Zehai Songab,
Hang Changa,
Na Hana,
Zhihui Liua,
Zhonglin Wanga,
Hao Gaoa and
Jun Yin*ab
aDevelopment and Utilization Key Laboratory of Northeast Plant Materials, School of Traditional Chinese Materia Medica 48#, Shenyang Pharmaceutical University, Wenhua Road 103, Shenhe District, Shenyang 110016, China. E-mail: yinjun2002@yahoo.com; Fax: +86-24-2398-6460; Tel: +86-24-2398-6491
bSchool of Chinese Materia Medica, Guangzhou University of Chinese Medicine, China
First published on 11th September 2017
As a common side effect of a variety of chemotherapy drugs, CINV severely limits the clinical use of chemotherapy drugs. Reducing the CINV levels may be useful to improve patient life quality and enable effective use of chemotherapy drugs. In this study, we investigated the protective effects of He-Wei granules (HWKL) against CINV using a cisplatin-induced (5 mg kg−1) pica model. HWKL consists of seven herbs: Pinellia ternata (Thunb.) Breit, Zingiber officinale Rosc, Panax ginseng C. A. Mey, Scutellaria baicalensis Georgi, Coptis chinensis Franch, Glycyrrhiza uralensis Fisch, and Ziziphus jujuba Mill. The CINV levels were measured based on the consumption of kaolin. Results showed that HWKL was able to reduce cisplatin-induced increase in kaolin consumption. Cisplatin-mediated increase in tryptophan hydroxylase1 (TPH1), TPH2, 5-hydroxytryptamine 3 receptors (5-HT3AR), neurokinin-1 receptors (NK-1R), 5-HT3AR mRNA, preprotachykinin A (PPTA) mRNA, and NK-1R mRNA was markedly suppressed by HWKL. In addition, cisplatin-induced inhibition of monoamine oxidase (MAOA) and serotonin reuptake transporter (SERT) mRNA was significantly increased after HWKL treatment. HWKL dramatically enhanced appetite by regulating obestatin (OB) and ghrelin (GH); this contributed to further inhibit CINV. The results obtained for G protein-coupled receptor (GPR39) mRNA also confirmed the abovementioned conclusion. HWKL also contributed towards an increase in epidermal growth factor receptor (EGFR) and phosphorylation of extracellular regulated protein kinases (p-ERK1/2) to help repair the damaged mucosa; this indicated that HWKL improved the gastrointestinal damage. Overall, our findings suggest that HWKL inhibit CINV by several mechanisms acting simultaneously and provide basic evidence to confirm that HWKL may be useful in clinical situations as a protective agent to prevent CINV. Moreover, 5-hydroxytryptamine (5-HT) and substance P (SP) result in inhibition of CINV.
HWKL (patent number: ZL2011102359 15.7) was derived from the formulation of Ban-Xia Xie-Xin Tang or Ban-Xia Xie-Xin decoction (BXXXT), which was first described in Shang-Han-Lun about 1700 years ago for the treatment of many different gastrointestinal symptoms and treating patients with a weak spleen and stomach, internal stasis of heat and cold glomus, and fullness below the heart without pain, vomiting, and borborygmus.5–10 Like BXXXT, HWKL consists of seven herbs: Pinellia ternata (Thunb.) Breit. (Pinelliae Rhizoma Praeparatum, Araceae, PR), Zingiber officinale Rosc. (Zingiberis Rhizoma Recens, Zingiberaceae, ZB), Panax ginseng C. A. Mey. (Ginseng Radix Et Rhizoma, Araliaceae, GR), Scutellaria baicalensis Georgi. (Scutellariae Radix, Labiatae, SR), Coptis chinensis Franch. (Coptidis Rhizoma, Ranunculaceae, CR), Glycyrrhiza uralensis Fisch. (Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle, Leguminosae, GHR), and Ziziphus jujuba Mill. (Jujubae Fructus, Rhamnaceae, JF). According to our previous study,11 HWKL could reduce high-dose cisplatin-induced nephrotoxicity and myelosuppression. A total of 37 major components, including ginsenosides, alkaloids, flavonoids, gingerols, and polysaccharides, were identified using UHPLC-Q-TOF-MS/MS, and 11 of these were confirmed and quantified using reference compounds.11
