Transient Receptor Potential Melastatin 8 Contributes to Cystitis-Induced Neuronal Sprouting and Pain Hypersensitivity Through AKT/Mtor Signaling Pathway in Interstitial Cystitis/ Bladder Pain Syndrome

Transient receptor potential melastatin 8 (TRPM8) is associated with the pathophysiology of interstitial cystitis (IC)/ bladder pain syndrome (BPS). We investigated the mechanism of TRPM8 in neuroproliferation and pain, as well as the relevance of the Akt/mTOR signaling pathway in mice with IC/BPS. The model of IC/BPS was established in wild and TRPM8 -/- mice. The mechanical sensitivity was measured. The number of neurite segments, length of neurites, and density of neurites were all counted. IL-6 and norepinephrine levels were detected by ELISA, Western blot was used to detect protein levels of TRPM8, Akt, p-Akt, mTOR, p-mTOR. Immunouorescence was used to detect TRPM8 expression and distribution in neurites, neurons, and sensory nerves in mouse bladder tissue. Pain threshold in the IC / BPS group was decreased, and neurite segments, length, and density were all signicantly enhanced when compared to the control group. The parameters in the TRPM8 agonists(menthol)+IC/BPS group were more statistically signicant. Neurite number and density were lower in TRPM8 -/- +IC/BPS mice than in IC/BPS mice. The expression of TRPM8 and the ratios of p-Akt/Akt and p-mTOR/mTOR rose in the IC/BPS group. In TRPM8 -/- +IC/BPS mice, the ratios of p-Akt/Akt and p-mTOR/mTOR were not substantially different from those in the control group. TRPM8 -/- +IC/BPS mice had considerably lower levels of serum IL-6 and urine norepinephrine than wild-type IC/BPS mice. TRPM8 can induce pain hypersensitivity and sensory nerve proliferation by activating Akt/mTOR pathway and raising the expression of IL-6 and norepinephrine in IC/BPS models. These ndings offer new perspectives on IC/BPS treatment. compared to IC/BPS visceral blockers can decrease normal to Lashinger et al . found a link between the relative of TRPM8 the and . Both of these corroborate what we've observed.


Introduction
The international continence society de nes interstitial cystitis (IC)/bladder pain syndrome (BPS) as recurrent exacerbation of pain in the bladder area with bladder lling and symptom relief after emptying, accompanied by other symptoms like frequency and nocturia, but without obvious evidence of urinary tract infection or other maladies 1 . "Suprapubic discomfort associated with bladder fullness" is a common symptom of IC/BPS. This pathological pain could be linked to the emergence or alteration of aberrant afferent signaling pathways linked to in ammation 2 . In both human and animal models, the following occurrences have been documented in IC/BPS: proteome modi cations 3 , ion channel sensitization, and enhanced neurotrophic factor release 4 , all occur as the neurons that innervate the bladder recombine 5 .
Transient receptor potential melastatin 8 (TRPM8) is a type of nonspeci c ion channel that has a high calcium permeability. A chilly environment (8°C-28°C) and cool substances can activate it, creating inward currents dominated by calcium ions 6 . TRPM8 may be linked to increased innervation of bladder afferent neurons in patients with IC/BPS, resulting in pain symptoms, according to our ndings 5 . Ciobanu AC et al revealed that a higher level of TRPM8 expression in dorsal root ganglion neurons lowers pain thresholds in sensory nerves in patients, despite the fact that many speci cs of the mechanism are unknown 7 . Furthermore, Lashinger et al found that TRPM8 blocker suppresses pain in normal rats, implying that TRMP8 blocker may play a role in the relief of IC/BPS pain symptoms 8 . However, the study did not demonstrate the e cacy of TRPM8 blockers in IC/BPS rat models. The role of TRPM8 in IC/BPS pain symptoms is currently unknown.
As a result, we evaluated the effects of TRPM8 on UPK3A65-84-induced wild and TRPM8 knockout IC/BPS mice models, as well as the role and mechanism of TRPM8 in IC/BPS treatment.

