Introduction
Hepatic ischemia-reperfusion (I/R) can not only cause damage to liver function [
1–
5], but may also be involved in other organ (such as the lung, kidney, digestive tract, etc.) dysfunction. The portal vein system gore caused by hepatoportal blood occlusion can exert a direct impact on the function of the digestive tract and result in endotoxemia. Therefore, it is particularly important to recognize the changes in gastrointestinal motility at the time of liver I/R so as to take measures to maintain or restore the motor function of the gastrointestinal tract at the earliest opportunity. The expression and significance of the upper jejunum inhibitory hormone cholecystokinin (CCK) and vasoactive intestinal peptide (VIP) were explored and the influence of salvia miltiorrhiza pretreatment on CCK and VIP at hepatic I/R injury was investigated as well.
Materials and methods
Grouping of rats
Eighty Sprague Dawley (SD) rats weighing 220±20 g, 3–4 months of age, purchased from the Hubei Provincial Academy of Medical Sciences (Wuhan, China) were randomly divided into four groups as follows: normal control group (CO group) (n = 5), sham-operated group (SO group) (n = 25), I/R injury group (IR group) (n = 25), and salvia miltiorrhiza pretreatment group (SM group) (n = 25). According to different time lengths after reperfusion (0, 3, 12, 24, and 72 h), each group was then divided into five sub-groups, each subgroup containing five rats.
Establishment of the rat I/R model
The rat I/R model had been preoperatively fasted for 12 h with free access to drinking water. Rats were anesthetized intraperitoneally with sodium pentobarbital (40 mg/kg body weight). After anesthesia was effectuated, iodine was used to sterilize the skin. An epigastric median incision of about 3 cm was then made and the liver was retracted upward. Ischemia was induced by exposing and dissociating the hepatoduodenal ligament, and then the hepatic pedicle at the distal end of the hepatoduodenal ligament was clamped for 45 min by a non-invasive artery clamp. Forty-five min later, the ischemic liver was reperfused by loosening the clamp, and the specimens were taken at different reflowing time points (0, 3, 12, 24, and 72 h, respectively). In rats of the SO group, the porta hepatis was dissected without clamping the hepatic pedicle after laparotomy. Rats in the SM group were injected with saline (40 mL/kg) and salvia miltiorrhiza (6 g/kg body weight) via the tail vein 30 min before blocking the flow. Anesthesia, surgical and sampling methods were the same as mentioned above.
Sampling and measurement parameters
The upper jejunum tissues at different time points were taken and fixed with 10% formalin, embedded and sliced conventionally. The CCK and VIP immunohistochemical test kits were purchased from Wuhan Boster Biological Products Company (China). Immunohistochemistry and staining were carried out in accordance with instructions of the kits. The positive standard was brown granules in the cytoplasm. Five non-consecutive sections were selected from each specimen. Under a 400× optical microscope, 10 vision fields were randomly selected from each section to make a statistical analysis on the positive unit (PU) [
6] and the average of five sections was calculated as final results.
Statistical analysis
The SPSS13.0 statistical software was applied to analyze data. Comparison among multi-groups was done by one-way ANOVA F-test, and the LSD test was performed to evaluate significant differences intra or between groups. Differences were considered significant when P<0.05.
Results
Detection of the CCK expression by immunohistochemistry and assessment of positive cells
There was a significant difference in the CCK expression between the CO group and SO group at every time limit point. The result of intra-comparison of the IR group (P<0.05) indicated the existence of differences. Through paired comparison of various time limit points (LSD test), it was found that paired comparison of each time limit point had significant differences. The result of intra-comparison of the SM group (P<0.05) indicated the existence of differences. Through paired comparison of various time limit points (LSD test), significant differences were also found.
Intra-group comparison
There was a significant difference in the CCK expression in the CO group and SO group at every time limit point. The result of intra-comparison of the IR group (P<0.05) indicated the existence of differences. Through paired comparison of various time limit points (LSD test), it was found that paired comparison of each time limit point had significant differences. The result of intra-comparison of the SM group (P<0.05) indicated the existence of differences. Through paired comparison of various time limit points (LSD test), significant differences were also found.
