4-PBA inhibits endoplasmic reticulum stress to improve autophagic flux in the treatment of protamine/lipopolysaccharide-induced interstitial cystitis in rats

Interstitial cystitis (IC) has severe clinical symptoms with unclear mechanism. The continuous inflammatory response of the bladder is the basis of its pathogenesis. Endoplasmic reticulum stress (ERS) is involved in the regulation and development of various inflammatory diseases. And autophagy plays an important role in IC. In this study, we mainly focus on the therapeutic effect of endoplasmic reticulum stress and autophagy on protamine/lipopolysaccharide-induced interstitial cystitis. Female Sprague–Dawley rats were randomized into three experimental groups as follows: sham controls(N), IC alone, and IC+4-PBA.Rats in group IC received 10 mg/ml PS in the urinary bladder, followed by 2 mg/ml LPS instillation after 30 min, IC+4-PBA group SD rats received 4-PBA solution administered intragastrically once a day for 5 days. ERS biomarker (GRP78), autophagy-related proteins (LC3I/II, and Beclin1), autophagic flux biomarker (P62), inflammatory biomarkers (IL-6, TNF-a, NF-κB), apoptotic biomarkers (Caspase 3, Bax) were highest in the IC group compared to IC+4-PBA group and N group and the biomarkers expression in IC+4-PBA group were lower than in the IC group, anti-apoptotic biomarker (Bcl-2) was highest in the N group compared to the IC group and IC+4-PBA group and lower in the IC group than in the IC+4-PBA group, oxidative stress biomarkers (HO-1, NQO-1) were remarkably lower in the control group than in the IC and IC+4-PBA groups and notably lower in the IC group than in the IC+4-PBA group. The histological score and mast cell count demonstrated most severe in the IC group than those in the IC+4-PBA group. TUNEL assay examined the level of apoptosis in IC group was higher than in the IC+4-PBA group. The bladder micturition function was significantly improved with 4-PBA treatment. 4-PBA inhibits ERS to recover autophagic flux, and then to suppress the bladder oxidative stress, the inflammatory reaction and apoptosis, finally improve the bladder urinary function in Protamine/Lipopolysaccharide (PS/LPS) induced IC.


Histological evaluation.
The bladder wall of rats (n = 5) of each group was sectioned (5 μm per slice) for histological analysis.HE and toluidine blue staining were applied.Subsequently, the histological score and mast cell counts were determined by an investigator in a blinded fashion.Histological slides were graded by a score of 0-5 as previous reported 16 .The quantification of mastocytes in the lamina propria and muscle layer was estimated at 200× magnification in five random sections from each group.

TUNEL staining. Detection of apoptosis by a TUNEL staining assay kit purchased from Roche Applied
Science on paraffin sections of the bladder of three groups.We carried out the quantitation of the positive cells number at a magnification of 200× in five randomly chosen fields of view on each slide.The apoptosis index was calculated using the ratio of the apoptotic cells to total cells.

Bladder cystometry.
Cystometry was performed in N, IC andIC+4-PBAgroups for 5 SD rats of each, similar to the method described previously 17 .First, we place the PE-50 catheter into the bladder, then body temperature (37-38 °C) saline was infused at a rate of 0.1 ml/min using a syringe pump.Intravesical pressure was recorded continuously, after a stabilizing time of approximately 30 min, urodynamic parameters including basal pressure (BP), maximum pressure (MP), and micturition frequency (MF)were evaluated.
Statistical analysis.SPSS16.0 (SPSS) was used for all analyses by an investigator blinded to the treatment groups.All outcomes were analyzed using one-way ANOVA, followed by Fisher's test to assess differences among treatment groups.A probability value of < 0.05 was considered significant.