Results
The number of CD3-positive cells (T cells) in mouse bladder tissue injected with UPK3A65-84 polypeptide was signi cantly higher (Fig. 1B) than in the control group (Fig. 1A), indicating that the model was successfully constructed.
In the control group, IC/BPS model group, IC/BPS model+ Menthol group, and TRPM8 −/− group, we examined TRPM8 mRNA ( Fig. 2A) and protein expression (Fig. 2B). TRPM8 mRNA and protein expression were substantially greater in the IC/BPS model group than in the control group (P<0.01). TRPM8 expression was signi cantly increased in the IC/BPS model+ menthol group. TRPM8 was absent in the TRMP8 −/− group. We also examined pain perception in the different groups, as shown in Figure 2C Neurites (positive for PGP9.5) and neurons (positive for Tuj1), as well as afferent sensory nerves (positive for CGRP), were identi ed using immuno uorescence. TRPM8 expression was enhanced in neurites, neurons, and afferent sensory nerves in the IC/BPS model group. In the IC/BPS model +menthol group, the results were clearer (Fig. 5). TRPM8 protein was absent in the TRPM8 −/− mice.
Western blots were used to detect the expression of Akt, p-Akt, mTOR, and p-mTOR in the bladder tissue of all groups (Fig. 6A). The p-Akt/Akt ratio (Fig. 6B) was considerably raised (P<0.01) in the IC/BPS model group and aggravated (P<0.01) in the IC/BPS model+ menthol group. The IC/BPS group had a higher ratio of p-mTOR/mTOR (P<0.05) (Fig. 6C), but there was no difference between IC/BPS and IC/BPS+ menthol group.

Discussion
There is evidence that the bladder neck and proximal urethra have the highest density of bladder nerves, with epithelial cells organized on the surface exhibiting neuronal-like features. The pathophysiology of UPK3A65-84-induced visceral pain in mice is strikingly similar to that of human IC/BPS 9 . The addition of a TRPM8 agonist or knocking out the TRPM8 gene dramatically increased or decreased visceral pain, suggesting that TRPM8 may play a role in IC/BPS visceral pain.
The pathogenesis of IC/BPS is complicated by in ammation. Chronic pain is frequently generated and maintained by peripheral or central in ammation, which is marked by high levels of in ammatory cytokines, including the well-known IL-6 5 . When infection and tissue damage occur, IL-6 is normally produced quickly. Changes in IL-6 were substantially linked with the regulation of TRPM8, suggesting that TRPM8 may cause IC/BPS by altering the microenvironment and in ammatory response, similar to our ndings.
Bladder in ammation also causes continual peripheral nerve stimulation, nociceptive nerve activation, neuron hypersensitivity, and spontaneous central nervous system neuronal activity. The release of neuropeptides is mediated by neural activity, which causes in ammation and creates a vicious cycle 10 .
The pain symptoms of IC/BPS rat model are in uenced by an increase in sympathetic activity and a rise in norepinephrine levels in the urine 7 . The activation of the α1A adrenoceptor in peripheral blood can cause chronic visceral discomfort by interacting with TRPV1 and ATP release, according to another study 8 . The shifting trend of IL-6 and epinephrine following TRPM8 regulation was similar to the sensitivity of pain, showing that TRPM8 activates epinephrine through an in ammatory response, resulting in chronic pain, and that reducing TRPM8 can considerably improve the above pathophysiological state.
Excessive peripheral signal input causes visceral pain sensitivity in peripheral neuroin ammatory states (such as IC/BPS). As a result, we looked at how neurite expression changed in the bladders of IC/BPS model animals. The number of neurite segments, neurite length, and neurite density are all highest in the bladder of IC/BPS model mice, indicating that increased neurite expression is proportional to increased visceral pain. The alterations in neurite quantity, length, and density after TRPM8 agonist or TRPM8 gene knockout con rmed that the role of TRPM8 in IC/BPS visceral pain was closely tied to peripheral nerve expression.
Furthermore, we discovered a substantial number of TRPM8 positive neurites, neurons, and sensory nerves, indicating that these nerves are important in visceral sensation. The number of TRPM8 positive nerve bers in the bladder wall of IC/BPS model mice rose considerably when compared to the control group. This behavior becomes more pronounced after TRPM8 agonist intervention. The ndings indicate that increased TRPM8 positive nerve bers in the bladder wall produce IC/BPS pathological alterations, which is consistent with our prior ndings in IC/BPS patients 5  In bladder tissue, we also found p-Akt and p-mTOR expression. Cell proliferation is linked to the p-Akt/p-mTOR pathway 13,14 . Furthermore, nociceptive processes are linked to mTOR 15,16 . The PI3K/Akt/mTOR signaling pathway is activated by afferent signals triggered by peripheral in ammation, and part of it is engaged in neural circuits that promote pain 17 . Sciatica caused by endometriosis can be relieved by inhibiting the PI3K/Akt/mTOR signaling pathway 18 . These ndings suggest that the PI3K/Akt/mTOR pathway is involved in pain. The trends of p-AKT/AKT and p-mTOR/mTOR across the groups in this investigation were similar to those of TRPM8.As a result, we believe that TRPM8-induced pain sensitization and neuroproliferation in IC/BPS mice is linked to the AKT/mTOR pathway. During the same period, the control group was given the same volume of saline once a day for 7 days in a row.
The mechanical sensitivity of mice was assessed after 35 days, and the mice's bladder tissues were removed to make para n sections for CD3 immunohistochemical labeling to identify T cell counts. The ndings were used to determine if the models had been developed correctly.

