Identification of key pathways and mRNAs in interstitial cystitis/bladder pain syndrome treatment with quercetin through bioinformatics analysis of mRNA-sequence data

Experimental rats and ethical approval

40 specific pathogen free female Sprague-Dawley (SD) rats (200 ± 20 g, n = 5) were provided by Hunan SiLaiKeJingDa Experimental Animal Co., Ltd. (SCXK (Xiang) 2019-0004). Rats were raised in the environment with 22 ± 1°C, 50 ± 5% humidity and 12-h light/dark cycle, and the rats had free access to water and food. Our study was approved by the Laboratory Animal Ethics Committee of the Kunming Medical University (IACUC Issue No: kmmu2021153). All experiments were performed in accordance with relevant guidelines and regulations. This study is reported in accordance with ARRIVE guidelines.

Construction of IC/BPS model

After feeding for one week, SD rats were randomly divided into 5 groups (Ctrl group n = 10, IC/BPS group n = 10, IC/BPS+QCT group n = 10, IC/BPS+QCT+pcDNA-NC group n = 5, IC/BPS+QCT+pcDNA-Lpl group n = 5). 150 μL LL-37 was slowly injected into rat bladder through catheter on the first, fourth and seventh day to establish rat model. On the eighth day, HE and Masson staining were used to identify the successful establishment of IC/BPS model.

QCT treatment and plasmid injection

One day after the construction of IC/BPS model, 20 mg/kg QCT was intraperitoneally injected to rats [9]. Then 20 mg/kg QCT was injected on the third and sixth day. Rats in the Ctrl groups were given the same amount of normal saline.

Then two hours later, the rats in IC/BPS+QCT+pcDNA-NC group and IC/BPS+QCT+pcDNA-Lpl group was injected pcDNA-NC and pcDNA-Lpl plasmid through tail vein, and then injected with plasmid again on the third and sixth days after the model was established.

Sample collection

Before the rats were sacrificed, the peripheral blood was collected, and serum was collected from whole blood by centrifugation for ELISA assay. Another part of whole blood from the IC/BPS+QCT group was used for HPLC assay. The 24-h urine volume (The seventh day) and the residual urine after bladder removal in the IC/BPS+QCT group were collected and used for HPLC assay.

The bladder tissues were divided longitudinally into 3 parts. One portion of the tissue was fixed with 4% paraformaldehyde for morphological testing. One portion of the tissue was frozen in liquid nitrogen for mRNA sequencing. One portion of the tissue was fixed with 2.5% glutaraldehyde and used for transmission electron microscopy.

HPLC assay

After acidification of 100 μL plasma or urine with 20% formic acid solution, 10 μL ascorbic acid was added to prevent oxidation of QCT. A total of 300 μL acetonitrile was added into the sample to extract QCT. After the QCT was centrifuged at 12000 r/min for 10 min, the supernatant was taken, and methanol was added into the residue to repeat the above extraction steps again. The mixed solution was air-dried by nitrogen flow, reconstituted by adding methanol solution, and filtered through a 0.22 μm microporous membrane. The samples were analyzed by Shimadzu LCMS-8040 triple and four-stage liquid chromatography-mass (LC-MS) spectrometry (Shimadzu, China).

ELISA

ELISA kit detected the concentration of IL-1β, IL-6, MPO and TNF-α (Shanghai Enzyme-linked Biotechnology Co., Ltd., China). According to the instruction of ELISA kit, the standard (50 μL) and sample (40 μL Sample diluent and 10 μL sample) were added to the standard holes and sample holes of microtiter plate, respectively. The culture plates were incubated at 37°C in 5% CO₂ for 30 min and then rinsed with washing solution. Then 50 μL enzyme-labeled reagent was added into each well, and the samples were incubated for 30 min and washed. Then 50 μL color developer A and color developer B were added, mixed, and stained in the dark for 10 min. After the reaction was stopped by adding stop solution, the absorbance density (OD) was measured at 450 nm by enzyme-labeled instrument (Molecular, CA, USA).

