyphylla Extract Significantly Atteunuated Repeated Nicotine-Induced Increase in Locomotor Activity in Rats
Since repeated nicotine exposure induces behavioral sensitization [2,5], we measured locomotor activity to examine whether L. platyphylla extract attenuated the nicotine-induced behavioral sensitization in rats. The timeline for measuring locomotor activity following repeated saline or nicotine administration after the pretreatment with L. platyphylla extract is shown in Figure 4A. The

Pretreatment with L. platyphylla Extract Significantly Rescued Repeated Nicotine-Induced Decrease in the Level of DAT Protein Expression in the NAc of Rats
Since pretreatment of 400 mg/kg L. platyphylla extract significantly attenuated repeated nicotine-induced behavioral sensitization, we performed western immunoblotting to investigate whether L. platyphylla extract mitigates the repeated nicotine-induced alterations in TH and DAT expression in the dSTR and NAc. Repeated nicotine administration significantly increased the level of TH protein expression in the dSTR (F(3, 12) = 3.74, p < 0.05), but not NAc, compared to the repeated saline group ( Figure 5A,B); however, there was no significant difference in the repeated nicotine-induced increase in TH protein expression of the dSTR between pretreatment of vehicle and L. platyphylla extract group ( Figure 5A). There was no difference in DAT expression in the dSTR among groups, but the level of DAT expression in the repeated nicotine-treated group was significantly decreased compared with the repeated saline-treated group (p < 0.05) ( Figure 5C,D).
Additionally, the pretreatment of L. platyphylla extract significantly rescued the repeated nicotine-induced decrease in DAT protein expression in the NAc compared to the repeated nicotine group (F(3, 12) = 6.60, p < 0.01) ( Figure 5D). Representative blots and quantification of expressed protein levels in TH and DAT of the dSTR and NAc following the final administration of saline or nicotine after the pretreatment of vehicle or 400 mg/kg L. platyphylla. One-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. * p < 0.05 versus VEH + RS group. ## p < 0.01 versus VEH + RN group. RS, repeated saline; RN, repeated nicotine; VEH, vehicle; 400 LP, 400 mg/kg L. platyphylla extract.
Additionally, the pretreatment of L. platyphylla extract significantly rescued the repeated nicotine-induced decrease in DAT protein expression in the NAc compared to the repeated nicotine group (F(3, 12) = 6.60, p < 0.01) ( Figure 5D).

Discussion
We evaluated the mitigating effects of L. platyphylla extract on: (1) nicotine-induced alterations in TH and DAT protein expression in vitro and in vivo, and (2) repeated nicotine-induced behavioral sensitization. Tyrosine hydroxylase (TH) and dopamine transporter (DAT) are well-known modulators of dopamine levels in the reward system of the brain. TH is involved in the synthesis of dopamine and DAT controls dopamine levels by reuptake into presynaptic terminals [6,7]. A previous study demonstrated that treatment of ginseng containing saponins significantly rescued the nicotine-induced increase in TH protein expression in PC12 cells [26]. Our results consistently demonstrated that treatment with L. platyphylla extracts significantly attenuates the nicotine-induced alterations in TH and DAT expression in PC12 cells. These findings suggest that L. platyphylla extract contains pharmacological compounds that affect the dopaminergic system. In the analysis of L. platyphylla extract using UPLC-MS/MS, we found that L. platyphylla extract contains a large amount of spicatoside A and spicatoside D. Thus, these two saponin compounds may have a mitigating effect on the dopaminergic system altered by nicotine exposure. Spicatoside A was previously reported to One-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. * p < 0.05 versus VEH + RS group. ## p < 0.01 versus VEH + RN group. RS, repeated saline; RN, repeated nicotine; VEH, vehicle; 400 LP, 400 mg/kg L. platyphylla extract.

