Nausea and vomiting are common problems in cancer patients throughout the trajectory of their illness, and emesis following high-dose cisplatin may be especially severe, greatly reducing the quality of life and affecting compliance in some patients.1 The cisplatininduced emesis in humans has been classified as either acute, delayed and anticipatory.2 Acute nausea and vomiting generally occurs within the first 24-hour period, delayed emesis occurs after this period, the anticipatory nausea and vomiting usually develops within four courses of chemotherapy in patients whose emesis is not controlled in previous cycles.3
The discovery of the 5-HT receptor antagonists has dramatically improved the treatment of emesis induced by anti-cancer therapy.4 However, 5-HT3 receptor antagonists fail to completely control the delayed nausea and vomiting associated with chemotherapy in man and there remain some patients who are resistant to 5-HT3 receptor antagonists.5,6 In recent years, substance P has been recognized as playing a pivotal role in delayed emesis, and NK1 receptor antagonists were reported to be effective for inhibiting emesis induced by central or peripheral emetogens in ferrets.7-9 In the clinic, NK1 receptor antagonists also improved emesis control, particularly when used in combination with 5-HT3 receptor antagonists and glucocorticoids.10,11 Nevertheless, the existing antiemetic drugs have high price, side effects and have to be given in combination.12,13 Therefore, it is reasonable to explore a natural antiemetic agent to treat vomiting.
Ginger as a traditional herb, has been used as an antiemetic drug for more than 2000 years in China, as well as a common food for cooking in East Asia. Ginger has been reported to be effective for inhibiting vomiting during pregnancy, for post-operative nausea and vomiting, and cisplatin-induced emesis.14-16 Ginger juice and ethanol extraction significantly inhibited the emesis induced by cisplatin, copper sulfate and motion sickness.17 Gingerol, as the generic term for pungent constituents in ginger,18 has been reported to inhibit contraction of isolated guinea-pig ileum by acting on 5-HT3 receptors and this is presumed to be related to its antiememtic activity.19 However, the antiemetic mechanism of gingerol remains unclear; particularly there are few reports of the effect on substance P and NK1 receptors.
This study was to investigate the mechanisms of the antiemetic effect of gingerol by studying, on both peripheral and central levels, the effects on substance P and NK1 receptor.
Adult castrated male minks (1.0 kg-1.8 kg) were purchased from Qingdao Special Animal Center. They were provided and used according to the Qingdao University Guide for the Care and Use of Laboratory Animals. All animals were housed individually in an iron cage of 75 cm × 50 cm × 50 cm with free access to water and food.
According to the previous study,20 minks were randomly divided into the following six groups (n=6): the blank control group, the model group, the ondansetron group and three gingerol groups. The blank control group were pretreated with sterile saline, the model group were preadministered with vehicle (1% tragacanth, 5 ml/kg, intragastrically (i.g.)), the ondansetron group were pretreated with ondansetron (2 mg/kg, i.p.), the gingerol groups were preadminstered with gingerol (50 mg/kg, 100 mg/kg or 200 mg/kg, i.g.), which was dissolved in 1% tragacanth. Cisplatin (7.5 mg/kg, i.p.) was administered 30 minutes after treatment with the antiemetic agent or its vehicle, except to the blank control group. Following administration of cisplatin, animals were observed continuously for 6 hours for the emetic responses, the time of onset of vomiting and the number of both retching and vomiting episodes were recorded.
Drugs and chemicals
Gingerol was purchased from Baoji Hongyuan Biotech Co. Ltd. and was dissolved in 1% tragacanth. Cisplatin came from Luoxin Pharmaceutical Co. Ltd., was prepared in normal saline at 70°C followed by gradual cooling to 40°C and was administered immediately. Ondansetron hydrochloride injection was from Qilu Pharmaceutical Co. Ltd. Anti-Substance P, anti-NK1 receptor antibody and goat anti-rabbit IgG were obtained from Sigma Chemical Co., USA.
