Protective effect of Aster tataricus extract on NLRP3‐mediated pyroptosis of bladder urothelial cells

Abstract Aster tataricus L.f. is a traditional Eastern Asian herbal medicine used for the relief of uroschesis‐related illnesses and has been demonstrated clinically to exert satisfied effects. However, the mechanism of its therapeutic action remains unclear. The present study aimed to evaluate the protective mechanism of Aster tataricus extract (ATE) on CYP or LPS + ATP‐induced interstitial cystitis (IC), we successfully constructed the induced IC Sprague‐Dawley (SD) rat model and IC human urothelium cell (SV‐HUC‐1) model. The main compounds of ATE were determined by LC‐MS. After intervention, the changes on the bladder wall morphology and inflammation were observed in each group. SV‐HUC1 cell viability was measured by MTT and double stained with Hoechst 33342 and propidium iodide (PI). The expression levels of NLRP3, Pro‐caspase‐1, Caspsae‐1 p20, GSDMD, GSDMD‐N and Cleave‐IL‐1β in vivo and in vitro in different groups were detected by Western blotting. ATE significantly alleviated oedema and haemorrhage and reduced the inflammation index and histopathological score in SD rat bladder. The results of cell revealed that ATE could improve cell viability and decrease pyroptosis ratio. The expression of NLRP3 and other pyroptosis‐related protein was remarkably decreased by ATE both in vivo and in vitro. ATE may be used as an inhibitor of NLRP3 in treating IC. The discovery of NLRP3/Caspase‐1/GSDMD‐N as a new protective pathway provides a new direction for protecting cell against IC.


| INTRODUC TI ON
Interstitial cystitis (IC) is a chronic condition characterized by chronic pelvic pain, pressure and/or discomfort perceived to be related to the urinary bladder. 1 The pathogenesis of IC is not clear enough. At present, the main treatment methods for IC include oral medication, bladder perfusion, bladder hydraulic dilatation and urinary diversion.
These treatments provide only partial relief after treatment, and there is no complete cure. 2  Nod-like receptor protein 3 (NLRP3) inflammasome is closely related to the progression of IC, and NLRP3 signalling pathway plays a key role in the pathogenesis of bladder injury disease. 5 NLRP3 inflammasome can be activated by a variety of substances, such as pathogen-associated components, injury-related molecules, environmental stimuli and pathogenic bacterium, and studies show that inflammatory markers such as NLRP3, Caspase-1 and GSDMD in patients with IC are elevated. 6,7 Aster tataricus L.f. is the dry root and rhizome of Chinese herb A tataricus. 8 A tataricus can be found in many regions in Eastern Asia including mainland China, South Korea and Japan. 9 Research reports that A tataricus has been used for the relief of coughs and as an expectorant and it possesses diuretic, anti-tumour and antibacterial activities. 10 A tataricus extract (ATE) could suppress the activation of pro-inflammatory cytokine and nuclear factor-kappa B (NF-κB) signalling pathway, so as to provide treatment for diabetic rat. 11 A study outlining the use of a methanol extract of A tataricus exhibited significant inhibitory activity against the production of inflammatory cytokines (prostaglandin E2, interleukin-6 and interleukin-1 beta) and the expression of inflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2) via the inhibition of NF-κB activation. 8 In the present study, we explored the effect of ATE on experimental models of interstitial cystitis in vivo and in vitro. We hypothesized that ATE might suppress NLRP3 expression in the urothelial cells and reduce the IC symptom, and investigate the effect of ATE isolated from A tataricus on urothelial cells infected with interstitial cystitis (IC) and the therapeutic effects and possible mechanism of ATE on the IC, which could provide valuable evidence supporting its traditional use in the treatment of dysuria.

| Experimental objects
40 specific pathogen free (SPF) grade healthy female SD rats (180-200 g in weight) were purchased from Qinglongshan Experimental Animal Center, Nanjing, China. Rats were housed in the feeding room with constant temperature (21-23°C) and humidity (45%-65%) and maintained in a 12 hours light-dark cycle with free access to eat and drink. All experiments followed the National Institute of Health Guidelines for the Care and Use of Animals.

| Preparation of ATE
After crushing, 0.5 kg A tataricus powder was poured into 95% ethanol for 1 hour and heat it at 90°C for 1 hour. After filtration, A tataricus fluid extract was evaporated into dried extract powder under vacuum of 60°C. 12 The dried powder was dissolved in doubledistilled water for next experiments.

| HPLC-MS analysis of ATE
For quantitative determination of compounds, chromatographic analysis was carried out in a Waters Acquity HPLC system (Waters, Milford, MA, USA). The chromatographic separation was achieved at 30°C on an ACQUITY C18 column (100 × 2.1 mm, 1.7 μm). The mobile phases consisted of A (water) and B (acetonitrile) both containing 0.1% formic acid and used a gradient elution of 30%-80% B at 0-9 minutes, 80%-95% B at 0-13 minutes. MS was performed on a Xevo ™ TQ-S system from Waters (USA) tandem quadrupole mass spectrometer using an ESI source operated in positive (Shionone) or negative-ion (others) mode. All the peaks of target compounds in the solution of A tataricus samples were unambiguously identified by the comparison of retention time, parent and product ions with standards. The product ions scan spectra are shown in Figure 1, and the concentrations of Shionone, Kaempferol, Quercetin, Luteolin, Ferulic acid were 3.78 mg/g, 0.34 mg/g, 3.98 × 10 −3 mg/g, 1.02 × 10 −1 mg/g, 3.12 × 10 −2 mg/g, respectively. All instrumentations were controlled and synchronized by MassLynx data systems (version 4.1) from Waters.