HE

The bladder tissue fixed with paraformaldehyde was placed in an embedding box, and xylene (XiLong Chemical Co., Ltd., China) was added for transparency after dehydration with ethanol (XiLong Chemical Co., Ltd., China). The tissue samples were cut into 5 μm thick sections after paraffin embedding. The paraffin sections were rinsed with dewaxing, hydration and distilled water, stained with hematoxylin (Sigma, MO, USA) for 10 min, and differentiated 2 s using 1% hydrochloric acid ethanol after rinsing. Subsequently, it was stained with eosin (Sigma, MO, USA) for 5 min. After stopping coloration, it was dehydrated with ethanol and transparent by adding xylene. The slices were sealed with neutral glue (Shanghai Aladdin, China). The staining results were observed by a microscope (JiNan DanJiEr Electronics Co., Ltd., China).

Masson

After dehydration, wax dipping and embedding of the fixed bladder tissue, the sections were placed in a 65°C incubator for 30 min. After dewaxing and hydration, the samples were stained with Weigert iron hematoxylin (Beijing Solarbio Science and Technology Co., Ltd., China) for 10 min. After differentiation with acidic ethanol (Xilong Chemical Co., Ltd., China), the sections were rinsed and stained with Masson Blue Solution (Beijing Solarbio Science and Technology Co., Ltd., China) for 3 min. After washing, Ponceau S was added for staining for 10 min. After the sections were rinsed again, they were made transparent by xylene, sealed with neutral glue and then observed under a microscope.

TB staining

Tissue samples were fixed, paraffin embedded, sectioned and then dewaxed and hydrated with gradient ethanol. After the slices were washed with distilled water, TB staining solution (Beijing Solarbio Science and Technology Co., Ltd., China) was added for staining for 5 min. The slices were rinsed, added with 0.5% acetic acid to differentiate into blue positive cells, dehydrated with gradient ethanol, and made transparent with xylene for 30 min. After the slices were sealed with neutral glue, then the scanning electron microscope was used to observe the results.

Electron microscopy

The bladder tissue was first fixed with glutaraldehyde solution and then treated with the addition of 1% OsO₄. After gradient ethanol dehydration, Quetol 812 resin was added to fix the bladder tissue again. After sectioning, the sections were stained with saturated uranyl acetate in 50% ethanol solution for 30 min and 0.1% lead citrate solution for 15 min at room temperature, washed and observed under a transmission electron microscope.

RNA extraction

The bladder tissue of rats was taken, cut into pieces with scissors and ground with liquid nitrogen. TRIzol reagent was added and mixed for complete lysis, and the supernatant was taken after centrifugation. After chloroform was added and shaken for 30 s, isopropanol and ethanol were added, respectively. The supernatant was discarded after centrifugation to obtain RNA. The OD value was detected by microplate reader.

Library construction

The transcription build library kit was NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^®. The first strand of cDNA was synthesized in M-MuLV reverse transcriptase system with segmented mRNA as template and random oligonucleotide as primer. Then RNA strand was degraded by RNaseH, and the second strand of cDNA was synthesized from dNTPs in DNA polymerase I system. The purified double-stranded cDNA was repaired at the end, poly (A) tail was added, and the sequencing adapter was connected. AMPure XP Beads were used to screen 250–300 bp cDNA. PCR amplification was performed, and AMPure XP beads were used to purify PCR products. Finally, the library was obtained.

mRNA-sequence

The Qubit 2.0 was used for initial quantification, and the library was diluted to 1.5 ng/μL. Agilent 2100 was used to test the distribution of inserts in the library. After the inserts met the expectation, RT-qPCR was performed to accurately quantitate the effective concentration of the library (the effective concentration of the library is higher than 3 nM) to ensure the quality of the library. For qualified libraries, a minimum of 6G of data was measured for each sample using the Illumina NovaSeq PE150.

Raw data filtering

To reduce data noise, unqualified sequential reads filters were performed on the original FastQ data, including filtered reads with dropped adapter, reads containing N (N indicates that no base information could be determined) and low-quality reads (Qphred ≤20 reads with bases accounting for more than 50% of the entire read length). The raw data were subjected to the above-mentioned filtering processing and becomes clean data for bioinformatics analysis [10].

Functional and pathway enrichment and construction of protein–protein interaction (PPI) network

The differential gene sets were enriched in the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases by the R package “cluster profiler” [11]. Protein-protein interaction (PPI) network was constructed by STRING and further visualized by Cytoscape.

Data availability

The mRNA sequencing raw data generated and analysed during the current study are available in the NCBI SRA repository (Sequence Read Archive; https://www.ncbi.nlm.nih.gov/sra/PRJNA898519). The communication author can be contacted for more information on the nine mRNA sequencing data presented herein.