Mechanical Sensitivity Testing
Von Frey laments were used to test mechanical sensitivity. Mice were examined in an individual cage with a stainless-steel wire grid oor after a 2-hour habituation interval. To avoid desensitization, stimulation was limited to the lower abdomen area in the general location of the bladder, with care given to excite diverse sites within this region. Positive responses to lament stimulation were de ned as quick retraction of the abdomen and immediate licking or scratching of the stimulation area. Each lament was used for 1-2 seconds, with a 3-5 second interstimulus interval.

Immunohistochemistry Staining
Slides were quenched in 0.3% hydrogen peroxide and rinsed in PBS before being blocked for 1 hour at room temperature in 5% normal goat serum in PBS. Sections were then incubated overnight at 4℃ in primary antibody (CD3, Abcam, 1:100) in blocking serum before being washed in PBS, and incubated in HRP labeled secondary antibody for 20min at 37℃. After that, the sections were washed and DAB solution was added. After counterstaining in hematoxylin, dehydrating, and clearing in xylene, the slides were mounted in di-n-butyl phthalate in xylene. A uorescent microscope was used to collect the images (Olympus, BX53).

qRT-PCR
Total RNA was isolated from bladder samples using TRizol® reagent (Invitrogen, Carlsbad, CA, USA), which was then used to make cDNA using a kit (Thermo, #K1622). The 7500 Real-Time PCR system (ROCHE, LC480) was used to evaluate quadruplicates of four different experiments using quantitative real-time polymerase chain reaction (qRT-PCR). Initial denaturation at 95°C for 10 minutes, 40 ampli cation cycles at 95°C for 15 seconds, and annealing and extension at 60°C for 60 seconds were the conditions. Each experiment was repeated three times, with the average values utilized for analysis.
Primer sequence: Western Blot The DC Protein Assay kit (Bio-Rad) and an ELx800 spectrophotometer were used to assess the protein concentration in cell lysates (Bio-TekTM). An equivalent number of proteins were isolated and blotted onto nitrocellulose sheets using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE

Neurite Quanti cation
Neurite (positive to PGP9.5) was detected visually as longer than 10µm with a thickness between 1 and 3µm. We used ImageJ software to analyze the images and measured bers on 8-10 images per bladder from three animals per condition. Results were expressed as neurite segments/µm 2 (the number of individual neurites per surface unit), the neurite length, and the neurite density (neurite length per surface unit).
Statistical Analysis SPSS software, version 22.0, was used for statistical analysis (SPSS Inc, Chicago, IL). All data is presented as Mean±SD. The LSD method of One-way ANOVA was used to compare the means observed in different groups. The signi cance level was established at P <0.05.
Statement for ARRIVE guidelines.
We have read the journal's policy and all the authors read and approve the manuscript. We declared that this study was carried out in compliance with the ARRIVE guidelines. The animal was raised in a stressfree environment and handled with extra care. They were euthanized peacefully. All the protocols were followed according to the ARRIVE guidelines for handling the animal.  Figure 1 Identi cation of the model. The UPK3A65-84 polypeptide was used to make the mouse IC BPS model, and the bladder was stained with CD3 immunohistochemistry.The scale bar represents 50μm.   Representative images of TRPM8-positive nerve sprouting in the bladder wall in various groups. TRPM8 expression increased in bladder neurites (positive for PGP9.5) and neurons (positive for Tuj1), as well as afferent sensory nerves (positive for CGRP), according to immuno uorescence data. After TRPM8 agonist intervention, these phenomena became more noticeable. After the TRPM8 gene was knockout, the protein TRPM8 was not expressed in the bladder tissue of mice. The scale bar represents 50μm.