Furthermore, it is generally known that nausea usually leads to emesis and emesis occurs in >90% of patients taking highly emetogenic chemotherapy drugs (such as cisplatin and carmustine) and 30–90% of those taking moderately emetogenic chemotherapy drugs (for instance, irinotecan and oxaliplatin) without appropriate prophylaxis.1,4 To study CINV in laboratory, several animal models, involving piglets, ferrets, and dogs, have been established. Due to their expense and difficult operating procedures, these animal models have not been widely used. Rats, one of the most common laboratory animals, have generally been considered unsuitable for studying CINV because they do not have a vomiting reflex.12 However, although rats lack a motor reflex of emesis, they display atypical feeding behavior, termed pica response.13–17 Pica behavior is an aversive/illness response that results in consumption of non-nutrient substances, such as kaolin, after an emetic stimulus.18,19 It has been confirmed that pica behavior can be used as a model of a nausea-like response or gastrointestinal malaise because it is induced by nausea stimuli and the amount of kaolin consumption is related to the severity of nausea in humans.13 In addition, it has been shown by the Japanese group Yamamoto et al.13–16 that the magnitude of pica response in rats is directly related to the emetogenic potential in humans. The highly emetogenic anti-cancer drug (such as cisplatin)-induced kaolin ingestion behavior pica in rats is analogous to emesis. As an antineoplastic agent with high risk of emetogenic level increase, cisplatin was used to simulate CINV in our study,1,14 and cisplatin-induced pica behavior was regarded as an acceptable model to measure the antiemetic effect of drugs according to kaolin consumption and other indices.1,13,16,18,20
As a classical formula that has been used for more than 1000 years, BXXXT is considered to be effective in controlling the emesis levels. In our previous study, HWKL has been found to reduce kaolin intake of rats in a pica model. However, there have been no reports on the pharmacological effects and proper mechanism of action of HWKL on CINV; thus, our present study was aimed at the investigation of the protective effects and possible mechanisms of action of HWKL on CINV.
Rats were randomly divided into the following 9 groups (n = 12): blank group, cisplatin group, cisplatin + ondansetron group, cisplatin + domperidone group, cisplatin + BXXXT group (1.38 g kg−1), cisplatin + HWKL low dose group (1.18 g kg−1), cisplatin + HWKL middle dose group (2.36 g kg−1, equal to the clinical dose of human beings), cisplatin + HWKL high dose group (4.725 g kg−1), and HWKL group (2.36 g kg−1). The clinical dose of BXXXT of human beings has been referenced in Shang-Han-Lun and other references.22–25 HWKL was used in the People's Liberation Army 463 Hospital for more than seven years. The clinical dose of HWKL for human beings was referenced to the dose used in the People's Liberation Army 463 Hospital. After 7 day acclimatization period, a measured quantity of kaolin pellets was filled in separate containers 3 days prior to the experiment to allow rats to psychologically adapt to its presence. After 3 days of habituation, rats were subjected to pica experiments. During the experimental observation period, rats in the blank and cisplatin groups were treated with pure water according to their bodyweight every day; the rats in the simple HWKL group were treated with HWKL (2.36 g kg−1, i.g. every day); rats in the cisplatin + ondansetron group were treated with ondansetron (1.3 mg kg−1, equal to the clinical dose of human beings, i.g. every day); rats in the cisplatin + domperidone group were treated with 3 mg kg−1 domperidone (equal to clinical dose of human beings), i.g. every day; rats in the cisplatin + BXXXT group were treated with 1.38 g kg−1 BXXXT (equal to the clinical dose of human beings) i.g. every day; rats in cisplatin + HWKL low dose, cisplatin + HWKL middle dose, and cisplatin + HWKL high dose groups were treated with 1.18, 2.36, and 4.725 g kg−1 HWKL, respectively (equal to 0.5-fold, 1-fold, and 2-fold clinical doses of human beings, respectively). The samples involved in the experiment were prepared in different concentrations to ensure that the same volume is administered. Cisplatin (5 mg kg−1, i.p.) was administered 1.5 h after each treatment with the antiemetic agent or its vehicle, except in the blank and HWKL groups. Following the administration of cisplatin, kaolin consumption, bodyweight, and food intake were continuously observed every 24 h for 168 h. Herein, 6 rats of each group were sacrificed after 72 h to obtain blood, serum, medulla oblongata, and ileum to measure the 5-HT relative targets such as 5-HT, 5-HIAA, TPH1, TPH2, MAOA, SERT mRNA, and 5-HT3A mRNA. Other 6 rats of each group were sacrificed after 168 h.