Results
Confirmation of the differences of autophagy and autophagic flux with western blot in N and IC groups.The expression of the autophagy biomarkers LC3I/II, and Beclin1 by western blot in the bladder tissue is higher in the IC group compared to control group.Also, the P62 expression, which is one indicator of autophagic flux, is higher in the IC group compared to control group (Fig. 1A-D).The results suggest that autophagy is enhanced in IC, but autophagic flux is inhibited.
Evaluation of ERS, autophagy and autophagic flux factors after 4-PBA treatment.The ERS biomarker (GRP78), autophagy biomarkers (LC3I/II, Beclin1) and autophagic flux biomarker (P62) expression using western blot are highest in the IC group.After 4-PBA treatment, the expressions of all former biomarkers reduce in IC + 4-PBA group (Fig. 2A-E).These results reveal that ERS is enhanced in IC, and autophagic flux is improved after inhibition of ERS.
Evaluation of inflammation-related factors and oxidative stress-related factors.The protein expression of three inflammation-related factors IL-6,TNF-a and NF-κB by western blot in the control group is remarkably lower than in the IC group and the figure inIC+4-PBA group is lower than inthe IC group.The two anti-oxidative indicators oxygenase (HO)-1 and NAD(P)H quinone oxidoreductase (NQO)-1 present in IC group is increased than in the control group, after 4-PBA treatment, the HO-1 and NQO-1expressionwere significantly enhanced compared to the IC group (Fig. 3A-F).These results reveal that the level of inflammatory reaction and oxidative stress are inhibited inhibition of ERS.The quantitative assessment of the histological score and mast cell count.There are massive ulcers, obvious edema, hemorrhage and increased inflammatory cell infiltration (particularly mast cells) in the sub-mucosal and muscular layer of the bladder tissue from the IC group rats compared with the control group.In the IC+4-PBA group, this situation is significantly improved (Fig. 4A-C).The quantitative of the mast cell count and histological score are reduced in the IC group than those in the IC+4-PBA group (Fig. 4D-H).This results demonstrate that the inflammatory response was the most severe in the IC group, it is significantly alleviated after inhibition of ERS.icantly higher in the IC group than in the N group by western blot.And the IC+4-PBA group is markedly lower than the IC group.Furthermore, the index of anti-apoptosis (Bcl-2 protein expression) was remarkably lower in the IC group, this indicator increases in the IC+4-PBA group and is lower than in the N group (Fig. 5A-D).We also use TUNEL staining to examine the apoptosis levels in each group.The most quantity of apoptotic nuclei are observed in the bladder sections of IC group rats, and fewer were present in the IC+4-PBAgroup and more present in the N group (Fig. 5E-H).These results reveal that inhibiting ERS could alleviate apoptotic effects of IC.
Changes in urodynamic parameters.Compared to the control group, basal pressure and micturition frequency per hour are significantly increased in IC group compare to the N group, they are decreased in the IC+4-PBA group than the IC group.No statistical significance was found in the maximum pressure compared between the three groups.These results indicate a recovered micturition function of IC after ERS impeded (Table 1 and Fig. 6).

Discussion
In this study, the expression of the autophagy biomarkersLC3I/II and Beclin1 by western blot in the bladder tissue is higher in the IC group.Also, the autophagic flux indicator P62 expression is higher in the IC group.So the autophagy was enhanced in IC, but autophagic flux was blocked.Autophagic flux refers to the complete process of autophagy including the delivery of cargoto lysosomes and its subsequent breakdown and recycling 8 .Autophagic flux blocking means that the function of autophagy scavenging waste was reduced.Research showed autophagy agonist RAPA could significantly decrease inflammation and improve bladder function of IC, and it may be used as a potential treatment for IC 18 .Based on our research, this result may be caused by the recovery of autophagic flux.Blocked autophagic flux participates in the occurrence and development of many diseases.In pregnancy, the blockade of autophagic flux takes part of disrupting homeostasis in trophoblasts 19 .In glioma, the autophagic flux

