Extracellular vesicles enhance oxidative stress through P38/NF‐kB pathway in ketamine‐induced ulcerative cystitis

Abstract Long‐term abuse of ketamine causes ketamine‐induced cystitis. The functional alterations of bladder epithelial cells in microenvironment during cystitis remain poorly understood. Here, we explored extracellular vesicles (EV) alteration in ketamine‐induced toxicity. To simulate the high‐concentration ketamine environment in vivo, we established an in vitro model of high ketamine using human uroepithelial cells (SV‐HUC‐1). Cell viability and proliferation were assessed to evaluate the effects of various concentrations (0, 0.25, 0.5, 1, 2, 4 and 8 mmol/L) of ketamine on SV‐HUC‐1 cells. The cell supernatant cultured at a concentration (0, 1, 2, 4 mmol/L) of ketamine was selected for EV extraction and identified. Subsequently, we assessed different groups (ketamine, ketamine plus EV blocker, EV, EV plus extracellular vesicles blocker) of oxidative stress and expression of inflammation. Last, luciferase reporter assay was performed to study the transcriptional regulation of EV on the NF‐kB and P38 pathway. The results of our study suggested that treatment with 0, 1, 2 or 4 mmol/L ketamine altered the morphology and secretion capacity of extracellular vesicles. As the concentration of ketamine increased, the average particle size of EV decreased, but the crest size, particle concentration and EV protein increased. Moreover, after the addition of EV blocker, EV secreted at different concentrations were blocked outside the cell membrane, and the degree of oxidative stress decreased. Our study provided evidence that ketamine alters the secretion of EV by directly stimulating cells in inflammation microenvironment and EV play significant roles in intercellular signal communication and the formation of KIC.EV


| INTRODUC TI ON
Ketamine, a derivative of phencyclidine, has been used as an anaesthetic in clinical and analgesic fields. Due to its intense euphoria and hallucinations, ketamine is prone to abuse by young people in entertainment settings. Chronic abuse often leads to bladder contracture and severe lower urinary tract symptoms, clinically called ketamine-induced cystitis, 1 which often manifests as mucosal ulcer with bleeding, similar to interstitium cystitis. Several studies have suggested that ketamine and its metabolites interact with bladder epithelium, leading to human uroepithelial cells' (SV-HUC-1) injury, triggering an oxidative stress cascade and, ultimately, cystitis formation. 2,3 The current treatment options for KIC include surgery and perfusion therapy among others. However, the available treatment strategies are not ideal for all patients. 4 A better understanding of the mechanism of KIC occurrence and development can, therefore, aid in the identification of new biomarkers, and the development of new KIC treatment strategies.
Being essential mediators of intercellular communication, extracellular vesicles (EV) are vital in the pathogenesis of many diseases. 5,6 EV (about 30-800 nm in diameter), such as exosomes and microvesicles, can affect physiological and pathological conditions through the transfer of various cargo molecules, including proteins, nucleic acids and lipids. 7 As lipid bilayer membrane vesicles, secretion of extracellular vesicles is closely related to cell viability. Numerous studies have reported the value of EV derived from urine, which now appears to be a very promising and completely non-invasive source of new information that accurately reflects the pathophysiology of the kidneys and other organ systems. 8 Besides, EV have been found in the urine of patients with interstitial cystitis and can be used as diagnostic biomarkers. 9,10 The presence of EV in urine reflects potential cellular processes that may be associated with cystitis. 11 Accumulating evidence indicates that oxidative stress injury plays an essential role in the prognosis of KIC. High levels of intracellular ROS activated the p38 signalling pathway 12 and stimulate the expression of nuclear factor kappa B (NF-kB). NF-kB, a member of a redox-sensitive transcription factor family, regulates the expression of pro-inflammatory factors, including TNF-α, iNOS and COX-2. 13,14 According to Juan et al, ketamine-induced cystitis enhances the translocation of NF-kB transport and the expression of cyclooxygenase-2 (COX-2) through oxidative stress in rats. 15 Khalyfa A and his team successfully isolated nanovesicles in the serum of patients with obstructive sleep apnoea, and the morphology of the nanovesicles was similar to that of exosomes. The production of such nanovesicles can be triggered by oxidative stress-related pathways, which also exacerbates cellular senescence. 16 Besides, a study led by Mizutani et al 17 also indicated that Akt and CD9 carried by EV derived from urothelial SV-HUC-1 cells may be useful markers for differential diagnosis of urinary tract infections and asymptomatic bacteriuria. We, therefore, postulate that EV play an essential role in the mechanism of KIC, although such research has not been previously reported. Extracellular vesicles extracted from the urine are susceptible to changes in cellular origin, temperature and pH, 18 rendering experimental results prone to variation. Therefore, we sought to optimize the extraction and identification of EV using SV-HUC-1 cells exposed to high concentrations of ketamine. In this study, we found that ketamine cell-derived EV can transport associated molecules to alter the physiology of target cells in a specific manner. These findings could allow us to explore the mechanisms of signal transduction and targeted regulation of EV, hence provide a new therapeutic strategy for clinical KIC patients.

