Effect and mechanisms of sacral nerve stimulation on visceral hypersensitivity mediated by nerve growth factor

Abstract To investigate the efficacy of sacral nerve stimulation (SNS) on nerve growth factor (NGF) mediated visceral sensitivity in normal rat and visceral hypersensitivity model rats. 120 male newborn rats were randomly divided into 6 groups: group A was normal model group; group B ~ F were all sensitized with acetic acid enema and grouped again. Group c2 was given NGF antagonist, d2 group was given NGF agonist, e2 group was given PI3K inhibitor, and f2 group was given PLC‐γ inhibitor. After treatment, the expression of NGF, TrKA, PI3K, AKT, PLC‐γ, NF‐κB, TRPV1, pTRPV1 and intracellular Ca2+ content were detected. The expression of protein TRPV1 and pTRPV1 was increased, and Ca2+ was increased in the visceral hypersensitive group. NGF, TrKA in NGF antagonist group, PI3K, AKT, NF‐κB in PI3K inhibitor group, PLC‐γ in PLC‐γ inhibitor group were all almost not expressed. The relative expression of NGF, TrKA, PI3K, AKT, PLC‐γ and NF‐κB in NGF antagonist group was lower than that in visceral hypersensitivity group and NGF activator group (P < .01). The relative expression of NGF, TrKA, PI3K and AKT mRNA in NGF antagonist group was lower than that in the normal model group (P < .01). There was no significant difference in the relative expression of PLC‐γ and NF‐κB mRNA (P > .05). The expression level of MAPK, ERK1 and ERK2 in visceral hypersensitivity group was higher than that in PI3K inhibitor group and PLC‐γ inhibitor group. The normal group Ca2+ curve was flat, and the NGF agonist group had the highest Ca2+ curve peak. Calcium concentration in visceral hypersensitivity group was higher than that in PI3K inhibitor group and that in PLC‐γ inhibitor group was higher than that in NGF antagonist group. The binding of TrkA receptor to NGF activates the MAPK/ERK pathway, the PI3K/Akt pathway and the PLC‐γ pathway, causing changes in the fluidity of intracellular and extracellular Ca2+, resulting in increased sensitivity of visceral tissues and organs.


| INTRODUC TI ON
Irritable bowel syndrome (IBS) is the most common digestive tract disease. 1 The prevalence of IBS in the general population is 3%-22%, 2,3 which seriously affects the quality of life of patients and expends a lot of medical resources. The mechanism of IBS is still unclear. Visceral hypersensitivity is considered to be one of the main cause of IBS. Visceral hypersensitivity is closely related to nerve plasticity in pain pathways of central, peripheral and enteric nervous system (ENS). 4 At present, the treatment of IBS is mainly to improve the symptoms, the curative effect is not satisfactory, and the symptoms often recur. Exploring the visceral hypersensitivity mechanism of IBS and finding new treatment methods are the research hotspots.
Sacral nerve stimulation (SNS) is a kind of peripheral nerve regulation. It was initially used for the treatment of urinary incontinence and retention. In 1995, it was used by Matzel for the minimally invasive treatment of faecal incontinence. 5 SNS was more and more widely used in the treatment of bladder dysfunction, faecal incontinence and some intractable constipation because of its minimally invasive, safe, effective and economical characteristics. [6][7][8] However, the high sensitivity of SNS to IBS or viscera has rarely been reported.
Fassov J performed SNS on 21 DIARRHEA-TYPE IBS patients and found that symptoms of some patients improved after treatment. 9 Langlois L reported that anorectal dilatation (acute visceral hypersensitivity model) and SNS on normal SD rats could improve their visceral hypersensitivity induced by anorectal dilatation. 10 Nerve growth factor (NGF) is one member of the neurotrophic factor family. It is widely distributed in the central nervous system, autonomic nervous system and intestinal nervous system. NGF has a stable synaptic nutritional role in regulating the transmission of synaptic signals. NGF mainly binds to two kinds of membrane receptors, usually with high-affinity receptor tyrosine kinase A (TrkA), regulates downstream signalling pathways, promotes neurotransmitter release, synaptic receptor expression and changes neuroplasticity, and plays an important regulatory role in the survival, growth, differentiation and function of neurons. 11 It was reported in IBS patients and visceral hypersensitivity animal models; NGF in serum and colon tissue was significantly higher than that in control group. Some scholars found that increased NGF could up-regulate the expression of TrkA receptor, while NGF antibody could improve visceral sensitivity and down-regulate the expression of TrkA receptor. [12][13][14] participates in the visceral hypersensitivity process, which leads to the decrease of visceral pain threshold. It may cause peripheral or central sensitization by regulating neuroplasticity and neurosynaptic transmitter secretion. 4,15,16 The specific mechanism is still unclear.
In this study, acetic acid enema-induced chronic visceral hypersensitivity rats were used as animal models. The expression of NGF, TrkA receptor, PI3K/Akt and PLC in colon and DRG before and after SNS were detected by Western blot and RT-PCR. After antagonizing NGF, the above contents were detected and the effect of SNS on visceral sensitivity was evaluated. The visceral sensitivity of rats was evaluated by blocking the downstream signal pathways separately.
To explore whether SNS can improve visceral sensitivity by reducing NGF down-regulation of TrkA receptor, and further study whether SNS can improve visceral sensitivity by affecting downstream signalling pathways (MAPK/ERK pathway, PI3K/Akt pathway and PLC pathway) through NGF/TrkA receptor. This study will help us to further understand the signal transduction mechanism of visceral hypersensitivity and provide a theoretical basis for the clinical application of neuromodulation in the treatment of IBS.

