Effect of MWCNTs on Gastric Emptying in Mice
© Li et al. 2010
Received: 6 July 2010
Accepted: 10 September 2010
Published: 7 October 2010
After making model of gastric functional disorder (FD), part of model mice were injected intravenously (i.v.) with oxide multi-walled carbon nanotubes (oMWCNTs) to investigate effect of carbon nanotubes on gastric emptying. The results showed that NO content in stomach, compared with model group, was decreased significantly and close to normal level post-injection with oMWCNTs (500 and 800 μg/mouse). In contrast to FD or normal groups, the content of acetylcholine (Ach) in stomach was increased obviously in injection group with 500 or 800 μg/mouse of oMWCNTs. The kinetic curve of emptying was fitted to calculate gastric motility factor k; the results showed that the k of injection group was much higher than FD and normal. In other words, the gastric motility of FD mice was enhanced via injection with oMWCNTs. In certain dosage, oMWCNTs could improve gastric emptying and motility.
Carbon nanotubes (CNT) represent the structural evolution of the archetypal molecular architecture consisting of pure carbon units, the C60 fullerene , CNT, or "buckytubes" , possess extraordinary properties, such as high electrical and thermal conductivity, great strength, and rigidity, and being developed for a wealth of applications, including field emission , energy storage, molecular electronics, and atomic force microscopy. These properties indicate diverse future biomedical uses in areas such as targeted chemotherapeutics, in vitro cell markers, diagnostic imaging contrast agents, biochemical sensors, and photoablative therapy agents . However, it was not found in previous works to use carbon nanotubes as single drug to therapy in clinical application, no works could prove that the carbon nanotubes can improve biological function of animals, and most papers only pay attention to toxicology of carbon nanotubes on tissues or cell [5–12]. In our long-term researches, we found that the oxide multi-walled carbon nanotubes (oMWCNTs) can improve gastric function of animals. Therefore, we attempt to investigate effect of oMWCNTs on gastric emptying in mice. If the CNTs alone have medicinal values, then it is very important for CNTs to develop in clinical application. It would be inevitable to widen the application prospects of CNTs in medical field.
Materials and Method
Preparation of Oxidized MWCNTs
MWCNTs commercially prepared by chemical vaporization deposition were obtained from Shenzhen Nanotech Port Co. Ltd. China. Determined with transmission electron microscopy (TEM), MWCNTs are several tens of micrometers in length, with a diameter of 10–30 nm. Purity was >96%, containing <3% amorphous carbon and ash < 0.2 wt%, according to thermal gravity analysis (TGA).
The as-grown MWCNTs (named as untreated MWCNTs) were added into the solution of 3 mol/L HNO3 to remove the hemispherical caps of the nanotubes. The mixture of 3 g MWCNTs and 400 mL 3 mol/L HNO3 was ultrasonically stirred for 24 h. The suspension was filtrated, and then dialyzed by dialysis bag for 2 weeks, and rinsed with deionized water until the pH of the suspension reached about 6, and then was dried at 80°C. Thus, prepared MWCNTs (named as oxidized MWCNTs or oMWCNTs) were calcined at 450°C for 24 h to remove the amorphous carbon . The oMWCNTs were dissolved in normal saline, and then ultrasonically treated before injection.
Preparation of the Test Meal
Hydrated diet was prepared by placing 45 g of pellets in 100 ml water and storing in the refrigerator at 4°C for at least 16 h. Before dispensing the food to the animals, a further 2 mL water was added to ensure that the food was saturated with water yet maintained a semi-solid state. The hydrated diet was initially introduced because it was observed that in mice fed on standard dry chow, gastric emptying was very slow. The weight of residual food in the stomach of the animals following 24 h of food deprivation was still very substantial, even with a grid floor in place, and this made evaluation of gastric emptying difficult [14, 15].