(F-H)
The TUNEL assay indicated that in sections from the IC group rats, the most apoptotic nuclei were observed, whereas fewer were present in the IC+4-PBA group and more were present than in the normal control group.* indicates a significant difference compared to the control group value (P < 0.05).# indicates a significant difference compared to the IC group value (P < 0.05). is impaired 20 .The autophagic flux damaged in former diseases are related to ERS.ERS can reduce the abnormal aggregation of proteins in cells by activating the unfolded protein response (UPR), thus playing a role in cell protection.However, long-term and severe ER stress can cause cell apoptosis or death.In our study, the expression of ER stress markerGRP78was enhanced in IC.This indicated that the ERS was enhanced in IC.Then we use ERS inhibitor, 4-Phenylbutyric acid, to treat the IC rats.We found autophagic flux biomarker (P62) reduced.This result indicated that inhibiting ERS could improve the autophagic flux in IC, subsequently the microenvironment of bladder became better, and then autophagy biomarkersLC3I/II and Beclin1 reduced.
ERS inhibiting autophagic flux is the key pathogenic factor of many diseases.HCV-induced ER stress correlates with autophagic flux impairment.Decrease of ER stress improved autophagic flux impairment is considered to be a promising therapeutic strategy for HCV-related chronic liver diseases 21 .Autophagy plays a critical role in the development of non-alcoholic fatty liver and steatohepatitis.ERS inhibiting autophagic flux contributes to high-fat, high-fructose, and high-cholesterol diet-induced liver injury and inflammatory response 22 .In our study, inhibition of ERS improved autophagic flux, the expression of inflammation-related factors (IL-6, TNF-a, NF-κB) and apoptosis indicator(Caspase 3 and Bax) were decreased.Anti-oxidative indicators(HO-1, NQO-1) and anti-apoptosis indicatorBcl-2 were remarkably increased.Histological score, mast cell and apoptotic nuclei count were declined.Finally, the micturition function of IC was recovered after ERS impeded.The application of 4-PBA inhibiting ER stress to restore autophagic flux could improve the bladder urination function of IC.But the specific mechanism is still unclear.
Disruption of ER homeostasis and the accumulation of unfolded or misfolded proteins elicit ERS, subsequently activate downstream signaling pathways: Protein Kinase R-like ER Kinase (PERK), Inositol Requiring Enzyme1α (IRE1α), and Activating Transcription Factor 6 (ATF6) 23,24 .Numerous studies have demonstrated that ERS affects autophagic flux through IRE1 signaling pathway.In the study of insulin resistance, activating ERS could inhibit autophagy flux through IRE1 signal pathway, leading to insulin resistance 25 .ERS inhibits autophagy flux by IRE1signaling pathway, leading to the accumulation of mutant huntingtin protein aggregates and neurotoxicity in Huntingtin 26 .IRE1 interferes with autophagic flux mainly through the following ways: 1. ERS makes autophagosomes and lysosomes blend through Rab7 27 .2. ERS inhibits autophagic flux by inhibiting lysosomal function through IRE1 pathway 28 .3. Since the double membrane structure of autophagosome is derived from the endoplasmic reticulum/Golgi theory, IRE1 regulates the production of endoplasmic reticulum/Golgi, and the ERS IER1 pathway regulates the formation of double membrane structure of autophagosome 29 .Therefore, ERS may block autophagic flux by IRE1 pathway to affect bladder function of IC.
In summary, the results of this study revealed that 4-PBA inhibiting ERS recover autophagic flux.The ability of autophagy removing waste was enhanced, which improved the microenvironment of bladder, ultimately significantly improved the bladder micturition function.It provides a new perspective and supplement for the pathogenesis and treatment of IC (Supplementary file).Table 1.Cystometric parameters changes in the sham control (N), IC, and IC+4-PBA groups.Data presented as means ± SD (n = 5).*Indicates a significant difference compared to the control group value (P < 0.05).# Indicates a significant difference compared to the IC group value (P < 0.05).

Figure 1 .
Figure 1.Expression of autophagy and autophagic flux factors in the urinary bladder at 5 days after IC induction (n = 5).(A) Expression of the autophagy and autophagic flux biomarkers LC3I/II, Beclin1, and P62 by western blot in the bladder tissue were higher in the IC group compared to normal control groups.All the blots were cut before hybridisation with antibodies.(B) A statistical chart of the relative optical density of Beclin1 / GAPDH in each group (n = 5).(C) A statistical chart of the relative optical density of LC3II / LC3I /GAPDH in each group (n = 5).(D) A statistical chart of relative optical density of P62/GAPDH in each group (n = 5).* indicates a significant difference compared to the control group value (P < 0.05).