Kaolin consumption was used as the measurable parameter of vomiting and nausea according to the previous research.14–16,21,26–31 Food intake was used as the measurable parameter of anorexia.
Tissue homogenate: tissue sample was immersed in 9-fold volume of PBS/0.9% saline and ground into a powder in ice by the Scientz-IID ultrasonic cell disruption system (Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China). The extracts were centrifuged at 3500 rpm for 15 min at 4 °C to remove insoluble materials.
The serum and supernatant fractions were analyzed using ELISA kits, MDA, GSH-PX, and SOD kits according to the manufacturer's instructions. After the experiment, the absorbance of each sample was measured at 450 nm (ELISA), 532 nm (MDA), 412 nm (GSH-PX), and 550 nm (SOD) using a microplate reader (Thermo Scientific L-117, USA).
Standard curves were prepared using diluted standard solutions to allow calculation of the relative target in the samples. All standards and samples were run in duplicate.
5-HT3A receptor antibody was purchased from abcam (Cambridge, MA, USA). NK-1 receptor antibody was purchased from ImmunoWay Biotechnology Company (Newark, DE, USA).
According to the kaolin consumption of the cisplatin group, cisplatin administration caused acute and delayed emesis (Fig. 1A and B). As shown in Fig. 1A and B, a small amount of kaolin was consumed by rats in −72 h because of their curiosity, but no kaolin was eaten by rats during the subsequent period. After cisplatin administration at 0 h, HWKL dose-dependently (except in the first 24 h) reduced kaolin consumption induced by cisplatin during the 168 h observation period, with significant reduction in each of the seven days in comparison with that in the cisplatin group. Kaolin consumption of the ondansetron group was found to be lower than that in any of the other groups after cisplatin injection at 24 h and increased steadily from 24 h to 72 h, whereas it decreased in the other groups. A peak kaolin consumption in the cisplatin and ondansetron groups occurred at 72 h, indicating delayed emesis although ondansetron contributed only slightly to it. Furthermore, HWKL treatment continuously reduced the kaolin consumption. Compared with those in the blank group, there were no significant differences in the food and kaolin consumption and body weight in the abovementioned groups; this suggested that HWKL alone did not affect the physical conditions (Fig. 1). Compared with that in the blank group, there was no significant difference in kaolin consumption in the HWKL group; this suggested that HWKL alone did not induce pica in rats (Fig. 1A and B).
Fig. 2 Effect of HWKL on 5-HT content (A–C), 5-HIAA content (D–F), and 5-HIAA/5-HT ratio (G–I) in cisplatin-treated rat serum, medulla oblongata, and ileum. |
According to the parts (E), (F), (G), and (H) of Fig. 3, the content of SERT mRNA in the medulla oblongata and ileum was reduced by cisplatin, whereas the 5-HT3AR mRNA content in the medulla oblongata and ileum increased following cisplatin administration in the cisplatin group. HWKL increased the content of SERT mRNA, whereas it reduced the 5-HT3AR mRNA content. After cisplatin administration, the TPH-1 content in the ileum and TPH-2 content in the medulla oblongata were found to sharply increase as compared to those in the blank group; this suggested that cisplatin could accelerate the synthesis of 5-HT by up-regulating the TPH content (Fig. 3C and D). HWKL can significantly reduce the content of TPH-1 and TPH-2 as compared to that in the cisplatin group. As a result of cisplatin administration, the MAO-A content in the ileum and medulla oblongata was reduced and inhibited in the cisplatin group as compared to that in the blank group (Fig. 3A and B), whereas HWKL increased the content of MAO-A to accelerate the metabolism of additional 5-HT caused by cisplatin administration. The relative content of 5-HT3AR protein in the medulla oblongata and ileum of the cisplatin group increased significantly after cisplatin administration in comparison with that in the blank group, whereas HWKL treatment markedly reduced the content of 5-HT3AR protein (Fig. 3J, K, and I). Ondansetron and domperidone positively regulated the content of MAO-A, but failed to regulate that of other biomarkers (Fig. 3).