Test of NO and ChAT
Female Kongming White mice weighing 16–20 g were obtained from laboratory Center for Medical Science, Lanzhou University, Gansu, China. All animals were introduced to hydrated diet (prepared as described above) 24–36 h before commencing the experiment. The animals were maintained on the hydrated diet for 24–36 h prior to commencement of the experiments in order to allow them to adapt to the new food. Water was provided ad libitum throughout the experimental period.
The four groups of mice (fifteen mice per group) were injected intraperitoneally (i.p.) with L-arginine  (6 mg/mouse) for 5 days to make model of FD. One additional group (fifteen mice) was injected intraperitoneally with normal saline as control. Until at 5 day, three groups of FD were injected intravenously (i.v.) with oMWCNTs for 3 days, the doses were 100, 500, and 800 μg/mouse, respectively. Another one was continuing to inject with L-arginine for 3 days. All mice were killed at 8 days; every stomach was collected, and then removed chyme. According to the procedures of specification, the ChAT and NO kits, purchased from Nanjing Jiancheng Bio-Technology Co., Ltd., were used to determine content of ChAT and NO in stomach tissues.
The Effect of oMWCNTs on the Secretion of Gastric Mucus  and the Activity of Pepsin
After making successful model of FD, one group of FD (fifteen mice) was injected intravenously with oMWCNTs (500 μg/mouse) for 3 days, the other group of FD was injected intraperitoneally with L-arginine (6 mg/mouse) for 3 days. Meanwhile, one normal group (fifteen mice) was injected intraperitoneally with normal saline as control. All mice were killed at 1 h after fed with hydrated diet to collect chyme and gastric tissues, and then the gastric mucosa was washed by 4 mL water, the flushing fluid was used to dissolve 1 g chyme. Suspension of 0.25 g/mL (solid–liquid ratio, S/L) was soaked for 24 h and centrifuged to measure pH values in supernatants. Meanwhile, the pepsin kits, purchased from Nanjing Jiancheng Bio-Technology Co., Ltd., were used to determine the pepsin activity in gastric tissues.
E emptying rate, Wst weighs of total stomach, Wsn weighs of net stomach, Wb body weighs.
Analysis of Data
The data were expressed as mean ± SEM, and statistical significance of differences was calculated using SPSS17.0 software to perform one-way ANOVA test.
Results and Discussion
Preparation of oMWCNTs
Distribution of oMWCNTs in Stomach and Chyme
The Effect of oMWCNTs on the Secretion of Gastric Mucus
The effect of oMWCNTs on pH in stomach
4.69 ± 0.04
4.75 ± 0.04*,&
4.68 ± 0.06
Test of NO and ChAT in Stomach
The choline acetyl transferase (ChAt) was synthase of acetylcholine (Ach), which indicated that ChAt content could represent Ach content in stomach; previous studies showed that gastric active function is complex physical process, which was regulated by body fluid and nerve . The coordination of excitatory and inhibitory neuron in midgut never regulates the gastrointestinal coordinated motion. The neurotransmitter released from excitatory neuron is Ach, which could promote contraction of gastrointestinal smooth muscle. The neurotransmitter released from inhibitory neuron is NO, which could induce relaxation of gastrointestinal smooth muscle. NO produced by nitric oxide synthase is the neurotransmitter of non-adrenergic and non-cholinergic nerves. The NO could promote the capacity relaxation of stomach and antagonize contraction of stomach induced by ChAt. In a word, the NO could affect on gastric peristalsis and emptying .
The L-arginine could induce synthesis of NO from nitric oxide synthase . The results showed that L-arginine has promoted increasing of NO in stomach of normal mice, so model of FD was made successfully for 5 days post-i.p. with L-arginine and that reported by literature . Lower dose of injection with oMWCNTs (100 μg/mouse) did not induce changes of NO and ChAT in stomach, but obvious effect has been observed post-i.v. with 500 or 800 μg/mouse (p < 0.01, Figures 5, 6). The higher content Ach could facilitate contraction of gastrointestinal smooth muscle, and lower content NO could inhibit relaxation of gastrointestinal smooth muscle [16, 21]. Therefore, the emptying force of stomach has been improved post-i.v. with higher dose of oMWCNTs. This implied that the gastric emptying could be enforced significantly after i.v. with higher dose of oMWCNTs (500, 800 μg/mouse).