Group comparison
There was no significant difference in the CCK expression between the CO group and the SO group at various time limit points. There was a significant difference at the 0, 3, 12, 24, and 72 h time limit points between the IR group and CO group or SO group (P<0.05). At the 72 h, the CCK expression level was clearly restored and returned to the normal level. Comparing the SM group with the CO group and the SO group at 0, 3, 12, and 24 h, there were significant differences (P<0.05). However, at the 72 h, there was no significant difference (P>0.05), indicating that the CCK expression level of the SM group returned to normal. There was a significant difference between the SM group and the IR group at 0, 3, 12, 24, and 72 h (P<0.05).
The results of VIP expression detected by immunohistochemical test and positive cell test
VIP was mainly expressed in the jejunum mucosa and submucosa. The VIP expression began to increase significantly at 0 h of reperfusion in the IR group and the SM group, reached the peak at 24 h, and then gradually declined to approximately baseline levels at 72 h. However, the expression of VIP in the IR group was higher than that in the SM group at the same time limit point. More detailed results are demonstrated in Table 2.
The immunohistochemical results of VIP expression at 24 h after reperfusion are shown in Fig. 2.
Intra-group comparison
There was no significant difference in the expression of VIP between the CO group and SO group at every time limit point. Intra-comparison of the IR group (P<0.05) indicated the existence of differences. Through paired comparison of various time limit points (LSD test), significant differences were found. Intra-comparison of the SM group (P<0.05) indicated the existence of differences. Through paired comparison of various time limit points (LSD test), significant differences were also found.
Group comparison
There was a significant difference in the expression of VIP at 0, 3, 12, 24, and 72 h time limit points between the IR group and the CO group or SO group. At 72 h of reperfusion, the expression was clearly restored and returned to the normal level. There was a significant difference in the VIP expression among the SM group, CO group and SO group at 0, 3, 12, and 24 h (P<0.05). But at 72 h, there was no significant difference (P>0.05) among these groups. This indicated that the CCK expression in the SM group returned to the normal level at 72 h of reperfusion. There was a significant difference in the expression of VIP between the SM group and IR group at 0, 3, 12, 24, and 72 h time limit points (P<0.05).
Discussion
There are a lot of researches on hepatic I/R injury and its protection [
7-
12]. However, there are few studies on the effect of hepatic I/R injury on extra-hepatic organs. The gastrointestinal tract is vulnerable to hepatic I/R injury, especially when the first hepatic portal is blocked. Gastrointestinal tract congestion and venous obstruction would happen and subsequently blood recanalization causes reperfusion injury.
CCK was discovered from the gastrointestinal mucosa of dogs in 1928 by Ivy and Oldberg [
13]. As a kind of gastrointestinal hormone and neuropeptide, CCK distributes widely in the digestive tract, central and peripheral nervous system, peripheral blood and other tissues [
14-
17]. It works primarily through two ways. First, through the blood circulation, CCK acts on the corresponding receptors of gastrointestinal smooth muscle cells in the form of endocrine. Second, it works as a gastrointestinal neuropeptide and regulates gastrointestinal motility. CCK in the gastrointestinal tract mainly exists in the mucous layer and in the form of a large molecule. Under physiological circumstances, the release of CCK in the gastrointestinal tract occurs in the feedback loop. A major factor causing the release of CCK is the fat hydrolysates and protein metabolites in the duodenum, and fat is a kind of powerful stimulus for the release of endogenous CCK. Intestinal CCK can regulate trypsin release and gallbladder contraction, inhibit gastric emptying, which would result in satiety and suppression of feeding behavior, and inhibit gastrointestinal motility. This study found that the positive expression of CCK in jejunum mucosa tissues of the IR group began to increase at 0 h of reperfusion 45 min after hepatic ischemia. At 0, 3, 12, 24, and 72 h after reperfusion, the expression of CCK in the IR group was significantly increased (
P<0.05) as compared with the other groups. This result suggests that in hepatic I/R injury, intestinal CCK gene is apparently activated. Its expression is increased, and thus gastrointestinal motility is inhibited. Tang
et al. [
18] reported that under the stomach expansionary mechanical stimulation, the cerebral cortex and hippocampus CCK mRNA expression was increased, indicating that the process of CCK synthesis in the central nervous system is involved in the regulation of intragastric pressure and gastric motility. Under the situation of gastrointestinal tract congestion, the gastrointestinal tract congestion expansion also leads to the activation of CCK expression in the brain. The peak time is at 24 h of reperfusion. Therefore, the first possible reason for the increase of CCK expression in the IR group might be brain feedback delay. The second reason might be the reaction of gastrointestinal tract congestion. These two factors may act collaboratively, leading to the increase of CCK secretion.