Discussion
We evaluated the mitigating effects of L. platyphylla extract on: (1) nicotine-induced alterations in TH and DAT protein expression in vitro and in vivo, and (2) repeated nicotine-induced behavioral sensitization. Tyrosine hydroxylase (TH) and dopamine transporter (DAT) are well-known modulators of dopamine levels in the reward system of the brain. TH is involved in the synthesis of dopamine and DAT controls dopamine levels by reuptake into presynaptic terminals [6,7]. A previous study demonstrated that treatment of ginseng containing saponins significantly rescued the nicotine-induced increase in TH protein expression in PC12 cells [26]. Our results consistently demonstrated that treatment with L. platyphylla extracts significantly attenuates the nicotine-induced alterations in TH and DAT expression in PC12 cells. These findings suggest that L. platyphylla extract contains pharmacological compounds that affect the dopaminergic system. In the analysis of L. platyphylla extract using UPLC-MS/MS, we found that L. platyphylla extract contains a large amount of spicatoside A and spicatoside D. Thus, these two saponin compounds may have a mitigating effect on the dopaminergic system altered by nicotine exposure. Spicatoside A was previously reported to promote neurite outgrowth in the PC12 cell line through the tyrosine kinase A receptor pathway [26]. Further study is required to understand the mechanisms underlying the mitigating effects of spicatoside A and spicateside D in L. platyphylla extract on the nicotine-induced stimulation of the dopaminergic system. Behavioral sensitization, an addictive phenomenon, refers to the hypersensitivity of motivated behaviors after repeated exposure to psychostimulants such as nicotine [9]. Many studies have shown that the development of psychostimulant-induced behavioral sensitization is closely related to the alteration in dopaminergic neurotransmission in the reward system including in the dSTR and NAc [2,3,9,10]. Ginseng saponins and herbal extracts significantly attenuate nicotine-induced behavioral sensitization in rodents [2,4,8]. Ginsenoside Rb2 was considered to inhibit adenylate cyclase related to nicotine-induced dopaminergic activation [8,27]. Ginsenoside Rc in ginseng regulates the GABA A receptor in the brain [4,28]. Tachikawa et al. reported that ginsenoside Rg2 decreased the secretion of catecholamines induced by nicotine from the adrenal chromaffin cells of guinea pigs [29]. In this study, pretreatment with L. platyphylla extract significantly and consistently attenuated the repeated nicotine-induced behavioral sensitization. Taken together, these finding suggested that L. platyphylla extract has a mitigating effect on nicotine-induced behavioral sensitization.
Unlike our in vitro results, the in vivo approaches in this study demonstrated that repeated nicotine administration significantly increased TH protein expression in the dSTR, but DAT protein expression in the dSTR was not altered. However, the level of DAT protein expression in the NAc was significantly decreased after repeated nicotine administration, but TH protein expression in the NAc was not altered. Previous findings demonstrate that nicotine-induced dopamine signaling and neuroadaptation are regulated by activation of nicotinic acetylcholine receptors in a region-specific manner [30][31][32]. Taken together, these findings suggest that nicotine may differentially regulate TH and DAT protein expression in the dopaminergic system in a region-specific manner, leading to behavioral sensitization. Additionally, consistent with the in vitro study, the results demonstrated that pretreatment with L. platyphylla extract significantly rescued the repeated nicotine-induced decrease in the level of DAT protein expression in the NAc. However, pretreatment with L. platyphylla extract did not mitigate the repeated nicotine-induced increase in TH protein expression in the dSTR. These findings suggest that the major saponin compounds in L. platyphylla extracts-spicatoside A and spicatoside D-may pharmacologically act on the dopaminergic system, mitigating the nicotine-induced behavioral sensitization through regulation of DAT protein expression in the NAc. Based on previous findings [33][34][35], it could be thought that the inconsistency between in vitro and in vivo results of this study was due to differences in the duration and frequency of nicotine exposure, doses of nicotine, metabolic rate, and other in vivo conditions. Additionally, there is a limitation of this study that the mitigating effects of L. platyphylla extract on nicotine-induced behavioral sensitization was investigated by only in the level of TH and DAT protein expression. It is well-known that level of mRNA expression is generally correlated to protein expression [36], and ginseng saponins were found to regulate nicotine-induced alteration in dopamine-related mRNA expression [26]. Therefore, in-depth approaches for exploring the effects of saponin compounds (spicatoside A and spicatoside D) on nicotine-induced changes in mRNA expression of the reward system with a study for the reward circuit are needed in further study.

Conclusions
In conclusion, this paper reported the activity of L. platyphylla extract that attenuated nicotine-induced behavioral sensitization. The nicotine-induced increase in TH protein expression was decreased and nicotine-induced decrease of DAT protein expression was increased by treatment with L. platyphylla extract in PC12 cells. L. platyphylla extract attenuated the repeated nicotine-induced locomotor hyperactivity in rats by regulating DAT protein expression in the NAc. UPLC-MS systems were used for the quantification of spicatoside A and D in L. platyphylla extract. These results suggest that it will be possible to develop L. platyphylla as a material for mitigating the effects of nicotine-induced psychomotor behaviors, and that spicatoside A and D would be good sources for quality control of L. platyphylla. Identifying the compounds of L. platyphylla that can contribute to the therapeutic effects provides important information for future drug development to treat nicotine dependence.