The Antiemetic Effect of Xiao-Ban-Xia-Tang Formula is Associated With Regulating CaM/CaMKII/ERK1/2 Signaling Pathway in Cisplatin and 1-phenylbiguanide Hydrochloride Induced Rat Pica Models

Background: Xiao-Ban-Xia-Tang (XBXT) formula is a traditional Chinese herbal formula for treating emesis. Chemotherapy-induced nausea and vomiting (CINV) are serious side effects of chemotherapy, which was closely related to the activation of 5-hydroxytryptamine 3 receptor (5-HT3R). In this paper, the effect of XBXT on cisplatin- and 1-phenylbiguanide hydrochloride (1-PBG, a selective 5-HT3R agonist)-induced pica behavior in male Wistar rats and inhibition of calmodulin/calmodulin-dependent protein kinase II/extracellular signal-regulated kinase 1/2 (CaM/CaMKII/ERK1/2) signaling pathway were investigated. Methods: XBXT (1.6 g/kg, twice daily) was orally administered from day 1 after intraperitoneal injection of cisplatin (6 mg/kg) and 1-PBG (25 mg/kg) to day 3. Pica behavior (consumption of kaolin, a type of clay) was recorded every 24 h. The expression and co-localization of CaM and 5-HT3R in small intestine and brain were detected by immunouorescence. The expression of CaMKII, pCaMKII, ERK1/2, and pERK1/2 proteins were detected by Western blot. Results: XBXT treatment signicantly decreased kaolin ingestion (pica) of rats after treatment of cisplatin and 1-PBG during both 0 - 24 h (respectively, from 1.57 g to 0.87 g; from 1.04 g to 0.11 g) and 0 - 72 h (respectively, from 2.98 g to 1.85 g; from 2.29 g to 0.35 g) periods. The uorescence expression of CaM and 5-HT3R and expression of CaMKII, pCaMKII, ERK1/2 and pERK1/2 in the small intestine and brain of cisplatin- and 1-PBG-treated rats were remarkably suppressed by XBXT. an underlying of for provide an experimental of

post chemotherapy [6]. Besides, it has been demonstrated the signi cant improvement of NEPA in the control of CINV in the period 24 h to 120 h post chemotherapy, which is the rst antiemetic combination product comprising of the new NK1 receptor antagonist and the 5-HT3R antagonist (netupitant plus palonosetron) [7]. There is great success in the treatment of CINV. But preclinical and clinical trials have shown that some of the 5-HT3R antagonists, main antiemetic agents, caused electrocardiogram interval changes, in particular, QTc prolongation [7]. What's more, the cost of treatment and prevention of CINV are expensive [8,9]. Therefore, it is necessary to develop some new treatments for treating CINV.
Xiao-Ban-Xia-Tang (XBXT) is a traditional Chinese herbal formula that is composed of the roots of ginger (Zingiber o cinale Rosc.) and the tubers of pinellia (Pinellia ternata (Thunb.) Breit.) in a weight ratio of 1: 2, which had been used for treating emesis in China for nearly 2000 years. Cisplatin is used for treating various types of cancer, which is usually accompanied by severe side effects, such as nausea and emesis [10]. Mink study has shown that XBXT ameliorated cisplatin-induced acute and delayed emesis, which is associated with its inhibited effects on central or peripheral neurokinin-1 receptor levels [11]. Rodents cannot vomit, but pica (consumption of non-nutritive substances such as kaolin, china clay) in rats is analogous to emesis [12]. Rat studies have shown that XBXT had a therapeutic effect on cisplatininduced pica behavior, which is possibly related to inhibit the activation of NLRP3 inflammasome and the increased level of 5-HT and expression of 5-HT3 receptor [11,13,14]. Ginger is a key component of XBXT, which is believed to play an important role in treating nausea and emesis. It is reported that ginger extract can counteract CINV in a randomized, double-blind, placebo-controlled, multicenter study [15]. Heba Abdel-Aziz et al considered that different extracts of ginger, such as [6]-, [8]-, [10]-gingerol and [6]-shogaol acted on the 5-HT3 receptor ion-channel complex and inhibited 5-HT3 receptor function to exert their antiemetic effect [16,17]. In addition, it is reported that the volatile oil from ginger and distinct compounds such as terpinolene, b-pinene, and a-phellandrene possess an antispasmodic effect at the rat ileum associating their functions on 5-HT3 receptor channel system [18]. Studies also have found that ginger and its pungent constituents non-competitively inhibited activation of human recombinant and native 5-HT3 receptors of enteric neurons as well as inhibited serotonin currents on visceral afferent neurons [19,20]. Gingerol, the main pungent constituents of ginger [21], is effective for inhibiting cisplatin-induced acute and delayed emesis in rat pica model and mink because of its regulation of central and peripheral 5-HT, SP, and DA systems [22,23]. [6]-gingerol is the most bioactive compound of gingerol [21], which can signi cantly improve the overall complete response (CR) rate in CINV as well as appetite and quality of life in cancer patients and can also ameliorate the alteration of gut microbiome induced by cisplatin in rats [24,25]. Additionally, study has shown that [6]-shogaol had a direct activation of vagal afferent Cber peripheral gastrointestinal endings and thereafter desensitized this activation, which shows potential anti-emetic effects [26]. As described above, XBXT exhibited the therapeutic effect of CINV, which is associated with several mechanisms. Among them, inhibiting activation of 5-HT3R is one of the mechanisms of XBXT for the prevention and treatment of CINV. Given the central role of 5-HT3 receptor in the pathways leading to CINV, the interaction of XBXT and central and peripheral emetic signaling components downstream of 5-HT3 receptor activation need to be further explored.