| IC rat model
After 1 week of adaptive feeding, animal modelling was carried out according to Table 1 below. Rats were divided into five groups with 8 in each group: control (no-CYP treatment + saline), saline (CYP treatment + saline), ATE-1.2 (CYP treatment + ATE-1.2, 1.

| Measurement of bodyweight, bladder wet weight, oedema and haemorrhage
Bodyweight and bladder wet weight of every rat from each group was measured by precision balance (Shimadzu, Japan). According to Gray's criteria, 14 oedema and haemorrhage indicators were scored macroscopically: Oedema is considered severe when fluid is seen inside and outside the bladder wall (3+); Moderate (2+) was limited to the internal mucosa; Mild (1+) indicated between normal and moderate. The haemorrhage score was as follows: There was a blood clot in the bladder (3+); Mucosal haematoma (2+); Telangiectasia or vesical vasectasia (1+); And normal (0).

| Histopathological assay
The weight of bladder tissue was weighed firstly, and pathological analysis of the bladder tissues after infection was carried out. The paraffin sections prepared for haematoxylin-eosin (H&E) staining by slicer (Histocore Biocut, Laica). The histological changes of the stained sections were observed using a light microscope equipped with a digital camera (Olympus, Japan) to observe the morphology of the cells, so as to evaluate bladder lesions, interstitial inflammation, oedema and mast cell infiltration. Histopathological score: normal epithelial cells, no inflammatory cell infiltration and ulcer

| Western blotting assay
The steps of protein extraction and Western blot analysis are referred to the previous literature. 15 In brief, the extracted bladder  protein tissue was loaded into a 10% SDS-polyacrylamide gel and transferred into a PVDF membrane after electrophoresis. After incubating with primary and secondary antibodies and washing (NLRP3, ASC, Pro-caspase-1, Caspsae-1 p20, GSDMD, GSDMD-N and Cleave-IL-1β, Affinity; Goat Anti-Rabbit IgG (H + L) HRP, Affinity), the ECL Plus assay Kit (Affinity, K002) was used for colour rendering. The gel analysis system scanned each strip protein, and the grey value of the strip was measured by image analysis software (Image J).

| Immunofluorescence assay
The 4 μm sections of bladder tissues were dehydrated with 100%,

| Modelling and administering of SV-HUC-1 cells
After 2 hours administration with different concentrations of ATE, 10 μg/L LPS and 2.5 mmol/L ATP were added to stimulate the cells for 12 hours. 16 The drug was administered in the following groups, as shown in Table 2.

| Assays cell activity by MTT and Hoechst33342 and PI double staining
The SV-HUC-1 cells were placed in a 96-well plate at a density of 1 × 10 4 cells/well, respectively. Different concentrations of ATE were added into medium, and samples were incubated for 24 hours.
After incubation, the cell cultures were mixed with MTT assay reagent for 4 hours and then read on microspectrophotometer (Nanodrop, Thermo, USA). After treatments, the cells in each group were stained with 5 mg/mL Hoechst 33342 and 10 mg/mL PI for 10 minutes in the dark. The stained cells were observed using fluorescence microscope (Olympus, Japan). The apoptotic cells were stained by Hoechst 33342 (blue) and pyroptosis cells by PI (red).

| Infection with overexpression (NLRP3) lentivirus vectors
The recombinant lentivirus vectors for NLRP3 and empty vector were provided by Genechem (Shanghai, China). The 293T cells overexpressing NLRP3 were transfected by HiTransG P (Genechem, Shanghai, China), and the grouping was shown in Table 3. The infection efficiency could be observed by fluorescence microscope at 72 hours after infection.

| Data analysis
Statistical analyses were performed using one-way ANOVA and were expressed as means ± standard error of the mean (SEM), followed by the Tukey-Kramer test. P < 0.05 was considered statistically significant.

| Component test of ATE
This study established and verified a UPLC-MS method for simultaneous determination of ATE ( Figure 1A,B), and chemical structures of the five compounds in A tataricus were shown in Figure 1C.

| ATE ameliorated bladder damage induced by CYP
There was no significant difference of bodyweight in control, saline and experimental groups (P > 0.05, Figure 2A). However, the wet weight of bladder in control group were significantly lower than that in saline (CYP-induced) group; Compare with the saline group, the wet weight of bladder in each medicated group decreased significantly (P < 0.05) to some extent, and bladder wet weight of ATE-2.4 was lower than that of ATE-1.2 ( Figure 2B). In Figure 2C,D, we found that in the macro score of bladder oedema and haemorrhage, the score of the saline group was generally about 2-3 points, while the score of each medicated group recovered to 0-2 points. The results suggested that CYP-induced modelling had been successful and the ATE could relieve the symptom of oedema and haemorrhage.