Immunohistochemistry (IHC)

Rat bladder tissue was fixed with 4% paraformaldehyde, embedded in paraffin and sliced. The slices were baked in an oven at 60°C for 30 min, and then dewaxed. Citric acid buffer (pH = 6.0) repaired antigen. The histochemical pen drew a circle on the section to prevent the liquid from leaking out during the dyeing process, and the section was sealed with 5% goat serum for 1 h. The slices were incubated with primary antibody (Lpl, 1:100) and secondary antibody (Lpl, 1:200) respectively, and reacted with DAB chromogenic solution for 1 min. The nuclei were stained with hematoxylin. The slices were treated alcohol and xylene, then sealed with neutral glue, and observed under a microscope.

Western blot

The total protein was extracted RIPA lysate (Beyotime), and it was denatured by boiling at 100°C for 10 min. IMPLEN ultra-micro spectrophotometer was used to detect the protein concentration. 80 μg samples were subjected to SDS-PAGE gel electrophoresis, and then transferred to PVDF membrane. PVDF membrane (Millipore, MA, USA) was sealed with 5% bovine serum albumin (Solarbio) for 2 h, rinsed with TBST, and then incubated with primary antibody and secondary antibody respectively. Enhanced chemiluminescence (Applygen, China) was used to show protein bands.

Statistical analysis

All data were analyzed by R software (version 4.2.2) or GraphPad Prism 8.0. One-way ANOVA was used to analyze the difference among multiple groups. The DESeq2 package (version 1.38.3) of R software is used for Wald test of sequencing data [12]. The p-values are calculated based on Fisher test of negative binomial distribution. The adjusted p-values and FDR values are corrected by the BH algorithm for multiple hypothesis testing. P < 0.05 was considered statistically significant. In the statistical analysis of mRNA sequencing results, when P.adj <0.05 it is considered to be statistically significant.

The concentration of QCT in the urine and plasma of rats

To investigate the mRNA expression in rats after QCT treatment, we first analyzed the concentration of QCT in plasma and urine of rats after QCT treatment by HPLC. In the QCT standard, (M-H)⁻ was used as the precursor ion, and the characteristic fragment ions were 150.85, 178.80, and 121.00, among which the quantitative ion was 150.85, and the retention time was 5.577 min, as shown in Figure 1A. Then, the standard curve of QCT in the plasma and urine is constructed respectively (Figure 1B, 1C and Table 1). It was confirmed that the retention time of QCT in plasma and urine was the same as that of QCT standard (Figure 1D–1L and Table 2). The concentration of QCT in 24-hour urine group was higher than that in the urine group and plasma group (Figure 1M), but the level of QCT in the urine group was not significantly different from that in the plasma group. These results showed that after injection of QCT, most QCT was excreted through the urinary system, and a small part was absorbed by the digestive system and entered the blood.

Figure 1. HPLC detected the concentration of QCT. (A) The HPLC chromatogram of QCT standard. (B) The standard curve of QCT in urine matrix. (C) The standard curve of QCT in plasma matrix. (D) The HPLC chromatogram of QCT in Urine-1 group. (E) The HPLC chromatogram of QCT in Urine-2 group. (F) The HPLC chromatogram of QCT in Urine-3 group. (G) The HPLC chromatogram of QCT in Urine 24 h-1 group. (H) The HPLC chromatogram of QCT in Urine 24 h-2 group. (I) The HPLC chromatogram of QCT in 24 h-3 group. (J) The HPLC chromatogram of QCT in plasma-1 group. (K) The HPLC chromatogram of QCT in plasma-2 group. (L) The HPLC chromatogram of QCT in plasma-3 group. (M) QCT concentration statistics of each group. ^*P < 0.05, ^**P < 0.01. Abbreviation: ns: no significant difference.

The effect of QCT on IC/BPS rats

Next, we observed the influence of QCT on IC/BPS model rats through experiments, and found that the level of IL-1β (Figure 2A), IL-6 (Figure 2B), MPO (Figure 2C) and TNF-α (Figure 2D) in the IC/BPS group increased compared with the control group. QCT treatment significantly inhibited the concentrations of IL-1β, IL-6, MPO and TNF-α in the serum of the IC/BPS rats. HE staining showed that LL-37 induced inflammatory infiltration of rat bladder tissue was significantly more than that of the control group (Figure 2E), and QCT reduced the inflammatory infiltration of IC/BPS rats. Masson and TB staining showed that the area of collagen fibers (Figure 2F, 2G) and the number of mast cells (Figure 2H, 2I) increased significantly in LL-37-induced rats, and the area of collagen fibers and the numbers of mast cells were inhibited by QCT treatment.