| Cell exposure
SV-HUC-1 cells (4 × 10 3 /well) were grown in 96-well plates. At 80% confluence, cells were exposed to different concentrations of ketamine working solution (100 µL/well) for 48 hours. The optimal time is 48 hours, the stimulation time is short, and the secretion of EV is insufficient. The long time will cause the cells to produce a large number of apoptotic bodies, which is not conducive to observation. We detected CCK-8 cell proliferation assay by cell supernatant. The SV-HUC-1 cells (4 × 10 4 /well) were also grown in 24-well plates. Then, we observed the survival of the cells at different concentrations of ketamine, and proteins from cell lysis were collected for the detection of oxidative stress indicators SOD, MDA and GSH.

| ELISA
We used the SOD, MDA and GSH ELISA kit (Nanjing Jiancheng Bioengineering Institute, China) to detect the proteins from cell lysis.
Quantitative assays were performed using the corresponding ELISA assay kit according to the manufacturer's protocol. The absorbance was measured at 550, 532 and 412 nm using a microplate reader (Thermo Fisher).

| CCK-8 determination
A Cell Counting Kit-8 (CCK-8) assay (Dojindo) was used to evaluate the cytotoxicity of ketamine on SV-HUC-1 cells. The CCK-8 solution (10 µL) added to each well, and the plate was incubated for 2.5 hours, at 37°C and an anaerobic condition of 5% CO 2 . The absorbance was determined at 450 nm. 1. The cell culture medium was placed in a spin column, and an equal amount of XBP was also added, which mixed together by inverting several times. Then, it was centrifuged at 500 g for 1 minute at 4°C using a Beckman Coulter Allegra X-15R centrifuge (Beckman Coulter), and the liquid was transferred in the spin column to a centrifuge tube. The spin column was taken out, all the liquid was discarded in the centrifuge tube, and the spin column was put back.

| Isolation of EV
2. 8 mL of XBP was added and centrifuged at 5000 g for 5 minutes at 4°C. The tube and all the liquid were discarded, the column was retained, and the column was placed into a new tube.
3. 400 µL of XE was added eluate to the spin column, incubated for 1-2 minutes, and was centrifuged at 500 g for 5 minutes at 4°C.
The liquid was collected in the centrifuge tube and placed on the spin column again. They were incubated for another 1-2 minutes and centrifuged at 5000 g for 5 minutes at 4°C. Finally, the liquid (20 µL) in the centrifuge tube was transferred to a new container. 80 µL cold PBS was added to each tube (100 µL/tube) for resuspension, and then, the EV were stored at −80°C until further use. 19

| Protein extraction and quantification
About 40 µL of lysis buffer (RIPA: PMSF = 100:1, Solarbio) was added to each EV sample (40 µL/tube). The EV were lysed on ice for 30 minutes. Protein concentration was then estimated using a BCA kit (Sigma).

| Electron microscope
A 20 µL drop of resuspended EV was placed on a sheet of Parafilm.
Grids were transferred to the drops of EV for 3 minutes, and then dried from the edge using filter paper (Solarbio). The sample side of the membrane was then transferred to a 30 µL drop of 3% phosphotungstic acid solution, which was negative stained at room temperature for 5 minutes. Then, negative staining solution was removed from the grids with filter paper, and grids were dried at room temperature. Grids were observed with an H-7650 transmission electron microscope (Hitachi).