Immunohistochemistry of substance P and NK1 receptor
Animals were sacrificed at 6 hours after administration of cisplatin. Tissues from the area postrema as well as the ileum were removed from every animal for immunohistochemical analysis of substance P and NK1 receptor. The ileum was excised at a distance of 20 cm from the pylorus. The samples were fixed in 10% buffered formalin at 4°C for 24 hours- 48 hours. This was followed by cryoprotection for 12 hours-18 hours at 4°C using 30% sucrose or until the samples equilibrated. The samples were sectioned (4 μm thick) using a freezing microtome (Leica, Germany; Lot. 200392900A) and standard immunohistochemical procedures were used to visualize substance P and NK1 receptor protein. Sections were washed 4×10 minutes in PBS (pH 7.4) at room temperature and were blocked with 10% normal goat serum. Sections were then incubated with rabbit polyclonal anti-substance P antibody or anti-NK1 receptor antibody (1:1000) overnight on a shaker at room temperature. Following washing (4×10 minutes) with PBS, sections were incubated for 2 hours with rat anti-rabbit antibody (1:300). Finally, sections were washed (3×10 minutes) in PBS, transferred onto gelatin-coated slides. Tissue processed without the primary substance P antibody or NK1 receptor antibody served as a negative control. Adjacent sections were mounted onto plus-coated slides and stained using Neutral Red (0.5%), washed through a series of increasing alcohol concentrations, cleared with xylene and coverslipped with Neutral Blasam.
Five fields from every section were randomly selected for examination using an Olympus CKX41-32PH microscope equipped with an imaging system (Olympus Optical Co., Japan). The pictures were reviewed and an overall semi-quantitative score,21 was assessed by integrating the extent and intensity of staining of substance P and NK1 receptors. Pictures were graded from minimally 0 to maximally 3 as follows: 0, maximally 10% of the picture stained weakly positive; 1, minimally 10% of the picture stained weakly positive and maximally 10% of the picture stained moderately or strongly positive; 2, 10%-40% of the picture stained moderately positive or 10%-20% stained strongly positive; 3, over 40% of the picture stained moderately positive or over 25% of the picture stained strongly positive.
Western blotting of NK1 receptor
Animals were sacrificed at 6 hours after administration of cisplatin. Tissues of the ileum and area postrema were removed for Western blotting assays.22 The pieces were stored at -80°C. The samples were lysed in cold lysis buffer (Na deoxycholate, 10 g/L; Triton X-100, 10 g/L; SDS, 0.1 g/L; NaCl, 150 mmol/L; Tris, 50 mmol/L, pH7.5; EDTA, 0.05 mmol/L; NaF, 50 mmol/L; Na pyrophosphate, 10 mmol/L; NaVO4, 0.5 mmol/L; PMSF, 1 mmol/L; aprotinin, leupeptin and pepstatin, 20 mg/L each), and sonicated for 10 seconds on ice. After being centrifuged for 10 minutes at 14 000 r/min at 4°C, the supernatants of all samples were assayed for NK1 receptor by Western blotting. Protein concentrations were measured by the Bradford method using bovine serum albumin as the standard. Equal amounts of protein from each sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes. Subsequently, membranes were blocked for one hour with 5% (w/v) non-fat dried milk in Tris-buffered saline containing 0.01% Tween-20 (TBS-T) and then incubated with the primary antibodies (diluted 1:1000) overnight at 4°C. Individual blots were probed with polyclonal anti-NK1 receptor and anti-GAPDH antibodies. After being washed, the membrane was incubated with goat anti-rabbit IgG conjugated to HRP. The bound antibody was detected using 3,3′-diaminobenzidine (DAB). Protein loading was monitored using immunodetection of GAPDH.
Data were expressed as mean±standard error (SE). Analysis of variance (ANOVA) was performed when more than two groups were compared. Multiple comparisons were performed with the Tukey test. Values of P <0.05 were considered statistically significant.
Effect of gingerol on emesis induced by cisplatin in minks
As shown in Table 1, cisplatin evoked a profound emetic response in minks and pretreatment with gingerol reduced the number of reteching and vomiting incidents induced by cisplatin in a dose-dependent manner during the 6 hour observation period (P <0.05). Although the latency of some groups could not be calculated because some minks did not retch or vomit, the latency of onset of cisplatin to induce emesis also increased significantly in the gingerol 50 mg/kg treatment group (P <0.05). Meanwhile, there was no significant difference in the number of reteching and vomiting episodes between the groups receiving gingerol 200 mg/kg and ondansetron (P >0.05). And from Figure 1A and 1B, the change trends in retching and vomiting of each group were more evident.