Figure 2. The effect of QCT on IC/BPS rats. ELISA kit detected the concentration of IL-1β (A), IL-6 (B), MPO (C), TNF-α (D). (E) HE staining was used to deserve the inflammatory of bladder tissue in rat. (F, G) Changes of collagen fibers in rat bladder tissue was performed by Masson staining. (H, I) TB staining observed the mast cells in bladder tissue of rat. (J) The degranulation of mast cells in rat bladder tissue was observed by transmission electron microscope. One-way ANOVA was used. Compared with Ctrl group, ^**P < 0.01, ^***P < 0.001. Compared with IC/BPS group, ^ΔP < 0.05, ^ΔΔP < 0.01, ^ΔΔΔP < 0.001. n = 3.

The results of the transmission electron microscope are shown in Figure 2J. In the bladder tissue of rats in the IC/BPS group, the mast cells infiltrated obviously, and the degranulated mast cells appeared vacancies. The particulate matter in the cytoplasm was released to the outside. The degranulation of mast cells was significantly improved in the QCT-treated group. The results demonstrated that QCT may alleviate LL-37-induced inflammation and the increase of collagen fibers and mast cells in rats.

The expression of mRNA in different groups

We collected the rat bladder tissue for mRNA sequencing, and found that there were 646 mRNAs differentially expressed (DE) (|log2 FC| ≥2, P.adj ≤0.01). Compared with the control group, there were 442 mRNAs up-regulated and 204 mRNAs down-regulated in the IC/BPS group (Figure 3A). Compared with IC/BPS group, there were 219 mRNAs DE in IC/BPS+QCT group, including 121 mRNAs up-regulated and 98 mRNAs down-regulated (Figure 3B). Tables 3 and 4 show the top 20 up-regulated mRNAs and top 20 down-regulated mRNAs. Venn analysis was performed on DE mRNA in the two comparison groups, there were 32 mRNAs co-expression in IC/BPS vs. control group and IC/BPS+QCT vs. IC/BPS group (Figure 3C). Clustering analysis was shown in heatmap (Figure 3D, 3E).

Figure 3. The expression of mRNA in different groups. (A) Volcano map of differential mRNA between B group and A group. (B) Volcano map of differential mRNA between B and C group. (C) Venn diagram of DE mRNAs in B vs. A and C vs. B group. (D) DE genes clustering heat map between B and A group. (E) Differential gene clustering heat map between B and C group. A: Ctrl group. B: IC/BPS group. C: IC/BPS+QCT group.

Pathway enrichment analysis

In order to further study the biological function of DE mRNA, GO enrichment analysis was carried out. The top 20 enriched GO terms in BP, CC and MF were shown in Figure 4A. Compared with control group, the terms of DE mRNA in the IC/BPS group mainly included organelle fission, cell division, nuclear division, external encapsulating structure, mitotic cell cycle phase transition, extracellular matrix, chromosome segregation, extracellular matrix organization, extracellular structure organization, external encapsulating structure organization (Figure 4B). Compared with the IC/BPS group, the GO terms of DE mRNA in the IC/BPS+QCT group mainly included ncRNA metabolic process, ribonucleoprotein complex biogenesis, ncRNA processing, small molecule catabolic process, positive regulation of proteolysis, ribosome biogenesis, response to insulin, rRNA metabolic process, rRNA process, cellular amino acid metabolic process (Figure 4C, 4D).

Figure 4. GO function enrichment analysis. (A) GO annotation analysis of differential mRNA in BP, CC, MF between B and A group. (B) Top 20 of GO annotation terms between B and A group. (C) GO annotation analysis of differential mRNA in BP, CC, MF between C and B group. (D) Top 20 of GO annotation terms between C and B group. A: Ctrl group. B: IC/BPS group. C: IC/BPS+QCT group.