| Nanoparticle tracking analysis
Each group of 20 µL EV was diluted 10-fold with cold PBS so that the particle concentration ranged from 1 to 10 × 10 8 particles/mL. Nanoparticle tracking analysis (NTA) measurements (NanoSight NS500, NTA 3.2 Dev Build 3.2.16) were used to estimate the size distribution and number of particles in each group of EV.

| Flow cytometry analysis of oxidative stress levels
Detection of active oxygen was performed using an active oxygen detection kit (Solarbio) according to the manufacturer's instruction.
The DCFH-DA fluorescent probe was diluted with a serum-free medium to a final concentration of 10 μmol/L at 1:1000. The cell culture medium was removed, and 1 mL of diluted DCFH-DA was added to a 12-well plate to fully cover the cells and incubated at 37°C for 20 minutes. Excess DCFH-DA was removed by washing the cells with serum-free cell culture thrice. Finally, the CytoFLEX (Beckman Coulter) was used to detect oxidative stress levels in cells, and the FITC channel was selected.

| Extracellular vesicle uptake experiment
The EV uptake kit PKH67 (Sigma) was used to determine cellular uptake of fluorescently labelled EV. 20 µL EV were mixed with diluent C according to the manufacturer's instructions. Then, 4 µL PKH67 probe was added, mixed and incubated for 3 minutes at room temperature. The reaction was stopped by adding an equal volume of exosome-free serum to complete the fluorescent labelling of EV.
The PKH67-labelled EV were then added to a spin column for reextraction. The same amount of binding buffer XBP was added and centrifuged at 500 g for 1 minute. Then, 8 mL of wash buffer XWP was added and centrifuged at 5000 g for 5 minutes. Then, 400 µL of XE eluate was added, and the mixture was incubated for 2 minutes and centrifuged again at 500 g for 5 minutes. The mixture was incubated for another 2 minutes and centrifuged at 5000 g for 5 minutes.
Finally, the isolated EV were added to the cells, and the EV uptake ability of the cells was observed using a fluorescence microscope after 24 hours.

| Western blotting analysis
Cell samples or ketamine-derived EV were lysed in RIPA buffer

| Real-time quantitative PCR
Cells total RNA extraction and reverse transcription PCR: The collected cells were treated with TRIzol reagent (Solebao, R1100), fully lysed and mixed. Thereafter, 50% by volume of chloroform was added, and the upper aqueous phase was collected after centrifugation. An equal volume of isopropanol was added, and RNA was precipitated by centrifugation. The RNA was washed with 70% alcohol, dried and dissolved in RNA-free enzyme water. Subsequently, RNA concentration and purity were detected using an ultra-micronucleic acid detector (SuiZheng, FC-1100), followed by reverse transcription using the reverse transcription kit (Mona, RN05004M) according to the instructions of the kit. 200 ng total RNA was used for cDNA preparation. The resulting cDNA was stored at −20°C until use.   Table 1.

| Statistical analysis
All statistical analyses in this study were conducted with GraphPad 8.0 (GraphPad Software Inc), and data were presented as the mean ± SEM. Comparisons between groups were performed using one-way ANOVA followed by LSD's post hoc test. P < .05 was considered statistically significant.

| High concentrations of ketamine decrease cell vitality
The Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell proliferation and cytotoxicity because it is very sensitive and does not involve the use of radioactive reagents. 22 Cell damage and cytotoxicity varied among groups but displayed similar trends over time.

| High concentration of ketamine enhances apoptosis
Our results of the Elisa test showed that enzymatic (SOD) (P < .05), non-enzymatic (GSH) (P < .05) antioxidant activity increased in cell supernatants with ketamine concentration ≥ 1 mmol/L. Meanwhile, lipid peroxidation (MDA formation) (P < .05) also increased, indicating that oxidative stress up-regulated in cells ( Figure 1B). After 48 hours of exposure to ketamine, apoptosis was increased in ≥1 mmol/L ketamine, which showed nuclear pyknosis, nuclear fragmentation and nuclear lysis ( Figure 1C). Cells exposed to 8 mmol/L ketamine showed significant necrosis. Therefore, lower concentrations of ketamine (0, 1, 2 and 4 mmol/L) were used in the subsequent experiments.

| Identification of EV
Electron microscopic images showed that EV derived from SV-  Figure S1).