The OB content in the serum and antrum of the cisplatin group increased sharply after cisplatin administration in comparison with that of the blank group (Fig. 5A and D). As demonstrated in (B), (C), (E), and (F) of Fig. 5, the GH content and the ratio of GH/OB of the cisplatin group in the serum and antrum were significantly reduced after cisplatin injection in comparison with those of the blank group. Based on these results, cisplatin was considered to increase the OB content and reduce the amount of GH to inhibit the gastrointestinal function to aggravate CINV. Compared with that of the cisplatin group, the GH content in the serum was up-regulated in all groups except in the cisplatin + domperidone group at 72 h. HWKL was able to simultaneously significantly increase the GH content and reduce the OB content, thus increasing the GH/OB ratio (Fig. 5). Furthermore, the GPR39 mRNA content in the antrum significantly increased in the cisplatin group after cisplatin administration as compared to that in the blank group; this indicated an increase in the OB receptors. HWKL efficiently reduced the marked increase in OB and GPR39 mRNA; this suggested that HWKL could combat the emesis induced by cisplatin administration by down-regulating the OB content and up-regulating the GH content.
In the blank and HWKL groups, the crypts in the glandular epithelium exhibited tight cylindrical structures and the lamina propria characterized by reticular connective tissue contained lymphatics and capillaries and was surrounded by individual lymphocytes, plasma cells, and eosinophils, as well as granulocytes that were not ungrouped (Fig. 7A). Moreover, high columnar absorptive cells stained with eosinophil were shown to have a regular arrangement. In addition, the villi were aligned in finger-like regular protrusions in the lumen, whereas the goblet cells with oval nuclei were found with basophilic staining (Fig. 7A). However, abscised intestinal mucosa, inflammatory cell infiltration of the intestinal mucosa, and intestinal mucosa necrosis were clearly present in the cisplatin, ondansetron, domperidone, and BXXXT groups (Fig. 7A). In these groups, the crypts had lost their cylindrical and tightly arrayed structures, and their lumens were enlarged. Furthermore, the villi had lost their finger-like structure and were oval in shape; their length reduced due to the effect of edema, and denuded villi were observed. Dense cell swelling and degeneration due to vacuolization were observed in columnar cells at the apex. The goblet cells had lost their oval structures, and intense basophilic staining showed that they were secreting their mucosal content (Fig. 7A). However, HWKL effectively protected the ileum from the damage caused by cisplatin administration (Fig. 7A and B). Scores for the damaged ileum were given on the basis of capillary congestion, epithelial sloughing, edema and dilatation, crypt degeneration, denuded villi, and lymphocyte infiltration according to the pathological evaluations described by Aydin et al.33 Details of the scores and their significance are given in Fig. 7B.
The content of EGFR and pERK1/2 in the cisplatin group increased significantly after cisplatin administration in comparison with those in the blank group; this suggested that extra EGFR and pERK1/2 were secreted to help recover the damaged tissue (Fig. 6E and F). Treatment with HWKL significantly contributed to the recovery of the damaged gastric mucosa via up-regulation of the EGFR and pERK1/2 content (Fig. 6E and F). BXXXT showed a tendency to increase the content of EGFR and pERK1/2, but there was no significant difference in comparison with that in the cisplatin group and ondansetron and domperidone showed very little effect (Fig. 6E and F).
As shown in (B) and (C) of Fig. 6 and (C) and (D) of Fig. 7, GSH-PX and SOD active levels of the antrum and ileum were found to be lowest in the cisplatin group as compared to those in the blank group. After HWKL administration, the GSH-PX and SOD active levels were found to be up-regulated; this indicated that HWKL could protect organs from the damage produced by cisplatin injection by reducing the oxidation level (Fig. 6 and 7). In addition, the content of MDA in the antrum and ileum decreased by HWKL treatment; this suggested that HWKL could reduce the oxidative level of the antrum and ileum after cisplatin injection (Fig. 6G and 7E). BXXXT had some effect on combatting the oxidative damage caused by cisplatin, whereas ondansetron and domperidone had a very little effect on the same (Fig. 6 and 7).