KEGG analysis showed that compared with the control group, human T-cell leukemia virus 1 infection pathways, focal adhesion, Epstein-Barr virus infection, cell cycle, cellular senescence were enriched in the DE mRNA of the IC/BPS group (Figure 5A). Compared with IC/BPS group, pathways of neurodegeneration - multiple diseases, amyotrophic lateral sclerosis, MAPK signaling pathway, protein processing in endoplasmic reticulum, lipid and atherosclerosis pathway were significantly enriched in the IC/BPS+QCT group (Figure 5B).

Figure 5. Analysis of pathway enrichment. (A) KEGG analyzed the top 20 enriched pathway between B and A group. (B) KEGG analyzed the top 20 enriched pathway between B and C group. (C) Reactome analyzed the top 20 enriched pathway between B and A group. (D) Reactome analyzed the top 20 enriched pathway between B and C group.

Reactome pathway analysis showed that cell cycle, “cell cycle, mitotic”, neutrophil degranulation, M Phase, cell cycle checkpoints, extracellular matrix organization were enriched in DE mRNAs of the IC/BPS (Figure 5C). And neutrophil degranulation, metabolism of carbohydrates, “metabolism of amino acids and derivatives”, “metabolism of vitamins and cofactors” were significantly enriched in DE mRNAs of the IC/BPS+QCT group (Figure 5D).

PPI network construction and the most significantly modules of genes

We intersected the mRNA that was DE at the same time in the IC/BPS vs. control group and the IC/BPS+QCT vs. IC/BPS group (|logFC| > 1 and P.adj <0.05). A total of 179 mRNAs were imported into the PPI network using STRING, there were 179 nodes and 435 edges in the PPI network (Figure 6A). We applied Cytoscape MCODE for further analysis and found that 13 central nods (Cyr61, Cd44, Spp1, Mmp7, Plau, Ldlr, Pcsk9, Mttp, Plat, Itgb1, Itga5, Vtn, Serpine1) formed the significantly modules among 179 nodes (Figure 6B). The hub gene of was confirmed by Cytobubba (a plug-in Cytoscape). The top ten genes included Serpine1, Itgb1, Vtn, Spp1, Cd44, Plau, Itga5, Mmp7, Apob and Lpl (Figure 6C). Among them, Serpine1, Spp1, Itga5, Mmp7 and Lpl were co-expressed in IC/BPS+QCT group and IC/BPS group (Table 5).

Figure 6. The PPI network and the most significant modules of genes. (A) PPI network was analyzed in B vs. A group and B vs. C group (|log2 Fold change| ≥ 1 and P.adj <0.05) by STRING software. (B) The most significant module was identified by Cytoscape MCODE (degree cutoff = 2, node score cutoff = −0.2, k-core = 2, and max. Depth = 100). (C) The top 10 hub genes were identified by Cytoscape Cytohubba.

Lpl involved in the therapeutic process of QCT on IC/BPS rats

According to Cytobubba’s prediction and mRNA sequencing results, we chose Lpl for further study. RT-qPCR results indicated that Lpl mRNA was decreased in IC/BPS group (Figure 7A), QCT treatment promoted Lpl expression. IHC staining displayed the same results with RT-qPCR (Figure 7B). Next, we further explore the regulatory effect of Lpl on IC/BPS rats. We injected Lpl overexpression plasmid and its negative control into rats through tail vein, and found that Lpl protein was significantly upregulated in Lpl overexpression group (Figure 7C). Pathological staining showed that compared with QCT+pcDNA-NC group, the infiltration of inflammatory cells in the overexpressed Lpl group was less than that in the control group, the cells were arranged neatly (Figure 7D) and the collagen fibers in the tissue were reduced (Figure 7E, 7F). ELISA kits were used to examine the level of GAG, MPO, IL-1β, IL-6 and TNF-α. And the results revealed that overexpression of Lpl increased GAG level in the bladder tissue of rats (Figure 7G). However, MPO, IL-1β, IL-6 and TNF-α were depleted in Lpl overexpression group (Figure 7H–7K). These results demonstrated that treatment with QCT upregulated Lpl expression, increased Lpl relieved the development process of IC/BPS.

Figure 7. Lpl was involved in the therapeutic process of QCT on IC/BPS in rats. (A) Lpl mRNA expression was detected using RT-qPCR. (B) The IHC staining of bladder tissues. (C) The protein expression of Lpl. (D) The HE staining of bladder tissues. (E, F) Masson staining was used to evaluate the collagen and smooth muscle in bladder tissues. The levels of GAG (G), MPO (H), IL-1β (I), IL-6 (J), TNF-α (K). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.