Effect Of Subcutaneous Injection Of "Ala"¹⁶-aFGF(1-29) On Slow Transit Constipation Mice

Background:Slow transit constipation is one of the most common clinical diseases, recent researches suggest that the enteric nervous system (ENS), intestinal cell of Cajal (ICC), and the change of intestinal neurotransmitter, closely related to the development of STC. However, as the etiology and pathogenesis remain unknown, the clinical treatments of STC mainly rely on various laxatives which always lack of effective treatment. Acidic fibroblast growth factor (aFGF) can promote cell growth, nutrition, nerve repair, and inhibition of apoptosis etc. Studies have reported that aFGF exist in the enteric nervous system and may play an important role in the maintenance the function of the enteric nervous system. [Ala]¹⁶-aFGF(1-29) is a short fragment of aFGF, which was more suitable for clinical use because of it's same biological activity with aFGF whole molecule. Therefore, to investigate the effect of [Ala]¹⁶-aFGF (1-29) on slow transit constipation may provide experimental evidence on a new treatmental strategy for STC.Aim:To investigate the therapeutical effect of [Ala]¹⁶-aFGF(1-29) on the slow transit constipation in the mice and its investigate its possible pathway by subcutaneous injection (SC).Methods:1. Sixty ICR mice were divided randomly into test group (Test) and control group (Control) (n=30, respectively). A STC mouse model was established by SC of morphine (2.5mg/kg/d) in Test for 45 days, and the Control was processed with normal saline at the same dosage. On the 45th day, the fecal character was observed according to Bristol Stool Form Scale (BSFS), BSFS 1 and 2 were considered as STC model and BSFS 4, 5 as normal stool, random 6 mice in each group were used to further confirm the model success by charcoal propulsion experiment.2. On the 45th day, the fecal character was observed according to BSFS. The residual stool in the colon was recorded after 24 hours of fasting. The intestinal propulsion rate of the two groups was detected by the charcoal propulsion experiment. Immunohistochemistry was used to detect the distribution of the enteric neurons and Casepase-3 (apoptin) positive cell, the expression of neuron-specific enolase (NSE, a neuronal maker) level was detected by Western blot. Apoptotic enteric neurons were identified and counted by double immunofluorescence with labeling for NSE and Caspase-3.3. Then the rest mice in Test and Control group were respectively divided into Test 1, Test 2 and Control 1, Control 2 (n=12, respectively). Test 1 and Test 2 were treated with [Ala]¹⁶-aFGF(1-29) (300ug/kg/d, SC) and the same volume solution respectively for 8 weeks, 2 times per week, to investigate the therapeutical effect of [Ala]¹⁶-aFGF(1-29) on STC; Control 1 and Control 2 were processed with [Ala]¹⁶-aFGF(1-29) and the solution at the same dosage to exclude the interference factors of the experiment. Fecal character of all mice was observed. Eight weeks later, for the mice in each group, the fecal character was observed based on BSFS; the residual stool in the colon was recorded after 24 hours of fasting; the intestinal propulsion rate was detected by the charcoal propulsion experiment. Immunohistochemistry was used to detect the distribution of the enteric neurons and Casepase-3 positive cell. The expression of NSE level was detected by Western blot. And apoptotic enteric neurons were identified and counted by double immunofluorescence with labeling for NSE and Caspase-3.Results:1. On the 45th day, the feces in Test were drier and harder (BSFS 1 and 2) than Control (BSFS 4 and 5). Compare with the Control, the residual stool was obviously increased (P<0.05), the intestinal propulsion rate was significantly declined (P<0.01) in Test group.2. On the 45th day, the distribution of enteric neurons, level of NSE and enteric neurons apoptosis1) The enteric neurons mainly distribute in myenteric plexus and submucosal plexus, especially in the myenteric plexus of the intestinal tract in mice. NSE level in Test was decreased compared with Control (P<0.05)2) Caspase-3 positive cell can be observed in the myenteric plexus and submucosal plexus of the intestinal tract. Expression of Caspase-3 was significantly increased in Test compared with the Control (P<0.05). The number of apoptotic enteric neurons was significantly increased in the Test compared with the Control (P<0.05).3. Eight weeks after chemical application, the fecal character of Test 1 became smooth and soft (BSFS 4 and 5) Control 1 and 2. Compared with Test 2, the residual stool was significantly declined and the intestinal propulsion rate was obviously increased in Test 1(P<0.05). The residual stool and the intestinal propulsion of Test 1 showed no significant difference with Control groups (P>0.05).4. Eight weeks after chemical application, the main results: 1) The enteric neurons mainly distributed in myenteric plexus and submucosal plexus, especially in the myenteric plexus of the mice intestinal tract. NSE level in Test 1 was increased compared with Test 2 (P<0.05), which showed no significantly difference with Control groups (P>0.05).2) Compared with the Test 2, the expression of Caspase-3 was significantly increased, and the number of apoptotic enteric neurons was significantly increased in Test 1 (P<0.05). Both the expression of Caspase-3 and the number of the apoptotic enteric neurons showed no significantly different between Test 1 and the two control groups (P<0.05).Conclusions:1) Animal model of slow transit constipation can be established by continuous subcutaneous injection of morphine for 45 days.2) There is a significant decline of enteric neurons in colonic tissue of slow transit constipation mice compared with the normal mice and the apoptosis of the enteric neurons is one of the pathogenesis.3) Subcutaneous injection of [Ala]¹⁶-aFGF(1-29) can improve the fecal character and intestinal motility of slow transit constipation mice.4) [Ala]¹⁶-aFGF(1-29) may improve the enteric neuropathy of STC mice by inhibiting the apoptosis of enteric neurons.