| ATE ameliorated SV-HUC-1 cells damage induced by ATP + LPS
We measured the cell viability of SV-HUC1 cells which were mod- The grouping case of overexpression F I G U R E 2 Effect of treatment with ATE on macroscopic evaluation of the bladder. A, Bodyweight. B, Wet weight of bladder. C, Oedema score of bladder was evaluated in CYP-induced interstitial cystitis in mice. D, Haemorrhage score of bladder was evaluated in CYP-induced interstitial cystitis in mice. E, Bladder sections were stained with haematoxylin and eosin (H&E). F, Histological evaluation was performed according to Gray's criteria. All values are expressed as mean ± SEM (n = 8 per group). *P < 0.05, **P < 0.01 compared with CYP group differences compared with control group (in Figure 4A). We detected the degree of pyroptosis of urothelial cells using Hoechst33342 + PI double staining method. As shown in Figure 4B, there were more PIpositive cells in IC model (no ATE) group compared with control and ATE medicated groups (P < 0.01, Figure 4C).

| ATE reduce the expression of pyroptosisrelated protein in cell
The expression of NLRP3, Pro-caspase-1, Caspsae-1 p20, GSDMD, and GSDMD-N in SV-HUC-1 cells in different groups was detected by Western blotting. In Figure 5B, the expression of NLRP3, GSDMD and GSDMD-N in ATE medicated groups was decreased compared with that in no medicated group, and the GSDMD-N group decreased most significantly (P < 0.01, Figure 5A,B).

| Immunofluorescence analysis of NLRP3 in bladder in vivo and vitro
The bladder tissues were observed under a fluorescence microscope, results showed that significant fluorescence signals were detected in the CYP-induced groups compared with the control group, and the NLRP3 fluorescence signals of ATE-1.2 and ATE-2.4 group was significantly lower than that of the saline group, respectively (P < 0.05, P < 0.01, in Figure 6A,B).
In order to investigate the mechanism of NLRP3 in pyroptosis signalling pathway, we over-expressed NLRP3 protein in SV-HUC1 cells.
Results showed that significant fluorescence signals were detected in the LPS + ATP-treated groups compared with the control group, but the NLRP3 expression level of the medicated groups was significantly lower than that of the LPS + ATP-treated groups; At the same time, the expression (NLRP3) of LPS + ATP-treated groups had increased compared with that of no-LPS + ATP-treated groups, and the expression (NLRP3) of ATE medicated group was lower than that of no medicated group ( Figure 6C,D).

| Effect of ATE on the pyroptosis-related proteins in SV-HUC1 cells
In Figure 7A, the relative expression levels of pyroptosis-related proteins were detected by Western blotting. The expression levels of NLRP3, ASC, Pro-caspase-1, Caspsae-1 p20, GSDMD, GSDMD-N and IL-1β in NLRP3-OE groups were increased significantly compared with that in control-OE groups (P < 0.05), and ATE-medicated groups showed lower expression level compared with no medicated groups ( Figure 7B).

F I G U R E 3
Effect of ATE on the pyroptosis-related proteins of bladder in mice with CYP-induced interstitial cystitis. A, Western blot analysis of the expression of pyroptosis-related proteins in bladder. B, Densitometric analysis of pyroptosis-related proteins relative expression. All values are expressed as mean ± SEM (n = 3 per group). *P < 0.05, **P < 0.01 compared with CYP group

| D ISCUSS I ON
The cause of IC is still unclear, because its treatment success rate is not high and easy to relapse, which is still challenging in clinical practice. The inflammation is one of the important factors of IC. 17 The present study investigated the potential therapeutic effects of ATE on IC. We found ATE has a therapeutic effect on Mesna, was positive medicine used for IC treatment, the continuous delivery of Mesna provides a constant source of thiol groups available to the bladder to bind acrolein and reduce local injury. 29 Previous study had reported the anti-inflammatory activity of A tataricus extract, and A tataricus could be used as a diuretic in clinic and has a good therapeutic effect. 30 In present study, five important Consistent with previous studies, our study indicates that NLRP3 plays a critical role in pyroptosis via NLRP3/GSDMD pathway. 32,33 ATE administration blocked the CYP-induced bladder inflammation and inhibited NLRP3 and GSDMD-N expression in the bladder and urothelial cell. We speculated that ATE could significantly inhibit the expression levels of NLRP3, Caspase-1 and GSDMD in the NLRP3 inflammatory pathway. Therefore, our results show that the anti-inflammatory effect of ATE on IC is achieved by inhibiting NLRP3/ GSDMD-N inflammatory pathway.
In conclusion, our results suggest that ATE has a potential pro-

ACK N OWLED G EM ENT
This work was financially supported by Basic Research on Medical and Health Application of Suzhou Science and Technology Program (SYS2019013).

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest in this work. Qingjiang Jin: Conceptualization (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be available from the corresponding author upon reasonable request.