Distribution of substance P in the area postrema and ileum
As shown in Figures 2 and 3, substance P staining intensities were mainly situated in the mucosa and submucosa of the ileum (Figure 2B) as well as in the neurons of the area postrema (Figure 3B). Compared with the blank control group, there was more substance P detected in the model group. Ondansetron, a selective 5-HT3 receptor antagonist, had no significant effect on the expression of substance P in either the area postrema or ileum (Figures 2C and 3C). Gingerol markedly suppressed the amount of substance P detected in a dose-dependent manner (Figures 2D-2F and 3D-3F).
As shown in Table 2, the substance P positive grades significantly increased after treatment with cisplatin (P <0.05). The increase of substance P positive grades in the area postrema and ileum induced by cisplatin was significantly inhibited by gingerol in a dose-dependent manner (P <0.05). Ondansetron had no significant effect on the elevated positive grades of substance P in either the area postrema or ileum (P >0.05).
Distribution of NK1 receptor in the area postrema and ileum
As shown in Figures 4 and 5, NK1 receptors were mainly situated in the muscular and submucosa of the ileum (Figure 4B) and the neurons of the area postrema (Figure 5B). There was more positive expression of NK1 receptors in the model group compared with the blank control group and gingerol markedly suppressed the number of NK1 receptors detected in both the area postrema and ileum in a dose dependent manner (Figures 4D-4F and 5D-5F). However, ondansetron had no significant effect on the expression of NK1 receptors in either the area postrema or ileum (Figures 4C and 5C).
As shown in Table 3, NK1 receptor positive grades significantly increased after treated with cisplatin (P <0.05). In both the area postrema and ileum the increase of NK1 receptor positive grades induced by cisplatin was significantly inhibited by gingerol in a dose-dependent manner (P <0.05). Ondansetron had no significant effect on the elevated positive grades of NK1 receptors in either the area postrema or ileum (P >0.05).
Expression of NK1 receptor in the ileum and area postrema
In Figure 6, the expression of NK1 receptors was analyzed by Western blotting. The expression levels of NK1 receptor significantly increased after treatment with cisplatin (P <0.05) and the elevated expression was inhibited by the pretreatment of gingerol in both the ileum and area postrema in a dose-dependent manner (P <0.05). However, ondansetron had no significant effect on the elevated level of NK1 receptor protein in either the area postrema or ileum (P >0.05).
Substance P has recently become a novel target in the study of antiemetic drugs. Substance P and substance P-like immunoreactivity have also been found at significant levels in the area postrema and nucleus tractus solitarius of rats and cats,23-25 both of which are associated with the emetic reflex. And it is reported that substance P induces physiological activity via the NK1 receptors.26 NK1 receptor antagonists also were reported to be effective for inhibiting emesis induced by central or peripheral emetogens in ferrets.7-9 Further studies have described an area in the dog which they term “the central pattern generator for vomiting” and have suggested that the antiemetic site of action for NK1 receptor antagonists is in the medullary area adjacent to the semi-compact part of the nucleus ambigus.27,28 It is possible that the site of action of NK1 receptor antagonists is located at or in the vicinity of the commissural subdivision of the nucleus tractus solitarius since it has been shown that activation of cardiorespiratory function in the cat induces an increase in the extraneuronal concentration of substance P in this brain region.29 Andrews30 reported that resiniferatoxin, an analogue of capsaicin, which releases and depletes substance P, prevented emesis caused by peripheral emetogenic stimuli such as radiation and copper sulphate and by the central emetogen loperamide in ferrets. This was confirmed further showing that the site of the anti-emetic effect of resinferatoxin is within the nucleus tractus solitarius and that the mechanism is relevant to the anti-emetic action of NK1 -receptor antagonists.31
In this study, we used minks as a vomiting model, which has been established as a new vomiting animal model by our laboratory.20 The results of this study showed that there was a significantly reduced frequency in cisplatin-induced emesis caused by gingerol in minks. And the study results also have found that cisplatin enhanced the expression of substance P and NK1 receptors in both the area postrema and ileum. Gingerol significantly reduced the detectable levels of substance P and NK1 receptor in the ileum as well as in the area postrema, and the elevated expression levels of NK1 receptor in both the area postrema and ileum were reduced by gingerol. It showed that gingerol exhibited an activity on secretion or production of substance P which can be explained by the similar structure between gingerol and capsaicin (Figure 7).32,33 The reduced levels of NK1 receptor may be related to the depletion of substance P or the inhibition of synthesis and expression of NK1 receptor.