Figure 2 .
Figure 2. Analysis of protein expression of ERS biomarker (GRP78), autophagy biomarkers (LC3I/II, Beclin1) and autophagic flux biomarker (P62) in the urinary bladder at 5 days after IC induction (n = 5).(A) Expression of the a ERS biomarker (GRP78), autophagy biomarkers (LC3I/II, Beclin1) and autophagic flux biomarker (P62) by western blot in the bladder tissue were highest in the IC group compared to IC+4-PBA group and normal control groups; the biomarkers expression in IC+4-PBA group was lower than in the IC group.All the blots were cut before hybridisation with antibodies.(B) A statistical chart of the relative optical density of GRP78/GAPDH in each group (n = 5).(C) A statistical chart of the relative optical density of Beclin1/GAPDH in each group (n = 5).(D) A statistical chart of the relative optical density of LC3II / LC3I /GAPDH in each group (n = 5).(E) A statistical chart of the relative optical density of P62/GAPDH in each group (n = 5).* indicates a significant difference compared to the control group value (P < 0.05).# indicates a significant difference compared to the IC group value (P < 0.05).

Figure 3 .
Figure 3. Expression of inflammatory-related factors and oxidative stress-related factors in the urinary bladder at 5 days after IC induction (n = 5).(A) Expression of the inflammation-related factors IL-6, TNF-a, NF-κB by western blot in the bladder tissue was highest in the IC group compared to IC+4-PBA group and normal control groups; the biomarkers expression in IC+4-PBA group was lower than in the IC group.The oxidative stressrelated factors HO-1 and NQO-1 by western blot was remarkably lower in the control group than in the IC and IC+4-PBA groups and notably lower in the IC group than in the IC+4-PBA group.All the blots were cut before hybridisation with antibodies.(B) A statistical chart of the relative optical density of IL-6/GAPDH in each group (n = 5).(C) A statistical chart of the relative optical density of NF-κB /GAPDH in each group (n = 5).(D) A statistical chart of relative optical density of TNF-a /GAPDH in each group (n = 5).(E) A statistical chart of relative optical density of HO-1/GAPDH in each group (n = 5).(F) A statistical chart of relative optical density of NQO-1/GAPDH in each group (n = 5).* indicates a significant difference compared to the control group value (P < 0.05).# indicates a significant difference compared to the IC group value (P < 0.05).

Figure 4 .
Figure 4.The quantitative assessment of the histological score and mast cell count in the urinary bladder at 5 days after IC induction (n = 5).(A-C) Photomicrograph images of H&E staining in rat bladder samples (scale bars are 200 μm).(D-F) Representative photomicrograph images of rat bladder samples stained with toluidine blue (arrows) demonstrate mast cells (scale bars are 200 μm).(G) The statistical chart demonstrates the inflammation grading (n = 5).(H) A statistical chart reveals the number of mast cells in the bladder of rats (n = 5).* Indicates a significant difference compared to the control group value (P < 0.05).# indicates a significant difference compared to the IC group value (P < 0.05).

Figure 5 .
Figure 5. Analysis of protein expression of the apoptosis biomarkers Bax and Caspase 3 and the anti-apoptosis indicator BCL-2 and a TUNEL assay in the urinary bladder at 5 days after IC induction (n = 5).(A) Expression of the apoptosis biomarkers Caspase 3 and Bax by western blot in the bladder tissue was highest in the IC group compared to the IC+4-PBA group and normal control group; the biomarker expression in the IC+4-PBA group was lower than in the IC group.Expression of anti-apoptosis indicator BCL-2 was highest in the normal control group compared to the IC group and IC+4-PBA group, lower in the IC group than in the IC+4-PBA group.All the blots were cut before hybridisation with antibodies.(B) A statistical chart of the relative optical density of Bax/GAPDH in each group (n = 5).(C) A statistical chart of the relative optical density of BCL-2/GAPDH in each group (n = 5).(D) A statistical chart of the relative optical density of Caspase 3/GAPDH in each group (n = 5).(E) A statistical chart reveals the index of apoptotic nuclei in all groups of bladder tissue (n = 5).(F-H)TheTUNEL assay indicated that in sections from the IC group rats, the most apoptotic nuclei were observed, whereas fewer were present in the IC+4-PBA group and more were present than in the normal control group.* indicates a significant difference compared to the control group value (P < 0.05).# indicates a significant difference compared to the IC group value (P < 0.05).