Serotonergic receptors are G-protein-coupled receptors excepted the 5-HT3 receptor that is a cysteine-loop ligand-gated ion channel receptor and exists throughout the brainstem dorsal vagal complex and GI tract [27]. As an ion channel that is permeable to the cations (Na + , K + , Ca 2+ ), the 5-HT3 receptor mediates fast depolarizing responses in pre-and post-synaptic neurons [28]. It is reported that 5-HT3R-mediated vomiting is associated with Ca 2+ -CaMKII-ERK1/2 cascade signaling [29,30]. To be speci c, the activation of 5-HT3R induces extracellular Ca 2+ in ux, which results in the activation of CaM, CaMKIIα and ERK1/2 proteins, eventually leads to the occurrence of CINV [31]. Additionally, studies have found that cisplatin caused dysregulation of calcium (Ca 2+ ) homeostasis and chemotherapeutics caused vomiting via Ca 2+ dependent release of multiple neurotransmitters [5,32]. What's more, exposure of dorsal root ganglion neurons to cisplatin for 24 -28 h in vitro signi cantly increased CaMKII-mediated I Ca(v) current density [32,33]. Study also has found that cisplatin elevated the phosphorylation of ERK1/2 [34]. It seems that chemotherapeutics caused the activation of 5-HT3R and subsequently results in the activation of CaM/CaMKII/ ERK1/2 signaling pathway, which eventually leads to the occurrence of CINV. Previous studies indicated that this signaling pathway triggered by the activation of 5-HT3R can be regarded as a therapeutic target for treating CINV. Thus, we hypothesize that inhibiting CaM/CaMKII/ERK1/2 signaling triggered by the activation of 5-HT3R could be one of the underlying mechanisms of XBXT for the prevention and treatment of CINV. 1-PBG is a kind of selective 5-HT3R agonist, which can cause vomiting in the cat [35]. We established both cisplatin-induced and 1-PBG-induced rat pica models to investigate the relationship between XBXT and CaM/CaMKII/ERK1/2 signaling pathway triggered by the activation of 5-HT3R. And ondansetron, a 5-HT3R antagonist, was used as a positive control, which is also widely used for treating CINV.
In the present study, we investigated the effect of XBXT in cisplatin-and 1-PBG-induced rat pica models and characterized the potential mechanisms of the antiemetic effect of XBXT against CINV by focusing on regulating CaM/CaMKII/ERK1/2 signaling pathway, which provides an experimental basis of XBXT for the clinical treatment of CINV Methods Drugs and reagents. Preparation of kaolin pellets and XBXT formula.
Kaolin pellets were prepared according to our previously reported method (25). The roots of ginger 200 g and the tubers of pinellia 400 g were soaked in 4800 mL distilled water overnight and boiled for 1.5 h at 100℃. After ltration, the dregs were boiled in distilled water one more time following the same procedure. The extract from two soaking and ltering was lyophilized to form a dried powder.

UPLC-Q-Orbitrap MS analysis of XBXT.