| Laboratory animals and groups
One-Twenty SPF-grade male newborn rats were fed at quiet environment with room temperature (22 ± 2℃), constant humidity (40%-50%) and 12/12 hours of circadian circulation. One week after adaptive feeding, 120 healthy and clean SD rats were randomly divided into 6 groups: group A, group B, group C, group D, group E and group F, 20 rats in each group.
Group A: Normal model group.
Group B: The visceral hypersensitivity model was established by acetic acid enema sensitization, and half of the model rats in group B were numbered b1/b2, respectively.
Group C: On the basis of the model group, half of the rats in group C were numbered c1/c2. Group C was given NGF antagonists.
Group D: On the basis of model group, half of the model rats in group D were numbered d1/d2, and group d2 was given NGF agonists.
Group E: On the basis of model group, half of model rats in group E were numbered e1/e2, and PI3K inhibitors were given in group e2.
Group F: On the basis of the model group, half of the rats in group F were numbered f1/f2, and the f2 group was given PLC-γ inhibitor.

| Method
At the age of 10 days, the neonatal rats were fed with 0.5% acetic acid solution 0.2 mL through intestinal perfusion with insertion of anus for 2 cm, while the neonatal rats in group A were fed with 0.2 mL saline through intestinal perfusion. Electrode implantation was performed at the age of 7 weeks in rats, and the experiment began at the age of 8-12 weeks. and d2, and fluorescence spectrophotometer was used to detect the intracellular Ca 2+ content in dorsal heel ganglion and colon tissues of animals in groups A, b2, c2, d2, E2 and f2. SPSS 18.0 statistical software was used to process and analyse the data, and the measurement data were described by mean ± standard deviation. Single factor analysis of variance was used for multi-group comparison, and correlation analysis was used for correlation analysis. P < .05 was statistically significant.

| Expression of TRPV1 and pTRPV 1 in rats of group A and group b1
As shown in Figure 1, the expression of TRPV1 and pTRPV1 was high in group b1 (visceral hypersensitivity group), and the expression of TRPV1 and pTRPV1 was low in group A (normal non-model group).

| Intracellular Ca 2+ content in dorsal calcaneal ganglion and colon tissues of rats in group 2A and group b1
The content of Ca 2+ in dorsal ganglion and colon tissue cells was detected by fluorescence spectrophotometer with fura-2 indicator.
The left figure in Figure 2 shows the intracellular Ca 2+ content in dorsal calcaneal ganglion cells, and the right figure shows the intracellular Ca 2+ content in colonic tissue cells. In both dorsal ganglion and colon tissues, the intracellular Ca 2+ content in group b1 (visceral hypersensitivity group) was higher than that in group A (normal nonmodel group).

| Expression of NGF, TrKA, PI3K, AKT, PLC-γ, NF-κB, TRPV1 and pTRPV1 in rats of group A, group b2, group c2, group d2, group e2 and group F2
As shown in Figure 3 were analysed by one-way ANOVA. The results in Figure 4 and Table 1 showed that the relative expression levels of NGF, TrKA, PI3K, AKT, PLC-γ and NF-κ B in each group were significantly different (P < .01).
The results of LSD showed that there was no significant difference in the relative expression of PLC-γ and NF-κ B between group A (normal non-model group) and group c2 (NGF antagonist group; P > .05).
There was no significant difference in the relative expression of PLC-γ between group A (normal non-model group) and group b2 (visceral hypersensitivity group; P = .08 > .05). There was no significant difference in the relative expression of PLC-γ between the other two groups (P = .08 > .05). The difference was statistically significant (P < .05).

| Expressions of MAPK, ERK1 and ERK2 in rats of 5b2, e2 and f2 groups
As shown in Figure 5, the expression levels of MAPK, ERK1 and ERK2 in group b2 (visceral hypersensitivity group) were higher than those in group e2 (PI3K inhibitor group) and group f2 (PLC-γ inhibitor group). The expression levels of MAPK, ERK1 and ERK2 in group e2 were similar to that in group f2.

| Intracellular Ca 2+ content in rats of group 6A, group b2, group c2, group d2, group e2 and group f2
The content of Ca 2+ in dorsal calcaneal ganglion and colon tissue cells was measured by fluorescence spectrophotometer with fura-2 indicator. As shown in Figures 6 and 7, the curve of Ca 2+ in both dorsal calcaneal ganglion and colon tissue cells was flat in the normal group in Figure 6. The curve peaks in group d2 (NGF agonist group) were the highest, while those in other groups were b2 (visceral hypersensitivity group) higher than that in e2 (PI3K inhibitor group) and f2 (visceral hypersensitivity group). Group A (PLC-γ inhibitor group) higher than that in group c2 (NGF antagonist group). NGF is a kind of protein that can induce the growth of menstruation and is closely related to the development of nervous system. 17 In recent years, it has been found that nerve growth factor also plays an important role in the generation and maintenance of body pain. 18   damage can lead to electrolyte disturbance inside and outside the cell, which can also lead to the opening of transport channel TRPV1, the change of fluidity of intracellular and extracellular Ca 2+ and the increase of the sensitivity of visceral tissues and organs.

CO N FLI C T O F I NTE R E S T
All of the authors have no conflict of interest in this research.

AUTH O R ' S CO NTR I B UTI O N
Each author has made an important scientific contribution to the study and has assisted with the drafting or revising of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are free access to available upon request.

S U M M A RY
TrkA receptors bind to NGF by activating the MAPK/ERK pathway, PI3K/Akt pathway and PLC-γ pathway, causing changes in

E TH I C S , CO N S E NT A N D PE R M I SS I O N S
Ethical approval was given by The First Affiliated Hospital of Nanjing Medical University.

CO N S E NT TO PU B LI S H
All of the authors have consented to publish this research.