Effect of oMWCNTs on the Secretion of Pepsin in Stomach
The pepsinogen that was secreted from gastric chief cells could be activated and transformed into pepsin in pH < 5.0, the pepsin could decompose the protein of chyme . Therefore, the activity of pepsin could be increased by improving the secretion of pepsinogen under stable pH values. Figure 7 showed that L-arginine could decrease significantly the activity of pepsin in FD group (p < 0.01) compared with normal mice, it was reported that L-arginine could increase NO content  so as to induce gastric functional disorder , but could not directly inhibit the secretion of pepsinogen, so these results implied that gastric functional disorder caused by L-arginine could inhibit the secretion of pepsinogen and decrease the pepsin activity in FD group. Hereby, we concluded that the secretion of pepsinogen in administration group could be increased slightly because of gastric functional disorder has been improved via injection with oMWCNTs. Therefore, the pepsin activity was increased to some extent in administration group due to the secretion of pepsinogen improved by carbon nanotubes (Figure 7).
Kinetic Study of Gastric Emptying
W is weighs of food residue, W = Wst - Wsn (Wst is total weighs of stomach, Wsn is net weighs of stomach); t is time; k gastric motility factor (equivalent to elastic coefficient); the k is related to gastric function and food state (liquid in here); when the same food was fed, the lager k is, the stronger emptying force is.
Figures 3 and 8 showed that the emptying force of injection group should be higher than FD and normal because of higher Ach and lower NO content in stomach, but the emptying force of FD was lower than normal mice for normal Ach and high-NO content in stomach. So the food was emptied rapidly from stomach in 1 h for injection groups, but for FD, slow emptying would be observed from 1 to 6 h for poor gastric emptying force.
According to above PM, gastric motility factor k was fitted to calculate in FD, normal and injection groups (Figure 8). When the same food was used in experiment, then the higher the k is, the stronger the gastric emptying is and the better the gastric function is. Figure 8 showed that the k of injection group with 500 μg/mouse was much higher than normal group and FD, the k of injection, FD and normal were 0.682, 0.531 and 0.432, respectively. It implied that oMWCNTs could improve gastric function of FD and enhance gastric motility and emptying.
As can be seen from Figures 3 and 4 and Table 1, the oMWCNTs could be accumulated in stomach and secreted into chyme as mucus bicarbonate barrier, so in the course of accumulation and secretion, oMWCNTs had to contact with gastric mucous, as one kind of foreign body, stimulated gastric tissues, caused a series of reactions, and decreased NO content and increased ChAT content in gastric tissues, sped up the gastric emptying, and improved to some extent the activity of pepsin. As a result, the physiological function of stomach was improved obviously post-i.v. with suitable dose of oMWCNTs. And we concluded that the high distribution of oMWCNTs in stomach was result from oxide treatment, so surface chemical groups on carbon nanotubes would be a key factor to affect on gastric physiological function. Therefore, after oxide treatment, the CNTs, injected into mice of functional disorder, could inhibit content NO and increase the content Ach in stomach, and it was more favorable for higher dose of CNTs to reinforce the effect.
The oMWCNTs can be secreted from mucus cells into chyme post-i.v. with oMWCNTs, and this course can increase pH in stomach.
The NO content can be decreased post-i.v. with carbon nanotubes into mice of functional disorder, and the Ach content can be increased, the effect is more obvious post-injection with higher dose of carbon nanotubes.
The carbon nanotubes can enhance gastric emptying and improve gastric function, and thus increase to some extent the activity of gastric pepsin.
This study was conducted with financial support from National Natural Science Foundation of China (20871062, J1030932, J0630962).
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