XBXT samples were analyzed by UPLC-Q-Orbitrap MS using ThermoFisher Scienti c (TF, Dreieich, Germany) Dionex UltiMate 3000. The separation was performed on a RP-C18 column (150 mm × 2.1 mm, 1.8 µm, Welch), operating at a ow rate of 0.3 mL/ min throughout the gradient. The column temperature was 35°C, and the injection volume was 10 μL, Eluent A was 0.1% formic acid in water, and eluent B was 0.1% formic acid in acetonitrile methanol. Separation was conducted under the following conditions: 0 -1 min, 2% B; 1 -5 min, 20% B; 5 -10 min, 50% B; 10 -15 min, 80% B; 15 -20 min, 95% B; 20 -25 min, 95% B; 25 -26 min, 2% B. 26 -30 min, 2% B. For the MS analysis, ESI source conditions were set as following: sheath gas ow rate as 40 Arb, aux gas ow rate as 15 Arb, capillary temperature 300℃, full mass resolution as 70000, MS/MS resolution as 17500, spray voltage as 3.8 kV (positive). The auxiliary gas heater temperature and capillary temperature were both controlled to 350°C. Nitrogen was used for spraying stabilization and damping gas in the Ctrap. The analysis was performed in the full scan mode with a positive ion swing. The resolution was 70,000 (full mass). The full MS scan ranges were set from 150 to 2000 m/z. All of the collected data in the pro le were acquired and processed using Thermo Kaolin pellets were added before drug administration in 3 days to make the rats familiar with the presence of kaolin pellets. Those rats that still took in large amounts of kaolin pellets on the third day were excluded. On the day of drug administration, the rats of C-XBXT and P-XBXT were given XBXT (1. the rats of C-Model, C-Ondansetron and C-XBXT were injected with cisplatin intraperitoneally (i.p.) at the concentration of 6 mg/kg (B.W.) and the rats of P-Model, P-Ondansetron and P-XBXT were injected with 1-PBG intraperitoneally at the concentration of 25 mg/kg (B.W.), while the rats of Control were injected with saline of equal volume intraperitoneally.
During the experiment, the spirit, activity, fur, appetite and stool of the rats were observed every day. The kaolin intake of rats was measured every 24 h. All animals were anesthetized by intraperitoneal injection of 2% nembutal (45 mg/kg B.W.) before sacri ce.
The co-localization of CaM and 5-HT3R in small intestine and brain were detected by immuno uorescence. The brain and small intestine tissues were embedded in para n and sliced into 4 μm sections. The slides were blocked with goat serum for 30 min at room temperature and incubated overnight at 4℃ with the following antibodies: rabbit anti-CaM (1: 2000, Abcam, #ab45689); rabbit anti-5HT3R (1: 2000, Abcam, #ab13897). After that, the slices were incubated with corresponding Fluorconjugated secondary antibody (1: 600, Servicebio, #GB21303) at room temperature for 1 h. Nuclei of cells were stained with DAPI. The expression of protein in cells was observed by uorescence microscope (Nikon, Japan).
Western blot analysis.
Total proteins of the brain and intestinal tissue were extracted using RIPA lysis buffer (P1003B, Beyotime, China), which containing protease and phosphatase inhibitor. The protein concentration was detected with the BCA protein quantitative kit (P0011, Beyotime, China). Proteins were electrophoresed by SDS-PAGE gel and transferred to a PVDF membrane. After blocked in 5% non-fat dry milk for 1h at room temperature, the membrane were incubated separately at 4℃ overnight with the following antibodies: CaMKII . Then membranes were washed and incubated for 1h at room temperature with an anti-rabbit or anti-mouse secondary antibody. Immunoreactive complexes were incubated in ECL kit and exposed for lm using BioSpectrum Imaging System (UVP, Upland, CA). The gray value of the protein was measured with Image-J software.
Statistical analysis.
Graphpad Prism 6 statistical software (San Diego, CA, USA) was used for data analysis. All data were expressed as mean ± SEM. Two-way analysis of variance (ANOVA) was used to compare differences between multiple groups, and the results were analyzed by Tukey's multiple comparisons test. P value less than 0.05 was considered to be signi cant.

Results
Effect of XBXT formula on kaolin intake of rats.
As showed in Fig. 1, the kaolin intake of rats of the C-Model was signi cantly increased during 0 -24 h, 0 -48 h and 0 -72 h periods, compared with the rats of Control (P < 0.001). The kaolin intake of rats of the C-XBXT was signi cantly decreased during 0 -24 h (P < 0.05) and 0 -72 h (P < 0.001) periods, compared with the rats of C-Model. The kaolin intake of rats of the C-Ondansetron was signi cantly decreased during 0 -24 h, 0 -48 h and 0 -72 h (P < 0.001) periods, compared with the rats of C-Model. The kaolin intake of rats of the P-Model was signi cantly increased during 0 -24 h, 0 -48 h and 0 -72 h periods, compared with the rats of Control (P < 0.001). The kaolin intake of rats of the P-XBXT and the P-Ondansetron was signi cantly decreased during 0 -24 h (both P < 0.001), 0 -48 h (P < 0.001, P < 0.01) and 0 -72 h (both P < 0.001) periods, compared with the rats of P-Model.