VIP, a kind of polypeptide composed by 28 amino acids, was first separated and purified from the small intestine of pigs by Said in 1970. In 1971, Cailquist also separated and purified VIP from the human body. It is a kind of gastrointestinal neuropeptide, widely existing in the digestive tract. It is secreted by endocrine cells of the gastrointestinal tract mucosa, small nerve fibers and myenteric plexus of the submucosa [
19-
25]. As a main transmitter of non-adrenergic non-cholinergic (NANC) nerves, it plays an inhibitory effect on digestive movement [
26]. This study found that, as compared with the CO, SO and SM groups, the VIP expression was increased at 0, 3, 12, and 24 h in the IR group (
P<0.05), indicating that the VIP gene in the digestive tract in hepatic I/R was obviously activated, and its expression was up-regulated. Therefore, the gastrointestinal motility was inhibited.
As gastrointestinal tract inhibitory hormones, CCK and VIP were increased to various degrees in the upper jejunum at different time points of hepatic reperfusion 45 min after ischemia. Their levels reached the peak at 24 h of reperfusion. It could be speculated that both the hepatic I/R injury and gastrointestinal congestion were stress factors; they inhibited the vagus nerve but excited the sympathetic nerve; these two types of hormones were regulated by the autonomic nerve. CCK and VIP secretion were increased by endocrine cells after sympathetic nerve excitation, while gastrointestinal motility was inhibited. At the same time, CCK and VIP are neurotransmitters that belong to the brain-gut peptides. They are released to the gastrointestinal tract through the regulatory action of the central nervous system and inhibit gastrointestinal motility.
The positive expression level of CCK and VIP in the SM group at each time limit point was obviously lower than that in the IR group (P<0.05). Further, there was no significant difference between the CO group and SO group at 72 h of reperfusion. However, the expression of CCK and VIP in the IR group at 72 h of reperfusion was still significantly higher than that in the CO group and SO group (P<0.05). It showed that when CCK and VIP expression had been restored to normal levels in the SM group at 72 h of reperfusion, while in the IR group it had not yet returned to normal levels. This suggests that SM possibly had an inhibitory effect on the CCK and VIP activation/expression; however, the mechanism is unclear. SM might improve the microcirculation and reduce gastrointestinal stasis, promote the restoration of gastrointestinal motility, and weaken the effects of stress factors caused by I/R injury and gastrointestinal stasis. Thus, it could indirectly down-regulate CCK and VIP expression and reduce their secretion. However, whether it has a direct inhibitory effect on CCK and VIP gene expression is unknown. The effects of SM have a positive significance in that it can reduce the injury caused by intestinal barrier function weakening, bacteria translocation and endotoxemia. It reduces the absorption of gut-derived endotoxin, which not only is helpful for the protection and recovery of liver function, but also contributes to the internal environment stability.
In summary, it was initially found that the expression of CCK and VIP was increased and gastrointestinal motility was inhibited after hepatic I/R injury. SM pretreatment can weaken the expression of CCK and VIP to some extent. The influence of hepatic I/R on gastrointestinal tract function at different reperfusion time limit points 45 min after hepatic ischemia was investigated. Further studies need to be performed regarding the molecular mechanisms, especially the signal transduction pathways and the differential activation of the gene map.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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