Fig. 8 Herbs involved in HWKL and proposed pathway of HWKL inhibiting chemotherapy-induced vomiting in rats. |
Cisplatin has been shown to induce a pica response in rats, and a cisplatin model has been developed in which CINV occurring within the first 24 hours is defined as acute and that occurring more than 24 hours later is defined as delayed.18,19,36 It has been reported that 3–6 mg kg−1 cisplatin can induce significant pica behavior and gastrointestinal mucositis in rats. However, 6 mg kg−1 cisplatin cannot induce delayed vomiting because of severe anorexia.13 In our previous experiment, 3, 4, 5, and 6 mg kg−1 doses of cisplatin have been screened. According to the kaolin consumption, the general status of the animals, and the levels of biomarkers, a 5 mg kg−1 dose was considered to be more appropriate due to its stability and significance.
CINV is a major adverse effect of cytotoxic chemotherapy and is one of the reasons for patient non-compliance with cancer treatment.37,38 Nausea is defined as an unpleasant feeling in the upper gastrointestinal tract with an involuntary urge to vomit, and vomiting involves the involuntary and forceful expulsion of stomach content through the mouth. It has been reported that CINV is a complicated phenomenon with a sophisticated mechanism. The central nervous system and gastrointestinal tract have been reported to play key roles in CINV. Neurotransmitters such as 5-HT and SP are released after enterochromaffin cells are stimulated by cytotoxic chemotherapeutic agents, and these neurotransmitters have an effect on related receptors. Activated receptors act on the vagus nerve, and then the nerve impulse is transmitted to the vomiting center; as a consequence, vomiting is induced. In addition, nerve impulse can be transmitted to the chemoreceptor trigger zone (CTZ). 5-HT and SP, located in the CTZ, are released to the active related receptors, and then, the signals reach the vomiting center to induce emesis. Moreover, cytotoxic chemotherapeutic agents can directly affect the CTZ through blood circulation because CTZ is located in the fourth ventricle and there is no blood–brain barrier.13–16,37,38
In addition, gastrointestinal damage, occurring as a result of exposure to toxic chemotherapeutic agents, triggers the release of neurotransmitters which activate the chemoreceptors at the end of abdominal vagal afferents. Cisplatin-induced acute emesis has been confirmed to be related to the release of 5-HT, whereas SP release is considered to be the key factor in cisplatin-induced delayed emesis. In clinical situations, 5-HT3 receptor antagonists, such as ondansetron and palonosetron, and NK1 receptor antagonists, such as aprepitant, are used alone or in combination with corticosteroids (for instance, dexamethasone) to treat CINV. Currently, antiemetic treatment involving 5-HT3 and NK1 receptor antagonists is used to alleviate acute emesis and some delayed emesis, but delayed emesis remains an issue.37,38 However, the high cost and adverse effects of chemical antiemetic drugs cause extra pain to patients and impose a great burden. Therefore, it is important to explore novel strategies to inhibit CINV.4
CINV is a sophisticated reflection. Nowadays, it is considered that acute CINV is predominantly due to the release of 5-HT within the gastrointestinal tract and nervous system, whereas SP plays an important role in delayed CINV.1,15,37–39 Unlike 5-HT and SP, OB and GH have been reported to be involved in CINV.40–44 Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of 5-HT, and the expression level of TPH can be considered as a marker for 5-HT synthesis. Moreover, two kinds of TPH, TPH-1 and TPH-2, have been identified due to their distribution in the body. TPH-1 is responsible for 5-HT synthesis in non-neuronal cells, whereas TPH-2 is expressed in neurons of raphe nuclei and myenteric plexus.39,45 As the main metabolic enzyme of monoamines in rats, MAO can be divided into two hypo-types: MAO-A and MAO-B. 5-HT is mainly oxidized by MAO-A.37–39 As the metabolite of 5-HT, 5-HIAA is considered to be an important indicator in the metabolism of 5-HT, and nowadays, 5-HIAA/5-HT ratio is used more and more frequently to indicate the real level of the effect of a drug on 5-HT. SERT is the main carrier of 5-HT, whereas PPTA is the main source of SP. 5-HT3A is the most common form of 5-HT involved in chemotherapy-induced emesis, and NK-1R is the most compatible receptor of SP. In addition, it has been shown that SP helps increase the release of 5-HT from enterochromaffin cells.15 GPR39 is considered to be the receptor of OB, and to measure the levels of all these biomarkers, serum and key organs have been studied in this experiment.1,15,37–40,42–49