Effect of XBXT formula on co-localization of CaM and 5-HT3R in small intestine and brain.
The co-localization of CaM and 5-HT3R in small intestine and brain were detected by immuno uorescence. The expression of 5-HT3R protein was red, the expression of CaM protein was green, and the co-locational tissue of CaM and 5-HT3R was yellow. Compared with the Control, the colocalization of 5-HT3R and CaM in the small intestine and brain tissues of rats of C-Model were increased (P < 0.05). As showed in Fig. 2 and Fig. 3, after treating with ondansetron and XBXT formula, the colocalization of 5-HT3R and CaM in the small intestine and brain tissues were signi cantly decreased (P < 0.05). Similarly, compared with the Control, the co-localization of 5-HT3R and CaM in the small intestine and brain tissues of rats of P-Model were increased (P < 0.05). After treating with ondansetron and XBXT formula, the co-localization of 5-HT3R and CaM in the small intestine and brain tissues were signi cantly decreased (P < 0.05) Effect of XBXT formula on the expression of CaMKII protein and the phosphorylation of CaMKII protein in small intestine and brain.
Western Blot was used to detect the expression levels of CaMKII and pCaMKII proteins. Compared with the Control, the expression of CaMKII and the phosphorylation of CaMKII proteins in the small intestine and brain tissues of rats of C-Model were increased (P < 0.05). After treating with ondansetron and XBXT formula, the expression of CaMKII and the phosphorylation of CaMKII proteins in the small intestine and brain tissues were decreased (Fig. 4). Similarly, compared with the Control, the expression of CaMKII and the phosphorylation of CaMKII proteins in the small intestine and brain tissues of rats of P-Model were increased (P < 0.05). After treating with ondansetron and XBXT formula, the expression of CaMKII and the phosphorylation of CaMKII proteins in the small intestine and brain tissues were decreased (P < 0.01) (Fig. 5).
Effect of XBXT formula on the expression of ERK1/2 protein and the phosphorylation of ERK1/2 protein in small intestine and brain.
Western Blot was used to detect the expression of ERK1/2 protein and the phosphorylation of ERK1/2 protein. The expression of ERK1/2 and the phosphorylation of ERK1/2 protein in the small intestine and brain tissues of the C-Model was increased compared with the Control (P < 0.05). After treating with ondansetron and XBXT formula, the expression of ERK1/2 protein and the phosphorylation of ERK1/2 protein in the small intestine and brain tissues was decreased (Fig. 6). Similarly, the expression of ERK1/2 and the phosphorylation of ERK1/2 protein in the small intestine and brain tissues of the P-Model was increased compared with the Control (P < 0.05). After treating with ondansetron and XBXT formula, the expression of ERK1/2 protein and the phosphorylation of ERK1/2 protein in the small intestine and brain tissues was decreased (P < 0.001) (Fig. 7).

Discussion
The development of antiemetics has substantially changed the landscape for treating CINV [36]. In consideration of the central role of 5-HT3 receptor in the pathways leading to CINV, the 5-HT3R antagonists are believed to play the most important role for treating CINV [37]. Especially, the development of palonosetron, a second generation of 5-HT3 receptor antagonists, exhibited superior effects in phase III clinical trials for the prevention of acute, delayed, and overall CINV comparing with other 5-HT3R antagonists because of its more potent binding a nity and longer half-life [38]. It is said that it can also affect the cross-talk between NK-1 and 5-HT3 receptors signaling pathway, which provides a further explanation for the demonstrated e cacy of this drug in controlling delayed CINV [37].
The occurrence of CINV is associated with several systems, thus, a single antiemetic agent has a limitation for treating CINV. Using a combination of antiemetic drugs (such as NEPA) is a potent strategy for treating CINV, but it also puts a heavy nancial burden on the patient [39]. Traditional Chinese medicines (TCMs) contain a variety of active ingredients, which could work on different target mildly. What's more, some TCMs are also food materials, such as ginger, the key antiemetic agent of XBXT, which is safer and cost-effective.