To conclude, this study demonstrated that gingerol could significantly inhibit cisplatin-induced retching and vomiting, and gingerol not only inhibited the increase of substance P from the area postrema and ileum, but also reduced the expression of NK1 receptor in the ileum as well as in the area postrema. Therefore, the mechanism underlying the antiemetic effect of gingerol may be related to the inhibitive activity on peripheral or central increase of substance P and NK1 receptor. We will soon carry out further investigations on the signal transduction modes to clarify the mechanism.
The first author of this article wants to express his special thanks to Professor WANG Kai of Department of Hepatology in Qilu Hospital of Shandong University for the suggestion and comments on experiments and manuscript. The author also thanks Qingdao University Guide for the Care and Use of Laboratory Animals for providing the animals.
1. Hickok TJ, Morrow GR. A biobehavioral model of patient reported nausea: implications for clinical practise. Adv Med Psychother 1993; 6: 227-230.
2. Fiore JJ, Gralla RJ. Pharmacologic treatment of chemotherapy-induced nausea and vomiting
. Cancer Invest 1984; 2: 351-361.
3. Morrow GR. Chemotherapy-related nausea and vomiting
: etiology and management. CA Cancer J Clin 1989; 39: 89-104.
4. Morrow GR, Hickok JT, Rosenthal SN. Progress in reducing nausea and emesis. Cancer 1995; 76: 343-357.
5. Gandara DR, Harvey WH, Monaghan GG, Perez EA, Hesketh PJ. Delayed emesis following high-dose cisplatin
a double-blind randomised comparative trial of ondansetron GR-38032F versus placebo. Eur J Cancer 1993; 29A: S35-S38.
6. Kris MG, Tyson LB, Clark RA, Gralla RJ. Oral ondansetron for the control of delayed emesis after cisplatin
. Report of a phase-II study and a review of completed trails to manage delayed emesis. Cancer 1992; 70: 1012-1016.
7. Bountra C, Bunce K, Dale T, Gardner C, Jordan C, Twissell D, et al. Anti-emetic profile of a non-peptideneurokinin NK1
receptor antagonist, CP-99,994, in ferrets. Eur J Pharmacol 1993; 249: R3-R4.
8. Tattersall FD, Rycroft W, Hill RG, Hargreaves RJ. Enantioselective inhibition of apomorphine-induced emesis in the ferret by the neuokinin1 receptor antagonist CP-99,994. Neuropharmacology 1994; 33: 259-260.
9. Watson JW, Gonsalves SF, Fossa AA, McLean S, Seeger T, Obach S, et al. The anti-emetic effects of CP-99,994 in the ferret and dog: role of the NK1
receptor. Br J Pharmacol 1995; 115: 84-94.
10. Hesketh PJ, Van Belle S, Aapro M, Tattersall FD, Naylor RJ, Hargreaves R, et al. Differential involvement of neurotransmitters through the time course of cisplatin
-induced emesis as revealed by therapy with specific receptor antagonists. Eur J Cancer 2003; 39: 1074-1080.
11. Andrews PLR, Rudd JA. The role of tachykinins and the tachykinin receptor in nausea and emesis. In: Holzer P (Ed.), Handbook of Experimental Pharmacology. Springer-Verlag Berlin Heidelberg: Germany; 2004: 359-440.
12. Leksowski K, Peryga P, Szyca R. Ondansetron, metoclopramid, dexamethason, and their combinations compared for the prevention of postoperative nausea and vomiting
in patients undergoing laparoscopic cholecystectomy: a prospective randomized study. Surg Endosc 2006; 20: 878-882.
13. Abali H, Celik I. Tropisetron, ondansetron, and granisetron for control of chemotherapy-induced emesis in Turkish cancer patients: a comparison of efficacy, side-effect profile, and cost. Cancer Invest 2007; 25: 135-139.
14. Vutyavanich T, Kraisarin T, Ruangsri R. Ginger for nausea and vomiting
in pregnancy: randomized double-masked placebo-controlled trial. Obstet Gynecol 2001; 4: 577-582.
15. Pongrojpaw D, Chiamchanya C. The efficacy of ginger in prevention of post-operative nausea and vomiting
after outpatient gynecological laparoscopy. J Med Assoc Thai 2003; 3: 244-250.
16. Sharma SS, Kochupillai V, Gupta SK, Seth SD, Gupta YK. Antiemetic effcacy of ginger against cisplatin
induced emsis in dogs. Ethnonharmacol 1997; 2: 93-96.