| Effects of ketamine concentration on EV protein
As EV are rich in protein, protein quantification can effectively indicate the secretory profiles of EV. The original sample was lysed, and the obtained protein (15 µL/sample) was subjected to Western blot analysis ( Figure 2B). Our results indicate that an increase in the F I G U R E 3 Bladder cells were co-cultured with ketamine. A, Flow cytometry analysis of oxidative stress. B, Determination of Nrf2, P-38, Cox-2, NF-kB and p-NF-kB expression by Western blot in different groups in cells treated with different ketamine concentrations. C, Changes in mRNA expression levels of Nrf2, P-38, Cox-2 and NF-kB after ketamine treatment. All mRNA levels were quantified by quantitative polymerase chain reaction. Data were expressed as the mean ± SEM of at least 3 number of biological replicates.*P < .05, **P < .01. (a), (b), (c) and (d) are similar to Figure 2 A B C concentration of ketamine increased the total protein levels of EV ( Figure 2F).

| Effects of ketamine group (K) on oxidative stress in SV-HUC-1
Our flow cytometry analysis data confirmed an increase in keta-  Figure 3C).

| Effects of ketamine group (K) + cytochalasin D (D) on oxidative stress in SV-HUC-1
We added cytochalasin D to different concentrations of ketamine-stimulated SV-HUC-1 cells. The results showed ( Figure 4A) that cytochalasin D significantly reduced intracellular ROS levels. Specifically, in the K + D group, the (b), (c) and (d) subgroups showed a certain amount of ROS peaks compared with (a) the control group, but they had decreased a lot compared with K group ( Figure 3A). In the K + D group, the (a) group was used as the control group, and the baseline was not significantly different from the group (a) in the K group. Western blot analysis ( Figure 4B) further showed that after the addition of cytochalasin D, the (d) subgroups in the K + D group showed the highest expression of inflammatory proteins compared with (a-c), which also decreased a lot (compared with K group). These indicated that although in the K group, the subgroup of (d) caused a decrease in inflammatory protein due to apoptosis. However, because of the addition of EV blocker, apoptosis was relatively reduced and inflammatory proteins were increased again. Besides, the same decrease trend was observed in the expression levels of inflammatory mRNA in the K + D group ( Figure 4C).

| Effects of extracted EV on oxidative stress in SV-HUC-1
We further explored the mechanism of action of EV in KIC. The derived EV were stained with PKH67. PKH67-labelled EV were incubated with bladder epithelial cells (nuclei labelled with DAPI (blue)). After 24 hours, we observed that PKH67-labelled EV were present in the cell membrane and cytoplasm ( Figure 5A). This result indicated that ketamine cell-derived EV could be incorpo- After incubation with the derived EV, we found the phosphorylation level of NF-kB was significantly increased and the expression of the anti-oxidative stress transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2) was decreased. These indicated that ketamine-induced EV can enhance P38 signal expression and further activate NF-kB signalling pathway and its phosphorylation level. Also, the downstream molecule COX-2 expression was up-regulated. We also used qPCR ( Figure 5D) to verify the expression of inflammatory protein factors, and the results were consistent with the above.
As we know that NF-kB and P38 are biologically activated after they are phosphorylated, it is important to investigate whether EV affect inflammation occurs through the phosphorylation of NF-kB or P38 signalling. To this end, QNZ and SB203580 were used to directly inhibit NF-kB and P38 signalling ( Figure S3). Western blotting analysis indicated that the inhibitor dramatically decreased the expression of phosphorylated NF-kB and P38 in cells ( Figure 5E).
We then performed a luciferase reporter assay with constructs driven by a human NF-kB and P38 promoter. As expected, inhibitor led to a significant transient expression decrease of NF-kB and P38 promoter activity in EV-induced cells ( Figure 5F). Interestingly, we also found that EV (2 mmol/L) group had higher NF-kB and P38 F I G U R E 4 Bladder cells were co-cultured with ketamine + cytochalasin D. A, Flow cytometry analysis of oxidative stress. B, Determination of protein expression of Nrf2, P-38, Cox-2, NF-kB and p-NF-kB in cells. C, Changes in mRNA expression levels of Nrf2, P-38, Cox-2 and NF-kB in cells. Data were expressed as the mean ± SEM of at least 3 number of biological replicates.*P < .05, **P < .01. (a), (b), (c) and (d) are similar to Figure 2 luciferase activity and significantly higher phosphorylation levels than EV (0 mmol/L) group.
Altogether, these data suggest that ketamine cell-derived EV played significant roles in KIC.