CINV is connected to gastrointestinal malaise,13 and cisplatin-induced gastrointestinal malaise including anorexia has been observed in our research. Therefore, reduction in cisplatin-induced gastrointestinal malaise may contribute to further inhibit chemotherapy-induced emesis. Regarding anorexia, GH and OB, two different peptides that are encoded by the same preproghrelin gene, are considered to have an important role in its control. Stimulation of feeding and body weight gain are the main effects of GH.17,50,51 In addition, GH can improve the activity of motilin and gastrin to increase the rate of gastric motility by regulating the secretion of gastric acid and digestive enzymes.52 On the contrary, OB inhibits food intake and gastrointestinal motility. A previous study has shown that OB overexpression plays a significant role in the development of CINV.41 GPR39 is considered to be the receptor of OB, and the level of GPR39 mRNA in the antrum is measured to examine the molecular relationship of OB at the gene level. According to the results obtained herein, we conclude that HWKL inhibits gastrointestinal damage by up-regulating GH and down-regulating OB. As a consequence of the protective effect of HWKL on the gastrointestinal tract, CINV is further inhibited.
Cisplatin-induced oxidative stress is regarded as one of the main causes of gastrointestinal tract injury. Therefore, reduction of cisplatin-induced oxidative stress has an important effect on the inhibition of gastrointestinal injury; this can further contribute to the suppression of CINV.53
Recovery of damaged gastrointestinal mucosa helps to maintain the function of the gastrointestinal tract; thus, further advantages are associated with the inhibition of CINV. Epidermal growth factor (EGF) is considered to promote the repair and proliferation of impaired gastrointestinal mucosa following the action of cytotoxic drugs such as cisplatin. Self-dimerization and self-phosphorylation of EGFR will be activated by combination with related ligands, which can activate the EGFR/ERK signal transduction pathway. In addition, the Ras/Raf/MEK signal transduction pathway will be mediated and activated followed by activation of ERK phosphorylation. As a consequence, there is accelerated repair of the damaged mucosa.54 Moreover, an increase in the phosphorylation status of ERK1/2 is regarded as a necessary factor for controlling the expression of PPTA and NK1 receptor mRNA.
In our experiment, we found a strange phenomenon that rats in the cisplatin + domperidone group were emaciated and weak and suffered mental fatigue. It is well known that domperidone can be used to treat gastric dysfunction and chemotherapy-induced emesis and nausea. However, domperidone was not effective in antagonising cisplatin-induced toxicity in this study. We believed that reduction in kaolin consumption in the cisplatin + domperidone group was not due to the efficiency of domperidone but because rats did not have enough energy to eat. However, this phenomenon requires further study.
Moreover, seven herbs are included in HWKL. Among them, PR and ZB are considered to be the main components on the basis of the traditional Chinese medicine theory. It has been reported that a water decoction of PR and ZB can inhibit cisplatin-induced pica by down-regulating the levels of obestatin.6,20,41 6-Gingerol in ZB is found to be effective in controlling CINV.55–57 GR is a well-known TCM that is believed to be the king of herbs in the Orient, especially in China, Korea, and Japan. Ginsenosides and polysaccharides are considered to be active constituents with anticancer, antioxidant, and immunomodulation effects, which contribute to enhance immunomodulation effect against CINV.21,58 Chrysin has been reported to attenuate cisplatin-induced colon toxicity via amelioration of oxidative stress.59 Its glucosides are present in HWKL.11 Baicalin in SR and berberine in CR are often used in clinical situations to treat gastrointestinal inflammation and have been reported to treat cancer.2,58,60,61 Chemical compositions in HWKL can work synergistically in regulating the biomarkers related to CINV. As a result, CINV is inhibited.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ra06312a |
This journal is © The Royal Society of Chemistry 2017 |