17. Zhao DX, Yang ZH, Li M, Xue M, Yue W. Anti-emetic effects of ginger on a nausea model of mink
. Chin Pharm Affairs (Chin) 2006; 10: 601-604.
18. Jiang SZ, Mi SZ, Wang NS. Survey of chemical constituents of gingerol
. Tradit Chin Drug Res Clin Pharmacol (Chin) 2006; 17: 386-389.
19. Abdel-Aziz H, Windeck T, Ploch M, Verspohl EJ. Mode of action of gingerols and shogaols on 5-HT3 receptors: binding studies, cation uptake by the receptor channel and contraction of isolated guinea-pig ileum. Eur J Pharmacol 2006; 530: 136-143.
20. Zhang F, Wang L, Yang ZH, Yue W. Value of mink
vomit model in study of anti-emetic drugs. World J Gastroenterol 2006; 12: 1300-1302.
21. Ferrier CM, de Witte HH, Straatman H, van Tienoven DH, van Geloof WL, Rietveld FJ, et al. Comparison of immunohistochemistry with immunoassay (ELISA) for the detection of components of the plasminogen activation system in human tumour tissue. Br J Cancer 1999; 79: 1534-1541.
22. Yuan ZL, Feng WY, Hong J, Zheng Q, Shuai J, Ge YB. p38MAPK and ERK promote nitric oxide production in cultured human retinal pigmented epithelial cells induced by high concentration glucose. Nitric Oxide 2009; 20: 9-15.
23. Leslie RA. Neuroactive substances in the dorsal vagal complex of the medulla oblongata: nucleus of the tractus solitarius, area postrema, and dosal motor nucleus of vagus. Neurochem Int 1985; 7: 191-211.
24. Nagashima A, Takano Y, Tateishi K, Matsuoka Y, Hamaoka T, Kamiya H. Cardiovascular roles of tachykinin peptides in the nucleus tractus solitarii of rats. Brain Res 1989; 487: 392-396.
25. Newton BW, Maley B, Traurig H. The distribution of substance P
, enkephalin and serotonin immunoreactivities in the area postrema of the rat and cat. J Comp Neurol 1985; 234: 87-104.
26. Ramírez MJ, Cenarruzabeitia E, Del Río J, Lasheras B. Involvement of neurokinins in the non-cholinergic response to activation of 5-HT3- and 5-HT4-receptors in guinea pig ileum. Br J Pharmacol 1994; 111: 419-424.
27. Fukuda H, Koga T, Furukawa N, Nakamura E, Shiroshita Y. The tachykinin NK1
receptor antagonist GR205171 abolishes the retching activity of neurons comprising the central pattern generator for vomiting
in dogs. Neurosci Res 1999; 33: 25-32.
28. Fukuda H, Nakamura E, Koga T, Furukawa N, Shiroshita Y. The site of the anti-emetic action of tachykinin NK1
receptor receptor antagonists may exist in the medullary area adjacent to the semicompact part of the nucleus ambiguus. Brain Res 1999; 818: 439-449.
29. Potts JT, Fuchs IE, Li J, Leshnower B, Mitchell JH. Skeletal muscle afferent fibres release substance P
in the nucleus tractus solitarii of anaesthetized cats. J Physiol 1999; 514: 829-841.
30. Andrews PLR, Bhandari P. Resinferatoxin, an ultrapotent capsaicin analogue, has anti-emetic properties in the ferret. Neuropharmacology 1993; 32: 799-806.
31. Andrews PLR, Bhandari P. The 5-hydroxytryptamine receptor antagonists as antiemetics: preclinical evaluation and mechanism of action. Eur J Cancer 1993; 29: S11-S16.
32. Andrews PLR, Okada F, Woods AJ, Hagiwara H, Kakaimoto S, Toyoda M, et al. The emetic and anti-emetic effects of the capsaicin analogue resiniferatoxin in, the Suncus murinus house musk shrew. Br J Pharmacol 2000; 130: 1247-1254.
33. Surh YJ. Anti-tumor promoting potential of selected spice ingredients with antioxidative and anti-inflammatory activities: a short review. Food Chem Toxicol 2002; 40: 1091-1097.
Keywords:© 2010 Chinese Medical Association
gingerol; vomiting; mink; cisplatin; substance P; NK1 receptors