| Effects of extracted EV + cytochalasin D on oxidative stress in SV-HUC-1
We then co-cultured the extracellular vesicle blocker (cytochalasin D) with EV and cells for 24 hours. It was found that in the presence of cytochalasin D, the passage of PKH67-labelled EV through the cell membrane was blocked, although some EV entered the cytoplasm ( Figure 6A).  The upstream P38 pathway was inhibited, and the expression of the downstream molecule COX-2 was also significantly reduced.
However, the expression of antioxidant transcription factor Nrf2 was increased. Although the method has lower cost, the extracted EV have higher purity but the operation is complicated and time-consuming, and the loss of EV is enormous. 26 We, therefore, used the exoEasy Maxi Kit (QIAGEN, Germany) to isolate ketamine-derived EV. 27 The elution of intact vesicles from the exoEasy membrane and the yield of EV were better than ultracentrifugation, which is particularly beneficial for subsequent studies of EV function. 28 Our results indicate that ketamine concentration affects the size and secretion of EV. The four EV subtypes may carry different signalling molecules and play different roles in a given inflammatory microenvironment. 29 EV are secreted by fusion with a membrane and secreted into the extracellular environment by exocytosis.

| D ISCUSS I ON
These endogenous nanocarriers typically carry biologically active molecules, such as RNA and proteins, which are stably present in the extracellular environment and regulate the metabolic pathways of target cells. 30 Moreover, the EV secreted by SV-HUC-1 cells in various environments vary greatly in shape, size distribution and secretion rate.
Previous studies have reported that a more limited particle size distribution is associated with faster absorption of target cells and greater efficacy in the microenvironment. 31 Therefore, we can assume that different concentrations of EV subtype trigger different signal molecules. For example, cells exposed to high concentrations of ketamine secrete many similarly sized exosomes, which facilitates the absorption of target cells. Our results showed that higher concentrations of ketamine damage SV-HUC-1 cells, inducing donor cells to secrete numerous exosomes. We considered that EV act as a 'messenger' in high-concentration ketamine environments, and we planned to further understand these signalling molecules by sequencing the EV proteins.
To verify the above conjecture, we did an EV function test. Most experimental evidence suggests that EV usually enter the endosomal compartment 21,32 by endocytosis. Ingestion can be very rapid, and EV can be found in cells as early as 15 minutes after initial introduction. Cytochalasin D is a metabolite known to depolymerize the actin filament network, leading to inhibition of the endocytic pathway. 33 Cytochalasin D treatment has been shown, on several occasions in various cell types, to significantly reduce, but not entirely prevent, EV uptake in a dose-dependent manner. 32,34 Our results showed that the addition of cytochalasin D confined most of the EV to the extracellular space, but some EV could still enter the cytoplasm and cannot be completely eliminated. This finding could be explained by postulating that EV uptake occurs through more than one mechanism. 35 Current research suggests that oxidative stress occurs when ketamine and its metabolites are toxic to the bladder urinary tract. Shu Mien team also found epigenetic regulation of COX-2 expression by DNA hypomethylation via NF-kB activation in ketamine-induced ulcerative cystitis. 38 In other studies, carbesolone could treat interstitial cystitis caused by cyclophosphamide, mainly by inhibiting P38 activation and reducing cyclophosphamide-induced bladder oxidative stress damage. 39 Nrf2 is a crucial transcription factor regulating cellular oxidative stress, which can alleviate cellular damage caused by reactive oxygen species and electrophiles and maintain the body's redox homeostasis. 40 In summary, we first extracted EV secreted by SV-HUC-1 cells exposed to high concentrations of ketamine, using a new protocol to distinguish the four subtypes of EV. Our study demonstrated an important mechanism in the development of KIC. That is, ketamine cell-derived EV mediate urinary epithelial cell-to-cell communication and enhance oxidative stress in ketamine-induced cystitis probably via the P38/NF-kB pathway. We have reason to believe that this study opens up new horizons for studying the mechanism of KIC formation.

CO N FLI C T O F I NTE R E S T
The authors have no competing interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support findings of the study are available from the corresponding author upon reasonable request.