XBXT has been used for treating emesis in a long history in China. In present study, we found that XBXT exhibited an ameliorative effect on both cisplatin-induced and 1-PBG-induced emesis in rats, which is consistent with previous research [11,14]. Some underlying mechanisms of XBXT for the prevention and treatment of CINV have been explored. Our previous studies have found that cisplatin caused gastrointestinal in ammation and altered gut microbiome in rats, while XBXT and [6]-gingerol ameliorated such situations [13,25]. Besides, study has revealed that serotonin and its receptors, mainly 5-HT3 receptor, is overexpressed in in ammatory bowel disease (IBD) and 5-HT3 receptor antagonists have anti-in ammatory properties in certain in ammatory conditions such as colitis [40]. Additionally, 5-HT3R-related pathway stands on the leading role in the occurrence of CINV. Thus, XBXT exhibited an inhibited effect on 5-HT3R. In this paper, we investigated the effects of XBXT on central and peripheral emetic signaling components downstream of 5-HT3R activation.
Weixia Zhong et al found that chemotherapeutics caused vomiting by activating 5-HT3 receptor and its downstream signaling pathway, Ca 2+ /CaMKII/ERK1/2 signaling pathway, while inhibitors of CaMKII (KN93) and ERK1/2 (PD98059) dose-dependently suppressed emesis [31]. And Nissar A. Darmani et al found that activation of L-type calcium channels (LTCC) also caused vomiting, whereas nifedipine-like drugs (LTCC antagonists) not only provide broad-spectrum antiemetic activity but also potentiate the antiemetic e cacy of well-established antiemetics such as palonosetron [29,41]. Study has found that cisplatin caused dysregulation of calcium homeostasis [32]. Intracellular receptor CaM triggers a variety of downstream processes and pathways after binding with calcium [42]. CaMKII mediated ERK1/2 activation in response to Ca 2+ -mobilizing stimuli [43]. Study has found that the toxicity of cisplatin was associated with the activation of ERK1/2 protein [44,45]. These studies provide potent evidence that Ca 2+ /CaMKII/ERK1/2 signaling pathway is a therapeutic target for treating CINV. In the present study, we found that XBXT inhibited CaM/CaMKII/ERK1/2 signaling pathway, which maybe its effective mechanism for treating CINV.
The relationship between CINV and CaM/CaMKII/ERK1/2 signaling pathway provides new insights into the pathophysiology of CINV. Ca 2+ is an important second messenger that participates in various physiological and pathological activities. For example, study has found that in ammation and NLRP3 in ammasome activation initiated by Ca 2+ /CaMKIIδ signaling in cardiomyocytes are essential for adverse cardiac remodeling [46]. What's more, study has found that cisplatin induced the accumulation of calcium ions in inner hair cells and impaired vesicles cycling [47]. Additionally, study has found that granisetron, a 5-HT3R antagonist, recti ed calcium dyshomeostasis in TgSwDI mice brains by restoring CaMKII/cAMP-response element binding protein pathway, which was associated with cognitive improvement [48]. Malik Bisserier et al found that Carabin, a protein expressed in cardiomyocytes, protects against cardiac hypertrophy partly by blocking Ca 2+ /CaMKII Signaling [47]. It is obvious that Ca 2+ and its relative signaling pathways can be regarded as therapeutic targets for so many diseases. Additionally, it is considered that CINV is mainly triggered by the activation of 5-HT3R, which subsequently activates Ca 2+ / CaMKII/ERK1/2 signaling pathway. In our present study, we considered that XBXT formula inhibits CINV partly by inhibiting CaM/CaMKII/ERK1/2 signaling pathway.
XBXT is composed of ginger and pinellia, which are cost-effective and easy to obtain from nature.
Especially, ginger is a key component of XBXT, which have been used for treating CINV in clinic [15]. Pinellia, another key component of XBXT, is used for the treatment of cough, phlegm, and vomiting in pregnant women [49]. TCMs have been widely used in treating CINV in China. And pinellia is one of the most frequently used herbs for treating CINV in China [50]. Based on the theory of TCMs, the combination of ginger and pinellia exhibits a stronger antiemetic effect. Ginger have been used for treating CINV in clinic, and the pungent constituents of ginger, such as gingerol and shogaol, are believed to play the most important role.
Ca 2+ plays an important role in stimulus-response reactions of cells as a second messenger, which involves in several elements, such as LTCC, mitochondria, transient receptor potential channel and ryanodine receptor [51][52][53][54]. In present study, we focused on the effects of XBXT on CaM/CaMKII/ERK1/2 signaling pathway. But whether XBXT has effects on LTCC, mitochondria, transient receptor potential channel and ryanodine receptor remain unclear, which should be further explored.

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